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WABRI Symposium 2005


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“A straight line may be the shortest distance between two points, but it is by no means the most

                                  - Dr Who, “The Time Monster”

                                  Welcome from the Director

Welcome to the inaugural WABRI Research Symposium.

This year marks the first “on campus” presentation of the range of research areas being undertaken at
the Institute.

The format is designed to provide an opportunity for colleagues, collaborators, visitors and students to
gain an overview of the exciting research and development activities being explored.

The Symposium will be of particular interest to students completing their degrees this year. They can
search for potential projects for Honours in 2006 and identify supervisors.           Similarly, Honours
candidates in the midst of fulfilling their course requirements should see opportunities for PhD topics.

The Symposium will also be a showcase for members of the WABRI Post-graduate Students Club.
Twenty-six poster presentations will be made in the foyer of the Norm Dufty Lecture Theatre each
highlighting their research results and the efforts of the Post-graduate students as they enter into their
chosen careers in science. I strongly recommend you take the opportunity to appreciate the work on

The WABRI Post-Graduate Students Club prepared this booklet. The thoughtful layout has been
designed to enable note taking and record keeping of the research and presentations. Their contribution
is a testament to their strong sense of engagement in their future. I am sure all post-graduate students
will benefit from being part of the Club.

I trust you will find the presentations stimulating, the sense of endeavour rewarding and the potential
outcomes of the research inspiring.

Professor Simon Carroll

               Welcome from the WABRI Postgraduate Student Club

The WABRI Postgraduate Student Club is proud to support the 2005 WABRI Research Symposium. As
an organisation created both for and by the postgraduate students of WABRI, it is our great pleasure to
be involved in co-ordinating an event aimed at showcasing the wonderful array of research undertaken
by the postgraduate students and WABRI reseachers as a whole.

It has been our belief that in order for us to fulfill our mission to promote and enhance the skills of our
research students, they must not only be exposed to cutting-edge science but must also be given a
platform to present their research. This Symposium provides that excellent oppurtunity.

While listening to the wide variety of talks today, please keep in mind that the students today are
researchers of the future. I invite you to encourage them on their journey.

Saima Majeed
President, WABRI Postgraduate Club

                 The Postgrad Club could not happen without the continued support of

                        2005 WABRI RESEARCH SEMINAR


9:00    Simon Carroll             Welcome

9:10    Ricardo Mancera           Biomolecular modelling in drug discovery
                                  Computer-aided design of small molecule inhibitors of Glycogen Synthase
9:20    Steve Bottomley
                                  Kinase 3β
9:30    Simon Fox                 Regulation of the Wnt signalling pathway in mesothelioma

9:40    Brett Dix                 Differential regulation of cell cycle arrest and apoptotic pathways by p53

9:50    Simon Carroll             Computer-aided drug design to accelerate drug discovery

               Short Break

10:10   Kevin Batty               Parasite Reduction Ratio in Murine Malaria Model
                                  Thrombosis research: It's a pain in the neck, and in the leg, and in the
10:20   Murray Adams
        Poster Presentation 1 –   Yeast two-hybrid identification of protein partners and new roles for human
        Sharyn Pope               sex hormone binding globulin

        Poster Presentation 2 –   Pharmacodynamic study of dihydroartemisinin in the asplenic murine
        Brioni Moore              malaria model

        Poster Presentation 3 –   A closer look at antigen presenting and effector cells within the tumour
        Biljana Koloska           microenvironment

               Morning Tea

11.10   Simon Carroll             Presentation of the 2005 WABRI Honours Scholarship
                                  Isolation and identification of bioactive compounds from natural sources, in
11:16   Cornelia Locher           particular from traditional medicinal plants
                                  Danger signals and the initiation of immuno-inflammatory responses -
11:28   Andrew McWilliam
                                  Platelets the neglected immune cells

11:40   Delia Nelson              IL-2 and/or agonist anti-CD40 antibody are able to induce regression of
                                  large tumours
11:52   Deirdre Coombe
                                  Facing the Chemistry Challenge in Developing Designer Antidiabetic
12:04   Erik Helmerhorst          Drugs
                                  Mapping the genetically specified rhombomeric domains in the mature
12:16   Charles Watson            mouse brain

12:28   Simon Carroll             Thanks and Close



                      Biomolecular modelling in drug discovery

                                      Ricardo Mancera

The methods of molecular modelling and computer simulation can describe in exquisite
atomistic detail the structure and dynamics of both small molecules (such as drugs) and large
biomolecules (such as proteins and DNA). Through the use of various computer algorithms, in
combination with theories of molecular structure, it is possible to track the time evolution of
the structure and interactions of biomolecules. These techniques can be applied to tackle
scientific problems in areas of importance for drug discovery and delivery.
Some of the topics that are currently being researched are: (1) the effect of water on the nature
and strength of drug-protein binding, (2) the modelling of protein conformational changes and
their role in protein function and drug design, and (3) the mechanism of drug loading and
delivery in biodegradable nanoparticles and dendrimers. These themes represent key areas
(hydration, protein flexibility and drug delivery) for the pharmaceutical industry, as more
complex therapeutic targets are being investigated.


 Computer-aided design of small molecule inhibitors of Glycogen Synthase
                               Kinase 3β

                                      Steve Bottomley

Structure-based drug design, using computer-aided molecular modelling, is an increasingly
important approach to drug design. The advantage of this approach is that it can save many
hours of laboratory work, and reduce cost, by effectively simulating drugs in silico before they
are used in expensive biological, or clinical, trials. The binding potency of small molecules is
one of the main criteria for assessing the potential of molecules as drug leads. However,
binding potency is only one aspect of drug design and development. We also have to consider
the physiological absorption, distribution (through tissue), metabolism, excretion, and
toxicology (ADMET) aspects of the small molecules. A high potency molecule is useless if it
does not have favourable ADMET characteristics. Computer-aided molecular modelling, and
simulation, can simultaneously combine the design of a drug with the evaluation of the
ADMET characteristics of a putative drug. We aim to design non-ATP competitive, small
molecule, inhibitors of GSK3β using structure-based molecular modelling and simulated
ADMET screening of modelled putative drugs.


           Regulation of the Wnt signalling pathway in mesothelioma

                                         Simon Fox

Malignant mesothelioma is an aggressive cancer which is highly resistant to both conventional
anti-cancer therapy such as radiotherapy/chemotherapy and biological therapies such as
immunotherapy. In our laboratory we are investigating mechanisms which contribute towards
the resistance of mesothelioma to therapy. Tumour cells produce a range of molecules which
enhance both their own growth and survival as well as that of normal tumour-associated cells.
One of the main focuses of our laboratory are components of the Wnt signalling pathway
which play a key role in cell proliferation, differentiation, apoptosis and tissue morphogenesis.
We have found that sFRP4 (secreted frizzled related protein 4), a negative regulator of Wnt
signalling, is expressed in mesothelial cells but not in mesothelioma cells. Further, sFRP4 can
suppress the growth of mesothelioma cells and render them more sensitive to cytotoxic drugs.
Using tissue array technology we have also investigated expression of sFRP4 in malignant
mesothelioma tumours. Current work in our laboratory is directed at better understanding
these mechanisms since if sFRP4 does regulate pro-tumorigenic pathways in mesothelioma
then the molecules with which it normally interacts are potential therapeutic targets.


