Journal of Biological Research, Vol. 1 (2000)

The Use of Cytogenetic Methodologies To Examine Human Chromosome Structure
Charles P. Catania
Advisor:  Dr. Stanley K. Sessions

I attempted to use cultured human white blood cells as a source of chromosomal material, to test cytogenetic methodologies, such as AgNOR banding and chromosome Karyotyping. Cultures were made in an RPMI growth media combined with a mixture of antibiotic reagents. The cells were treated with colchicine, centrifuged, separated, and fixed in a 3:1 fixative (Methanol: Glacial Acetic Acid). Karyotypes were attempted from the giemsa stained cells, but were marginally unsuccessful. Cells were stained by the AgNOR technique to show the Nucleolus Organizing Regions (NOR) within Interphase cells. A correlation was made between the amount of NOR’s found within the different Interphase cells and the given number of NOR’s within the human genome. The number of NOR’s varied from 1 to 6 sites inside the nucleolus. The known number of NOR’s within the human genome was observed to be 5 pairs or 10 regions, so I showed the variance is due to fusion or interconnection of the chromosomes, by using a chi-squared test.

Green Tea Polyphenols Inhibit Growth and Induce Apoptosis in Mouse Myeloma and Neuroblastoma Cells In Vitro
Kevin C. Collins
Advisors:  Dr. Linda Swift, and Prof. M. Richard Segina

The polyphenol compounds of green tea (Camellia sinensis (L.) Kuntze) have gained increasing attention as a chemopreventive agent. Tea is the second most popular beverage in the world with green tea comprising 20% of all tea consumed. The polyphenol compounds of green tea have demonstrated growth inhibitory effects in numerous cancer cell types. We studied the effects of whole green tea polyphenols (20, 70, and 120 ug/mL) on mouse myeloma and neuroblastoma cell lines. Green tea polyphenols displayed strong growth inhibitory effects against mouse myeloma cell lines MPC 11 and Sp2/0-Ag14, and mouse neuroblastoma cell line NB41A3. Normal mouse liver cell line NCTC clone 1469 showed no significant growth inhibitory effects. In another experiment, apoptosis was assessed by the formation of internucleosomal DNA fragments in gel electrophoresis. MPC 11, Sp2/0-Ag14, NB41A3, and NCTC clone 1469 cells were treated with 70 ug/mL. Internucleosomal DNA fragments, characteristic of apoptosis, were observed in MPC 11, Sp2/0Ag14, and NB41A3 cells, but not in NCTC clone 1469 cells. These results suggest that green tea polyphenols may induce apoptosis and inhibit the growth of myeloma and neuroblastoma cells. The effects of green tea polyphenols may also be cancer-specific.

Anthelmintic Activity of Oroxylum indicum Against Equine Strongyles in vitro Compared to the Anthelmintic Activity of Ivermectin
Jessica E. Downing
Advisor:  Dr. Linda A. Swift

This experiment assessed the anthelmintic activity of Oroxylum indicum against equine strongyle eggs in vitro and compared it to that of ivermectin, one of the most effective deworming agents on the market today. Seven Oroxylum indicum treatment groups were established, using seven different extract dosages: 2x10-7 g/mL, 2x10-6 g/mL, 2x10-5 g/mL, 2x10-4g/mL, 2x10-3g/mL, 2x10-2g/mL, and 2x10-1 g/mL. Two additional groups of equine strongyle eggs were treated with EQVALAN (ivermectin) Liquid® at dosages of: 2x10-7 g/mL (the product's recommended dosage) and 2x10-4g/mL, a dosage capable of prohibiting hatching. A non-treated group consisting of equine strongyle eggs in distilled water served as the control. The eggs in each group were incubated at 25°C for two days to promote hatching. The development of the strongyle eggs was then analyzed under 400x magnification. The percentage of hatched larvae and the percentage of viability of both the larvae and the eggs were calculated for each group. At dosages of 2x10-5 g/mL and greater, hatching of the strongyle eggs was delayed using Oroxylum indicum. 0% hatching was achieved at 2x10-1 g/mL Oroxylum indicum. At dosages of 2x10-4 g/mL and greater, 0% viability of the strongyle eggs and larvae was achieved. The results of this study suggest that Oroxylum indicum may be an appropriate anthelmintic against equine strongyles.

