Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Growth promotion of the edible fungus Pleurotus ostreatus by fluorescent pseudomonads

Bacteria were isolated from the mycelial surface of Pleurotus ostreatus and their role in fruiting body induction (fructification) of the edible mushroom P. ostreatus was investigated. Analysis of the bacterial community that colonized the mycelium showed that the species composition and numbers of culturable bacteria differed according to the developmental stage of P. ostreatus. In particular, the population size of fluorescent pseudomonads increased during fruiting body induction. An experiment showed that inoculation of pure cultures of the mycelium with strains of fluorescent Pseudomonas spp. isolated from the mycelial plane of commercially produced mushrooms promoted the formation of primordia and enhanced the development of the basidiome of P. ostreatus. Results of this research strongly suggest that inoculation of the mycelium with specific bacteria may have beneficial applications for mushroom production.     

The action of Cu2+ on Bacillus thuringiensis growth investigated by microcalorimetry

By using an LKB-2277 Bioactivity Monitor, ampoule mode, the heat output of Bacillus thuringiensis growth metabolism has been determined at 28 degrees C and effect of Cu2+ on B. thuringiensis growth was studied. Copper has been regarded as an essential trace element for life. Its deficiency may be the cause of diseases. Cu2+ of different concentration have different effects on B. thuringiensis growth metabolism, Cu2+ of low concentration (0-30 micrograms/ml) can promote the growth of B. thuringiensis, and Cu2+ of high concentration (40-120 micrograms/ml) is able to inhibit its growth and B. thuringiensis can't grow at all when the concentration of Cu2+ is up to 130 micrograms/ml.     

Catalase, but not MnSOD, inhibits glucose deprivation-activated ASK1-MEK-MAPK signal transduction pathway and prevents relocalization of Daxx: hydrogen peroxide as a major second messenger of metabolic oxidative stress

Overexpression of catalase, but not manganese superoxide dismutase (MnSOD), inhibited glucose deprivation-induced cytotoxicity and c-Jun N-terminal kinase 1 (JNK1) activation in human prostate adenocarcinoma DU-145 cells. Suppression of JNK1 activation by catalase overexpression resulted from inhibition of apoptosis signal-regulating kinase 1 (ASK1) activation by preventing dissociation of thioredoxin (TRX) from ASK1. Overexpression of catalase also inhibited relocalization of Daxx from the nucleus to the cytoplasm as well as association of Daxx with ASK1 during glucose deprivation. Taken together, hydrogen peroxide (H(2)O(2)) rather than superoxide anion (O(2) (*-)) acts as a second messenger of metabolic oxidative stress to activate the ASK1-MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-mitogen-activated protein kinase (MAPK) signal transduction pathway.     

High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines

A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/.     

Vernalization-induced flowering in wheat is mediated by a lectin-like gene VER2

A vernalization-related gene VER2 was isolated from winter wheat ( Triticum aestivum L.) using a differential screening approach. The deduced VER2 is a lectin-like protein of 300 amino acids, which contains the presence of a jacalin-like GWG domain. RNA in situ hybridization results demonstrated that VER2 gene expression is restricted to the marginal meristems of immature leaves in vernalized wheat seedlings. No hybridization signal was detected in the epidermal tissue and vascular bundles. However, "devernalization" resulted in the silencing of VER2 gene activity. The gene expression pattern of VER2 induced by jasmonate was similar to that induced by vernalization. Antisense inhibition of VER2 in transgenic wheat showed that heading and maturation time were delayed up to 6 weeks compared with non-transformed wheat and the pBI121empty-vector-transformed wheat. Tissue degeneration at the top of the spike was also noticed in the antisense inhibited transgenic wheat. These results suggest that VER2 plays an important role in vernalization signaling and spike development in winter wheat.     

Polymerization of cardanol using soybean peroxidase and its potential application as anti-biofilm coating material

Soybean peroxidase (20 mg) catalyzed the oxidative polymerization of cardanol in 2-propanol/phospate buffer solution (25 ml, 1:1 v/v) and yielded 62% polycardanol over 6 h. Cobalt naphthenate (0.5% w/w) catalyzed the crosslinking of polycardanol and the final hardness of crosslinked polycardanol film exceeded 9 H scale as pencil scratch hardness, which shows a high potential as a commercial coating material. In addition, it showed an excellent anti-biofouling activity to Pseudomonas fluorescens compared to other polymeric materials such as polypropylene.     

Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.