The involvement of nitric oxide,nitric oxide synthase,neutrophil elastase,myeloperoxidase and proinflammatory cytokines in the acute lung injury caused by phorbol myristate acetate

Phorbol myristate acetate (PMA) causes acute lung injury (ALI). The present study was designed to elucidate the role of nitric oxide (NO), inducible NO synthase (iNOS), neutrophil elastase (NE) and other mediators in the ALI caused by PMA. In isolated rat’s lungs, PMA at various doses (1, 2 and 4 μg/g lung weight) was added into the lung perfusate. Vehicle group received dimethyl sulfoxide (the solvent for PMA) 100 μg/g. We measured the lung weight changes, pulmonary arterial pressure, capillary filtration coefficient, exhaled NO, protein concentration in bronchoalveolar lavage (PCBAL) and Evan blue dye leakage. Nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and myeloperoxidase (MPO) in lung perfusate were determined. Histopathological examination was performed. We detected the iNOS mRNA expression in lung tissue. PMA caused dose-dependent increases in variables for lung changes, and nitrate/nitrite, methyl guanidine, proinflammatory cytokines, NE and MPO in lung perfusate. The pathology was characterized by alveolar hemorrhagic edema with inflammatory cell infiltration. Scanning electron microscopy revealed endothelial damage. PMA upregulated the expression of iNOS mRNA. Our results suggest that neutrophil activation by PMA causes release of NE, upregulation of iNOS and a series of inflammatory responses leading to endothelial damage and ALI.     

Surface plasmon resonance detection of interactions between peptide fragments of N-telopeptide and its monoclonal antibodies.

As populations age, osteoporosis is becoming an important public health care problem. Urinary level of the cross-linked N-telopeptide of type I collagen has been reported to be a sensitive marker of bone resorption. Recently, we synthesized and characterized 10 overlapping peptides covering the N-telopeptide of alpha-2 type I collagen and reported their relative binding response to anti-type I collagen cross-linked N-telopeptide (NTX) antibodies determined by a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). In this study, we design an assay based on the surface plasmon resonance (SPR) technology to detect binding interaction of each peptide fragment of NTX with the anti-NTX monoclonal antibodies. Anti-NTX monoclonal antibodies were immobilized on the surface of sensor chip by amine-coupling procedure. Serial dilutions of each peptide were prepared and injected separately onto the antibodies-immobilized sensor chip. The real-time association and dissociation interactions of each peptide were detected and reported as sensorgrams. Binding response of each peptide to the monoclonal antibodies was determined, and the SPR results were compared with the ELISA results. We demonstrate that the trends of binding potency of peptide fragments detected by SPR are in good correlation to the results obtained by ELISA, indicating that our developed SPR-based method can be further applied to detect the NTX fragments in urine and to monitor the bone loss in humans. The potent peptide fragments identified by both assays are promising for further preparation of specific monoclonal antibodies in order to develop bioassays for bone loss in humans.     

Cytochrome P450 (CYP105F2) from Streptomyces peucetius and its activity with oleandomycin

The cytochrome P450 enzyme is one of the most versatile redox proteins and it is responsible for the oxidative metabolism of a wide variety of endogenous and exogenous compounds. The cytochrome P450 gene, CYP105F2, from Streptomyces peucetius was subcloned into the pET-32a(+) vector to overexpress the protein in E. coli BL21 (DE3) pLysS. The expressed enzyme was purified by fast protein liquid chromatography with a DEAE and UNO Q column. A 3D model was constructed based on the known crystallographic structures of cytochrome P450, and comparison with PikC and MoxA signified broad substrate specificity toward structurally diverse compounds. In addition, the in vitro hydroxylation of oleandomycin by purified CYP105F2 observed in liquid chromatography/mass spectrometry and mass/mass spectrometry indicated its flexibility towards alternative polyketides for the structural diversification of the macrolide by post-polyketide synthase hydroxylation.     

Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein

Using Caenorhabditis elegans genetic screens, we identified receptor-mediated endocytosis (RME)-4 and RME-5/RAB-35 as important regulators of yolk endocytosis in vivo. In rme-4 and rab-35 mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME-4 and RAB-35 function downstream of clathrin, upstream of RAB-7, and act synergistically with recycling regulators RAB-11 and RME-1. We find that RME-4 is a conserved DENN domain protein that binds to RAB-35 in its GDP-loaded conformation. GFP-RME-4 also physically interacts with AP-2, is enriched on clathrin-coated pits, and requires clathrin but not RAB-5 for cortical association. GFP-RAB-35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in rme-4 mutants. We propose that RME-4 functions on coated pits and/or vesicles to recruit RAB-35, which in turn functions in the endosome to promote receptor recycling.     

