Suppressor of cytokine signaling-3 is a biomechanical stress-inducible gene that suppresses gp130-mediated cardiac myocyte hypertrophy and survival pathways.

The gp130 cytokine receptor activates a cardiomyocyte survival pathway during the transition to heart failure following the biomechanical stress of pressure overload. Although gp130 activation is observed transiently during transverse aortic constriction (TAC), its mechanism of inactivation is largely unknown in cardiomyocytes. We show here that suppressor of cytokine signaling 3 (SOCS3), an intrinsic inhibitor of JAK, shows biphasic induction in response to TAC. The induction of SOCS3 was closely correlated with STAT3 phosphorylation, as well as the activation of an embryonic gene program, suggesting that cardiac gp130-JAK signaling is precisely controlled by this endogenous suppressor. In addition to its cytoprotective action, gp130-dependent signaling induces cardiomyocyte hypertrophy. Adenovirus-mediated gene transfer of SOCS3 to ventricular cardiomyocytes completely suppressed both hypertrophy and antiapoptotic phenotypes induced by leukemia inhibitory factor (LIF). To our knowledge, this is the first clear evidence that these two separate cardiomyocyte phenotypes induced by gp130 activation lie downstream of JAK. Three independent signaling pathways, STAT3, MEK1-ERK1/2, and AKT activation, that are coinduced by LIF stimulation were completely suppressed by SOCS3 overexpression. We conclude that SOCS3 is a mechanical stress-inducible gene in cardiac muscle cells and that it directly modulates stress-induced gp130 cytokine receptor signaling as the key molecular switch for a negative feedback circuit for both myocyte hypertrophy and survival.

Induction of JAB/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3 is involved in gp130 resistance in cardiovascular system in rat treated with cardiotrophin-1 in vivo.

CIS (cytokine-inducible SH2 protein), SOCS (suppressor of cytokine signaling), or SSI (signal transducers and activators of transcription [STAT]-induced STAT inhibitor) proteins are a family of cytokine-inducible negative regulators of cytokine signaling via Janus kinase (JAK)-STAT pathways. Given the evidence that the JAK-STAT pathway plays a critical role in the cardiovascular system, the primary objective of this study was to assess the effects of the CIS family on JAK-STAT signaling in the cardiovascular system in rats treated with cardiotrophin-1 (CT-1), an interleukin-6 family of cytokines. Intravenous injection of 20 microgram/kg body weight of CT-1 induced a transient, marked increase in STAT3 activation in various tissues, including heart and lung, and subsequent upregulation of 2 members of the CIS family, JAK-binding protein (JAB)/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3, in the same tissues. It was also observed that CIS3 was directly associated with JAK2 in vivo. Pretreatment with the same dose of CT-1 60 minutes before significantly attenuated the STAT3 activation induced by a second injection of CT-1. We previously reported that intravenous injection of CT-1 results in the nitric oxide (NO)-dependent hypotension accompanied by the induction of inducible NO synthase mRNA. In rats pretreated with CT-1, the induction of inducible NO synthase mRNA or hypotension by subsequent CT-1 injection was not observed. Forced expression of JAB or CIS3, but not other CISs, directly blocked CT-1-induced STAT3 activation in 293 cells. These results suggest that JAB and CIS3 serve as endogenous inhibitors of CT-1-mediated JAK-STAT signaling in the cardiovascular system in vivo.

Interleukin-6-induced activation of signal transducer and activator of transcription-3 in ruptured rotator cuff tendon.

The aim of this study was to examine interleukin-6 production and the activation of signal transducer and activator of transcription-3 (STAT3) in ruptured rotator cuff tendon. Specimens of ruptured rotator cuff tendons were analysed using real-time reverse transcriptase polymerase chain reaction, Western blotting and immunohistochemistry. Specimens of co-existing inflammatory subacromial synovia were examined for comparison. The level of interleukin-6 messenger RNA was increased in ruptured rotator cuff tendon as well as in subacromial synovium. Western blot analysis showed constitutive production of activated, phosphorylated STAT3 in ruptured rotator cuff tendon and co-existing subacromial synovium. Immunohistochemical examination detected cells producing interleukin-6, interleukin-6 receptor and phosphorylated STAT3 in ruptured rotator cuff tendon, mainly in proliferative vessels and, to a lesser extent, in tendon fibroblasts around the vessels. This study demonstrates that activation of STAT3 induced by interleukin-6 is promoted mainly by proliferative vessels in ruptured rotator cuff tendon.

CIS3 and JAB have different regulatory roles in interleukin-6 mediated differentiation and STAT3 activation in M1 leukemia cells.

