Water Balance in F344 Rats During Acute Heat Exposure
Ms. Cynthia L. Shriver* and Dr. Annette M. Gabaldon
Department of Biology
Research Project #19 – Abstract
The goal of this research project is to investigate thermoregulation in senescent Fischer 344 (F344) rats spontaneously losing body weight near the end of their natural life span. Previous work has been done on this animal as a potential model for understanding the neurological and endocrine causes for geriatric failure to thrive (FTT) syndrome that occurs in many elderly humans. Hallmarks of geriatric FTT syndrome are decreased food intake (anorexia), rapid body weight loss, impaired cognitive function, and physical inactivity followed by death. Despite the impact FTT will have on the rapidly growing population of elderly people in the U.S., relatively little is known about the mechanisms that are responsible for the rapid functional decline. The thermoregulatory system involves neural and hormonal signaling pathways and thus provides a good model for understanding the impact of FTT on basic regulatory mechanisms. In previous studies, it was shown that senescent F344 rats become severely hypothermic during acute cold exposure, partly due to decreased heat production. Here, we will test the hypothesis that heat-induced thermoregulation is impaired in senescent F344 rats. A series of bi-weekly acute heat exposure tests (1.5 h at 35°C) will be performed on each animal while it is weight-stable, and a final experiment will be performed 3-4 days after transition to the “end-of-life” physiological state marked by the onset of spontaneous rapid weight loss. The ability to maintain a constant body temperature during acute heat exposure and effectiveness of cooling mechanisms will be investigated by measuring total evaporative water loss (TEWL) from the animal. This includes any water lost from the respiratory passages and skin, both of which can effectively cool the blood during heat exposure. Following each heat exposure test, the drinking (thirst) response will be monitored using a computer automated system. The pilot studies for this research are described here. Initial studies on the rats will begin with young animals (age 12 months) and continue with the senescent rats. This study will provide information about brain function in senescence (FTT), and specifically the hypothalamus, which regulates numerous homeostatic functions including thermoregulation, food intake, body weight, and drinking behavior.
Toxicity of 8-MOP to Embryos of the African Clawed Frog, Xenopus Laevis
Ms. Jennifer M. Cozzetta*, Dr. Cassandra Osborne and Dr. Moussa M. Diawara
Department of Biology
Research Project #17 – ABSTRACT
The psoralens are found in produce and used in skin photochemotherapy; they have been shown to compromise reproductive function in rats. We assessed the toxicity of 8-methoxypsoralen (8-MOP) on early embryo development of the African clawed frog (Xenopus laevis) by examining the developmental epoch spanning gastrulation to the freely swimming tadpole. Male frogs were injected twice (24 hours apart) with 0.35cc of human chorionic gonadotropin (hCG) and the female frogs were injected once with 0.65cc of hCG to induce mating. The frogs were placed in a separate mating aquarium in the dark overnight. Fertilized eggs were harvested from the aquarium the next day. After the embryos reached gastrulation, they were reared in 400mL of distilled water, pH 7.2, at room temperature (controls), or distilled water supplemented with DMSO (vehicle), 1, 10, or 100µM 8-MOP, with 30 embryos per treatment. Mortality was evaluated and the embryos were fixed in MEMFA (100mM MOPS, pH 7.2, 2mM EGTA, 1mM MgSO4, 4% paraformaldehyde) for 1 hour at room temperature and then dehydrated and stored in methanol at -20°C. Using a dissecting microscope equipped with a micrometer, body length and interocular distance (the distance between the medial sides of the eyes) were taken. A dose response relationship was observed for body length with the higher concentrations of 8-MOP stunting embryonic growth in all experiments. Mortality also increased in a dose-dependant manner. Other deformities included kinked tails, underdeveloped eyes, misshapen dorsal fins, swollen guts, and undefined spinal cords. Additional experiments using tunnel essay showed 8-MOP-induced apoptosis in exposed embryos in a dose-dependent manner, confirming our previous observations using rodent models.
