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									          Catalyst
The




An Oregon State University Publication
              Volume 3
                                                        The
                                                           Catalyst




              Catalyst
        The

                                                                                                         Staff


         A letter from the editors:
                                                                                                         Editors
                                                                                                       Megan Cook

                                                                                                      Carly Dougher
                  As scientists, we conduct research in an effort to explain natural phenomena
         by piecing together models that best fit our observations. Because these models are

                                                                                                     Katy McHenry
         only our best explanation of how our world works, many times our research results
         do not match our predictions. It is with these unexpected findings that we are able to
         shape, fine tune, and discover the limits of our current scientific models. New discov-
         eries not only allow for shifts in paradigms but present opportunity for expansion of
         scientific application as well. What is a widely accepted scientific theory today may
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         be considered tomorrow as an out-dated and flawed hypothesis. It is for this reason
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                                                                                                         Tari Tan
         that we must always keep open minds in science and remain flexible to the possibilities
         of new modes of thinking. As editors of the Catalyst, we encourage you to think ten,
         fifty, even one hundred years down the road, and ponder on what new discoveries will
         be made, which models will be replaced, and which will weather the decades and remain
                                                                                                     Faculty Advisor
                                                                                                       Kevin Ahern
         our best explanation.


         Keep on discovering!


         Katy, Megan, and Carly




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                                               The
                                                 Catalyst




Table of Contents

Short-term Impacts of the Invasive Indo-Pacific Lionfish on
Bahamian Coral reef Fish Communities ............................4

Sources of Variability in the Duration of Anesthesia in
Snakes....................................................................................7

Detection          of         Leaching               Organic               Migrants                from
Polyethylene Terephthalate and Polycarbonate Water
Bottles................................................................................................13

Testing the Role of Conserved Genes in Pollen
Development...............................................................................................15

The Characterization of Oxytocin Receptors on Corpus Luteum
Endothelial Cells........................................................................18

An Investigation on the Toxicity of Aluminum in
Zebrafish.....................................................................................21




                                                     Publication of The Catalyst
                                                     was made possible by HHMI
                                                     grant#52005883 and the University
                                                     Honors College at OSU.

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                                                                                           Volume 3
                                                  The
                                                    Catalyst

          Short-term Impacts of the Invasive Indo-Pacific Lionfish on
                   Bahamian Coral Reef Fish Communities
                                         Megan Cook, Mark Albins, Mark Hixon
                                                     Zoology Department
                                                    Oregon State University


    Introduction                                               Bahamian grouper feeding on lionfish9.
         As people and goods move worldwide, intro-                 During the summer of 2008, I participated in a
    duced species become a key problem. In the early           research program investigating the effects of lionfish
    1990’s the red lionfish (Pterois volitans) was intro-      on native coral reef fishes. This project is headed by
    duced to the Atlantic from its native range in the In-     Dr. Mark Hixon, who has been conducting research
    do-Pacific. Highly desired for their bright colors and     at the Perry Institute for Marine Science, Lee Stock-
    exotic appeal, this predatory fish has been shipped        ing Island Bahamas, since the early 1990s. The Hix-
    around the world, and is displayed in numerous pub-        on team has observed lionfish populations increase
    lic and private aquarium collections. Introduction of      at an explosive rate at their field sites over the last
    this species occurred in 1992 when several individu-       several years and have initiated a series of experi-
    als were released near Biscayne Bay, Florida after         mental and observational investigations into their ef-
    a small aquarium was destroyed by Hurricane An-            fects on native fish communities. My independent
    drew1, 2. However, it is likely that the lionfish inva-    research project served as a significant contribution
    sion was a result of multiple intentional and uninten-     to this overall goal. I sought to answer the following
    tional introduction events throughout the region. In       questions: (1) do lionfish have predators in this new
    less than two decades, lionfish have expanded north        ecosystem, and (2) are lionfish competing with simi-
    as far as Rhode Island, south to Colombia, east to         larly sized native predators (specifically grouper).
    Bermuda and St. Croix, and west to the Yucatan Pen-
    insula and Belize3,4,5,6. Lionfish are voracious preda-
    tors that have been shown to have a large impact on
                                                               Methods
                                                                    In order to answer the predation question, I con-
    native fishes. Recent research demonstrates that sin-
                                                               ducted a series of feeding trials. I collected several
    gle lionfish on small patch reefs reduce the recruit-
                                                               large predatory species found locally and placed
    ment of native fishes by an average of 79% in only
                                                               them in large holding tanks (600 ft3 or 108 ft3). The
    five-weeks7. Addition of a novel predator can have
                                                               predators ranged from 7 to 46 cm (total length- TL)
    devastating impacts on native ecosystems by causing
                                                               and included one Caribbean octopus (Octopus bri-
    extinctions and disrupting trophic structure of native
                                                               areus), two nurse sharks (Ginglymostoma cirratum),
    communities. In other ecosystems, prey density has
                                                               one red hind (Epinephelus guttatus), three Nassau
    declined by five-fold to ten-fold within three years
                                                               grouper (Epinephelus striatus), and three graysby
    of the introduction of an invasive predator8. The full
                                                               grouper (Epinephelus cruentatus). Each predator
    impact of the lionfish invasion is not yet known. The
                                                               was acclimated to its holding tank for 72 hours, fed
    expansion of lionfish populations throughout the Ca-
                                                               at least one live native fish to ensure that it would,
    ribbean could add further strain to coral reef ecosys-
                                                               in fact, eat in captivity, and then offered a small li-
    tems in the region. These systems are already highly
                                                               onfish (4-10cm TL). Lionfish remained in the tanks,
    degraded by human-caused disturbance, including
                                                               which were monitored twice a day, for the presence
    climate change, pollution and overfishing.
                                                               and health of the predator and lionfish.
         As with any invasive species, in addition to iden-
                                                                    I also conducted a lab experiment designed to
    tifying the effects that lionfish are having on native
                                                               examine potential competition between lionfish and
    species, it is also important to identify factors that
                                                               a similarly sized native predator. I used coney grou-
    may control their populations. One such factor may
                                                               per (Cephalopholis fulva) as my native predator for
    be predation. Unfortunately little is known about
                                                               several reasons: (1) coney and lionfish have similar
    potential predators of lionfish in their native range.
                                                               diets, feeding predominantly on small fishes, (2) co-
    However, there is one published account of native

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                                                          The
                                                              Catalyst

ney and lionfish reach similar maximum sizes, and                   of time each feeder fish remained in the tank before
(3) coney are readily collected using SCUBA in sizes                being consumed. All predators were also weighed
comparable to lionfish. In addition, my competition                 and measured again at the end of the experiment to
experiment was designed to complement a similar                     determine growth rate over the experimental period.
field experiment that was being conducted at the same               I hypothesized that the growth rate and average con-
time. The experiment included three different pred-                 sumption per feeding of the coney would be lower
ator-combination treatments, a single coney alone, a                in the presence of lionfish than in the coney-alone
single lionfish alone, and a single coney and a single              treatment.
lionfish together in one tank. All predators (coney
and lionfish) were measured and weighed, and were                   Results
assigned to experimental blocks based on size simi-                      In the predation trials, different predatory spe-
larity. Predators ranged in size from 5–11cm total                  cies were enclosed with a lionfish for variable dura-
length (TL). There were six blocks, each of which                   tions due to logistical constraints. In all trials, which
contained each of the three treatments. Each treat-                 ranged in duration from 5 to 39 days, no lionfish
ment was housed in its own 6.5 foot3 aquarium with                  were consumed by any of the native predators. Nas-
rock habitat in the center of each to standardize the               sau groupers feeding trials were the longest. Nas-
amount of shelter each predator was provided during                 saus spent 39 days in the presence of lionfish with
feeding. Each block was fed feeder fish (Gambusia                   no direct predation or lionfish mortality observed.
sp.) every other day. Two feeder fish were provided                 All Nassau groupers were offered a single small na-
to each predator at each feeding (i.e. single predator              tive prey fish at the midpoint of the feeding trial. All
treatments received two feeder fish and two predator                Nassau ate the native prey fish within 2 minutes. One
treatments received four feeder fish). During each                  native predator, the red hind, never ate a lionfish, but
feeding, I recorded feeding efficiency (ratio of suc-               was observed eating a noxious native soapfish that
cessful strikes to attempted strikes), and the amount               was introduced to its tank. Native predators were


