Inhibitory effect of platinum and ruthenium bipyridyl complexes on porcine pancreatic phospholipase A2

Pancreatic phospholipase A(2) (PLA(2)) plays an important role in cellular homeostasis as well as in the process of carcinogenesis. Effects of metallo-drugs used as chemotherapeutics on the activity of this enzyme are unknown. In this work, the interaction between porcine pancreatic PLA(2) and two selected transition metal complexes--tetrachloro(bipyridine) platinum(IV) ([PtCl(4)(bipy)]) and dichloro (bipyridine) ruthenium(III)chloride ([RuCl(2)(bipy)(2)]Cl)--was studied. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and fluorescence spectroscopy have been used to analyse the enzyme activity in the absence and presence of metal complexes and to verify potential binding of these drugs to the enzyme. The tested metal complexes decreased the activity of phospholipase A(2) in an uncompetitive inhibition mode. A binding of the ruthenium complex near the active site of the enzyme could be evidenced and possible modes of interaction are discussed.     

Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes

The effects of schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in H9c2 cardiomyocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transition (MPT), and mitochondrial membrane potential (Deltapsi(m)) values, were examined in (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch and (-)Sch B (2.5-5.0 microM) pretreatments protected against hypoxia/reoxygenation-induced apoptosis of H9c2 cells in a concentration-dependent manner, with (-)Sch B being more potent. The degrees of protection decreased, however, at the higher drug concentrations of 7.5 microM in both (+/-)gamma-Sch-pretreated and (-)Sch B-pretreated cells. The anti-apoptotic effects of the drugs were further evidenced by the suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and the subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase after (-)Sch B pretreatment. Both (+/-)gamma-Sch and (-)Sch B pretreatments increased GSH levels in H9c2 cells, with (-)Sch B being more potent. Hypoxia/reoxygenation challenge caused a depletion in cellular GSH and the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B was associated with enhancement of cellular GSH in H9c2 cells, as compared to the drug-unpretreated control. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening and decreased Deltapsi(m) in H9c2 cardiomyocytes, cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by (+/-)gamma-Sch/(-)Sch B pretreatments was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, as compared to the respective drug-unpretreated controls. The degrees of protection against apoptosis correlated negatively with the extents of Ca(2+)-induced MPT (r=-0.615, P<0.01) and positively with the values of Deltapsi(m) (r=0.703, P<0.01) in (+/-)gamma-Sch/(-)Sch B-pretreated and hypoxia/reoxygenation challenged cells. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in cellular sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.     

A Robust Method for Large‐Scale Multiple Hypotheses Testing

When drawing large‐scale simultaneous inference, such as in genomics and imaging problems, multiplicity adjustments should be made, since, otherwise, one would be faced with an inflated type I error. Numerous methods are available to estimate the proportion of true null hypotheses π₀, among a large number of hypotheses tested. Many methods implicitly assume that the π₀ is large, that is, close to 1. However, in practice, mid‐range π₀ values are frequently encountered and many of the widely used methods tend to produce highly variable or biased estimates of π₀. As a remedy in such situations, we propose a hierarchical Bayesian model that produces an estimator of π₀ that exhibits considerably less bias and is more stable. Simulation studies seem indicative of good method performance even when low‐to‐moderate correlation exists among test statistics. Method performance is assessed in simulated settings and its practical usefulness is illustrated in an application to a type II diabetes study.     

Alterations in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats

Changes in tissue glutathione antioxidant system in streptozotocin-induced diabetic rats for a period of 15 weeks were examined. Total glutathione level was significantly increased in kidney tissue, but were slightly decreased and increased in liver and heart tissues, respectively. The small changes in total glutathione level in the liver and heart, though not statistically significant, were associated with reciprocal alterations in the activity Of -glutamylcysteine synthetase (GCS). While the GCS activity was not changed in kidney tissue, the activity of -glutathione peroxidase was significantly increased in kidney tissue. Insulin treatment could completely or partly normalize almost all of these changes induced by diabetes. However, the decrease in hepatic glutathione S-transferases activity in diabetic rats was not reversed by the insulin treatment. The ensemble of results suggests that the diabetes-induced alterations in tissue glutathione antioxidant system may possibly reflect an inter-organ antioxidant response to a generalized increase in tissue oxidative stress associated with diabetes.Abbreviations AGES advanced glycosylation end-products - EDTA ethylenediamine tetraacetic acid - GCS -glutamylcysteine synthetase - GlyHb glycated hemoglobin - GPX Se-glutathione peroxidase - GRD glutathione reductase - GSH reduced glutathione - GSSG oxidized glutathione - GST glutathione S-transferases - SSA sulfosalicylic acid - STZ streptozotocin     