An Analysis of the Genetic Variation Within and Between Populations of the Threatened Canadian River Spiny Aster (Asteracers: Eurybia Horrida) Using ISSR Fragments
Mr. Matthew S. Garcia* and Dr. Brian D. Vanden Heuvel
Department of Biology
Research Project #2 - ABSTRACT
We undertook an analysis of the genetic variation within and between populations of the Canadian River Spiny Aster (Asteraceae: Eurybia horrida). This plant species is of interest due to the fact that it has been slated by the Nature Conservancy and other agencies as threatened and endangered due to its small distribution (along the Canadian River in New Mexico and along Raton Pass). The small number of populations, although they have many individuals within them, could have very little or no genetic variance making the plant more susceptible to extinction. Previous experiments within Eurybia horrida had found difficulty in DNA extraction, and our goals were two fold: 1) To assess DNA quality in our collections, and 2) To assess genetic variation within and between populations. To address the first goal, plant DNA extractions were tested for quality by using PCR and chloroplast primers (trnS and trnG). These primers showed some results and indicated which plants contained quality DNA present to test with the ISSR primers. The first round of amplifications using the ISSR primers failed. We attributed this result to the state of the DNA within preserved specimens collected by Carmen Lara in the previous year. These specimens were dried in such a way in which the chloroplast DNA might survive yielding strong amplification using chloroplast primers, but not the nuclear DNA (ISSR primers). These results prompted a new collection of fresh specimens from two sites along Interstate 25, one at Colorado mile marker 6 and the other at New Mexico mile maker 456. Around thirty samples were collected from each of these sites for a total of sixty individuals. With the new samples, an extraction of the DNA was carried out using a combination of CTAB technique, and further cleaned using glass bead technology (QIAGEN). A subset of DNA samples were used to scan the 100 possible ISSR primers for a set that would reliable amplify Eurybia horrida DNA. At the same time, the same DNA samples were scanned using nuclear ribosomal primers ITS4 and ITS5. Once viable primers and DNA were identified with positive amplification, the PCR reaction was modified to maximize the products using the ISSR primers. After scanning the primers, two primers (UBC 809 and UBC 814) were chosen and ran in PCR reaction with each of the DNA samples. The resulting PCR products were separated by gel electrophoresis. Banding patterns for each individual were scored presence/absence, and analyzed for population specificity/ population structure in the two populations collected. WE found a surprising amount of variation within and between populations, signaling that there is indeed genetic variation within the two Eurybia horrida populations tested.
Diversity of Frankia Associating with the Noxious Weed Elaeagnus Augustifolia (Russian Olive)
Ms. Jennifer H. Kedward* and Dr. Brian D. Vanden Heuvel
Department of Biology
Research Project #3 - ABSTRACT
This experiment investigated the diversity of Frankia strains using DNA sequence data in variably aged stands. Frankia are nitrogen-fixing bacteria that associate with members of eight plant families, including the Colorado noxious weed Russian Olive (Elaeagnus angustifolia L.). In past experiments, it was determined that Elaeagnus did not select specific Frankia strains, yet age of the stand was not investigated. We were interested in exploring if selection was occurring on Frankia strain diversity over time, specifically if one strain came to dominate in a stand of Elaeagnus through accelerated reproduction. We sampled both nodules and soil from three different aged stands: 10 years, 20 years, and 30 years as determined by tree rings. By comparing Frankia strains found in Russian Olive nodules with Frankia strains found in soil samples around Russian Olive trees, the impact of age within a Russian Olive stand on Frankia strain diversity can be established. Nodules lobes harbor only one strain of Frankia due to the infection pathway. The glutamate synthatase gene (glnA) was used to estimate strain diversity using the criterion that different glnA sequences suggested different Frankia strains. Five lobes per nodule and 5 nodules per plant were collected and analyzed for the glnA sequence in each lobe. DNA was extracted from the soil around the nodules and the glnA region specific to Frankia genomic DNA was amplified. We found no significant difference in the strain diversity found in the nodules as compared to the soil samples, nor significant differences in the diversity from different aged stands. It appears that Elaeagnus is non-specific with respect to Frankia strains, and there is no evidence for strong selective pressure reducing Frankia strain diversity over time in Elaeagnus stands
Does the Ryanodine Receptor Play a Central Role in Efferent Inhibition
of the Chicken Cochlea?
