The aim of this study was to design a resolution typing system for the HLA-B gene. This technique involves a one-step PCR reaction utilizing genomic DNA and sequence-specific primers to determine the specificity of each allele and to produce a larger primer data base ideal for serological analysis. The application of this technique to serological analysis can improve serology detection which is currently hindered by antibody cross-reactivity and the unavailability of useful typing reagents.

Natural transformation is the process by which cells take up DNA from the surrounding medium under physiological conditions, altering the genotype in a heritable fashion. This occurs without chemical or physical treatment of the cells. Certain Acinetobacter strains exhibit a strong tendency to incorporate homologous DNA into their chromosomes by natural transformation. Transformation in Acinetobacter exhibits several unique properties that indicate this system's superiority as a model for transformation studies or studies which benefit from the use of transformation as an experimental method of gene manipulation. Pseudomonas putida is the natural host of TOL plasmids, ranging between 50 kbp and 300 kbp in size and encoding genes for the catabolism of toluene, meta-toluate, and xylene. These very large, single-copy plasmids are difficult to isolate, manipulate, or modify in vitro. In this study, the TOL plasmid pDKR1 was introduced into Acinetobacter calcoaceticus strains and genetically engineered utilizing natural transformation as part of the process. Following engineering by transformation, the recombinant DNA molecule was returned to the native genetic background of the original host P. putida strain. Specific parameters for the successful manipulation of large plasmids by natural transformation in Acinetobacter were identified and are outlined. The effects of growth phase, total …

The most apparent symptom of boron deficiency in higher plants is a cessation of growth. Deficiency causes a reduction in ascorbate concentration and the absorption of nutrient ions. Addition of ascorbate temporarily relieves deficiency symptoms. In boron sufficient plants the addition of ascorbate to media causes an increased uptake of nutrients. In an attempt to discover if ascorbate addition to deficient plants causes increased ion uptake, radish plants were grown hydroponically in four different strengths of boron solution. A colorimetric assay for phosphorus was performed both before and after supplementation. Results, however, were inconclusive.

The enzyme, aspartate transcarbamoylase (ATCase) from Moraxella (Branhamella) catarrhalis, has been purified. The holoenzyme has a molecular mass of approximately 510kDa, harbors predominantly positive charges and is hydrophobic in nature. The holoenzyme possesses two subunits, a smaller one of 40 kDa and a larger one of 45 kDa. A third polypeptide has been found to contribute to the overall enzymatic activity, having an approximate mass of 55 kDa. The ATCase purification included the generation of cell-free extract, streptomycin sulfate cut, 60 °C heat step, ammonium sulfate cut, dialysis and ion, gel-filtration and hydrophobic interaction chromatography. The enzyme's performance throughout purification steps was analyzed on activity and SDS-PAGE gradient gels. Its enzymatic, specific activities, yield and fold purification, were also determined.

The question of post-mortem interval (PMI) or time since death is often the most sought after piece of information associated with a medical death investigation. Based on the observation that DNA degradation disproportionately affects the analysis of larger genetic loci, it was proposed that DNA degradation, as a result of autolysis or putrefaction, could prove suitable as a potential rate-of-change indicator of PMI. Nine randomly amplified polymorphic DNA (RAPD) analysis primers and three sets of directed amplification primers were evaluated to determine their suitability for use in assessing the degree of DNA fragmentation in tissue samples. They were assessed for amplicon specificity, total DNA target sensitivity, allele monomorphism and the observance of degradation-based profile changes. Markers meeting the requisite criteria were then used to assess a range samples degraded under controlled and uncontrolled conditions. Tissue samples collected from seven domestic pigs (Sus scrofa) were incubated under controlled laboratory or uncontrolled field conditions to produce samples simulating those potentially collected in a forensic case. DNA samples isolated from these specimens were then analyzed at those loci which had been determined to meet the requisite criteria. Collectively, data generated from these analyses indicate that genetic profiles generated by this approach can provide …

