Efficient Down-Regulation of Glia Maturation Factor Expression in Mouse Brain and Spinal Cord

Long-lasting siRNA-based down-regulation of gene of interest can be achieved by lentiviral-based expression vectors driving the production of short hairpin RNA (shRNA). We investigated an attractive therapeutic approach to target the expression of proinflammatory GMF by using lentiviral vector encoding GMF-specific shRNA to reduce GMF levels in the spinal cord and brain of mice. To determine the effect of GMF-shRNA on GMF protein levels, we performed quantitative ELISA analysis in brain and in thoracic, cervical and lumbar regions of spinal cord from mice followed by GMF-shRNA (G-shRNA) or control shRNA (C-shRNA) treatments. Our results show a marked reduction of GMF protein levels in brain and spinal cord of mice treated with GMF-shRNA compared to control shRNA treatment. Consistent with the GMF protein analysis, the immunohistochemical examination of the spinal cord sections of EAE mice treated with GMF-shRNA showed significantly reduced GMF-immunoreactivity. Thus, the down-regulation of GMF by GMF-shRNA was efficient and wide spread in CNS as evident by the significantly reduced levels of GMF protein in the brain and spinal cord of mice.     

Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph-1 mice

J. Neurochem. (2012) 122, 1211-1218. ABSTRACT: In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4) ) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-) ) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4) . In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4) , increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.     

Gut mucosal injury in neonates is marked by macrophage infiltration in contrast to pleomorphic infiltrates in adult: evidence from an animal model

Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. In tissue samples of NEC, we identified numerous macrophages and a few neutrophils but not many lymphocytes. We hypothesized that these pathoanatomic characteristics of NEC represent a common tissue injury response of the gastrointestinal tract to a variety of insults at a specific stage of gut development. To evaluate developmental changes in mucosal inflammatory response, we used trinitrobenzene sulfonic acid (TNBS)-induced inflammation as a nonspecific insult and compared mucosal injury in newborn vs. adult mice. Enterocolitis was induced in 10-day-old pups and adult mice (n = 25 animals per group) by administering TNBS by gavage and enema. Leukocyte populations were enumerated in human NEC and in murine TNBS-enterocolitis using quantitative immunofluorescence. Chemokine expression was measured using quantitative polymerase chain reaction, immunoblots, and immunohistochemistry. Macrophage recruitment was investigated ex vivo using intestinal tissue-conditioned media and bone marrow-derived macrophages in a microchemotaxis assay. Similar to human NEC, TNBS enterocolitis in pups was marked by a macrophage-rich leukocyte infiltrate in affected tissue. In contrast, TNBS-enterocolitis in adult mice was associated with pleomorphic leukocyte infiltrates. Macrophage precursors were recruited to murine neonatal gastrointestinal tract by the chemokine CXCL5, a known chemoattractant for myeloid cells. We also demonstrated increased expression of CXCL5 in surgically resected tissue samples of human NEC, indicating that a similar pathway was active in NEC. We concluded that gut mucosal injury in the murine neonate is marked by a macrophage-rich leukocyte infiltrate, which contrasts with the pleomorphic leukocyte infiltrates in adult mice. In murine neonatal enterocolitis, macrophages were recruited to the inflamed gut mucosa by the chemokine CXCL5, indicating that CXCL5 and its cognate receptor CXCR2 merit further investigation as potential therapeutic targets in NEC.     

Oxidative stress response and fatty acid changes associated with bioaccumulation of chromium [Cr(VI)] by a fresh water cyanobacterium Chroococcus sp.

Cr(VI) at 2.5, 5, 7.5 and 10 mg/l was removed over 1–5 days by a freshwater cyanobacterium, Chroococcus sp. 2.5 mg Cr(VI)/l gave the optimum rate. With 5 mg Cr(VI)/l, activities of superoxide dismutase and catalase were increased. Amounts of palmitic (16:0), stearic (18:0) and oleic acid (18:1) in the cell also increased after exposure to Cr(VI).     

Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis

A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.     

Advanced glycation endproducts: from precursors to RAGE: round and round we go

The formation of advanced glycation endproducts (AGEs) occurs in diverse settings such as diabetes, aging, renal failure, inflammation and hypoxia. The chief cellular receptor for AGEs, RAGE, transduces the effects of AGEs via signal transduction, at least in part via processes requiring the RAGE cytoplasmic domain binding partner, diaphanous-1 or mDia1. Data suggest that RAGE perpetuates the inflammatory signals initiated by AGEs via multiple mechanisms. AGE–RAGE interaction stimulates generation of reactive oxygen species and inflammation—mechanisms which enhance AGE formation. Further, recent data in type 1 diabetic kidney reveal that deletion of RAGE prevents methylglyoxal accumulation, at least in part via RAGE-dependent regulation of glyoxalase-1, a major enzyme involved in methylglyoxal detoxification. Taken together, these considerations place RAGE in the center of biochemical and molecular stresses that characterize the complications of diabetes and chronic disease. Stopping RAGE-dependent signaling may hold the key to interrupting cycles of cellular perturbation and tissue damage in these disorders.     

Design of Carbon Nanotube‐Based Gas‐Diffusion Cathode for O2 Reduction by Multicopper Oxidases

Multicopper oxidases, such as laccase or bilirubin oxidase, are known to reduce molecular oxygen at very high redox potentials, which makes them attractive biocatalysts for enzymatic cathodes in biological fuel cells. By designing an enzymatic gas‐diffusion electrode, molecular oxygen can be supplied through the gaseous phase, avoiding solubility and diffusion limitations typically associated with liquid electrolytes. In doing so, the current density of enzymatic cathodes can theoretically be enhanced. This publication presents a material study of carbon/Teflon composites that aim to optimize the functionality of the gas‐diffusion and catalytic layers for application in enzymatic systems. The modification of the catalytic layer with multiwalled carbon nanotubes, for example, creates the basis for stronger π–π stacking interactions through tethered enzymatic linkers, such as pyrenes or perylene derivates. Cyclic voltammograms show the effective direct electron contact of laccase with carbon nanotube‐modified electrodes via tethered crosslinking molecules as a model system. The polarization behavior of laccase‐modified gas‐diffusion electrodes reveals open‐circuit potentials of +550 mV (versus Ag/AgCl) and current densities approaching 0.5 mA cm² (at zero potential) in air‐breathing mode.