Enhancement of muscarinic receptor-coupled phosphatidyl inositol hydrolysis in diabetic bladder

We previously have shown an increase in muscarinic receptor density in streptozotocin (STZ)-induced diabetic and sucrosefed diuretic rat detrusor that correlates with an increase in the contractile response to muscarinic agonist (J Pharmacol Exp Ther 248: 81, 1989; Diabetes 40: 265, 1991). To investigate the signal transduction pathway involved in this altered functional response, we examined muscarinic receptor-coupled phosphatidylinositol metabolism in STZ-diabetic, sucrose-fed diuretic and age-matched control rat bladders. [³H]myo-inositol uptake was similar in all groups, but incorporation of myo-inositol into phosphatidylinositol (PI) was significantly increased in the diabetic bladder compared to the sucrose-fed and control rat bladders. Carbachol-induced increase in inositol phosphate (IPs) production was higher in the diabetic bladder than in bladders from control and sucrose-fed animals although the EC₅₀ values were similar for all groups. Enhanced inositol phosphate production after muscarinic agonist stimulation may be due not only to the upregulation of muscarinic receptors but also to the increased incorporation of myo-inositol into PI in the STZ-induced diabetic bladder.     

Hepatitis B Virus with X Gene Mutation Is Associated with the Majority of Serologically “Silent” Non-B,Non-C Chronic Hepatitis

Hepatitis B virus (HBV) with X gene mutations has been a putative pathogen of chronic hepatitis without serological markers of known hepatitis viruses. The aim of this study was to reconfirm whether the HBV with the X gene mutation is associated with these serologically “silent” non-B, non-C (NBNC) chronic hepatitis, alcoholic liver disease (ALD) and autoimmune hepatitis (AIH). HBV DNA was amplified from serum and sequenced in 30 patients with NBNC chronic hepatitis in comparison with 20 patients with ALD and 5 patients with AIH. HBV DNA was identified in 21 patients (70%) in NBNC chronic hepatitis by nested polymerase chain reaction while only one patient (5%) in ALD and none in AIH showed HBV DNA. Eighteen (85.7%) of the 21 identified HBV DNAs had an identical 8-nucleotide deletion mutation at the distal part of the X region. This mutation affected the core promoter and the enhancer II sequence of HBV DNA and created a translational stop codon which truncated the X protein by 20 amino acids from the C-terminal end. All the HBV DNAs had a precore mutation at the 83rd nucleotide resulting in disruption of HBe antigen synthesis. These results indicate that HBV mutants are closely associated with the majority of serologically “silent” NBNC chronic hepatitis cases and the population of such mutant HBV DNAs is not uniform.     

Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation

Cell-cell interaction via the gap junction regulates cell growth and differentiation, leading to formation of organs of appropriate size and quality. To determine the role of connexin43 in salivary gland development, we analyzed its expression in developing submandibular glands (SMGs). Connexin43 (Cx43) was found to be expressed in salivary gland epithelium. In ex vivo organ cultures of SMGs, addition of the gap junctional inhibitors 18α-glycyrrhetinic acid (18α-GA) and oleamide inhibited SMG branching morphogenesis, suggesting that gap junctional communication contributes to salivary gland development. In Cx43^−/− salivary glands, submandibular and sublingual gland size was reduced as compared with those from heterozygotes. The expression of Pdgfa, Pdgfb, Fgf7, and Fgf10, which induced branching of SMGs in Cx43^−/− samples, were not changed as compared with those from heterozygotes. Furthermore, the blocking peptide for the hemichannel and gap junction channel showed inhibition of terminal bud branching. FGF10 induced branching morphogenesis, while it did not rescue the Cx43^−/− phenotype, thus Cx43 may regulate FGF10 signaling during salivary gland development. FGF10 is expressed in salivary gland mesenchyme and regulates epithelial proliferation, and was shown to induce ERK1/2 phosphorylation in salivary epithelial cells, while ERK1/2 phosphorylation in HSY cells was dramatically inhibited by 18α-GA, a Cx43 peptide or siRNA. On the other hand, PDGF-AA and PDGF-BB separately induced ERK1/2 phosphorylation in primary cultured salivary mesenchymal cells regardless of the presence of 18α-GA. Together, our results suggest that Cx43 regulates FGF10-induced ERK1/2 phosphorylation in salivary epithelium but not in mesenchyme during the process of SMG branching morphogenesis.     

