Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

The family 42 carbohydrate-binding module of family 54 alpha-L-arabinofuranosidase specifically binds the arabinofuranose side chain of hemicellulose

Alpha-L-arabinofuranosidase catalyses the hydrolysis of the alpha-1,2-, alpha-1,3-, and alpha-1,5-L-arabinofuranosidic bonds in L-arabinose-containing hemicelluloses such as arabinoxylan. AkAbf54 (the glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Aspergillus kawachii) consists of two domains, a catalytic and an arabinose-binding domain. The latter has been named AkCBM42 [family 42 CBM (carbohydrate-binding module) of AkAbf54] because homologous domains are classified into CBM family 42. In the complex between AkAbf54 and arabinofuranosyl-alpha-1,2-xylobiose, the arabinose moiety occupies the binding pocket of AkCBM42, whereas the xylobiose moiety is exposed to the solvent. AkCBM42 was found to facilitate the hydrolysis of insoluble arabinoxylan, because mutants at the arabinose binding site exhibited markedly decreased activity. The results of binding assays and affinity gel electrophoresis showed that AkCBM42 interacts with arabinose-substituted, but not with unsubstituted, hemicelluloses. Isothermal titration calorimetry and frontal affinity chromatography analyses showed that the association constant of AkCBM42 with the arabinose moiety is approximately 10(3) M(-1). These results indicate that AkCBM42 binds the non-reducing-end arabinofuranosidic moiety of hemicellulose. To our knowledge, this is the first example of a CBM that can specifically recognize the side-chain monosaccharides of branched hemicelluloses.     

Identification and partial characterization of soluble and membrane-bound KDN(deaminoneuraminic acid)-glycoproteins in human ovarian teratocarcinoma PA-1, and enhanced expression of free and bound KDN in cells cultured in mannose-rich media

KDN (Deaminoneuraminic acid, or deaminated neuraminic acid) is a minor but biosynthetically independent member of the sialic acid. Human occurrence of KDN has already been established, although its level is so little that it is often undetectable by conventional sialic acid analysis. Elevated expression of KDN in fetal cord blood cells and some malignant tumor cells have been reported. However, in mammalian cells and tissues KDN mostly occurs as the free sugar and little occurred conjugated to glycolipids and/or glycoproteins. A positive correlation between the ratio of free KDN/free Neu5Ac in ovarian adenocarcinomas and the stage of malignancy has been noted for diagnostic use. We hypothesized that elevated expression of KDN in mammalian systems may be closely related to elevated activities of enzymes involved in the formation of sialoglycoconjugates and/or aberrant supply of the precursor sugar, mannose, used in the biosynthesis of KDN. In this study we used human ovarian teratocarcinoma cells PA-1 to further analyze KDN expression in human cells. Major findings reported in this paper are, (i) a 30 kDa KDN-glycoprotein immunostainable with monoclonal antibody, mAb.kdn3G, (specific for the KDNα2 → 3Galβ1→ epitope) and sensitive to KDNase was identified in the membrane fraction of the cell: (ii) a 49 kDa KDN-glycoprotein that is not reactive with mAb.kdn3G but is sensitive to KDNase was identified in the soluble fraction: and (iii) PA-1 cells showed unique response to mannose added to the growth medium in that the levels of both free and bound forms of KDN are elevated. This is the first report on the identification of mammalian KDN-glycoproteins by chemical and biochemical methods.     

Ganglioside GD1a regulation of caveolin-1 and Stim1 expression in mouse FBJ cells:Augmented expression of caveolin-1 and Stim1 in cells with increased GD1a content