  Differential regulation of cell cycle arrest and apoptotic pathways by p53

                                          Brett Dix

The p53 tumour suppressor protein detects damaged or abnormally proliferating cells and
signals cell cycle arrest or apoptosis to prevent continuation of these potentially tumourigenic
clones. In the majority of human tumours p53 is eliminated either by direct mutation or by
mutation of downstream effector pathways. This allows tumour cells to proliferate
unencumbered by the tumour suppressor activities of p53.
In normal cells p53 expression is maintained at low levels by an interaction with it auto
regulatory partner MDM2. As p53 expression increases it transcriptionally activates MDM2,
which in turn binds to p53, transporting it out of the nucleus and targeting it for ubiquitin
mediated degradation. Phosphorylation at key sites on the p53 molecule interferes with MDM-
2 binding and this leads to an accumulation of p53 and an increase in expression of p53
responsive genes. However, how p53 differentially regulates cell cycle arrest and apoptotic
genes remains to be adequately explained.
In an attempt to study these separate functions effectively we have been working on ways of
activating alternate pathways individually. Here we present work aimed at separating these
alternate pathways.


            Computer-aided drug design to accelerate drug discovery

                    Simon Carroll, E. Helmerhorst and M. Makhija

The time and cost of discovery of new therapeutics is estimated to take 12-15 years and some
US$500 million. This cost estimation includes the cost of success and failures. Increasing the
rate of success is a critical goal for getting new medicines to the community. Important
considerations for drug discovery include target validation, drug-like chemical characteristics,
synthetic processes, toxicity assessments and proof-of-concepts ahead of testing in humans.

The “Rapid Drug Discovery” program aims to identify drug-like compounds with qualities
selected to increase the speed and accuracy in compound choice. Using qualified compound
databases, protein structures, computer algorithms and insights into drug binding compounds
have been identified, sourced and supplied to laboratories for testing in 8 weeks. To
demonstrate the utility of this approach, projects which have good market opportunity (eg
unmet medical need, large addressable market, commercial interest), protein structures and
detailed biological data supporting the validity of the target, ease of proof-of-concept,
including animal studies. The first project embarked on is an antiviral project targeting herpes
viruses – HSV 1 and 2, CMV and VZV. Further projects being undertaken are addressing
drug-resistant TB and apoptosis.


               Parasite reduction ratio in a murine malaria model

    Kevin Batty, P. Gibbons, B. Moore, C. Andrzejewski, J.R. Stoney, J. Jago

The parasite reduction ratio (PRR) has been proposed as a model of pharmacodynamic
response to antimalarial drugs. Described as a fractional reduction of parasite bio-burden
during one asexual cycle, it has been suggested that the PRR for antibiotics, quinolines and
artemisinin drugs is ten, 100-1,000 and 10,000 respectively. As the PRR model may be useful
in predicting successful antimalarial combination therapy, we are investigating the response to
antimalarial drugs in mice, to determine the value of a murine malaria model in pre-clinical
development of antimalarial drug regimens. The parasite used in our murine studies
(Plasmodium berghei) has a 24-hour asexual cycle and does not normally sequester, hence all
stages of the parasite are visible in peripheral blood smears. In the present studies, total
parasite density-time data has been used as an estimate of PRR in the murine model, because
parasite bio-burden in a host cannot be accurately determined. Following single-dose
administration of dihydroartemisinin, chloroquine and piperaquine, we have shown that the
PRR is 12.5, 9 and 7 respectively. Despite the differences in PRR data between the murine
model and the conceptual model in humans, further pre-clinical PRR studies are warranted in
the quest for effective antimalarial drug combination regimens.


    Thrombosis research: It's a pain in the neck, and in the leg, and in the

                                       Murray Adams

Thrombosis is manifested in a variety of diseases including cancer, diabetes, stroke and renal
failure and is a leading cause of morbidity and mortality in western culture. Direct and indirect
costs of thrombosis represent a significant financial burden to governments, health
organizations and hospitals. This presentation will discuss how abnormalities of blood
coagulation, in particular anomalies in the regulation of the tissue factor (TF) pathway, may
lead to the development of thrombosis. Ongoing work in the Haemostasis Research
Laboratory of the School of Biomedical Sciences will also be discussed. Current research
includes 1) investigation of the release from endothelial cells of the regulator of the TF
pathway, tissue factor pathway inhibitor (TFPI), in response to heparins, and 2) investigation of
interactions between TFPI and antibody markers of the antiphospholipid syndrome (a disease
characterized by frequent episodes of thrombosis).


Isolation and identification of bioactive compounds from natural sources, in
                particular from traditional medicinal plants

                                       Connie Locher

The area of natural product chemistry is emerging as an important activity in particular the
isolation and identification of bioactive compounds from traditional medicinal plants. Current
and previous projects undertaken in our laboratory include the investigation of Scaevola
spinescens, a traditional Aboriginal medicinal plant with reputation as an anti-cancer treatment,
and Carissa lanceolata, a northern Australian shrub with interesting anti-bacterial activities.
Furthermore, bioactive compounds from Tinospora smilacina, another important Aboriginal
bush medicine from tropical Australia attributed with a variety of pharmacological effects,
have been isolated.

Current investigations centre on Kinos, astringent exudates mainly from Eucalyptus species,
which have a long tradition as Aboriginal but also early settler bush cures. The antibacterial
effects of Centella asiatica, an Asian food and medicinal herb, known in Australia as anti-
arthritis plant have been investigated, as well as Curcuma longa as it is used traditionally in
many Asian countries in external preparation for the treatment of acne.

Dr Locher’s laboratory is also involved in a multi-group initiative under the umbrella of the
Cooperative Research Centre Desert Knowledge, which looks at traditional Aboriginal food
and medicinal plants from four different communities across desert Australia in an attempt to
identify future economically viable enterprises. Other interesting projects included the
confirmation of betel nut residue on an archaeological tooth sample from Viet Nam as well as
the screening for the presence of nephrotoxic aristolochic acid in a variety of food and
medicinal herb samples from a community in Sarawak with a high incidence rate of renal
failure believed to be due to Chinese Herb Nephropathy.


    Danger signals and the initiation of immuno-inflammatory responses -
                     Platelets the neglected immune cells

                            Andrew McWilliam and Siew Lai

Platelets are known for their function in clotting, however there is evidence they are also
involved in the pathogenesis of asthma. Thus, platelets numbers are increased in the
bronchoalveolar lavage (BAL) fluid in asthmatics and are larger in size in atopic asthmatics. In
a rabbit model, platelet depletion results in reduced airway hyper responsiveness following
allergen exposure. However, whether or not platelets have direct contact with the airway
epithelium has not been investigated. To date we have shown
   1. A novel interaction between non-activated and thrombin-activated platelets and airway
      epithelial (A549) cells.
   2. This interaction involves surface attachment only and not internalisation of the
   3. The interaction between non-activated platelets and A549 cells triggers a dramatic
      increase in IL-8 production by the A549 cells.
These results suggest that platelet interactions at epithelial surfaces may play a role in the
generation of inflammatory reactions.


