Most bacteriophage escape the confines of the host bacterium by compromising the integrity of its cell wall, an event that results in rupture (lysis) of the cell. The lysis strategy of bacteriophage Qβ is inhibition of cell wall biosynthesis while the cell is growing. To elicit lysis, the maturation protein (A2) of Qβ inhibits the catalytic activity of MurA, an essential, induced fit enzyme in the cell wall biosynthetic pathway. Consequent lysis releases progeny phage into the environment. The research in this dissertation addresses how lysis timing is integrated into Qβ’s life cycle and discerns the molecular basis of the lytic event. Working off the notion that, as displayed by the mature virion, A2 inhibits MurA, we developed an in vivo bioassay to resolve the amount of inhibitory A2 during infection. We found that the amount of free A2 is vastly greater than the amount of virion-associated A2 and that both forms inhibit MurA. Additionally, the amount of A2 correlates to lysis time and the burst size, as mutant Qβ with upregulated expression of A2 (Qβpor) elicit host cell lysis faster and release fewer mature virions than with the wildtype level of A2. This further suggests that protection from Qβ lysis afforded by MurAL138Q is due to perturbed affinity between A2 and MurA. Yeast two-hybrid analysis supports that A2 and MurAL138Q interact with weaker affinity by rendering small colonies compared to yeast containing interacting A2-MurAwt. Scanning mutagenesis of MurA’s surface near L138 identified residues that may be important for A2 contact in the inhibitory complex. Potentially important residues map to a contiguous area on the surface of MurA that spans both lobes on the flexible loop face of the enzyme, suggesting that A2 prevents the induced fit mechanism of MurA in an uncompetitive manner. Subsequent truncation analysis reveals that the aminoterminal half of A2 is sufficient to mediate host cell lysis. Together, these findings insinuate a model in which the amino-terminus of free A2 interacts with, and inhibits MurA. Then, when the infected cell initiates division, septal catastrophe ensues causing the cell to lyse and liberate progeny bacteriophage Qβ.

The goal of the present study is to explore the functional involvement of the N-terminal region in the biological activity of phospholipase A2 (PLA2) enzyme. Native PLA2 from the venoms of Naja naja atra and Bungarus multicinctus and N-terminally mutated N. naja atra PLA2, i.e. an additional Met before Asn-1(M-PLA2), substitution of Asn-1 with Met-1(PLA2(N1M)) and removal of N-terminal seven residues (PLA2(¡µN7)), were employed in this study. Mutations on the N-terminal region insignificantly perturbed the binding ability of PLA2 for Ca2+ and ANS, but the enzymatic activity of mutants drastically decreased. Moreover, an alteration in the secondary structure was observed as revealed by CD spectra. Compared to other mutants, the fine structure of Ca2+-binding site within PLA2(¡µN7)) changed. Additionally, removal of the N-terminal region caused significant alternation in the structures of active site and substrate-binding site as evidenced by the results of fluorescence measurement, chemical modification and denaturation with detergents. In all N-terminal mutants, substituting Ans-1 with Met-1 affected the NNA-PLA2 structure to a least extent. The membrane-damage activity of PLA2(N1M) and M-PLA2 was 89% and 34% that of NNA-PLA2, respectively. PLA2(¡µN7) did not exhibit the membrane-damage activity. Studies on the biological activities of chemically modified N. naja atra PLA2 reflected a dissociation of the enzymatic activity from membrane-damage activity, and suggested the involvement of Trp-18, Trp-61, Lys-65, Tyr-3 and Tyr-63 in membrane-damage activity. Collectively, our data indicate that the intact N-terminus was crucial for maintaining of the functional conformation of PLA2 in the manifestation of the enzymatic activity and membrane-damage activity, and the enzymatic activity of PLA2 is in aid of but not exclusively essential for the membrane-damage effect.

No description available.