Immunodominant IgM and IgG Epitopes Recognized by Antibodies Induced in Enterovirus A71-Associated Hand,Foot and Mouth Disease Patients

Enterovirus A71 (EV-A71) is one of the main causative agents of hand, foot and mouth disease (HFMD). Unlike other enteroviruses that cause HFMD, EV-A71 is more frequently associated with severe neurological complications and fatality. To date, no effective licensed antivirals are available to combat EV-A71 infection. Little is known about the immunogenicity of viral non-structural proteins in humans. Previous studies have mainly focused on characterization of epitopes of EV-A71 structural proteins by using immunized animal antisera. In this study, we have characterized human antibody responses against the structural and non-structural proteins of EV-A71. Each viral protein was cloned and expressed in either bacterial or mammalian systems, and tested with antisera by western blot. Results revealed that all structural proteins (VP1-4), and non-structural proteins 2A, 3C and 3D were targets of EV-A71 IgM, whereas EV-A71 IgG recognized all the structural and non-structural proteins. Sixty three synthetic peptides predicted to be immunogenic in silico were synthesized and used for the characterization of EV-A71 linear B-cell epitopes. In total, we identified 22 IgM and four IgG dominant epitopes. Synthetic peptide PEP27, corresponding to residues 142–156 of VP1, was identified as the EV-A71 IgM-specific immunodominant epitope. PEP23, mapped to VP1 41–55, was recognized as the EV-A71 IgG cross-reactive immunodominant epitope. The structural protein VP1 is the major immunodominant site targeted by anti-EV-A71 IgM and IgG antibodies, but epitopes against non-structural proteins were also detected. These data provide new understanding of the immune response to EV-A71 infection, which benefits the development of diagnostic tools, potential therapeutics and subunit vaccine candidates.     

Serogroup,Virulence, and Genetic Traits of Vibrio parahaemolyticus in the Estuarine Ecosystem of Bangladesh