Mr. Kevin D. Marquez*, Mr. Hakim Hiel and Mr. Paul A. Fuchs,
Johns Hopkins University School of Medicine
Department of Biology
Research Project #4 - ABSTRACT
Cochlear hair cells receive efferent input from the superior olivary complex in the brainstem. These efferent axons release the neurotransmitter acetylcholine (ACh), leading to the influx of calcium into the hair cells. Electron microscopy revealed a synaptic cistern on the post-synaptic side coextensive with the efferent terminal. Recent evidence suggests that this synaptic cistern serves as a calcium store and may mediate the hair cell’s response to ACh via the Ryanodine receptor (RyR). Prior research on the mutant crooked neck dwarf (CND) chicken has shown that they express abnormal RyRs in skeletal muscle; however, it is not known whether the protein is present in the cochlear hair cells. Using immunohistochemical techniques, we were able to determine that the CND mutant expresses the Ryanodine receptor protein in cochlear hair cells; however, it is unclear whether the protein is functional. We were also able to discover that there is a lack of choline acetyltransferase – the enzyme responsible for making ACh – in the mutant efferent axons, which suggests that there is no efferent innervation or that the efferent axons fail to express this protein.
Identifying Suppressors of Segregation Distortion in Drosophila Melanogaster
Ms. Angela M. Miller*, Ms. Micaela E. Ponce* and Dr. Janna R. McLean
Department of Biology
Research Project #6 – ABSTRACT
According to Mendelian Genetics all alleles have an equal opportunity to be transferred to the progeny. Segregation Distortion is a violation of this principle, whereby only one allele is transmitted. This phenomenon has been seen in male Drosophila melanogaster that are heterozygous for the SD/SD+ chromosome and have a Responder Super-Sensitive (Rspss) locus. Although the Responder locus has no known gene product, when it is present on the chromosome bearing the Sd+ gene it behaves as a target of Sd activity. When distortion occurs, two primary phenotypes can be observed: 1) sperm dysfunction (failure of the spermatid bearing the SD+ chromosome to undergo chromatin condensation), and 2) interruption of the Ran nuclear transport (RanGTPase-activating protein). To try to understand how these two phenotypes relate to Segregation Distortion, isolation of interacting mutations will be performed. Identification of other gene products involved Segregation Distortion should help clarify the mechanism behind these phenotypes as well. Although the mutagenesis was not completed, all of the preliminary work need to proceed to the P-element mutagenesis has been.
Impact of 8-MOP and Heavy Metals on the Wistar Rat Embryo
Mr. Dave D. Unis* and Dr. Moussa M. Diawara
Department of Biology
Research Project #7 – ABSTRACT
The psoralens, compounds found in produce and used in skin thereapy, have been shown to affect reproductive function in rats. Recent studies have shown that 8-methoxypsoralen (8-MOP) is a reproductive toxicant. The litter size (average girth of the fetuses analyzed as litter units) of females bred to 8-MOP-treated males was significantly smaller compared with controls, suggesting an indirect effect on embryo through the seminal fluid or direct effect on sperm DNA. We evaluated herein the reproductive toxicity of 8-MOP and three heavy metals (arsenic, cadmium, and lead) on early embryo development of the Syrian golden hamster (Mesocricetus auratus) by examining the time frame during which the blastocysts escape from the zona pellucida, which corresponds to the blastocyst attaching to the uterine endometrium (in vivo) and exhibiting trophectoderm outgrowth. This consequently leads to the formation of the placenta and fetus. Females were superovulated with 50 IU of pregnant mare-serum gonadotropin (PMSG) and mated during estrus to ensure pregnancy. Embryos were collected by excising uterine horns and flushing them with culture medium. Embryos were then grown on non-treated culture medium, DMSO vehicle control or 8-MOP (0-433 nM), arsenic (0-750nM), lead (0-100uM) and cadmium (0-50uM) for 4 four days. For each embryo, initial and final sizes were measured under the light microscope. Treated embryos and blastocysts were compared with control groups for deformities and/or morphological evidence of atrisia. 8-MOP and all three heavy metals showed lethality in a dose-dependent manner. In addition all lethal doses caused major deformities expressed as dissociation of blastomeres, deformed blastomeres, and failure of the zona pellucida to undergo lysis. This suggests that the compounds are having an adverse affect on tight junctions between blastomeres, blastomere gap junctions responsible for early cell to cell signaling, and a failure of the blastomere expression of the zona lytic protease. These findings are consistent with previous effects of other xenobiotics on development of murine embryos. Future research will involve qualitative and quantitative analysis of embryonic proteins.