                                        Average Length Growth Rate (SL)

            0.03




           0.025




            0.02




           0.015




            0.01




           0.005



         Growth Rate (cm/day)
                   0

                          Coney Alone                  LC Coney                   LC Lionfish               Lionfish Alone


          -0.005




 Figure 1: Average Length growth rate of experimental treatments: (Left to Right) the single coney grouper alone, the coney
 grouper from the single lionfish + single coney treatment, the lionfish from the single lionfish + single coney treatment, and
 the single lionfish alone. Error bars represent +/- one standard deviation in average length.



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    acclimated to their tanks and were obviously hun-           Medical Institute provided me with the opportunity
    gry (i.e. they readily ate live native prey fish) but did   to work side-by-side with experts to understand one
    not show any interest, whatsoever in the lionfish that      of the world’s emergent ecological problems. I en-
    were placed in their tanks. Native predators do not         joyed the chance to literally get my feet wet every
    seem to recognize lionfish as a potential prey item.        day in an intensive fieldwork season. Now I can
         The competition experiment demonstrated that           make informed decisions about the direction I would
    lionfish consumed significantly more prey than              like to take with my education and my career.
    similarly sized coney across all treatments (t=-1.88,
    p=0.08), and that lionfish grew at a faster rate than       References
    similarly sized coney grouper held under identical          1. Courtenay WR (1995) Marine fish introductions
    conditions across all treatments (t=2.71, p=0.014)               in southeastern Florida. Am Fish Soc Introd Fish
    (Fig. 1). Coney grouper alone grew slightly faster,              Sect Newsl 14:2-3
    on average, than coney in the presence of lionfish,         2. Hamner RM, Freshwater DW, Whitfield PE (2007)
    although this difference was not statistically sig-              Mitochondrial cytochrome b analysis reveals two
    nificant (t = 0.08, p= 0.935). While the difference              invasive lionfish species with strong founder ef-
    was not significant, this trend lends support to the             fects in the western Atlantic. J Fish Biol 71:214-
    hypothesis that lionfish compete with coney. In the              222
    presence of lionfish, coney ate fewer mosquito fish         3. Hare JA, Whitfield PE (2003) An integrated as-
    per feeding than the coney without lionfish. Again,              sessment of the introduction of lionfish (Pterois
    this difference was not statistically significant (t=            volitans/miles complex) to the western Atlantic
    1.48, p=0.563), although the trend may help explain              Ocean. NOAA Tech Memo NOS NCCOS 2:1-
    the decreased growth rates observed for coney with               21
    lionfish.                                                   4. Snyder D, Burgess G (2007) The Indo-Pacific red
                                                                     lionfish, Pterois volitans (Pisces: Scorpaenidae),
    Conclusion                                                       new to Bahamian ichthyofauna. Coral Reefs
         The introduction of lionfish to the Caribbean may           26:175
    have serious implications for the health of native cor-     5. Whitfield PE, Hare JA, David AW, Harter SL,
    al reef ecosystems. The high growth rate observed                Muñoz RC, Addison CM (2007) Abundance es-
    for lionfish suggests they not only grow larger faster           timates of the Indo-Pacific lionfish Pterois voli-
    than native predators, but may increase predation on             tans/miles complex in the Western North Atlan-
    prey populations. Lionfish may increase their con-               tic. Biol Invasions 9:53
    sumption rates more quickly than native predators           6. Schofield, P. J., J. N. Langston and P. L. Fuller.
    in the same age class. Lionfish may even grow fast               2009. Pterois volitans/miles. USGS Nonindige-
    enough to prey upon native species in the same age               nous Aquatic Species Database, Gainesville, FL.
    class, impacting a single cohort of native predator         < h t t p : / / n a s . e r. u s g s . g o v / q u e r i e s / F a c t S h e e t .
    fishes as first a competitor and later, as a predator.           asp?speciesID=963> Revision Date: 2/27/2009
    In addition to faster growth and higher consumption         7. Albins MA, Hixon MA (2008) Invasive Indo-
    rates, my experiments demonstrated that lionfish are             Pacific lionfish (Pterois volitans) reduce recruit-
    extremely resilient and resistant to stress. Only one            ment of Atlantic coral-reef fishes. Marine Ecol-
    single lionfish died during the entire process of col-           ogy Progress Series 367: 233–238
    lection, transport, and feeding trials. With an appar-      8. Grosholz ED, Ruiz GM, Dean CA, Shirley KA,
    ent lack of predators in the introduced range, resource          Maron JL, Connors PG (1999) The impacts of
    availability remains one of the few limiting factors in          a nonindigenous marine predator in a California
    the growth of lionfish populations. Complete eradi-              bay. Ecology 81:5
    cation is unlikely now that lionfish are numerous and       9. Maljkovic A, Van Leeuwen TE, Cove SN (2008)
    widespread. It will be important to monitor the eco-             Predation on the invasive red lionfish, Pterois
    logical impacts in the region in order to determine              volitans (Pisces: Scorpaenidae), by native grou-
    the full effect of this invasive predator.                       pers in the Bahamas. Coral Reefs DOI 10.1007/
         The funding provided by the Howard Hughes                   s00338-008-0372-9

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         Sources of Variability in the Duration of Anesthesia in Snakes
                                  Daniel Preston1, Craig Mosley2 and Robert Mason1
                                   1Department    of Zoology, 2College of Veterinary Medicine
                                                      Oregon State University

    Introduction                                                   physiological parameters may influence the duration
         Surgical procedures requiring anesthesia are of-          of anesthesia in snakes. Separate experiments were
    ten necessary to study aspects of reptilian anatomy,           conducted to asses the effects of 1) body temperature,
    physiology and behavior [e.g. 1,2]. An ideal anes-             2) body condition, 3) gravidity, and 4) time post-
    thetic agent should take effect rapidly, create a suf-         feeding on duration of brevital sodium anesthesia in
    ficient depth of anesthesia for the desired procedure,         Thamnophis sirtalis parietalis.
    and allow reasonably fast and predictable recovery
    after surgery has ended.                                       Methods
         Brevital sodium (methohexital sodium) is a bar-                       Thirty male and 45 female T. s. parietalis were
    biturate anesthetic that has been used in numerous                   collected from over-wintering hibernacula near In-
    reptile taxa [3]. Research conducted by Mason and                    wood, Manitoba during May of 2007 and 2008. Gar-
    collaborators has utilized brevital sodium in the field              ter snakes were transported to Oregon State Univer-
    and laboratory for over 20 years to anesthetize red-                 sity and maintained in a microprocessor controlled
    sided garter snakes (Thamnophis sirtalis parietalis)                 environmental chamber. During the summer months,
    prior to surgical procedures. While brevital sodium                  snakes were fed an alternating diet of earthworms
    is an effective injectable agent in T .s. parietalis, a              and trout weekly. Water was provided ad libitum.
    high degree of variability in the duration of anesthe-                     One hour prior to anesthesia, snakes were re-
    sia has been observed between individuals.                           moved from the environmental chamber and placed
         Identifying sources of variability in the duration              individually into ten gallon glass aquaria lined with
    of anesthesia in reptiles will allow the improvement                 newspaper. Brevital sodium (methohexital sodium,
    of anesthetic protocols and will increase understand-                JHP Pharmaceuticals, Parsippany, NJ) was diluted to
    ing of the mechanisms that terminate the action of                   a 0.5% solution (5 mg/ml) in deionized water and
    anesthetics in reptiles. We hypothesized that several                                              was stored at 4°C.
                                                                                                       Dosages of 15 mg/kg
                                                                                                       (equivalent to 0.003
                                                                                                       ml of 0.5% brevital
                                                                                                       sodium/g body mass)
                                                                                                       were administered sub-
                                                                                                       cutaneously between
                                                                                                       the dorsal and ventral
                                                                                                       scales at a distance
                                                                                                       approximately 20 cm
                                                                                                       from the head of the
                                                                                                       snake. Quantifying the
                                                                                                       duration of anesthesia
                                                                                                       was accomplished by
                                                                                                       measuring the time af-
                                                                                                       ter anesthetic adminis-
                                                                                                       tration at which each
                                                                                                       snake regained its
        Figure 1: Mean time to righting ability (+/- SE) for T. s. parietalis (n =20 per temperature,  ability to right its en-
        repeated measures) at 21°C, 26°C and 31°C.                                                     tire body when placed