Forty-two strains of Vibrio parahaemolyticus were isolated from Bay of Bengal estuaries and, with two clinical strains, analyzed for virulence, phenotypic, and molecular traits. Serological analysis indicated O8, O3, O1, and K21 to be the major O and K serogroups, respectively, and O8:K21, O1:KUT, and O3:KUT to be predominant. The K antigen(s) was untypeable, and pandemic serogroup O3:K6 was not detected. The presence of genes toxR and tlh were confirmed by PCR in all but two strains, which also lacked toxR. A total of 18 (41%) strains possessed the virulence gene encoding thermostable direct hemolysin (TDH), and one had the TDH-related hemolysin (trh) gene, but not tdh. Ten (23%) strains exhibited Kanagawa phenomenon that surrogates virulence, of which six, including the two clinical strains, possessed tdh. Of the 18 tdh-positive strains, 17 (94%), including the two clinical strains, had the seromarker O8:K21, one was O9:KUT, and the single trh-positive strain was O1:KUT. None had the group-specific or ORF8 pandemic marker gene. DNA fingerprinting employing pulsed-field gel electrophoresis (PFGE) of SfiI-digested DNA and cluster analysis showed divergence among the strains. Dendrograms constructed using PFGE (SfiI) images from a soft database, including those of pandemic and nonpandemic strains of diverse geographic origin, however, showed that local strains formed a cluster, i.e., “clonal cluster,” as did pandemic strains of diverse origin. The demonstrated prevalence of tdh-positive and diarrheagenic serogroup O8:K21 strains in coastal villages of Bangladesh indicates a significant human health risk for inhabitants.Vibrio parahaemolyticus, a halophilic bacterium, is a causative agent of seafood-related gastroenteritis worldwide (5, 13, 41) and one of the major causes of seafood-associated gastroenteritis in the United States, Asia, Europe, and countries where sporadic cases and outbreaks occur regularly (12, 13). The bacterium is prevalent in brackish and marine waters (43). Historically first identified as the causative agent of a gastroenteritis outbreak in Japan in 1950 (14), V. parahaemolyticus is now recognized as one of the most important food-borne pathogens in Asia, causing approximately half of food poisoning outbreaks in Taiwan, Japan, Vietnam, and Southeast Asian countries.The gene encoding the thermostable direct hemolysin (TDH)—manifested as beta-hemolysis when V. parahaemolyticus is plated onto Wagatsuma blood agar (43), i.e., the Kanagawa phenomenon (KP)—has been shown to be present in more than 90% of clinical strains and less than 1% of environmental strains (31, 39). Some strains also possess the gene trh, encoding the TDH-related hemolysin (TRH), or both tdh and trh (18, 43). Another gene, the thermolabile hemolysin gene (tlh), was reported to be present in V. parahaemolyticus (36) and subsequently in all V. parahaemolyticus strains tested (38).V. parahaemolyticus gastroenteritis is a multiserogroup affliction, with at least 13 O serogroups and 71 K serotypes detected (19, 42). In 1996, serogroup O3:K6 was first reported from diarrhea patients in Kolkata, India (32), and subsequently worldwide, as an increasing incidence of gastroenteritis caused by the serogroup O3:K6 was reported in many countries (41). Rapid spreading of serogroup O3:K6 infections in Asia (27, 32), and subsequently in the United States (12), Africa (3), Europe (25), and Latin America (15), indicated its potential as a pandemic pathogen (34, 43). In addition, V. parahaemolyticus serogroup O3:K6 possesses the group-specific (GS) gene sequence in the toxRS operon and ORF8, of the 10 known open reading frames (ORFs) of the O3:K6-specific filamentous phage f237. The GS gene and ORF8 provide genetic markers distinguishing O3:K6 from other serogroups (27, 29). Recent studies have shown O4:K68, O1:K25, O1:K26, O1:K untypeable (O1:KUT), and O3:K46 serogroups to share genetic markers specific for the pandemic serogroup O3:K6 (7, 10, 27, 34, 41). The non-O3:K6 serogroups with pandemic traits are increasingly found worldwide, and therefore, their pandemic potential cannot be ruled out.In Bangladesh, strains of different serogroups having genetic markers for the serogroup O3:K6 of V. parahaemolyticus were reported to have been isolated from hospitalized gastroenteritis patients in Dhaka (7). A systematic surveillance of the coastal areas bordering the Bay of Bengal where diarrheal disease is endemic (1) has not been done. This study, the first of its kind, was undertaken to investigate virulence potential, as well as phenotypic and genotypic traits of V. parahaemolyticus strains occurring in the estuarine ecosystem of Bangladesh.     

Slits and Robo-2 regulate the coalescence of subsets of olfactory sensory neuron axons within the ventral region of the olfactory bulb

Olfactory sensory neurons (OSNs) project their axons to second-order neurons in the olfactory bulb (OB) to form a precise glomerular map and these stereotypic connections are crucial for accurate odorant information processing by animals. To form these connections, olfactory sensory neuron (OSN) axons respond to axon guidance molecules that direct their growth and coalescence. We have previously implicated the axon guidance receptor Robo-2 in the accurate coalescence of OSN axons within the dorsal region of the OB (Cho et al., 2011). Herein, we have examined whether Robo-2 and its ligands, the Slits, contribute to the formation of an accurate glomerular map within more ventral regions of the OB. We have ablated expression of Robo-2 in OSNs and assessed the targeting accuracy of axons expressing either the P2 or MOR28 olfactory receptors, which innervate two different regions of the ventral OB. We show that P2-positive axons, which express Robo-2, coalesce into glomeruli more ventrally and form additional glomeruli in the OB of robo-2^lox/lox;OMP-Cre mice. We also demonstrate that Robo-2-mediated targeting of P2 axons along the dorsoventral axis of the OB is controlled by Slit-1 and Slit-3 expression. Interestingly, although MOR28-positive OSNs only express low levels of Robo-2, a reduced number of MOR28-positive glomeruli is observed in the OB of robo-2^lox/lox;OMP-Cre mice. Taken together, our results demonstrate that Slits and Robo-2 are required for the formation of an accurate glomerular map in the ventral region of the OB.     