During mitogen-induced transformation of human lymphocytes, phosphoglycerate kinase (PGK) exhibits new electrophoretic forms (pl=8.5-8.9). Electrophoresis and electrofocusing showed that the new forms are not due to expression of the autosomally linked isozyme found in semen (PGK-B; pl=9.7). The multiple electrophoretic forms are the result of protease modification of sex-linked PGK-A isozyme.When peripheral lymphocytes from young persons are stimulated in vitro with phytohemagglutinin, a selective increase in the levels of the glycolytic enzymes occurs concomitantly with blastogenesis. Human lymphocytes from a geriatric population were also subjected to mitogen stimulation. The initial levels of the enzymes were essentially identical in lymphocytes from young and old subjects as were mitogenfree cultured controls. However, during mitogen stimulation the cells from the old subjects failed to increase the glycolytic enzymes. This inability to activate glycolysis may be related to the decline in cell-mediated immunity which occurs with advancing age. Triosephosphate isomerase (TPI) has an increased thermolabile component in skin fibroblasts from patients with progeria (41.4 per cent)and Werner's syndrome (20.1 per cent) when compared with normal fibroblasts (0-3 per cent). The incorporation of various protease inhibitors failed to affect the percentages of heat-labile triosephosphate isomerases. The labile component appears to be identical to the deamidated …

Three populations of Southern flying squirrels were studied in the Ouachita Mountains of Arkansas to assess the impact of population subdivision-due to island formation--on the population genetics of Glaucomys volans. One island, one mainland, and one open population were investigated. A 367 nucleotide hypervariable region of mitochondrial DNA was sequenced in individuals from each population. Individuals and populations were compared to assess relatedness. Higher sequence diversity was detected in the open and island populations. One island individual shared characters with both the island and mainland populations. Results support the hypothesis that the mainland population may have reduced gene flow. Also, the island population may have been originally founded by at least two maternal lineages.

The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.

The regulatory gene for pyrimidine biosynthesis has been identified and designated pyrR. The pyrR gene product was purified to homogeneity and found to have a monomeric molecular mass of 19 kDa. The pyrR gene is located directly upstream of the pyrBC' genes in the pyrRBC' operon. Insertional mutagenesis of pyrR led to a 50- 70% decrease in the expression of pyrBC', pyrD, pyrE and pyrF while pyrC was unchanged. This suggests that PyrR is a positive activator. The upstream regions of the pyrD, pyrE and pyrF genes contain a common conserved 9 bp sequence to which the purified PyrR protein is proposed to bind. This consensus sequence is absent in pyrC but is present, as an imperfect inverted repeat separated by 11 bp, within the promoter region of pyrR. Gel retardation assays using upstream DNA fragments proved PyrR binds to the DNA of pyrD, pyrE, pyrF as well as pyrR. This suggests that expression of pyrR is autoregulated; moreover, a stable stem-loop structure was determined in the pyrR promoter region such that the SD sequence and the translation start codon for pyrR is sequestered. β-galactosidase activity from transcriptional pyrR::lacZ fusion assays, showed a two-fold in increase when expressed in a …

The initial purpose of this work was to investigate the interaction of methylmercuric chloride (MMC) and X-irradiation given as a co-insult upon the rat blood-brain barrier (BBB). The indicators used to determine BBB alterations were mortality and the in vivo tissue uptake of radioactive sulfate administered as 3 5S-sodium sulfate. The results of the interaction studies indicated a neutralization of effects when MMC and X-irradiation were given together. X-irradiation as a single insult generally caused an increase in sulfate uptake by the brain regions monitored, whereas MC treatment generally resulted in decreased sulfate uptake. The neutralization patterns following co-insult treatments were somewhat varied in the different brain regions, exhibiting cancellation of effects in some cases and overriding by one insult in other eases. From the data obtained by this work and in the literature, it is hypothesized that the P-L organelle system of the perivascular glia serves as a trap for MMC, preventing MMC from reaching the neurons. The system appears to proliferate in response to increased peroxides in the body fluids, thereby increasing tolerance to larger doses of MMC.

