Characterization of the flgG operon of Rhodobacter sphaeroides WS8 and its role in flagellum biosynthesis

The effects of water-soluble nonstarch polysaccharides (sNSP) on human metabolism are considered to be beneficial because they decrease postprandial glycaemia and insulinaemia following ingestion of starch-rich foods. The mechanisms by which sNSP attenuate the postprandial rise in blood glucose are not well understood but their presence increases the viscosity of gastrointestinal contents, which affects physiological functions, e.g. gastric emptying and peristalsis. Increased viscosity and decreased water activity during hydrothermal treatment of starch could influence alpha-amylase action.Using guar galactomannan as a representative of sNSP, we found that galactomannan has a direct noncompetitive inhibitory effect on alpha-amylase with a K(i) value of approximately 0.5% (3.3 microM). The inhibition is not time dependent and studies suggest direct binding of the enzyme to galactomannan; the resulting galactomannan-amylase complex being inactive. Processing of starch at low water levels greatly affects the catalytic efficiency of alpha-amylase. The Km value for starch heat treated in limited water is raised and kcat is lowered relative to starch gelatinised in excess water. Since galactomannan has no effect on the Km of alpha-amylase, we conclude that the inhibitory action of the polymer is not secondary to a decrease in available water. Neither does it seem to be a consequence of impaired diffusion of enzyme, substrate and products because of an increase in viscosity of the medium.Thus, the effects of sNSP in lowering postprandial glycaemia not only involve modifications of gut physiology, but also include direct inhibition of the first stage in the biochemical degradation of starch.     

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline

Although cis-diamminedichloroplatinum (II) (cisplatin) is a potent anticancer drug, clinical use of this agent is highly limited predominantly because of its strong side effects on the kidney and gastrointestinal tracts. We found that cisplatin impaired respiratory function and DNA of mitochondria in renal proximal tubules and small intestinal mucosal cells, thereby inducing apoptosis of epithelial cells. Cisplatin-induced mitochondrial dysfunction and DNA (mtDNA) injury, lipid peroxidation, and apoptosis of epithelial cells in the kidney and small intestine were strongly inhibited by L-carnitine. However, carnitine had no appreciable effect on the tumoricidal action of cisplatin against cancer cells inoculated in the peritoneal cavity. These results indicate that L-carnitine may have therapeutic potential for inhibiting the side effects of cisplatin and other anticancer agents in the kidney and small intestine.     

Three different binding sites of Cks1 are required for p27-ubiquitin ligation

Previous studies have shown that the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) is targeted for degradation by an SCF(Skp2) ubiquitin ligase complex and that this process requires Cks1, a member of the highly conserved Suc1/Cks family of cell cycle regulatory proteins. All proteins of this family have Cdk-binding and anion-binding sites, but only mammalian Cks1 binds to Skp2 and promotes the association of Skp2 with p27 phosphorylated on Thr-187. The molecular mechanisms by which Cks1 promotes the interaction of the Skp2 ubiquitin ligase subunit to p27 remained obscure. Here we show that the Skp2-binding site of Cks1 is located on a region including the alpha2- and alpha1-helices and their immediate vicinity, well separated from the other two binding sites. All three binding sites of Cks1 are required for p27-ubiquitin ligation and for the association of Skp2 with Cdk-bound, Thr-187-phosphorylated p27. Cks1 and Skp2 mutually promote the binding of each other to a peptide similar to the 19 C-terminal amino acids of p27 containing phosphorylated Thr-187. This latter process requires the Skp2- and anion-binding sites of Cks1, but not its Cdk-binding site. It is proposed that the Skp2-Cks1 complex binds initially to the C-terminal region of phosphorylated p27 in a process promoted by the anion-binding site of Cks1. The interaction of Skp2 with the substrate is further strengthened by the association of the Cdk-binding site of Cks1 with Cdk2/cyclin E, to which phosphorylated p27 is bound.     

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.     

Ineffective humoral immunity in the elderly

As individuals age, dysfunction of the immune system leads to an increased incidence of infectious disease. Due to the complex network of cellular interactions and the multi-factorial process of aging, numerous impairments in humoral immunity have been reported. Advances in technology have allowed scientists to begin to identify the molecular mechanisms behind the age-associated decline.     

