Free-range layer chickens as a source of <Emphasis Type="Italic">Campylobacter</Emphasis> bacteriophage

Bacteriophage specific for Campylobacter were isolated from chicken excreta collected from established free-range layer breed stock. Bacteriophage were either propagated on a Campylobacter jejuni host with broad susceptibility to bacteriophage (NCTC 12662) or on Campylobacter isolates from the same samples. Campylobacters were confirmed as being C. jejuni and or C. coli, using a combination of standard biochemical tests and PCR analysis with genus and species specific primers. The bacteriophage displayed differential patterns of susceptibility against reference NCTC strains and contemporary C. jejuni /C. coli isolates from chicken excreta. Electron microscopy demonstrated that the phage possessed icosahedral heads and rigid contractile tails. Pulsed-field gel electrophoresis revealed the bacteriophage genomes to be double stranded DNA in the range of 140 kb in size and the restriction enzyme patterns of the DNAs indicate they are genetically related members of the Myoviridae family. This study showed that Campylobacter bacteriophage could easily be isolated from free-range chickens and form part of their normal microbiological biota of environmentally exposed birds.     

Staurosporine differentiation of NPFF2 receptor-transfected SH-SY5Y neuroblastoma cells induces selectivity of NPFF activity towards opioid receptors

Activation of the NPFF(2) receptor reduces the inhibitory effect of opioids on the N-type Ca(2+) channel. Although this anti-opioid effect is specific for opioid receptors in neurons and tissues, it also affects NPY Y2 and alpha(2)-adrenoreceptors in undifferentiated SH-SY5Y cells stably expressing the NPFF(2) receptor. To test whether this difference could be due to the immaturity of these cells, they were differentiated to a noradrenergic neuronal phenotype with staurosporine. The differentiated cells ceased to divide and grew long, thin neurites. The inhibition of the depolarization-triggered Ca(2+) transient by activation of G(i)-coupled receptors was either unaffected (micro-opioid), increased (NPY), reduced (NPFF(2)) or lost (alpha(2)-adrenoreceptors). Following a 20 min incubation with 1DMe, the effect of DAMGO was reduced, as in undifferentiated cells, but the effect of NPY was no longer affected. Staurosporine differentiation did not modify the coupling of the micro-opioid and NPFF(2) receptors to the G(i/o) proteins. We suggest that the specificity of the effect of NPFF may not reside in the molecular mechanism of its anti-opioid activity itself but in the organization of receptors within the membrane.     

Opposite regulation of endothelial NO synthase by HSP90 and caveolin in liver and lungs of rats with hepatopulmonary syndrome

The hepatopulmonary syndrome is a complication of cirrhosis that associates an overproduction of nitric oxide (NO) in lungs and a NO defect in the liver. Because endothelial NO synthase (eNOS) is regulated by caveolin that decreases and heat shock protein 90 (HSP90) that increases NO production, we hypothesized that an opposite regulation of eNOS by caveolin and HSP90 might explain the opposite NO production in both organs. Cirrhosis was induced by a chronic bile duct ligation (CBDL) performed 15, 30, and 60 days before sample collection and pharmacological tests. eNOS, caveolin, and HSP90 expression were measured in hepatic and lung tissues. Pharmacological tests to assess NO released by shear stress and by acetylcholine were performed in livers (n = 28) and lungs (n = 28) isolated from normal and CBDL rats. In lungs from CBDL rats, indirect evidence of high NO production induced by shear stress was associated with a high binding of HSP90 and a low binding of caveolin to eNOS. Opposite results were observed in livers from CBDL rats. Our study shows an opposite posttranslational regulation of eNOS by HSP90 and caveolin in lungs and liver from rats with CBDL. Such opposite posttranslational regulation of eNOS by regulatory proteins may explain in part the pulmonary overproduction of NO and the hepatic NO defect in rats with hepatopulmonary syndrome.     