Forensic STR analysis is limited by the quality and quantity of DNA. Significant damage or alteration to the molecular structure of DNA by depurination, crosslinking, base modification, and strand breakage can impact typing success. Two methods that could potentially improve STR typing of challenged samples were explored: an in vitro DNA repair assay (PreCR™ Repair Mix) and whole genome amplification. Results with the repair assay showed trends of improved performance of STR profiling of bleach-damaged DNA. However, the repair assay did not improve DNA profiles from environmentally-damaged bloodstains or bone, and in some cases resulted in lower RFU values for STR alleles. The extensive spectrum of DNA damage and myriad combinations of lesions that can be present in forensic samples appears to pose a challenge for the in vitro PreCR™ assay. The data suggest that the use of PreCR™ in casework should be considered with caution due to the assay’s varied results. As an alternative to repair, whole genome amplification (WGA) was pursued. The DOP-PCR method was selected for WGA because of initial primer design and greater efficacy for amplifying degraded samples. Several modifications of the original DOP-PCR primer were evaluated. These modifications allowed for an overall more robust amplification …

Fatty acid desaturase 2 (FAD2) enzymes are phosphatidylcholine desaturases occurring as integral membrane proteins in the endoplasmic reticulum membrane and convert monounsaturated oleic acid into polyunsaturated linoleic acid. The major objective of this research was to study the expression and function of two cotton FAD2 genes (the FAD2-3 and FAD2-4 genes) and their possible role in plant sensitivity to environmental stress, since plants may increase the polyunsaturated phospholipids in membranes under environmental stress events, such as low temperature and osmotic stress. Two FAD2 cDNA clones corresponding to the two FAD2 genes have been isolated from a cotton cDNA library, indicating both genes are truly expressed in cotton. Model yeast cells transformed with two cotton FAD2 genes were used to study the chilling sensitivity, ethanol tolerance, and growth rate of yeast cells. The expression patterns of the two FAD2 genes were analyzed by reverse transcription polymerase chain reactions (RT-PCR) and Western blot analyses in cotton plants under different treatment conditions. The coding regions of both FAD2 genes were inserted downstream from the CaMV 35S promoter in the pMDC gateway binary vector system. Five different FAD2/pMDC constructs were transformed into the Arabidopsis fad2 knockout mutant background, and multiple potential transgenic Arabidopsis plant …

It was hypothesized that chronic hypoxia will affect various biological processes including developmental trajectory and behavior. To test this hypothesis, embryos were raised to adulthood in severe hypoxic environments (0.5% O2 or 1% O2, 22°C) and analyzed for survival rate, developmental progression, and altered behaviors. Wildtype hermaphrodites survive chronic hypoxia yet developmental trajectory is slowed. The hermaphrodites raised in chronic hypoxia had different phenotypes in comparison to the normoxic controls. First, hermaphrodites exposed to chronic hypoxia produced a significantly lower number of embryos and had a slight increase in male progeny. This suggests that chronic hypoxia exposure during development affects the germline. Second, animals raised in chronic hypoxia from embryos to young adults have a slight increase in lifespan when re-exposed to a normoxic environment, indicating that chronic hypoxia does not negatively decrease lifespan. Finally, hermaphrodites that were raised in hypoxia will lay the majority of their eggs on the area of the agar plate where the bacterial lawn is not present. This is in contrast to animals in normoxia, which lay the majority of their eggs on the bacterial lawn. One hypothesis for this hypoxia-induced egg-laying behavior is that the animal can sense microenvironments in hypoxia. To examine if …