   IL-2 and/or agonist anti-CD40 antibody are able to induce regression of
                               large tumours

                                       Delia Nelson

We have used a variety of immune-based regimens that aim to enhance the host anti-tumour
immune response such that it eradicates established tumours. We have shown that delivering
immune-activating agents directly into a tumour can be highly effective. Herein, we show that
IL-2 and/or agonist anti-CD40 antibody induce the regression of large tumours. Anti-CD40
had variable responses on tumour growth. Complete tumour regression was seen in 50% of
mice with small tumours when treated with IL-2. Regressing tumours were infiltrated by CD8+
T cells in association with a marked reduction in associated-associated vascularity. Tumour
progression inevitably occurred in mice with large tumours at the start of IL-2 treatment.
However, combining anti-CD40 and IL-2 generated severe necrosis at the tumour site resulting
in complete tumour rejection in 90% of mice and was highly effective in mice with fast
growing, large, tumours in which the IL-2 therapy had failed. This response was critically
dependent upon granulocytes.
Acknowledgements: NH&MRC and the Cancer Council of Western Australia


                                      Deirdre Coombe

Abstract unavailable at time of printing


  Facing the chemistry challenge in developing designer antidiabetic drugs

                                       Erik Helmerhorst

Diabetes is one of the most serious metabolic diseases. Its incidence is rapidly rising with
more than 150 million people or 5.4% of the world's population affected by the disease. Over
the next 20 years, the number of diabetics will double worldwide as the incidence of diabetes
in developing countries such as India and China continue to grow. Diabetes and its
complications are estimated to consume 1 out of every 7 health care dollars. This social and
economic burden has made diabetes a priority area for health and funding agencies in Australia
and worldwide.
Insulin is the mainstay therapy for treating type I diabetics, and at least 20% of type II diabetics
also would benefit from insulin therapy. Insulin has traditionally been delivered by injection.
This is inconvenient, has significant lifestyle implications, can cause local pain, itching,
lipodystrophy and infection. There is a substantial demand for alternative technologies.
Our research group at Curtin University of Technology has used a pharmacophore approach,
based on the structure of insulin, and database searching strategies to discover small molecule
insulin mimetics that can be delivered orally or by other convenient routes of administration.
These discovered insulin mimetics and "active" pharmacophore are protected through
international patenting strategies. This seminar will describe our strategies to develop more
potent insulin mimetics suitable for clinical trials. The research entails a multidisciplinary
approach involving computer-aided drug discovery approaches, pharmacokinetic and toxicity
studies, and a significant chemistry effort so that quantitative structure activity relationship
studies (QSAR) can be undertaken. This seminar will highlight some of our successes and
difficulties encountered in our research. The need for close integration of knowledge across a
number of disciplines will be emphasised.


   Mapping the genetically specified rhombomeric domains in the mature
                               mouse brain

                   Charles Watson (with George Paxinos (UNSW)
                            and Luis Puelles (Murcia))

We are investigating the relative rhombomeric topography of neural structures in the mature
mouse hindbrain. Our first step has been to establish the homologies that exist between the
chick and mouse hindbrain. We were then able to transfer the information from detailed
segmental fate maps available for the avian hindbrain to the mouse. We have used this and
other data to fomulate a map of putative rhombomeric domains for the entire mouse hindbrain.
Finally, we will test the validity of this map with gene expression studies in adult mice. We
have access to transgenic mice expressing lacZ conjugates of Hoxa2 (which is expressed in the
r2 rhombomeric derivatives in the adult) and Krox20 gene (r3 and r5). The results from the
Hoxa2 trangenic mice show that embryonic r2 pattern is maintained in the adult, except for two
small migrations that contribute to the superior olive and part of the rostral raphe. The
rhombomeric model provides a new window through which the organization and function of
the hindbrain can be viewed.



 1    Pharmacodynamic study of dihydroartemisinin in the asplenic murine malaria model
                               Brioni Moore, Kevin Batty1, Jeffrey Jago2
                                         School of Pharmacy
                                     School of Biomedical Sciences

The aims of this study were to provide a greater understanding of the role of the spleen after
antimalarial treatment and to compare the efficacy of dihydroartemisinin (DHA) in asplenic and control
mice. Our hypothesis was that host mechanisms compensate for asplenic status in malaria infections.
Splenectomised, Swiss mice and age-matched controls received a standard inoculum of 107 parasitised
red blood cells (Plasmodium berghei ANKA) and were given single, i.p. doses of 0, 10, 30 or 100
mg/kg of DHA, 57 hours after inoculation (n=8 asplenic and n=8 controls at each dose). Thin films
were prepared at regular intervals and examined by oil immersion light microscopy. The time to reach
5% total parasitaemia (t5%) after DHA was determined by linear interpolation of the parasite density-
time profile. For histopathological examination, key organs (liver, spleen and kidney) were harvested
from untreated control Swiss mice at increasing parasitaemia. Slides were prepared using 5µm sections
of each organ, which were subsequently stained and evaluated (Giemsa for parasite visualisation and
Haematoxylin and Eosin for tissue damage) by light microscopy.
The parasite density nadir occurred 20 h after DHA, falling 2.8, 4.4 and 6.0 fold in asplenic mice, and
2.7, 5.1 and 6.9 fold in controls, at 10, 30 and 100 mg/kg respectively. The mean ± s.d. t5% was 39 ± 5
h, 34 ± 8h and 48 ± 5 h (P = 0.003; ANOVA) in asplenic mice, and 32 ± 3 h, 43 ± 7h and 62 ± 24 h (P =
0.004; ANOVA) in controls, at 10, 30 and 100 mg/kg respectively. In the histopathological study,
stimulation of host defence mechanisms and immune responses (increased numbers of macrophages
and phagocytes in all organs studied and an increased ratio of white to red pulp of the spleen) was
observed in infected mice. After 96hrs of infection the liver appeared to take over the majority of
parasite removal from peripheral circulation (increased deposition of pigment in Kupffer cells and
sinusoidal spaces in comparison to the spleen).
DHA is effective in asplenic mice. The liver plays an important role in the removal of circulating
parasites from the blood circulation in advanced stages of infection.

 2     A comparative analysis of various anti-bacterial bioassays using Tinospora smilacina
                               Dhanushka Hettiarachchi, Connie Locher
                                          School of Pharmacy

Tinospora smilacina, which is the Australian member of the genus Tinospora, is also known as “snake
vine” and used in indigenous medicine by various Aboriginal communities in Northern and Western
Australia. A literature survey revealed that little scientific work has to date been undertaken with this
particular species, which is in contrast to the well researched Asian members of the family, T. cordifolia
and T. sinensis. In the present study methanol and hexane extracts of T. smilacina as well as selected
extract fractions separated by column chromatography were subjected to anti-microbial activity studies
using three different methods, namely disc diffusion, well diffusion and micro-broth dilution methods.
One aim was to compare the results obtained with the three methodologies and, based on this, assess
their suitability for bioassay guided fractionations. Out of the three methods trailed micro-broth
dilution was found to be the most convenient and reliable. Five fractions isolated by column
chromatography from the methanol extract showed promising antimicrobial activity, particularly
against S. aureaus, and should be investigated further in a follow up study.
 3    A closer look at antigen presenting and effector cells within the tumour microenvironment
                            Biljana Koloska, Bruce Robinson, Delia Nelson
                                     School of Biomedical Sciences

Solid tumours contain not only tumour cells, but also a significant number of immune cells. These
intra-tumoural (i.t.) cells represent potential targets for anti-cancer therapies. Although our knowledge
of basic cellular tumour biology is inconsistent, it is evident that professional antigen presenting cells
(APCs), dendritic cells (DCs), B cells and macrophages, which can process and present tumour-antigen
to naïve T cells, and tumour-specific effector T cells, need to be present and functionally active, within
the tumour microenvironment so that immune mediated tumour destruction can occur.
We used a murine lung carcinoma cell line to examine tumours for the presence of DCs and T cells.
From our previous work it was evident that during tumour growth, APCs and T cells don’t merely
travel through blood vessels, they enter the tumour microenvironment. Therefore, given that these
APCs and T cells can be found in tumours and it is well known that their interactions play a crucial role
in the induction and maintenance of an immune response, this project monitors their relationship with
each other and other cells within the tumour microenvironment.
Intra-tumoural, APC and T cells can be juxtaposed to each other, as well as to other immune cells,
indicating that they could be interacting with a number of cells.