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                                                     The
                                                        Catalyst
                                                                                                      increased, times to right-
                                                                                                      ing ability decreased
                                                                                                      (Figure 2, r2 = 0.252,
                                                                                                      P<0.001). Differences
                                                                                                      in time to righting abili-
                                                                                                      ty were statistically sig-
                                                                                                      nificant between body
                                                                                                      condition groups (Fig-
                                                                                                      ure 3, ANOVA, F2,42
                                                                                                      = 5.026, P = 0.011).
                                                                                                      Thin snakes regained
                                                                                                      righting ability 60%
                                                                                                      more slowly on average
                                                                                                      than fat snakes (Figure
                                                                                                      3, posthoc Tukey, q =
     Figure 2: Time to righting ability versus mass/SVL ratio of T. s. parietalis. Squares represent  4.482, P = 0.008). In the
     snakes in the thin group, circles represent snakes in the medium group and triangles represent   gravidity experiment,
     snakes in the fat group (n = 15 per group).                                                      there was a statistically
                                                                                                      significant effect of gra-
    on its back (hereafter called righting ability).                                                  vidity overall (Figure 4,
         For the body temperature experiment, twenty ANOVA, F1,3 = 6.241, P = 0.047) but not of time
    male T. s. parietalis were anesthetized at body tem-                or time and gravidity. Within the month of August,
    peratures of 21°C, 26°C and 31°C in a repeated mea-                 gravid snakes took twice as long to recover as non-
    sures experimental design. In the body condition                    gravid snakes (Figure 4, posthoc Tukey, q = 4.439, P
    experiment, forty five female T. s. parietalis were = 0.009). In the feeding experiment, snakes ate 31%
    anesthetized after being divided into three groups                  of their body mass in food during the single feeding
    based on body condition (mass/SVL ratio was used                    event (Figure 5). There was not a statistically signifi-
    to quantify body condition). Of the 45 female T. s.                 cant difference between the time to righting ability
    partietalis that were used in the body condition ex-                on day 1, 3 or 10 (Figure 6, ANOVA, F2,17 = 3.091,
    periment, four became gravid after mating in the P = 0.057).
    wild prior to collection. The four gravid females and
    four nongravid females of similar size were anesthe-
    tized in June and again in August to test for effects               Discussion
                                                                             The shortened duration of sodium brevital anes-
    of gravidity. Lastly, ten male T. s. parietalis and ten
                                                                        thesia at higher temperatures in garter snakes is con-
    female T. s. parietalis were anesthetized one, three
    and ten days after feeding in a repeated measures sistent with previous reports in lizards anesthetized
    experimental design. The dosage of sodium brevital with ketamine [4] and isoflurane gas [5]. Elevated
    administered to each snake was calculated based on body temperatures likely reduce duration of anesthe-
                                                                        sia in all reptile species, whether inhalant or inject-
    the snake’s mass one day prior to feeding.
                                                                        able anesthetics are used. The respiration and heart
                                                                        rates of reptiles increase with body temperatures [6],
    Results                                                             and this likely increases rates of distribution and me-
         Higher temperatures resulted in shorter times tabolism of injectable anesthetics and decreases their
    to righting ability (Fig.1, ANOVA, F2,17 = 12.71, duration of action.
    P<0.001 ). The mean time to righting ability at 31°C                     On average, thinner garter snakes took longer
    was nearly twice as high (half as high?) as 21°C (Fig-              to recover from anesthesia than heavier snakes. In
    ure 1, posthoc Tukey, q = 7.129, P<0.001) and the                   mammals, barbiturate anesthetics are known to dis-
    difference in mean times to righting ability was sta-               tribute sequentially from the blood pool into viscera,
    tistically significant between all three temperatures. lean tissue and adipose tissue and are metabolized
    In the body condition experiment, as mass/SVL ratio hepatically [7]. It is largely redistribution from blood


8   Volume 3                                                                                                                       7
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                                                              Catalyst

                                                                                                           the increased time to
                                                                                                           righting ability ob-
                                                                                                           served in this group.
                                                                                                           A similar increased
                                                                                                           effect of brevital sodi-
                                                                                                           um has been anecdot-
                                                                                                           ally observed in grav-
                                                                                                           id crotalines [11].
                                                                                                                 A     statistically
                                                                                                           significant effect of
                                                                                                           time post-feeding on
                                                                                                           duration of anesthesia
                                                                                                           was not detected de-
                                                                                                           spite the considerable
                                                                                                           physiological chang-
                                                                                                           es, such as increased
                                                                                                           liver size, that snakes
                                                                                                           undergo during diges-
    Figure 3: Mean time to righting ability (+/- SE) of T. s. parietalis in three body condition groups (n tion [12]. This finding
    = 15 per group). Body condition groups are based on mass/SVL ratios.                                   supports the idea that
                                                                                                           it is primarily redistri-
    into other tissues that terminates the action of the                                                   bution from the blood
    anesthetic [8]. The shorter duration of anesthesia in pool that terminates the action of the anesthetic, as
    fatter snakes may be the result of a greater percent- opposed to hepatic metabolism [8].
    age of the dosage becoming bound in adipose tissue                          In order to produce more consistent durations of
    during the early distribution of the drug.                            anesthesia between individual garter snakes, a linear
         The longer time
    to righting ability ob-
    served in gravid snakes
    compared to nongravid
    snakes in August may
    also be the result of dif-
    ferences in body com-
    position. Gravid garter
    snakes may be anorexic
    shortly before giving
    birth [9] and viviparous
    snakes are often emaci-
    ated after parturition due
    to the high energetic cost
    of reproduction [10]. By
    August, it is likely that
    the gravid females had
    burned much of their
    adipose tissue to meet
    the energetic demands
    of developing offspring Figure 4: Mean time to righting ability (+/- SE) of gravid and nongravid snakes in June and
    and this reduction in ad- August (n = 4 per group). All gravid snakes gave birth within five weeks of being anesthetized
                                   in August.
    ipose tissue may cause


8                                                                                                                     Volume 3         9
                                                          The
                                                             Catalyst

     equation to predict
     the dosage for surgi-
     cal anesthesia based
     on body temperature
     and body condition
     was created:

         Dose = 5.2 + 0.4T
     + 2.4(M/SVL),

          where dose is
     in mg/kg, T is body
     temperature in de-
     grees Celsius, M is
     mass in grams and
     SVL is snout-vent
     length in cm. The
     dosages predicted by Figure 5: Relative body masses (+/- SE) of T. s. parietalis (n = 20) over 12 days. Snakes were
     this equation were fed on day zero. Yellow points indicate days when snakes were anesthetized. Snakes were given
     tested in 30 snakes dosages of anesthetic based on their mass one day prior to feeding.
     ranging in mass/SVL
     ratio from 0.7 to 1.8                                     perature, body condition and gravidity affect the du-
     and at temperatures of 21°C to 29°C. Results of these ration of anesthesia with sodium brevital in snakes.
     trials suggest that the equation predicts safe dosages    Other factors undoubtedly play a role in creating
     that produce more consistent durations of anesthesia variability and future research, such as pharmacoki-
     than using 15 mg/kg in all snakes regardless of body      netic studies, will allow the further improvement of
     temperature or body condition.                            anesthetic protocols and a better understanding of the
          Of the factors evaluated in this study, body tem-    physiological processes that terminate the duration
                                                                                                    of action of anesthetics
                                                                                                    in reptiles.

