Rab27b localizes to zymogen granules and regulates pancreatic acinar exocytosis

To understand the function of pancreatic zymogen granules, we performed a proteomics analysis to identify ZG membrane components. Here we report the identification of Rab27b through this proteomics study and validate its role in granule function. MALDI-MS peptide mass fingerprint was matched to rat Rab27b with 43% sequence coverage, and the identification was also confirmed by tandem mass spectrometry. The localization of Rab27b on ZGs was confirmed by Western blotting and immunocytochemistry. To examine the function of Rab27b in acinar secretion, we overexpressed wild type and mutant Rab27b protein in pancreatic acini using recombinant adenoviruses. Wild type Rab27b had no effect on amylase secretion, while Rab27b Q78L enhanced, and Rab27b N133I inhibited, CCK-induced amylase release by 92+/-13% and 53+/-8%, respectively. This enhancement and inhibition occurred at all points on the CCK dose-response curve and over a 30min time course. These results demonstrate that Rab27b is present on ZGs and plays an important role in regulating acinar exocytosis.     

Expression of glucose transporter 4 in the human pancreatic islet of Langerhans

Glucose transporter 4 (GLUT4) is the main insulin-responsive glucose transporter in skeletal muscle and adipose tissue of human and rodent, and is translocated to the plasma membrane in response to insulin. GLUT2 is well known as the main glucose transporter in pancreatic islets and could highly regulate glucose-stimulated insulin secretion by B-cells as a glucose sensor. We confirmed the presence of GLUT4 mRNA and GLUT4 protein in pancreas in the human. Indirect immunohistochemistry showed that the pancreatic islets of human and rat were conspicuously labeled by anti-GLUT4 antibody. The presence of placental leucine aminopeptidase (P-LAP), a homologue of insulin-regulated aminopeptidase (IRAP), was also shown in the human pancreatic islet. IRAP/P-LAP is thought to be involved in glucose metabolism. This study provides the first evidence that GLUT4 is present in human and rat pancreatic islets and may suggest its specific role in glucose homeostasis in conjunction with IRAP/P-LAP.     

E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.     

Cell elongation and cell death of helicobacter pylori is modulated by the disruption of cdrA (cell division-related gene A)

The cell division-related gene A (cdrA) of Helicobacter pylori is dispensable in vivo and unique in having a repressive role on cell division and long-term survival. To clarify its role, comparisons of the wildtype HPK5 and isogenic cdrA-disrupted mutant HPKT510 were examined by ultrastructural morphology, PBP profiles, and susceptibility to beta-lactam antibiotics during long-term cultivation. Ultrastructural analyses revealed that the shorter rods of HPKT510 had a slightly wider periplasmic space between the inner and the outer membrane than those of HPK5. Cell division of HPKT510 cells was complete even under high-salt conditions in which HPK5 cells became filamentous due to inhibition of division. The filamentous HPK5 cells constructed an inner membrane without a cell wall at the presumed division site. After 4 days of cultivation (the late stationary phase), most of the HPK5 cells turned into ghosts and aggregates, while some of the HPKT510 cells remained as curved rods, which coincided with the results of cell viability. HPKT510 cells became resistant to ampicillin killing compared to HPK5 cells, although their minimum inhibitory concentrations (MICs) and PBP profiles were not significantly different. These results suggest that the cdrA product represses cell division via inhibiting cell wall synthesis at division site. During infection in both mice and humans, inactivation of cdrA eventually gains biological aspects such as increased viability, long-term survival and tolerance to antibiotics and high-salt condition, which might enhance a persistent infection.     

Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.     

Stereoselective reduction of ketones by various vegetables

Reduction of (+)-and (−)-camphorquinones (1a, 1b) by various vegetables (carrot, potato, sweet potato, apple, Japanese radish, cucumber, burdock and onion) gave -hydroxycamphor selectively. Using burdock, (+)-camphorquinone was reduced to give (−)-3S-exo-hydroxycamphor (4a) as major product in high stereoselectivity with high yield. Moreover, 1,2-cyclohexanedione (1c) and 2-methylcyclohexanone (1d) with various vegetables afford enantiomerically pure trans- and/or cis-alcohol, respectively. Various vegetable reduction gave a new idea of a biotechnological process.     

TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents

TRPA1 is an excitatory ion channel targeted by pungent irritants from mustard and garlic. TRPA1 has been proposed to function in diverse sensory processes, including thermal (cold) nociception, hearing, and inflammatory pain. Using TRPA1-deficient mice, we now show that this channel is the sole target through which mustard oil and garlic activate primary afferent nociceptors to produce inflammatory pain. TRPA1 is also targeted by environmental irritants, such as acrolein, that account for toxic and inflammatory actions of tear gas, vehicle exhaust, and metabolic byproducts of chemotherapeutic agents. TRPA1-deficient mice display normal cold sensitivity and unimpaired auditory function, suggesting that this channel is not required for the initial detection of noxious cold or sound. However, TRPA1-deficient mice exhibit pronounced deficits in bradykinin-evoked nociceptor excitation and pain hypersensitivity. Thus, TRPA1 is an important component of the transduction machinery through which environmental irritants and endogenous proalgesic agents depolarize nociceptors to elicit inflammatory pain.     

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.