 4      Yeast two-hybrid identification of protein partners and new roles for human sex hormone
                                         binding globulin
                                        Sharyn Pope and Ian Lee
                                     School of Biomedical Sciences

Sex hormone binding globulin (SHBG) binds and transports circulating sex steroids, regulating
bioavailable concentrations and access to target cells. Recognition that sex steroid target cells both
synthesise and specifically bind SHBG suggests it may also have autocrine and/or paracrine effects on
these cells. The cell surface receptor responsible for SHBG binding has not been isolated and there are
no reports of proteins with which SHBG may interact to exert intracellular effects on sex steroid target
cells. Human prostatic proteins interacting with SHBG have been identified using a yeast two-hybrid
(Y2H) system. Screening of about 3x 106 independent clones from a prostate cDNA library yielded
230 positive interactions. Of the 50 or so interacting proteins identified to date, those of particular
interest are flotillin-1, cathepsin D and kallikrein 4 (KLK 4).
Flotillin-1 contributes to the structure of caveolae and has roles in cell signalling and in plasma
membrane rafts. As its molecular weight is only 45kDa, it is unlikely to be the elusive SHBG receptor
which is thought to be considerably larger than this. However, as non-genomic actions of the androgen
receptor are exerted through interaction with flotillin-1 signalling pathways, even if it is not the SHBG
receptor, it may be involved in the interaction of SHBG with its receptor.
KLK 4 a member of the kallikrein family of serine proteases, is expressed in sex steroid target tissues.
It is over-expressed in prostate cancer along with lysosomal protease cathepsin D, implicating them in
the development and/or progression of breast and prostate cancer. As the intracellular expression of
SHBG is associated with non-invasive carcinomas, elucidation of the nature of its interaction with these
proteases is of significance in understanding these diseases.

 5                Identification of cosmid clones spanning the sheep MHC region
                                Jinyi Qin, David Groth, John Wetherall
                                     School of Biomedical Science

Previous research completed at Curtin and other places have demonstrated an association between
genetic markers within the class I region of the sheep major histocompatibility complex (MHC) and
resistance to parasitic nematodes. This project aims to extend the study of these associations by using
single nucleotide polymorphisms (SNP’s) as markers for haplotypic variation. A novel method has
been employed to identify sheep cosmid clones containing genes from the MHC region. Mouse mRNA
sequences, were aligned with human MHC genomic DNA sequences. Primers were identified on the
basis of highly conserved regions within a single exon and sheep DNA amplified using PCR. Nine
probes including CAT56 and UK genes in the class I region, RING3 and TAPBP genes in the class II
region and BAT3, BAT4, NTH, G6D, and G7c genes in the class III region. Hybridisation of sheep
cosmid library with TAPASINBP probe has identified cosmid clones containing the TAPBP gene.
Subsequent sequencing has revealed that this cosmid spans the region from B3GALT4, BING4, HKE2,
RGL2, TAPBP, and BING1 gene. Meanwhile, another cosmid clone containing CAT56 gene in the
class I region has also been identified. Cosmids spanning the class III region from C2 to TNXB gene
have also previously been identified. Primers have been designed based on the end sequences of these
known cosmid clones and will be used to generate probes to identify overlapping regions. More
detailed sequencing of these sheep genes will then facilitate the detection of polymorphic genetic
markers referred to as short tandem repeats (STR) and single nucleotide polymorphisms (SNPs).

  6         Melanoma Cell Adhesion Molecule in cell-cell and cell-matrix interactions
                            Danielle Dye, Chiara Aquilia, Deirdre Coombe
                                     School of Biomedical Science

The incidence of melanoma continues to increase in Australia and around the world. In addition,
melanoma metastasizes early in the disease process and is often unresponsive to chemotherapy. As
such, a new approach to melanoma therapeutics is desirable. One possible target is the cell-cell and
cell-matrix interactions of melanoma cells; as disrupting these interactions would interfere with cell
invasion, migration and thus metastasis.
Melanoma Cell Adhesion Molecule (MCAM) is highly expressed in more than 70% of metastatic
melanoma and its expression is correlated with metastatic potential. Recent studies also suggest that
MCAM plays a role in the invading trophoblast in pregnancy and in neurite outgrowth; both of which
are under tight developmental control. Thus, the evidence suggests that MCAM is important in cell
invasion and migration; a process which in melanoma has become de-regulated.
Over-expression of MCAM in melanoma cells results in changes in cell-cell and cell-matrix
interactions. Our results suggest that high expression of cell surface MCAM leads to stronger cell-cell
and weaker cell-matrix adhesion. This data is compatible with the theory of “collective cell migration”;
where cells migrate in groups and strings rather than alone. We propose that the invasive potential
conferred on cells by MCAM over-expression is mediated, at least in part, by an increase in the ability
of cells to migrate collectively.

 7              Characterisation of recombinant human insulin-degrading enzyme
                         Denis Ballantyne, David Groth and Erik Helmerhorst
                                     School of Biomedical Sciences

The insulin-degrading enzyme (IDE) is an evolutionary conserved metalloprotease. Whilst it has been
known for over fifty years that this enzyme degrades insulin, it is now apparent that it also degrades a
number of other hormones and peptides that share a consensus sequence1. This includes substrates as
diverse as insulin, glucagon1 and the b-amyloid peptide associated with Alzheimer's disease. Having
additional biological roles in development, cellular differentiation and steroid mediated signalling, IDE
may also influence insulin signalling. Recently, low levels of IDE have been implicated in the onset of
neurodegenerative disease. Indeed, it has been suggested that IDE may be an Alzheimer's gene
whereby mutation or certain alleles may predispose to neurodegenerative disease. Thus, IDE presents a
novel target for drug design and therapeutic intervention in Type II diabetes and Alzheimer's disease.
We aim to characterise the biological and biochemical properties of the recombinant IDE. Ultimately,
the 3D molecular structure will be resolved by X-ray diffraction techniques. Computer-based
molecular modelling will be exploited in a rational drug discovery program to develop novel drugs to
treat the diabetic condition or Alzheimer's disease. To date, the gene sequence encoding for human and
mouse IDE have been cloned. The mouse and human constructs have been transformed into a bacterial
host and high level expression of a recombinant enzyme induced. Biological assays have been
developed to quantitate IDE activity. Methodology for the isolation and purification of large quantities
of homogenous insulin-degrading enzyme is being established.