We have reported JAK-signaling modulators, CIS1 (cytokine-inducible SH2 protein-1), CIS3 and JAB (JAK2 binding protein), which are structurally related. In M1 myeloid leukemia cells, CIS3 was induced by neither interleukin 6 (IL6) nor interferon gamma (IFNgamma), while JAB was induced strongly by IFNgamma and slightly by IL6 and leukemia inhibitory factor (ILF). Forced expression of CIS3 and JAB in M1 cells prevented IL6- or LIF-induced growth arrest and differentiation, even when their expression levels were comparable to endogenous ones in several cell lines such as HEL, UT-7, IFNgamma-treated M1, and CTLL2 cells. Pretreatment of parental M1 cells with IFNgamma but not IFNbeta resulted in suppression of LIF-induced STAT3 activation and differentiation, further supporting that physiological level of JAB is sufficient to inhibit LIF-signaling. However, unlike JAB, CIS3 did not inhibit IFNgamma-induced growth arrest, suggesting a difference in cytokine specificity between CIS3 and JAB. CIS3 inhibited STAT3 activation with slower kinetics than JAB and allowed rapid c-fos induction and partial FcgammaRI expression in response to IL6. In 293 cells, CIS3 as well as JAB bound to JAK2 tyrosine kinase domain (JH1), and inhibited its kinase activity, however, the effect of CIS3 on tyrosine kinase activity was weaker than that of JAB, indicating that CIS3 possesses lower affinity to JAK kinases than JAB. These findings suggest that CIS3 is a weaker inhibitor than JAB against JAK signaling, and JAB and CIS3 possess different regulatory roles in cytokine signaling.

APS, an adaptor protein containing PH and SH2 domains, is associated with the PDGF receptor and c-Cbl and inhibits PDGF-induced mitogenesis.

Previously we cloned a novel adaptor protein, APS (adaptor molecules containing PH and SH2 domains) which was tyrosine phosphorylated in response to c-kit or B cell receptor stimulation. Here we report that APS was expressed in some human osteosarcoma cell lines, markedly so in SaOS-2 cells, and was tyrosine-phosphorylated in response to several growth factors, including platelet derived growth factor (PDGF), insulin-like growth factor (IGF), and granulocyte-macrophage colony stimulating factor (GM-CSF). Ectopic expression of the wild type APS, but not C-terminal truncated APS, in NIH3T3 fibroblasts suppressed PDGF-induced MAP kinase (Erk2) activation, c-fos and c-myc induction as well as cell proliferation. In vitro binding experiments suggest that APS bound to the beta type PDGF receptor, mainly via phosphotyrosine 1021 (pY1021). Indeed, tyrosine phosphorylation of PLC-gamma, which has been demonstrated to bind to pY1021, but not that of PI3 kinase and associated proteins, was reduced in APS transformants. PDGF induced phosphorylation of the tyrosine residue of APS close to the C-terminal end. In vitro and in vivo binding experiments indicate that the tyrosine phosphorylated C-terminal region of APS bound to c-Cbl, which has been shown to be a negative regulator of tyrosine kinases. Since coexpression of c-Cbl with wild type APS, but not C-terminal truncated APS, synergistically inhibited PDGF-induced c-fos promoter activation, c-Cbl could be a mechanism of inhibitory action of APS on PDGF receptor signaling.

Determination of the single strand origin of Shigella sonnei plasmid pKYM.

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechanism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cloned the fragment into a -ori defective M13 vector phage, M13 delta lac183. The chimera phage, MDKY5, showed a larger plaque size, and increased phage yield and rate of progeny replicative form DNA (RF) synthesis. Rifampicin reduced rate of conversion of the single- to double-stranded RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hairpin loops and formed smaller plaques. The in vivo analyses revealed that a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single strand origin, referred to as SSO) and harbors a recognition site(s) by host E. coli RNA polymerase, for primer RNA synthesis. Moreover, we found a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RSA) which functions in plasmid sitespecific recombination and/or transfer.

FebA: a gene for eukaryotic translation initiation factor 4E-binding protein (4E-BP) in Dictyostelium discoideum.

We have identified a gene encoding a eukaryotic initiation factor 4E-binding protein (4E-BP) in the EST database of the Dictyostelium cDNA project. The Dictyostelium 4E-BP, designated febA (four e-binding), showed significant similarity to mammalian 4E-BPs. Northern blot analysis revealed that febA was expressed at a high level in the vegetative growth phase but the level of expression decreased during late development. The gene was shown to be non-essential since disruption of the gene had no severe effect; the null mutant proliferated normally and formed normal fruiting bodies. However, strains overexpressing the gene could not be established, suggesting that an excess of FebA protein may have a lethal effect on the cells.

Peripheral blood levels of matrix metalloproteases-2 and -9 are elevated in patients with acute coronary syndromes.