Lungworms of Untreated Rocky Mountain Bighorn Sheep From the
Sangre de Cristo Mountains
Ms. Rosemary L. Townsend* and Mr. Ron Rankin, Trinidad State Junior College
Department of Biology
Research Project #8 – ABSTRACT
Current numbers of the rocky mountain bighorn sheep (Ovis canadensis) are probably only 2%-8% of their numbers at the time of European settlement. Unregulated harvesting, habitat destruction, overgrazing of rangelands, and diseases contracted from domestic livestock have all contributed to their decline. The major problem today is mortality precipitated by parasitic (Protostrongylus spp.) lungworms. Parasite caused lesions often lead to Pasturella spp. bacterial infections that end with pneumonia and death. The prevalence of first-stage larvae of parasitic lungworms in the feces of bighorn sheep from the Sangre de Cristo Mountains was evaluated using the Baermann funnel technique. Since this herd has never been treated with antihelminthics, we hypothesized that the majority would be infected with lungworms. Nine fecal groupings were collected from wild sheep along the upper Huerfano River. Long-axis diameter per fecal pellet, per cent moisture, dry weight, and first stage lungworm larvae per gram of dry weight equivalent (lpg) of feces were determined. The parasites were also stained using the carmine-propionic technique and photographed. Two species of nematodes were found. 100% (n = 9) of the sheep were infected with lungworms, with rates ranging from 11- 1776 lpg. The mean was 370 lpg. These rates are high and represent a threat to the health and survival of the herd. Per cent moisture ranged from 10 – 45% with a mean of 26%. Per cent moisture and size were not correlated with lungworm density. This research supported by a NIGMS Bridges to the Biomedical Career Grant.
M1 Aminopeptidase Cellular Localization
Mr. Joshua M. Shaink* and Dr. Daniel R. Caprioglio
Department of Biology
Research Project #9 – ABSTRACT
M1 aminopeptidases (M1APs) are enzymes that have been correlated to cancer, immune cell development and cell cycle. The M1 aminopeptidase genes are found in a wide variety of organisms such as plants, animals, fungus, and bacteria. The specific aminopeptidase genes that are being studied belong to the metallo or M1 family of aminopeptidases which means that the enzyme requires a divalent metal ion. The enzymatic activity of these enzymes is to cleave one to three amino acids from the N-terminus of proteins by hydrolysis. In order to understand their role in cancer and cell cycle, this study uses the Green Fluorescent Protein fusion technology to localize the protein in a cell.
In order to monitor the localization of the protein, a green fluorescent protein sequence is fused to the M1 aminopeptidase genes. This will create a fusion protein that has two parts, the M1AP gene sequence and a fluorescent protein coding sequence at the end. This allows tracking of protein localization by the fluorescent signal. In order to create this fusion protein, a fluorescent protein coding sequence must first be generated for fusion into the M1 aminopeptidase gene. Using PCR (Polymerase Chain Reaction) and hybrid primers, the fluorescent protein coding sequence was amplified along with a selectable marker. The significance of the selectable marker allows selection of yeast that integrated in the fluorescent protein coding sequence from yeast that did not.
The yeast that took in the fluorescent protein coding sequence were then tested for the correct placement of the fluorescent protein using PCR and gel electrophoresis. All the yeast that had the correct placement of the fluorescent protein coding sequence were further studied for protein localization. The gene activity was also tested to make sure that the gene was functioning properly using resistance to caffeine. The yeast containing the correct fusion protein were then fixed on a slide and there DNA was stained using a fluorescent stain called DAPI. The fusion protein localization were analyzed by fluorescence microscopy. The result of this indicated that the protein was localizing at the nucleus.
Since the protein is localizing at the nucleus, it may suggest a mechanism for the involvement of M1APs in cell cycle. To further study if this protein is localized to the nucleus at specific times during the cell cycle, a strain of yeast is being developed that contains the fusion protein a Bar 1 mutation (for synchronization) and is of mating type a. After this strain of yeast has been developed cultures of the yeast will be synchronized, and at various time points samples from the culture will be removed and their nucleus will be stained to localize the fusion protein product at the various stages of the cell cycle.