Journal of Biochemical Technology

25 August, 2008 Volume 1, Issue 1 ISSN 0974-2328 Journal of Biochemical Technology J. Biochem. Tech Editor: R.R. Siva Kiran Associate Editors: Brijesh Eugenia Xu http://jbt.biodbs.info SEVAS Publishing Journal of Biochemical Technology Editor R R Siva Kiran Dept. of Biotechnology, M S Ramaiah Institute of Technology, MSR Nagar, Bangalore-560054, India (editor@biodbs.info) SEVAS Publishing Editorial Board Advisory David Wood Dept. of Chemical Engineering, Princeton University, Princeton, NJ 08544, USA (dwood@princeton.edu) G Hanumantha Rao Dept. of Chemical Engineering, Andhra University, Visakhapatnam, 530003, India (ghrao_777@yahoo.co.in) Hideaki Shiraishi Graduate School of Biostudies, Kyoto University, Kyoto 606 8502, Japan (siraisi@kuchem.kyoto-u.ac.jp) Lawrence K Duffy Dept. of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK 99775 6160, Alaska (fflkd@uaf.edu) Alex Georgakilas Department of Biology, East Carolina University, Greenville, NC 27858, United States of America (georgakilasa@ecu.edu) Archna Dept of Biotechnology, M S Ramaiah Insitute of Technology, MSR Nagar, Bangalore - 560054, Karnataka, India (archna@msrit.edu) Anthony A Capehart Department of Biology, East Carolina University, Greenville, NC 27858, United States America (capehartt@ecu.edu) Associate Editors Brijesh Dept. of Chemical Engineering, M S Ramaiah Institute of Technology, Bangalore-560054, India (brijesh@biodbs.info) Eugenia Xu Dept. of Molecular Biology, Princeton University, Lewis Thomas Lab, Washington Rd., Princeton, NJ 08544, USA (eugenia@biodbs.info) Associate Editors Advisory Marcelo Fernandez Lahore School of Engineering and Science, Biochemical Engineering, Jacobs University, P.O. Box 750561, Bremen, 28725, Germany (m.fernandez-lahore@jacobs-university.de) V Padmaja Dept. of Botany, College of Sciences and Technology, Andhra University, Visakhapatnam 530003, India (vpadmaja4@yahoo.com) Rene Kizek Mendel University of Agriculture and Forestry, Laboratory of Molecular Biochemistry and Bioelectrochemistry, Zemedelska 1, 61300 Brno, Czech Republic (kizek@sci.muni.cz) Sevas Publishing Staff R N Lakshmi Naidu, Publishing Editor (info@sbioinformatics.com) K Sharada (sharadabioinf@gmail.com) Scope “Journal of Biochemical Technology” is a quarterly journal published by “Sevas Educational Society”. The journal publishes advances in the field of biochemical research, biochemical engineering & bioinformatics. It provides a medium for the rapid publication of full-length articles, mini-reviews of new and emerging products and short communications on all aspects of biochemical technology. It is a unique source for scientists interested in both engineering as well as basic biological research. Cover Coverage includes enzymes and proteins; applied genetics and molecular biotechnology; computational biology, genomics and proteomics; metabolic & tissue engineering; medical, environmental, food and agro biotechnology; biodiversity, reactor design, modeling. Major Keywords: 1. Biochemical Research: Molecular Mechanisms, Biophysics, Biomolecular Associations, Systems Biology, Chemical Biology, Membrane Biology, Proteomics, Drug Targets, G-Proteins, Enzymology, Bioenergetics, Stem Cells, Photosynthesis, Signalling, Nitrogen fixation, Plant Disease, Circadian Controls, Hormones, Cellular Metabolism, Cell Growth, Cell Cycle, Cell Migrations, Oxidative Stress, Endo/Exocytosis, Trafficking. Computational & Nanobiology Research: Structure, Proteins, Nucleic Acids, Amino Acids, Database Developments, Nanobiotechnology, Software Tools Development, System Biology, Algorithms, Advances in Bioinformatics, Chemoinformatics, Genomics, Proteomics, Drug Design, Drug Targets, Statistical Modeling, Biophysics, Chemical Biology, Biomolecules, Molecular Mechanisms, Enzymology, Lipids & Carbohydrates. Biochemical Engineering Research: Industrial Biotechnology, Bioreactors, Modeling & Simulation Software, Statistical Modeling, Design of Experiments, Artificial Neural Networks, Advances in Bioprocess Design Engineering, Enzyme Technology, Environmental Biotechnology, Metabolic Engineering, Tissue Engineering, Agro Biotechnology & Medical Biotechology. 2. 3. i Sevas Publication Division “Sevas Publication Division” is maintained by a non-governmental, not-for-profit organization “Sevas Educational Society”. "Seva" means selfless service in Sanskrit (An Ancient Indian Language), “The Society" was established on August 4th, 2006 at Komatipalli, a remote village near Visakhapatnam, South India. The society aims at the development of villages by educating people in various fields like biotechnology and bioinformatics. The society concentrates on distributing technologies for producing cost effective biofertilizers, bioinsecticides, single cell protein (Spirulina) and biodiesel. The funds generated from this journal will be directed towards village development. The society has started this journal to increase the standards of faculties in developing countries. This journal maintains “Authors Advisory Board” which will help authors to reach the Journal standards. Research articles not reaching a satisfactory standard will be given to the advisory board by the editor. Advisory Board will assist authors in experimental protocols, analytical procedures, presentation style, formatting, submitting articles online, understanding author instructions etc. Authors Advisory Board Carlo Zambonelli Liangran Zhang Jin Ah Kwon Gil Blander Kenneth Wiley Olivier Binda Emily Abrash Ruhul H Kuddus Farzin Roohvand Yeoh Hock Hin Ahalya N Suwanchai Nitisoravut John K Sakellaris Manoj Tyagi Abdel-Badie Salem Oganesyan Vaheh Serwan M J Baban Neal L First Pavol Miskovský Manjunath R Gaolin Zheng Milledge Christina A Binag Annie M Jarabek Odeta Laknori Pattle P T Pun Ing. Hana Stritecka Chandraprabha M N Aleksandra B Korac Constantin Volosencu Elbrus Jafarov Roberto Revetria Mirela Lika Pierre Schembri Wismaye Ezendu Ariwa Antonio Tiezzi John Caulfield Aleksander F Sikorski Arunrat Chaveerach Sanjeevani Arora Ioan Turcu Razimah Abdullah Mohd Saidfudin Bin Masodi Somsong Nanakorn Ivana Maksimovic Lucia Altucci Ramesh Sistla Zakir Husain Anninos P Calin Ciufudean Omar Badran Dana Simian Manoj K Jha Srinivasan N Arun Sharma Ashok Kumar S Rene Kizek Vojtech Adam Mario Spremic Ranjit Prasad Bahadur Yeoh Hock Hin Farzin Roohvand Zeljko Tomanovic Miroslav Pohanka Di Wu Cui Tao Sagar Chittori Dhamodhar P Walter J Banta M Shiva Naresh Sevas Educational Society Council Vice Chairman G Swami Naidu Secretary R N Lakshmi Naidu Treasurer G Appa Rao Deputy Secretary S Saradha Society Advisory Board R V R Naidu Brijesh Y M K Prasad Members A Srinivasa Rao M Shiva Naresh V Appla Naidu K Hemantha Kumar Society Office Address Komatipalli, Bobbili Mandal, Vizianagaram (Dt), Andhra Pradesh (St), India Pin: 535558 Tel: 0091 8944 255595 http://www.sbioinformatics.com Email: info@sbioinformatics.com Registration & Affiliation The society was registered under “The Societies Registration Act”, 1860, Indian Government. Registration Number: 165/2006 Journal of Biochemical Technology (ISSN 0974 – 2328) is published quarterly by the Sevas Publication Division, Sevas Educational Society, Komatipalli 535558, Bobbili, Andhra Pradesh for delivery in the India at $115* and for all other foreign countries at $125* per calendar year. Periodicals postage paid at Bobbili-535558, Andhra Pradesh, India. (*Postage Extra) Copyright © 2008 by the Sevas Educational Society. Printed in India. Subscriptions to Journal of Biochemical Technology: All members of the Sevas Educational Society receive J Biochem Tech Online as a benefit of membership for $12 (All countries). Institutional & Industrial subscriptions are available according to a tiered rate structure; please visit http://jbt.biodbs.info/Subscription.html for additional information. 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A possible role of repair proteins BRCA1 and DNA-PK in the processing of oxidative DNA damage Alexandros G Georgakilas 09-10 Review Articles 4. How effective is T-DNA insertional mutagenesis in Arabidopsis? Yi Hong Wang 11-20 Special Contributions 5. Hatching enzyme of Volvox: a possible implication in the evolution of multicellularity Hideaki Shiraishi In search of best impact factor and citation indexed journals towards achieving the goals of universities Hj. Kamaruzaman Jusoff 21-22 6. 23-29 Recent Abstracts in Biochemical Technology Acknowledgment 30-37 38 J Biochem Tech (2008) 1(1):1-05 ISSN: 0974-2328 Mapping the chromatographic behavior of a cell proteome utilizing orthogonal routines: the influence of feedstock pH Rosa Cabrera and Marcelo Fernandez-Lahore* Received: 3 August 2008 / Received in revised form: 15 August 2008, Accepted: 19 August 2008 Published online: 20 August 2008 © Sevas Educational Society 2008 Abstract Surface charge, molecular weight, and folding state are known to influence protein chromatographic behavior onto ion-exchangers. Experimentally, information related to such factors can be gathered via two-dimensional electrophoretic (2-DE) methods. The separation behavior depicted by the insect cultured-cells proteome, which is an important host for recombinant protein production, was explored in this study. Experimental evidence showed a correlation between apparent isoelectric point distributions and the mobile phase conductivity. It was observed that the information contained in the isoelectric point (pI) value(s) obtained with a 2-DE routine showed a good correlation with the IEX chromatographic behavior, for a number of commercial adsorbents. This correlation was observed irrespective of the pH of the feedstock within the range 6 to 8. An initial prediction of protein ion-exchange chromatographic behavior could be possible utilizing an experimental approach based on the mentioned orthogonal methods. This technique is providing information that more closely resembles the separation behaviour observed with a complex biotechnological feedstock. Keywords: Insect cells, proteome, chromatography, ion-exchange, bioprocessing consequence, bioprocess technology options have became limited considering both total available manufacturing capacity and process efficiency and cost. The main obstacle to simplified and costeffective bioproduct processing are the inefficiencies associated with the recovery and purification of these macromolecules. Chromatography, usually employed sequentially in various operational modes, is a wide spread method which represents the major costs is downstreaming of biotechnological relevant proteins and products (Novais et al. 2001). Some recent progress has been made to better understand bioproduct chromatographic behaviour and to improve the modelling of this type of unit operations (Salgado et al. 2006; Salgado et al. 2006). Certainly, there are many possible routes when selecting a sequence of high-resolution chromatographic steps, especially when dealing with a complex biological protein mixture i.e. a crude or partially purified real feedstock. Expert systems have been proposed in an attempt to link protein physicochemical properties and the ability of a defined unit operation to separate the product from discrete number of contaminants. Most of these approaches to understand bioproduct behaviour alongside the downstream pathway have focused on the targeted species and often were limited to the utilisation of pure proteins as a model (Asenjo et al. 2004). In a previous communication (Cabrera and Fernandez-Lahore, 1997), our group has described a routine to define the contaminant proteome of real recombinant expression hosts in order to provide an appropriate description of individual contaminant behaviour during purification. The development of databases describing such purification behaviour, as a function of the nature of the expression system, and in relation with the chromatographic mode utilised might open the way for easy in silico downstream processing development. A change in the current paradigm i.e. changing from focusing on the protein-product to defining the purification profile of the main protein-contaminants would make this novel approach attractive (Cabrera et al. 2007). Analytical proteomics tools, originally developed to study the complexity of biological systems in the post-genomic era (Elrick et al. 2006), are now available to help in understanding complex separation process situations (Cabrera et al. 2008). The underlying mechanisms governing the chromatographic separation of proteins and other bioproducts is not completely understood, particularly when complex protein mixtures are involved. Introduction The modern life science industry is facing a real challenge to deliver new products to a highly demanding and regulated market. As a Rosa Cabrera and Marcelo Fernandez-Lahore* Downstream Processing Laboratory. School of Engineering and Science. Jacobs University gGmbH. Campus Ring 1, D-28759, Bremen, Germany *Tel: 00494212003172, Fax: 00494212003660 Email: m.fernandez-lahore@jacobs–university.de 2 J Biochem Tech (2008) 1(1):1-05 Ion-exchange chromatography (IEC) is nowadays a widely utilized chromatographic method within the pharmaceutical and biotech industry due to its good resolution capability, high binding capacity, and versatility. In this chromatographic mode, macromolecule retention is manly determined by Coulomb-type interactions. However, with macromolecules such as proteins, interaction mechanisms with charged surfaces are often complex and the fundamental understanding of such processes is limited (Yao et al. 2005; Malmquist et al. 2006). In addition, non-electrostatic interactions, such as hydrophobic interactions and hydrogen bonding, or other factors like the nature of the buffer ions are also known to influence protein separation with ion-exchangers. Moreover, protein-protein association may lead to non-expected protein chromatographic behavior in a chemical environment supporting native protein conformation. This paper further advanced previous studies aimed at the evaluation of proteomic tools in order to understand global separation performance in anion-exchangers. Particularly, the influence of sample pH was evaluated employing a complex protein mixture obtained from D. melanogaster S2 cells in culture. This expression system is gaining importance for the production of biopharmaceutics in the light of recent European legislation which favors intellectual property protection for insect-cell derived bioproducts. Chromatographic fractionation: Chromatographic experiments were performed in an AKTA FPLC system equipped with a Frac900 fraction collector and UNICORN 4.10 Software for data collection and analysis (GE Healthcare Europe GmbH, Munich, Germany). The HiTrap™ IEX Selection Kit (1ml matrix bed volume) was employed as adsorbent phase. The kit included prepacked columns containing Q- and DEAE- Sepharose™ Fast Flow, and ANX Sepharose™ 4 Fast Flow high sub. Chromatography was performed at a flow rate of 117 cm/h and at 20°C. The mobile phase consisted in 20 mM sodium phosphate buffer (pH 5 to 8; 4 mS/cm). Samples (1.0 ml; 40 mg protein) were loaded onto the columns. Unbound biological material was washed out with the same buffer solution and retained protein fractions were eluted stepwise utilizing 0.1, 0.3, 0.6 and 1 M sodium chloride in running buffer as eluent. Collected fractions were desalted by size exclusion chromatography in an open column packed with Sephadex™ G-25 Medium and subsequently concentrated at 40°C under vacuum (Vacufuge Concentrator 5301 Eppendorf AG, Hamburg, Germany). Chromatographic fractionation experiments were performed in triplicate and fraction pooled before analysis. Two-dimensional polyacrylamide gel electrophoresis: Chromatographic samples were solubilized in a lysis buffer (7 M urea, 2 M tiourea, 4% w/v CHAPS, 1 % w/v DTT, and 2% v/v PharmalyteTM 3-10). First dimension isoelectrofocusing (IEF) was performed using 7-cm ImmobilineTM DryStrips pH 3–10 NL which were placed in a flatbed Multiphor II unit (GE Healthcare Europe GmbH, Munich, Germany) at 20°C. The IPG strips were hydrated for 12 h in Rehydration buffer (6 M urea, 2 M thiourea, 1% CHAPS, 0.4 % DTT and 0.5 % v/v PharmalyteTM 3-10). Fifty ml of sample was applied in the strip by cup loading. IEF was performed for 10,000–20,000 Vh. Before running the second SDS-PAGE dimension, the Immobiline strips were first equilibrated for 15 min in buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% w/v SDS, 10 mg/ml DTT) and then for 15 min in the same buffer with 25 mg/ml iodoacetamide instead of DTT. After equilibration, the strips were placed on top of a12.5 % SDS vertical slab and embedded in 0.5 % hot low-melting point agarose in SDS electrophoresis running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). SDS-PAGE was performed in a HoeferTM SE260 gel electrophoresis unit in 0.75 mm thick 10cm x 10.5cm gels at 20 mA per gel. The gels were stained with colloidal Coomassie blue (Neuhoff et al. 1988). Stained 2-D gels were scanned using a 48-bit (400 dpi) Epson color scanner (Epson perfection 4990 Photo) and images analyzed with ImageMaster 2D platinum 6.0 (GE Healthcare Europe GmbH, Munich, Germany). Electrophoretic evaluation of chromatographic fractions was performed in triplicate. Protein content of the samples: The protein concentration in the samples was determined by the BCA protein assay kit (Pierce, Rockford, IL) as per the manufacturer instructions. This procedure is based on the well known bicinchoninic acid assay (Smith assay), which is a colorimetric method for determining the total level of protein in a solution. Materials and methods Materials The insect cell line, the cultivation media, and other cultivation additives were from Invitrogen Europe (Karlsruhe, Germany). Acrylamide, Dithiothreitol (DTT) and (3-[(3-Cholamidoprpyl) dimethylammonio]-1-prpanesulfonate) (CHAPS) were purchased from Carl-Roth GmbH (Karlsruhe, Germany). The molecular weight markers were purchased from Pierce Biotechnology Inc. (Rockford, IL). Chromatographic materials and pre-casted gels were purchased from GE Healthcare Europe GmbH (Munich, Germany). All other buffer-salts and process chemicals were analytical grade and were obtained from AppliChem (Darmstadt, Germany). Methods Cell cultivation procedure: Drosophila melanogaster S2 cells (D.Mel-2 ATCC #CRL 1963) were grown under aerobic conditions in liquid media by incubating in 1 L baffled flasks at 150 rpm, in 300 ml serum free medium with 5% GlutaMAXTM, 10,000 U/ml Penicillin G (sodium), and 10 mg/ml Streptomycin sulphate at 27°C ± 0.5°C. Cells were harvested in the late exponential growth phase (1-2 x 107 cell/ml) by centrifugation at 200 g for 15 min. The resulting cell pellet was stored at –20°C. Preparation of the crude cell extract: One gram (wet weight) of the cell pellet was suspended in 8 ml of chromatographic mobile phase. One hundred μl of protease inhibitor cocktail (Sigma P 8849, St. Louis, MO) was added per gram of cells. Cells were disrupted by sonication with pulses having duration of 10 s and which were delivered at 30s intervals. Cell disruption was performed at 4°C. Cell debris was separated by centrifugation at 3220 g for 20 min. The soluble fraction remaining in the supernatant was subsequently filtered with a 0.45 μm membrane (Minisart Sartorius, Göttingen, Germany) and utilized as sample for further chromatographic fractionation. Results and discussion Chromatographic experiments Scouting experiments were performed to evaluate the chromatographic behavior of the insect cell soluble proteome obtained as described in the materials and methods section. In doing so, a variety of ion-exchange materials were employed. All the materials under study represent beaded hydrogel-type supports based on agarose. Weak and strong anion-exchangers were J Biochem Tech (2008) 1(1):1-05 3 represented among the materials tested. The characteristics of the mentioned ion-exchange supports are given by the provider. On the other hand, other chromatographic conditions were kept the same so as to assure standard operational conditions and parameters. Elution of bound protein fractions were performed stepwise thus resembling industrial practice were gradient elution is not favored. The experimental approach presented here opens the opportunity for a direct comparison between adsorbents in terms of selectivity towards protein naturally occurring in a complex feedstock. Information of this kind is seldom available in the open literature since most studies on material selectivity or protein chromatographic behavior are usually restricted to one or a few purified model proteins (Lagerlund et al. 1998). -angers tended to behave alike at alkaline pH values. On the other hand, DEAE-Sepharose showed better resolution at acidic pH e.g. 5 peaks can be noticed at pH 6. Proteomic routines The chromatographic fractions obtained by fractionation of the total soluble Drosophila cell proteome were analyzed on the basis of 2DPAGE, as well as, the crude feedstock loaded into this system. Fig. 2 shows, as an example, the 2D-PAGE analysis of the Q-Sepharose fractions. These studies, when applied to the various adsorbents and feedstock pH values, gave information on the overall number of potential contaminants present in each of these fractions and on their distribution in a proteome space defined by coordinates of apparent isoelectric point values and protein molecular weights, Figure 1: Chromatographic profiles obtained on various commercial adsorbents and with several initial feedstock pH values. Anion exchanger materials were comparatively studied in relation with the Drosophila cell soluble proteome. Dynamic adsorption experiments were performed in phosphate buffer at pH 7.5 at a superficial velocity of 117 cm/h. Fig. 1 depicts the separation attained utilizing weak (DEAE- and ANX-) or strong (Q-) anionexchange chemical moieties onto agarose beads. Total protein load in such systems was limited to approximately the expected average dynamic binding capacity for these materials. It was observed that ∼ 70 % of the protein load in all cases was lost in the column flowthrough as non retained components of the mixture. On the other hand, the bound material was fractionated by elution via stepwise increase in the mobile phase ionic strength by adding increasing concentrations of sodium chloride i.e. measured conductivity was 11, 29, 54, and 86 mS/cm in the four collected fractions, respectively. As judged by UV absorbance @ 280 nm, four main eluting peaks were distinguished for ANX- and Q- adsorbents but only tree in the case of the DEAE- support. This is not anticipated on the basis of the functional ligands involved since DEAE- and ANX- (tertiary ammonium group) but not Q- (quaternary ammonium group) are considered to be weak anion-exchangers. However, there is a clear match between the separation profiles obtained for ANX- and Q- materials as opposed to DEAESepharose FF. Changes introduced in the initial pH of the mobile phase would alter chromatographic separation not only due to changes in protein charge but as a function of the titration profiles of the different materials that could be utilized to exert a certain purification task. As shown in Fig. 1, chromatographic experiments were performed at three levels of pH: acidic (pH 6), neutral (pH 7), and basic (pH 8). It was observed that the fraction of protein material, as judged by @ 280 nm, which is very weakly retained (i.e. because elutes at very low salt concentration) was increased at acidic pH values. Differences between materials were exacerbated at such pH. Increasing pH values to 7 and further to 8 has resulted in a much smaller weakly-retained protein fraction and in chromatographic profiles more closely resembling each others. Therefore, anion-exch distribution in a proteome space defined by coordinates of apparent isoelectric point values and protein molecular weights, under chemically denaturing conditions. Each sample, which represents a set of potential contaminants within a certain operational window, was subjected to concentration keeping an identical concentration factor. This would directly reflect the total mass of the contaminating material in each recovered fraction. Similarly, protein load onto the 2D gels was related to the total protein concentration in each of the collected chromatographic fractions. This allowed for direct comparison between 2D gels after staining with Colloidal blue. The selection of this particular staining method reflected the intention of revealing major protein spots since priority should be given during any downstream process for the removal of major Figure 2: Analysis of the chromatographic fractions obtained after separation with Q FF- Sepharose at different pH. Each fraction was obtained by pooling three different chromatographic runs. An orthogonal proteomic routine was utilized to fractionate the proteins existing in each of the mentioned samples. 