                                                                                                         References
                                                                                                         1. Blouin-Demers, G.,
                                                                                                         Weatherhead, P.J., Shil-
                                                                                                         ton, C.M., Parent, C.E.
                                                                                                         and G.P. Brown. 2000.
                                                                                                         Use of inhalant anese-
                                                                                                         thetics in three snake
                                                                                                         species. Contemporary
                                                                                                         Herpetology 2000.
                                                                                                         2. Krohmer, R.W.,
                                                                                                         Martinez, D. and R.T.
                                                                                                         Mason. 2004. Develop-
                                                                                                         ment of the renal sex-
                                                                                                         ual segment in imma-
                                                                                                         ture snakes: Effect of
     Figure 6: Mean time to righting ability (+/- SE) of T. s. parietalis one, three and ten days post   sex steroid hormones.
     feeding (n=20 per group, repeated measures).                                                        Comparative Biochem-


10   Volume 3                                                                                                                       9
                                                     The
                                                        Catalyst
         istry and Physiology 139:55-64.                      9. Gregory, P.T., Crampton, L.H. and K.M. Skebo.
     3. Wang, R.T., Kubie, J.L. and M. Halpern. 1977.             1999. Conflicts and interaction among reproduc-
         Brevital sodium: An effective anesthetic agent           tion, thermoregulation and feeding in viviparous
         for performing surgery on small reptiles. Copeia         reptiles: are gravid snakes anorexic? Journal of
         1977:738-743.                                            Zoology 248:231-241.
     4. Arena, P.C., Richardson, K.C. and L.K. Cullen.        10. Madsen, T. and R. Shine. 1993. Costs of re-
         1988. Anaesthesia in two species of large Austra-        production in a population of European adders.
         lian skink. The Veterinary Record 123:155-158.           Oecologia 94: 488-495.
     5. Dohm, L. and D. Brunson. 1998. Effective dose of      11. Miller, L.R. and W.H.N. Gutzke. 1998. Sodium
         isoflurane for the desert iguana                         brevital as an anesthetizing agent for crotalines.
     (Dipsosaurus dorsalis) and the effect of hypothermia         Herpetological Review 29:16.
         on effective dose. Proceedings American College      12. Starck, J.M. and K. Beese. 2002. Structural flex-
         of Veterinary Anesthetists 1998:543.                     ibility of the small intestine and liver of garter
     6. Greenwald, O.E. 1971. The effect of body tem-             snakes in response to feeding and fasting. The
         perature on oxygen consumption and heart rate            Journal of Experimental Biology 205:1377-
         in the Sonora gopher snake, Pituophis catenifer          1388.
         affinis Hallowell. Copeia 1971: 98-106.
     7. Bickel, M.H. 1984. The role of adipose tissue in      Acknowledgements
         the distribution and storage of drugs. Progress in        M. Parker and C. Friesen provided assistance
         Drug Research 28:273-303.                            with most aspects of this study. Funding for this
     8. Hudson, R.J., Stanski, D.R. and P.G. Burch. 1983.     project has come from a Howard Hughes Medical
         Pharmacokinetics of methohexital and thiopen-        Institute Fellowship to D. Preston, a National Sci-
         tal in surgical patients. Anesthesiology 59: 215-    ence Foundation Grant to R. Mason and College of
         219.                                                 Veterinary Medicine Research Funds to C. Mosley.




10                                                                                                     Volume 3        11
                                                         The
                                                            Catalyst

                          Detection of Leaching Organic Migrants from
                                 Polyethylene Terephthalate and
                                  Polycarbonate Water Bottles
                                            Paul Dornath and Dr. Skip Rochefort
                            School of Chemical, Biological, and Environmental Engineering (CBEE)
                                                   Oregon State University

     Abstract: In recent years, various plastics have been criticized for their safety due to chemicals that could leach from the
     material. Many studies show conflicting results between the toxicity of the chemicals and the actual concentrations of these
     chemicals in the material. The goal of this study was to identify organic migrants from two types of commodity plastic: poly-
     ethylene terephthalate (PETE) and polycarbonate (PC). Both plastics were exposed to unique conditions. The study on PETE
     aimed to determine the effect of sunlight on the polyester backbone. New, unopened water bottles were left on the southern
     roof of a building for two months, July through August. The goal of this study was to identify any organic fragments and
     plasticizers that may release from the plastic into the water. The study on the PC bottles was aimed at determining the extent
     of bisphenol-A (BPA) leaching when PC is exposed to extreme heat conditions. New, unwashed PC water bottles were filled
     with water and then autoclaved at 121 degrees Celsius at two bars of pressure. Since both studies involve detecting organic,
     aromatic molecules, the same organic solid phase extraction, concentration and detection methods were used for both plastics.
     After running tests using a gas chromatograph/mass spectrometer system (GC/MS), results were compiled for each plastic. In
     the case of PETE bottles, no significant concentrations of any one backbone fragment were found. The only significant find
     was a small amount of di-(2-ethylhexyl) phthalate (DEHP, a common plasticizer used to soften plastics) in an unknown con-
     centration. In the case of PC, standard solutions of known concentration of BPA were run through the extraction, concentration
     and detection processes. After comparing these control tests to the samples taken with from the autoclave, the amount of BPA
     leaching from the PC bottles was found to be between 1 to 10 ppb. There is a series of tests waiting to be run to show the exact,
     statistically accurate average amount of BPA leaching, but the current results seem to indicate that the levels are well below
     those for toxicity.

     Background                                                         ing into the body are usually toxic, and it is impor-
          Both polyethylene terephthalate (PETE) (Figure                tant to know the identity and concentration of these
     1) and polycarbonate (PC) (Figure 2) are plastics                  molecules, if they exist, before this process is to be
     that contain aromatic esters in their backbones. In                considered safe.
     recent years, rural citizens in India and other devel-                  Nalgene and Camelback, and other producers of
     oping nations have been using a process called so-                 polycarbonate water bottles have come under heavy
     lar disinfection. Solar disinfection uses PETE water               criticism from the press and public because of the po-
     bottles left out in the sun (usually on corrugated alu-            tential for leaching of a chemical called bisphenol-A
     minum roofing) using the UV light from the sun to                  from their bottles. Bisphenol-A is one of two mono-
     kill microbes found in the water. Some studies have                mers that combine to form the backbone of polycar-
     shown, however, these bottles can break down due                   bonate. According to a study done at the Washington
     to a process called                                                State University from 1999 to 2003 by Patrica Hunt
     Photo-Fries rear-                                                                                   entitled: Bisphenol
     rangement (Fig-                                                                                      A Causes Meiotic
     ure 3). This rear-                                                                                   aneuploidy in the
     rangement causes                                                                                     female mouse, mice
     a        photolytic                                                                                  housed in cages
     splicing of the                                                                                      made out of poly-
     backbone into an                                                                                     carbonate experi-
     aromatic organic                                                                                     enced     endocrine
     fragment and ac-                                                                                     cycle problems due
                                       Figure 1:                                                          to BPA they ingest-
     etaldehyde. Un-                 Polyethylene                                     Figure 2:
     checked aromatic                                                             Polycarbonate           ed after being ex-
                                     Terephthalate
     molecules enter-

