Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha

Mammalian tissues express three immunologically distinct peroxiredoxin (Prx) proteins (Prx I, II, and III), which are the products of distinct genes. With the use of recombinant proteins Prx I, II, and III, all have now been shown to possess peroxidase activity and to rely on Trx as a source of reducing equivalents for the reduction of H2O2. Prx I and II are cytosolic proteins, whereas Prx III is localized in mitochondria. Transient overexpression of Prx I or II in cultured cells showed that they were able to eliminate the intracellular H2O2 generated in response to growth factors. Moreover, the activation of nuclear factor kappaB (NFkappaB) induced by extracellularly added H2O2 or tumor necrosis factor-alpha was blocked by overproduction of Prx II. These results suggest that, together with glutathione peroxidase and catalase, Prx enzymes likely play an important role in eliminating peroxides generated during metabolism. In addition, Prx I and II might participate in the signaling cascades of growth factors and tumor necrosis factor-alpha by regulating the intracellular concentration of H2O2.     

Understanding the role of the essential Asp251 in cytochrome p450cam using site-directed mutagenesis, crystallography, and kinetic solvent isotope effect

Proton transfer in cytochromes P450 is a critical step in the activation of molecular oxygen. Extensive study of the P450cam active site has identified several residues that play a central role in dioxygen bond scission. A highly conserved carboxylate, aspartate-251 in P450cam in the distal helix I, participates in a series of hydrogen-bond/ion pairs near the molecular surface and has been implicated in the catalytic mechanism. Mutation of Asp251 is known to lower activity by 2 orders of magnitude and change the rate-limiting step in the catalytic cycle, suggesting a role for an acid functionality in generation of iron-oxygen reactive intermediates. The turnover rates of the Asp251Asn mutant in various protium-deuterium mixtures have been determined and show a significantly larger kinetic solvent isotope effect, with an overall magnitude of 10 compared to 1.8 for the wild-type P450cam. In addition, a much larger number of protons are involved in the rate-limiting step for the Asp251Asn mutant than in the wild-type enzyme. These results indicate that Asp251 is an essential part of the normal proton delivery machinery required for O-O bond scission. The crystal structure of the Aps251Asn mutant obtained from data collected at cryogenic temperatures has been refined to 1.9 A. Key hydrogen bonds required to hold Asp251 in position have been broken which allows the mutant Asn251 side chain to swing out and away from the O2 binding site leading to a more open active site. This change could allow easier access by water and thus contribute to the observed kinetic solvent isotope effects.     

SNS Na+ channel expression increases in dorsal root ganglion neurons in the carrageenan inflammatory pain model

In contrast to conventional T cells, natural killer (NK) 1.1+ T cell receptor (TCR)-alpha/beta+ (NK1+T) cells, NK cells, and intestinal intraepithelial lymphocytes (IELs) bearing CD8-alpha/alpha chains constitutively express the interleukin (IL)-2 receptor (R)beta/15Rbeta chain. Recent studies have indicated that IL-2Rbeta/15Rbeta chain is required for the development of these lymphocyte subsets, outlining the importance of IL-15. In this study, we investigated the development of these lymphocyte subsets in interferon regulatory factor 1-deficient (IRF-1-/-) mice. Surprisingly, all of these lymphocyte subsets were severely reduced in IRF-1-/- mice. Within CD8-alpha/alpha+ intestinal IEL subset, TCR-gamma/delta+ cells and TCR-alpha/beta+ cells were equally affected by IRF gene disruption. In contrast to intestinal TCR-gamma/delta+ cells, thymic TCR-gamma/delta+ cells developed normally in IRF-1-/- mice. Northern blot analysis further revealed that the induction of IL-15 messenger RNA was impaired in IRF-1-/- bone marrow cells, and the recovery of these lymphocyte subsets was observed when IRF-1-/- cells were cultured with IL-15 in vitro. These data indicate that IRF-1 regulates IL-15 gene expression, which may control the development of NK1+T cells, NK cells, and CD8-alpha/alpha+ IELs.     