4 J Biochem Tech (2008) 1(1):1-05 contaminants at first. Ion exchange is a moderate resolution method, which usually finds place as an early-to-intermediate isolation step and thus, optimization of this method on the basis of major contaminants accompanying a certain product, is duly justified. A first insight on overall chromatographic behavior can be gathered after analyzing the number of spots revealed by 2D electrophoresis. Fig. 3 depict the spot number as a function of material type, feedstock pH, and concentration of sodium chloride required for elution. After colloidal blue staining, the total number of spots observed in the crude feedstock was 824. considering the distribution of isoelectric point values -among the observed spots and in relation to the ionic strength levels required for elution. A second level of data evaluation can be done on the basis of apparent isoelectric point distributions. These could be linked to desorption behaviour in a way that not necessarily reflects total (or major) spot number(s). Figure 4 shows the relationship between pI value distributions and salt concentration relationship between pI value distributions and salt concentration in the eluting buffer. Overall, a tendency was observed for the pI values to be displaced to the range of acidic pH values. This was a general trend observed from very rich data obtained after analyzing a large Figure 3: Spot number in each chromatographic fraction, as reveled by 2D SDS-PAGE analysis, as a function of adsorbent type, feedstock pH, and mobile phase conductivity. 2D gel electrophoresis was performed in triplicate, for each of the samples under analysis. Stained 2D gels were scanned using a 48-bit (400 dpi) Epson color scanner (Epson perfection 4990 Photo) and images analyzed with Image Master 2D platinum 6.0 (GE Healthcare Europe). observed in the crude feedstock was 824. Fractions obtained after elution of weakly bound material i.e. the material eluting from the column a low salt concentration (0.1 M sodium chloride in running buffer) presented a limited number of spots. In Table 1 it can be observed that, in most cases, the number of spots is maximum in the fraction eluted with 0.33 M of sodium chloride. The DEAEmaterial showed a more even distribution in spot number among the fractions analyzed. On the contrary, ANX- and Q- showed a more unequal distribution: a) a larger number of spots was observed at acidic pH values and low salt concentration for ANX-Sepharose, and b) a larger number of spots was observed at neutral-alkaline pH values and high salt concentration. Table 1: Spot number in each chromatographic fraction. Adsorbent Material DEAE ClNa (M) 0.1 0.33 0.66 0.1 0.33 0.66 0.1 0.33 0.66 6 37 77 19 113 155 28 39 147 19 pH (-) 7 35 80 27 25 96 80 60 328 128 8 41 192 27 33 86 150 38 238 112 observed from very rich data obtained after analyzing a large number of chromatographic fractions: samples (3 initial pH values / 3 pH values for the mobile phase) were run onto 3 types of anionexchangers, and fractions collected at 3-5 conductivity intervals. These observations are in agreement with the known retention principle of proteins onto ion-exchangers which is manly determined by Coulomb-type interactions with a charged surface: for a fixed adsorbent type it is expected to have strong-negatively charged proteins binding with more affinity to the positively charged anion-exchangers. Consequently, it is expected that more acidic proteins i.e. those with the lower pI values would elute at 6.5 6.3 6.1 5.9 5.7 pI (-) 5.5 5.3 5.1 4.9 4.7 4.5 0 0.2 0.4 NaCl (M) 0.6 0.8 ph 6 ph 8 ph 7 ANX Q Apparent pI distributions The quantitative and qualitative information obtained after the 2D electrophoretic evaluation of the chromatographic fractions in terms of spot number and their mass-relevance can be complemented by Figure 4: Isoelectric point value distribution as a function of salt required for elution. 3 anion-exchangers and three sample pH values are considered. 2D gels were run in triplicate and scanned images statistically analyzed with Image Master 2D platinum 6.0 (GE Healthcare Europe). Each point represents the mean isoelectric point for a certain spot population, i.e. fractions obtained as a function of buffer conductivity required for elution and pH of the mobile phase. The trend line was drawn by averaging such mean isoelectric point values, at each buffer conductivity value. J Biochem Tech (2008) 1(1):1-05 5 higher salt concentrations. However, non-electrostatic interactions can also affect protein separation during ion-exchange chromatography. Solid phase effects, as well as, nonspecific interactions with the immobilization chemical structures or the ligand itself can not be ruled out. Ion exchange chromatography at near-neutral pH values, in relatively low salt polar buffers is non-denaturing and therefore, proteins in a real sample might have the ability to form complexes (Butt et al. 2001). Protein-protein complexes usually will have a different pI than the one of the individual proteins. Association of two or more proteins can also mask charged patches at the surface of these proteins thus, impacting on the nature of the interaction with the solid phase. Interaction of proteins with chromatographic supports is an often complex physicochemical event(s) and little is known about the fundamental mechanism behind protein binding to charge surfaces. It is still uncertain which model best describes the ionic strength dependence of this interaction since retention changes caused by changing the salt concentration in the eluent depend mainly on the protein charge or the charge distribution on its surface (Hallgren et al. 2000) as well as the geometry (Malmquist et al. 2006). The nature of the chromatographic support also influences protein interaction and separation. Conclusion Chromatographic runs performed with a cell proteome utilizing 3 types of anion-exchangers and mobile phases having pH values within the range 6 to 8 were analysed by orthogonal proteomic techniques. A clear tendency between isoelectric point distributions and the amount of salt required to elute sample sub-proteomes was clearly observed. The information provided in this work can help in understanding the chromatographic behavior of natural (complex) protein mixtures, usually found in a real process feedstock. . Our studies could be particularly helpful for the design of separation process of recombinant proteins produced in the intracellular space in cultured insect cells. However, the observed trends in separation behavior might be partially extended to other cell proteomes. Studies are under way in our laboratory to more closely understand the chromatographic behavior of proteins derived from microbial and mammalian cell systems. cell proteome Towards in silico downstream processing of bioproducts. J Chromatogr A 1161(1-2):41-50 Cabrera R, Zhelyazkova P et al (2008) Tailoring orthogonal proteomic routines to understand protein separation during ion exchange chromatography. J Sep Sci 31(13):2500-10 Elrick MM, Walgren JL et al (2006) Proteomics: Recent applications and new technologies. Basic & Clinical Pharmacology & Toxicology 98(5):432-441 Hallgren E, Kalman F et al (2000) Protein retention in ion-exchange chromatography: effect of net charge and charge distribution. J Chromatogr A 877(1-2):13-24 Lagerlund I, Larsson E et al (1998) Characterisation of ANX Sepharose (R) 4 Fast Flow media. J Chromatogr A 796(1):129140 Malmquist G, Nilsson UH et al (2006) Electrostatic calculations and quantitative protein retention models for ion exchange chromatography. J Chromatogr A 1115(1-2):164-186 Neuhoff V, Arold N et al (1988) Improved Staining of Proteins in Polyacrylamide Gels Including Isoelectric-Focusing Gels with Clear Background at Nanogram Sensitivity Using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9(6):255-262. Novais JL, Titchener-Hooker NJ et al (2001) Economic comparison between conventional and disposables-based technology for the production of biopharmaceuticals. Biotechnol Bioeng 75(2):143-153. Salgado JC, Rapaport I et al (2006) Predicting the behaviour of proteins in hydrophobic interaction chromatography 2. Using a statistical description of their surface amino acid distribution. J Chromatogr A 1107(1-2):120-129. Salgado JC, Rapaport I et al (2006) Predicting the behaviour of proteins in hydrophobic interaction chromatography. 1: Using the hydrophobic imbalance (HI) to describe their surface amino acid distribution. J Chromatogr A 1107(1-2):110-9. Yao Y, Lenhoff AM (2005) Electrostatic contributions to protein retention in ion-exchange chromatography. 2. Proteins with various degrees of structural differences. Anal Chem 77(7):2157-2165. Acknowledgements This work was partially financed by Jacobs University Bremen, start-up funds. Reference Asenjo JA, Andrews BA (2004) Is there a rational method to purify proteins? From expert systems to proteomics. J Mol Recognit 17(3):236-247 Butt A, Davison MD et al (2001) Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis. Proteomics 1(1):42-53 Cabrera R, Fernandez-Lahore M (2007) Global screening of protein chromatographic behavior on ion exchangers from a complex J Biochem Tech (2008) 1(1):06-08 ISSN: 0974-2328 Yeast That Smell Eugenia Y Xu, Addison D Ault, James R Broach* Received: 31 July 2008 / Received in revised form: 6 August 2008, Accepted: 13 August 2008, Published online: 17 August 2008 © Sevas Educational Society 2008 Abstract The fundamental mechanism of olfactory receptor activation has been conserved from yeast to humans. Engineered yeast cells can smell some of the same odorants as humans can, which makes yeast an ideal model system for studying human olfaction. Furthermore, if engineered yeast cells are incorporated into sensory arrays, they can be used as biosensors or artificial noses. Keywords: Yeast, olfactory receptor, G protein-coupled receptor, biosensor, smell of cyclic nucleotide-activated, nonselective cation channels (Fig. 1A) (Mombaerts 2004). Electrical signals generated from OR activation are propagated in turn to the main olfactory bulb, the olfactory cortex, higher cortical areas and limbic structures of the brain, where eventually a perception of odor is formed (Reed 2003). The olfactome The sense of smell is one of the most complex and elaborate physiological systems in humans and other animals. Based on genome sequences analysis, humans have about 350 olfactory receptors (ORs) and mice have about 1000 ORs (Malnic 2007). Nonetheless, humans can sense as many as 10,000 to 100,000 chemicals as distinct odors. This discrepancy between number of receptors and number of odors perceived suggests that the mammalian olfactory system uses a combinatorial code, such that each OR recognizes multiple odorants and most odorants are recognized by several ORs (Malnic et al. 1999). The limited pharmacological studies conducted to date on ORs support this hypothesis. Perception of odorants starts with the stimulation of ORs on sensory neurons within the nasal olfactory epithelium, resulting in the activation of an adenylyl cyclase (AC), and subsequent opening Eugenia Y Xu, Addison D Ault, James R Broach* Department of Molecular Biology, Princeton University, Princeton, NJ 08544 *Tel: 001 609 258 5981, Fax: 001 609 258 1975 E-mail: jbroach@princeton.edu Figure 1: Conservation of OR-activated signal transduction pathways between yeast and mammals. (a) Schematic of the mammalian olfactory signal transduction pathway in the olfactory epithelium. Binding of odorants to olfactory receptor activates heterotrimetric G protein, which subsequently results in activation of the AC activity, elevated cAMP level, and opening of cAMP-gated ion channels. Synthesis of cAMP activates PKA and in turn upregulates many genes. OR, olfactory receptor; ACIII, adenylyl cyclase; PKA, protein kinase A; α, β, γ, heterotrimeric G protein α subunit, β subunit, γ subunit; CRE, cAMP responsive element. (b) Schematic of the yeast mitogen-activated protein kinase signal transduction pathway. Activation of pheromone receptor results in activation of heterotrimetric G protein, stimulation of MAP kinase signaling cascade and in turn upregulation of many genes. J Biochem Tech (2008) 1(1):06-08 7 OR genes are members of the G protein coupled receptor (GPCR) superfamily, which are cell surface proteins with a conserved structure of seven transmembrane domains (Buck and Axel 1991). Understanding OR activation is central to decoding human olfaction; the combinatorial map of OR-odorant pairs that determine the perception of distinct odors is far from complete. Given the difficulty of in situ pharmacology in the olfactory epithelium, a practical approach to obtaining a comprehensive ORodorant coding map would be to determine the interaction of odorants and ORs in a simple heterologous cell system. Because the basic molecular mechanism of OR activation is conserved evolutionarily from yeast to humans, yeast has attracted attention as a potential platform for ‘deorphanizing’ the human olfactome. Pheromone sensing in yeast Haploid yeast cells exist in two cell types — a or α cells. a and α cells can mate with each other to form diploids. Haploid yeast cells initiate mating with the opposite cell type upon “smelling” the pheromone produced by mating partners. The protein that “smells” the pheromone released from the opposite cell type is a cell typespecific GPCR, Ste2 in a cells or Ste3 in α cells. Stimulation of the yeast GPCR by pheromone leads to activation of a mitogenactivated protein (MAP) kinase signal transduction pathway and the induction of several genes, including FUS1, a gene involved in cell fusion (Fig. 1B) (King et al. 1990; Dohlman 2002). Many mammalian GPCRs have been functionally expressed in the yeast Saccharomyces cerevisiae. Stimulation of such heterologous GPCRs by ligands leads to activation of the MAP-kinase pathway in yeast, which can be monitored in colorimetric, fluorometric or growth assays using appropriate transcriptional reporters fused to the FUS1 promoter (Klein, Paul et al. 1998). The significant conservation of GPCR-activation-machinery between yeast and mammalian cells, the null background of mammalian GPCRs in the yeast system, the easy manipulation of the yeast genome genetically and molecularly, and the robust growth of yeast cells, all render yeast an excellent platform for pharmacological studies of mammalian GPCRs (Silverman et al. 1998). in Y3623 showed 50-100 fold dose-dependent response to NECA (Sigma-Aldrich), but not to non-specific ligands, as determined by measuring the β-galactosidase activity with expression of the PFUS1LacZ reporter gene on a vector (Fig. 2B). Thus, a yeast-based assay was developed to study the olfactory receptors. Rat OR I7 or human OR 17-40 expressed under the control of a strong yeast promoter PGK1 in a yeast LEU2-marked 2µ vector was transformed into Y3623, respectively. The functional activation of rat OR I7 or human OR 17-40 by its reported odorants was examined. Although we were only able to detect moderate (~ 2 fold) activation sporadically and could not observe the dosedependent response consistently, Minic et al. reported agonistdependent activity in a similarly engineered yeast system (Minic et al. 2005). These authors found that both rat OR I7 and human OR 17-40 functionally respond to odorants as they do in mammalian cells. The response of ORs to their ligands in yeast was enhanced when the receptors were induced to a high level at 15oC. Since the concentrations of the odorant responses by the ORs Olfactant sensing in yeast Study OR-activation in yeast, an analogous approach was taken as that of studying the GPC activation in yeast: Two key mammalian olfactory components, an OR and the G protein α subunit, Gαolf, expressed in the olfactory epithelium, were engineered into a yeast strain to replace yeast GPCRs STE2 and STE3 genes and the yeast Gα gene, GPA1. GPCRs couple to downstream effects through a heterotrimeric complex consisting of three subunits-Gα, Gβ and Gγ. Gα mediates the interaction between the receptor and the Gβγ heterodimer. To maximize Gα subunit interaction with heterologously expressed mammalian ORs while maintaining its association with yeast Gβγ heterodimer, a sandwich Gα protein was constructed by replacing the receptor-coupled domain of yeast Gpa1 with the corresponding rat Gαolf (Fig. 2A). This sandwich Gα gene was used to replace the genomic copy of GPA1 to create yeast strain Y3623*. Since amino acid sequences of Gαolf show high similarity to that of Gαs, which is also a Gα subunit that activates AC in mammals, the functional integrity of the engineered Y3623 yeast strain can be examined by determining whether the Gαs– coupled human adenosine receptor 2B (A2B) can couple to Gαolf and be activated by its specific ligand 5’-N-ethylcarboxamidoadenosine (NECA) (Mirabet et al. 1999). The human A2B receptor expressed Figure 2: Engineering of the olfactory yeast strain Y3623. (a) Schematic illustration of sandwich Gα subunit in Y3623. Amino acids (aa) SKTAEDQGVDEKERREANKKIEKQLQKERLAYKATHR from 8 to 44 and the last 5 aa QYELL of rat Gαolf replaced corresponding GPA1 domain. This was done by fusion PCR. PCR primers used to amplify rat Gαolf (a gift from Dr. Randall Reed) and yeast GPA1 are available upon request. Open rectangle represents the aa sequences of Gpa1p and shaded rectangle represents the aa sequences of rat Gαolf. (b) Response of human A2B receptor expressing yeast strain to NECA, but not non-specific ligands. Human A2B receptor and a vector containing the FUS1p-LacZ reporter gene were transformed into the olfactory yeast strain Y3623. The resulting yeast cells were grown overnight to exponential phase, seeded to a 96-well plate in 100 μl, and treated with NECA at increasing concentrations for 4 hours at 30oC. β-galactosidase activity was determined by addition of 0.5 mM fluorescein di-β-D-galactopyranoside (FDG, Molecular Probes). Fluorescence was measured in a Wallac Victor microplate reader with 485-nm excitation and 535-nm emission. 8 J Biochem Tech (2008) 1(1):06-08 concentrations of the odorant responses by the ORs lie in a very narrow range in Minic et al.’s paper, we speculate that the range of ligand concentration could be critical for observing activation of the OR, or that the engineered yeast strain background influences the specificity of the odorants. Using the engineered olfactory yeast cells made by Minic et al., Marrakchi et al. developed an olfactory biosensor (Marrakchi et al. 2007). Yeast cells expressing OR 17-40 immobilized onto interdigitated thin film microelectrodes specifically recognize the ligand for OR 17-40. Thus, yeast cells could potentially be used as artificial noses. Recently, Radhika et al. developed “olfactory yeast” by engineering a multistep mammalian olfactory signaling pathway into the yeast Saccharomyces cerevisiae (Radhika et al. 2007). In the mammalian olfactory system, stimulation of ORs by odorants activates the Gαolf subunit, which interacts with type III adenylyl cyclase (ACIII) and leads to synthesis of cyclic AMP (cAMP). Besides opening cyclic nucleotide gated channels, cAMP can activates CREBP, a DNA binding factor that activates transcription via cAMP-response elements (CREs) (Fig. 1A). To establish an olfactory signal transduction pathway independent of any yeast signal transduction pathways, Radhika et al. cloned all the main components of the mammalian cAMP reponse pathway into yeast — ACIII and CREBP — along with mammalian ORs and three mammalian subunits of the olfactory heterotrimeric G protein (rat Gαolf,, Gβ2 and Gγ5),. In addition, a GFP reporter gene expressed under the control of human CRE elements was integrated into the yeast genome so that activation of the olfactory signaling pathway in yeast could be monitored by the expression of GFP. In this olfactory yeast, activation of the OR by odorants only functions through the artificial olfactory signaling pathway. Functional expression of rat OR I7 and response to its odorants were observed. Interestingly, Radhika et al. identified a receptor that responds to 2, 4-dinitrotoluene, a mimic for the explosive trinitrotoluene (TNT), through screening a library of “orphan” receptors. This demonstrates that olfactory yeast could be adapted to highthroughput screens to identify ligands for orphan ORs and it suggests that the olfactory yeast could be used as biosensors for environmental monitoring. *Y3623 is made from a yeast strain derivative of Cy1141 (Klein et al. 1998). The genotype of Y3623 is: Matα FUS1p-HIS3 gpa1Δ::gpa1(7)gαolf(8-48)-gpa1- gαolf(5) ste18g6-3841 ste3Δ1156 far1Δ1442 sst2Δ2 tbt1-1 ste14::trp1::LYS2 his3 leu2 lys2 trp1 ura3 can1 Acknowledgements We would like to thank previous and current members of the Broach laboratory for helpful discussions for this project. This work was supported by National Institutes of Health Grants GM48540 to J.R.B. and F32 DC 005580 to A.D.A. References Buck L, Axel R (1991) A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell 65(1):175-187 Dohlman H (2002) G proteins and pheromone signaling. Annu Rev Physiol 64:129-152 King K, Dohlman HG et al (1990) Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit. Science 250(4977):121-123 Klein C, Paul JI et al (1998) Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast. Nat Biotechnol. 16(13):1334-1337 Malnic B (2007) Searching for the ligands of odorant receptors. Mol Neurobiol 35(2):175-181 Malnic B, Hirono J et al (1999) Combinatorial receptor codes for odors. Cell 96(5):713-723 Marrakchi M, Vidic J et al (2007) A new concept of olfactory biosensor based on interdigitated microelectrodes and immobilized yeasts expressing the human receptor OR17-40. Eur Biophys J 36(8):1015-1018 Minic J, Persuy MA et al (2005) Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening. FEBS J 272(2):524-537 Mirabet M, Herrera C et al (1999) Expression of A2B adenosine receptors in human lymphocytes: their role in T cell activation. J Cell Sci 112( Pt 4):491-502 Mombaerts P (2004) Genes and ligands for odorant, vomeronasal and taste receptors. Nat Rev Neurosci 5(4):263-278 Radhika V, Proikas-Cezanne T et al (2007) Chemical sensing of DNT by engineered olfactory yeast strain. Nat Chem Biol 3(6):325-330 Reed RR (2003) The contribution of signaling pathways to olfactory organization and development. Curr Opin Neurobiol 13(4): 482-486 Silverman L, Campbell R et al (1998) New assay technologies for high-throughput screening. Curr Opin Chem Biol 2(3):397-403 Perspective With recombinant DNA and genetic engineering technologies, olfactory yeast strains have been produced that can “smell” some of the same chemicals that humans do. As additional receptors are expressed and profiled in these strains, it will become possible to address the question of how the combinatorial olfactory mechanism allows such a large ensemble of receptors to function seamlessly together. These strains will provide invaluable clues as to the encoding and decoding mechanisms required for discrimination of distinctive odors, along with perception of spatial and temporal cues. Yeast cells have a long life-time and have the characteristics of low-cost preparation and robust growth, which makes olfactory yeast attractive candidates for development as biosensors or an artificial nose. The problems with the current generation of olfactory yeast are their sensitivity, specificity and versatility. However, successful construction of mammalian olfactory signaling pathway in yeast portends that fine-tuning the pathway for yeast that smell better is possible. To this end, it is anticipated that in the future having a yeast-based biosensor or yeast nose in daily life will not be science fiction. J Biochem Tech (2008) 1(1):09-10 ISSN: 0974-2328 A possible role of repair proteins BRCA1 and DNA-PK in the processing of oxidative DNA damage Alexandros G Georgakilas Received: 6 June 2008, Accepted: 10 August 2008 Published online: 18 August 2008 © Sevas Educational Society 2008 Abstract BRCA1 and DNA-PK are two significant multifunctional proteins involved primarily in the processing of double strand breaks (DSBs). BRCA1 participates actively in homologous recombination (HR) while DNA-PK in non-homologous end joining (NHEJ). In this mini review, we discuss all recent evidence for a possible involvement of these repair proteins also in the processing of oxidatively-induced DNA damage. or reduced expression of the protein (DiBiase et al. 2000; Peng et al. 2002; Salles et al. 2006). The BRCA1 gene is located on the long (q) arm of chromosome 17 at position 21 and provides instructions for making the corresponding protein that is directly involved in repairing DSBs through HR repair (Holt et al. 1996; Zhong et al. 1999). Interestingly, deficiencies of both proteins (DNA-PK and BRCA1) have been associated with elevated breast (Yu et al. 2001; Fu at al. 2003) or ovarian cancer risk (Futreal et al. 1994; Turner et al. 2006). BRCA1 and DNA-PK involvement in the processing of DSBs DSBs are considered highly genotoxic DNA lesions because they can potentially lead to chromosomal breakage (Burma et al. 2006). DSBs can arise as the result of exposure to exogenous sources like natural and medical radiation or other environmental exposures to chemicals, when chromosomes exchange genetic material in preparation for cell division and finally endogenously either by free radicals that emerge as byproducts of normal cellular metabolism or as repair intermediates during the processing of clustered DNA lesions (Georgakilas 2008). Two major pathways have been implicated in the processing of DSBs: non-homologous end joining (NHEJ) and homologous recombination repair (HR) (Kanaar et al 1998). One of the key protein complexes in the NHEJ pathway is the DNA-PK holoenzyme consisting of the Ku70/80 heterodimer and the catalytic subunit of DNA-PK (DNA-PKcs) (Meek et al. 2004). The central role of DNA-PK in the processing of DSBs is underlined by several studies showing a severe radiosensitivity and decreased DSB repair in cells with compromised DNA-PK activity An alternative role of BRCA1 and DNA-PKcs in the processing of oxidative DNA lesions Recent evidence from our laboratory suggests an additional and alternative role of these two traditional DSB repair proteins in the processing of non-DSB lesions induced