Characterization of Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Expressed in Pichia pastoris

Oxalate oxidase (E.C. 1.2.3.4) catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase fromCeriporiopsis subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. Interestingly, CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC). We show that CsOxOx activity directly correlates with Mn content and other metals do not appear to be able to support catalysis. EPR spectra indicate that the Mn is present as Mn(II), and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of OxDC from Bacillus subtilis. EPR spin-trapping experiments support the existence of an oxalate-derived radical species formed during turnover. Acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. The pH dependence of this reaction suggests that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated

Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

<p>Abstract</p> <p>Background</p> <p>Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis <it>in vivo </it>unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that <it>chronic </it>HIV-<it>replication in non-endothelial cells </it>may produce novel factors that provoke angiogenic pathways.</p> <p>Methods</p> <p>Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth <it>in vitro </it>over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses.</p> <p>Results</p> <p>By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development.</p> <p>Conclusion</p> <p>Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p = 10^-4to 10^-12). Bioinformatics analyses suggest that overproduction of PTKs and other kinases in HIV-infected cells has <it>suppressed </it>VEGF/VEGFR-PTK expression and promoted <it>VEGFR-independent </it>pathways. This unique mechanism is similar to that observed in neovascularization and angiogenesis during embryogenesis. Validation of clinically relevant proteins by gene-silencing and translational studies <it>in vivo </it>would identify specific targets that can be used for early diagnosis of angiogenic disorders and future development of inhibitors of angiopathies. This is the first comprehensive study to demonstrate that HIV-infection alone, without any co-infection or treatment, can induce numerous "embryonic" proteins and kinases capable of generating novel <it>VEGF-independent </it>angiogenic pathways.</p

Family Therapy

Family therapy is psychotherapy that directly involves family members and attends explicity to interactions among family members. In family therapy, the relational and communicational processes of families are utilized as the primary context for treating psychiatric disorders or solving clinical problems. During the latter 20th century, psychodynamic, structural, strategic, cognitive-behavioral, and postmodern family therapies matured as distinct clinical traditions. More recently, family psychiatry has emerged as a compilation of skills that psychiatrists can utilize in the biopsychosocial treatment of individual patients. Family psychiatry emphasizes family assessment, psychoeducation, and family interventions that reduce symptoms and build resilience against illness recurrence. © 2008 John Wiley & Sons, Ltd

Number of OX1138B and APHIS male moths released over all fields during the trial period.

<p>Black line = OX1138B; grey line = APHIS.</p

Number of moths of both strains released in Fields 1, 2 and 3 during the trial period.

<p>Field 1 = diamond data points with solid line; Field 2 = square data points with dotted line; and Field 3 = triangular data points with dotted line.</p

In-field recapture data during release period.

<p>Mean number of OX1138B and APHIS moths caught per trap is shown for each field separately, and for all three fields combined. Lower recapture rates in Fields 2 and 3 than in Field 1 were likely a result of pesticide treatment in the former. Filled bars = OX1138B, white bars = APHIS moths; error bars indicate Standard Error of Mean. The recapture rates of OX1138B and APHIS moths were significantly different (Poisson regression model, 95% CI: 7.8–33.3%; p<0.01).</p

Moth mating performance.

<p>Initiation of mating between (a) sentinel wild type [APHIS or University of Arizona (UoA)] female moths and released OX1138B, APHIS males or wild males present in the field; and (b) sentinel OX1138B or APHIS female moths and wild males. Initiation of mating was defined as a male and female joined together in the tail-to-tail position typical of mating in Lepidoptera. Wild males also initiated mating with the females in this experiment: three on 26 July, 2007, four on 3 August, 2007 and 59 males on 15 September, 2007 (the high number on this last date reflects the typical wild pink bollworm recapture in traps late in the growing season).</p