Thesis (M.S.)--University of Texas at Dallas, 2007. / Includes vita. Includes bibliographical references (leaves 62-64)

Two of the existing assay procedures for dopamine B-hydroxylase were studied with respect to their suitability in the measurement of low levels of activity. Specifically, the spectrophotometric method and the coupled radioenzymatic methods were assessed relative to their ability to detect DBH activity in the perfusate of isolated rat tail arteries incubated under conditions known to inhibit the sodium pump. The spectrophotometric method , was the initial choice because of its low cost, greater ease of performance, and the fact that DBH is assayed under saturating conditions. Although it was found that this method suffered from a lack of sensitivity, attempts were made to improve this. The blank value was reduced and stabilized by the introduction of a mixture of 5 X 10⁻⁴ M fusaric acid and 5 X 10⁻⁴ M EDTA, which produced routine blank levels of ΔA= 0.005, equal to the lowest reported literature values. Also, the effect of ADP activation was investigated for its suitability in the procedure. Despite a three-fold increase in measured DBH activity, however, this method was still not sensitive enough to detect the enzyme activity in tissue incubates, although these improvements made the method more suitable for the assessment of levels of DBH activity in the serum of laboratory animals. The second choice was the coupled radioenzymatic method of Molinoff, because of its inherently greater sensitivity. While initial experiments proved this procedure effective in the detection of the release of DBH from rat vas deferens under conditions known to stimulate maximum exocytosis it was felt that problems could be encountered in the measurement of DBH activities obtained under less than optimal conditiona. Therfore, the procedure was investigated with respect to maximizing the absolute sensitivity. Preliminary studies on the feasibility of extended incubation in the first step, led to a rationale in which the two enzymic reactions were isolated and studied separately. In the second step, it was found that tyramine as expected, inhibited the PNMT reaction, although with sigmoidal kinetics. Paradoxically, non-dialyzable and heat stable impurities that inhibited PNMT were found in commercial preparations of catalase, although the addition of ascorbate eliminated this effect. Bovine serum albumin was found to selectively activate PNMT in a highly concentration dependent manner, with a five-fold maximum activation resultant from the inclusion of 0.14% BSA in the sample aliquot. Blank mixture and reaction time were investigated, and a doubling of sensitivity was found to result from limiting reaction time for the second step to 25 minutes. The PNMT was also shown to be unsaturated with respect to SAM, and an increase in the total concentration of SAM by the addition of labelled and unlabelled SAM to a concentration 40 times normal, was found to make the second reaction linear with respect to octopamine concentration. Fin ally, the [PNMT] could be increased up to 20 times without affecting linearity, and this produced an increase in sensitivity in direct proportion to the increase in enzyme concentration. These modifications were sufficiently effective so as to allow an increase in the concentration of tyramine in the first reaction mixture without too much of a loss of activity. This made the DBH reaction linear with respect to both time and enzyme concentration, which greatly improves accuracy and correlatability of results. Trial experiments proved that the combination of all these modifications into an improved procedure resulted in an assay with an improvement in sensitivity of at least two orders of magnitude over the standard method, with vastly improved characteristics of time and concentration linearity. The suitability of this method for our planned tissue release studies was also confirmed. It is hoped that the improvement in sensitivity and linearity of this modified procedure will allow its use in new experimental situations. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Prolyl Hydroxylase Domain 2 (PHD2) has been identified as a key oxygen sensor in humans along with Factor Inhibiting Hypoxia Inducible Factor (FIH). As such PHD2 and FIH play critical roles in myriad pathways of medical relevance by hydroxylation of their target substrate hypoxia inducible factor (HIF), a transcription factor responsible for the regulation of over 100+ genes. With such critical roles in human physiology the ability to selectively regulate these two enzymes could potentially lead the way for novel therapeutic treatments of a vast array of disease states from cancer to myocardial infarction. We report on three classes of iron chelators which show promise for independent regulation of the HIF hydroxylases. Compounds representing the pyrones/pyridinones, pyridines and catechols were tested and found to have differential impacts on PHD2 and FIH under the same experimental conditions. The mode of inhibition is the result of binding to the active site iron and is supported by UV-visible and electroparamagnetic resonance spectroscopy. PHD2 at the current time does not have a well resolved mechanistic understanding regarding its catalytic cycle and subsequent rate determining steps. We have employed pH, solvent isotope, and X-ray absorption studies in an effort to gain further understanding regarding PHD2's overall mechanism. Our data support that dissociation of an iron(II)-OH2 bond centered about the active site contributes to a portion of the overall rate determining steps in the catalytic reaction of PHD2 that activates oxygen and ends with the production of hydroxylated substrate.

No description available.

<p>Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion, steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By immunoscreening human cDNA libraries derived from normal human duodenum and scalp with APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with gastrointestinal dysfunction (p<0.0001) and TH-Abs were significantly correlated to alopecia (p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity <i>in vitro</i> was reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH) constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed <i>in vitro</i> showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH reactivity reflects a cross-reactivity with TPH.</p><p>In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.</p>