12   Volume 3
                                                                                                                                         11
                                                       The
                                                          Catalyst

                                                                      times, and was ready to be analyzed by GC/
                                                                      MS. The GC in this experiment also uses a
                                                                      silica fused C8 column. 1 microliter of liquid
                                                                      sample is pushed through the gas chromato-
                                                                      graph (GC) to determine peak height and re-
                                                                      tention time for certain compounds. The com-
                     Figure3: Photo-Fries Rearrangement               pounds are then fed to the mass spectrometer
      Figure 3: Photo-Fries Rearrangement
                                                                      (MS) to determine the identity of the molecules
     posed to the polycarbonate cages and food contain-               and match them to the GC peaks.
     ers. Since then, many studies have shown that BPA
     itself is harmful at relatively low levels, especially     Results
     for younger children in developmental stages. How-              PETE Water Bottles: No significant backbone
     ever, few studies have focused on the amount of BPA        fragments of PETE were identified. There were,
     that is actually released from ordinary polycarbon-        however, some GC peaks that the MS was not able
     ate water bottles and the minimum exposure limits          to identify, and these peaks could need further study
     needed to cause harm to a human being. EPA’s mini-         to show if they are the fingerprints of harmful mol-
     mum hazardous exposure of BPA is 0.05 mg/kg/day.           ecules. The only aromatic molecule found was a
     This means that an 80 kg person (180 pounds) would         small amount of di-(2-ethylhexyl) phthalate (DEHP,
     have to drink 1 liter of water with a concentration of     a common plasticizer used to soften plastics) in an
     4 ppm per day to experience any harmful effects.           unknown concentration (Figure 4). DEHP was iden-
                                                                tified to be present with a 91 percent confidence, but
     Methods                                                    there wasn’t sufficient time or resources to develop
          The process starts with the exposure of the water     a calibration curve for DEHP since it was a surprise
     inside the bottles to their respective conditions. New,    result to find this molecule leaching from PETE wa-
     unopened PETE bottles are left on the roof for a pe-       ter bottles.
     riod of two summer months (July – August). These                PC Water Bottles: The study of bisphenol-A
     bottles are being exposed to UV light from the sun.        leaching from polycarbonate yielded significant re-
     New, unwashed PC bottles were filled with water            sults. Solutions with known concentrations of BPA
     and put into an autoclave at 121 degrees Celsius at 2      were run through the same extraction, concentration
     bars of pressure for 25 minutes, and allowed to cool       and detection processes. The water bottles were run
     inside the autoclave for 30 minutes. The bottles were      through the autoclave with two sets of water each.
     then quench cooled in ice.                                 Both the first and second pass showed concentrations
          The organic extraction, concentration and detec-      of BPA between 1 and 10 parts per billion (ppb). The
     tion method is the same for both PC and PETE. The          bisphenol-A was identified to 93 percent confidence
     process starts with one liter of water that have been      (figure 5).
     exposed with the plastic. Silica bonded C8 reverse
     phase solid phase extraction (SPE) filters were used       Conclusion
     to extract the organic fragments from the water. After          After exposing PETE bottles to sunlight for two
     the filters had been partially dried, 3 ml of the strong   summer months, no significant amounts of known ar-
     organic solvent dichloromethane (DCM) are pushed           omatic molecules were found. Only a small amount
     through the filter, removing the organic compounds         of DEHP of unknown concentration was identified.
     by dissolving them in DCM. Dry sodium sulfate salt         An alternative experiment to determine the extent of
     is added to this 3 ml solution to precipitate any re-      degradation would be to look for the molecule ac-
     maining water out of solution. The remaining DCM           etaldehyde. This would require a more complicated
     is removed, placed into a clean container and placed       method, as acetaldehyde is very soluble in water and
     under a stream of nitrogen to evaporate the solution       is smaller than the DCM solvent, making it impossi-
     down to 200 microliters.                                   ble to identify in trace concentrations in the GC/MS.
          The solution at that point was concentrated 5000      In the case of PC bottles, concentrations of 1 to 10


12                                                                                                       Volume 3
                                                                                                                         13
                                                      The
                                                    Catalyst
                          Figure 4: Mass Spectrometry (MS), Gas Chromatography
                                           (GC) results for PETE




         MS: Identified DEHP to 91% confidence in 2              GC: DEHP of unknown concentration
                month exposure water sample                             highlighted in red
     Figure 4: Mass Spectrometry (MS), Gas Chromatography (GC) results for PETE


     PPB were identified. According to the EPA standard,            Acknowledgements
     BPA exposure limit of 0.05 mg/kg/day, one would This study would not have been possible without the
     have to drink over 1,000 liters of this water a day to
                                                     generous support from the Howard Hughes Medical
     see any harmful effects. It appears that polycarbon-
                                                     Institute and coordinator head Dr. Kevin Ahern, Dr.
     ate is safe for consumer use. More tests need to be
                                                     Skip Rochefort, Dr. Mohammad Azizian and Brian
     run on PETE to reach a definite conclusion.     Maloney of OSU’s CBEE department, Moey Hand-
                                                     loser of the Apprenticeships in Science and Engineer-
                                                     ing, and Kristy Edwards and Greg Jones of OSU’s
                     Figure 5: Mass Spectrometry (MS), Gas
                                                     Chemistry Department.
                 Chromatography (GC) results for PC water bottles




        Control results at a concentration            BPA leaching levels highlighted in
          of 10 ppb (parts per billion).              red are well below the level of the
                                                      10ppb autoclaved control samples.

      Figure 5: Mass Spectrometry (MS), Gas Chromatography (GC) results for PC water bottles



14   Volume 3
                                                The
                                                   Catalyst

   Testing the Role of Conserved Genes in Pollen Development
                               Luisa Snyder, Rex Cole, and John Fowler
                                  Department of Botany and Plant Pathology
                                          Oregon State University

Introduction
     Pollen is important in plant reproduction as it
is indispensible for the process of sexual reproduc-
tion. Plants are non-motile, so pollen allows them
to exchange gametes across distances allowing for
sexuality-associated genetic variation. Pollen, across
angiosperms (flowering plants), shows many dif-
ferences including variance in their shape and their
ability to survive in extreme environments. Howev-
er, there are also many similarities in pollen shared
among all angiosperms. Examples of such similari-
ties include strong cell walls and the development       Figure 1
of a pollen tube upon germination. We know that
biological functions are due to genes that code for
proteins involved in a specific pathway. If all angio-   of Putative Orthologous Group (POGs), as defined
sperms share similar functions in their pollen, we can   by the group of Dr. Alice Barkan at the University
hypothesize that they share a set of genes respon-       of Oregon (Walker et al., 2007, and http://plantrbp.
sible for these. We call this set of shared genes the    uoregon.edu/index.php); they were highly expressed
“Core Pollen Transcriptome” as it represents the set     in pollen of Arabidopsis, rice, and maize; a mutant
of genes transcribed in all pollen.                      version of the genes was available in Arabidopsis;
                                                         and no duplicate gene was present in the Arabidopsis
                                                         genome.
Objective
                                                              After finding four genes that met the criteria
     The objective of this research was to identify
                                                         mentioned above, seeds that contained the mutant
genes involved in pollen function and development.
                                                         version of the list of the candidate genes were ob-
The hypothesis of this project was that genes in the
                                                         tained from the Salk collection of T-DNA insertion
Core Pollen Transcriptome provide important func-
                                                         mutants (Alonso et al., 2003). The plants were geno-
tions in all angiosperm pollen. The predictions are as
                                                         typed using Polymerase Chain Reaction (PCR), and
follow:
                                                         gel electrophoresis, and heterozygous plants contain-
                                                         ing a copy of the mutant allele and one copy of the
   1. The genes in the pollen core set should be
                                                         wild type allele were found.
      conserved and highly expressed in pollen
                                                              Two different sets of experiments were performed
      across Arabidopsis, rice, and maize (i.e., in
                                                         during this research project: The first experiments
      both dicots and monocots).
                                                         were done to determine if the presence of mutation in
   2. Mutation in the genes in the pollen core set
                                                         the chosen genes would cause a transmission defect,
      should cause a pollen defect.
                                                         which is the inability for a specific trait to be passed
                                                         onto the next generation, and the second experiments
Methodology                                              were done to determine if this same mutation in the
    The plant that was used in the research this sum-    chosen genes would cause a phenotypic defect. It
mer was Arabidopsis thaliana. The first step taken       is important to keep in mind that pollen is haploid.
was to find a group of genes that were possible can-     Therefore, mutant effects are shown in 50% of pollen
didates to be part of the pollen core set. Each candi-   derived from a mutant heterozygote. The first sets
date met the following criteria: They were all part      of experiments performed involved the use of the