Myzus persicae (Sülzer), commonly known as green peach aphid (GPA), utilizes its slender stylet to penetrate the plant tissues intercellularly and consume copious amounts of photoassimilates present in the phloem sap causing extensive damage to host plants. The compatible interaction between GPA and Arabidopsis thaliana enabled us to characterize plant response to aphid infestation. Upon GPA infestation, Arabidopsis PAD4 (PHYTOALEXIN DEFICIENT4) gene modulates premature leaf senescence, which is involved in the programmed degradation of cellular components and the export of nutrients out of the senescing leaf. Senescence mechanism is utilized by plants to limit aphid growth. In addition, PAD4 provides antixenosis (deters insect settling and feeding) and antibiosis (impair aphid fecundity) against GPA and adversely impact sieve element availability to GPA. Basal expression of PAD4 contributes to antibiosis, and the GPA-induced expression of PAD4 contributes to antixenosis. Mutation in the Arabidopsis stearoyl-ACP desaturase encoding SSI2 (suppressor of SALICYLIC ACID [SA] insensitivity2) gene that results in an accelerated cell death phenotype and dwarfing, also conferred heightened antibiosis to GPA. Results of this study indicate that PAD4 is required for the ssi2-mediated enhanced antibiosis to GPA. The PAD4 protein contains conserved Ser, Asp and His residues that form the catalytic triad of …

Ischemic events of even a very short duration are not tolerated Ill in humans. The human cost of ischemia, when looked at as combined cardiovascular disease, dwarfs all other causes of death in the United States. Annually, CVD kills as many people in the US as does cancer, chronic lower respiratory disease, accidents, and diabetes mellitus combined. In 2005 (the latest year for which final statistics are available), CVD was responsible for 864,480 deaths or 35.3 percent of total deaths for the year. In my study, I have used the nematode Caenorhabditis elegans to determine genetic and environmental modulators of oxygen deprivation a key component of ischemia. I have found that animals with mutations in insulin like signaling pathways, neuronal function, electron transport chain components, germline function, and animals that are preconditioned by being raised on a diet of E. coli HT115 bacteria at 25°C have an enhanced ability to survive long-term (>72 hours) anoxia (<.005 kPa O2) at 20°C. The enhanced anoxia survival phenotype partially correlates with increased levels of carbohydrate stores in the nematodes. Suppression of this enhanced anoxia survival phenotype is possible by altering expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase, the FOXO transcription factor DAF-16, and …

In humans, von Willebrand factor (vWF) is a key component in hemostasis and acts as a 'cellular adhesive' by letting the circulating platelets bind to exposed subendothelium. It also acts as a carrier and stabilizer of factor VIII (FVIII). A dysfunction or reduction of vWF leads to von Willebrand disease (vWD), resulting in bleeding phenotype which affects 1% of the population. Currently there are a variety of animal models used for the study of vWF and vWD; however, they do not possess the advantages found in zebrafish. Therefore, we set out to establish zebrafish as a model for the investigation of vWF and vWD through the use of bioinformatics and various molecular techniques. Using bioinformatics we found that the vWF gene is located on chromosome 18, that the GPIb? protein sequence is conserved. Confirmation of vWF production was shown by means of immunostaining and by RT-PCR, in thrombocytes as well as in veins and arteries. Evidence of vWF involvement in hemostasis and thrombosis was shown using MO and VMO technology to produce a vWD like phenotype, resulting in an increase in TTO and TTA, as well as a reduction in FVIII when blood was tested using the kPTT assay, coinciding …

The pyrimidine biosynthetic pathway is essential and similar in all bacteria. The pathway from Pseudomonas is regulated by nucleotides which bind to the upstream region of the pyrBC’ gene complex. Work in our lab mapped the genes and showed that the pyrB and pyrC’ were part of an overlap complex. The Pseudomonas aeruginosa has one circular chromosome. A former Pseudomonas now called Burkholderia cepacia is similar to P. aeruginosa except that it contains three circular chromosomes (CI, CII, CIII) and one large plasmid. The primary chromosome named CI contains the pyrBC’. To our knowledge there has been no report of the activity of ATCase in Pseudomonas and contrasted with that of Burkholderia. Here, we compare the activity of ATCase in P. aeruginosa and B .cepacia. Cells of both organisms were grown in Pseudomonas minimal medium and in Enriched medium. The ATCase was extracted and partially purified from each sample. It is hypothesized that the B. cepacia has greater activity for ATCase than do the Pseudomonas.