 8    Successful synthesis of 1-substituted 1,2,3,4-tetrahydroisoquinoline derivates from N-
                                 Andreas Hartkorn, Dr. Connie Locher
                                          School of Pharmacy

Since, according to previous research, the intramolecular cyclisation of N-benzotriazol-1-ylalkyl-β-
phenylethylamines does not work due to steric factors, their ring closure is attempted by exploiting the
influence of a bulky N-(p-toluenesulfonyl) substituent.
The reactions were carried out in a classic one-pot reaction. Benzotriazole, various alipathic or aromatic
aldehydes and tosylated phenylethylamine were reacted in dichloromethane at room temperature,
followed by a Fridel-Crafts cyclisation with either anhydrous AlCl3 or conc. H2SO4 The obtained solids
were recrystallised from ethanol and characterised by 1H- and 13C-NMR spectroscopy, elemental
analysis and their melting points.
The synthetic protocol worked very well with a variety of aliphatic and also aromatic aldehydes
resulting in high yields of 1-substituted 1,2,3,4-tetrahydroisoquinoline derivatives using mild reaction
It appears that the bulkiness of the tosyl substituent facilitates the intramolecular cyclisation of N-
benzotriazol-1-ylalkyl-N-tosyl-β-phenylethylamines to their corresponding 1-substituted 1,2,3,4-
tetrahydroisoquinoline derivatives. Further research now needs to establish if the tosyl group is also
able to exert stereochemical control on position 1 of the obtained heterocycle.

 9       Expression of the soluble receptor for advanced glycation end-products (sRAGE) for 3D
          structure determination: an opportunity for novel inflammatory disease therapeutics
                         David Chandler, Mike Garlepp2 and Erik Helmerhorst1
                                      School of Biomedical Sciences
                                           School of Pharmacy

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super
family that has been implicated in a variety of pathologies such as diabetes, amyloidoses, tumour
metastasis and inflammation.
On a molecular level, prolonged RAGE-ligand binding encourages cellular dysfunction through
deregulation of the transcription factor NFκ-B. It has been demonstrated that blockades against RAGE
using a secretory form of the receptor (sRAGE), impair tumour tissue invasion, reduce vascular
hyperpermeability in diabetics and promote a return to normal cellular conditions in inflammatory
The development of potent, small-molecule antagonists of RAGE capable of crossing the blood-brain
barrier would provide a novel strategy for reducing the progression of those diseases associated with
RAGE activation. A powerful strategy for the design of such molecules is through the elucidation of
the receptor for advanced glycation end products atomic structure, for application to rational drug
design methodologies.
In this project, we have developed recombinant constructs of human sRAGE for expression in
Spodoptera frugiperda 21 (Sf21) insect cells. We are now developing strategies to enable sRAGE to be
purified to homogeneity and in sufficient amounts so that we can attempt to grow crystals of the
protein. Given that quality crystals are obtained, we will resolve the 3D structure of sRAGE by X-Ray
diffraction studies. The developed 3D structure would then form the basis of a rational drug design
program for RAGE.

 10          Understanding how insulin, the antidiabetic hormone, acts on cells using
                                state-of-the-art BIAcore™ technology
                        Bradley Shelton, Brian Plewright and Erik Helmerhorst
                                     School of Biomedical Sciences

The rational development of new therapeutics for diabetes demands an intimate understanding of how
insulin works at the molecular level. However, the first step in insulin signalling, the binding of insulin
to a cell-surface protein known as the insulin receptor, is not clear. Current models of this interaction
are based on equilibrium binding data suggesting that insulin binds to its receptor with two different
affinities. This and the knowledge that the insulin receptor is a preformed dimer, have led to the belief
that two insulin molecules may bind to one receptor molecule. If this stoichiometry is correct, it greatly
complicates the design of drugs that mimic insulin. In this study, we have determined the binding
stoichiometry of insulin to its receptor using BIAcore's surface plasmon resonance technology, a
revolutionary and powerful method that enables real-time visualisation of molecular interactions
without the need to chemically label the reactants. For the first time, we unequivocally show that only
one insulin molecule binds to a single receptor molecule, despite still showing two binding affinities by
equilibrium binding analysis. These results suggest that the two observed affinities arise as a result of a
conformational change in the receptor from a low to high affinity state, rather than through the binding
of two insulin molecules. This is a significant finding that helps us better understand the mechanism of
insulin action.
 11                       Insulin and Insulin-like Growth Factor-I receptors:
                Preset molecular “ mouse traps” triggered by ligands or serine proteases
                                       Jie Liu and Erik Helmerhorst
                                       School of Biomedical Sciences

Insulin and Insulin-like Growth Factor I (IGF-1) are vitally important for normal metabolic and
mitogenic function of mammalian cells. In order to perform these functions, they first bind to and
activate their respective cellular receptors. Both the insulin receptor (IR) and the insulin-like growth
factor I receptor (IGF-IR) are (αβ)2 heterotetrametrc glycoproteins that are structurally and functionally
related.     Ligand binding to the extracellular α-subunits of each receptor promotes their
autophosphorylation and inherent tyrosine kinase activities. Interestingly, limited trypsin treatment of
the IR that results in cleavage of the α-subunit at three sites, abolishes insulin binding, and
simultaneously promotes its autophosphorylation and tyrosine kinase activity. This suggests that the α-
subunits of the IR act as a structural constraint on its kinase activity that is released by insulin or trypsin
treatment. In this study, we demonstrate that mutations at each individual site of trypsin cleavage in the
IR did not prevent the constitutive activation of the IR by trypsin. Inhibition of the proteolytic activity
of trypsin with the inhibitor benzamidine abolished the effect of trypsin. We conclude that the
proteolytic cleavage of the IR is necessary for the effect of trypsin, but that no specific site of cleavage
in the IR is needed to relieve the inhibitory constraint of α-subunits on the IR kinase activity. This
conclusion was supported by the observation that chymotrypsin, free of trypsin activity, also
constitutively activated the IR under carefully controlled conditions. Finally, we demonstrate that the
IGF-1R is also constitutively activated by trypsin. Taken together, this study strongly supports the
concept that the α-subunits of the IR and IGF-1R act as a general structural constraint on their kinase
activity that is released by ligand binding.

 12                  Identification of clones spanning the sheep MHC gene region
                    Jinyi Qin1,2, Chee Yang Lee2, John Wetherall1,2 and David Groth1,2
                                    School of Biomedical Science
             Centre for High Throughput Agricultural Genetic Analysis, Murdoch University

This project aims to study the association between parasitic nematodes and sheep MHC class III region
by using single nucleotide polymorphisms (SNP’s) as markers for haplotypic variation.
Mouse mRNA sequences were aligned with human MHC genomic DNA sequences to identify highly
conserved exons, which served as DNA templates for designing PCR primers as well as overlapping
primers (“overgo primers”). 9 probes developed from PCR of a merino sheep DNA were hybridised
with sheep cosmid library, while 20 sets of overgo primers were hybridised with sheep BAC library. 2
positive cosmid colonies and108 positive BAC colonies have been identified containing the entire MHC
class III region and parts of class I and II regions. Subcloning and sequencing of 1 cosmid colony has
revealed the end part of MHC class II region spanning from B3GALT4, BING4, HKE2, RGL2,
TAPBP, and BING1 gene. Previously identified cosmids containing genes from C2 to TNXB within
the class III region have also been subcloned and sequenced, upon which a SNP marker within Tumour
Necrosis Factor (TNFα) gene has been discovered. Identification of additional SNPs markers is