Ecology of the Pitch Pine (Pinus rigida) on the Vermont Sandplains
Kristin E. Hall
Advisor:  Dr. Michael T. Murphy

Sandplains communities in Vermont are unique habitats that were formed at the end of the last glacial period as a part of the Winooski River Delta by glacial runoff. The largest remaining site is located on the property of the Camp Johnson National Guard Base in Colchester, Vermont and the Vermont Non-Game and Natural Heritage Program is attempting to restore the community. As part of that effort, I mapped, tagged and measured height and diameter at breast height (DBH) for individual pitch pines (Pinus rigida). Pitch pines are considered to be an “occasional species” and the Vermont population appears to be undergoing a decline. I also took core samples from approximately 10% of individuals. From these data I formulated a size class distribution and growth history for the site. Size and age were poorly correlated and growth rate differed greatly among trees. I examined possible ecological determinants of growth, but found no correlation of growth with precipitation. Surprisingly, I found that there was no difference in long term growth rate between living and dead trees. However, the last 10 years of growth of a tree was significantly related to a tree's entire previous growth history. This particular site appears to not have any site wide determinant of growth. The survival and growth of the trees is probably related to site-specific phenomenon such as light availability, soil quality, burn history and possibly competition.

Bony Triangles in Deformed Frogs: Are They Diagnostic of Retinoids?
Louise Hecker
Advisor:  Dr. Stanley K. Sessions

Currently, two leading hypotheses have emerged to explain the occurrence of deformed amphibians with extra limbs in the wild: biochemical perturbation induced by environmental retinoids, and mechanical perturbation induced by parasite cysts. I have tested the idea that mechanical perturbation is sufficient to induce the observed deformities including extra limbs. This was done by using microsurgery to mechanically perturb the developing limbs of frog tadpoles. The range of morphologies produced through microsurgical-induced mechanical perturbation is indistinguishable from the range of morphologies produced by cysts and those found in the wild. The deformities include bony triangles, which were formerly thought to be diagnostic of retinoids. I conclude that mechanical perturbation is sufficient to produce limb deformities, including outgrowth of supernumerary limbs and bony triangles. This indicates that many of the deformities seen in the wild may be produced by mechanical perturbation induced by parasite cysts.

The Origin and Migration of Progenitor Red Blood Cells in Living Transgenic Zebrafish
Vanessa Horner
Advisors:  Dr. Douglas Hamilton and Dr. Stanley Sessions

 GATA-1 is a transcription factor which is critical for the production of progenitor red blood cells in developing vertebrates. In order to determine the precise point of stem cell commitment to progenitor red blood cells an in vivo labeling system involving green fluorescent protein was used. The GFP-GATA-1 construct was obtained from the University of Ottawa and amplified using E. coli cloning vectors. The construct was injected into 207 zebrafish embryos at the one or two cell stage. The final 49 embryos were injected with the construct and a molecular probe, Texas Red dextran. Embryos were examined at varying developmental stages under a UV microscope in order to detect fluorescence. Fluorescence due to Texas Red dextran was observed in 36.8% of the injected embryos. In addition, fluorescence like that of GFP was seen, but it was not distinct enough from uninjected controls to warrant a positive identification. Previous experiments which have elucidated GATA-1 expression patterns can help to shed light on the origin and migration of progenitor red blood cells.

The Effects of pH and Cadmium Uptake on Plant Growth: Exploring Phytoremediation Capabilities and Possible Effects of Acid Rain.
Ginneh Lewis
Advisor:  Dr. Douglas Hamilton

