2003 Research Fair Archive - Biology Abstracts
Protein-Protein Interactions downstream of LRP in Drosophila
The Low Density Lipoprotein (LRP ) family proteins have been shown to be important during the embryonic development of fruit flies, frogs, mice, and humans. Previous research has shown that LRP's are important for Wnt family protein signal transduction. Using the yeast two-hybrid assay to further investigate the protein-protein interactions of the Drosophila LRP protein. We have focused on interactions of homologous proteins found in Drosophila that have been previously found in mice between the LRP and Vinexin, Axin and Drk (downstream kinase receptor). Particular interest was given to the possibility of an interaction between the LRP with the Drosophila Drk (Fly equivalent of mouse GRB2) which had not been previously linked in the Wnt signaling pathway. The Drosophila Drk was found to have a strong interaction with Vinexin and weak but apparent interaction with Axin but to our surprise there was no interaction found between the LRP and the Drk. These results were interesting in comparison to previous experiments with the equivalent mouse protein that showed that there was interaction between Axin, Vinexin, and GRB2 with LRP 5/6. The results indicate that although there is a conservation of the proteins and some of the interactions across the two species there is also some divergence in the pathways.
Molecular Phylogeny of Chordeiles minor
The phylogenies of most orders of birds have not been investigated on the molecular level. Salvage samples of Chordeiles minor (Nighthawk) obtained from the Tracy Aviary in Salt Lake City have provided us with a unique opportunity to study this species. With these samples, I was able to investigate the Nighthawks evolutionary relationship to other birds such as chicken, and to its currently accepted phylogeny. First, a protocol was developed using muscle tissue from chicken. This species was chosen because its mitochondrial genome was readily available for comparison. The genomic DNA from Nighthawk tissue was isolated using a Qiagen DNA extraction kit with a refined protocol. Next, the mitochondrial gene Cytochione B was amplified with PCR and sequenced.
Evidence for Interspecific Competition in Subfossil Gastropod Assemblages, Salt Lake City, Utah
Interspecific competition occurs between the Neogene stream snails Pyrgulopsis transversa and Pyrgulopsis kolobensis in post-Bonneville deposits along Emigration Creek in Salt Lake City, Utah. We collected samples from eighteen fossil horizons, sorted and photographed them. From the photographs, we applied shape-describing software to examine small differences and changes over time between the two species. Finally, we used multivariate statistical techniques to discern patterns of competition. In the end, we conclude that character displacement occurs between these two species. This displacement has occurred multiple times in the geologic past, and each instance of displacement shows a different pattern, suggesting that there is no preferential partitioning of resources.
Refinement of Protocol: Molecular Phylogeny of Hornbills and
The phylogeny of most bird orders has not been examined at the molecular level. In addition, the Tracy Aviary in Salt Lake City houses four species of hornbill. This presents a unique opportunity to create a molecular phylogenetic tree of these birds and various outgroups.
The protocol was refined from a Qiagen DNeasy Tissue extraction kit. Using G.gallus (muscle tissue) as a control, the appropriate cytochrome B fragment was PCR amplified using primers designed from the G.gallus mitochondrial genome samples were sequenced at Iowa State University and matched the expected fragment. Other avian DNA is being extracted and PCR amplified to continue sequencing.
Adherence of Enterococcus Faecalis to Human Bladder Epithelium and
Enterococcus faecalis is a common cause of nosocomial infections in humans. We have been studying the adherence of these bacteria to human urinary bladder cells. Our long term goal is to devise an alternate treatment modality which does not require antibiotics and which utilizes our current knowledge of adherence genes (e.g. antisense technology). Numerous studies suggest that adherence of enterococci is driven by specific surface proteins carried in the genomic and/or plasmid DNA. We characterized twenty-three human enterococcal isolates from urinary tract infections and confirmed their identity using standard biochemical tests. We tested the strains for adherence to human urinary bladder (HTB-5) and MDCK cells under standardized conditions. We then completed PCR with primers for several previously reported enterococcus genes (ace, esp, asc 10, efa Afs); and for similar adherence genes from other Gram + organisms (scl, sclB, hsa, fibri). Both genomic and plasmid DNA were tested for these genes. We found a wide range of adherence characteristics to human bladder cells under the conditions used in our assay (percentage of cells with 20 or more adherent bacteria ranged from 11.2±1.0 to 71.6±3.1%). However, all strains were non-adherent on MDCK cells. Our confidence in the adherence assay was supported by using coded samples and blindly scoring the results. PCR products of adherence genes ace, asc 10 and efa Afs were found in all strains tested, but the esp gene was found less frequently. The identity of PCR products was confirmed by sequencing studies. Adherence genes were not found in plasmids isolated from strains used in this study. Several additional targets were identified using primers for non-enterococcal adherence proteins. In summary, we have identified suitable gene targets and isolates for development of a model for alternate treatment techniques, and a test system for analysis of altered strains.