A Comparative Analysis of the Molecular Features of MANF and CDNF

Cerebral dopamine neurotrophic factor (CDNF) is a paralogous protein of mesencephalic astrocyte-derived neurotrophic factor (MANF). Both proteins have been reported to show a common cytoprotective effect on dopaminergic neurons as a secretory protein containing the KDEL-like motif of the ER retrieval signal at the C-terminus, RTDL in MANF and [Q/K]TEL in CDNF among many species, although functions of paralogous proteins tend to differ from each other. In this study, we focused on post-translational regulations of their retention in the endoplasmic reticulum (ER) and secretion and performed comparative experiments on characterization of mouse MANF and mouse CDNF according to our previous report about biosynthesis and secretion of mouse MANF using a NanoLuc system. In this study, co-expression of glucose-regulated protein 78 kDa (GRP78), KDEL receptor 1 or mutant Sar1 into HEK293 cells similarly decreased MANF and CDNF secretion with some degree of variation. Next, we investigated whether CDNF affects the secretion of mouse cysteine-rich with EGF-like domains 2 (CRELD2) because mouse wild-type (wt) MANF but not its KDEL-like motif deleted mutant (ΔC_MANF) was found to promote the CRELD2 release from the transfected cells. Co-expressing CRELD2 with wt or ΔC CDNF, we found that CDNF and ΔC_MANF hardly elevated the CRELD2 secretion. We then investigated effects of the four or six C-terminal amino acids of MANF and CDNF on the CRELD2 secretion. As a result, co-transfection of mouse CDNF having the mouse MANF-type C-terminal amino acids (CDNF_RTDL and CDNF_SARTDL) increased the CRELD2 secretion to a small extent, but mouse CDNF having human CDNF-type ones (CDNF_KTEL and CDNF_HPKTEL) well increased the CRELD2 secretion. On the other hand, the replacement of C-terminal motifs of mouse MANF with those of mouse CDNF (MANF_QTEL and MANF_YPQTEL) enhanced the CRELD2 secretion, and the mouse MANF having human CDNF-type ones (MANF_KTEL and MANF_HPKTEL) dramatically potentiated the CRELD2 secretion. These results indicate that the secretion of mouse MANF and mouse CDNF is fundamentally regulated in the same manner and that the variation of four C-terminal amino acids in the MANF and CDNF among species might influence their intracellular functions. This finding could be a hint to identify physiological functions of MANF and CDNF.     

A high-throughput sequence analysis of Japanese patients revealed 11 candidate genes associated with type 1 autoimmune pancreatitis susceptibility

The pathogenesis of autoimmune pancreatitis is unknown. In the present study we used high-throughput sequencing with next generation sequencing to identify the candidate genes associated with AIP. A total of 27 type 1 AIP patients and 30 healthy blood donors were recruited, and DNA samples were isolated from their mononuclear cells. A high-throughput sequencer with an original custom panel of 1031 genes was used to detect the genetic variants in each sample. Polymorphisms of CACNA1S (c.4642C>T), rs41554316, rs2231119, rs1042131, rs2838171, P2RX3 (c.195delG), rs75639061, SMAD7 (c.624delC) and TOP1 (c.2007delG), were identified as candidate genetic variants in patients with type 1 AIP. P2RX3 and TOP1 were significantly associated with AIP, even after adjusting bay means of Bonferroni's correction. In addition, we also identified eight candidate genetic variants that were associated with the relapse of type 1 AIP, namely: rs1143146, rs1050716, HLA-C (c.759_763delCCCCCinsTCCCG), rs1050451, rs4154112, rs1049069, CACNA1C (c.5996delC) and CXCR3 (c.630_631delGC). Finally polymorphisms of rs1050716 and rs111493987 were identified as candidate genetic variants associated with extra-pancreatic lesions in patients with type 1 AIP. These candidates might be used as markers of AIP susceptibility and could contribute to the pathogenesis of type 1 AIP.     

Modification of the anticodon triplet of E.coli tRNAMetf by replacement with trimers complementary to non-sense codons UAG and UAA.

E. coli tRNAMetf was hydrolyzed with RNase A using a limited amount of the enzyme to give two half molecules lacking the anticodon trimer and 3'-terminal dimer. Chemically synthesized trimers CUAp and UUAp were joined to the 5'-half molecules by phosphorylation with polynucleotide kinase plus ATP followed by treatment with RNA ligase. These modified tRNAMetf species had anticodons complementary to the termination codons UAG and UAA. Two half fragments were joined by a similar procedure to yield a molecule lacking the anticodon trimer and the 3'-dimer. Methionine acceptor activity of these tRNA was tested under conditions in which the CAU inserted control tRNAMetf accepted methionine. It was found that all three modified molecules were not recognized by the methionyl-tRNA synthetase from E.coli. The other sixteen amino acids were not incorporated with partially purified aminoacyl-tRNA synthetases.     

Synthesis of 5' fragments of formylmethionine transfer ribonucleic acid and their reconstitution with a natural three-quarter molecule

An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.