Prostaglandin E2, postulated to be immunosuppressive to the tumor bearing host, is produced and excreted in elevated quantities by many tumors. Arachidonic acid, the precursor molecule for PGE2, is released from membrane phospholipids by phospholipase A2. Phospholipase A2 has been proposed as the rate limiting enzyme in the production of prostaglandin E2.Phospholipase A2 from different sources varies in substrate specificities, pH optima, and Ca ++ concentration requirements. Therefore, the determination of its specific activity depends on the development of appropriate incubation, extraction, and identification methodologies.This study attempted to develop methodologies for determination of PLA2 activity using enzymes from snake venom, mouse liver, and normal and tumored mouse mammarytissue. The method of substrate preparation, kind of substrate, amount of protein, length of incubation, and addition of KC1 and deoxycholate were varied. Reaction products were extracted and isolated with hexame, and methylated with diazomethane. The methyl esters were identified by gas liquid chromatography. Quantitative analyses were based on proportionality of experimental peak areas to internal standard peak area.Activity could not be demonstrated with snake venom or liver PLA2 preparations. Low specific activity was obtained in some tumor and normal mammary tissue extracts. These studies will be used as a basis for developing an optimal assay system for PLA2 from normal and tumored mouse mammary tissue.

Thromboxane receptor (TPr) activity is elevated in diabetes and contributes to complications of diabetic kidney disease (DKD). TPr blockade appears to have therapeutic potential. Several rodent models of DKD show attenuation of renal damage and proteinuria upon administration of the TPr antagonist, S18886. However, the cellular targets that underlie the injurious effects of TPr activation in DKD remain to be elucidated. A pilot study in our laboratory subjected a conditionally-immortalized mouse podocyte cell line to high glucose (25 mM D-glucose) and equibiaxial mechanical stretch (an in vitro simulator of increased glomerular capillary pressure associated with glomerular hyperfiltration in early diabetes). qRT-PCR revealed that exposure of podocytes to mechanical stretch (10% elongation) and high glucose for 6 hours yielded a 9-fold increase in TPr mRNA levels vs. controls (non-stretch, 5mM D-glucose + 25mM L-glucose) (p<0.05, n=5). We hypothesized that TPr expression and activity are increased in podocytes during the onset of DKD resulting in maladaptive effects on this key glomerular filtration barrier cell type. We showed that enhanced TPr signaling threatens podocytes viablility. Cultured podocytes treated with the TPr agonist, U-46619 (1 μM) for 24 hours are more vulnerable to apoptosis as quantified by Hoescht 33342 (20% cell death p<0.001, n=3) , TUNEL (30-fold increase, ns, n=3) and Annexin-V labeling (3-fold increase, p <0.001, n=3). To further support these in vitro findings, we developed a transgenic mouse with podocyte-specific overexpression of TPr. A construct consisting of a desensitization resistant mutant of the human TPr with both N- and C-terminal HA-epitope tags under the control of an 8.3 kb fragment of the immediate 5’ mouse NPHS1 promoter was cloned, isolated and injected into FVB/n oocytes that were implanted into pseudopregnant CD1 females. Founders were characterized for TPr transgene expression, and TPr transgene mRNA levels were detected by qRT-PCR. Our in vitro results suggest that increased TPr expression in podocytes of diabetic mice may contribute to filtration barrier damage and have important implications in the development and progression of DKD.

Lysis in cells infected with the ssRNA phage Qbeta is effected by the A2 protein. It was previously shown that a single copy of A2 assembled on the surface of the Qbeta virion inhibited the activity of MurA, which catalyzes the first committed step of murein biosynthesis. This led to a model for lysis timing in which A2 is not active as a MurA inhibitor until assembled into virion particles. Here we report that MurA inactivates purified Qbeta particles. Moreover, over-expression of MurA does not inactivate particles during the Qbeta infection cycle; thus, casting doubt on the notion that completed virions could be the lytic agent in vivo and also that the MurA-virion interaction does not occur in the infected cell. Furthermore, RNA released from particles was found to protect virions from inactivation by MurA in vitro, suggesting that Qbeta RNA might serve as the protective element during the infection cycle. Comparison of A2 accumulation between Qbeta and Qbeta^por mutants, which are Qbeta A2 mutants with a shorter infection cycle and reduced burst size, reveals that a delicate balance between assembled and unassembled A2 levels regulates lysis timing. A new model is proposed in which "free", unassembled A2 inhibits MurA. From in vitro binding studies and genetic analyses it was determined that A2 binds MurA in a closed conformation with UDP-N-acetylglucosamine bound.

Univ., Diss.--Münster, 2004.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 195-227). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 156 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Th. 3e cycle--Pharm.--Paris 5, 1980. N°: 10.