                                                                                                    Volume 3        15
                                                The
                                                   Catalyst

                                                                 observed in the progeny. This is due to the fact that if
                                                                 the mutated gene is important for pollen function as
                                                                 hypothesized, the pollen that possesses the mutation
                                                                 will not be as competitive as the wild-type pollen,
                                                                 thus reducing its ability to produce progeny.
                                                                     The second sets of experiments were performed
                                                                 using pollen germination media and a light micro-
                                                                 scope to observe the pollen grains from heterozygous
                                                                 plants with the mutation in chosen genes. Because the
       Figure 2                                                  quartet1 mutant was also used in all of these lines,
                                                                 pollen was released in connected sets of four pollen
     dissecting microscope to emasculate heterozygous            grains, each with two mutant pollen grains and two
     mutant and homozygous wild-type flowers, in order           wild-type pollen grains (Johnson-Brousseau and Mc-
     to reciprocally cross them with each other’s pollen.        Cormick, 2004). Thus, a phenotypic effect due to
     Reciprocal genetic crosses between heterozygous             the mutation would be evident in two out of the four
     plants and wild type plants were performed. When            pollen grains in a quartet.

                                                                 Results
                                                                      The results obtained from these two sets of ex-
                                                                 periments did not prove or disprove the hypothesis.
                                                                 First, when the progeny that resulted from the recip-
                                                                 rocal crosses were processed, there was no pollen-
                                                                 associated transmission defect found as the result of
                                                                 the mutation in any of the four chosen genes.
                                                                      Figure 4 is an example of my results, showing a
       Figure 3                                                  1:1 ratio from the PCR done on progeny segregating
                                                                 for a mutation in the At1g64110 gene. These results
                                                                 were consistent throughout most of the chosen genes.
     a flower is emasculated, the anthers are removed,           The exception to these results was found in the fam-
     to avoid the presence of the flower’s own pollen.           ily carrying a mutant in At5g17290, which encodes
     After the heterozygous flowers were crossed with            the autophagy protein ATG5. When PCR was used
     wild-type pollen, and the wild-type flowers were            to genotype the progeny obtained from the recipro-
     crossed with the same heterozygous plant’s pollen,          cal cross between the heterozygous stigma and the
     the plants were covered to avoid cross pollination.         wild-type pollen, a nine mutant to twenty wild-type
     After about sixteen days, the siliques were elongat-        ratio was found, which has a χ2 value of 4.17 and a
     ed and mature enough for the seeds to be collected.         p-value greater than 0.05, making these results statis-
     In order to determine if a transmission defect was          tically significant. These results were unexpected be-
     present, the seeds that were collected from the
     reciprocal crosses were planted and the plants
     were genotyped. As Figure 2 shows, when a
     transmission defect is not present, a one to one
     ratio of homozygous wild-type and heterozy-
     gous mutant plants in the progeny would be
     observed. This is because there would not be
     any problem passing on these genotypes to the
     next generation. In contrast, if a transmission
     defect is present, as observed in Figure 3, a
     higher number of wildtype plants would be
                                                      Figure 4

16   Volume 3
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                                                       The
                                                          Catalyst

                                                                on the results of these experiments, we conclude that
                                                                the four candidate genes that were chosen for this
                                                                project do not show either a transmission defect or a
                                                                phenotypic defect. A possible explanation for these
                                                                results is that, in contrast to our original prediction,
                                                                each plant had duplicate genes were also highly ex-
                                                                pressed in Arabidopsis pollen. This could have af-
                                                                fected the results, because the duplicate gene could
                                                                be accomplishing the functions that the mutated gene
      Figure 5                                                  cannot accomplish.
                                                                     Future research will include the testing of more
                                                                candidate genes to be part of the pollen core set. In
     cause the chosen genes were hypothesized to have           addition, the gene that resulted in the female specific
     an effect in pollen, and not an effect in the stigma       transmission defect will be further studied by repeat-
     of a plant containing the mutation in these genes.         ing these experiments and confirming that the results
     Second, when the pollen grains were observed under         are consistent across multiple generations.
     the light microscope in order to determine if a phe-
     notypic defect was caused by these mutations, there
     was no difference found in the pollen quartet grains       References
     or in the development of a pollen tube after germina-      1. Alonso, J.M. et al. (2003). Genome-wide inser-
     tion (Figure 5).                                               tional mutagenesis of Arabidopsis thaliana.
                                                                    Science 301: 653-7
                                                                2. Johnson-Brousseau, S.A. and McCormick, S.
     Discussion & Conclusion                                        (2004). A compendium of methods useful for
         As the importance of pollen has become more                characterizing Arabidopsis pollen mutants and
     evident in recent years, it is important that scientists       gametophytically-expressed genes. Plant J. 39:
     know and understand the processes that explain the             761-75.
     way pollination works and functions. The objective         3. Walker, N., Stiffler, N. and Barkan, A. (2007).
     for this research project was to identify genes in-            POGs/PlantRBP: a resource for comparative ge-
     volved in pollen function and development. Based               nomics in plants. Nucleic Acids Res. 35: D852-
                                                                    D856.




16                                                                                                         Volume 3
                                                                                                                           17
                                                     The
                                                       Catalyst

      The Characterization of Oxytocin Receptors on Corpus Luteum
                            Endothelial Cells
                                  Kathryn McHenry and Dr. Fredrick Stormshak
                                        Department of Animal Sciences
                                           Oregon State University
     Introduction
          The reproductive cycle of nonpri-
     mate mammalian species is referred to
     as the estrous cycle. In the cow, the es-
     trous cycle is 21 days long with day 0
     being designated as estrus. Estrus is the
     only phase during the estrous cycle when
     the cow is receptive to mating. During
     this 12-15 hour window, there is a re-
     lease of gonadotropin releasing hormone
     (GnRH) from the hypothalamus, which
     acts on the anterior pituitary to stimulate
     a surge release of luteinizing hormone
     (LH). Luteinizing hormone acts on ma-
     ture ovarian follicles to cause ovulation,
     or the release of an ovum. If mating Figure 1. The changes in an ovary during the bovine estrous cycle.
     occurs, sperm deposited in the anterior
     vagina by a bull is transported by con-                         ters the uterus where it will mature into a fully devel-
     tractions of the reproductive tract and enters into the oped calf to be born 283 days later.
     oviduct to await the arrival of the ovum. Fertilization              Following ovulation, the empty follicle begins
     occurs in the oviduct and the developing embryo en- to undergo angiogenesis, or the production of blood
                                                                                                vessels. This process re-
                                                                                                sults in the formation of
                                                                                                the corpus luteum (CL), a
                                                                                                temporary endocrine gland
                                                                                                (figure 1) made up of 50%
                                                                                                endothelial cells. Once
                                                                                                fully developed, the CL
                                                                                                releases hormones, such
                                                                                                as progesterone, that are
                                                                                                necessary for the uterus to
                                                                                                maintain an embryo.
                                                                                                     The hormone oxytocin
                                                                                                (OT) is normally released
                                                                                                as the result of childbirth
                                                                                                in mammals, causing uter-
                                                                                                ine contractions and stimu-
                                                                                                lating milk letdown. How-
                                                                                                ever, OT is also secreted
     Figure 2. The physical and hormonal changes during the bovine estrous cycle.               by the bovine CL in large
                                                                                                amounts, the reason for
                                                                                                which is unknown. Secre-

