Leptin suppression of insulin secretion and gene expression in human pancreatic islets: implications for the development of adipogenic diabetes mellitus.
Previously we demonstrated the expression of the long form of the leptin receptor in rodent pancreatic beta-cells and an inhibition of insulin secretion by leptin via activation of ATP-sensitive potassium channels. Here we examine pancreatic islets isolated from pancreata of human donors for their responses to leptin. The presence of leptin receptors on islet beta-cells was demonstrated by double fluorescence confocal microscopy after binding of a fluorescent derivative of human leptin (Cy3-leptin). Leptin (6.25 nM) suppressed insulin secretion of normal islets by 20% at 5.6 mM glucose. Intracellular calcium responses to 16.7 mM glucose were rapidly reduced by leptin. Proinsulin messenger ribonucleic acid expression in islets was inhibited by leptin at 11.1 mM, but not at 5.6 mM glucose. Leptin also reduced proinsulin messenger ribonucleic acid levels that were increased in islets by treatment with 10 nM glucagon-like peptide-1 in the presence of either 5.6 or 11.1 mM glucose. These findings demonstrate direct suppressive effects of leptin on insulin-producing beta-cells in human islets at the levels of both stimulus-secretion coupling and gene expression. The findings also further indicate the existence of an adipoinsular axis in humans in which insulin stimulates leptin production in adipocytes and leptin inhibits the production of insulin in beta-cells. We suggest that dysregulation of the adipoinsular axis in obese individuals due to defective leptin reception by beta-cells may result in chronic hyperinsulinemia and may contribute to the pathogenesis of adipogenic diabetes. (+info)
Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.
The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D. (+info)
Membrane deinsertion of SecA underlying proton motive force-dependent stimulation of protein translocation.
The proton motive force (PMF) renders protein translocation across the Escherichia coli membrane highly efficient, although the underlying mechanism has not been clarified. The membrane insertion and deinsertion of SecA coupled to ATP binding and hydrolysis, respectively, are thought to drive the translocation. We report here that PMF significantly decreases the level of membrane-inserted SecA. The prlA4 mutation of SecY, which causes efficient protein translocation in the absence of PMF, was found to reduce the membrane-inserted SecA irrespective of the presence or absence of PMF. The PMF-dependent decrease in the membrane-inserted SecA caused an increase in the amount of SecA released into the extra-membrane milieu, indicating that PMF deinserts SecA from the membrane. The PMF-dependent deinsertion reduced the amount of SecA required for maximal translocation activity. Neither ATP hydrolysis nor exchange with external SecA was required for the PMF-dependent deinsertion of SecA. These results indicate that the SecA deinsertion is a limiting step of protein translocation and is accelerated by PMF, efficient protein translocation thereby being caused in the presence of PMF. (+info)
Human papillomavirus DNA in adenosquamous carcinoma of the lung.
AIM: To investigate the presence of human papillomavirus (HPV) DNA in adenosquamous carcinoma of the lung--which is relatively common in Okinawa but not in mainland Japan--and examine its histological features. METHODS: Of 207 cases where primary lung cancers were surgically removed between January 1995 and June 1997 in Okinawa, 23 were adenosquamous carcinoma. HPV was detected by non-isotopic in situ hybridisation (NISH) and polymerase chain reaction (PCR) amplification with primers specific for E6 and E7 regions of the HPV genome. PCR products were analysed by Southern blotting. Immunohistochemical determination of high molecular weight cytokeratin (HMC) and involucrin was also carried out. RESULTS: 18 cases were positive for HPV DNA by PCR and NISH. HPV types 6, 11, 16, and 18 were found. Seven cases were dual positive for different types of HPV. Using NISH, HPV was also found in the squamous cell components and in neighbouring enlarged adenocarcinoma cells. The HMC and involucrin were demonstrated immunohistochemically in the same areas. CONCLUSIONS: HPV DNA was found in a high proportion (78.3%) of adenosquamous carcinomas in Okinawa, a region where HPV has previously been shown to be prevalent in squamous cell carcinoma of the lung. The adenocarcinoma cells adjacent to the squamous cell carcinoma component were enlarged and positive for HPV, HMC, and involucrin. This is thought to indicate the transition from adenocarcinoma to squamous cell carcinoma. (+info)
Physical interaction of the bHLH LYL1 protein and NF-kappaB1 p105.