Current energy and environmental challenges are driving the use of cellulosic materials for biofuel production. A major obstacle in this pursuit is poor ethanol tolerance among cellulolytic Clostridium species. The first objective of this work was to establish a potential upper boundary of ethanol tolerance for the cellulosome itself. The hydrolytic function of crude cellulosome extracts from C. cellulolyticum on carboxymethyl cellulose (CMC) with 0, 5, 10, 15, 20 and 25% (v/v) ethanol was determined. Results indicated that the endoglucanase activity of the cellulosome incubated in 5% and 10% ethanol was significantly different from a control without ethanol addition. Furthermore a significant difference was observed in endoglucanase activity for cellulosome incubated in 5%, 10%, 15%, 20% and 25% ethanol in a standalone experiment. Endoglucanase activity continued to be observed for up to 25% ethanol, indicating that cellulosome function in ethanol will not be an impediment to future efforts towards engineering increasing production titers to levels at least as high as the current physiological limits of the most tolerant ethanologenic microbes. The second objective of this work was to study bioethanol production by a microbial co-culture involving Clostridium cellulolyticum and a recombinant Zymomonas mobilis engineered for the utilization of oligodextrans. The …

A new rapid computer assisted micro-titer plate interferon assay system was developed and characterized for use in high capacity clinical and research applications. The biological aspect of the assay was a modification of the assay methods of Finter, Armstrong and McManus. It was an application of spectrophotometric quantification of the reduction of viral cytopathic effect (CPE) as reflected by neutral red dye uptake by viable cells. A computer program was developed for the extrapolation of raw data to reference interferon units.

The ability to synthesize pyrimidine nucleotides is essential for most organisms. Pyrimidines are required for RNA and DNA synthesis, as well as cell wall synthesis and the metabolism of certain carbohydrates. Recent findings, however, indicate that the pyrimidine biosynthetic pathway and its intermediates maybe more important for bacterial metabolism than originally thought. Maksimova et al., 1994, reported that a P. putida M, pyrimidine auxotroph in the third step of the pathway, dihydroorotase (DHOase), failed to produce the siderophore pyoverdin. We created a PAO1 DHOase pyrimidine auxotroph to determine if this was also true for P. aeruginosa. Creation of this mutant was a two-step process, as P. aeruginosa has two pyrC genes (pyrC and pyrC2), both of which encode active DHOase enzymes. The pyrC gene was inactivated by gene replacement with a truncated form of the gene. Next, the pyrC2 gene was insertionally inactivated with the aacC1 gentamicin resistance gene, isolated from pCGMW. The resulting pyrimidine auxotroph produced significantly less pyoverdin than did the wild type. In addition, the mutant produced 40% less of the phenazine antibiotic, pyocyanin, than did the wild type. As both of these compounds have been reported to be vital to the virulence response of P. aeruginosa, …

Murine neuronal networks, derived from embryonic frontal cortex (FC) tissue grown on microelectrode arrays, were used to investigate zinc toxicity at concentrations ranging from 20 to 2000 mM total zinc acetate added to the culture medium. Continual multi-channel recording of spontaneous action potential generation allowed a quantitative analysis of the temporal evolution of network spike activity generation at specific zinc acetate concentrations. Cultures responded with immediate concentration-dependent excitation lasting from 5 to 50 min, consisting of increased spiking and enhanced, coordinated bursting. This was followed by irreversible activity decay. The time to 50% and 90% activity loss was concentration dependent, highly reproducible, and formed linear functions in log-log plots. Network activity loss generally preceded morphological changes. 20% cell swelling was correlated with 50% activity loss. Cultures pretreated with the GABAA receptor antagonists bicuculline (40 mM) and picrotoxin (1 mM) lacked the initial excitation phase. This suggests that zinc-induced excitation may be mediated by interfering with GABA inhibition. Partial network protection was achieved by stopping spontaneous activity with either tetrodotoxin (200 nM) or lidocaine (250 mM). However, recovery was not complete and slow deterioration of network activity continued over 6 hrs. Removal of zinc by early medium changes showed irreversible, catastrophic …