Imbalance between interstitial collagenase and tissue inhibitor of metalloproteinases 1 in synoviocytes and fibroblasts upon direct contact with stimulated T lymphocytes: involvement of membrane-associated cytokines

OBJECTIVE: To determine whether direct cell-cell contact with stimulated T lymphocytes (a) differentially modulates the production of interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) and tissue inhibitor of metalloproteinases 1 (TIMP-1) on human synoviocytes and dermal fibroblasts, and (b) induces the production of prostaglandin E2 (PGE2); and to identify the membrane-associated factors on T cell surfaces involved in these mechanisms. METHODS: Dermal fibroblasts and fibroblast-like synovial cells (synoviocytes) were cultured with fixed T cells, isolated plasma membranes from T cells, interleukin-1beta (IL-1beta; 250 pg/ml), or transforming growth factor beta (TGFbeta; 5 ng/ml). Culture supernatants were assayed for the production of MMP-1, TIMP-1, and PGE2. The expression of MMP-1 and TIMP-1 messenger RNA was analyzed by Northern blot of total fibroblast RNA. RESULTS: Membranes of stimulated T cells, i.e., human peripheral blood T lymphocytes (PBTL) and the human T cell line HUT-78, induced the production of PGE2 and MMP-1 on both synoviocytes and dermal fibroblasts. TIMP-1 production was enhanced upon contact with PBTL stimulated for short periods of time (2-4 hours) but not for longer periods. Similar results were obtained with CD4+ and CD8+ synovial tissue T cell clones (TCCs), which induced the production of TIMP-1 by fibroblasts when stimulated for short (2-4 hours), but not long, periods of time. This time dependency was not observed with HUT-78 cells. The production of MMP-1 by fibroblasts and synoviocytes upon cellular contact with stimulated T cells was higher than that induced by an optimum concentration of IL-1beta, whereas the production of PGE2 was equivalent or slightly lower. Cell membrane-associated IL-1alpha and tumor necrosis factor a, but not CD69, CD40 ligand, or CD11b, were involved in the induction of MMP-1 and PGE2 production, as shown by blockade experiments using monoclonal antibodies and cytokine antagonists. CONCLUSION: Synovial tissue TCCs and PBTL stimulated for long periods of time trigger the production of PGE2 and MMP-1, but not TIMP-1, in synoviocytes and dermal fibroblasts, thus inducing an imbalance between the metalloenzyme and its inhibitor. These results demonstrate that T cells may affect fibroblast and synoviocyte functions directly (i.e., by contact activation) and indirectly (i.e., by activation of cytokine production in monocyte/macrophages, which in turn, trigger stromal cell functions). Since the production of MMPs in monocyte/macrophages is also induced upon contact with stimulated T cells, our results strongly suggest that contact of synovial cells with chronically stimulated T lymphocytes favors matrix catabolism. By analogy, this mechanism may trigger tissue destruction in vivo and, thus, may potentiate tissue destruction in chronic inflammatory diseases such as RA.     

Infant feeding practices in a tribal community of Melghat region in Maharashtra state

1. Out of 494 infants none was breastfed within 2 hours of delivery. 36% infants received first breastfeed after 24 hours of delivery 91.2% of the mothers used prelacteal feeds. 2. Only 31.06% of the infants were weaned, out of these only 7 received complementary foods between 4-6 months. 3. Thus, it can be concluded that though breastfeeding is practiced by all mothers, there is need for early initiation of breastfeeding and proper weaning habits.     

Three-dimensional structure of meso-diaminopimelic acid dehydrogenase from Corynebacterium glutamicum

