Cardiolipin induces premature senescence in normal human fibroblasts

Lipids seem to have various roles in cellular senescence. We found that cardiolipin very sensitively inhibits growth of normal human fibroblasts, whereas other phospholipids do not at 100 times higher concentrations. Growth arrested cells showed morphology similar to those of normally senesced cells and strongly induced senescence-associated beta-galactosidase. Senescence markers such as the p21(waf1/sdi-1), fibronectin, and collagenase-I genes were significantly upregulated by cardiolipin. In addition, caldiolipin significantly increased in normally senesced human fibroblasts leaving other phospholipids unaltered. These results suggest that accumulation of cardiolipin is one of the causes for replicative senescence.     

Low- and high-level expressions of heme oxygenase-1 in cultured cells under uninduced conditions

Heme oxygenase-1 (HO-1) degrades heme into biliverdin, iron, and CO. The enzyme participates in adaptive and protective responses to oxidative stress and various inflammatory stimuli. We examined the regulation of HO-1 expression in culture cells under uninduced conditions. Observations by in situ hybridization and immunostaining showed that in cultured mouse fibroblast Balb/3T3 cells not subjected to treatment, 10-15% of cells highly expressed HO-1. The similar pattern of the expression of HO-1 was observed with mouse embryo liver BNL-CL2 cells and Chinese hamster ovary cells. The marked expression of HO-1 was related to the activation of stress-activated protein kinase and to the expression of cyclooxygenase (Cox)-2. When the cells were treated with arachidonic acid, a precursor of prostaglandin, induction of HO-1 in the HO-1-expressing cells but not in the low-expressing cells occurred. This increase was abrogated by the treatment with the Cox inhibitors, indomethacin, and dexamethasone. Neither prostaglandin H2, E2 nor F2a induced HO-1 expression. These results suggest that some cells respond to the cellular stress and intermediates of prostaglandin biosynthesis may act as endogenous stressors to induce HO-1.     

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.     

General enzymatic screens identify three new nucleotidases in Escherichia coli. Biochemical characterization of SurE, YfbR, and YjjG

To find proteins with nucleotidase activity in Escherichia coli, purified unknown proteins were screened for the presence of phosphatase activity using the general phosphatase substrate p-nitrophenyl phosphate. Proteins exhibiting catalytic activity were then assayed for nucleotidase activity against various nucleotides. These screens identified the presence of nucleotidase activity in three uncharacterized E. coli proteins, SurE, YfbR, and YjjG, that belong to different enzyme superfamilies: SurE-like family, HD domain family (YfbR), and haloacid dehalogenase (HAD)-like superfamily (YjjG). The phosphatase activity of these proteins had a neutral pH optimum (pH 7.0-8.0) and was strictly dependent on the presence of divalent metal cations (SurE: Mn(2+) > Co(2+) > Ni(2+) > Mg(2+); YfbR: Co(2+) > Mn(2+) > Cu(2+); YjjG: Mg(2+) > Mn(2+) > Co(2+)). Further biochemical characterization of SurE revealed that it has a broad substrate specificity and can dephosphorylate various ribo- and deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates with highest affinity to 3'-AMP. SurE also hydrolyzed polyphosphate (exopolyphosphatase activity) with the preference for short-chain-length substrates (P(20-25)). YfbR was strictly specific to deoxyribonucleoside 5'-monophosphates, whereas YjjG showed narrow specificity to 5'-dTMP, 5'-dUMP, and 5'-UMP. The three enzymes also exhibited different sensitivities to inhibition by various nucleoside di- and triphosphates: YfbR was equally sensitive to both di- and triphosphates, SurE was inhibited only by triphosphates, and YjjG was insensitive to these effectors. The differences in their sensitivities to nucleotides and their varied substrate specificities suggest that these enzymes play unique functions in the intracellular nucleotide metabolism in E. coli.     

SYNCRIP, a member of the heterogeneous nuclear ribonucleoprotein family, is involved in mouse hepatitis virus RNA synthesis

Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5' and 3' untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5'-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5'-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5'-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.     

Threshold-dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. Here we ask whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. We have addressed this question in two ways: First, we asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and second, we examined whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm. In this study, we show that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. We also provide evidence for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. We propose that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes.     

Immunohistochemical Localization of Mitochondrial Fatty Acid ��-Oxidation Enzymes in Rat Testis

The testis consists of two types of tissues, the interstitial tissue and the seminiferous tubule, which have different functions and are assumed to have different nutritional metabolism. The localization of enzymes of the mitochondrial fatty acid β-oxidation system in the testis was investigated to obtain a better understanding of nutrient metabolism in the testis. Adult rat testis tissues were subjected to immunoblot analysis for quantitation of the amounts of enzyme proteins, to DNA microarray analysis for gene expression, and to immunofluorescence and immunoelectron microscopy for localization. Quantitative analysis by immunoblot and DNA microarray revealed that enzymes occur abundantly in Leydig cells in the interstitial tissue but much less so in the seminiferous tubules. Immunohistochemistry revealed that Leydig cells in the interstitial tissue and Sertoli cells in the seminiferous tubules contain a full set of mitochondrial fatty acid β-oxidation enzymes in relatively plentiful amounts among the cells in the testis, but that this is not so in spermatogenic cells. This characteristic localization of the mitochondrial fatty acid β-oxidation system in the testis needs further elucidation in terms of a possible role for it in the nutritional metabolism of spermatogenesis. (J Histochem Cytochem 58:195–206, 2010)     

Arkadia complexes with clathrin adaptor AP2 and regulates EGF signalling

Arkadia is a positive regulator of transforming growth factor (TGF)-β signalling that induces ubiquitin-dependent degradation of several inhibitory proteins of TGF-β signalling through its C-terminal RING domain. We report here that, through yeast-two-hybrid screening for Arkadia-binding proteins, the μ2 subunit of clathrin-adaptor 2 (AP2) complex was identified as an interacting partner of Arkadia. Arkadia was located in both the nucleus and the cytosol in mammalian cells. The C-terminal YXXΦ-binding domain of the μ2 subunit associated with the N-terminal YALL motif of Arkadia. Arkadia ubiquitylated the μ2 subunit at Lys130. In addition, Arkadia interacted with the AP2 complex, and modified endocytosis of epidermal growth factor receptor (EGFR) induced by EGF. Arkadia thus appears to regulate EGF signalling by modulating endocytosis of EGFR through interaction with AP2 complex.