The use of plants to take up pollutants is not a new idea, but specialization of the field to increase phytoremediative efficiency is a rising science. It is believed that different plants may be suited to different sites of cleanup. Several hyperaccumulating plant species have been identified, but many require chemicals (e.g. chelators) to make the metal more available to the plant for uptake. Lower pHs are thought to mobilize metals, which is one of the problems of acid rain. In order to test the effect of pH on plant hyperaccumulation and show potential effects of acid rain, plants were placed in pHs 4, 5, 6, 7, and 9 with varying cadmium concentrations. Plant growth was recorded and metal uptake was determined by subsequent atomic absorption analysis. Growth and metal uptake were recorded and calculated. pH and cadmium both had a significant effect on plant growth (F = 3.844, df = 4, 158, p = 0.005 for pH and  = 11.833, df = 3, 158, p = 0.000 for Cd). The extreme pHs, 4 and 9, seemed to have the greatest effect on growth, especially pH 9. pH also had a significant effect on final cadmium concentration in the plant (F = 5.25, df = 4, 49, p = 0.0025 for pH). The highest final Cd concentrations were found in pH 5, followed closely by pH 6, then pH 7, pH 4, and pH 9. This may indicate that using a lower pH may mobilize Cd and increase phytoremediative efficiency. The lowest pH (4) caused growth inhibition and a decline in Cd uptake, but may demonstrate the more severe effects of acid rain on plant growth.

A Comparison of Biodiversity and malnutrition Between a Subsistence and a Market-Oriented Agroecosystem in Pakhasukjai and Ahbae Akha Villages, Chiang Rai, Thailand Kristine Neuhaus
Advisor:  Dr. Linda Swift

This study examines the biodiversity patterns of cultivated crops in each of two Akha villages in Northern Thailand and the prevalence of malnutrition amongst preschool children up to 5 years of age within those communities. Pakhasukjai is a larger, more dynamic community, where the families supplement staples grown in the gardens and fields of the village with cash crops regularly sold at the market place to produce income that might supply the rest of their necessities. Ahbae Village is a smaller, more isolated community near Pakhasukjai that uses traditional subsistence agricultural techniques to supply their household food needs. They do cultivate some market crops, but being more geographically isolated from the nearest market, most of their nutritional needs originate within the village. It was hypothesized that Ahbae would have a greater diversity of crops and a lower prevalence of wasting and stunting than Pakhasukjai. The results showed that while the predominant crops in each village were different (rice and corn in Ahbae vs. Cabbage in Pakhasukjai), the basic crop make-up was similar between the two villages. In other words, there was no indication that biodiversity was affected in Pakhasukjai. There has been a shift in Pakhasukjai to plant fewer household crops in the fields, less rice for household consumption, and cabbage to sell at the market. The preschool children in Pakhasukjai were significantly more stunted than those in Ahbae indicating a poor long-term nutritional history, at least for the past five years. The children in Ahbae showed a significantly greater prevalence of wasting, indicating a recent lack of food or presence of disease. 

Control of the Developmental Expression of the ZM13 Gene in Pollen
Jennifer Peat
Advisor:  Dr. Douglas Hamilton

A pollen grain is the microgametophyte generation of higher plants. Inside a pollen grain there is DNA that contains genes, and roughly 20% of these genes have been found to be pollen specific. The promoter region of a particular pollen-specific gene ( called Zm13), a gene that was first found in maize and later in most other plants, has been isolated and deletion mutations made. While overall expression of these deletion mutants of the Zm13 gene has been examined, their expression during the course of development has not. So in my thesis, I have examined the amount of expression using two methods. The first method is a GUS staining technique and the second is a flourometric assay.

The Effects of 3,4-Methylenedioxymethamphetamine (MDMA; Ecstasy) on the Rat Kidneys: An Evaluation of Renal Clearance and Histopathology
John Podraza
Advisor:  Dr. Allen Crooker

The effects of MDMA (3,4-methylenedioxymethamphetamine) on the renal system of the male laboratory rat were studied. Six animals (three experimental and three control) were used with the test group receiving approximately 5.0 mg/kg MDMA via gastric gavage once a week for three weeks. Urine output was monitored daily and serum urea nitrogen (SUN) levels were measured one and two weeks following the last dose. Renal clearance (RC) measurements were then calculated using these values. The two-sample t-test showed a statistically significant decrease in SUN and an increase in RC levels in the test group after one week (95% confidence interval; CI). SUN levels underwent a statistically significant reversal after week two, i.e. they were elevated in the test group (95% CI). However, renal clearance measurements were not significantly different between the test and control groups at week two. These results do not support MDMA-induced impairment of the rodent renal system. Renal tissues observed by light microscopy demonstrated no gross abnormalities.