Phosphoinositide-dependent activation of the ADP-ribosylation factor GTPase-activating protein ASAP1. Evidence for the pleckstrin homology domain functioning as an allosteric site

The ADP-ribosylation factor (Arf) family of GTP-binding proteins are regulators of membrane traffic and the actin cytoskeleton. Both negative and positive regulators of Arf, the centaurin beta family of Arf GTPase-activating proteins (GAPs) and Arf guanine nucleotide exchange factors, contain pleckstrin homology (PH) domains and are activated by phosphoinositides. To understand how the activities are coordinated, we have examined the role of phosphoinositide binding for Arf GAP function using ASAP1/centaurin beta4 as a model. In contrast to Arf exchange factors, phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P(2)) specifically activated Arf GAP. D3 phosphorylated phosphoinositides were less effective. Activation involved PtdIns-4,5-P(2) binding to the PH domain; however, in contrast to the Arf exchange factors and contrary to predictions based on the current paradigm for PH domains as independently functioning recruitment signals, we found the following: (i) the PH domain was dispensable for targeting to PDGF-induced ruffles; (ii) activation and recruitment could be uncoupled; (iii) the PH domain was necessary for activity even in the absence of phospholipids; and (iv) the Arf GAP domain influenced localization and lipid binding of the PH domain. Furthermore, PtdIns-4,5-P(2) binding to the PH domain caused a conformational change in the Arf GAP domain detected by limited proteolysis. Thus, these data demonstrate that PH domains can function as allosteric sites. In addition, differences from the published properties of the Arf exchange factors suggest a model in which feedforward and feedback loops involving lipid metabolites coordinate GTP binding and hydrolysis by Arf.     

Overexpression of epidermal growth factor receptor in NIH-3T3- transfected cells slows its lateral diffusion and rate of endocytosis

Interactions between membrane proteins are believed to be important for the induction of transmembrane signaling. Endocytosis is one of the responses which is regulated by both intracellular and extracellular signals. To study such interactions, we have measured the lateral mobility and rate of endocytosis of epidermal growth factor receptor in three transfected NIH-3T3 cell lines (HER84, HER22, and HER82) expressing 2 X 10(4), 2 X 10(5) and 1.5 X 10(6) EGF-receptors per cell, respectively. Using rhodamine-labeled EGF (Rh-EGF) and rhodamine-labeled monoclonal anti-EGF-receptor antibody (Rh-mAb-108), we measured twofold decreases in the lateral diffusion coefficients for each approximately 10-fold increase in EGF-receptor concentration. Since steric effects cannot account for such dependence, we propose that protein mobility within the membrane, which is determined by the rate of motion between immobile barriers, decreases due to aggregate formation. The rate of endocytosis also decreases twofold between the HER84 (2 X 10(4) receptors/cell) and HER22 (2 X 10(5) receptors/cell) cell lines, suggesting that it is diffusion limited. The comparable rates of endocytosis of the HER82 and HER22 cell lines suggest that at high receptor density endocytosis may be limited by the total number of sites for receptors in coated-pits and by their rate of recycling.     

Pasteurella multocida sialic acid aldolase: a promising biocatalyst

Sialic acid aldolases or N-acetylneuraminate lyases (NanAs) catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-D: -mannosamine (ManNAc). A capillary electrophoresis assay was developed to directly characterize the activities of NanAs in both Neu5Ac cleavage and Neu5Ac synthesis directions. The assay was used to obtain the pH profile and the kinetic data of a NanA cloned from Pasteurella multocida P-1059 (PmNanA) and a previously reported recombinant Escherichia coli K12 NanA (EcNanA). Both enzymes are active in a broad pH range of 6.0-9.0 in both reaction directions and have similar kinetic parameters. Substrates specificity studies showed that 5-O-methyl-ManNAc, a ManNAc derivative, can be used efficiently as a substrate by PmNanA, but not efficiently by EcNanA, for the synthesis of 8-O-methyl Neu5Ac. In addition, PmNanA (250 mg l(-1) culture) has a higher expression level (2.5-fold) than EcNanA (94 mg l(-1) culture). The higher expression level and a broader substrate tolerance make PmNanA a better catalyst than EcNanA for the chemoenzymatic synthesis of sialic acids and their derivatives.