GD1a was previously shown responsible for regulating cell motility, cellular adhesiveness to vitronectin, phosphorylation of c-Met and metastatic ability of mouse FBJ osteosarcoma cells. To determine the particular molecules regulated by GD1a, FBJ cells were assessed for tumor-related gene expression by semi-quantitative RT-PCR. Caveolin-1 and stromal interaction molecule 1 (Stim1) expression in FBJ-S1 cells, rich in GD1a, were found to be 6 and 4 times as much, respectively, than in FBJ-LL cells devoid of GD1a. Enhanced production of caveolin-1 in protein was confirmed by Western blotting. A low-metastatic FBJ-LL cell variant, having high GD1a expression through β1-4GalNAcT-1 (GM2/GD2 synthase) cDNA transfection (Hyuga S, et al, Int J Cancer 83: 685-91, 1999), showed enhanced production of caveolin-1 and Stim1 in mRNA and protein, compared to mock-transfectant M5. Incubation of FBJ-M5 cells with exogenous GD1a augmented the expression of caveolin-1 in mRNA and protein and Stim1 in mRNA as well. Treatment of FBJ-S1 with fumonisin B1, an inhibitor of N-acylsphinganine synthesis, for 15 days caused the complete depletion of gangliosides and suppressed the expression of caveolin-1 and Stim1. St3gal5 siRNA transfected cells showed decreased expression of caveolin-1 and Stim1 mRNA, as well as St3gal5 mRNA. These findings clearly indicate ganglioside GD1a to be involved in the regulation of the transformation suppressor genes, caveolin-1 and Stim1. Moreover, treatment with GD1a of mouse melanoma B16 cells and human hepatoma HepG2 cells brought about elevated expression of caveolin-1 and Stim1. Li Wang and Shizuka Takaku are equal contributors to the present work     

Association of the -112A>C polymorphism of the uncoupling protein 1 gene with insulin resistance in Japanese individuals with type 2 diabetes

The -112A>C polymorphism (rs10011540) of the gene for uncoupling protein 1 (UCP1) has been associated with type 2 diabetes mellitus in Japanese individuals. The aim of the present study was to investigate the effects of this polymorphism, as well as the well-known -3826A>G polymorphism (rs1800592), on clinical characteristics of type 2 diabetes. We determined the genotypes of the two polymorphisms in 93 Japanese patients with type 2 diabetes. Intramyocellular lipid content and hepatic lipid content (HLC) were measured by magnetic resonance spectroscopy. No significant differences in age, sex, BMI, or HbA1c level were detected between type 2 diabetic patients with the -112C allele and those without it. However, homeostasis model assessment for insulin resistance (p=0.0089) and HLC (p=0.012) was significantly greater in patients with the -112C allele. We did not detect an association of the -3826A>G polymorphism (rs1800592) of UCP1 gene with any measured parameters. These results suggest that insulin resistance caused by the -112C allele influences the susceptibility to type 2 diabetes.     

Rho2 is a target of the farnesyltransferase Cpp1 and acts upstream of Pmk1 mitogen-activated protein kinase signaling in fission yeast

We have previously demonstrated that knockout of the calcineurin gene or inhibition of calcineurin activity by immunosuppressants resulted in hypersensitivity to Cl- in fission yeast. We also demonstrated that knockout of the components of the Pmk1 mitogen-activated protein kinase (MAPK) pathway, such as Pmk1 or Pek1 complemented the hypersensitivity to Cl-. Using this interaction between calcineurin and Pmk1 MAPK, here we developed a genetic screen that aims to identify new regulators of the Pmk1 signaling and isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vic1-1, carried a missense mutation in the cpp1+ gene encoding a beta subunit of the protein farnesyltransferase, which caused an amino acid substitution of aspartate 155 of Cpp1 to asparagine (Cpp1(D155N)). Analysis of the mutant strain revealed that Rho2 is a novel target of Cpp1. Moreover, Cpp1 and Rho2 act upstream of Pck2-Pmk1 MAPK signaling pathway, thereby resulting in the vic phenotype upon their mutations. Interestingly, compared with other substrates of Cpp1, defects of Rho2 function were more phenotypically manifested by the Cpp1(D155N) mutation. Together, our results demonstrate that Cpp1 is a key component of the Pck2-Pmk1 signaling through the spatial control of the small GTPase Rho2.     