The LYL1 gene was first identified upon the molecular characterization of the t(7;9)(q35;p13) translocation associated with some human T-cell acute leukemias (T-ALLs). In adult tissues, LYL1 expression is restricted to hematopoietic cells with the notable exclusion of the T cell lineage. LYL1 encodes a basic helix-loop-helix (bHLH) protein highly related to TAL-1, whose activation is also associated with a high proportion of human T-ALLs. A yeast two-hybrid system was used to identify proteins that specifically interact with LYL1 and might mediate its activities. We found that p105, the precursor of NF-kappaB1 p50, was the major LYL1-interacting protein in this system. The association between LYL1 and p105 was confirmed both in vitro and in vivo in mammalian cells. Biochemical studies indicated that the interaction was mediated by the bHLH motif of LYL1 and the ankyrin-like motifs of p105. Ectopic expression of LYL1 in a human T cell line caused a significant decrease in NF-kappaB-dependent transcription, associated with a reduced level of NF-kappaB1 proteins. (+info)
Probing the function of Bordetella bronchiseptica adenylate cyclase toxin by manipulating host immunity.
We have examined the role of adenylate cyclase-hemolysin (CyaA) by constructing an in-frame deletion in the Bordetella bronchiseptica cyaA structural gene and comparing wild-type and cyaA deletion strains in natural host infection models. Both the wild-type strain RB50 and its adenylate cyclase toxin deletion (DeltacyaA) derivative efficiently establish persistent infections in rabbits, rats, and mice following low-dose inoculation. In contrast, an inoculation protocol that seeds the lower respiratory tract revealed significant differences in bacterial numbers and in polymorphonuclear neutrophil recruitment in the lungs from days 5 to 12 postinoculation. We next explored the effects of disarming specific aspects of the immune system on the relative phenotypes of wild-type and DeltacyaA bacteria. SCID, SCID-beige, or RAG-1(-/-) mice succumbed to lethal systemic infection following high- or low-dose intranasal inoculation with the wild-type strain but not the DeltacyaA mutant. Mice rendered neutropenic by treatment with cyclophosphamide or by knockout mutation in the granulocyte colony-stimulating factor locus were highly susceptible to lethal infection by either wild-type or DeltacyaA strains. These results reveal the significant role played by neutrophils early in B. bronchiseptica infection and by acquired immunity at later time points and suggest that phagocytic cells are a primary in vivo target of the Bordetella adenylate cyclase toxin. (+info)
The integrin alpha v beta 6 binds and activates latent TGF beta 1: a mechanism for regulating pulmonary inflammation and fibrosis.
Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin. (+info)
Canine preprorelaxin: nucleic acid sequence and localization within the canine placenta.
Employing uteroplacental tissue at Day 35 of gestation, we determined the nucleic acid sequence of canine preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Canine preprorelaxin cDNA consisted of 534 base pairs encoding a protein of 177 amino acids with a signal peptide of 25 amino acids (aa), a B domain of 35 aa, a C domain of 93 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the canine relaxin B domain GRDYVR contained two substitutions from the classical motif (E-->D and L-->Y). Canine preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a 1-kilobase transcript present in total RNA of canine uteroplacental tissue but not of kidney tissue. Uteroplacental tissue from two bitches each at Days 30 and 35 of gestation were studied by in situ hybridization to localize relaxin mRNA. Immunohistochemistry for relaxin, cytokeratin, vimentin, and von Willebrand factor was performed on uteroplacental tissue at Day 30 of gestation. The basal cell layer at the core of the chorionic villi was devoid of relaxin mRNA and immunoreactive relaxin or vimentin but was immunopositive for cytokeratin and identified as cytotrophoblast cells. The cell layer surrounding the chorionic villi displayed specific hybridization signals for relaxin mRNA and immunoreactivity for relaxin and cytokeratin but not for vimentin, and was identified as syncytiotrophoblast. Those areas of the chorioallantoic tissue with most intense relaxin immunoreactivity were highly vascularized as demonstrated by immunoreactive von Willebrand factor expressed on vascular endothelium. The uterine glands and nonplacental uterine areas of the canine zonary girdle placenta were devoid of relaxin mRNA and relaxin. We conclude that the syncytiotrophoblast is the source of relaxin in the canine placenta. (+info)