by oxidative stress (base damage and single strand breaks-SSBs) (Francisco et al. 2008; a Peddi et al. 2008; bPeddi et al 2008). In support of this idea, several other laboratories have shown a compromised repair of non-DSB oxidative DNA damage in cells or tissues with DNA-PK or BRCA1 deficiencies. Rodriguez et al. have recently shown a deficient processing of oxidative DNA lesions in lymphoblasts from women with BRCA1 mutations. Additional support for the role of BRCA1 in DNA DSB repair and potentially oxidative lesions, comes from the finding that BRCA1 plays a coordinator role of multiple activities required for maintenance of genomic integrity interacting with various DNA damage repair proteins like MSH2, RAD51, ATM, BLM and RAD50-MRE11-NBS1 (Zhong et al 1999; Wang et al. 2000) and actively participates in transcription coupled repair (Le Page et al. 2000). Reduced levels of DNA-PKcs maybe compromising the processing of base damages through base excision repair (BER) since DNA-PKcs has been shown recently to interact with many BER proteins suggested actually forming a repairsome (Levy et al. 2006; Parlanti et al. 2007) i.e., direct effect. Another possible explanation can be the strong inhibitory action of existing SSBs (forming a DSB) towards the processing of neighboring base damages i.e., indirect effect. Many in vitro studies suggest that the existence of a single SSB can significantly inhibit or delay the processing of opposing base damages (Dianov et al. 2001). Alexandros G Georgakilas Department of Biology, Thomas Harriot College of Arts and Sciences, East Carolina University, Greenville, NC 27858 USA Tel: 001-252 3285446, Fax: 001-252 3284178 E-mail: georgakilasa@ecu.edu 10 J Biochem Tech (2008) 1(1):09-10 Conclusion Accumulating evidence from different laboratories suggests an alternative (direct or indirect) effect of BRCA1 and DNA-PK in the processing of oxidatively-induced DNA damage like abasic sites, altered bases and/or single strand breaks. This potential function of these proteins in the repair of non-DSB DNA lesions in addition to their verified involvement in the repair of DSBs, raises significant issues in the predisposition of individuals to several diseases like breast and ovarian cancer since both of these genes have been involved in increased breast or ovarian cancer susceptibility. a References Burma S, Chen BPC, Chen DJ (2006) Role of non-homologous end joining (NHEJ) in maintaining genomic integrity. DNA Repair 5:1042-1048 Georgakilas AG (2008) Processing of DNA damage clusters in human cells: Current status of knowledge. Mol Biosyst 4:30-35 Kanaar R, Hoeijmakers JH, Van Gent DC (1998) Molecular mechanisms of DNA double strand break repair. Trends Cell Biol 8:483-489 Meek K, Gupta S, Ramsden DA, Lees-Miller SP (2004) The DNAdependent protein kinase: the director at the end. Immunological Reviews 200:132-141 DiBiase SJ, Zeng ZC, Chen R et al (2000) DNA-dependent protein kinase stimulates an independently active nonhomologous, end-joining apparatus. Cancer Res 60:1245-1253 Peng Y, Zhang Q, Nagasawa H et al (2002) Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiationinduced chromosome damage, cell killing, and mutation. Cancer Res. 62:6400-6404 Salles B, Calsou P, Frit P, Muller C (2006) The DNA repair complex DNA-PK a pharmacological target in cancer chemotherapy and radiotherapy. Pathologie Biologie 54:185193. Holt JT, Thompson ME, Szabo C et al (1996) Growth retardation and tumour inhibition by BRCA1. Nat Genet 12:298-302 Zhong Q, Chen CF, Li S et al (1999) Association of BRCA1 with the hRad50-hMre11-p95 complex and the DNA damage response. Science 285:747-750 Yu Y, Okayasu R, Weil MM et al (2001) Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene. Cancer Res. 61:18201824 Fu YP, Yu JC, Cheng TC et al (2003) Breast cancer risk associated with genotypic polymorphism of the nonhomologous endjoining genes: a multigenic study on cancer susceptibility. Cancer Res 63:2440-2446 Futreal PA, Liu Q, Shattuck-Eidens D et al (1994) BRCA1 Mutations in Primary Breast and ovarian carcinomas. Science 266:120-122 Turner NC, Reis-Filho JS, Russell AM (2006) BRCA1 dysfunction in sporadic basal-like breast cancer. Oncogene 26:2126-2132 Francisco DC, Peddi P, Hair JM et al (2008) Induction and processing of complex DNA damage in human breast cancer cells MCF-7 and non-malignant MCF-10A cells. Free Radic Biol Med 44:558-569. Peddi P, Francisco DC, Cecil A et al (2008) Deficient processing of clustered DNA damage in human breast cancer cells MCF-7 with silenced DNA-PKcs expression. Cancer Lett.:In press b Peddi P, Francisco DC, Hair JM et al. (2008) Accumulation of complex DNA damage in human breast cancer cells MCF-7 deficient in DNA-PKcs. In: Meeting, A. A., ed. AACR Annual Meeting, volume 49. San Diego, California, USA. Rodriguez H, Jaruga P, Leber R et al (2007) Lymphoblasts of women with BRCA1 mutations are deficient in cellular repair of 8,5'-cyclopurine-2'-deoxynucleosides and 8-hydroxy-2'deoxyguanosine. Biochemistry 46:2488-2496 Wang Y, Cortez D, Yazdi P et al (2000) BASC, a super complex of BRCA1-associated proteins involved in the recognition and repair of aberrant DNA structures. Genes Dev 14:927-939 Le Page F, Randrianarison V, Marot D et al (2000) BRCA1 and BRCA2 are necessary for the transcription-coupled repair of the oxidative 8-oxogunanine lesion in human cells. Cancer Res. 60:5548-5552 Levy N, Martz A, Bresson A et al (2006) XRCC1 is phosphorylated by DNA-dependent protein kinase in response to DNA damage. Nucleic Acids Res 34:32-41 Parlanti E, Locatelli G, Maga G, Dogliotti E (2007) Human base excision repair complex is physically associated to DNA replication and cell cycle regulatory proteins Nucleic Acids Res. 35:1569-1577 Dianov GL, O'Neill P, Goodhead DT (2001) Securing genome stability by orchestrating DNA repair: removal of radiationinduced clustered lesions in DNA. BioEssays 23:745-749 J Biochem Tech (2008) 1(1):11-20 ISSN: 0974-2328 How effective is T-DNA insertional mutagenesis in Arabidopsis? Yi Hong Wang Received: 2 June 2008 / Received in revised form: 13 June 2008, Accepted: 20 June 2008, Published online: 18 August 2008 ©Sevas Educational Society 2008 Abstract To evaluate the effectiveness of T-DNA insertion in knocking out a gene, 1084 published Arabidopsis thaliana insertion mutants representing 755 genes in 648 publications were reviewed. Insertion in the protein-coding region of a gene generates a knockout at least 90% of the time or 25% of the time if it is before the start codon which also produces a knockdown 67% of the time. Insertion after the stop codon had no effect on transcription of the upstream gene 17% of the time compared to 8% in insertion before the start codon. T-DNA insertion can also cause deletion and translocation. Keywords: Arabidopsis thaliana, deletion, expression, knockout, T-DNA insertion, translocation homozygous mutant plants in most cases examined. Even if mRNA is transcribed, the T-DNA sequence may contains stop codons, resulting in early translation termination (Krysan et al. 1999). Although it is not a perfect technique (Alonso and Ecker 2006; Østergaard and Yanofsky 2004), T-DNA insertion mutagenesis has been a powerful tool to link genes to phenotypes. Thanks to the availability of these insertion mutants and the complete genome sequence, the number of reports characterizing Arabidopsis genes has increased tremendously since 2000. The trend is projected to continue because only a small portion of these mutants have been characterized so far. The purpose of this paper is to summarize the effect of these insertions on gene knockout based on published insertional mutants. To do this, position of insertion site, its effect on transcript/protein level were collected from 648 reports on 755 Arabidopsis genes published from 1997 to January 2007 which includes 1084 insertion mutants. This is by no means a complete coverage because not all reports monitored the expression of mutated genes and this may be biased toward insertions that knocked out the target genes. But it includes approximately 90% of all papers characterizing these mutants and may be useful for investigators working with T-DNA mutants. Introduction Transfer-DNA (T-DNA) insertion is a highly effective mutagen for genome-wide mutagenesis (Krysan et al. 1999). It has been widely used to produce insertion mutants in Arabidopsis thaliana (Alonso et al. 2003; Bechtold et al. 1993; Feldmann 1991; Galbiati et al. 2000; Koncz et al. 1989; Krysan et al. 1999; Rosso et al. 2003; Sessions et al. 2002) for functional characterization of every gene in the genome. Over 360,000 insertions have been mapped in the Arabidopsis genome, covering >90% of the genes (Alonso and Ecker, 2006). Because it tends to insert as concatemers (Krysan et al. 1999), most T-DNA insertions result in the loss-of-function alleles although semi-dominant T-DNA mutation has been reported (Bolle et al. 2000) and functional protein is absent in the Effect of T-DNA insertion on expression of mutated genes The effect of T-DNA insertion is most commonly evaluated by monitoring expression of mutated genes in homozygous insertion mutants. But as described in more detail below, this can be tricky because the transcript level may not be correlated with protein level (Delatte et al. 2005; Pastuglia et al. 2006) which may also depend on position of the insertion in a gene. In some cases, even if the transcript level does not differ significantly from the wild type, still no protein is produced in the mutant (e.g., Monte et al. 2003), complicating the evaluation of T-DNA insertion especially using reverse transcription-polymerase chain reaction (RT-PCR). For RNA gel blot and RT-PCR analyses, probes/primers from downstream and upstream of the insertion site should be used or at least the downstream probe/primers should be used if the insertion is toward the middle of the gene. It is also notable that for all 24 reports examined that employed RT-PCR using primers spanning the insertion site to monitor expression of mutated genes, none Yi Hong Wang Penn State University Behrend College School of Science, 4205 College Dr., Erie, PA 16563, USA Email: yxw23@psu.edu Phone: 001 814 898 6276 Fax: 001 814 898 6213 12 J Biochem Tech (2008) 1(1):11-20 produced any product, presumably because the inserted T-DNA sequence often larger than 5 kb, if transcribed, is too long for normal DNA polymerase to amplify. And if the T-DNA is not transcribed as one unit, it is not usually possible to amplify it by RT-PCR. The exception is that if the insertion is in an intron and the T-DNA has been spliced out (see below). It is also possible that T-DNA inserted into an exon be processed out generating a shorter altered transcript with a very low efficiency (Lehti-Shiu et al. 2005) or only a small part of the T-DNA was inserted (Shin et al. 2004). These are rare cases but RT-PCR using primers spanning the insertion site will yield a product. Overall, insertion in exons or introns can almost always knock out or knock down the target gene (Table 1). Table 1: Effect of published T-DNA insertion on transcript level of mutated genes in Arabidopsis Insertion site Number of No effect on Increased insertion transcript transcript mutants level level Exon Intron Before start codon After stop codon Major Deletion Total a level was similar to that in wild type in annAt2-2 (Lee et al. 2004). The insertion in annAt2-2 was more toward the 3’ end of the gene but still some distance before the stop codon. It is possible that the transcript detected by Northern in annAt2-2 is chimeric and just happens to be the similar size as in the case of ebf2-3 (Gagne et al. 2004). And in two insertion mutants of AnnAt4, annAt4-1 and annAt4-2, insertions were also in the similar positions in the last exon and transcript of AnnAt4 was not detected by RT-PCR in either mutant (Lee et al. 2004). Table 2: Effect of T-DNA insertion on protein expression of inserted genes in Arabidopsis Insertion site Exon Intron Before Total start codon No protein 70(88%) 32(82%) 7(41%) 109(80%) expression Reduced protein 7(8%) 5(13%) 9(53%) 21(16%) expression No effect on 1(1%) 1(3%) 1(6%) 3(2%) protein expression Truncated protein 2(2%) 1(3%) 0(0%) 3(2%) expression Total 80 39 17 136 It is also puzzling that insertion in the second exon of CSN5B failed to alter protein level compared to the wild type although the insertion only changed the phenotype slightly (Dohmann et al. 2005). If insertion is into an intron, wild type transcript may be produced in such mutants because intron can be spliced out together with the inserted T-DNA sequence. This indeed occurred in insertion mutants of AGL104 (Verelst et al. 2007), ASP2 (Miesak and Coruzzi 2002), AtEXP7 (Cho and Cosgrove 2002), AtGA2ox6 (Wang et al. 2004), AtMIS12 (Sato et al. 2005), ETA2 (Chuang et al. 2004), FATB (Bonaventure et al. 2003), MRH1 (Jones et al. 2006), SPDS2 (Imai et al. 2004), and ULT2 (Carles et al. 2005). Although wild type transcripts were produced, it was with decreased efficiency and the level of correctly spliced transcript was reduced. But this reduced level of wild type transcript can be made into protein as demonstrated in AtMIS12 (Sato et al. 2005) and ETA2 (Chuang et al. 2004) but not always (e.g., Bonaventure et al. 2003). Such leaky expression of mutated genes due to intron splicing is useful in rescuing an otherwise lethal mutation (Sato et al. 2005). But not all T-DNA insertions into an intron are spliced out (Hurtado et al. 2006) and only a small percentage of insertions in introns produce a reduced level of wild type transcript (10 out of 263 insertions or 4%); the rest are knockouts. Based on protein level, this number is 13% or 5 out of 39 insertions in introns produced reduced levels of wild type protein (Table 2). If insertion is toward the middle of the gene in an intron or exon, then transcript levels both upstream and downstream of the insertion can be monitored as described earlier. When 32 genes were surveyed for which transcript level was monitored both upstream and downstream of the insertion site, upstream transcript was detected in 29 genes while downstream transcript was detected in only 12 genes. In two cases, neither transripts were detected (Shen et al. 2006; Shimotohno et al. 2006). This is one reason that for insertions in exons/introns, knockout and knockdown are not separated in Table 1. But when truncated transcript is produced due to transcription termination by the insertion (Noh and Amasino 609 263 155 23 34 1084 7 (1.1%) 2 (0.7%) 17 (11%) 4 (17%) 0 (0%) 25 (2.3%) 0 (0%) 0 (0%) 5 (3%) 2 (8%) 0 (0%) 7 (0.6%) For each category of insertions, only insertions that showed no or increased effect on expression are listed. The rest are knockout/knockdowns which are not distinguished in exon/intron insertion mutants because a lot of reports used RT-PCR. RT-PCR can show a reduced level of transcript upstream from an insertion site and increased transcript downstream from the insertion site or vice versa, making it difficult to assign the mutant as knockdown or knockup. For “Major Deletions”, See Table 5. Insertions before the start codon or after stop codons are slightly less effective. Because of low correlation between transcript level and protein level as described above, the impact of insertion on expression is more accurately reflected on protein levels, which was used in about 12% (136) of all insertion mutants evaluated. Protein expression of the 136 insertion mutants indicates that the effectiveness for insertions into exon and intron are similar and both are more effective than insertions into either promoter or 5’ untranslated regions (5’ UTRs---Table 2). When T-DNA is inserted before the start codon, the translation of the gene is most likely reduced rather than completely knocked out (Table 2), hence producing a leaky mutant phenotype. This is illustrated by six insertions in GPT 1 in which three insertions in the coding region were lethal, while the other three insertions in either 5’ or 3’ noncoding regions had no effect on the transcript level of GPT1 (Niewiadomski et al. 2005). Insertions in exons and introns As indicated in Tables 1 and 2, insertion into either exon or intron in the protein-coding region is equally effective in knocking out/down the target gene. Among the cases in which insertion failed to change expression pattern of mutated genes, the T-DNA was inserted 39 bp upstream of the stop codon in BRM and normal transcription of the gene was not affected (Hurtado et al. 2006). It is not clear, however, why insertion into the exon did not knock out/down the target gene in the other cases. For example, in both annAt2-1 and annAt2-2 mutants, the T-DNA was inserted in the last exon of Arabidopsis annexin 2 (AnnAt2) gene. While no transcript was detected by RNA gel blot analysis in annAt2-1, the transcript J Biochem Tech (2008) 1(1):11-20 13 Similarly, insertion in the promoter significantly reduced AGP17 2003; Schnurr et al. 2002), a truncated protein can also be produced transcript in the roots but not in the leaves compared to the wild (Gusmaroli et al. 2004; Henderson et al. 2005; Kim et al. 2004; type (Gaspar et al 2004), similar to the reduced MIF1 transcript in Ohtomo et al. 2005; Okushima et al. 2005) which may not be the root not the stem when an insertion was 246 bp upstream of the functional (Gusmaroli et al. 2004; Okushima et al. 2005) even start codon (Hu and Ma 2006). So it is possible that insertion in a though in most cases truncated transcript is not translated (e.g., promoter could change expression pattern of the downstream gene. Dieterle et al. 2005; Doelling et al. 2001; Hashimoto et al. 2005; Schuster et al. 2006; Ullah et al. 2001). Similarly, even if transcripts Effectiveness of insertion decreases as it moves further upstream of both up- and down-stream of the insertion site are expressed, still the 5’ end in the promoter/5’ UTR based on its effect on transcript no protein can be produced due to interruption of the coding region level. If insertion before the start codon is grouped according to its by T-DNA (Delatte et al. 2005). In addition, truncated transcript distance (in bp) from ATG (Table 3), then knockout rate decreases may contain deletions altering protein sequence (see below; Bostick et al. 2004). Finally, it is not clear why one mutant would show no from 54% to 31% as insertion moves from 1-50 bp toward 201-500 detectable transcript and the others would have truncated transcripts bp upstream of the ATG and knockdown increases at the same time while they were all insertions in the same exon (Noh and Amasino from 33% to 53%. 2003). Table 3: Position effect of T-DNA insertion before the start codon in Arabidopsis on transcript level of affected genesa Insertion site 1-50 bp 51-200 bp upstream 201-500 bp 501-1000 bp Total upstream of of start codon upstream of start upstream of start codon codon start codon No transcript 21(54%) 20(42%) 10(31%) 9(50%) 60(44%) Reduced transcript 13(33%) 23(48%) 17(53%) 5(28%) 58(42%) No effect 3(8%) 2(4%) 4(13%) 3 (17%) 12(9%) Increased transcript 2(5%) 3(6%) 1(3%) 1 (5%) 7(5%)b Total 39 48 32 18 137 a Because 5’ UTR and promoter region were not clearly indicated in a lot of characterized insertion mutants, they are listed together. Data gathered from mutants for which an insertion position was given or can be estimated based on information given. b The percentage is higher than that listed in Table 1 because only mutants with insertion position given and less than 1 kb upstream of the start codon were counted here. Insertion toward the 3’ end of a gene but before the stop codon can also effectively knock out the function of the target gene. For example, the insertion at 47 bp upstream of the stop codon in AHA3 was found to be lethal (Robertson et al. 2004) and a T-DNA insertion at 13 bp before the stop codon also knocked out transcription in RCI2A (Mitsuya et al. 2005). On the other hand, as mentioned earlier, insertion at 39 bp before the stop codon in BRM did not affect transcript level (Hurtado et al. 2006). Similarly, insertion in the 5’ end of a coding region does not necessarily knock out the gene even if it is six bp after start codon. It only led to reduced protein production and less severe phenotype (Pružinská et al. 2007). But overall, this does appear to be the exception rather than the rule. Insertion toward the 3’ end of a gene can also produce a weaker phenotype (e.g., Chelysheva et al. 2005; Chen et al 2005; Guo and Ecker 2003; Yoine et al. 2006) although this is not always a case (Robertson et al. 2004). But the trend is reversed when insertion beyond 500 bp is considered, i.e., knockout rate increases to 50% and knockdown rate decreases to 28% (Table 3). Based on this set of data, the number of no-effect insertion also increases (Table 3). This difference in insertion effectiveness explains why 80% of characterized mutants harbor T-DNA insertion in the coding region and only 15% were focused on insertions before the start codon (Table 1). The total knockout rate based on transcript level is 45% and knockdown rate is 41% if all insertions within 1000 bp upstream of the start codon are considered (Table 3). Overall, 11% of insertions before the start codon did not affect transcription of the downstream gene while that number is less than 1% for insertion into the coding region (Table 1). Based on protein level, 6% (1 out of 17) insertions before ATG had no effect on protein expression of the mutant allele (Table 2). The fact that insertion closer to the start codon is more effective might be because some promoter elements essential for transcription are close (within ~500 bp) to the start codon (Table 4). If T-DNA is inserted into a promoter element such as TATA box (Novillo et al. 2004) or between TATA box and ATG (Nakajima et al. 2004), the gene is likely to be knocked out. The same is true for other elements such as AuxREs (Tatematsu et al. 2004) and Gboxes (Ito et al. 2003). T-DNA insertion between these elements and ATG knocked out or significantly reduced transcription of the respective genes. Even if these elements are farther upstream from the start codon, insertion could still be effective. For example, insertions more than 3 kb upstream of the UFO start codon but in CArG box-like sequences that are recognition elements for MADS box DNA-binding proteins disrupted petal development due to reduced UFO expression (Durfee et al. 2003). However, insertion of T-DNA among AuxREs in the promoter of the auto regulated ARF8 increased its transcript level (Goetz et al. 2006). Insertion upstream of the start codon increased transcription of the downstream gene in several cases. But it is not clear if the increase Insertions before the start codon Insertion into the promoter region also produces more knockdowns than knockouts. Based on protein expression, among 17 mutants with the T-DNA inserted in the promoter region, 7 showed no (knockout rate 41%) and 9 showed reduced (knockdown rate 53%) expression of mutated genes (Table 2). In the remaining one mutant there was no effect. Compared to the combined knockout (86%) or knockdown (10%) rates in the coding region (both introns and exons) based on protein expression (Table 2), the knockout rate is lower and knockdown rate is higher. T-DNA insertion into the promoter region can cause misexpression of the downstream gene. Such insertion resulted in fewer GTE6 transcripts in leaf 7 to a level similar to leaf 4 of the wild-type which caused round laminae in leaf 7, rather than the elliptical laminae of leaf 7 of the wild-type plants. Thus, elevated expression of GTE6 in leaves 6 and 7 of wild-type plants is important for the development of the elliptical leaf lamina (Chua et al. 2005). 