Copper deprivation in rats induces islet hyperplasia and hepatic metaplasia in the pancreas

BACKGROUND INFORMATION: Prolonged copper deprivation in rats followed by refeeding with a normal diet has previously been used to induce the appearance of hepatocyte-like cells in the pancreas, but the effects on islet size and morphology have not been determined. RESULTS: In the present study we investigated the distribution of pancreatic alpha- and beta-cells and of hepatocytes in adult rats fed a copper-deficient diet followed by refeeding with a normal diet. Immunohistochemical staining for insulin and glucagon showed that the islets of the copper-deficient group were up to 2.4 times larger in mass compared with controls. The islets were disorganized, with alpha-cells found in multiple layers at the periphery of the islet and sometimes deep in the core. Isolated alpha- and beta-cells were also found in increased numbers in the ductular system. Copper deprivation caused almost complete ablation of the acinar cells, and refeeding induced adipogenesis, acinar regeneration and hepatocyte-like cells. Ductular proliferation and nerve hyperplasia were also present. The hepatocytes tended to be associated with islets or with ducts, rather than with residual pancreatic exocrine tissue. CONCLUSIONS: These data show that copper deficiency in rats, as well as inducing the appearance of hepatocytes, is capable of causing islet hyperplasia.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance

UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC(-/-) embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC(-/-) embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC(-/-) fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC(-/-) phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes.     

Casein kinase 1 is a novel negative regulator of E-cadherin-based cell-cell contacts

Cadherins are the most crucial membrane proteins for the formation of tight and compact cell-cell contacts. Cadherin-based cell-cell adhesions are dynamically established and/or disrupted during various physiological and pathological processes. However, the molecular mechanisms that regulate cell-cell contacts are not fully understood. In this paper, we report a novel functional role of casein kinase 1 (CK1) in the regulation of cell-cell contacts. Firstly, we observed that IC261, a specific inhibitor of CK1, stabilizes cadherin-based cell-cell contacts, whereas the overexpression of CK1 disrupts them. CK1 colocalizes with E-cadherin and phosphorylates the cytoplasmic domain of E-cadherin in vitro and in a cell culture system. We show that the major CK1 phosphorylation site of E-cadherin is serine 846, a highly conserved residue between classical cadherins. Constitutively phosphorylated E-cadherin (S846D) is unable to localize at cell-cell contacts and has decreased adhesive activity. Furthermore, phosphorylated E-cadherin (S846D) has weaker interactions with beta-catenin and is internalized more efficiently than wild-type E-cadherin. These data indicate that CK1 is a novel negative regulator of cadherin-based cell-cell contacts.     

Localisation of caveolin in mammary tissue depends on cell type

Caveolins, components of caveolae, are expressed in mammary tissue. In order to determine whether caveolins are present in different mammary cell types and whether their localisation depends on the physiological stage or species, cav-1 and cav-2 were characterised by immunoblotting in mammary tissues from the mouse, ewe and rabbit and localised, by immunofluorescence and electron microscopy, in mammary tissues from the mouse and ewe. At all the physiological stages studied, cav-1 and cav-2 were present in endothelial and myoepithelial cells in which flask-shaped caveolae were abundant. However, labelling of cav-1 and cav-2 associated with small vesiculo-tubular structures (including those close to lipid droplets) was low in epithelial cells. To study the possible association of cav-1 with lipid droplets, lactating ewe mammary fragments were treated in vitro with brefeldin A. This treatment did not modify the association of cav-1-labelled structures with lipid droplets. Finally, HC11 and MCF-10A mammary cell lines were treated with oleic acid. The total quantity of cav-1 was little affected by the treatment, although the lipid droplet labelling of cav-1 was amplified in MCF-10A cells. Thus, the synthesis and localisation of caveolins are mostly dependent upon the cell types of mammary tissue and upon their state of differentiation.     

Diverse functions of RNase L and implications in pathology

The endoribonuclease L (RNase L) is the effector of the 2-5A system, a major enzymatic pathway involved in the molecular mechanism of interferons (IFNs). RNase L is a very unusual nuclease with a complex mechanism of regulation. It is a latent enzyme, expressed in nearly every mammalian cell type. Its activation requires its binding to a small oligonucleotide, 2-5A. 2-5A is a series of unique 5'-triphosphorylated oligoadenylates with 2'-5' phosphodiester bonds. By regulating viral and cellular RNA expression, RNase L plays an important role in the antiviral and antiproliferative activities of IFN and contributes to innate immunity and cell metabolism. The 2-5A/RNase L pathway is implicated in mediating apoptosis in response to viral infections and to several types of external stimuli. Several recent studies have suggested that RNase L could have a role in cancer biology and evidence of a tumor suppressor function of RNase L has emerged from studies on the genetics of hereditary prostate cancer.