18   Volume 3
                                                                                                                                18
                                                        The
                                                           Catalyst
    tion of OT begins fol-
    lowing ovulation and
    continues until the CL
    is fully formed around
    day 7 or 8 of the es-
    trous cycle, at which
    point levels of the hor-
    mone decrease (figure
    2). It was hypothesized
    that oxytocin produced
    by the CL may act in a
    paracrine fashion to en-
    hance luteal angiogen-
    esis. An experiment was
    conducted to determine
    whether luteal endothe-
    lial cells were endowed
    with OT receptors.
                               Figure 3. The amount of tritium present in samples of endothelial cells and non-endothelial cells as
                               shown by six runs of the experiment. The first two runs, only endothelial cells were tested.
    Significance
         If oxytocin is the
    hormone causing an-                                            g and 105 x g respectively, resuspended in 1 ml of
    giogenesis to occur in the corpus luteum and the               0.1% BSA medium and filtered through a 53 micron
    hormonal receptors on the endothelial cells were               nylon mesh. Total cell quantity was determined us-
    blocked, the CL would not form. Without the CL,                ing a hemocytometer.
    secretion of progesterone would be inhibited thus                   Tosyl-activated magnetic beads were covalently
    preventing pregnancy and, consequently, acting as a            bound to Griffonia Bandeiraea Simplicifolia lectin
    form of birth control. Furthermore, the growth of tu-          1, a ligand with an affinity for endothelial cells. The
    mors is almost identical to that of the corpus luteum.         beads were added to the digested CL tissue in a ratio
    Again, if oxytocin receptors are causing angiogenesis          of 20 beads per steroidogenic luteal cell and incu-
    in the CL, then it is also possible that oxytocin may          bated for 25 minutes at room temperature. The sam-
    be causing angiogenesis to occur in tumors as well.            ple was placed next to a magnet and the supernatant
    The following research was designed to support the             containing the non-endothelial cells was removed
    hypothesis by showing that oxytocin was specifically           from the sample. Finally, the endothelial cells were
    binding to oxytocin receptors on endothelial cells of          removed from the beads using a fucose solution. The
    the CL.                                                        cells were recounted on a hemocytometer to ensure
                                                                   separation of endothelial and non-endothelial cells.
    Methods                                                             The endothelial and non-endothelial cells were
         Five heifers were checked for behavioral estrus           homogenized in order to lyze them and then centri-
    twice each day using a vasectimized bull. The corpus           fuged at 1000 x g in order to separate out the nucleus.
    luteum was removed via a trans-vaginal lutectomy               The remaining supernatant containing the cell mem-
    on day 8 of the cycle, when it would be at the peak            branes was then re-centrifuged at 100,000 x g in or-
    of its growth. Collagenase and DNAse were incu-                der to concentrate the sample and the membrane pro-
    bated with the tissue until it was fully dispersed. The        tein concentration of endothelial and non-endothelial
    cells were washed by centrifugation at 160 x g for             cells was determined using a spectrophotometer. The
    ten minutes at 4°C and removal of the supernatant.             two samples were diluted until in equal concentration
    Ten ml of 0.1% BSA medium were added to break                  and 100 µl of both membrane samples were added to
    up the pellet. The cells were washed again at 125 x            four test tubes each.


8                                                                                                                   Volume 3          19
                                                        The
                                                           Catalyst

                                                                                            of samples to which only [3H]-
                                                                                            oxytocin was added provided
                                                                                            an estimate of total binding of
                                                                                            ligand to the hormone recep-
                                                                                            tors. Radioactivity of samples
                                                                                            to which both labeled and un-
                                                                                            labeled ligand were added pro-
                                                                                            vided an estimate of nonspecific
                                                                                            binding of labeled oxytocin. The
                                                                                            difference in radioactivity be-
                                                                                            tween these samples represented
                                                                                            oxytocin specifically bound to
                                                                                            the oxytocin receptor.


                                                                                            Results
     Figure 4. The total amount of specifically bound oxytocin on endothelial and non-en-        The first two runs of the
     dothelial cells tested.                                                                experiment, only endothelial
         Membranes of both endothelial and non-en-                                          cells were tested and the results
     dothelial cells were then tested for oxytocin bind-               showed a slightly higher amount of tritium present in
     ing using tritium-labeled oxytocin ([3H]-oxytocin).               the sample of endothelial cells with only [3H]-oxyto-
     20 nM [3H]-oxytocin was added to half of the tubes                cin present, giving the impression that the hypothesis
     of both types of cell membranes. To the remaining                 was correct. However, subsequent studies included
     half of the membranes, the same amount of [3H]-                   samples of the non-endothelial luteal cells. In these
     oxytocin was added as well as a 200 fold excess of                studies, it was observed that non-endothelial cells
     a non-labeled oxytocin. After incubating the tubes at             actually had much more oxytocin specifically bind-
     room temperature for 60 minutes the unbound (free)                ing than in endothelial cells (Figures 3 and 4). These
     ligand was removed and the radioactivity determined               results, while disproving the hypothesis, suggest the
     by use of a liquid scintillation counter. Radioactivity           presence of oxytocin receptors on other cell types
                                                                       within the corpus luteum.




20   Volume 3
                                                                                                                                19
                                                      The
                                                         Catalyst
           An Investigation on the Toxicity of Aluminum in Zebrafish
                                 Carly Dougher, Megan Hirko, Dr. Joseph S. Beckman
                          Linus Pauling Institute, Department of Biochemistry and Biophysics
                                                Oregon State University