DNA Repair as an Extreme Sport
Extremely halophilic Archaebacteria may exhibit DNA repair of ultraviolet (UV) lesions through the use of photoreactivation as well as "dark-repair," or nucleotide excision repair (NER). These repair mechanisms are critical to the survival of the organisms due to the harsh conditions that their habitat exerts. The halophilic repair systems that have been studied are similar to Eubacterial systems. However, unlike Eubacteria, the literature on the halophilic Archaebacterial species has yielded controversial data concerning which strains repair by which means. In addition, there are a number of interesting extreme halophilic species that have not been analyzed in terms of DNA repair mechanisms, including Haloferax mediterranei. This strain of bacteria requires upwards of 15% sodium chloride in their media as do other extreme halophiles, but setting them apart, they also require a significant amount of magnesium. In testing for the presence of NER, Hfx. mediterranei showed a four-fold greater efficiency over E.coli according to their respected lethal dose for 50% of the cells (LD50). A higher tolerance to ultraviolet exposure was also observed in Hfx. mediterranei as opposed to the negative control strain E. coli.
Media Effects on Enterococcal Adhesion
E. faecalis is a common cause of nosocomial infections in immunocompromised patients and are becoming increasingly antibiotic resistant. Clinical strains were previously shown to be E. faecalis using standard biochemical tests. The goal of this mini-project was to determine the effect of growth medium on enterococcal adherence to mammalian cells in vitro. We cultured human bladder cells (HTB-5) and canine kidney cells (MDCK) in DMEM with 10% Fetal bovine serum, and calf serum respectively. We then transferred the cells to chamber slides at a concentration of 3.25 X 105 cells per chamber. Human urine, calf serum, and tryptic soy broth were used to grow the bacteria overnight. E. faecalis were then pelleted out of the growth media and resuspended in 0.5 mL of DMEM with 1% calf serum. The eukaryotic cells were rinsed twice with the DMEM with 1% calf serum, and then inoculated with bacteria, and left to incubate for an hour with occasional agitation. At the end of the incubation period the bacteria containing medium was removed, and the cells were rinsed twice with the DMEM with 1% calf serum. The cells were fixed and stained with Giemsa-Wright stain. Cells with >20 bacteria were counted as positive; at least 100 cells were counted in each chamber. In this study, HBT-5 cells had adherent bacteria but MDCK did not. Initial tabulations have shown that enterococci grown in urine have an increased adhesiveness (as compared to serum or TSB), consistent with their common behavior in vivo.
Isolation of Phage in Raw Sewage and Human Saliva
E. faecalis is a common cause of nosocomial infections in immunocompromised patients. Clinical isolates are becoming increasingly antibiotic resistant. For this study 23 isolates from patients with urinary bladder infections were obtained. The strains have previously been shown to be E. faecalis, using standard biochemical tests. The goal of this project was to find an alternate approach for treating E. faecalis infections, specifically by using bacteriophage. We attempted to isolate phage utilizing raw sewage from the SLC treatment facility, and from human saliva. An E. faecalis specific bacteriophage from ATCC (Enterococcus faecalis 42) was also obtained. To isolate phage from raw sewage, bacteria and other impurities were "salted out" using NaCl and centrifugation, then precipitated the virus using PEG. To isolate phage from human saliva, a combined sample from approximately 30 donors was collected. Bacteria and human cells were pelleted at 14,000 rpm for 10 minutes, and then filtered. The phage samples from sewage and saliva, plus the positive control, were tested in an overlay plaque assay. Conditions for the assay were adjusted to detect any E. faecalis specific bacteriophage.
Planarian Central Nervous System Regeneration
Since recent research on planarians has focused on stem cells, there is little known concerning at what point during regeneration the CNS of the planarian starts to regenerate. We would like to determine this because, although regeneration is interesting, the order of CNS regeneration in the planarian model could be helpful in further studies of CNS regeneration and in the understanding of the regenerative process.
To determine when CNS regeneration occurs, a sample from each of the matching head population and the tail population will be killed and stored every three days. At the end, the samples will be stained and analyzed. Samples will be killed, stained and analyzed similar to standards found in Identification of Immunological Reagents for Use in the Study of Freshwater Planarians by Means of Whole-Mount Immunofluorescence and Confocal Microscopy (Robb and Alvarado 2002).
Materials and methods from the above research article:
Freshwater planarians will be cultured at room temperature in pond water in a petri dish. Planarians are fed beef liver puree twice a week and starved for 1 week prior to experimentation.
