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Catalyst
The
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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
Layout & Logo
be considered tomorrow as an out-dated and flawed hypothesis. It is for this reason
Design By
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
Now accepting submissions for the next issue of
*Scientific Articles
* Scientific Articles
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Plus Any Other Ideas You May Have!
Plus any other ideas you may have!!!
Submit articles to:
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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.
3
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
4 Volume 3
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.
Volume 3
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Catalyst
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
6 Volume 3
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Catalyst
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
6 Volume 3
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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
The
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
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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-
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
122 Volume 3 23
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24 Volume 3