Composition and Stability of the Upper Tract Forest Tree Community
Elissa J. Quattrini
Advisor:  Dr. Michael T. Murphy

Patterns of change in forest community structure can be caused by repeated disturbance or normal succession. Approximately 100 hectares of forest in the Upper Tract were sampled and assessed for species composition and importance. To evaluate stability of species composition, data were collected for DBH (diameter breast height), species, and height class of standing trees at 744 data points. Different species had centers of distribution that were separated spatially, particularly chestnut oak (Q. prinus). The five species with the greatest importance values were red maple (Acer rubrum), red oak (Quercus rubra), hemlock (Tsuga canadensis), big-tooth aspen (Populus grandidentata), and beech (Fagus grandifolia.) I used principal components analysis to identify major gradients of distribution of trees based on basal area of canopy trees. The most important gradient identified was one of red and white oak (Q. alba) vs. red maple and hemlock. The distribution seems to be related to a moisture gradient created by the stream in the middle of the sample area. Three additional gradients were also identified. To assess species stability, the canopy gradients were compared to the basal areas of understory trees that were potential candidates for the canopy of the future forest. The only species that seemed to be replacing itself was eastern hemlock, i.e. areas dominated by hemlocks are relatively stable communities. All other communities will undergo drastic changes in species composition in the next few decades. Red and white oak are failing completely to replace themselves. If this trend continues, the red, white, and chestnut oak may become permanently absent from the forest of the Upper Tract.

The Effect of Acorn Granivores on Oak Regeneration
 Kristen L. Schlansker
Advisor:  Dr. Michael T. Murphy

Evidence suggests that oaks in northeastern deciduous forests are failing to regenerate. Most suggest that this could be due to a lack of forest fires, but the impact of granivores has not been investigated fully. I tested the effect of granivores on acorn availability by controlling the vertebrate species allowed to consume acorns in a northeast deciduous forest. I collected red oak (Quercus rubra) acorns from the Upper Tract of the Pine Lake Environmental Center, West Davenport, NY and constructed exclosures that controlled access to acorns by three groups of consumers, according to size: (1) all vertebrate granivores excluded, (2) access to only small mammals, (3) access to small and mid-sized mammals, and (4) access to small, medium, and large mammals, and turkeys (Meleagris gallopavo). Each exclosure contained 25 acorns in a 5 X 5 array. Consumers were allowed access to the exclosures during the winter and early spring 2000. I found that the small mammal community alone was capable of consuming all of the acorns, and that the small mammal community combined with white-tailed deer (Odocoileus virginianus) and turkey probably have an important negative effect on oak regeneration.

Analysis of Avian Community Structure in the Bahamas with Special Reference to Andros Island and San Salvador
Jonathan R. Shoen
Advisor:  Dr. Michael T. Murphy

INTRODUCTION: I compare the avifauna of two Bahamian islands as part of a larger effort to quantify the effects of biogeography and interspecific competition on species richness, abundance, and diversity among both migrant and resident bird populations in the Bahamas. I captured birds on San Salvador and Andros Island during January 2000. On San Salvador I captured nine resident species and17 migrant species, while on Andros, I captured nearly twice as many resident species as on San Salvador (17) and fewer migrant species (13). On both islands, the majority of the winter residents were parulid warblers. For both migrants and residents, Andros had higher diversity and evenness. Capture rates were higher on San Salvador, partly due to the high abundance of the Bananaquit (33.33/100 net h). San Salvador was also segregated into three distinct habitats: mangrove, scrub, and early succession. The early succession, or disturbed habitat, had the highest capture rate, mostly due to the fact that it contained the most Bananaquits. Similarity indices (Morisita's index, Cm) indicated that while all of the habitats on San Salvador were similar to each other, none were similar to Andros, particularly with respect to residents. Biogeographic comparisons of bird species richness and composition for eight islands showed that area (km2) had no impact on richness, but that distance from Florida negatively impacted species richness. Resident species richness also tended to decline as the distance from Cuba increased, but migrant species richness was independent of the distance from Cuba. Those islands with the highest resident richness (Andros) are closest to Cuba, while those islands with the highest proportion of migrants to total species (San Salvador) are the farthest away from Cuba. 