An XML message broker framework for exchange and integration of microarray data

MOTIVATION: Microarrays are an important research tool for the advancement of basic biological sciences. However this technology has yet to be integrated with clinical decision making. We have implemented an information framework based on the Microarray Gene Expression Markup Language (MAGE-ML) specification. We are using this framework to develop a test-bed integrated database application to identify genomic and imaging markers for diagnosis of breast cancer. RESULTS: We developed extensible software architecture for retrieving data from different microarray databases using MAGE-ML and for combining microarray data with breast cancer image analysis and clinical data for correlation studies. The framework we developed will provide the necessary data integration to move microarray research from basic biological sciences to clinical applications. AVAILABILITY: Open source software will be available from SourceForge (http://sourceforge.net/projects/microsoap/).     

Detection of bacterial contamination in starch and resin-based papermaking chemicals using fluorescence techniques

Rapid fluorescence techniques were evaluated for the detection of bacterial contaminants in papermaking chemicals including starch and the resin-based sizes and starch slurries used in the paper industry. Viable and non-viable bacterial cells were visualised by fluorescent probes and detected by epifluorescence microscopy and flow cytometry. The best discrimination ability was obtained with the fluorescent probes LIVE/DEAD and SYBR Green, based on the staining of cellular nucleic acid, and ChemChrome V3, which demonstrated cellular enzymatic activity. The process samples had to be diluted and filtered before fluorescence staining and analysis because they were viscous and contained solid particles. Fluorescence microscopic counts of bacteria in highly contaminated process samples were similar to plate counts, but flow cytometric enumeration of bacterial cells in process samples yielded 2- to 10-fold lower counts compared with plate counts, depending on the consistency of the sample. The detection limits in flow cytometric analysis and in epifluorescence microscopy were 10³–10⁶ cells ml⁻¹ and 10⁵–10⁶ cells ml⁻¹, respectively. Intrinsic bacterial contamination was detectable with fluorescence techniques and highly contaminated process samples could be analysed with fluorescence methods. Electronic Publication     

Tyrosine phosphorylation of the beta3-subunit of the alphaVbeta3 integrin is required for embrane association of the tyrosine phosphatase SHP-2 and its further recruitment to the insulin-like growth factor I receptor

Ligand occupancy of the alphaVbeta3 integrin is required for IGF-I receptor (IGF-IR) phosphorylation of an appropriate duration and for stimulation of IGF-I actions. In vascular smooth muscle cells (SMCs), the tyrosine phosphatase SHP-2 regulates the duration of IGF-IR phosphorylation and biological actions. We determined the role of ligand occupancy of the alphaVbeta3 integrin on beta3 phosphorylation and studied the role of beta3 phosphorylation in regulating both SHP-2 recruitment to the cell membrane and IGF-I-dependent biological responses. Vitronectin binding to alphaVbeta3 induced tyrosine phosphorylation of the beta3-subunit in subconfluent SMCs and was accompanied by increased association of SHP-2 with beta3. In confluent SMCs, the beta3-subunit was constitutively phosphorylated leading to basal binding of SHP-2. The Src kinase inhibitor PP2 caused a concentration-dependent decrease in beta3 phosphorylation and resulted in decreased SHP-2 association with beta3 and with the cell membrane. In contrast to control cells, SMCs expressing a mutant beta3 that had two tyrosines changed to phenylalanines showed a 89.9 +/- 1.2% decrease in beta3 phosphorylation. This decrease was associated with reduced SHP-2 binding to nonphosphorylated beta3 and a corresponding decrease in the membrane association of SHP-2. When IGF-I was added to cells expressing mutant beta3, SHP-2 was not recruited to the Src homology 2 domain-containing tyrosine phosphatase substrate-1 or to IGF-IR. This was associated with prolonged IGF-IR phosphorylation and an impaired cellular DNA synthesis response to IGF-I. These results define a mechanism by which ligand occupancy of alphaVbeta3 regulates the SMC response to IGF-I.