Carbocysteine lysine salt monohydrate (SCMC-LYS) is a selective scavenger of reactive oxygen intermediates (ROIs)

Carbocysteine lysine salt monohydrate (SCMC-Lys) is a well-known mucoactive drug whose therapeutic efficacy is commonly related to the ability of SCMC-Lys to replace fucomucins by sialomucins. The aim of this study was to determine if SCMC-Lys could exert an anti-oxidant action by scavenging reactive oxygen intermediates (ROIs). Our results show that SCMC-Lys proved effective as a selective scavenger of hypochlorous acid (HOCl) and hydroxyl radical (OH.), this effect being related to the reactivity of the SCMC tioether group. The scavenger activity of SCMC-Lys was observed in free cellular system as well as in activated human polymorphonuclear neutrophils (PMNs). SCMC-Lys scavenger activity on HOCl was paralleled by a powerful protection from HOCl-mediated inactivation of alpha1-antitripsin (alpha1-AT) inhibitor, the main serum protease inhibitor. Production of interleukin-(IL-)8, a major mediator of PMN recruitment in inflammatory diseases, is known to be mediated by intracellular OH. SCMC-Lys significantly reduced IL-8 production on stimulated human peripheral blood mononuclear cells (PBMCs) in the same range of concentrations affecting OH. activity. It is concluded that SCMC-Lys could exert, in addition to its mucoactive capacity, an anti-oxidant action, thus contributing to the therapeutic efficacy of SCMC-Lys.     

Interleukin-6 N-terminal peptides modulate the expression of junB protooncogene and the production of fibrinogen in HepG2 cells

Interleukin-6 (IL-6) is a helical cytokine exerting pleiotropic activities including the regulation of hematopoiesis, B cell activation and acute-phase reaction. The structure-function relationship of the molecule is the subject of intensive investigation using point and deletion mutants. Our objective was to analyse the role of the N-terminal 18-46 region in IL-6-mediated expression of junB protooncogene and fibrinogen production, reflecting the acute phase response, with synthetic overlapping peptides. mRNA expression of junB was monitored by competitive RT-PCR, while sandwich ELISA was used for the detection of fibrinogen in the supernatant of HepG2 human hepatoma cells. We found that even short synthetic octapeptides can be stimulatory (in the absence of IL-6) or inhibitory (in the presence of IL-6) in both assays. To establish the molecular mechanism by which synthetic peptides exert their biological effects electromobility shift assay was carried out using HepG2 nuclear extracts. Peptides inducing junB expression initiate gel shifts of STAT3/DNA complexes, which may indicate the involvement of this signal transduction pathway. Circular dicroism spectroscopy data suggest that 8-11-mer peptides representing different parts of the 18-46 region have a marked tendency to adopt ordered conformations in a water/trifluoroethanol (1:1 v/v) mixture. Competition studies with rhIL-6 and selected fluorophore-labelled peptides indicate the presence of more than one binding site on soluble IL-6 receptor. Considering the possible multiple etiologic role of IL-6 in the pathogenesis of various diseases, these peptides could be useful for dissection of IL-6 related biological effects.     

Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph

The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied. Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively. In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA. In artificial seawater (ASW), no significant differences between the two strains were observed. N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively. The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant. A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria. In contrast, serum adsorbed with either wild-type V. cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes. The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

GacS sensor domains pertinent to the regulation of exoproduct formation and to the biocontrol potential of Pseudomonas fluorescens CHA0

In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.     

Lipase-catalyzed enantiotope selective acetylation of 2-acyloxypropane-1,3-diols. Influence of the acyl moiety on the selectivity

Preparation and lipase-catalyzed enantiotope selective acetylation of the prochiral 2-acyloxypropane-1,3-diols (1a–h) including sulfonic ester (1a–c) and carboxylic ester (1d–h) series is described. A strong influence of the acyl moiety in these diols on the enantiotope selectivity of the porcine pancreatic lipase (PPL)-catalyzed reaction with vinyl acetate was observed. The best results were achieved with the 2-(4-methylbenzoyl)oxy- and cyclohexanecarbonyloxypropane-1,3-diols (1g and 1h) resulting in acetylated products (2g) of ≥98% e.e. in 77% yield and (2h) of 95% e.e. in 66% yield, respectively.     

Calcium signal transmission between ryanodine receptors and mitochondria

Control of energy metabolism by increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) may represent a fundamental mechanism to meet the ATP demand imposed by heart contractions, but the machinery underlying propagation of [Ca(2+)] signals from ryanodine receptor Ca(2+) release channels (RyR) to the mitochondria remains elusive. Using permeabilized cardiac (H9c2) cells we investigated the cytosolic [Ca(2+)] ([Ca(2+)](c)) and [Ca(2+)](m) signals elicited by activation of RyR. Caffeine, Ca(2+), and ryanodine evoked [Ca(2+)](c) spikes that often appeared as frequency-modulated [Ca(2+)](c) oscillations in these permeabilized cells. Rapid increases in [Ca(2+)](m) and activation of the Ca(2+)-sensitive mitochondrial dehydrogenases were synchronized to the rising phase of the [Ca(2+)](c) spikes. The RyR-mediated elevations of global [Ca(2+)](c) were in the submicromolar range, but the rate of [Ca(2+)](m) increases was as large as it was in the presence of 30 microm global [Ca(2+)](c). Furthermore, RyR-dependent increases of [Ca(2+)](m) were relatively insensitive to buffering of [Ca(2+)](c) by EGTA. Therefore, RyR-driven rises of [Ca(2+)](m) appear to result from large and rapid increases of perimitochondrial [Ca(2+)]. The falling phase of [Ca(2+)](c) spikes was followed by a rapid decay of [Ca(2+)](m). CGP37157 slowed down relaxation of [Ca(2+)](m) spikes, whereas cyclosporin A had no effect, suggesting that activation of the mitochondrial Ca(2+) exchangers accounts for rapid reversal of the [Ca(2+)](m) response with little contribution from the permeability transition pore. Thus, rapid activation of Ca(2+) uptake sites and Ca(2+) exchangers evoked by RyR-mediated local [Ca(2+)](c) signals allow mitochondria to respond rapidly to single [Ca(2+)](c) spikes in cardiac cells.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.