14 J Biochem Tech (2008) 1(1):11-20 is solely driven by the promoter that drives the antibiotic resistance gene in the T-DNA vector (see below). It has been shown that when T-DNA was inserted into the promoter region of KIS the fused 35SBasta-KIS fusion transcript abundance increased (Kirik et al. 2002). Similarly, an insertion 100 bp upstream of the start codon in ARF17 increased its transcript by 7-12-fold compared to wild type, possibly due to the 35S promoter in the T-DNA vector (Sorin et al. 2005). So it is possible that promoter in the T-DNA sequence plays an important role in increasing transcript level of the downstream gene. downstream gene. A T-DNA was inserted downstream of AT1G65250 but upstream of AT1G65260 (VIPP1). Northern blot analysis using both genes as probes detected only significant reduction in VIPP1 transcription (Kroll et al. 2001). On the other hand, when a T-DNA was inserted 525 bp downstream of IRT2 but 2440 bp upstream of the start codon of IRT1, the expression of IRT1 was almost completely abolished while that of IRT2 was also impaired (Varotto et al. 2002). The latter may be caused by T-DNA disruption of the 3’ sequence of a gene as discussed above. Table 4: T-DNA insertion between promoter elements and coding region increases knockout effectiveness in Arabidopsis Gene Promoter element Distance to T-DNA insertion Effect on Transcript Reference start codon (in level bp) CBF2 TATA box 179 Within TATA box Expression not detected Novillo et al. (2004) SPR1 TATA box Within 200 Between TATA and ATG Expression not detected Nakajima et al. (2004) IAA19 AuxREs 165 Within AuxREs Expression not detected Tatematsu et al. (2004) APRR9 G-boxes 434-511 Between G-boxes and ATG Expression severely Ito et al. (2003) reduced. Hsp101 Heat-shock elements 320-400 Within HSEs but upstream No effect on Hong and Vierling (HSEs) and TATA of TATA transcription but protein (2001) box severely reduced UFO CArG box-like >3 kb Within CArG box-like Expression severely Durfee et al. (2003) sequences sequences reduced. Insertions after the stop codon T-DNA insertion after the stop codon is least effective compared to insertion in other parts of a gene. Based on 23 characterized insertions, 17% (or 4) insertions after the stop codon had no effect on transcript level of upstream genes compared to ~1% in insertions in the coding region (Table 1). Its knockout and knockdown rates of 37.5% each are lower than the 45% and 41% for the insertion in before the start codon, respectively. But insertion at some distance downstream from the stop codon still disrupts the transcription of the upstream gene such as TFL1 (650-700 bp after stop; Ohshima et al. 1997), CSLA9 (260 bp after stop; Zhu et al. 2003) and F5H (283 bp after stop; Ruegger et al. 1999) or interfere with the transcription of the gene such as LOL1 (629 bp after stop; Epple et al. 2003) and IRT2 (525 downstream; Varotto et al. 2002). The reason could be that sequence downstream the insertion site contains enhancer or other regulatory sequence essential for expression of the upstream gene. This was demonstrated in GL1 that harbored a T-DNA insertion 658 bp after the stop codon that separated the coding region from an enhancer element 900 bp downstream of the stop codon, causing a partial phenotype (Larkin et al. 1993; Oppenheimer et al. 1991). Although it is possible that insertion after the stop codons in the above genes were due to separation of downstream regulatory elements by T-DNA, no evidence was reported. Insertion after stop codons may also knock up the expression of the upstream gene such as PIP5K9 (Lou et al. 2007) and ACT7 (Gilliland et al. 2003) or have no apparent effect on the level of transcript such as ATEM6 (Manfre et al. 2006). Nevertheless, no or reduced level of proteins were detected in these mutants, probably because these mutants also produced shorter than expected transcripts (Gilliland et al. 2003; Manfre et al. 2006). Insertion in the 3’ end of a gene could increase its transcript level due to 35S enhancer present in the T-DNA region. When a T-DNA is inserted in the intergenic region between two genes in the same orientation, it is more likely to disrupt the T-DNA promoter can drive expression of downstream gene Most T-DNA sequences contain a selection marker driven by the Cauliflower Mosaic Virus 35S or other promoters which can in turn drive expression of the downstream gene producing a chimeric transcript as mentioned above in ARF17 (Sorin et al. 2005) and KIS (Kirik et al. 2002). This is one factor that is responsible for increased transcript abundance. The 35S promoter was suggested to be responsible for increased SWP transcript abundance and size although the mutant phenotype was still recessive, suggesting that the fused T-DNA-SWP transcript was not translated or the protein was not functional (Autran et al. 2002). The fused transcript was ~7 kb compared to 5.5 kb in the wild type. Since the insertion was 250 bp upstream of the start codon and at least the first intron was spliced out based on RT-PCR (Autran et al. 2002), it is puzzling as to what factors caused the fused transcript not to be translated other than stop codons. But in arf19, the T-DNA was inserted at 12 bp upstream of the start codon and a larger and more abundant mutant transcript was produced which fueled reduced ARF19 synthesis (Willmoth et al. 2005). So transcribed genes driven by the T-DNA promoter may or may not lead to protein synthesis. Over expression of a mutated gene due to fusion with T-DNA sequence can also occur in insertions in the coding region. In a swi1 mutant that carried an insertion at 2 bp after the start codon, a TDNA-SWI1 fusion transcript was apparently translated with a low efficiency because the mutant phenotype is leaky (Mercier et al. 2001). If T-DNA is inserted into the middle of a gene such as in exon 13 (out of 18) in AtISA1 as described earlier, the endogenous promoter drives the transcription of sequence upstream the insertion site while promoter in the T-DNA may drive the downstream sequence transcription (Delatte et al. 2005). The downstream transcript was more abundant compared to that of wild type, indicating an over expression due to the T-DNA promoter (see also Bertrand et al. 2005). Despite normal transcription of up- and downstream sequence, no protein was detected because the gene was interrupted by the insertion (Delatte et al. 2005). Over J Biochem Tech (2008) 1(1):11-20 ISSN: 0974-2328 Table 5: Larger deletions caused by T-DNA insertion in Arabidopsis genes. Gene Function DNA damage repair ARS27A Ribosomal protein S27 /elimination of damaged mRNA AtLIG4 DNA damage repair AtMSH2 Mismatch repair AtRAD17 DNA damage repair AtRAD51C DNA damage repair DME Mismatch/ damage repair/ Female Gametophyte Development MRE11 DNA damage repair/ chromosome integrity WEE1 DNA damage response WEE1 DNA damage response WEE1 DNA damage response Transport AtMRP4 Guard cell ABC transporter AtNrt2.2 Nitrate transporter AtSUC5 Endosperm sucrose transporter AtSUC5 Endosperm sucrose transporter GORK Guard cell K channel /transporter Cell division AGM Mitosis/Gamete Development ANQ1 MAPKK/ Cytokinesis ASY Synaptonemal complex assembly AtTop6B Topoisomerase TUBG2 Spindle formation/ microtubule organization Light signaling APRR5 Circadian rhythm/light sensing PHYB Phytochrome B PIL5 Phytochrome interacting/seed germination Chromatin remodeling LHP1 Chromatin remodeling/gene silencing Mom1 SWI2/SNF2 chromatin remodeling Plastid biogenesis Alb4 atTOC34 Miscellaneous AtAPG9 BAM2 NFD1 PRL1 Plastid membrane protein insertion/plastid biogenesis Plastid protein import/plastid biogenesis Protein degradation/ autophagy Receptor kinase/male gamete development Mitochondrial ribosomal protein L21/female gametophyte development Glucose/ hormone response Insertion Before ATG Exon 1 Exon 7 Exon 4 Intron 2 Before ATG Intron 9 Exon 1 Intron 7 Exon 9 Exon 1 850 after ATG -1061-+1665 -58-+499 -2280 - +1464 6 bp before ATG Intron 1 5’UTR Exon 12 Exon 2 Exon 5 Exon 3/Intron 3 Exon 2 Up ATG 3’ end Intron 6 Exon 6 Intron 3 Exon 1 Intron 1 Exon 15 Deletion 1287 bp including promoter and the 5’ UTR 329 bp upstream of insertion including the first 37 codons 1,510 bp from exons 7 to 13 (insertion to the end) 172 bp encompassing part of exon 4, intron 4 and a part of exon 5 141 bp of intron 4 177 bp before insertion site The entire exon 10 and portions of introns 9 and 10 Most of the coding region (~1.5 kb) From insertion to the end (~600 bp) >330 bp −588 to +1545 including a significant portion of the promoter, the entire TMD0, and most of TMD1 domains 25 kb from the insertion on including at least one other gene AtNrt2.1 -1061 - +1665 including exons 1 and 2 -58 - +499 including most of exon 1 -2280 - +1464 including exon 1 and most of intron 1 148 bp including 127 bp 5’UTR and 21 bp promoter 5’ UTR and exon 1 were deleted and part of ANQ1 was duplicated 1.1 kb including 74 bp 5’ UTR and ~1. 0 kb promoter 268 bp consisting of intron 12 to intron 13 (total 18 exons) 2440 bp from the insertion to the end of the gene including eight exons From the insertion to the end and also include At5G24460 downstream ~150 bp in the border of exon 3 and intron 3 From insertion to the end of PIL5 1.2 kb upstream of the insertion 1,980 bp from the insertion to the end (last 4 exons) to part of promoter of the next gene Exon 7 and intron 7 The remaining gene downstream from the insertion (~600 bp) From insertion to the end covering seven exons >670 bp upstream of the insertion deleted and/or rearranged 138 bp including part of intron 1 and entire exon 2 ~300 bp from inside exon 15 into intron 16 Reference Revenkova et al. 1999 Friesner and Britt 2003 Leonard et al. 2003 Heitzeberg et al. 2004 Li et al. 2005 Choi et al. 2002 Puizina et al. 2004 De Schutter et al. 2007 Klein et al. 2004 Filleur et al. 2001 Baud et al. 2005 Baud et al. 2005 Hosy et al. 2003 Sorensen et al. 2004 Soyano et al 2003 Caryl et al. 2000 Hartung et al. 2002 Pastuglia et al. 2006 Yamamoto et al. 2003 Reed et al. 1993 Oh et al. 2004 Gaudin et al 2001 Amedeo et al. 2000 Gerdes et al. 2006 Constan et al. 2004 Hanaoka et al. 2002 DeYoung et al. 2006 Portereiko et al. 2006 Németh et al. 1998 J Biochem Tech (2008) 1(1):11-20 16 ISSN: 0974-2328 J Biochem Tech (2008) 1(1):11-20 expression of upstream sequence by endogenous promoter because of the insertion was also reported although the mechanism was not clear (Okushima et al. 2005). Insertion leads to chimeric transcript T-DNA insertion can interfere with mRNA processing of affected genes, particularly intron splicing. When the insertion was into an intron within the 5' UTR of ERS1, the resulting chimeric transcript contained a segment of T-DNA sequence, a segment of unspliced UTR intron, a segment of native UTR, and the native coding sequence which could not possibly be translated into the functional protein (Wang et al. 2003). Insertion into intron 2 of WRKY33 produced a larger transcript due to the deletion and duplication at the 3'-end of intron 2 that prevented proper splicing (Zheng et al. 2006). Insertion before the start codon also interferes with correct intron splicing. PORC mutant contained an insertion at 26 bp before the start codon and RT-PCR with primers spanning first three exons showed that the introns were not properly spliced (Masuda et al. 2003). In a mutant with a T-DNA insertion in intron 4 (out of 9), the upstream transcript included the first four ALD1 exons and TDNA border sequence and that the downstream transcript included a T-DNA sequence at the 5' end and the six 3' exons of ALD1 at the 3' end (Song et al. 2004). On the other hand, insertion in intron 3 of SCA3 at +1894 bp produced a transcript with 1667 bp SCA3 mRNA and more than 270 bp T-DNA left border (Hricova et al. 2006). Insertion into exons can produce wild type transcript up to the insertion point followed by sequence addition/deletion and T-DNA. If the insertion is in the very last exon, i.e., 47 bp before the stop codon as in AHA3 (Robertson et al. 2004), the resulting transcript could include the wild type mRNA without the last 65 bases followed by >200 bases of T-DNA. Another insertion in exon 4 (~1/3 of the coding region) yielded a transcript with the first 1/3 wild type mRNA with 17 bases of unknown sequence followed again by >200 bases of T-DNA (Robertson et al. 2004). Longer TDNA can also be transcribed and attached to the transcript. For example, an insertion at +1787 bp position of EBF2 had a transcript with 1787 bp EBF2 mRNA plus ~800 bases of T-DNA (Gagne et al. 2004). In most cases, these chimeric transcripts were not translated or the proteins were not fully functional. and meiosis/mitosis and five (16%) into transporter genes. Four of the genes also played roles in male or female gamete development. Similarly, among the 82 mutants with deletions smaller than 100 bp, 29 (39%) insertions were into genes related to DNA repair, replication, meiosis and chromosome remodeling and eight (10%) into transporter genes. These numbers are a lot higher than those expected from random deletions. It is known that repair genes are essential for T-DNA integration which double-stranded DNA breaks can attract (reviewed by Tzfira and Citovsky 2006) and the female reproductive tissues are the primary target of transformation by the Arabidopsis floral-dip method (Desfeux et al. 2000). But why are these genes more likely to suffer larger deletions as a result of T-DNA insertion? The answer is not clear although it is conceivable that because T-DNA tends to insert into transcriptionally active regions (Alonso et al. 2003) and these genes are consistently transcribed in the actively-dividing tissue that open themselves up for the T-DNA molecule. The prolonged exposure to T-DNA somehow leads to incorrect double-stranded break which results in larger deletion of the target site. Incidentally, 11 of the 31 mutants had insertions in chromosome V while there was only one in chromosome IV. But the distribution of the other 19 insertions seems to be random among the other three chromosomes. T-DNA insertion also triggered reciprocal translocation and duplication. Reciprocal translocation was reported for T-DNA insertion into ARL2 (Guan et al. 2003) and RHD3 (Yuen et al. 2005) while translocation was observed in atToc33 (Gutensohn et al. 2004). Insertion in an intron of WRKY33 caused duplication of the entire gene as described earlier (Zheng et al. 2006). More dramatic rearrangements were reviewed by Latham et al. (2006). Conclusion Insertion in the protein-coding region of a gene generates a knockout 86% of the time whether it is in the introns or exons; or 41% of the time if the insertion is before the start codon. Insertion before the start codon produces a knockdown 53% of the time. Insertion after the stop codon had no effect on transcription of the upstream gene 17% of the time compared to 11% in insertion before the start codon. In 10 out of 263 mutants that had an insertion in an intron, the intron was spliced out together with the T-DNA producing wild type transcript at a reduced level. While the absence of the transcript of a gene carrying a T-DNA indicates the gene has been knocked out, it is more complex when an increased transcript level of the gene is detected. Based on this survey, it is more likely than not that protein level is also reduced if it is still synthesized. So it is always advisable to check the transcript level of the mutant allele, especially when a putative homozygous mutant did not show discernible phenotypes. Compared to Northern analysis, RT-PCR can not determine the size of a large transcript created by T-DNAplant gene fusion produced by insertion upstream of the start codon. The fused transcript can be driven by promoter in the T-DNA and can be chimeric. The event of T-DNA insertion is known to cause additional changes to the host genome such as deletion and translocation. In these cases, the mutated gene suffers both deletion and insertion, effectively disrupting its expression. Among insertion mutants with reported deletions of the genomic sequence, three quarters of them had deletions smaller than 100 bp. For the large deletions, a disproportionate number (17 out of 31) of them are in genes related to damage repair, replication, chromatin remodeling, meiosis/mitosis, and transport. This may warrant further study to identify possible links between this and mechanism of T-DNA insertion. However, because these conclusions are based on Insertion causes deletion T-DNA insertion can often cause deletion and rearrangements of the host genome (reviewed by Latham et al. 2006). Out of 1084 insertion mutants surveyed in this paper, about 10% (113) reported deletions although this number can be as high as 87% (Forsbach et al. 2003). But most deletions were small. Among the 113 T-DNA insertion mutants with reported deletions, 82 (73%) had deletions smaller than 100 bp, similar to that reported earlier (78% by Forsbach et al. 2003; 81% by Meza et al. 2002). Of the 82 mutants, 77 (94%) had deletions smaller than 60 bp and 68 (83%) less than 50 bp. With a sample size of 22, Meza et al. (2002) reported that 18 were smaller than 100 bp and 14 of the 18 (78%) were smaller than 60 bp. The average size of deletions is 30 bp among the 82 mutants. The size of deletion for the other 31 mutants ranged from 138 bp to 25 kb but majority was below 2 kb while Forsbach et al. (2003) reported 1 out of 64 (1.6%) and Meza et al. (2002) found 4 out of 22 (18%) deletions are larger than 100 bp. 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Plant Physiol 133:1000-1010 J Biochem Tech (2008) 1(1):21-22 ISSN: 0974-2328 Hatching enzyme of Volvox: a possible implication in the evolution of multicellularity Hideaki Shiraishi Received: 6 June 2008, Accepted: 10 August 2008, Available online: 26 August 2008 © Sevas Educational Society 2008 Volvox as a model organism A green alga Volvox carteri is an attractive model organism for studying the evolution of multicellularity because of its simple body plan consisting of only two cell types. A typical young spheroid of V. carteri consists of about 16 reproductive cells called gonidia and approximately 2000 biflagellate somatic cells that are morphologically similar to unicellular green alga Chlamydomonas reinhardtii. Since the divergence time of V. carteri and C. reinhardtii estimated by molecular phylogenetic analysis was as recently as 50-75 million years ago (Rausch et al. 1989), it is considered that relatively simple alterations in the genetic system of the unicellular ancestor gave rise to multicellular organisms (Kirk 2005). Development of tools for molecular genetic study in V. carteri, such as genetic transformation by microprojectile bombardment and gene cloning by transposon-tagging, enabled researchers to use this organism as a model for studying the genetic and cytological control of morphogenesis (Kirk 2005; Kirk and Nishii 2001; Miller 2002). the daughter organisms have matured (Fig. 1). In C. reinhardtii, hatching of daughter cells from their mother cell walls occurs within several hours after cell division. In contrast, in V. carteri, hatching of juveniles occurs after more than a day after the first cell division of the reproductive cells. Various developmental events including cleavage divisions to increase cell number take place before hatching. Also, in colonial relatives of Volvox, cell cleavages to increase cell numbers occur before hatching in individual organisms. Thus, heterochronic delay in the timing of hatching that allows embryos to increase cell Extracellular matrix and hatching protease V. carteri belongs to the order Volvocales that include a group of closely related organisms that range in complexity from unicellular organisms (e.g. C. reinhardtii), through colonial organisms with various cell numbers (e.g. Gonium and Pandorina), to multicellular organisms (e.g. V. carteri) (Kirk 2005). Individual cells of many of these algae are held together in the body of adult organisms by extracellular matrix (ECM), the major components of which are glycoproteins (Hallmann 2006). The cells of unicellular species are also surrounded by ECM, that is usually called ‘cell wall’, that is homologous to the ECM of the colonial and multicellular relatives. In the life cycle of volvocine algae, daughter organisms hatch out of parental bodies degrading the parental ECM after Figure 1: Mature spheroid of Volvox carteri (left) and a daughter spheroid in the process of hatching (right). Bars = 0.1 mm. Hideaki Shiraishi, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan Tel: 008 175 753 3998, Fax: 008 175 753 3996 E-mail: siraisi@kuchem.kyoto-u.ac.jp number is one prerequisite for the evolution of multicellularity in volvocine algae. Concerning the hatching process in Volvox carteri, it had been known that an enzymatic activity that degrades parental ECM is detected in the culture medium during and after hatching. Recently, molecular details of the enzyme were revealed (Fukada et al. 2006). This hatching enzyme is a serine protease that accumulates in the embryos as a precursor containing N-terminal transmembrane domain. The accumulated enzyme is secreted exactly at the timing of hatching after removal of the N-terminal domain, suggesting that post-translational regulation is involved in the strict control of the timing of hatching. Also suggested in the study is that the substrate of the protease is synthesized and integrated into the ECM only after spheroids are liberated from parental spheroids, since ECM of young spheroids just after hatching is resistant to the enzyme whereas ECM of the spheroids in later stages become susceptible. Thus, hatching of V. carteri is regulated at two levels: one is at the level of the activation and secretion of the hatching enzyme and the other is at the Level of the synthesis of the substrate and its integration into ECM. 22 It is known that, in mammals, degradation of ECM by proteases secreted from cancer cells are involved in invasion and metastasis of cancer. Although these events are not welcome for higher organisms, the combination of ECM connecting cells and proteases degrading ECM may have played an important role in the evolution of multicellularity in volvocine algae and possibly in the early life on earth. J Biochem Tech (2008) 1(1):21-22 References Fukada K, Inoue T, Shiraishi H (2006) A posttranslationally regulated protease, VheA, is involved in the liberation of juveniles from parental spheroids in Volvox carteri. Plant Cell 18:2554-2566 Hallmann A (2006) The pherophorins: common, versatile building blocks in the evolution of extracellular matrix architecture in Volvocales. Plant J 45:292-307 Kirk DL (2005) A twelve-step program for evolving multicellularity and a division of labor. BioEssays 27:299-310 Kirk DL, Nishii I (2001) Volvox carteri as a model for studying the genetic and cytological control of morphogenesis. Dev Growth Differ 43:621-631 Miller SM (2002) Taming the fierce roller: an “enhanced” understanding of cellular differentiation in Volvox. BioEssays 24:3-7 Rausch H, Larsen N, Schmitt, R (1989) Phylogenetic relationship of the green alga Volvox carteri deduced from small-subunit ribosomal RNA comparison. J. Mol. Evol. 29:255-265 J Biochem Tech (2008) 1(1):23-29 ISSN: 0974-2328 In search of best impact factor and citation indexed journals towards achieving the goals of universities Hj. Kamaruzaman Jusoff Received: 16 July 2008 / Received in revised form: 22 July 2008, Accepted: 18 August 2008, Published online: 20 August 2008 © Sevas Educational Society 2008 Abstract This paper provides an introduction to the concept and importance of journal impact factor (JIF), citation indexes. The paper takes the form of a short history of JIF, the importance of journal performance indicators, about calculating and determining JIF and the significance of using Citation Indexes. Elements of the factors affecting impact factor namely scientometrics and journalology were also highlighted. A guide to locating the best impact factor journals in various research fields was demonstrated. Despite that JIF is still a dilemma for some fields of research, academic members are still subjected to the Key Performance Index (KPI) criteria compliance for further promotion and excellence service awards on top of the need to generate high impact research publications. Keywords: Impact factor, Research University, Citation indexes, biochemical journals One of the ways to maintain Research University status is to ensure that all academic staff do research and publish in scholarly journals. Most academic work is published in journal article or book form. In journal publishing, despite some researchers feel that Impact Factor of journals should not be used for evaluating research quality and performance (Seglen 1997; Smith 2006; Hobbs 2007), we have to make sure that the journals are of citation indexed, have a high impact factor and refereed since one of the university requirements is to quantify the number of such journals meet the target of the minimal requirement of a research status. Perhaps, impact factor of a journal is more valid measure of journal quality for specific fields such as medicines and biomedical (Saha et al. 2003). Under such a dilemma, all academic staff, does not matter of the fields of research, have to abide by the Key Performance Index (KPI) which requires that at least a Professor must have three journals a year, two for Associate Professors and one for a lecturer which of Citation Indexed and of High Impact Factor. Most established academic fields have their own journals and other outlets for publication, though many of these academic journals are somewhat interdisciplinary, and publish work from several distinct fields or subfields. The kinds of publications that are accepted as contributions of knowledge or research vary greatly between fields. The objective of this paper is therefore to give a greater in-sight of what an impact factor journal is and the significance of publishing research papers in high impact factor journals. An overview of citation indexed journal is explained with other related factors like Immediacy Index (II) in relation to the need of a researcher to have his or her publications as Open Access (OA). Introduction The inspirational motto, 'With Knowledge We Serve', reflects the full commitment of Universities in developing countries to contribute towards the discovery of knowledge and the exploration of human endeavor as well as the creation of wealth and nation building. Today, we are faced with complex challenges and only those who persevere will survive. In order to face these challenges, the generation of knowledge and life-long learning is crucial and need to be reinforced. Universities must therefore visionalized to actively participate in new adventures of ideas, experiment with innovative methods, and take intellectual initiatives to further discover and expand the frontiers of knowledge Hj. Kamaruzaman Jusoff Yale University Yale’s Centre for Earth Observation, Environmental Science Centre 21 Sachem St, New Haven, CT 06511, USA Tel: 001 203 432 1384 Email: jusoff.kamaruzaman@yale.edu The need to publish and get accessed openly Academic publishing describes a system that is necessary in order for academic scholars to peer review the work and make it available for a wider audience or make it opened to a bigger access. The system which is probably disorganized enough not to merit the title, varies widely by field, and is always changing, if often slowly. Meanwhile, 'Open access' (OA) means that a reader of a scientific publication can read it over the Internet, download and even further distribute it for non-commercial purposes without any payments or restrictions. The four most important OA channels are electronic- J Biochem Tech (2008) 1(1):23-29 ISSN: 0974-2328 24 J Biochem Tech (2008) 1(1):23-29 refereed-scientific periodicals, research-area-specific archive (eprint) servers (in this paper called subject-specific repositories), institutional repositories of individual universities, and self-posting on authors' home pages (Bjork 2004). Research & Development policy makers around the world such as that of the American Association for the Advancement of Science (AAAS), an international non-profit organization dedicated to advancing science around the world by serving as an educator, leader, spokesperson and professional association that publishes “SCIENCE” have recommended mandating that researchers provide Open Access (OA) to their research articles by self-archiving them free for all on the Web. Another renowned publishing group based in the UK, Nature. Com for instance publishes the world's best science and medicine abstracts on your desktop is now firmly on the agenda for funding agencies, universities, libraries and publishers. David’s article entitled “the Counting House” that appeared in Nature should be an interesting one to be read (Adam 2002). What is needed now is objective, quantitative evidence of the benefits of OA to research authors, their institutions, their funders and to research itself. OA articles have significantly higher citation impact than non-OA articles (Harnad et al. 2004). Brody (2004) was also supported in a web-based analysis of usage and citation patterns. One universally important factor for all authors is impact made by their research papers, typically measured by the number of times a paper is cited. Now the Open Archives (OA) era has revolutionized with new ideas about starting a global database for finding the number of citations received to the OA submissions. Citebase and Citeseer are two such web tools, which serve this partially (Brody 2003). Studies have begun to show that open access increases impact, although more studies and more substantial investigations are needed to confirm the effect. Hitchcock (2004) has given the progress in these directions in the form of a chronological bibliography with some explanation. The citation analysis in the fields of high-energy physics and astrophysics, indicates that the number of citations to traditional preprints has gradually declined over the past 10 years, and that citations to electronic preprints have nearly doubled every year since 1992 (Youngen 1998a & 1998b). The electronic preprint servers are often the first choice of physicists and astronomers for finding information on current research, breaking scientific discoveries, and keeping up with colleagues (and competitors) at other institutions (Prakasan et al. 2004; Prakasan and Kalyane 2004). In addition to these benefits, electronic preprints allow the free, unrestricted access to scientific information without concern for international, institutional, or political barriers. Recently, Laurence (2001) and Brody et al., (2004) have demonstrated that articles which are available on-line at no charge are cited at substantially higher rates than those which are not. Kurtz (2004) has shown that restrictive access policies can cut article downloads to half the free access rate (Kurtz et al. 2004). A new measure that becomes possible with online publication is the number of downloads or 'hits', opening a new line of investigation. Although many authors believe that their work has a greater research impact if it is freely available, studies to demonstrate that impact are few (Antelman 2004). The latest example of such on-line free journals are that of the Canadian Centre for Science & Education (CCSE), World Scientific Engineering Academy and Society (WSEAS) and World Academy for Science, Engineering & Technology (WASET) to name a few. Brody et al. (2004) have been prominent in showing there is a correlation between higher downloads and higher impact, particularly for high impact papers, holding out the promise not just for higher impact resulting from open access but for the ability to predict high impact papers much earlier, not waiting years for those citations to materialize (Brody and Harnad 2004). The effect can be verified with the Correlation Generator. Citation analysis can be used to find emerging fields, to map the time-course and direction of research progress, and to identify synergies between different disciplines (Brody 2004). Citation analysis is being mainly used for measuring the impact made by journal articles. But Rousseau (1997) has attempted to compare the impact made by the ‘first and second international conferences on bibliometrics, scientometrics and informetrics’ with some top journals in the field. Information scientists are already computing web impact factors (Bjorneborn and Ingwersen 2001). Definition and history of journal impact factor (JIF) The analysis of citations is among the means by which policymakers, scientists, and information professionals seek to achieve a greater understanding of the qualitative forces that affect communications in science (Tomer 1986). Like nuclear energy, the two measures have become a mixed blessing, expected that it would be used constructively while recognizing that in the wrong hands it might be abused (Garfield 1999a). As long as scientists publish articles containing lists of cited references, it will be possible to calculate impact factors (Garfield 2001). Garfield (2004) has also stated that “it has been demonstrated that on line access improves both readership and citation impact”. The same impact factor can indicate the ‘influence’ and ‘performance’ of e-print archives they make among scientists. Dr. Garfield, the current Chairman Emeritus of Thomson Scientific, Philadelphia first mentioned the idea of an impact factor in Science in 1955. (Garfield 1955). With support from the National Institutes of Health, the experimental Genetics Citation Index was published, and that led to the 1961 publication of the Science Citation Index. Garfied and Sher (1963) then created the journal impact factor to help select additional source journals. To do this, the author citation index was simply re-sorted into the journal citation index. From this simple exercise, it can be learned that initially a core group of large and highly cited journals needed to be covered in the new Science Citation Index (SCI). Consider that, in 2004, the Journal of Biological Chemistry published 6500 articles, whereas articles from the Proceedings of the National Academy of Sciences were cited more than 300 000 times that year. Smaller journals might not be selected if we rely solely on publication count, thus Garfield (1972) created the journal impact factor (JIF). Tables providing a selective list of journals ranked by impact factor for 2004 or even 2005 can be easily obtained from the web. Such tables also includes the total number of articles published in 2004, the total number of articles published in 2002 plus 2003 (the JIF denominator), the citations to everything published in 2002 plus 2003 (the JIF numerator), and the total citations in 2004 for all articles ever published in a given journal. Sorting by impact factor allows for the inclusion of many small (in terms of total number of articles published) but influential journals. Obviously, sorting by total citations or other provided data would result in a different ranking. The term "impact factor" has gradually evolved to describe both journal and author impact. Journal impact factors generally involve relatively large populations of articles and citations. Individual authors generally produce smaller numbers of articles, although some have published a phenomenal number. Even before the Journal Citation Reports (JCR) appeared, Garfield (1972) sampled the 1969 SCI to create the first published ranking by impact factor. Today, the JCR includes every journal citation in more than 6000 journals about 15 million citations from 1 million source items per year. The precision of impact factors is questionable, but reporting to 3 decimal places reduces the number 25 J Biochem Tech (2008) 1(1):23-29 of journals with the identical impact rank. However, it matters very little whether, for example, the impact of “Journal of American Medical Association” (JAMA) is quoted as 24.8 rather than 24.831. A journal's impact factor is based on two elements: the numerator, which is the number of citations in the current year to items published in the previous two years, and the denominator, which is the number of substantive articles and reviews published in the same two years. The impact factor could just as easily be based on the previous year's articles alone, which would give even greater weight to rapidly changing fields. An impact factor could also take into account longer periods of citations and sources, but then the measure would be less current. months, then 776 times in 2003 and 862 times in 2004. The second, more recent, hot paper has already been cited in 300 articles. Calculation and determination of Impact Factor (IF) Garfield, probably the world’s foremost proponent of citation analysis through two measures: impact factor (IF) and immediacy index (II), first mentioned the ideas in 1955. The analysis of citations is among the means by which policy-makers, scientists, and information professionals seek to achieve a greater understanding of the qualitative forces that affect communications in science (Tomer 1986; Bauer and Bakkalbasi 2005). Like nuclear energy, the two measures have become a mixed blessing, expected that it would be used constructively while recognizing that in the wrong hands it might be abused (Garfield 1999a). As long as scientists publish articles containing lists of cited references, it will be possible to calculate impact factors (Garfield 2001). Garfield (2004) has also stated that “it has been demonstrated that on line access improves both readership and citation impact”. The same impact factor can indicate the ‘influence’ and ‘performance’ of eprint archives they make among scientists. According to Institute of Scientific Information (ISI), the ‘Impact Factor’ and ‘Immediacy Index’ of a journal are calculated as follows: Impact Factor= No. of citations to the previous two years articles in the calculating year No. of articles published in the previous two year citable The significance of journal performance indicators Many of the discrepancies inherent in JIFs are eliminated altogether in another Thomson Scientific database called Journal Performance Indicators (JPI). Unlike the JCR, the JPI database links each source item to its own unique citations. Therefore, the impact calculations are more precise. Only citations to the substantive items that are in the denominator are included. And it is possible to obtain cumulative impact measures covering longer time spans. For example, the cumulated impact for JAMA articles published in 1999 was 84.5. This was derived by dividing the 31 257 citations received from 1999 to 2004 by the 370 articles published in 1999. That year JAMA published 1905 items, of which 680 were letters and 253 were editorials. Citations to these items were not included in the JPI calculation of impact. In addition to helping libraries decide which journals to purchase, JIFs are also used by authors to decide where to submit their articles. As a general rule, the journals with high impact factors include the most prestigious. Some would equate prestige with high impact. The use of JIFs instead of actual article citation counts to evaluate individuals is a highly controversial issue. Granting and other policy agencies often wish to bypass the work involved in obtaining citation counts for individual articles and authors. Allegedly, recently published articles may not have had enough time to be cited, so it is tempting to use the JIF as a surrogate evaluation tool. Presumably, the mere acceptance of the paper for publication by a high-impact journal is an implied indicator of prestige. Typically, when the author's work is examined, the impact factors of the journals involved are substituted for the actual citation count. Thus, the JIF is used to estimate the expected count of individual papers, which is rather dubious considering the known skewness observed for most journals. Today, so-called Webometrics are increasingly brought into play, though there is little evidence that this approach is any better than traditional citation analysis. Web "citations" may occur a little earlier, but they are not the same as "citations." Thus, one must distinguish between readership or downloading and actual citation in new published papers. But some limited studies indicate that Web citation is a harbinger of future citation. The assumption that the impact of recent articles cannot be evaluated in the SCI is not universally correct. While there may be several years' delay for some topics, papers that achieve high impact are usually cited within months of publication and certainly within a year or so. This pattern of immediacy has enabled Thomson Scientific to identify "hot papers" in its bimonthly publication, Science Watch. However, full confirmation of high impact is generally obtained 2 years later. The Scientist waits up to 2 years to select hot papers for commentary by authors. Most of these papers will eventually go on to become "citation classics." Two recent examples of hot papers published in JAMA are those on the benefits and risks of estrogen in postmenopausal women. The first was cited in 132 articles after 6 Immediacy Factor= No. of citations to the articles published in the calculating year No. of citable articles published in the calculating year Similarly, the OA (Open Access) Impact Factor (IF) and the OA Immediacy Index (II) can be calculated as follows: OAIF= Number of citations received to the previous two years submissions in the calculating year (without self citations) Number of submissions in the previous two years OAII= Number of citations received to the submissions in the calculating year (without self citations) Number of submissions in the same years The minimum Impact Factor and Immediacy Index for Open Archives as calculated for journals are usually done by Institute of Scientific Information (ISI) without the first factor x2. The Science Citation Index data is used for computing the Impact Factors and Immediacy Index for Open Archives. Then the Open Archive Classes are compared with the journals included in the Science Citation Index. Refining the computation of topic based impact factors can be done through the computation of impact factors for individual research papers (Garfield 1999b). Citation and publication patterns differ between disciplines, so the Impact Factor is only meaningful when it is used to compare journals within a discipline (Testa and McVeigh 2004). Relationship between high impact and citation index journals What are citation indexes and why use it? Citation indexes track references that authors put in the bibliographies of published papers. They provide a way to search for and analyze the literature in a way not possible through simple keyword/topical searching. It also enables users to gather data on the "impact" of individual authors and journals, as well as assessing particular areas of research activity and publication. This latter field is called bibliometrics. J Biochem Tech (2008) 1(1):23-29 26 Citation indexing began in the 1950s, and has long been dominated by the Institute for Scientific Information (now Thomson Scientific), the creator and publisher of the three citation indexes available today: Science Citation Index (SCI), Social Sciences Citation Index (SSCI), and Arts & Humanities Citation Index (AHCI). This page focuses on the use of SCI in the "hard" sciences, which have the longest track record in citation studies. SCI covers nearly 6,000 journals across all science and engineering disciplines. All three ISI citation indexes back to 1975 are available online to users via the Web of Science system. Scientometrics and journalology Citation analysis has blossomed over the past four decades. The field now has its own International Society of Scientometrics and Informetrics. Lock (1989) aptly named the application of bibliometrics to journals evaluation "journalology." All citation studies should be adjusted to account for variables such as specialty, citation density, and half-life (Pudovkin and Garfield 2004). The citation density is the average number of references cited per source article and is significantly lower for mathematics journals than for molecular biology journals. The half-life (i.e. number of retrospective years required to find 50% of the cited references) is longer for physiology journals than that for physics journals. For some fields, the JCR's 2-year period for calculation of impact factors may or may not provide as complete a picture as would a 5- or 10year period. Nevertheless, when journals are studied by category, the rankings based on 1-, 7-, or 15-year impact factors do not differ significantly (Garfield 1998a and 1998b). When journals are studied across fields, the ranking for physiology journals improves significantly as the number of year’s increases, but the rankings within the category do not significantly change. There are exceptions to these generalities. Critics of the JIF will cite all sorts of anecdotal citation behavior that do not represent average practice. Referencing errors abound, but most are variants that do not affect journal impact, since only variants in cited journal abbreviations matter in calculating impact. These are all unified prior to issuing the JCR each year. The impact factors reported by the JCR tacitly imply that all editorial items in BMJ, JAMA, Lancet, New England Journal of Medicine, etc, can be neatly categorized, but such journals publish large numbers of items that are not substantive in regards to citations. Correspondence, letters, commentaries, perspectives, news stories, obituaries, editorials, interviews, and tributes are not included in the JCR's denominator. However, they may be cited, especially in the current year. For that reason, they do not significantly affect impact calculations. Nevertheless, since the numerator includes later citations to these ephemera, some distortion will result, although only a small group of leading medical journals are affected. The assignment of publication codes is based on human judgment. A news story might be perceived as a substantive article, and a significant letter might not be. Furthermore, no effort is made to differentiate clinical versus laboratory studies or, for that matter, practice-based versus research-based articles. All these potential variables provide grist for the critical mill of citation aficionados. cross-disciplinary impact. It is well known that there is a skewed distribution of citations in most fields. The so-called 80/20 phenomenon applies, in that 20% of articles may account for 80% of the citations. The key determinants of impact factor are not the number of authors or articles in the field but, rather, the citation density and the age of the literature cited. The size of a field, however, will increase the number of "super-cited" papers. And while a few classic methodology papers exceed a high threshold of citation, thousands of other methodology and review papers do not. Publishing mediocre review papers will not necessarily boost a journal's impact. Some examples of super-citation classics include the Lowry method, cited 300 000 times (Lowry et al. 1951), and the Southern Blot technique, cited 30 000 times (Southern 1975). Since the roughly 60 papers cited more than 10 000 times are decades old, they do not affect the calculation of the current impact factor. Indeed, of 38 million items cited from 1900-2005, only 0.5% were cited more than 200 times. Half were not cited at all and about one quarter was not substantive articles but rather the editorial ephemera mentioned earlier. The skewness of citations is well known and repeated as a mantra by critics of the impact factor. If manuscript refereeing or processing is delayed, references to articles that are no longer within the JCR's 2year impact window will not be counted (Yu et. al. 2005). Alternatively, the appearance of articles on the same subject in the same issue may have an upward effect, as shown by Opthof (1999). For greater precision, it is preferable to conduct item-by-item journal audits so that any differences in impact for different types of editorial items can be taken into account (Garfield 1986). Some editors would calculate impact solely on the basis of their most-cited papers so as to diminish their otherwise low impact factors. Others would like to see rankings by geographic or language group because of the SCI's alleged English-language bias, even though the SCI covers European—largely German, French, and Spanish—medical journals. Other objections to impact factors are related to the system used in the JCR to categorize journals. The heuristic methods used by Thomson Scientific (formerly Thomson ISI) for categorizing journals are by no means perfect, even though citation analysis informs their decisions. The collective work by Pudovkin and Garfield (2002) was an attempt to group journals objectively. They relied on the 2-way citational relationships between journals to reduce the subjective influence of journal titles such as the Journal of Experimental Medicine - one of the top 5 immunology journals (Garfield 1972). The Importance of using citation indexes Finding papers that cite earlier papers Citation indexing is a way to look forward in the literature from the starting point of a particular paper or group of papers. This is a different and complementary approach to ordinary word-based literature searching, which looks backward in the literature from the present time. For example, if you have an excellent paper on a particular topic that was published in 2007, you can use Science Citation Index (via Web of Science) to find papers published after 2007 that cited that paper. Citation implies a direct subject relationship between the papers. So, by searching for later papers citing your known paper, you can find more documents on the same or similar topic without using any keywords or subject terms. Lately, newly established journal publishers encourage their authors to cite their own papers from the publisher’s journals to increase the impact factor of their own journals since journal paper publishing nowadays tend to be business oriented. Size versus citation density There is a widespread belief that the size of the scientific community that a journal serves significantly affects impact factor. This assumption overlooks the fact that while more authors produce more citations, these must be shared by a larger number of cited articles. Most articles are not well-cited, but some articles may have unusual 27 J Biochem Tech (2008) 1(1):23-29 Procedures of citing papers You can easily find out how many times your papers have been cited. Citation searching allows you to move forward in time by finding newer papers that cite earlier papers. SCI is part of the Web of Science online system produced by Thomson-ISI. For information on counting your total citations, see the Counting Your Citations page. As an example, for information on searching for citing articles in google scholars (http://scholar.google.com/) can be demonstrated below: Google Scholar includes a "Cited by" (Fig. 1) count in its display of individual entries. This is calculated from citations appearing in other articles indexed by Google Scholar. Clicking on this link will take you to the list of citing articles. Since it is impossible to determine with any accuracy what publications Google Scholar does or does not index, this is not a reliable figure but it is one of the best open source citation search found in web. Scholarly journals contain articles written by, and addressed to, experts in a discipline. They are concerned with academic study, especially research, and demonstrate the methods and concerns of scholars. The main purpose of a scholarly journal is to report original research or experimentation and to communicate this information to the rest of the scholarly world. The language of scholarly journals reflects the discipline covered, as it assumes some knowledge or background on the part of the reader. Scholarly journals always rigorously cite their sources in the form of footnotes or bibliographies. Many scholarly journals are published by professional organizations. While not all scholarly journals go through the peer-review process, it is usually safe to assume that a peer-reviewed journal is also scholarly. Some of the databases which allow you to limit your search to referee or peer reviewed journals are: • • • • EBSCOhost (multidisciplinary) InfoTrac (multidisciplinary) ProQuest (business and criminal justice) CINAHL (nursing & allied health) - select "Peer Reviewed" in the Journal Subset Phrase dropdown box. Other options include "blind peer reviewed", "double blind peer reviewed", "editorial board reviewed" and "expert peer reviewed". You may find that most or all of the journals included in a particular database are scholarly, refereed journals, so that proper selection of the database may automatically eliminate the less scholarly sources. • • • • • • • A few examples are: JSTOR (multidisciplinary) Science Direct (multidisciplinary) Project MUSE (humanities, social sciences, mathematics) PsycINFO (psychology) Web of Science (multidisciplinary) Cambridge Scientific Abstracts (multidisciplinary) Figure 1: A Sample of citation Impact Factor for Kamaruzaman Jusoff from the Google Scholar (http://scholar.google.com/) Finding the "Best" journals in your field of expertise Before I go further discussing about research paper publications in high impact or citation indexes journals, the term Refereed Journal must first be understood. The terms "refereed" and "peer reviewed" mean essentially the same thing. In The Oxford English Dictionary, "peer review" is defined as the process by which a learned journal passes a paper received for publication to outside experts for their comments on its suitability and worth; refereeing. To identify scholarly journals which are refereed, the Ulrich's International Periodicals Directory, available as a Web LUIS database, is a unique, current, comprehensive and continuously updated source of information on selected periodicals and serials published in the United States and throughout the world. The Advanced Search screen allows you to limit Document Type to "Academic/Scholarly Publication" and Special Features to "Refereed". On the other hand, non-refereed materials such as Trade Journals or Magazines use less rigorous standards of screening prior to publication. In some publications, each article may be only screened by the publication's editor. While knowledgeable, no editor can be an authority on all the subject matter printed in a journal. Other non-refereed materials accept almost anything submitted in order to have something to print. Meanwhile, the term "scholarly materials" is often used to describe refereed materials, but this term is not exclusive to refereed material. Non-refereed materials may not by scrutinize as intensely as refereed materials, but they can still be considered scholarly. However, there are cases whereby some researchers get confused between Peer-Reviewed and Scholarly Journals. Publishing your research papers either in Refereed or Non-Refereed journals are still a subject of debate by some scientists and researchers. For instance, there is a significant decrease of Indians and Chinese researchers publishing or reporting their “commercial valued” papers in the American or EU based High Impact Journals for the scare and threats of having their products patented by others outside their countries. Citations have long been used to rank journals within particular subject areas, usually based on the ISI Impact Factor. The impact factor is simply a numerical ratio of the total number of citations a journal receives in ISI Source Journals in one year to the total number of "citable" articles it published in the previous two years. It is a useful way to see how journals perform in relation to others in the same subject area. It is not particularly useful in comparing journals across subject areas, and the number taken out of this context is essentially meaningless. Citation behavior varies considerably from field to field. Thus, impact factors are only meaningful in context with other journals in the same field. Impact factor can also vary based on the number and types of articles a journal publishes. Review articles tend to be more heavily cited than full papers or communications, so journals and annuals that publish mostly reviews will often have high impact factors. Similarly, journals that publish only a few articles in a given year may have disproportionately high impact factors. Which ever J Biochem Tech (2008) 1(1):23-29 28 way you want to rank or count your journal impact factor, I still believe that it is all that “money matters” to some newly established publishers or even the old established ones. Seglan (1997) and Walter (2003) for example discussed in details on the flaws of why the impact factor of journals should not be used for evaluating research articles. While Impact Factors are useful within certain limits, they are somewhat arbitrary and subject to manipulation by editors and publishers. Despite university requirements to evaluate one of the KPIs of an individual academic staff using Impact Factors alone to make personal performance rating and funding decisions, is still a question mark to me. Impact factors for journals covered by ISI are published annually in an electronic compilation called Journal Citation Reports. JCR also contains data on historical trends, immediacy index, cited half-life, etc… References Antelman GK (2004) Do Open-Access Articles Have a Greater Research Impact?. College & Research Libraries 9:372-382 Bauer K, Bakkalbasi N (2005) An examination of citation counts in a new scholarly communication environment. D-Lib Magazine. doi:10.1045/september2005-bauer Björk BC (2004) Open access to scientific publications - an analysis of the barriers to change. Information Research, Available via DIALOG. http://informationr.net/ir/9-2/paper170.html of of subordinate document. Cited 27 Oct 2005 Bjorneborn L, Ingwersen P (2001) Perspectives of webometrics. Scientometrics 50:65-82. Brody T (2003) Citebase Search: Autonomous Citation Database for e-Print Archives SINN 03 conference on Worldwide Coherent Workforce, Satisfied Users - New Services For Scientific Information. Germany. Available via DIALOG http://physnet.physik.uni-oldenburg.de/projects/SINN/sinn03/pr oceedings/brody of subordinate document. Cited 27 Oct 2005. Brody T (2004) Citation Analysis in the Open Access World. Interactive Media International. Available via DIALOG. http://eprints.ecs.soton.ac.uk/10000/01/tim_oa.pdf of subordinate document. Cited 27 Oct 2005 Brody T, Stamerjohanns H, Harnad S, Gingras Y, Vallieres F, Oppenheim C (2004) The effect of Open Access on Citation Impact. National Policies on Open Access (OA) Provision for University Research Output: an International meeting. Southampton University, Southampton UK. Available via DIALOG. http://opcit.eprints.org/feb19oa/brody-impact.pdf of subordinate document. Cited 27 Oct 2005. Title of subordinate document. In: Citation Classics. Available via DIALOG. http://www.citationclassics.org of subordinate document. Cited 14 Nov 2005 Garfield E (1955) Citation indexes to science: a new dimension in documentation through association of ideas. Science 122:108111 Garfield E, Sher IH (1963) Title of subordinate document. In: Genetics Citation Index. Philadelphia, Pa: Institute for Scientific Information. Available via DIALOG. http://www.garfield.libra ry.upenn.edu/essays/v7p515y1984 pdf of subordinate document. Cited 29 Nov 2005 Garfield E (1972) Journal Citation Studies, III: Journal of Experimental Medicine compared with Journal of Immunology: or, how much of a clinician is the immunologist?. Curr Contents Clin Med 5-8 Garfield E (1972) Citation analysis as a tool in journal evaluation. Science 178:471-479 Garfield E (1986) Which medical journals have the greatest impact? Ann Intern Med 105:313-320 Garfield E (1998a) Long-term vs. short-term journal impact: does it matter? Scientist 12:10-12. Available via DIALOG. http://www.garfield.library.upenn.edu/commentaries/tsv12(03)p 10y19980202.pdf of subordinate document. Cited 25 Oct 2005 Garfield E (1998b) Long-term vs. short-term journal impact, II: cumulative impact factors. Scientist 12:12-13. Available via DIALOG. http://www.garfield.library.upenn.edu/commentari es/ts v12(14)p12y19980706.pdf of subordinate document. Cited 25 Oct 2005 Garfield E (1999a) Journal Impact Factor - A Brief Review. Canadian Medical Association Journal 161: 979-980 Garfield E (1999b) Refining the Computation of Topic Based Impact Factors: Some Suggestions. Occupational Medicine 49:571 Available via DIALOG. http://www.garfield.librar Conclusion In my humble opinion, there are many conflicting opinions about impact factors and citation indexing. Both Impact Factor and Citation Indexes is not a perfect tool to measure the quality of articles but there is nothing better and it has the advantage of already being in existence and is, therefore, a good technique for scientific evaluation. Based on my little experience submitting papers to a citation index and/or impact factor journals has shown that in each specialty the best journals are those in which it is most difficult to have an article accepted and/or need to pay a subscription or membership fee, and these are the journals that have a high impact factor. It may not be true since some of these journals existed long before the impact factor was devised. The use of impact factor/citation index as a measure of quality is getting popular and widespread, because it fits well with the opinion we have in each field of the best journals in our specialty. The author personally feels a bit uncomfortable with the use of journal impacts in evaluating academic staff because it has its inherent dangers. However, we academicians, especially in the specialized fields have to go with it and prove to the university’s top management that you can do it as others have done it. In an ideal world, evaluators would read each article and make personal judgments. The recent International Congress on Knowledge Peer Review (KPR) held in Florida, USA last year in 2006, where I was one of the Peer Reviewer Assessor demonstrated the difficulties of reconciling such peer judgments. Most individuals do not have the time to read all the relevant articles in addition to the personal bias and conflicts of self-interest of some reviewers. Even if they do, their judgment surely would be tempered by observing the comments of those who have cited the work. Online full-text access has made that practical. Last but not least, somehow or rather, academicians must adhere strictly to the policy that you must publish (or perish) in open accessed journals as one of the universities requirements. Once the impact and immediacy in citations of subject open archives are compared, you should submit your research documents in the open archive categories with high impact factors and immediacy index. In that case, the continued emphasis on ‘Impact Factors’ will not be misguided the readers. The readers can make a comparison of sources they want to publish considering impact factors as the criteria; they may slant towards the high impact side. 29 J Biochem Tech (2008) 1(1):23-29 y.upenn.edu/papers/occupmed49(8)p571y1999.html as on subordinate document. Cited 25 Oct 2005. Garfield E (2001) Impact Factors, and Why They Wont Go Away. Nature 411:522-522 Garfield E (2004) Title of subordinate document. In: Post to American Scientist - Open Access Forum listserv. Available via DIALOG. http://listserver.sigmaxi.org/sc/wa.exe?A2=ind04& L=american-scientistopen-access-forum&F=l&S=&P=9766 of subordinate document. Cited 18 Jan 2004 Harnad S (2001) Research access, impact and assessment. Times Higher Education Supplement 1487:16 Harnad S, Brody T, Vallieres F, Carr L, Hitchcock S, Gingras Y, Oppenheim C, Stamerjohanns H, Hilf E (2004) The Access/Impact Problem and the Green and Gold Roads to Open Access. Serials Review 30. Available via DIALOG. http://www.ecs.soton.ac.uk/~harnad/Temp/impact.html of subordinate document. Cited 18 Jan 2004 Lock SP (1989) Journalology: are the quotes needed? CBE Views 12:57-59. Available via DIALOG. http://garfield.libr ary.upenn.edu/essays/v13p019y1990.pdf of subordinate document. Cited 25 Oct 2005 Lowry OH, Rosebrough NJ, Farr,AL (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275 Opthof T (1999) Submission, acceptance rate, rapid review system and impact factor. Cardiovasc Res. 41:1-4 Prakasan ER, Sagar A, Kumar A, et al (2004) Nuclear Theory and Nuclear Experiment E-print Archives: Science Citation Index based Analysis. Proc. of the 22nd Annual Convention & Conference on Digital Information Exchange: Pathways to Build Global Information Society. IIT Madras, Chennai, India 172-183. Pudovkin AI, Garfield E (2004) Rank-normalized impact factor: a way to compare journal performance across subject categories. Proc of the 67th Annual Meeting of the American Society for Information Science & Technology, Silver Spring, USA 1:507515. Available via DIALOG. http://garfield.library.upe nn.edu/papers/ranknormalizationasist2004published.pdf of subordinate document. Cited 25 Oct 2005 Rousseau R (1997) The proceedings of the first and second international conferences on bibliometrics, scientometrics and informetrics: A data analysis. Proc of the Sixth Conference of the International Society for Scientometrics and Informetrics, Jerusalem, Israel 4:22-35. Saha S, Saint S, Christakis DA (2003) Impact factor: a valid measure of journal quality? J Med Libr Assoc. 91:42–46 Seglen O (1987) Education and Debate. Why the impact factor of journals should not be used for evaluating research. BMJ 314:497 Seglen PO (1997) Why the impact factor of journals should not be used for evaluating research. BMJ 314:498-502 Smith R (2006) Commentary: The power of the unrelenting impact factor-is it a force for good or harm? Int. J Epidemiol 35:11291130 Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel-electrophoresis. J Mol Biol 98:503517 Testa J, McVeigh ME (2004) The Impact of Open Access Journals: A Citation Study from Thomson ISI, Available via DIALOG. http://www.isinet.com/media/presentrep/acropdf/impact-oa-jou rnals.pdf of subordinate document. Cited 14 Apr 2004 . Thomson Scientific. Journal Performance Indicators. Available via DIALOG. http://scientific.thomson.com/products/jpi/ of subordinate document. Cited 14 Nov 2005 Tomer C (1986) A statistical assessment of two measures of citation: the impact factor and the immediacy index. Information Processing and Management 22:251-258 Walter G (2003) Counting on citations: a flawed way to measure quality. Medical Journal of Australia, 178:280-81 Youngen GK (1998a) Citation Patterns to Electronic Preprints in the Astronomy and Astrophysics Literature. Library and Information Services in Astronomy 153. Youngen GK (1998b) Citation Patterns of the Physics Preprint Literature with Special Emphasis on the Preprints Available via DIALOG. http://gateway.library.uiuc.edu/phx/preprint.html of subordinate document. Cited 5 Nov 2005. Yu G, Wang XY, Yu DR (2005) The influence of publication delays on impact factors. Scientometrics 64:25-246. J Biochem Tech (2008) 1(1):30-37 ISSN: 0974-2328 Recent abstracts in biochemical technology R R Siva Kiran, Brijesh © Sevas Educational Society 2008 “Recent abstracts in biochemical technology” is a collection of interesting research articles published in “List of biochemical technology journals” (Table 1). The abstracts are most likely to report significant results in biochemical technology. • Sylvain P, Alamgir Md, Green JR et al (2008) Computational Methods For Predicting Protein–Protein Interactions, Advances in Biochemical Engineering/Biotechnology Protein–protein interactions (PPIs) play a critical role in many cellular functions. A number of experimental techniques have been applied to discover PPIs; however, these techniques are expensive in terms of time, money, and expertise. There are also large discrepancies between the PPI data collected by the same or different techniques in the same organism. We therefore turn to computational techniques for the prediction of PPIs. Computational techniques have been applied to the collection, indexing, validation, analysis, and extrapolation of PPI data. This chapter will focus on computational prediction of PPI, reviewing a number of techniques including PIPE, developed in our own laboratory. For comparison, the conventional large-scale approaches to predict PPIs are also briefly discussed. The chapter concludes with a discussion of the limitations of both experimental and computational methods of determining PPIs. January 18, 2008, Advances in Biochemical Engineering/Biotechnology. • Beutling U, Stading K, Stradal T, Frank R (2008) Large-Scale Analysis of Protein–Protein Interactions Using Cellulose-Bound Peptide Arrays. Advances in Biochemical Engineering/Biotechnology Peptide arrays for screening large numbers of peptide fragments and probing with large numbers of samples is discussed. April 17, 2008, Advances in Biochemical Engineering/Biotechnology. • Takaaki I, Murayama Y, et al (2008) Probing force-induced unfolding intermediates of a single staphylococcal nuclease molecule and the effect of ligand binding, Biochemical and Biophysical Research Communications Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics. August 26, 2008, Communications Biochemical and Biophysical Research • Taru T, Tuulia T, et al. (2008) A multi-metabolite analysis of serum by 1H NMR spectroscopy: Early systemic signs of Alzheimer’s disease. Biochemical and Biophysical Research Communications A three-molecular-window approach for 1H NMR spectroscopy of serum is presented to obtain specific molecular data on lipoproteins, various low-molecular-weight metabolites, and individual lipid molecules together with their degree of (poly)(un)saturation. The multiple data were analysed with self-organising maps, illustrating the strength of the approach as a holistic metabonomics framework in solely data-driven metabolic phenotyping. We studied 180 serum samples of which 30% were related to mild cognitive impairment (MCI), a neuropsychological diagnosis with severely increased risk for Alzheimer’s disease (AD). The results underline the association between MCI and the metabolic syndrome (MetS). Additionally, the low relative amount of ω-3 fatty acids appears more indicative of MCI than low serum ω-3 or polyunsaturated fatty acid concentration as such. The analyses also feature the role of elevated glycoproteins in the risk for AD, supporting the view that coexistence of inflammation and the MetS forms a high risk condition for cognitive decline. R R Siva Kiran* Department of Biotechnology, M S Ramaiah Institute of Technology, MSR Nagar, Bangalore – 560054, India Brijesh Department of Chemical Engineering, M S Ramaiah Institute of Technology, MSR Nagar, Bangalore – 560054, India *Tel: 0091 80 23600822, Fax: 0091 80 23603124 E-mail: rrskiran@msrit.edu Table1: List of Biochemical Technology Journals Journal Name ADVANCES IN BIOCHEMICAL ENGINEERING BIOCHEMICAL AND BIOPHYSICAL RESEARCH BIOCHEMICAL ENGINEERING JOURNAL BIOCHEMICAL GENETICS BIOCHEMICAL JOURNAL BIOCHEMICAL PHARMACOLOGY BIOCHEMICAL SOCIETY SYMPOSIUM BIOCHEMICAL SOCIETY TRANSACTIONS BIOCHEMICAL SYSTEMATICS AND ECOLOGY CHEMICAL AND BIOCHEMICAL ENGINEERING QUARTERLY JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY JOURNAL OF BIOCHEMICAL TECHNOLOGY Impact Factor 3.253 Journal Description Advances in Biochemical Engineering/Biotechnology reviews actual trends in modern biotechnology. Its aim is to cover all aspects of this interdisciplinary technology where knowledge, methods and expertise are required for chemistry, biochemistry, microbiology, genetics, chemical engineering and computer science. Biochemical and Biophysical Research Communications is the premier international journal devoted to the very rapid dissemination of timely and significant experimental results in diverse fields of biological research. The Biochemical Engineering Journal aims to promote progress in the crucial chemical engineering aspects of the development of biological processes associated with everything from raw materials preparation to product recovery, relevant to industries as diverse as medical/healthcare, food and environmental protection. Biochemical Genetics links the sciences of biology, chemistry and genetics by offering an interdisciplinary forum for the discussion of new developments. The coverage includes different knowledge environments: Cell, Disease, Energy, Gene, Plant, Signal, Structure Biochemical Pharmacology is an international journal devoted to publishing original work on the interaction of drugs and nontherapeutic xenobiotics with biological systems. Journal covers selected topic in the forefront of research in any aspect of the cellular and molecular life sciences. The journal captures the exciting science presented at the Biochemical Society Annual Symposium, Focused Meetings and Independent Meetings supported by the Society as a series of mini-reviews. The application of biochemistry to problems relating to systematic biology of organisms (biochemical systematics) & the role of biochemistry in interactions between organisms or between an organism and its environment (biochemical ecology). The scope of the journal is wide and no limitation except relevance to chemical and biochemical engineering is required. Website http://www.springer.com/series/10 2.749 1.872 http://www.sciencedirect.com/science/jo urnal/0006291X http://www.sciencedirect.com/science/jo urnal/1369703X http://www.springer.com/biomed/human +genetics/journal/10528 http://www.biochemj.org/bj/default.htm http://www.sciencedirect.com/science/jo urnal/00062952 http://symposia.biochemistry.org/ http://www.biochemsoctrans.org/ http://www.sciencedirect.com/science/jo urnal/03051978 http://www.fkit.hr/cabeq/index.html 0.8761 4.1002 4.006 3.300 3.447 1.048 0.632 1.4033 1.4104 The development of new methods or the significant modification of existing techniques to solve theoretical and experimental problems in the field of life sciences, particularly biochemistry and biophysics. The scope includes effects on the organism at all stages of development, on organ systems, tissues, and cells as well as on enzymes, receptors, hormones, and genes. Coverage includes enzymes and proteins; applied genetics and molecular biotechnology; computational biology, genomics and proteomics; metabolic & tissue engineering; medical, environmental, food and agro biotechnology; biodiversity, reactor design, modeling. The journal is a unique source for scientists interested in both engineering as well as basic biology http://www.sciencedirect.com/science/jo urnal/0165022X http://as.wiley.com/WileyCDA/WileyTit le/productCd-JBT.html http://jbt.biodbs.info/ N/A research. JOURNAL OF COMPARATIVE PHYSIOLOGY BBIOCHEMICAL MOLECULAR AND BIOCHEMICAL PARASITOLOGY PHYSIOLOGICAL AND BIOCHEMICAL ZOOLOGY TRENDS IN BIOCHEMICAL SCIENCES CHEMBIOCHEM 2.209 The journal publishes topics related to comparative physiology of invertebrate and vertebrate animals. Special emphasis is placed on integrative studies that elucidate mechanisms at the whole-animal, organ, tissue, cellular and/or molecular levels. The journal provides a medium for rapid publication of investigations of the molecular biology and biochemistry of parasitic protozoa and helminths, and their interactions with both the definitive and intermediate host. The journal publishes the results of original investigations in animal physiology and biochemistry at all levels of organization, from the molecular to the organismic, focusing on adaptations to the environment. TiBS covers discoveries in the fields of biophysics, biochemistry, genetics, microbiology, and cell biology. http://www.springerlink.com/content/10 0425/ http://www.sciencedirect.com/science/jo urnal/01666851 http://www.journals.uchicago.edu/page/ pbz/brief.html http://www.sciencedirect.com/science/jo urnal/09680004 2.896 2.0105 13.90 3.446 ChemBioChem is a source for important primary and secondary information across the whole field of chemical biology, http://www3.interscience.wiley.com/jou bio(in)organic chemistry, and biochemistry. rnal/72510898/home 1 http://www.ovid.com/site/catalog/Journal/1447.jsp, 2http://en.wikipedia.org/wiki/Biochemical_Journal, 3http://www.speciation.net/Appl/Literature/Source/sources.html?id=1237 4 5 http://www.genebee.msu.su/journals/if01j1.html, http://ww.genebee.msu.su/journals/if01p.html and impact factors of other journals are taken from corresponding journal website N/A-Not Available J Biochem Tech (2008) 1(1):30-37 J Biochem Tech (2008) 1(1):30-37 ISSN: 0974-2328 31 August 26, 2008, Communications Biochemical and Biophysical Research August 24, 2008, Communications Biochemical and Biophysical Research • Yun X, Simon G et al (2008) Rapid detection and identification of a pathogen’s DNA using Phi29 DNA polymerase. Biochemical and Biophysical Research Communications Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. A number of these agents have been further weaponized and are widely recognized as being potentially significant biothreat agents. We describe a novel method based on multiply-primed rolling circle in vitro amplification for profiling genomic DNAs to permit rapid, cultivationfree differential detection and identification of circular plasmids in infectious agents. Using Phi29 DNA polymerase and a two-step priming reaction we could reproducibly detect and characterize by DNA sequencing circular DNA from Borrelia burgdorferi B31 in DNA samples containing as little as 25 pg of Borrelia DNA amongst a vast excess of human DNA. This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments. August 26, 2008, Communications Biochemical and Biophysical Research • Kenneth LS, Caleb BM et al. Thermodynamic analysis of the heterodimerization of leucine zippers of Jun and Fos transcription factors. Biochemical and Biophysical Research Communications Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain αhelical conformations in isolation and that the formation of the native coiled-coil α-helical dimer is attained through a coupled foldingdimerization mechanism. August 24, 2008, Communications Biochemical and Biophysical Research • Xing Y, Lu X et al (2008) The effect of high intensity focused ultrasound treatment on metastases in a murine melanoma model.. Biochemical and Biophysical Research Communications This study aims to assess the risk of high intensity focused ultrasound (HIFU) therapy on the incidence of distant metastases and to investigate its association with HIFU-elicited anti-tumor immunity in a murine melanoma (B16-F10) model. Tumor-bearing legs were amputated immediately after or 2 days following HIFU treatment to differentiate the contribution of the elicited anti-tumor immunity. In mice undergoing amputation immediately after mechanical, thermal, or no HIFU treatment, metastasis rates were comparable (18.8%, 13.3%, and 12.5%). In contrast, with a 2-day delay in amputation, the corresponding metastasis rates were 6.7%, 11.8%, and 40%, respectively. Animal survival rate was higher and CTL activity was enhanced in the HIFU treatment groups. Altogether, our results suggest that HIFU treatment does not increase the risk of distant metastasis. Instead, HIFU treatment can elicit an anti-tumor immune response that may be harnessed to improve the overall effectiveness and quality of cancer therapy. August 24, 2008, Communications Biochemical and Biophysical Research • Jorge P, Romero F et al (2008) Some effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. Biochemical and Biophysical Research Communications A study was made of the effects of the venom of the Chilean spider Latrodectus mactans on endogenous ion-currents of Xenopus laevis oocytes. 