Diaminopimelate dehydrogenase catalyzes the NADPH-dependent reduction of ammonia and L-2-amino-6-ketopimelate to form meso-diaminopimelate, the direct precursor of L-lysine in the bacterial lysine biosynthetic pathway. Since mammals lack this metabolic pathway inhibitors of enzymes in this pathway may be useful as antibiotics or herbicides. Diaminopimelate dehydrogenase catalyzes the only oxidative deamination of an amino acid of D configuration and must additionally distinguish between two chiral amino acid centers on the same symmetric substrate. The Corynebacterium glutamicum enzyme has been cloned, expressed in Escherichia coli, and purified to homogeneity using standard biochemical procedures [Reddy, S. G., Scapin, G., & Blanchard, J. S. (1996) Proteins: Structure, Funct. Genet. 25, 514-516]. The three-dimensional structure of the binary complex of diaminopimelate dehydrogenase with NADP+ has been solved using multiple isomorphous replacement procedures and noncrystallographic symmetry averaging. The resulting model has been refined against 2.2 A diffraction data to a conventional crystallographic R-factor of 17.0%. Diaminopimelate dehydrogenase is a homodimer of structurally not identical subunits. Each subunit is composed of three domains. The N-terminal domain contains a modified dinucleotide binding domain, or Rossman fold (six central beta-strands in a 213456 topology surrounded by five alpha-helices). The second domain contains two alpha-helices and three beta-strands. This domain is referred to as the dimerization domain, since it is involved in forming the monomer--monomer interface of the dimer. The third or C-terminal domain is composed of six beta-strands and five alpha-helices. The relative position of the N- and C-terminal domain in the two monomers is different, defining an open and a closed conformation that may represent the enzyme's binding and active state, respectively. In both monomers the nucleotide is bound in an extended conformation across the C-terminal portion of the beta-sheet of the Rossman fold, with its C4 facing the C-terminal domain. In the closed conformer two molecules of acetate have been refined in this region, and we postulate that they define the DAP binding site. The structure of diaminopimelate dehydrogenase shows interesting similarities to the structure of glutamate dehydrogenase [Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., & Stillmann, T.J. (1992a) J. Mol. Biol. 228, 662-671] and leucine dehydrogenase [Baker, P.J., Turnbull, A.P., Sedelnikova, S.E., Stillman, T. J., & Rice, D. W. (1995) Structure 3, 693-705] and also resembles the structure of dihydrodipicolinate reductase [Scapin, G., Blanchard, J. S., & Sacchettini, J. C. (1995) Biochemistry 34, 3502-3512], the enzyme immediately preceding it in the diaminopimelic acid/lysine biosynthetic pathway.     

Knowledge and attitudes of health-care providers toward cancer pain management: a comparison of physicians, nurses, and pharmacists in the state of New Hampshire

The knowledge and attitudes toward cancer pain management of physicians, nurses, and pharmacists in the state of New Hampshire were examined through the use of a statewide survey. Many of the providers who completed the survey, and thus indicated that they treated patients with cancer pain on a regular basis, were not pain or oncology specialists. Most of these providers were quite well informed about the fundamentals of cancer pain management. Approximately 90% of providers in all three groups were not concerned about addiction among cancer patients. Yet, there was a small percentage of providers who responded in less than optimal ways to items dealing with opioid pharmacology, pain assessment, and the importance of pain relief. Comparison of responses among provider groups indicated that nurses were the most knowledgeable and pharmacists the least knowledgeable about pain assessment. Physicians were the most knowledgeable regarding opioid pharmacology but seemed the least committed to providing optimal pain relief. Further analysis identified a small group of physicians that included a disproportionately high percentage of family practitioners and surgeons who consistently responded in less than optimal ways to items dealing with the importance of pain relief. The results of this study indicate a continuing need for broad-based educational programs in cancer pain management and for new initiatives focused on practitioners who see relatively few cancer patients and may have difficulty accessing traditional educational programs.     

Comparative evaluation of National Committee for Clinical Laboratory Standards broth macrodilution and agar dilution screening methods for testing fluconazole susceptibility of Cryptococcus neoformans

A simple screening method for fluconazole susceptibility of Cryptococcus neoformans using 2% dextrose Sabouraud dextrose agar (SabDex) with fluconazole was compared to the National Committee for Clinical Laboratory Standards (NCCLS) broth macrodilution method. By this method, fluconazole-susceptible C. neoformans isolates are significantly smaller on medium with fluconazole than on fluconazole-free medium. Isolates with decreased susceptibility have normal-size colonies on medium containing fluconazole. The 48-h NCCLS broth macrodilution MICs (NCCLS MICs) for isolates with normal-size colonies on 8- or 16-microg/ml fluconazole plates were predicted to be > or =8 or > or =16 microg/ml, respectively. On medium with 16 microg of fluconazole per ml, all strains (84 of 84) for which the NCCLS MICs were <16 microg/ml were correctly predicted, as were all isolates (7 of 7) for which the MICs were > or =16 microg/ml. Agar dilution appears to be an effective screening method for fluconazole resistance in C. neoformans.