Role of connexin-43 in protective PI3K-Akt-GSK-3β signaling in cardiomyocytes

Sarcolemmal connexin-43 (Cx43) and mitochondrial Cx43 play distinct roles: formation of gap junctions and production of reactive oxygen species (ROS) for redox signaling. In this study, we examined the hypothesis that Cx43 contributes to activation of a major cytoprotective signal pathway, phosphoinositide 3-kinase (PI3K)-Akt-glycogen synthase kinase-3β (GSK-3β) signaling, in cardiomyocytes. A δ-opioid receptor agonist {[d-Ala(2),d-Leu(5)]enkephalin acetate (DADLE)}, endothelin-1 (ET-1), and insulin-like growth factor-1 (IGF-1) induced phosphorylation of Akt and GSK-3β in H9c2 cardiomyocytes. Reduction of Cx43 protein to 20% of the normal level by Cx43 small interfering RNA abolished phosphorylation of Akt and GSK-3β induced by DADLE or ET-1 but not that induced by IGF-1. DADLE and IGF-1 protected H9c2 cells from necrosis after treatment with H(2)O(2) or antimycin A. The protection by DADLE or ET-1, but not that by IGF-1, was lost by reduction of Cx43 protein expression. In contrast to Akt and GSK-3β, PKC-ε, ERK and p38 mitogen-activated protein kinase were phosphorylated by ET-1 in Cx43-knocked-down cells. Like diazoxide, an activator of the mitochondrial ATP-sensitive K(+) channel, DADLE and ET-1 induced significant ROS production in mitochondria, although such an effect was not observed for IGF-1. Cx43 knockdown did not attenuate the mitochondrial ROS production by DADLE or ET-1. Cx43 was coimmunoprecipitated with the β-subunit of G protein (Gβ), and knockdown of Gβ mimicked the effect of Cx43 knockdown on ET-1-induced phosphorylation of Akt and GSK-3β. These results suggest that Cx43 contributes to activation of class I(B) PI3K in PI3K-Akt-GSK-3β signaling possibly as a cofactor of Gβ in cardiomyocytes.     

Intricate interactions between the bloom-forming cyanobacterium Microcystis aeruginosa and foreign genetic elements, revealed by diversified clustered regularly interspaced short palindromic repeat (CRISPR) signatures

Clustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.     

A strong exonic splicing enhancer in dystrophin exon 19 achieve proper splicing without an upstream polypyrimidine tract

Proper splicing is known to proceed under the control of conserved cis-elements located at exon-intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in 'in vitro splicing'. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.     

Induction of human UGT1A1 by a complex of dexamethasone-GR dependent on proximal site and independent of PBREM

UDP-glucuronosyltransferase 1A1 (UGT1A1) plays a key role to conjugate bilirubin and prevent jaundice. There are two major elements for the induction of UGT1A1, such as PBREM (-3483/-3194), far from the promoter site, and HNF1 (-75/-63), near to the promoter site. In a previous report, we showed that the proximal HNF1 site is essential for the induction of UGT1A1 by glucocorticoid receptor (GR). In this report, we investigated the influence of PBREM on the induction of the UGT1A1 reporter gene by GR and PXR with dexamethasone (DEX). We confirmed that GR was transferred from cytosol into the nucleus in 15-30 min by DEX stimulation, but HNF1 was not. We constructed a reporter gene containing PBREM to compare the induction of the reporter gene without PBREM by DEX-GR. The results show that induction of the reporter gene with PBREM by DEX at 100 muM is the same level as the induction of the reporter gene without PBREM, although PBREM contains GRE. Co-transfection of hGR with the reporter gene did not show any influence of the induction of the reporter gene between the vector with and without PBREM. Meanwhile, by co-transfection of hPXR, the induction of the reporter gene with PBREM was significantly more than the induction of the reporter gene without PBREM at 100 muM DEX. This supports that hPXR induced UGT1A1 through PBREM by DEX. These results showed that PBREM has no relation with the induction by DEX-GR but the proximal site of UGT1A1 may function in stimulation by DEX-GR.