The planarians are killed in 2% HC1 for 30 sec and then fixed in Carnoy's (six parts ethanol, three parts chloroform, one part glacial acetic acid) for 2 hours at room temperature followed by three methanol rinses. The animals then are bleached overnight in 6% hydrogen peroxide in methanol, rinsed briefly, and stored in 100% methanol (at -20°C). 75%; 50%; 25% methanol in phosphate-buffered solution + 0.3% Triton X-100 (PBST) are used to rehydrate planarians. PBST is used to rinse the planarians and then they are blocked for 2 hours at room temperature in PBST + 0.25% bovine serum albumin (PBSTB). Samples are separated into small 8-ml scintillation glass vials and incubated overnight at room temperature on a rotating platform in 500µl of primary antibodies (22C10) diluted in PBSTB. The planarians are rinsed again and washed in PBSTB for 6-8 hours at room temperature. They are then incubated overnight in secondary antibodies diluted in PBSTB [1:400 Alexa 488 anti-mouse]. After incubation, the planarians are washed and observed under a fluorescence microscope.
Condoms: Iatrogenic Transmission, or Causes for HIV Transmission in Africa
AIDS has become one of the largest epidemics of our time. A disease that its transmission started out very slow and increased exponentially to disastrous proportions. While every sector of the world is affected by the disease, one area continues to get hit the hardest – sub-Saharan Africa. 29.4 million people in sub-Saharan Africa are believed to be HIV positive (UNAids, 2002) with an annual increase of 12%. (Brewer, et al., 2003). This accounts for 70% of the 42 million cases of HIV world wide (UNAids, 2002). This raises an important question that must be asked – why Africa? Many researchers attribute this to a lack of education and prevention programs in Africa. According to the World Health Organization's (2002) World Health Report 2002, "current estimates suggest that more than 99% of HIV infections are attributable to unsafe sex" (p. 9). That, coupled with severe lack of funds for medication and health care, yield the staggering numbers we are currently seeing. A small group of researchers, however, disagree with this cause. They believe that the majority of HIV was transmitted not through unsafe sexual practices, but rather unhygienic medical practices. These researchers have stated that unprotected sexual activity with infected people is not enough to maintain the epidemic numbers of HIV cases and transmission methods must be coupled with more higher risk activity such as intravenous drug use, unsafe medical care, or anal sex. This is a highly debated issue within scientific and political communities. I would like to write a thesis that explores unsafe medical practices that plague sub-Saharan Africa with its biological and political implications for why this is a continuing problem.
UNAids (2002) rejects this and states that sexual transmission and medical care transmissions account for 90% and 5%, respectively, of HIV occurrences in sub-Saharan Africa. Other researchers think that injections make up 40-60% of cases and sexual transmission is about one third (Gisselquist, Potterat, Brody, and Vachon, 2003). These researchers believe that the epidemic was started and has been maintained by the poor medical care that litters Africa. They point to the mass polio vaccination campaigns in the 1950's for the beginning of this debacle (Elswood and Stricker, 1994). At the time, the newly formed World Health Organization (WHO), began humanitarian relief by providing mass polio vaccinations for those in Africa to prevent its spread and ultimately lead to its elimination. It is thought that the needles that were used to give mass vaccination and antibiotic treatment were unclean and unsterile. These infected needles were able to infect hundreds of people within a few hours.
One of the first issues that must be addressed is when and where did HIV originate. While no one theory has been accepted universally, the majority of researchers agree that HIV-1 derived from a simian progenitor. According to Elswood et al. (1994) Africa has had a long history of butchering and eating of monkeys. The simian progenitor could have mutated and crossed into a different host during this type of overlap. The question that follows is why, during this century, did the progenitor mutate. One reason that has been suggested points to the unsterile used needles. The simian progenitor could be ingested into the body and lived for a couple of weeks. Since the virus lacks the specificity for the host, eventually that virus would be eliminated by the immune system. However, if there were many people who were sharing blood, which basically occurs when hypodermic needles are shared, the virus in one person would have
Unhygienic medical practices are not a problem of the 1950s and 1960s though. Researchers argue that a majority of HIV infections actually come from medical procedures rather than sexual transmission (Brewer, et al. 2003). It is thought that poor hygiene in hospitals is linked with needles contaminated with HIV. Iatrogenic transmission of HIV is very controversial. Giseelquist et al. (2003), have demonstrated the disconnect between data that suggest `heterosexual cause' of HIV transmission and the epidemic numbers of HIV in sub-Saharan Africa.
Gissellquist et al. (2003) have suggested that iatrogenic transmission of HIV is more prevalent given the data previously published works. They pointed to a number of factors: `discontinuity between HIV and STIs' (144), `transmission efficiency' (144), `reported sexual activity'(145), `transmission dynamics' (145), and other `anomalous findings' (145).
In the thesis, I would like to explore the connection(s) between medical care and HIV prevalence as related to humanitarian efforts of non-government organization in sub-Saharan Africa. This connection between politics and biology has not been elucidated. Moreover, this is a critical piece because it informs and explains why sub-Saharan Africa has reached epidemic proportions of HIV cases.