      Background                                               fare. It does this by acting as a strong adjuvant that
           Amyotrophic Lateral Sclerosis (ALS), more           stimulates a prolonged immune response, but ques-
      commonly known as Lou Gehrig’s Disease, is a fa-         tions remain about its other possible effects. In this
      tal neurodegenerative disease. This disease affects      investigation, the toxicity of the aluminum hydroxide
      individuals via motor neuron death, which leads to       component of the anthrax vaccination in zebrafish
      paralysis, muscle wasting, and eventual respiratory      was explored.
      failure. It is estimated that ALS affects two out of          Zebrafish are an efficient vertebrate model with
      every 100,000 individuals globally. At this point, the   many advantages for studying neurotoxicants. Ze-
      molecular pathology of ALS is not entirely under-        brafish are highly prolific and their small size and
      stood and there is no effective cure for the disease.    low maintenance cost allows for large sample sizes.
      Approximately ten percent of all cases of ALS are        The zebrafish genome is completely sequenced and
      familial with the remaining 90 percent being spo-        transgenic lines allow for in vivo analysis. Use of
      radic. Causes of the sporadic forms of ALS are not       a green fluorescent protein (GFP)-expressing trans-
      completely understood.                                   genic line allows for visualization of motor neuron
           The Gulf-War Illness (GWI) developed in Amer-       status without having to sacrifice the animal (Figure
      ican troops after the Persian Gulf War of the early      1). By using zebrafish as a model to determine alu-
      1990s. GWI typically has a late onset, appearing in      minum toxicity, the effects of aluminum on neurode-
      most of the effected troops years after the war. It      generation can rapidly be assessed. In investigating
      has been estimated that as many as 29 percent of de-     the toxicity of aluminum, the ultimate goal is to gain
      ployed veterans of the Gulf War have presented with      further insight into the pathways by which motor
      symptoms linked to GWI. This illness presented           neurodegenerative diseases (such as ALS, Parkin-
      with a wide range of symptoms including those asso-      son’s Disease, and Alzheimer’s Disease) progress.
      ciated with neurodegeneration. It is believed that the
      GWI contains an ALS component. Both deployed             Hypothesis
      and non-deployed veterans reportedly developed           Zebrafish injected with comparable amounts of
      such symptomology. Similar to the varying forms          aluminum hydroxide, Al(OH)3, to that found in the
      of sporadic ALS, the cause and pathology of this ill-    human anthrax vaccine, will exhibit signs of motor
      ness remains unknown. Many possible causes have
      been suggested ranging from environmental factors
      to vaccinations. One such suspect vaccine is the An-
      thrax Vaccine.
           The Anthrax Vaccine was one of many vaccina-
      tions administered to the US troops during the first
      Gulf War to protect against biological warfare. It is
      unclear whether this vaccination was beneficial to
      the troops as both its efficacy and safety have been
      in question ever since the war. The controversy sur-
      rounding the vaccine involves, but is not limited to,
      insufficient testing, possible impurities, likely ex-
      piration, and missing records. Recently, suspicion
      has risen around the vaccine’s aluminum adjuvant.
      The Anthrax vaccine has a notably high aluminum          Figure 1. Motor neuron of a GFP-expressing zebrafish imaged
      hydroxide content in order to make the vaccination       using an Axiovert 200M Zeiss microscope (Carl Zeiss International,
      more effective in protection against biological war-     Germany) with a 546 nm filter and AxioVision software (Carl Zeiss
                                                               International, Germany).


920                                                                                                                Volume 3         21
                                                   The
                                                      Catalyst

     neuron degeneration and neurotoxicity.                     following months and assessed based on behavioral
                                                                and physiological changes indicating signs of motor
     Materials and Methods                                      neuron degeneration. Secondary motor neurons
     For this investigation, three sets of fish were injected   of the GFP transgenic groups were also imaged
     to mimic both adult exposure to the vaccine (2 month       using an Axiovert 200M Zeiss microscope (Carl
     old non-transgenic fish) and adolescent exposure           Zeiss International, Germany) with a 546 nm filter
     (one set of 3 week old non-transgenic fish and one         and AxioVision software (Carl Zeiss International,
     set of 3 week old GFP expressing transgenic fish).         Germany).
     Each set of fish was divided into five groups of 10
     fish. Four groups within each set were assigned a          Results
     different concentration of Al(OH)3: 1X, 10X, 100X,         No changes or differences between groups in behavior
     and 1000X (1X = 1.186 x 10-5 mg Al / g fish). The          or physiology were observed. Discrepancies between
     fifth group remained a control group and was injected
     with PBS. The injections contained phenol red to
     allow for visualization of injection. Each fish was
     injected twice intramuscularly into the tailfin, and
     each injection was two days apart, again mimicking
     the anthrax vaccine. The time between injections
     was adjusted to accommodate the shorter lifespan of
     the fish. Each fish was anesthetized in tricaine water
     prior to injection.




                                                                    Figure 3. Injection of a 2 month old zebrafish.



                                                                motor neuron images of the transgenic line were not
                                                                found between groups.

                                                                Discussion
                                                                Although the fish used in this experiment have not
                                                                yet shown any adverse effects, the study has so far
                                                                lasted only 6 weeks post injections. An experiment
                                                                published in Neuromolecular Medicine in 2006
                                                                completed by Petrik, et al showed that mice injected
                                                                with comparable amounts of aluminum demonstrated
          Figure 2: Zeiss Imaging Microscope                    a significant decrease in strength after 10 weeks. It
                                                                is also possible that the zebrafish immune system is
                                                                sufficiently different from those of mice and humans
                                                                and thus aluminum may not be as effective as an
     After injection, the fish were monitored at Oregon
                                                                adjavent.
     State University’s central facility of the Aquatic
     Pathology and Toxicology Facility Core, the
     Sinnhuber Aquatic Research Laboratory (SARL),              Acknowledgements
     in tanks housing each group individually (10 fish          I thank Dr. Joe Beckman and the Beckman lab for
     per tank). The fish were then observed for the             their guidance throughout my research experience

22   Volume 3
                                                                                                                        21
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                                                        Catalyst

      this summer and Megan Hirko for her unwavering          4. Oyanagi, K. “Magnesium deficiency over genera-
      support and direction. I would also like to thank the       tions in rats with special references to the patho-
      Howard Hughes Medical Institute and Oregon State            genesis of the Parkinsonism-dementia complex
      University for this invaluable research opportunity.        and amyotrophic lateral sclerosis of Guam.”
                                                                  Neuropathology 26 (2006): 115-28.
      References                                              5. Petrick, Michael S., Margaret C. Wong, Rena
      1. Bharathi, P. “Molecular toxicity of aluminium in         C. Tabata, Robert F. Garry, and Christopher A.
          relation to neurodegeneration.” Indian J Med Res        Shaw. “Aluminum adjuvant linked to gulf war
          128 (2008): 545-56.                                     illness induces motor neuron death in mice.”
      2. He, Bei Ping, and Michael J. Strong. “A morpho-          NeuroMolecular Medicine 9 (2007): 83-100.
          logical analysis of the motor neuron degenera-      6. Rijpkema, Sjoerd G. “Investigation in a model
          tion and microglial reaction in acute and chronic       system of the effects of combinations of anthrax
          in vivo aluminum chlorie neurotoxicity.” Journal        and pertussis vaccines administered to service
          of Chemical Neuroanatomy 17 (2000): 207-15.             personnel in the 1991 Gulf War.” Human Vac-
      3. Kihira, Tameko. “Chronic low-Ca/Mg high-Al               cines 1 (2005): 34-38.
          diet induces neuronal loss.” Neuropathology 22      7. Verstraeten, Sandra V. “Aluminium and lead: mo-
          (2002): 171-79                                          lecular mechanisms of brain toxicity.” Archives
                                                                  of Toxicology 82 (2008): 789-802.



                                      HHMI at OSU
      OSU’s Summer Undergraduate Research Program, sponsored by grant #52005883 from the Howard
      Hughes Medical Institute (HHMI) is grateful for generous support from many sources. They include

                                    HHMI (http://www.hhmi.org/)
                    University Honors College (http://oregonstate.edu/dept/honors/)
         OSU’s Undergraduate Research, Innovation, Scholarship, Creativity (URISC) Program
                            (http://oregonstate.edu/research/incentive/awards.htm#URISC)
                                        Ernest and Pauline Jaworski Fund
                   (http://www.science.oregonstate.edu/bpp/ernest_and_pauline_jaworski_fund.htm)
                 DeLoach Undergraduate Research Fund in the University Honors College
                 Ray, Frances, and Dale Cripps Scholarship Fund in the College of Science


      The HHMI Web site (http://oregonstate.edu/dept/biochem/hhmi/summerresearch.html) features additional
      information about the program, including PowerPoints, streaming videos of student presentations and
      downloadable versions of Catalyst issues. Applications for the 2010 program will be available for down-
      load from the site in late January, 2009.


                                       Instructions to Authors
      The Catalyst has recently updated its instructions to authors. These can now be downloaded online at the
      following URL:

               http://www.oregonstate.edu/dept/biochem/hhmi/undergradresearch/catalystguidelines.doc


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