1 μg/ml of the venom made the resting plasma membrane potential more negative in cells voltage-clamped at −60 mV. The effect was potentially due to the closure of one or several conductances that were investigated further. Thus, we determined the effects of the venom on the following endogenous ionic-currents: (a) voltage-activated potassium currents, (b) voltage-activated chloride-currents, and (c) calcium-dependent chloride-currents (Tout). The results suggest that the venom exerts its action mainly on a transient outward potassium-current that is probably mediated by a Kv channel homologous to shaker. Consistent with the electrophysiological evidence we detected the expression of the mRNA coding for xKv1.1 in the oocytes. August 24, 2008, Communications Biochemical and Biophysical Research • Takahashi S, Sakakibara Y et al (2008) Molecular cloning, expression, and characterization of mouse amine N-sulfotransferases. Biochemical and Biophysical Research Communications By searching the GenBank database, we recently identified a novel mouse cytosolic sulfotransferase (SULT) cDNA (IMAGE Clone ID 679629) and a novel mouse SULT gene (LOC 215895). Sequence analysis revealed that both mouse SULTs belong to the cytosolic SULT3 gene family. The recombinant form of these two newly identified SULTs, designated SULT3A1 and SULT3A2, were expressed using the pGEX-4T-1 glutathione S-transferase fusion system and purified from transformed BL21 Escherichia coli cells. Both purified SULT3A1 and SULT3A2 exhibited strong amine N-sulfonating activities toward 1naphthylamine among a variety of endogenous and xenobiotic compounds tested as substrates. Kinetic constants of the sulfation of 1naphthylamine and 1-naphthol by these two enzymes were determined. Collectively, these results imply that these two amine-sulfonating SULT3s may play essential roles in the metabolism and detoxification of aromatic amine compounds in the body. • Laura Barsanti, Coltelli P et al (2008) Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor. Biochemical and Biophysical Research Communications This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90 Ǻ). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our 32 J Biochem Tech (2008) 1(1):30-37 opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor. August 21, 2008, Communications Biochemical and Biophysical Research pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21Cip1 expression in a p53-dependent manner. August 13, 2008, Communications Biochemical and Biophysical Research • John E McRory, Rehak R et al (2008) Syntaxin 1A is required for normal in utero development. Biochemical and Biophysical Research Communications We have generated a syntaxin 1A knockout mouse by deletion of exons 3 through 6 and a concomitant insertion of a stop codon in exon 2. Heterozygous knockout animals were viable with no apparent phenotype. In contrast, the vast majority of homozygous animals died in utero, with embryos examined at day E15 showing a drastic reduction in body size and development when compared to WT and heterozygous littermates. Surprisingly, out of a total of 204 offspring from heterozygous breeding pairs only four homozygous animals were born alive and viable. These animals exhibited reduced body weight, but showed only mild behavioral deficiencies. Taken together, our data indicate that syntaxin 1A is an important regulator of normal in utero development, but may not be essential for normal brain function later in life. August 14, 2008, Communications Biochemical and Biophysical Research • Salvatore Patané, Pietrancosta N et al (2008) A new Met inhibitoryscaffold identified by a focused forward chemical biological screen. Biochemical and Biophysical Research Communications The receptor tyrosine kinase Met is crucial for the genetic program causing cancer progression and metastasis. Its nodal function during aggressiveness and resistance acquisition poses Met inhibition as an obligatory step in anti-cancer targeted therapy. Here, we applied a “Metfocussed” forward chemical biological screen to discover new agents antagonizing Met-triggered biological functions. The identified new scaffold, JLK1360, has a dual mechanism of action towards Met: it impairs Met signalling and also prevents its restoration after degradation. Docking and molecular dynamics provide evidences on the interacting mode of JLK1360 within the Met ATP-binding pocket. Moreover, computational and biochemical studies also highlighted that JLK1360 has a good degree of selectivity towards Met than other RTKs tested. Altogether, these findings demonstrate that the approach we have applied is a powerful strategy to identify compounds with combined properties towards a chosen target. Our studies show how integration of chemistry, biology and computational analysis can provide robust strategies to identify new inhibitory scaffolds suitable for further development of anti-cancer targeted therapies. August 12, 2008, Communications Biochemical and Biophysical Research • Hidenori Nonaka, Watabe T et al (2008) Development of stabilin2+ endothelial cells from mouse embryonic stem cells by inhibition of TGFβ/activin signaling. Biochemical and Biophysical Research Communications To understand the endothelial cell (EC) development, arterial, venous, and lymphatic EC (LEC) have been successfully induced from embryonic stem cells (ESC). However, tissue-specific EC, such as hepatic sinusoidal EC (HSEC), have never been generated from ESC. Based on the findings that TGFβ/activin signaling negatively regulates differentiation of both LEC and HSEC, and that HSEC and LEC are distinguishable by the expression of marker genes, we assessed the role of TGFβ/activin signaling in EC development from ESC. Here we show that the inhibition of TGFβ/activin signaling by a TGFβ receptor I (TGFβRI) kinase inhibitor increased the expression of Lyve1 and stabilin2 but not podoplanin in CD31+CD34+ EC derived from ESC. EC generated by the inhibition of TGFβRI signaling also exhibited stronger endocytic activity than control EC, indicating that their phenotype is similar to fetal HSEC. Our results reveal that TGFβ/activin signaling negatively regulates the early events of HSEC differentiation. August 14, 2008, Communications Biochemical and Biophysical Research • Helena Mistry, Gibson L et al (2008) Interplay between Np95 and Eme1 in the DNA damage response. Biochemical and Biophysical Research Communications Mus81 (methyl methansulfonate UV sensitive clone 81) and Eme1 (essential meiotic endonuclease 1, also known as MMS4) form a heterodimeric endonuclease that is critical for genomic stability and the response to DNA crosslink damage and replication blockade. However, relatively little is known as to how this endonuclease is regulated following DNA damage. Here, we report mammalian Eme1 interacts with Np95, an E3 ubiquitin ligase that participates in chromatin modification, replication-linked epigenetic maintenance and the DNA damage response. Np95 and Eme1 co-localize on nuclear chromatin following exposure of cells to camptothecin, an agent that promotes the collapse of replication forks. The observed co localization following DNA damage was found to be dependent on an intact RING finger, the structural motif that encodes the E3 ubiquitin ligase activity of Np95. Taken together, these findings link Mus81–Eme1 with the replicationassociated chromatin modifier functions of Np95 in the cellular response to DNA damage. August 8, 2008, Communications Biochemical and Biophysical Research • Xiaobing Han, Xi L et al (2008) The potassium ion channel opener NS1619 inhibits proliferation and induces apoptosis in A2780 ovarian cancer cells. Biochemical and Biophysical Research Communications Diverse types of voltage-gated potassium (K+) channels have been shown to be involved in regulation of cell proliferation. The maxiconductance Ca2+-activated K+ channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC50 = 31.1 μM, for 48 h • Soumen Mukherjee, Das P et al (2008) Enhanced production of biosurfactant by a marine bacterium on statistical screening of nutritional parameters. Biochemical Engineering Journal Marine microorganisms can serve as rich sources for novel biosurfactants with diverse biological activities. In the present investigation, the nutritional requirement for the growth and biosurfactant production by a marine bacterium was determined using a J Biochem Tech (2008) 1(1):30-37 33 Plackett–Burman-based statistical screening procedure. Six out of the eleven factors of a reported production medium were found to be critically affecting the biosurfactant metabolism. Glucose, the carbon substrate of the medium was the most influential factor with an effect contribution of 78.13% and a very low p-value of <0.001. Glucose, NH4NO3 and FeSO4·7H2O had a direct proportional correlation with biosurfactant production while, K2HPO4, KH2PO4 and MgSO4·7H2O showed inversely proportional relationship with biosurfactant production in the selected experimental range. A simpler modified medium (MM) was formulated based on the statistical screening results. Modified medium combination (MM-1) having the following composition in g l−1: glucose 30; NH4NO3 6.0; K2HPO4 1.1; MgSO4·7H2O 0.3; KH2PO4 2.8 × 10−2 and FeSO4. 7H2O 6 × 10−2 showed 84.7% increase in biosurfactant yield over the reported medium (SM). Fourier transform infrared spectroscopy and thin layer chromatography studies showed that the biosurfactants produced in the modified medium (MM) were similar to those produced in the reported medium (SM). July 15, 2008, Biochemical Engineering Journal • Lianghua Wang, Shen S et al (2008) Adsorption and elution behaviors of bovine serum albumin in metal-chelated affinity cryogel beds. Biochemical Engineering Journal Metal-chelated supermacroporous cryogels are effective supports for affinity chromatographic separation of biomolecules in downstream processes. In this work, polyacrylamide cryogel beds were prepared in glass columns (16 mm inner diameter) and coupled with iminodiacetic acid (IDA). These cryogels were loaded with Zn2+ and Ni2+ and the socalled Zn2+-IDA-cryogels and Ni2+-IDA-cryogels were obtained. Permeabilities and height equivalent to theoretical plates (HETPs) of these cryogel beds were measured and the cryogel structure was analyzed using scanning electron microscopy (SEM). Bovine serum albumin (BSA) was employed as a model protein to elucidate the adsorption and elution behaviors of these cryogels under various conditions, such as different flow rate, solution pH, and composition of the eluents. The results showed that the Zn2+-IDA-cryogels and Ni2+IDA-cryogels in this study had interconnected supermacropores and high water permeabilities ( 10−11 m2). The loading flow velocity had a weak influence on the breakthrough curves and binding capacities for BSA, while the solution pH had an evident effect on the binding capacities for BSA in these cryogels. Maximum binding capacity for BSA was observed near the isoelectric point of BSA. The bound BSA can be eluted effectively using an imidazole solution. A low-eluting flow rate was found to be beneficial to the elution process. Possible mechanisms were proposed and discussed. July 15, 2008, Biochemical Engineering Journal • Teresa Lopes da Silva, Reis A et al (2008) Physiological effects of the addition of n-dodecane as an oxygen vector during steady-state Bacillus licheniformis thermophillic fermentations perturbed by a starvation period or a glucose pulse. Biochemical Engineering Journal The effect of the presence of n-dodecane as a potential oxygen vector during oxygen-limited continuous cultures of a Bacillus strain was studied, under extreme nutrient supply conditions: glucose excess, limitation and starvation. The addition of n-dodecane to the aqueous phase of a mechanically agitated and aerated fermentation increased the kLa by up to 35%. The n-dodecane additions to Bacillus licheniformis cells during starvation (oxygen limitation with concomitant glucose starvation) caused a severe detrimental progressive change in cell physiological state with respect to cytoplasmic membrane polarisation and permeability which was mitigated against by alleviating either the oxygen limitation (by increasing the mean energy dissipation rate or by the addition of n-dodecane as an oxygen vector) or by alleviating the carbon limitation (by resuming the carbon feed or by the addition of a glucose pulse). Further that during periods of excess glucose (glucose pulse) a much higher kLa was required to prevent the onset of anaerobic mixed acid fermentation than could be provided by the addition of ndodecane alone. n-Dodecane can be used to increase the kLa when added in sufficient quantities to the aqueous phase of a mechanically agitated and aerated bioreactor but the magnitude of this increase is process and vessel geometry specific. July 9 2008, Biochemical Engineering Journal • Syed Asif Nizam, Shimizu K (2008) Effects of arcA and arcB genes knockout on the metabolism in Escherichia coli under anaerobic and icroaerobic conditions. Biochemical Engineering Journal The Arc system is a two-component regulatory system composed of ArcA and ArcB in Escherichia coli. In the present study, the effects of arcA and arcB genes knockout on the TCA cycle activation in E. coli were investigated for the anaerobic and microaerobic conditions. Under anaerobic condition, the TCA cycle was up-regulated along with high lactate production, together with up-regulation of LDH for arcB mutant as compared with the parent strain. Due to down-regulation of aceE, aceF and lpdA genes which code for PDHc and low activity of Pfl in arcB mutant, the glycolysis as well as oxidative pentose phosphate pathway was down-regulated under anaerobic condition. The TCA cycle enzymes were further up-regulated when nitrate was added by modifying the redox state along with lower lactate production for arcB mutant. Different from the case of anaerobic condition, the glycolysis was activated under microaerobic condition, which may be partly due to the increased activity of PDHc encoded by aceE, F and lpdA genes. Under microaerobic condition, the TCA cycle genes together with their corresponding enzymes were up-regulated for arcB mutant as compared with the parent strain. These characteristics were further enhanced in arcA mutant as compared with the case of arcB mutant. The upregulation of the TCA cycle together with down-regulation of cydB gene expression caused higher redox state in the arcA/B mutants, which in turn repressed the TCA cycle. Then the TCA cycle could be further increased by the addition of nicotinic acid (NA). July 3, 2008, Biochemical Engineering Journal • Serpil Ozmihci, Kargi F (2008) Ethanol production from cheese whey powder solution in a packed column bioreactor at different hydraulic residence times. Biochemical Engineering Journal Cheese whey powder (CWP) solution containing 50 g L−1 total sugar was fermented to ethanol in a continuously operated packed column bioreactor (PCBR) using olive pits as support particles for cell attachment. Pure culture of Kluyveromyces marxianus (DSMZ 7239) was used in the PCBR for ethanol formation from lactose content of CWP solution. Sugar utilization and ethanol formation were investigated as function of the hydraulic residence time (HRT) between 17.6 and 64.4 h. Sugar concentration decreased with increasing ethanol concentration along the height of the column. Percent sugar utilization increased while effluent sugar concentration was decreasing with HRT between 17.6 and 50 h. Similarly, effluent ethanol concentration increased while ethanol productivity was decreasing with increasing HRT up to 50 h. Further increases in HRT above 50 h resulted in decreases in effluent ethanol concentration. The ethanol yield coefficient also increased with increasing HRT and reached the highest level of 0.54 gE g−1S at an HRT of 50 h. Due to cell settling to the bottom of the column, high fermentation rates were obtained in the lower section of the system. Therefore, the system can be operated with a height of 36 cm from the inlet to obtain high ethanol contents in the effluent with an HRT of 18 h. July 1, 2008, Biochemical Engineering Journal • Xiaojuan Yang, Zhang X et al (2008) The leaching of pentlandite by Acidithiobacillus ferrooxidans with a biological–chemical process. 34 J Biochem Tech (2008) 1(1):30-37 Biochemical Engineering Journal The Fe3+ leaching solution which was produced by Acidithiobacillus ferrooxidas was used to leach pentlandite. The effects of several kinetic parameters including temperature, Fe3+ concentration and Cl− on Ni recovery were investigated in a container independently. The results showed that the leaching rate of Ni was favored by the rise of temperature, the increase of Fe3+ concentration and the partial replacement of SO42− by Cl−, all of which could prove the indirect mechanism for sulfides leaching. Then a bioleaching process comprising biooxidation and chemical leaching is proposed. In the biooxidation process, A. ferrooxidans was used to produce Fe3+ at 30 °C, which was then fed to the chemical process to oxidize pentlandite at 50 °C. This biological–chemical leaching process features the continuous production of Fe3+ in the bioreactor and the high temperature leaching of pentlandite in the chemical reactor. By using this process, Ni leaching rate of 83.8% was obtained at 5 days. According to the results, the present heap leaching process could be improved by the results obtained by this biological–chemical leaching process. July 28 2008, Biochemical Engineering Journal • Jean-Marc Engasser, Chamouleau F et al (2008) Kinetic modeling of glucose and fructose dissolution in 2-methyl 2-butanol. Biochemical Engineering Journal With the objective of developing simulation models for sugar bioconversion processes in non-aqueous media, the present study investigated the dissolution kinetics of glucose and fructose in 2-methyl 2-butanol at temperatures between 20 °C and 80 °C. For both sugars a two-phase dissolution process was observed, characterized by an initially fast dissolution lasting a few minutes, followed by a much slower dissolution phase extending up to 24 h. The experimental results are described by a combined sugar dissolution and mutarotation kinetic model that considers the dissolution of the sugar anomeric form present in the solid particles, namely α-d-glucopyranose and ß-d-fructopyranose, and its subsequent mutarotation in solution. The initial dissolution step is assumed limited by the solute transport from the surface to the bulk solution, and the corresponding sugar transport coefficient evaluated from established mass transport correlations. The second slower dissolution phase is solely controlled by the sugar mutarotation rate, and modeled as a first-order reversible reaction. The determined values of the mutarotation rate and equilibrium constants can be related to the solution temperature by an Arrhenius and Van’t Hoff relationship, respectively. July 27 2008, Biochemical Engineering Journal • Dumont E, Andrès Y et al (2008) Evaluation of a new packing material for H2S removed by biofiltration. Biochemical Engineering Journal This study aims to evaluate the feasibility of using a new packing material (UP20) in treating H2S. Three identical laboratory-scale biofilters, filled with, respectively, UP20 alone, pine bark, and a configuration made of two layers of pozzolan/UP20 (80/20, v/v), were used for critical comparison. Various concentrations of H2S (up to 100 ppmv) were used to determine the optimum biofilter performances. The superficial velocity of the polluted gas on each biofilter was 65 m h−1 (0.018 m s−1; gas flow rate 0.5 N m3 h−1) corresponding to an empty bed residence time of 57 s. Changes in elimination capacity, removal efficiency, moisture content, temperature and pH were tracked during 95 days. The pressure drops along each biofilter were also measured by varying the gas flow rate from 0.5 to 4 N m3 h−1. After 63 days of operation, the loading rate was significantly increased to 10 g m−3 h−1 and the UP20 biofilter retained a removal efficiency of more than 93%, indicating a strong ability to stimulate microbial activity (compared to 69% for the pine bark biofilter and 74% for the biofilter filled with a configuration of two layers of pozzolan/UP20). A Michaelis–Menten type equation was applied and the maximum removal rate (Vm) and saturation constant (Ks) were calculated. Vm was evaluated at 35gH2S m-3biofilterh-1 for UP20 (14 and 15gH2S m-3biofilterh-1 for pine bark and pozzolan/UP20, respectively). The saturation constant Ks was 70 ppmv for UP20 (18 ppmv for pine bark and 20 ppmv for pozzolan/UP20) indicating that the new packing material will be effective in treating large pollutant concentrations. At low concentrations of pollutant, the results suggest that a biofilter with a configuration of two layers of pozzolan/UP20 is the most suitable choice for treating H2S. July 22 2008, Biochemical Engineering Journal • Haifeng Pan, Xie Z et al (2008) Optimization of culture conditions to enhance cis-epoxysuccinate hydrolase production in Escherichia coli by response surface methodology. Biochemical Engineering Journal The effect of culture conditions on the cis-epoxysuccinate hydrolase (CESH) production in recombinant Escherichia coli BL21 was investigated using response surface methodology (RSM), which was based on rotatable central composite design. The optimization of seed conditions consisted of a total of 13 experiments involving 4 star points and 5 replicates at the central points, while the optimization of induction conditions consisted of a total of 31 experiments involving 8 star points and 7 replicates at the central points. The optimum predicted culture conditions for maximum expression of recombinant CESH were found to be comprised of 11.75 h seed age, 4.13% (v/v) inoculation level, OD600 0.2 induction-starting time, 2.53% (w/v) lactose, 24.29 °C postinduction temperature and 27.56 h post-induction time, with a predicted CESH activity of 40,460 U/g, which was very close to the experimental CESH activity of 40,129 U/g resulting in 4.6-fold increment after optimization. July 20 2008, Biochemical Engineering Journal • Fernando GF, Bartacek J et al (2008) Supplementation of cobalt to UASB reactors by pulse dosing: CoCl2 versus CoEDTA2− pulses. Biochemical Engineering Journal The effect of chelation on the dosing strategy of cobalt to restore the performance of a cobalt limited methanol-fed bioreactor was investigated. Three upflow anaerobic sludge bed (UASB) reactors (30 °C, pH 7.0) were operated with methanol as the substrate at an organic loading rate of 8.5 g COD L−1 d−1. One UASB reactor was supplied with several pulses of cobalt bound to EDTA, and its operation was compared to that of another UASB reactor to which several pulses of CoCl2 were given. The addition of cobalt (5 μmoles cobalt per litre of reactor volume) in the form of CoCl2 creates a pool of cobalt in the granular sludge matrix due to the high cobalt retention (around 90%). The methanogens present in the granular sludge are able to use that cobalt pool for stable methane formation from methanol during the subsequent 15 days. When added as Co-EDTA2−, only around 8% of the cobalt added is retained. The small amount of retained cobalt in case of Co-EDTA2− addition supports methylotrophic methanogenesis only a few operational days. Furthermore, the side-effects EDTA has on the granule matrix or microbial cells make EDTA an unsuitable ligand for cobalt dosage in full-scale applications. July 17 2008, Biochemical Engineering Journal • Ka F Luk, Ko MK, Ng KM (2008) Separation and purification of (−)schisandrin B from schisandrin B stereoisomers. Biochemical Engineering Journal Schisandrin B (Sch B), consisting of a mixture of its stereoisomers, namely (−)Sch B and (±)γ-schisandrin, is the most abundant and biologically active dibenzocyclooctadiene lignan present in Fructus J Biochem Tech (2008) 1(1):30-37 35 Schisandrae (FS). The objective of this study is to develop a process for large-scale separation and purification of a single stereoisomer of Sch B, (−)Sch B, which offers the highest desirable bioactivities. To this end, a crystallization-based separation and purification process has been conceptualized. Bench-scale crystallization experiments guided by experimental solid–liquid equilibrium phase diagrams were performed to verify process feasibility. A (−)Sch B product with a purity of 98.5 wt% and a (±)γ-schisandrin-enriched product with a purity of 65.0 wt% were obtained. The (−)Sch B product caused a 32% increase in cellular glutathione level and the (±)γ-schisandrin-enriched product a 26% increase, indicating a potentially more efficacious pharmaceutical preparation. July 5 2008, Biochemical Engineering Journal • Karunakaran S, Umapathy NS et al (2008) Interaction of tryptophan derivatives with SLC6A14 (ATB0,+) reveals the potential of the transporter as a drug target for cancer chemotherapy. Biochemical Journal 414: 343-355 ATB0,+ [SLC6A14 (solute carrier family 6 member 14)] is an Na+/Cl−coupled amino acid transporter whose expression is upregulated in cancer. 