Early Stages of Supernumerary Limb Development in Trematode Infected Frogs
Geffrey F. Stopper
Advisor:  Dr. Stanley K. Sessions

We tested the idea that trematode cyst infestation induces supernumerary limbs in frogs by perturbation of cellular positional relationships in developing limb buds. Patterns of mitotic cellular growth and Fgf2 expression were examined in early stages of rear limb development in two species of Rana using Anti-BrdU and Anti-FGF2 immunocytochemistry. No obvious differences were observed in density of cell division or Fgf2 expression among normal limbs, bead implanted limbs, or cyst-infected limbs. However, both a massive disruption of normal limb growth and large amounts of extra tissue growth were observed in and around the limb buds of cyst infected individuals, but not in normal or bead implanted limbs. The effect of Ribeioria sp. cysts on limbs may be more than purely mechanical.

The Effects of Coccina indica and Alluim sativum on Alloxan Induced Diabetic Rats
Janis K. Sullivan
Advisor:  Dr. Linda Swift

Anti-diabetic properties of the medicinal plant Coccinia indica and Allium sativum (garlic) were compared to a reference drug, Tolbutamide. The lypholized drug of the extract of the medicinal plant, garlic, and the reference drug was administered to alloxan induced chronic diabetic rats. A .25mg/kg dose of these three drugs was administered to three groups of rats and no drug was given to the control group. Blood glucose levels were taken prior to, during, and after the experiment. Coccinia indica and Tolbutamide significantly decreased blood glucose levels over time (p< 0.05), while the blood glucose levels of the control group increased with time (p<0.01). Coccinia indica, Tolbutamide, and Allium sativum decreased blood glucose levels 34.89%, 21.15%, and 7.14% respectively (p<0.01) as compared to the increased blood glucose levels in the control group (58.0%). When Coccinia indica and Garlic are compared to Tolbutamide the decrease in blood sugar levels was not significant (p<0.05). Coccinia indica showed the greatest decrease in blood glucose levels, Tolbutamide second, and Allium sativum showed the smallest decrease in blood glucose levels. 

A Study of Abundance, Density, and Habitat Use by an Avian Community in Southern New York
Christopher D. Valligny
Advisor:  Dr. Michael Murphy

Population declines have been reported for many North American migrant bird species. Many hypotheses have been proposed to explain these declines, but small-scale studies are needed to determine the causes. One hypothesis that has received serious consideration is the negative impact of forest fragmentation of a migrant species’ breeding and/or wintering habitat. I performed the first two years of a long-term breeding bird census and territory mapping project in a highly fragmented landscape in southern New York, compared the breeding bird density in the forest at this study site to that of other sites, and examined the change in numbers of all species as a whole and according to guilds based on life history traits. Based on these data, short-distance migrants breeding at the study site appeared to decline between 1998 and 1999, while populations of residents and long-distance remained constant. Species did not show any statistically significant change in numbers based on preference of interior forest, forest edge, successional-shrub, or grassland breeding habitat. Population trends were not associated with breeding habitat and therefore breeding habitat preference did not seem to have a significant effect on population status. The density of breeding birds at Vassar Farm was significantly lower than that of similar yet unfragmented forests. In addition, several species that are considered to be typical of forest interior habitat were either absent or exhibited very low abundance. These data support the claim that fragmented forests provide poor breeding habitat for forest interior species.

The Long-Term Effects of Caffeine on Gastrocnemius Muscle Strength and Fatigue in Swiss Webster Mice
Victor A. Willingham
Advisor:  Dr. Allen Crooker

The effects of caffeine on gastrocnemius muscle strength and fatigue were observed in 30 Swiss Webster mice administered 0(control), 5, and 10 mg/kg body mass of caffeine in their drinking water for 14 weeks. Using a force transducer and a Lafayette Minigraph to measure contraction strength and fatigue factor, the contraction factor and fatigue factor were determined. Concentrations of blood caffeine measured by HPLC were used to evaluate variations in the contraction and fatigue factors. Analysis showed no statistically significant relationship between caffeine blood concentration and strength (p=0.4040) or fatigue (p=0.1506). Analysis of the fatigue factor did suggest a negative correlation between the time until fatigue and the amount of caffeine present in the body; however, linear regression indicated this was insignificant. Mice receiving no caffeine 24 hours prior to sacrifice had a significantly reduced contraction factor (p=0.0488) compared to those kept on caffeine until sacrifice.