Functional dissection of the promoter of the interphotoreceptor retinoid-binding protein gene: the cone-rod-homeobox element is essential for photoreceptor-specific expression in vivo.

The essential control elements in the interphotoreceptor retinoid-binding protein gene (IRBP) promoter are located between -156 and +19. The -156/-109 sequence contains a retina-specific DNAse I footprint and shows a positive regulatory activity in transiently transfected retinoblastoma cells. The -105/-85 sequence is G/C rich, shows a non-tissue specific DNAse I hypersensitivity, and a negative regulatory activity in retinoblastoma cells. The -76/-42 sequence shows a retinal-specific footprint and contains a "cone-rod-homeobox element" (CRXE) and a "photoreceptor conserved element" (PCE). IRBP promoter fragments with mutations in either CRXE, PCE or in both were linked to reporter genes and analyzed both by transient transfection and in transgenic mice. In retinoblastoma cells, the mutated CRXE-containing promoter shows a 60% repression of the CAT activity whereas the mutated PCE-containing promoter shows a 30% repression. In HeLa cells transfected with these promoters, co-transfection of a Crx expression vector with wild-type, but not with CRXE mutant promoter, activates CAT activity 20-fold over the background activity. Mutation of PCE alone or conversion of CRXE to PCE reduces this Crx-activated CAT activity to only 4-fold over the background activity. In the transgenic mouse experiments, none of the 12 lines with CRXE mutant promoter show significant expression of lacZ in the retina. In contrast, 9 of the 17 transgenic lines with PCE mutant promoter show photoreceptor-specific lacZ expression. Thus the Crx interaction with CRXE is essential for the photoreceptor-specific activity of the IRBP promoter in vivo. This interaction does not appear to require PCE, but is enhanced when PCE is present.     

Functional identification of rub52 gene involved in the biosynthesis of rubradirin

An open reading frame, rub52, has been identified as a gene encoding thymidine diphospho-glucose 2,3-dehydratase by sequence analysis of the rubradirin biosynthetic gene cluster of Streptomyces achromogenes var. rubradiris NRRL3061.The gene codes for a protein consisting of 458 amino acids with calculated molecular mass of 50862 Da. The gene was amplified and heterologously expressed in Escherichia coli as a soluble His-tagged fusion protein. C-2 deoxygenation functionality of thymidine diphospho-4-keto-6-deoxyglucose was assigned to the rub52 gene product from in vitro enzyme assay.     

Decolorization of the textile dyes by newly isolated bacterial strains

Six bacterial strains with the capability of degrading textile dyes were isolated from sludge samples and mud lakes. Aeromonas hydrophila was selected and identified because it exhibited the greatest color removal from various dyes. Although A. hydrophila displayed good growth in aerobic or agitation culture (AGI culture), color removal was the best in anoxic or anaerobic culture (ANA culture). For color removal, the most suitable pH and temperature were pH 5.5-10.0 and 20-35 degrees C under anoxic culture (ANO culture). More than 90% of RED RBN was reduced in color within 8 days at a dye concentration of 3,000 mg l(-1). This strain could also decolorize the media containing a mixture of dyes within 2 days of incubation. Nitrogen sources such as yeast extract or peptone could enhance strongly the decolorization efficiency. In contrast to a nitrogen source, glucose inhibited decolorization activity because the consumed glucose was converted to organic acids that might decrease the pH of the culture medium, thus inhibiting the cell growth and decolorization activity. Decolorization appeared to proceed primarily by biological degradation.     

Effect of ozone treatment on Eimeria colchici oocysts

The effect of ozone on the inhibition of the sporulation of Eimeria colchici oocysts in vitro was examined. Lower sporulation ratios were found to correspond directly to longer ozone exposure time. In pheasants, Phasianus colchicus, orally inoculated with ozone-treated oocysts, lower mortality and lower oocysts per gram of feces were observed as compared with birds given untreated oocysts. Thus, treatment of E. colchici oocysts with ozone alone was observed to partially inhibit the growth and infectivity of the oocysts.