Mastoparan induces oscillations of cytosolic Ca2+ in rat pancreatic acinar cells

Microfluorimetry of fura-2 was used to monitor [Ca2+]i in single cells stimulated with the G-protein activating agent mastoparan. Mastoparan induced the generation of [Ca2+]i oscillations, which in contrast to oscillations induced by low concentrations of CCK were acutely dependent on the presence of extracellular Ca2+. Oscillations were inhibited by phorbol ester. Sodium fluoride, a known activator of G-proteins, gave similar results. Both mastoparan and CCK induced turnover of inositol phosphates, at concentrations higher than necessary to induce oscillations.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29321/1/0000386.pd

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y2 receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y2 receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca2+ mobilization indicating that nucleotides cause Ca2+ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca2+ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca2+ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca2+ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca2+ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca2+ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y2 receptor can cause Ca2+ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins

Association Analysis of the Extended MHC Region in Celiac Disease Implicates Multiple Independent Susceptibility Loci

Celiac disease is a common autoimmune disease caused by sensitivity to the dietary protein gluten. Forty loci have been implicated in the disease. All disease loci have been characterized as low-penetrance, with the exception of the high-risk genotypes in the HLA-DQA1 and HLA-DQB1 genes, which are necessary but not sufficient to cause the disease. The very strong effects from the known HLA loci and the genetically complex nature of the major histocompatibility complex (MHC) have precluded a thorough investigation of the region. The purpose of this study was to test the hypothesis that additional celiac disease loci exist within the extended MHC (xMHC). A set of 1898 SNPs was analyzed for association across the 7.6 Mb xMHC region in 1668 confirmed celiac disease cases and 517 unaffected controls. Conditional recursive partitioning was used to create an informative indicator of the known HLA-DQA1 and HLA-DQB1 high-risk genotypes that was included in the association analysis to account for their effects. A linkage disequilibrium-based grouping procedure was utilized to estimate the number of independent celiac disease loci present in the xMHC after accounting for the known effects. There was significant statistical evidence for four new independent celiac disease loci within the classic MHC region. This study is the first comprehensive association analysis of the xMHC in celiac disease that specifically accounts for the known HLA disease genotypes and the genetic complexity of the region

Hepatic safety of antibiotics used in primary care

Antibiotics used by general practitioners frequently appear in adverse-event reports of drug-induced hepatotoxicity. Most cases are idiosyncratic (the adverse reaction cannot be predicted from the drug's pharmacological profile or from pre-clinical toxicology tests) and occur via an immunological reaction or in response to the presence of hepatotoxic metabolites. With the exception of trovafloxacin and telithromycin (now severely restricted), hepatotoxicity crude incidence remains globally low but variable. Thus, amoxicillin/clavulanate and co-trimoxazole, as well as flucloxacillin, cause hepatotoxic reactions at rates that make them visible in general practice (cases are often isolated, may have a delayed onset, sometimes appear only after cessation of therapy and can produce an array of hepatic lesions that mirror hepatobiliary disease, making causality often difficult to establish). Conversely, hepatotoxic reactions related to macrolides, tetracyclines and fluoroquinolones (in that order, from high to low) are much rarer, and are identifiable only through large-scale studies or worldwide pharmacovigilance reporting. For antibiotics specifically used for tuberculosis, adverse effects range from asymptomatic increases in liver enzymes to acute hepatitis and fulminant hepatic failure. Yet, it is difficult to single out individual drugs, as treatment always entails associations. Patients at risk are mainly those with previous experience of hepatotoxic reaction to antibiotics, the aged or those with impaired hepatic function in the absence of close monitoring, making it important to carefully balance potential risks with expected benefits in primary care. Pharmacogenetic testing using the new genome-wide association studies approach holds promise for better understanding the mechanism(s) underlying hepatotoxicity

MOLECULAR PHARMACOLOGY, 55:279–287 (1999). A Novel Positive Regulatory Element That Enhances Hamster CYP2A8 Gene Expression Mediated by Xenobiotic Responsive Element

CYP2A8 is a major form of cytochrome P-450 inducible by 3-methylcholanthrene in Syrian hamster liver. To identify DNA elements necessary for the transcriptional activation of the CYP2A8 gene, we analyzed the regulatory region of the CYP2A8 gene and conducted transient transfection experiments of CYP2A8-luciferase fusion plasmids in primary cultures of hamster hepatocytes. We analyzed up to �5 kbofthe 5�-flanking region and found the region sufficient for the 3-methylcholanthrene-inducible gene expression. This region contained a consensus sequence for xenobiotic responsive element (XRE) between �2366 and �2349, which was shown to be essential for induction of the gene expression. Furthermore, we found a novel positive regulatory element for XRE-The cytochrome P-450 (CYP) superfamily consists of variou

Mastoparan, a peptide toxin from wasp venom, stimulates glycogenolysis mediated by an increase of the cytosolic free Ca2+ concentration but not by an increase of cAMP in rat hepatocytes

AbstractA wasp venom, mastoparan, rapidly increased the cytosolic free Ca2+ concentration ([Ca2+]i) and activated phosphorylase in rat hepatocytes in a concentration-dependent manner. Mastoparan could increase [Ca2+]i even in the absence of extracellular Ca2+, but a larger increase was observed in the presence of extracellular Ca2+. Thus, mastoparan mobilized Ca2+ from intracellular and extracellular Ca2+ stores. It also activated inositol triphosphate (IP3) accumulation, but did not stimulate cAMP production. From these results, we conclude that mastoparan activates rat hepatic glycogenolysis mediated by the accumulation of IP3, which causes an increase of [Ca2+]i but not that mediated by cAMP

Supplementary_Appendix_1 - Possible Causes of Failing to Meet Primary Endpoints

<p>Supplementary_Appendix_1 for Possible Causes of Failing to Meet Primary Endpoints by Mitsugu Ikeda, Tatsuya Ochibe, and Masahiro Tohkin in Therapeutic Innovation & Regulatory Science</p