Mercury Detoxification Effects of Allium sativumand Silymum marianum in Livers and Kidneys in Rats
Svetlana Zakharchenko
Advisor: Dr. Linda Swift

This study examines the detoxifying effects of Allium sativum (garlic) and Silybum marianum (milk thistle) on ingested mercury chloride in rats. Previous studies have shown that garlic contains thiol compounds which detoxify heavy metals, while milk thistle contains silymarin which protects the liver against damage from environmental toxins. Since heavy metals accumulate in the liver and kidneys, I hypothesize that the livers and kidneys of the rats treated with garlic and milk thistle will contain less mercury. Three groups of 8 rats were treated for 7 weeks with mercury chloride (conc.50 ppm.) in drinking water. The Garlic Group was intubated with 0.0028gm per body weight of garlic twice daily, the Milk Thistle Group was intubated with 0.0085 gm of milk thistle twice daily. The liver and kidney mercury content was measured using Atomic Absorption Spectrophotometry. Results indicate that the liver to body weight ratio in the control group was significantly greater than those found in garlic (P<0.01) and milk thistle (P<0.001) groups. Also, the kidney contained significantly more mercury (mg)/organ weight (gm) than the liver in both control and the experimental groups (P<0.001). Further, the ratio of the kidney to liver mercury concentration was greatest in the Milk thistle group (59.3) (P<0.05). These results indicate that the livers of rats treated with milk thistle were somewhat protected from the mercury effects.

The Prevalence of Mastitis Pathogens and Bacterial Resistance to Antibiotics Used to Control Bovine Mastitis
Brent Zogg
Advisor: Dr. Mary Allen

Bovine mastitis is inflammation of the mammary glands of cattle. It is caused by a variety of pathogens and is the leading cause of financial loss in the dairy industry. This is a rigorous study designed to study mastitis pathogens so that potential solutions may be concluded. Mastitis records were obtained from three New York dairy farms to observe the prevalence of the pathogens causing mastitis. The first farm owned and run by the VanAlthuis family is approximately a 200 cow dairy and during the span of approximately 11 months, from May 15, 1999 to April 17, 2000, 32 cases of mastitis were discovered. Also, an approximately 300 cow dairy, owned by Stewart Young, had 174 cases during 2 years and 5 months, from September 7, 1997 to February 11, 2000. Lastly, a 50 cow dairy operated by Carl J. Zogg had 11 cases of mastitis during 2 years and 2 months, from November 14, 1998 to February 15, 2000. Some cases, from each farm, were obviously difficult to treat indicating the possible existence of resistant pathogens. The Kirby-Bauer Antibiotic Disc Susceptibility test, a standardized procedure, was used to determine whether recurrent mastitis cases were more resistant to antibiotics than new cases. The bacterial strains used, Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli, were obtained from Cornell University. A total of 6 bacterial samples were obtained, 3 of each strain isolated from new cases and 3 of each strain isolated from recurrent cases, and lincomycin (2ug), gentamicin (10ug) and amoxicillin clavulanic/acid (20/10ug) antibiotic discs were used in the susceptibility tests. Streptococcus agalactiae was more resistant to all the antibiotic discs used in the recurrent form while the new form was always susceptible. Both Escherichia coli forms were resistant to lincomycin discs and susceptible to gentamicin and amoxicillin clavulanic/ acid discs. Both Staphylococcusaureus forms were susceptible to gentamicin and amoxicillin clavulanic/acid discs while being intermediate to lincomycin in both forms. Conclusions suggest microorganisms should be isolated and identified in cases of mastitis before treatment, especially chronic cases, because in the case of Streptococcus agalactiae the recurrent form became more resistant to the antibiotic concentration being used.