 13       Optimising the expression of genes important for bioleaching processes within
                                      iron-sulphur oxidising microbes
                        Misty-Lee Palmer, Suzanne Keeling2, David Townsend1
                                         Biomedical Sciences
                                   CSIRO Minerals, Curtin University

Techniques from microbiology, molecular genetics and chemistry were combined to examine the
chalcopyrite leaching capacities of native microbial isolates. This ore has proven difficult to leach
using laboratory microbial strains but as it is the most common form of copper found world-wide,
substantial interest has been generated to improve leaching rates using indigenous microbes.
Enrichment of native bioleaching microbes from a spent chalcopyrite/pyrite test heap in W.A. recovered
seven new isolates. The diversity of the native isolates was examined by determining their growth
temperature range, pH tolerance and energy substrate metabolism. All seven isolates were iron-
oxidisers, five were able to grow in pH 1 and two of the isolates were capable of growth in 80 g/L of
copper sulphate. All were moderate thermophiles, capable of growing between 30-50°C. Other native
isolates (previously isolated from the same test heap and part of CSIRO Minerals’ culture collection)
were examined for resistance to four metals commonly found in ore; cobalt, copper, nickel and zinc.
One isolate, N39-30-03, was capable of growing in high concentrations of all four metals (>35 g/L)
whilst the other isolates were capable of growth in at least two of the metals (>10 g/L). Pairs of these
isolates were tested for their ability to exchange genes for metal resistance by conjugation but the
results were inconclusive and further research is required to determine the exchange of genetic material.
Additionally, these isolates were screened for the presence of the rusticyanin gene, an electron transport
protein involved in iron-sulphur oxidation and high expression thought to increase the cell’s
leaching capacity.

 14     Development and validation of in vitro HPLC assay of Loperamide Hydrocholride
                        P. Patel, Gautam Dalwadi, Heather Benson, Yan Chen,
                                            School of Pharmacy

The aim of the project was to develop and validate the HPLC assay for loperamide HCl.
The HPLC method was developed using a reverse phase C18 column (length 250 mm x 4.6 mm ID)
connected to the HPLC system which comprised of a pump (Waters 501), UV detector and Integrator
(Hewlett Packard, HP 3386).
Plate count for the stated column was found to be 2000 which was unaffected by concentration of the
samples. The peak of loperamide which eluted at 11 minutes was well resolved from the solvent front
with capacity factor value 4.4. The calibration curve was found to be linear over a concentration range
from 1 to 50 μg/ml (R2= 0.9998). The lower quantitation limit (LQL) was 1 μg/ml (RSD = 1.76, n= 6).
The lower detection limit (LDL) was found to be 50 ng/ml derived from signal to noise ratio (S/N) 6.5
of at least five injections (RSD = 6.5 %, n=5). The RSD values of precision were always less than 2 %
for each concentration point over 1 to 50 μg/ml. The precision at 100 % assay concentration was RSD
0.78 %, (n=5). The method was found to be an accurate in quantifying the known amount of drug from
nanoparticles matrix with an accuracy value 100 ± 2.0 % (n=3).
From the assay validation data, the method seems reasonable to adopt for further in-vitro formulation
evaluation studies.

 15          Synthesis of some novel biphenyl xanthone and biphenyl bipheny derivatives with
                                        antidiabetic activity
                                  Robert Peter Herman and Hugo Huixiang Diao
                              Department of Applied Chemistry, Curtin University

Discovery of an effective insulin mimetic that can be administered orally would be a major advance in
the treatment of diabetes mellitus, providing a less expensive therapy whilst alleviating the discomfort,
inconvenience and risks associated with injections. Recent research at Curtin University has identified
a compound (IM140) that could be further developed into a suitable insulin mimetic1. The aim of this
research is the synthesis of IM140 analogues and to test their activity in a range of bioassays.

                                             remove or modify                       replace scaffold with
                                                                                       more amenable
                         F                                                                 target
                                        OH                             O


                                             O            O            O

             change substituent
              type and position                                             relpace COOH with
                                                                           SO3H, tetrazole and/or
                                             IM140                            extend C chain

Synthetic targets summarised in the figure above are currently being undertaken with the aim of
identifying a clinically suitable orally administrable insulin mimetic.

 16      A descriptive analysis on the utilization and interpretation of serum drug assays (SDAs)
                                    data of perhexiline therapy
                              Zaiton Kamaruddin, Bruce Sunderland, Peter Tenni
                                             School of Pharmacy

The study was aim to determine the current practice involved in the utilization and interpretation of
SDAs data for perhexiline at Royal Perth Hospital based on the established criteria of (1) ordering of
SDAs, (2) time of blood sampling and (3) use of assay results for dose adjustment.
A list of patients prescribed perhexiline from Jan. 2001 to March 2003 retrieved from the pharmacy
database (ASCRIBE ®) were selected and patient’s medical record were requested. All relevant data
were collected in a standard pre-designed form.
A total of 127 SDAs were assessed (N=125 charts). The overall appropriateness in compliance to
SDAs criteria was 42.5% (54/127) and subsequent dose changed of 87% (47/54) were appropriate. The
criteria for ordering, sampling time and use of results were appropriate in 96.9%, 62.6% and 70.1%
respectively. Common reasons for inappropriateness were (a) unnecessary repeated levels, 1.6%
(2/127) (b) ordering less than 1 week after dose commencement, 1.6% (2/127) (c) sampling time not
indicated, 17.3%(22/127) and (d) sampling done >2 hours post-dose, 22%(28/127). No statistically
significant relationship found between all the three criteria (Chi-square; p>0.05).
The inappropriateness of SDAs (57.5%) and dose changes (13%) of perhexiline therapy could
contribute to wastage of assays and a negative impact on patient outcomes.
 17      High Performance Liquid Chromatographic assay for the simultaneous determination of
                     Prilocaine, Lignocaine and Tetracaine Hydrochloride
                                     Vaishaali Munot, Heather Benson
                                            School of Pharmacy

A simple, sensitive and selective isocratic high performance liquid chromatographic method was
developed for the simultaneous determination of Prilocaine Hydrochloride (PH), Lignocaine
Hydrochloride (LH) and Tetracaine Hydrochloride (TH).
The drugs were separated by reversed phase C-18 column using a mobile phase of 60:40
methanol:20mM ammonium acetate buffer, pH 7.4 at 1.5mL/min and UV detection was at 230nm. The
method was reproducible and fully validated.
A linear relationship for all the three local anaesthetic drugs was obtained in the concentration range of
4 to 10 µg/mL. The precision and accuracy was obtained in the range between 4 to 10µg/mL. The
limit of quantitation was 1µg/mL for all drugs and the limit of detection was 20ng/mL, 40ng/mL, and
50ng/mL for PH, LH, and TH respectively.
The assay is suitable for studies into the development of novel local anaesthetic injection formulations.

