Ecophysiological factors affecting growth,sporulation and survival of the biocontrol agent Penicillium oxalicum

The effect of temperature, pH, water potential and sources of nitrogen and carbon on the biocontrol agent Penicillium oxalicum were studied in vitro. The fungus is xerotolerant, mesophillic and has a wide pH tolerance. The parameters evaluated (germination, germ tube length, growth rate and sporulation) showed different sensitivities to the environmental factors. Peptone and free amino acids gave the highest growth rates and high levels of sporulation. Xylose, mannose and fructose gave the highest growth rates and mannose induced strong sporulation. The effect of nutrients (mannose + arginine) and water potential was also studied in vivo. The xerotolerant character of the fungus was confirmed. From this study we consider Penicillium oxalicum ecologically competent to perform effectively as a biocontrol agent in the soil environment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.     

Regulation of Hoxb4 induction after neurulation by somite signal and neural competence

Background  

While the body axis is largely patterned along the anterior-posterior (A-P) axis during gastrulation, the central nervous system (CNS) shows dynamic changes in the expression pattern of Hox genes during neurulation, suggesting that the CNS refines the A-P pattern continuously after neural tube formation. This study aims at clarifying the role of somites in up-regulating Hoxb4 expression to eventually establish its final pattern and how the neural tube develops a competence to respond to extrinsic signals.     

Advanced glycation end-products disrupt human endothelial cells redox homeostasis: new insights into reactive oxygen species production

Advanced glycation end-products (AGEs) trigger multiple metabolic disorders in the vessel wall that may in turn lead to endothelial dysfunction. The molecular mechanisms by which AGEs generate these effects are not completely understood. Oxidative stress plays a key role in the development of deleterious effects that occur in endothelium during diabetes. Our main objectives were to further understand how AGEs contribute to reactive oxygen species (ROS) overproduction in endothelial cells and to evaluate the protective effect of an antioxidant plant extract. The human endothelial cell line EA.hy926 was treated with native or modified bovine serum albumin (respectively BSA and BSA-AGEs). To monitor free radicals formation, we used H₂DCF-DA, dihydroethidium (DHE), DAF-FM-DA and MitoSOX Red dyes. To investigate potential sources of ROS, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and mitochondrial inhibitors were used. The regulation of different types of ROS by the polyphenol-rich extract from the medicinal plant Doratoxylon apetalum was also studied for a therapeutic perspective. BSA-AGEs exhibited not only less antioxidant properties than BSA, but also pro-oxidant effects. The degree of albumin glycoxidation directly influenced oxidative stress through a possible communication between NADPH oxidase and mitochondria. D. apetalum significantly decreased intracellular hydrogen peroxide and superoxide anions mainly detected by H₂DCF-DA and DHE respectively. Our results suggest that BSA-AGEs promote a marked oxidative stress mediated at least by NADPH oxidase and mitochondria. D. apetalum plant extract appeared to be an effective antioxidant compound to protect endothelial cells.     

A novel metalloprotease from Bacillus cereus for protein fibre processing

A novel protease produced by Bacillus cereus grown on wool as carbon and nitrogen source was purified. B. cereus protease is a neutral metalloprotease with a molecular mass of 45.6 kDa. The optimum activity was at 45 °C and pH 7.0. The substrate specificity was assessed using oxidized insulin B-chain and synthetic peptide substrates. The cleavage of the insulin B-chain was determined to be Asn³, Leu⁶, His¹⁰-Leu¹¹, Ala¹⁴, Glu²¹, after 12 h incubation. Among the peptide substrates, the enzyme did not exhibit activity towards ester substrates; with p-nitroanilide, the kinetic data indicate that aliphatic and aromatic amino acids were the preferred residues at the P₁ position. For furylacryloyl peptides substrates, which are typical substrates for thermolysin, the enzyme exhibited high hydrolytic activity with a K_m values of 0.858 and 2.363 mM for N-(3-[2-Furyl]acryloyl)-Ala-Phe amide and N-(3-[2-Furyl]acryloyl)-Gly-Leu amide, respectively. The purified protease hydrolysed proteins substrates such as azocasein, azocoll, keratin azure and wool.     

Effect of vasoactive intestinal peptide, peptide histidine isoleucine and growth hormone-releasing factor-40 on bombesin-like immunoreactivity, somatostatin and gastrin release from the perfused rat stomach

Bombesin-like immunoreactivity (BLI) has been demonstrated in neurons of the gastrointestinal tract and gastric BLI secretion can be demonstrated in response to the classical neurotransmitter acetylcholine. Since structurally related peptides VIP, PHI and GRF have to be considered as peptidergic neurotransmitters it was of interest to determine their effect on gastric BLI secretion. Additionally, somatostatin (SLI) and gastrin secretion was examined. The isolated stomach of overnight fasted rats was perfused with Krebs-Ringer buffer via the celiac artery and the effluent was collected via the portal vein. The gastric lumen was perfused with isotonic saline at pH7 or pH2. All four peptides were tested at a dose of 10(-11) M and 10(-8) M at both pH levels and in addition the effect of VIP and PHI was examined at 10(-14) M and 10(-12) M during luminal pH2. At luminal pH7 VIP and PHI stimulated SLI release at 10(-8) M but had no effect on BLI or gastrin secretion. rGRF and hpGRF were both ineffective on SLI and gastrin release while rGRF inhibited and hpGRF stimulated BLI secretion. This effect was not dose related. At luminal pH2 all four peptides stimulated BLI secretion. Stimulation by PHI was already observed at a dose of 10(-14) M while VIP elicited a stimulatory effect at 10(-12) M. PHI at the two lowest concentrations of 10(-14) and 10(-12) M elicited a stimulation of SLI and gastrin release while the same doses of VIP and the higher doses of all four peptides had no effect on SLI and gastrin secretion at an acidic intraluminal pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure-function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the ¹⁴C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer.