Knockdown of tight junction protein claudin-2 prevents bile canalicular formation in WIF-B9 cells

The polarization of hepatocytes involves formation of functionally distinct sinusoidal (basolateral) and bile canalicular (apical) plasma membrane domains that are separated by tight junctions. Although various molecular mechanisms and signaling cascades including polarity complex proteins may contribute to bile canalicular formation in hepatocytes, the role of tight junction proteins in bile canalicular formation remains unclear. To investigate the role of the integral tight junction protein claudin-2 in bile canalicular formation, we depleted claudin-2 expression by siRNA in the polarized hepatic cell line WIF-B9 after treatment with or without phenobarbital. When WIF-B9 cells were treated with phenobarbital, claudin-2 expression and tight junction strands were markedly increased together with induction of canalicular formation with a biliary secretion function. Knockdown of claudin-2 prevented bile canalicular formation after treatment with or without phenobarbital. Furthermore, knockdown of claudin-2 caused a change from a hepatic polarized phenotype to a simple polarized phenotype, together with upregulation of pLKB1, pMAPK, pAkt and pp38 MAPK, but not pMLC, PTEN or cdc42, and an increase of intracellular vacuoles, which were present before bile canalicular formation. These results suggest that claudin-2 may affect not only the bile canalicular seal but also bile canalicular formation.     

Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation

Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.     

Pharmacological preconditioning with resveratrol: an insight with iNOS knockout mice

Resveratrol, a natural antioxidant and polyphenol found in grapes and wine, has been found to pharmacologically precondition the heart through the upregulation of nitric oxide (NO). To gain further insight of the role of NO in resveratrol preconditioning, mouse hearts devoid of any copies of inhibitory NO synthase (iNOS) (iNOS knockout) and corresponding wild-type hearts were perfused with 10 microM resveratrol for 15 min followed by 25 min of ischemia and 2 h of reperfusion. Control experiments were performed with wild-type and iNOS knockout hearts that were not treated with resveratrol. Resveratrol-treated wild-type mouse hearts displayed significant improvement in postischemic ventricular functional recovery compared with those of nontreated hearts. Both resveratrol-treated and nontreated iNOS knockout mouse hearts resulted in relatively poor recovery in ventricular function compared with wild-type resveratrol-treated hearts. Myocardial infarct size was lower in the resveratrol-treated wild-type mouse hearts compared with other group of hearts. In concert, a number of apoptotic cardiomyocytes was lower in the wild-type mouse hearts treated with resveratrol. Cardioprotective effects of resveratrol was abolished when the wild-type mouse hearts were simultaneously perfused with aminoguanidine, an iNOS inhibitor. Resveratrol induced the expression of iNOS in the wild-type mouse hearts, but not in the iNOS knockout hearts, after only 30 min of reperfusion. Expression of iNOS remained high even after 2 h of reperfusion. Resveratrol-treated wild-type mouse hearts were subjected to a lower amount of oxidative stress as evidenced by reduced amount of malonaldehyde content in these hearts compared with iNOS knockout and untreated hearts. The results of this study demonstrated that resveratrol was unable to precondition iNOS knockout mouse hearts, whereas it could successfully precondition the wild-type mouse hearts, indicating an essential role of iNOS in resveratrol preconditioning of the heart.     

Marked increase in gastric histidine decarboxylase activity in patients with hypergastrinemia.

Histidine decarboxylase (HDC) activity and histamine content were measured in endoscopic gastric biopsy specimens of 19 control subjects with normogastrinemia and 6 patients with hypergastrinemia. In controls, the HDC activity was 3 fold higher in fundic mucosa (120 +/- 13 fmol/min/mg protein, mean +/- S.E.) than in antral mucosa (39 +/- 5 fmol/min/mg protein). In patients with hypergastrinemia, an extremely high HDC activity (713 +/- 181 fmol/min/mg protein) was observed in fundic mucosa, although the HDC activity in antral mucosa was not significantly different from that of controls. The histamine content in fundic mucosa was also significantly higher in patients with hypergastrinemia than in controls but no significant difference was seen in histamine content in antral mucosa between the two groups. These results are compatible with the hypothesis that in man, as well as in rat, histamine synthesis in fundic mucosa is enhanced by gastrin.     

Detection and Typing of Human Rotavirus in Reference to Repeated Acute Gastroenteritis in Infants

Stool specimens from infants who visited a clinic because of acute gastroenteritis were tested for the presence of human rotavirus. Among the samples obtained were specimens taken from seven patients who had visited the clinic at two different times. In six of these seven children, human rotavirus (HRV) was detected in only one of the specimens taken (i.e. during only one of the two visits). One patient was shown to have excreted HRV twice; in both cases the HRV was serotyped to be type 1. The present results indicate that the symptomatic reinfection of HRV was not a widely occurring phenomenon in the group of infants tested.     

Activation of prothrombin by ASP, a serine protease released from Aeromonas sobria

The effect of a serine protease (ASP) secreted from Aeromonas sobria on plasma coagulation was investigated. Proteolytically active ASP promoted human plasma coagulation in a dose-dependent manner. Consistent with the preference for a factor Xa-specific oligo-peptide substrate, ASP produced enzymatic activity from human prothrombin but not from factors IX and X. ASP cleaved prothrombin to produce enzymatically active 37 kDa-fragment displaying the same molecular mass as alpha-thrombin. ASP is the first bacterial serine protease that produces alpha-thrombin, through which ASP may contribute to the induction of thrombotic tendency in disseminated intravascular coagulation complicated with sepsis caused by A. sobria infections.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.     

beta -Arrestin-mediated recruitment of the Src family kinase Yes mediates endothelin-1-stimulated glucose transport

The insulin and the endothelin type A (ETA) receptor both can couple into the heterotrimeric G protein alpha(q/11) (Galpha(q/11)), leading to Galpha(q/11) tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, and subsequent stimulation of glucose transport. In this study, we assessed the potential role of Src kinase in ET-1 signaling to glucose transport in 3T3-L1 adipocytes. Src kinase inhibitor PP2 blocked ET-1-induced Src kinase activity, Galpha(q/11) tyrosine phosphorylation, and glucose transport stimulation. To determine which Src family kinase member was involved, we microinjected anti-c-Src, -c-Fyn, or -c-Yes antibody into these cells and found that only anti-c-Yes antibody blocked GLUT4 translocation (70% decreased). Overexpression or microinjection of a dominant negative mutant (K298M) of Src kinase also inhibited ET-1-induced Galpha(q/11) tyrosine phosphorylation and GLUT4 translocation. In co-immunoprecipitation experiments, we found that beta-arrestin 1 associated with the ETA receptor in an agonist-dependent manner and that beta-arrestin 1 recruited Src kinase to a molecular complex that included the ETA receptor. Microinjection of beta-arrestin 1 antibody inhibited ET-1- but not insulin-stimulated GLUT4 translocation. In conclusion, 1) the Src kinase Yes can induce tyrosine phosphorylation of Galpha(q/11) in response to ET-1 stimulation, and 2) beta-arrestin 1 and Src kinase form a molecular complex with the ETA receptor to mediate ET-1 signaling to Galpha(q/11) with subsequent glucose transport stimulation.