 18      Does Residue 270 of the insulin receptor, a tryptic cleavage site, play a key role in insulin
                                Liu Jie, Brad Shelton and Erik Helmerhorst
                                       School of Biomedical Sciences

Insulin, the antidiabetic hormone, is able to initiate its cellular effects via interaction with a cell surface
protein, known as the insulin receptor (IR). The binding of insulin to the extracellular portion of the IR
initiates a conformation change in the receptor that allows activation of its intercellular tyrosine kinase
activity. This active tyrosine kinase initiates the insulin signalling cascade, which inevitably leads to
insulin’s biological effects.
Limited treatment of the IR with the protease trypsin has previously been shown to mimic the effects of
insulin, by constitutively activating the IR. Four major sites of trypsin cleavage have been identified:
the extracellular residues Lys164, Arg270, and Lys 582, and the intercellular residue Lys1115.
Previous studies have eliminated Lys164 and Lys582 as the sites of tryptic activation of the IR.
Lys1115 has likewise been discounted due to its intercellular location. This leaves Arg270 as the only
remaining possibility. This residue is located within a region of the IR that is thought to play a
significant role in insulin binding and activation of the receptor. Investigation of the role of this residue
in trypsin activation of the IR will help to improve our understanding of the mechanism by which
insulin is able to activate its receptor.
In this study, we investigate the role of residue Arg270 in the tryptic activation of the IR, by creating a
mutant IR that is no longer sensitive to trypsin cleavage at this site. The ability of trypsin to activate
this mutant IR was assessed, and found to be similar to that of the wild-type receptor. This
demonstrates that Arg270 is not involved in the tryptic activation of the IR. The results of this study
will necessitate the development of new theories concerning activation of the IR, both in response to
trypsin and insulin stimulation.

 19      One-pot synthesis of N-benzotriazol-1-ylalkyl-β-phenylethylamine derivates as potential
             intermediates in the synthesis of 1,2,3,4-tetrahydroisoinoline derivates
                                  Wolfgang Wimmer, Connie Locher
                                          School of Pharmacy

This project extends the well-known reaction between benzotriazole (Bt), formaldehyde and
phenylethylamine (PEA) by using various aldehydes as starting materials. In a one-pot reaction it aims
to prepare different N-benzotriazol-1-ylalkyl-β-phenylethylamines as potential intermediates in the
synthesis of 1-substituted 1,2,3,4-tetrahydroisoquinolines.
To synthesise the various N-benzotriazol-1-ylalkyl-β-phenylethylamines two different reaction
conditions, both ‘one-pot but two-step reactions’, were trialled.
A) Benzotriazole and the aldehyde were mixed without a solvent to give the corresponding adduct in
excellent yields. PEA was then added and, after a period of time, the anticipated product detected by
 H-NMR spectroscopy.
B) The aldehyde and PEA were stirred at room temperature yielding the corresponding imine, which
was then reacted with benzotriazole to obtain the same product as in Method A.
The intramolecular cyclisation of these compounds using a Friedel-Crafts catalyst (e.g. AlCl3, H2SO4),
was also attempted.
A wide variety of aldehydes gave the expected product in good yields, although Method B generally
resulted in higher quantities than Method A. The attempted cyclisation reaction, however, failed with all
obtained intermediates.
The establishment of a protocol for the preparation of various N-benzotriazol-1-ylalkyl-β-
phenylethylamines was very successful, but the intramolecular ring-closure of these products failed,
most likely the result of steric influences. Based on the findings of this work it is suggested that the
presence of a second bulky group on the PEA nitrogen is required for a successful ring closure.

 20             Comparison of molecular typing methods for Moraxella catarrhalis
                      Nevada Pingault1, Deborah Lehmann2, and Thomas Riley3,4.
                                    School of Biomedical Sciences
                          Telethon Institute for Child Health Research, Perth,
                 Western Australian Centre for Pathology and Medical Research, Perth
              School of Biomedical and Chemical Sciences, University of Western Australia

A number of molecular typing techniques were investigated to determine which method was the most
discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. Twenty five M.
catarrhalis isolates obtained from nasopharyngeal aspirates from Aboriginal and non Aboriginal
children were subjected to random amplified polymorphic DNA analysis (RAPD), automated
ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two M. catarrhalis
types, automated ribotyping with Pst1 determined four M. catarrhalis ribogroups and PFGE analysis
with Not1 determined 21 pulse field groups within the 25 isolates examined. This study confirms that
PFGE is the most discriminatory method for the typing of M. catarrhalis

 21       Development of a pharmacokinetic-pharmacodynamic model of dihydroartemisinin in
                                      murine malaria
              Peter Gibbons1, Kevin Batty1, Hugh P Barrett2, Tim ME Davis2, Ken F Ilett2
                                          School of Pharmacy,
                School of Medicine and Pharmacology, University of Western Australia

The aim of this study was to investigate the pharmacokinetic-pharmacodynamic (PK-PD) relationship
for dihydroartemisinin (DHA) in a murine malaria model.
Plasmodium berghei (ANKA) infection was established in 5-week old Swiss mice. Pharmacodynamic
parameters were determined by examination of thin blood films via light microscopy to determine the
parasite density profile of infected mice treated with DHA administered intraperitoneally at 0, 1, 3, 10,
30 and 100mg/kg. Parasites were classified into ring, early trophozoite, late trophozoite and schizont
stages based on microscopic observation of morphological features. Pharmacodynamic data was
modelled using SAAM II software package.
Pharmacokinetic data were obtained from age-matched malaria-infected Swiss mice using 30 and
100mg/kg DHA doses administered intraperitoneally. Serum concentrations of DHA were measured by
high performance liquid chromatography and PK parameters determined by non-compartmental
analysis of the DHA concentration-time data. In malaria infected mice DHA had a tmax of 15 mins and
an elimination t1/2 of 12 mins. PD experiments showed a graded relationship between dose and
response, with a maximal decrease in parasite density occurring at approximately 24 hours. Parasite
staging data indicated that all parasite stage populations were affected by DHA treatment.
These results provide the basis for future studies aimed at building a stage specific PK-PD model for
DHA and other antimalarial drugs. The model could be used prospectively to simulate the effects of
multiple dosage regimens and drug combinations.

 22      Development and application of advanced molecular methods for the rapid and sensitive
                                 detection of infectious agents
                          Carmel Hancock, David Townsend, Debby Cousins
       AB-CRC, Department of Agriculture Western Australia, Curtin University of Technology

There are many infectious agents, which affect both animal and man that require fast and reliable
detection methods so as to prevent epidemics. This presentation will focus on the development of
advanced molecular detection techniques for such pathogens. The suitability of real-time PCR using
LightUp probes and immuno-PCR will be discussed. Ideally, these new methods will allow faster
identification of serious pathogens, combined with sensitivity, specificity and reliability of results.
Initially, this study will assess the suitability and development of these methods using Campylobacter
coli, Campylobacter jejuni. This initial pilot study using the Campylobactor spp. was performed in
Gothenburg, Sweden and will also be discussed in this presentation. Further development of the
techniques will be performed using Newcastle disease virus, which is a disease of interest for end users
of any developed methods due to the severe economic effects of any NDV epidemic.