Autoimmune polyendocrine syndrome type I (APS) is a monogenous, recessively inherited disease characterised by endocrine and non-endocrine autoimmune manifestations. One fifth of APS I patients suffer from periodic intestinal dysfunction with varying degrees of malabsorbtion, steatorrhea and constipation. Alopecia areata is found in one third of APS I patients. By immunoscreening human cDNA libraries derived from normal human duodenum and scalp with APS I sera, we identified tryptophan hydroxylase (TPH) as an intestinal autoantigen and tyrosine hydroxylase (TH) as a dermal autoantigen. Forty-eight percent (38/80) of the APS I patients had TPH antibodies (Ab) and 44% (41/94) showed TH immunoreactivity. No reactivity against TPH or TH was seen in healthy controls. TPH-Abs showed a statistically significant correlation with gastrointestinal dysfunction (p&lt;0.0001) and TH-Abs were significantly correlated to alopecia (p=0.02). TPH-Ab positive APS I sera specifically immunostained TPH containing enterochromaffin cells in normal duodenal mucosa. In affected mucosa a depletion of the TPH containing EC cells was seen. In enzyme inhibition experiments TPH and TH activity in vitro was reduced by adding APS I sera. TPH and TH together with phenylalanine hydroxylase (PAH) constitute the group of pteridine dependent hydroxylases. These are highly homologous enzymes involved in the biosynthesis of neurotransmitters. Immunoprecipitation of PAH expressed in vitro showed that 27% (25/94) of APS I patients had antibodies reacting with PAH, but no associations with clinical manifestations was observed. An immunocompetition assay showed that the PAH reactivity reflects a cross-reactivity with TPH. In conclusion, we have identified TPH and TH as intestinal and dermal autoantigens in APS I, coupled to gastrointestinal dysfunction and alopecia. We have also demonstrated immunoreactivity against PAH in APS I patient sera reflecting a cross-reactivity with TPH.

PhD (Microbiology) / Department of Microbiology / Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity. / NRF

No description available.

Cupins are a large superfamily of enzymatic and non-enzymatic members that contain a conserved β-barrel domain, or double-stranded β-helix (DSBH) fold. The cupin superfamily is one of the most functionally diverse groups of proteins known to exist. The vast majority of cupins contain a mononuclear metal binding site at the core of the DSBH fold capable of binding different metal ions. One of the largest cupin subfamilies is known as the Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases. Prolyl 4-hydroxylases (P4Hs) belong to the group of Fe(II)/αKG-dependent dioxygenases and catalyze the formation of 4R-hydroxyproline (Hyp) from various proline-containing substrates. The formation of Hyp is an important post-translational modification to many different proteins involved in essential biochemical pathways. Abnormalities in these pathways can lead to diseases such as cancer, fibrosis, respiratory issues, scurvy, and stroke. An Fe(II)/αKG-dependent prolyl hydroxylase from Bacillus anthracis (BaP4H) was investigated to understand its substrate recognition ability and catalytic properties. Novel crystal structures were solved that revealed conformational changes upon substrate binding and key interactions of various ligands in the active site for different catalytic steps. Although the majority of cupin family enzymes catalyze a reaction using iron as an essential cofactor, other metal cofactors can allow the diverse biological transformations carried out by this group of enzymes. A class of enzymes known as dimethylsulfoniopropionate (DMSP) lyases uses different metal ions to catalyze the formation of acrylate and dimethylsulfide (DMS) from DMSP. DMSP is one of the most prevalent and significant molecules to the life and biogeochemistry of the oceans. The products DMS and acrylate are environmentally significant and industrially valuable. DMSP is predominantly catabolized by marine bacteria and can serve different functions. One of the most abundant bacteria in the ocean, Pelagibacter, was determined to contain a DMSP lyase DddK. The DddK catalyzed DMSP lyase activity in the presence of different metal ions has shown that it catalytically prefers Ni(II) compared to other transition metal ions examined. Spectroscopic, site-directed mutagenesis, and crystallographic studies illustrate central residues responsible for metal ion binding and possible roles in transition state stabilization. A greater mechanistic understanding of DMSP lyases will lead to more impactful information about global environmental climate regulation.

<i>Claviceps purpurea</i>, a fungal pathogen, of ergot diseases in agriculturally important cereal crops, produces high levels of glycerides containing ricinoleic acid (12-hydroxyoctadec-cis-9-enoic acid) in its sclerotia. A fatty acid hydroxylase (CpFAH) involved in the biosynthesis of ricinoleic acid was recently identified from <i>C. purpurea</i>. This research describes the biochemical characterization of a type II diacylglycerol acyltransferase (CpDGAT2) involved in the assembly of this fatty acid into triglycerides from <i>C. purpurea</i>. Expression of CpDGAT2 in a quadruple mutant <i>Saccharomyses cerevisiae</i> H1246, in which all four triacylglycerol (TG) biosynthesis genes (DGA1, LOR1, ACAT1 and ACAT2) were disrupted, restored the ability of the mutant to synthesize TGs <i>in vivo</i>. <i>In vitro</i> enzymatic assays of microsomal preparations of the transformants indicated that CpDGAT2 preferentially use ricinoleic acid over linoleic acid, oleic acid and linolenic acids as acyl donor, and 1,2-dioleoyl-sn-glycerol over 1,2-dipalmitoyl-sn-glycerol as acyl acceptor. CpDGAT2 did not show any activities for the formation of wax esters and estolides when 1-hexadecanol and triricinolein were used as acyl acceptors. Co-expression of CpFAH and CpDGAT2 in yeast resulted in increased accumulation of ricinoleic acids compared to expression of CpFAH along with the yeast native DGAT2 (ScDGA1) or expression of CpFAH alone. Northern blot analysis indicated that CpFAH is solely expressed in sclerotium cells and no transcripts of this gene were detected in mycelium and conidium cells. CpDGAT2 is more widely expressed in cell types examined except for conidiospores where the expression is low. The highest expression of CpDGAT2 was detected in 20 day-old sclerotium cells where the highest levels of ricinoleate glycerides are accumulated. Collectively, these data indicate CpDGAT2 and CpFAH are two key enzymes coordinating the biosynthesis and bioassembly of ricinoleic acid in <i>C. purpurea</i>.