1-Methyltryptophan is an inducer of immune surveillance against tumour cells through its ability to inhibit indoleamine dioxygenase. In the present study, we investigated the role of ATB0,+ in the uptake of 1-methyltryptophan as a potential mechanism for entry of this putative anticancer drug into tumour cells. These studies show that 1-methyltryptophan is a transportable substrate for ATB0,+. The transport process is Na+/Cl−-dependent with an Na+/Cl−/1-methyltryptophan stoichiometry of 2:1:1. Evaluation of other derivatives of tryptophan has led to identification of α-methyltryptophan as a blocker, not a transportable substrate, for ATB0,+. ATB0,+ can transport 18 of the 20 proteinogenic amino acids. α-Methyltryptophan blocks the transport function of ATB0,+ with an IC50 value of ∼250 μM under conditions simulating normal plasma concentrations of all these 18 amino acids. These results suggest that α-methyltryptophan may induce amino acid deprivation in cells which depend on the transporter for their amino acid nutrition. Screening of several mammary epithelial cell lines shows that ATB0,+ is expressed robustly in some cancer cell lines, but not in all; in contrast, non-malignant cell lines do not express the transporter. Treatment of ATB0,+-positive tumour cells with α-methyltryptophan leads to suppression of their colony-forming ability, whereas ATB0,+negative cell lines are not affected. The blockade of ATB0,+ in these cells with α-methyltryptophan is associated with cell cycle arrest. These studies reveal the potential of ATB0,+ as a drug target for cancer chemotherapy. June 3 2008, Biochemical Journal • Alphey MS, Janine K et al (2008) Structural and mechanistic insights into type II trypanosomatid tryparedoxin-dependent peroxidases. Biochemical Journal 414: 375-381 TbTDPX (Trypanosoma brucei tryparedoxin-dependent peroxidase) is a genetically validated drug target in the fight against African sleeping sickness. Despite its similarity to members of the GPX (glutathione peroxidase) family, TbTDPX2 is functional as a monomer, lacks a selenocysteine residue and relies instead on peroxidatic and resolving cysteine residues for catalysis and uses tryparedoxin rather than glutathione as electron donor. Kinetic studies indicate a saturable Ping Pong mechanism, unlike selenium-dependent GPXs, which display infinite Km and Vmax values. The structure of the reduced enzyme at 2.1 Å (0.21 nm) resolution reveals that the catalytic thiol groups are widely separated [19 Å (0.19 nm)] and thus unable to form a disulphide bond without a large conformational change in the secondary-structure architecture, as reported for certain plant GPXs. A model of the oxidized enzyme structure is presented and the implications for small-molecule inhibition are discussed. June 3 2008, Biochemical Journal • Lue X, Lin F et al (2008) Characterization of the topology and functional domains of RKTG. Biochemical Journal 414: 399-406 RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seventransmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolicside. Two short stretches of amino acid sequences at the membrane Proxximal to the N- and C-termini (amino acids 61–71 and 299–303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling. June 11 2008, Biochemical Journal • Ilhan A, Gartner W et al (2008) Localization and characterization of the novel protein encoded by C20orf3. Biochemical Journal 414: 485495 In the present study, we characterized the gene product of open reading frame 3 encoded at human chromosome 20 (C20orf3), which represents a member of the lactonohydrolase super family. Multiple-tissue Northern blot analysis showed ubiquitous expression of the 2.4 kb transcript coding for 416 amino acids, with highest levels in human liver, placenta and kidney. After recombinant production of protein variants in Escherichia coli and insect cells, antibodies directed against different epitopes within the C20orf3 gene product were generated. Using these immunoreagents, protein expression was demonstrated in the liver, and glomerular and tubular structures of the kidney, as well as in endothelial cells and arterial wall. Positive staining was also observed at the pancreatic islets of Langerhans. Using immunoblotting, we identified three size variants. In line with the results of in silico analysis demonstrating a single transmembrane sequence (amino acids 40–61) at the N-terminus of the full-length protein, FACS cell-surface staining confirmed a mainly extracellular localization of the full-length protein. Sucrose density gradient cell fractionation revealed membrane association of the dominant 50 kDa variant in HepG2 and Rin-5F cells. The finding of a strong arylesterase activity with β-naphthyl acetate and phenyl acetate of the C20orf3 protein-containing fractions suggests potential involvement of this protein in enzymatic processes. C20orf3 promoter-driven reporter assays, which were verified by gene-specific RT-qPCR (real-time quantitative PCR) showed a strong inhibitory effect of human serum on transcription using the HEK-293 human embryonic kidney cell line. In conclusion, we characterized the structure and expression pattern of the C20orf3 gene product. According to a series of analogies with PON (paraoxonase) family members, we speculate that the C20orf3 gene product represents a new member of this important protein family present at the cellular level. May 30 2008, Biochemical Journal 36 J Biochem Tech (2008) 1(1):30-37 • Ercisli S, Orhan1 E et al (2008) Genetic Diversity in Grapevine Germplasm Resources in the Coruh Valley Revealed by RAPD Markers. Biochemical Genetics Random amplified polymorphic DNA analysis was carried out in 35 autochthonous table grapevine cultivars grown in Coruh valley. Fifty five oligonucleotide primers were screened on cultivars, and among them, 12 primers showed clear polymorphic patterns. PCR amplification with 12 primers generated a total of 157 polymorphic bands. There was genetic variation among the cultivars with values of genetic diversity ranging from 0.19 to 0.72 using the Jaccard coefficient. UPGMA analysis of the distance matrix resulted in a dendrogram with two main clusters. The first cluster included 28 cultivars and the second 7 cultivars. The greatest genetic similarity was observed between cultivars Gah and Kolik, while the greatest dissimilarity was observed between cultivars Gah and Siyah Kus Uzumu. The dendrogram revealed that the cultivars present in Coruh valley can be distinguished to a relatively high degree. May 30 2008, Biochemical Genetics • Sha L, Yang R et al (2008) Phylogenetic Analysis of Leymus (Poaceae: Triticeae) Inferred from Nuclear rDNA ITS Sequences. Biochemical Genetics To investigate the phylogenetic relationships of polyploid Leymus (Poaceae: Triticeae), sequences of the nuclear rDNA internal transcribed spacer region (ITS) were analyzed for 34 Leymus accessions representing 25 species, together with three Psathyrostachys species (Ns genome), two Pseudoroegneria (St genome) species, Lophopyrum elongatum (Ee genome), and Thinopyrum bessarabicum (Eb genome). The phylogenetic analyses (maximum likelihood and Bayesian inference) supported two major clades, one including 21 Leymus species and three Psathyrostachys species, the other with nine Leymus species and four diploid species. The ITS RNA secondary structure of the Leymus species was compared with that of their putative diploid donor. It is suggested that (1) the species from the same areas or neighboring geographic regions are closely related to each other; (2) L. coreanus, L. duthiei, L. duthiei var. longearistatus, and L. komarovii are closely related to other Leymus species, and it is reasonable to transfer these species from the genus Hystrix to Leymus; (3) the ITS sequences of Leymus are evolutionarily distinct; (4) the different Leymus species and different distribution of a species derived their Ns genome from different Psathyrostachys species; and (5) there is a close relationship among Leymus, Pseudoroegneria, Lophopyrum, and Thinopyrum, but it is difficult to presume that the St, Ee, and Eb genome may be the Xm genome donor of the Leymus species. August 15 2008, Biochemical Genetics • Arora R, Bhatia S et al (2008) Genetic Polymorphism of Type 1 Intermediate Filament Wool Keratin Gene in Native Indian Sheep Breeds. Biochemical Genetics Information is presented on the genetic polymorphism of the Type 1 intermediate filament wool keratin gene in 15 native Indian sheep breeds belonging to different agro-ecological regions of India. The study analyzed random blood samples of the 638 sheep by the PCR-RFLP technique. Restriction digestion analysis of a 480 bp PCR fragment of the first exon region with MspI revealed two allelic variants (M = 0.748 and N = 0.252) and three genotypes (MM = 0.543, MN = 0.410, and NN = 0.047) across the 15 sheep breeds. The allelic frequency differences for both alleles across the Indian breeds, irrespective of their geographic distribution, color pattern, and utility traits, were observed to be statistically insignificant by a chi-square test (P > 0.05). According to the pattern of occurrence of allelic variants (M > N), the Indian breeds exhibited similarity to some of the reported European sheep breeds. The average heterozygosity was 0.420, and none of the breeds deviated from Hardy-Weinberg equilibrium. The predominance of the M over the N allele supported its ancestry in Indian sheep too. May 30 2008, Biochemical Genetics • Zhu H, Wu H et al (2008) Role of MicroRNA miR-27a and miR-451 in the regulation of MDR1/P-glycoprotein expression in human cancer cells. Biochemical Pharmacology 76(5):582-588. MicroRNAs are short non-coding RNA molecules able to affect stability and/or translation of mRNA, thereby regulating the expression of genes involved in many biological processes. We report here that microRNAs miR-27a and miR-451 are involved in activating the expression of Pglycoprotein, the MDR1 gene product that confers cancer cell resistance to a broad range of chemotherapeutics. We showed that expressions of miR-27a and miR-451 were up-regulated in multidrug resistant (MDR) cancer cell lines A2780DX5 and KB-V1, as compared with their parental lines A2780 and KB-3-1. Treatment of A2780DX5 cells with the antagomirs of miR-27a or miR-451 decreased the expression of Pglycoprotein and MDR1 mRNA. In contrast, the mimics of miR-27a and miR-451 increased MDR1 expression in the parental cells A2780. The sensitivity to and intracellular accumulation of cytotoxic drugs that are transported by P-glycoprotein were enhanced by the treatment with the antagomirs of miR-27a or miR-451. Our results demonstrate for the first time the roles of microRNAs in the regulation of drug resistance mediated by MDR1/P-glycoprotein, and suggest the potential for targeting miR-27a and miR-451 as a therapeutic strategy for modulating MDR in cancer cells. September 1 2008, Biochemical Pharmacology • Hollenbach M, Hintersdorf A et al (2008) Ethyl pyruvate and ethyl lactate down-regulate the production of pro-inflammatory cytokines and modulate expression of immune receptors. Biochemical Pharmacology 76(5):631-644 Esters of α-oxo-carbonic acids such as ethyl pyruvate (EP) have been demonstrated to exert inhibitory effects on the production of antiinflammatory cytokines. So far, there is no information about effects, if any, of ethyl lactate (EL), an obviously inactive analogue of EP, on inflammatory immune responses. In the present study, we provide evidence that the anti-inflammatory action of α-oxo-carbonic acid esters is mediated by inhibition of glyoxalases (Glo), cytosolic enzymes that catalyse the conversion of α-oxo-aldehydes such as methylglyoxal (MGO) into the corresponding α-hydroxy acids using glutathione as a cofactor. In vitro enzyme activity measurements revealed the inhibition of human Glo1 by α-oxo-carbonic acid esters, whilst α-hydroxycarbonic acid esters such as EL were not inhibitory. In contrast, both EP and EL were shown to suppress the Lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines such as tumor necrosis factorα, interleukin (IL)-1β, IL-6 and IL-8 from human immunocompetent cells, and modulated the expression of the immune receptors HLA-DR, CD14 and CD91 on human monocytes. Here, we show a crossing link between glyoxalases and the immune system. The results described herein introduce glyoxalases as a possible target for therapeutic approaches of immune suppression. September 1 2008, Biochemical Pharmacology • Cui X, Thomas A et al (2008) Application and interpretation of hPXR screening data: Validation of reporter signal requirements for prediction of clinically relevant CYP3A4 inducers. Biochemical Pharmacology 76(5):680-689 A human pregnane X receptor (PXR) reporter-gene assay was established and validated using 19 therapeutic agents known to be clinical CYP3A4 inducers, 5 clinical non-inducers, and 6 known inducers in human hepatocytes. The extent of CYP3A4 induction J Biochem Tech (2008) 1(1):30-37 37 (measured as RIF ratio in comparison to rifampicin) and EC50 was obtained from the dose–response curve. All of the clinical inducers (19/19) and human hepatocyte inducers (6/6) showed positive responses in the PXR assay. One out of five clinical non-inducers, pioglitazone, also showed a positive response. An additional series of 18 commonly used drugs with no reports of clinical induction was also evaluated as putative negative controls. Sixteen of these were negative (89%), whereas two of these, flutamide and haloperidol showed 16-fold (RIF ratio 0.79) and 10-fold (RIF ratio 0.48) maximal induction, respectively in the reporter-gene system. Flutamide and haloperidol were further demonstrated to cause CYP3A4 induction in human cryopreserved hepatocytes based on testosterone 6β-hydroxylation activity. The induction potential index calculated based on the maximum RIF ratio, EC50, and in vivo maximum plasma concentration was used to predict the likelihood of CYP3A4 induction in humans. When the induction potential index is greater than 0.08, the compound is likely to cause induction in humans. A high-throughput screening strategy was develop -ed based on the validation results at 1 μM and 10 μM for the same set of drugs. A RIF ratio of 0.4 was set as more practical screening cut-off to minimize the possibility of generating false positives. Thus, a tiered approach was implemented to use the human PXR reporter-gene assay from early lead optimization to late lead characterization in drug discovery. September 1 2008, Biochemical Pharmacology • Sheng MY, Chen QF, Yang QX (2008) Variation in icariin and flavonoid contents of barrenwort accessions native to Guizhou, China. Biochemical Systematics and Ecology Flavonoid and icariin contents of 87 samples drawn from 21 accessions of seven species native to Guizhou, China, of the genus Epimedium were determined by means of ultraviolet and high-performance liquid chromatography. The contents differed significantly among the species and accessions. E. acuminatum, E. yinjiangense, E. myrianthum, E. wushanense, and E. simplicifloum had greater amounts of flavonoids and icariin than E. letorrhizum and E. luodianense. July 23 2008, Biochemical Systematics and Ecology • Onifade AK, Fatope MO et al (2008) Nematicidal activity of Haplophyllum tuberculatum and Plectranthus cylindraceus oils against Meloidogyne javanica. Biochemical Systematics and Ecology The potentials of Haplophyllum tuberculatum and Plectranthus cylindraceus oils to control Meloidogyne javanica were investigated in vitro and in a greenhouse. A mixture of Haplophyllum and Plectranthus oils (1:1) was highly toxic to M. javanica in vitro, as it killed all nematode juveniles and inhibited hatching of eggs at 12.5 μg/ml concentration after 24 h exposure time, as did carbofuran at the same concentration. In the green-house, tomatoes grown in soil treated with a combination (1:1) of the two oils developed fewer root galls than those grown in soil treated with higher doses of either oil. The oil mixture, at 2.5 and 5.0 μg/ml of soil, was not phytotoxic to tomato plants as evident from the appearance and height of plants after 12 weeks exposure time, compared to treatment over the same period at lower effective doses. The nematicidal activity of the combined essential oils was suggested by the presence of C10 dienes, C10 trienes and C10 phenol. July 1 2008, Biochemical Systematics and Ecology • Gordiyenko Y, Robinson CV (2008) The emerging role of MS in structure elucidation of protein–nucleic acid complexes. Biochemical Society Transactions 36:723-731 Developments in MS enable us to apply this technique to non-covalent complexes, defining their stoichiometry, subunit interactions and architectural organization. We illustrate the application of this non- covalent MS approach to uncovering the overall topological arrangements of subunits and interactions within RNA–protein complexes studied in our laboratory over the last 5 years. These studies exemplify the emerging role and potential of MS as a complementary structural biology methodology and demonstrate its unique niche in investigations of dynamic or heterogeneous protein–nucleic acid complexes, which are not accessible to classical high-resolution structural biology techniques. April 6 2008, Biochemical Society Transactions • Curticapean C, Muntean D et al (2008) Optimized HPLC method for tramadol and O-desmethyl tramadol determination in human plasma. Journal of Biochemical and Biophysical Methods 70(6):1304-1312 The optimized method for HPLC determination of tramadol and its metabolite O-desmethyl tramadol in human plasma using sotalol as internal standard has been developed and validated by a new approach. The determination by fluorescence detection was performed on re-eluted solution, obtained after liquid–liquid extraction with ethyl acetate of the three analytes from plasma. The chromatographic separation of tramadol under a gradient elution was achieved at a temperature of 15 °C with a RP-18 column, guarded by a C18 precolumn. The mobile phase was a mixed aqueous solution containing ortho-phosphoric acid, triethylamine, acetonitrile and methanol in a complex gradient mode. The quantitative determination of tramadol was performed at different successive pairs of excitation/emission wavelengths (200/300 nm, 200/295 nm, 212/305 nm) with lower limits of quantification: LLOQ = 4.078 ng/ml for tramadol, respectively LLOQ = 3.271 ng/ml for O-desmethyl tramadol. For the LLOQ limits, were calculated the values of the coefficient of variation and difference between mean and the nominal concentration. For tramadol analyte they were CV% = 5.147% and bias% = − 7.273% in the intra-days and CV% = 4.894% and bias% = 0.836% in the between-days assay, respectively for the metabolite O-desmethyl tramadol they were CV% = 11.517% and bias% = 0.337% in the intra-days and CV% = 6.41% and bias% = 3.259% in the between-days assay. In addition, the stabilities of the analytes were verified in different conditions. Both, tramadol and its metabolite proved to be stable in plasma for four weeks, frozen at − 20 °C, but also for 48 h at 15 °C in the re-eluted solution after liquid–liquid extraction. February 9 2008, Journal of Biochemical and Biophysical Methods • Fenton AW (2008) Allostery: an illustrated definition for the ‘second secret of life’. Trends in Biochemical Sciences Although allosteric regulation is the ‘second secret of life’, the molecular mechanisms that give rise to allostery currently elude understanding. In my opinion, experimental progress is hampered by a commonly used but misleading definition of allostery as protein structural changes that are elicited by the binding of a single ligand. Allostery is more strictly defined in functional terms as a comparison of how one ligand binds in the absence, versus the presence, of a second ligand. Therefore, as each of the two binding events involves two protein complexes, a study of allostery must consider four complexes and not just two. Such a comparison can distinguish allosteric from nonallosteric protein changes, the importance of which is frequently overlooked. When a study of all four complexes is not feasible, an alternative, albeit limited, strategy can identify subsets of allostericspecific changes. August 15 2008, Trends in Biochemical Sciences Journal of Biochemical Technology Acknowledgments to Reviewers SEVAS Publishing The Editors acknowledge with gratitude the assistance of the following individuals, who reviewed manuscripts for the Journal of Biochemical Technology for first issue. The scientific standards of the journal rely upon their contribution of time and expertise and for this we are sincerely grateful. Kenneth Wiley Department of Molecular & Cellular Biology, Harvard University, MA-02138, USA E-mail: klwiley@mcb.harvard.edu Tel: 001-617-495-2358 Liangran Zhang Department of Molecular & Cellular Biology, Harvard University, MA-02138 E-mail: zhanglran@mcb.harvard.edu Tel: 001-617-495-4396 Alex Georgakilas Department of Biology, East Carolina University, Greenville, NC 27858, USA Email: georgakilasa@ecu.edu Tel: 001-252 3285446 Serwan M. J. Baban Ruhul H Kuddus Department of Biology, Utah Valley State College, 179, SB-202e, 800 West University Parkway - Orem, UT 84058 E-mail: kuddusru@uvsc.edu Tel: 001-801-863-6260 School of Environmental Science and Management, Southern Cross University, P. O. Box 157, Lismore, NSW 2480, Australia E-mail: sbaban2001@yahoo.com Tel: 006-126-620-3766 Rudolf Emil Department of Medical Biology and Genetics, Faculty of Medicine in Hradec Králové, Charles University, Prague 2, Czech Republic E-mail: rudolf@lfhk.cuni.cz Tel: 042-049-581-6393 Pirkko Heikinheimo Emily Abrash Department of Biological Sciences, Stanford University, Stanford, CA 94305 E-mail: eabrash@stanford.edu Tel: 001-425-785-9149 Structural Biology and Biophysics, Institute of Biotechnology, P. O. Box 65, University of Helsinki, Finland - 00014 E-mail: pirkko.heikinheimo@helsinki.fi Tel: 0035 -891-915-8957 Antonio Tiezzi Dept. of Environmental Sciences, University of Tuscia, Largo dell'Università blocco D 01100 Viterbo, Italy E-mail: antoniot@unitus.it Tel: 003-976-135-7102 Acknowledgments to “Authors Advisory Board” Members The Editor kindly acknowledges the support and encouragement of “Authors Advisory Board” members. Their suggestions helped improve the overall quality of this journal. Emily Abrash Department of Biological Sciences, Stanford University, Stanford, CA 94305 E-mail: eabrash@stanford.edu Tel: 001-425-785-9149 Ruhul H Kuddus Department of Biology, Utah Valley State College, 179, SB-202e, 800 West University Parkway - Orem, UT 84058 E-mail: kuddusru@uvsc.edu Tel: 001-801-863-6260 Special Acknowledgments The Editor acknowledges the immense effort put in by Ms Ahalya N and Mr Brijesh in proofreading and editing the first print of the Journal issue. Brijesh Dept. of Chemical Engineering, MS Ramaiah Institute of Technology, MSR Nagar, Bangalore-560054, India E-mail: brijesh@biodbs.info Tel: 0091-802-360-0822 Ahalya N Dept. of Biotechnology, MS Ramaiah Institute of Technology, MSRIT, Bangalore-560054, India E-mail : ahalya.naidu@gmail.com Tel: +91 80 23600822 38 SEVAS EDUCATIONAL SOCIETY "Seva" means selfless service in Sanskrit (An Ancient Indian Language), "Sevas Educational Society" is established on August 4th, 2006 at Komatipalli, a remote village near Visakhapatnam, South India. The society aims at the development of villages by educating people in various fields like biotechnology & bioinformatics. The society has placed 35 design related Chemical Engineering Thesis, village scale industrial protocols in website (For helping industries which cannot afford funds to design consultancy). The society has also started certified courses in bioinformatics and nanotechnology (with minimum fee) for the development of students studying in Biotechnology. “Sevas Publication Division” has started this Journal to encorage biochemical research. The idea is to contribute to the fraternity of scientists in the area of Biotechnology, Bioinformatics & Chemical Engineering sciences and encourage genuine research necessary for the development of mankind. “Authors Advisory Board” service was started by the society to help authors to reach journal standards. Research articles not reaching a satisfactory standard will be given to the advisory board by the editor. Advisory Board will assist authors in experimental protocols, English language, analysis procedures, presentation style, formatting, submitting articles online, understanding author instructions etc...... We are very much pleased to invite you to submit the articles to “Journal of Biochemical Technology”. Thanking you Sincerely, Secretory R N Lakshmi Naidu Publishing Editor Sevas Educational Society Publishing