 23       Serum enhances the heparin dependent release of tissue factor pathway inhibitor from
                            human umbilical vein endothelial cells
                      Paul Ellery1, Kathy Hardy1, 2, Robert Oostryck1, Murray Adams1
                   Haemostasis Research Laboratory, School of Biomedical Sciences
                                         Cytolabs, Bentley

Recent evidence suggests that two forms of Tissue Factor Pathway Inhibitor (TFPI), TFPIα and TFPIβ,
are synthesised by the endothelium. TFPIα is released by heparin, thereby partially contributing to its
anticoagulant effect. The aim of this study was to further characterise the release of TFPIα from
endothelial cells after exposure to heparin.
Human umbilical vein endothelial cells (HUVEC’s) were isolated and grown to confluence using
conventional culture methods. Cells were exposed to 0, 1 or 10 U/ml of heparin (UFH or LMWH) for 1
and 24 hours, the supernatants collected and then assayed for TFPI activity.
Compared to the 0 U/mL control, UFH and LMWH (1 U/mL and 10 U/mL) generated statistically
significant increases in TFPI activity after 1 hour when diluted in DMEM containing 5% FCS (UFH:
9.0 mU/ml vs 18.3 and 18.4 mU/ml, p<0.0001; LMWH: 8.8 mU/ml vs 13.3 and 21.4 mU/ml, p<0.05).
A statistically significant increase in TFPI was not demonstrated after 24 hours using 1 U/mL and 10
U/mL UFH (93.6 mU/ml vs 131.8 and 121.2 mU/ml, p>0.05) or LMWH (100.3 mU/ml vs 115.2 and
137.7 mU/ml, p>0.05) under the same conditions. TFPI activity levels between the different
concentrations of both heparins, at both time points tested, were not statistically different when FCS
was absent.
These results demonstrate that serum enhances the heparin-induced release of TFPIα from HUVEC’s.
Further studies are required to elucidate the serum component(s) that are required in this mechanism.

 24                          Investigation of the mysterious exudate known as kino
                                     Laura Currie and Connie Locher
                                           School of Pharmacy

A ‘kino’ is a type of wood extractive, unique from any other by virtue of its deep rich colouration, high
tannin content and characteristic astringency. Historically, kinos from various botanical sources
throughout the world have been used medicinally, on account of their astringent property, to arrest a
range of secretions and emissions in conditions such as diarrhoea, dysentery and haemorrhage.
Although many have been described, the most abundant kino of Australian origin is that of the Eucalypt
and this too has been exploited for its healing properties, first by the Indigenous Aborigines and then
later by early European settlers. Whilst the various medicinal uses of Eucalyptus kino by the white
settlers have been well documented, much of the information regarding their medicinal employment by
native Aborigines has been lost. In recent years however, with the rise in ethnobotanical exploration,
some of this information has been retrieved, and of that so far uncovered there is an indication that
some may actually possess antibacterial, antifungal or antiviral properties. Moreover, the fact that kinos
still appear to be used today for medicinal purposes by Aboriginal communities in country Australia
further supports the potential of these compounds in providing a source of effective, new therapeutic
agents and thus warrants further investigation of both chemical and biological aspects of these
interesting exudates.
In this project a preliminary chemical investigation as well as screening for antimicrobial activity of
various kino samples collected from different parts of Western Australia will therefore be undertaken.

 25        The expression of receptor for advanced glycation endproducts (RAGE) in sporadic
                                     inclusion body myositis
                             Saima Majeed, Michael Garlepp, Simon Fox
                                         School of Pharmacy

Sporadic inclusion body myositis (sIBM) is an inflammatory myopathy. The pathological hallmark of
this autoimmune disease are muscle fibers containing rimmed vacuoles with abnormal protein
aggregates. The RAGE is expressed in regions of ongoing tissue dysfunction and is relevant to the
progression of sIBM pathogenesis. A coordinated signalling pathway downstream of RAGE-ligand
interaction may trigger abnormal protein production and accumulation. This project aims to determine
effects of RAGE-ligand interaction on muscle cell gene expression and the influence of RAGE
promoter region polymorphisms on its expression.
To study RAGE gene expression, skeletal muscle cell lines of human and murine origin were used
either as myoblasts or terminally differentiated myotubes. Human macrophages were used as positive
controls for RAGE up-regulation. B cell lines with different RAGE promoter region polymorphisms
were used to study the differential expression of RAGE. Total RNA extracted was reverse transcribed
and real-time quantitative PCR performed to determine gene expression levels normalised against stable
housekeeping genes.
RAGE mRNA present at comparable levels in the B cell lines was down regulated 2-fold by endotoxin
exposure (10ng/mL) in a manner independent of MHC haplotype. Increasing endotoxin challenge (0 –
1µg/mL) also down regulated RAGE mRNA levels dose-dependently in myoblasts . In myotubes,
RAGE mRNA levels were completely inhibited at the lowest dose of endotoxin. Interestingly, RAGE
expression was significantly down regulated (6-fold) upon amphoterin treatment (0 – 1µg/mL). This
was independent of differentiation stage of the muscle cells. While increasing amyloid concentrations
(0 – 250nM) resulted in 2-fold decrease in myoblast RAGE gene expression, an initial 2-fold up
regulation in myotubes returned to baseline as amyloid burden increased. As expected, RAGE mRNA
levels in macrophages increased in a dose-dependent manner upon exposure to amyloid.
The results obtained show that muscle cell RAGE expression decreases in the presence of endotoxin
and amphoterin but shows a cell-type specific response to amyloid exposure. Taken together, the data
suggest a previously unseen cell-type specific and ligand-specific regulation of the RAGE gene. This
discovery has the potential to greatly improve our understanding of sIBM pathogenesis.

 26     Molecular analysis of genes encoding resistance to cationic compounds in Staphylococci
                                   Dale Morgan and David Townsend
                                     School of Biomedical Sciences

Antibiotic resistance in bacteria is a worldwide health issue with an increasing number of human
pathogens acquiring resistance to nearly all available antibiotics. Pre-eminent among these is
Staphylococcus aureus, one of the major causes of bacterial infection, particularly in hospitalised
patients. The increasing emergence of epidemic strains of multi-resistant S. aureus (MRSA) in recent
years has highlighted an urgent need to learn more about the properties of these organisms that
encourage their spread and survival.
Most epidemic strains of MRSA express resistance to a range of cationic compounds including
quaternary ammonium compounds (QAC). In addition to providing pathogenic bacteria with protection
against antiseptics and disinfectants, this resistance may also encode resistance to antimicrobial cationic
peptides that form part of the body’s innate defence mechanism. These preliminary observations
suggest that epidemic MRSA within hospitals have become resistant not only to antimicrobial
chemotherapy but also to one of the body’s most important primary defence mechanisms. This
hypothesis is controversial. The resistance mechanism does not provide a high level of resistance to
QACs in common use within hospitals and there has been no confirmation of resistance to human
cationic peptides.
This study will focus on characterising the genetic determinants of cationic resistance in staphylococci
with a view to understanding the roles they may play in the survival and spread of staphylococci within
hospitals as well as the virulence and pathogenicity of the staphylococci.
Several genes have been identified and sequenced in staphylococci that encode resistance to cationic
compounds including qacA, qacB, qacC, qacH and qacJ. These genes differ in the range of cationic
compounds to which they express resistance and all seem to encode for a membrane complex that
“pumps” cationic compounds out of the cell. As the first stage of investigating the role of these genes,
clinical isolates of staphylococci are being screened for the presence of cationic resistance and qac
genes using specific PCR probes. Isolates resistant to cationic compounds but not targeted by the PCR
probes will be investigated for the presence of novel resistance genes. These will be transferred to a
laboratory strain, cloned and sequenced so that they can be compared with the known qac genes. It is
hoped that a picture may emerge that correlates the distribution of cationic-resistant staphylococci, type
of resistance gene, site of infection and patient medication. This may help to identify the role and
significance of these controversial resistance genes.



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