OBJECTIVES: This study was sought to investigate whether peripheral blood levels of matrix metalloproteases (MMPs) are affected in patients with acute coronary syndromes (ACS). BACKGROUND: Synthesis of MMPs has been reported in coronary atherosclerotic lesions in patients with unstable angina (UA), suggesting a pathogenic role of MMPs in the development of ACS. METHODS: Using sandwich enzyme immunoassay, serum MMP-2 and plasma MMP-9 were measured in 33 patients with ACS (22 with acute myocardial infarction [AMI], 11 with UA), 17 with stable effort angina (EA) and 17 normal control subjects. RESULTS: Serum MMP-2 in patients with UA and AMI on day 0 was two times greater than that in control subjects, and patients with EA showed higher MMP-2 levels than those in control subjects. Plasma MMP-9 in patients with UA and AMI on day 0 was elevated by threefold and twofold versus that in control subjects, respectively. In patients with UA and AMI who underwent medical treatment (n = 11 and 13, respectively), MMP-2 elevation was sustained until day 7. In patients with UA, MMP-9 elevation on day 0 was followed by a gradual decrease toward the control range up to day 7. Some patients with AMI showed a transient MMP-9 elevation with a peak on day 3, whereas in others, MMP-9 levels were significantly elevated on day 0 and remained higher than those in control subjects up to day 3. CONCLUSIONS: Serial changes in serum MMP-2 and plasma MMP-9 were documented in patients with ACS. These findings provide an insight into the molecular mechanism of plaque destabilization.

Asymmetrical dimethylarginine, an endogenous nitric oxide synthase inhibitor, in experimental hypertension.

NG,NG-dimethyl-L-arginine (ADMA) is an endogenously synthesized nitric oxide (NO) synthase inhibitor which has potent pressor/vasoconstrictor effects. Dimethylargininase metabolizes ADMA to L-citrulline and plays a key role in determining the in vivo levels of ADMA. To investigate the role of ADMA in the pathogenesis of hypertension, we measured 24-hour urinary excretion of ADMA (UADMA) and nitrate/nitrite (NOx) in Dahl salt-sensitive hypertensive rats and spontaneously hypertensive rats (SHR). In Dahl salt-resistant rats, high-salt diet (8% NaCl) did not increase blood pressure and increased urinary NOx (P < .01) without changes in UADMA compared with low-salt diet (0.3% NaCl). In contrast, in Dahl salt-sensitive rats, high-salt diet increased blood pressure (P < .01), did not change urinary NOx excretion, and increased UADMA (P < .01). There was a significant (r = .65, P < .01) correlation between UADMA and the level of blood pressure in Dahl salt-sensitive rats. Plasma levels of NOx and ADMA and renal dimethylargininase content were comparable among them. These results may suggest that in Dahl salt-resistant rats, blood pressure is kept constant during high-salt intake, possibly due to the compensatory increased production of NO, and that in Dahl salt-sensitive rats, high-salt intake increases the production of ADMA, attenuates the compensatory increases in NO, and increases blood pressure. These results also suggest that the systemic production of ADMA is not dependent on renal dimethylargininase. SHR had significantly greater urinary NOx excretion (P < .05) and smaller UADMA than Wistar-Kyoto rats (P < .05), and UADMA was inversely correlated with their mean arterial pressure (r =.64, P < .05). In conclusion. ADMA, independently of the renal dimethylargininase content, may play a role in the pathogenesis in Dahl salt-sensitive hypertensive rats but not in SHR.

Rolling-circle plasmid pKYM re-initiates DNA replication.

It is believed that rolling-circle plasmids are incapable of re-initiation since they have to maintain their copy number and this is one of the differences between plasmids and phages as phi-x174. To examine whether a rolling-circle plasmid pKYM is incapable of re-initiating DNA replication, we constructed a plasmid that carries both the pKYM origin (fragment 13, 173 bp) and its truncated origin (fragment 32, 56 bp) in the same orientation. This plasmid yielded two smaller plasmids in the presence of RepK, an initiator protein. We showed that RepK can bind to the fragment 13 but not to fragment 32 which lacks the 3'- moiety of fragment 13. These results imply that RepK initiates DNA replication from fragment 13 and terminates at fragment 32, then the same RepK is used for re-initiation of replication from the fragment 32 region. pKYM is likely to be a unique plasmid that re-initiates DNA replication like a phase phi-x174.

Identification of the region required for monomerization of the rolling circle plasmid pKYM.

Plasmid pKYM, isolated from the gram-negative bacterium Shigella sonnei, is a small multicopy plasmid which replicates by a rolling circle mechanism. The formation of multimers has been observed in a derivative of pKYM which lost a part of the origin region, and the loss of the monomerization mechanism would have led to these multimers. By analyzing the constructs of several mutants, we discovered that a DNA region required for monomerization was present upstream of the RepK binding site in the replication origin. As either of the T-rich sequence or the inverted repeat sequences which were seen in that region have been lost in the multimer-forming plasmids, these sequences may be necessary for monomerization.

Synthesis of RepK of rolling circle plasmid pKYM is regulated by countertranscript and HU protein.

Replication of rolling circle plasmid pKYM was regulated by RepK, a plasmid-encoded initiator protein, with HU protein and antisense RNA (copRNA) that block the expression of RepK. HU protein bound to the repK promoter in the presence of RepK protein and inhibited the transcription of repK mRNA. The copRNA would hybridize to repK mRNA and induce a stem-loop structure in which the repK Shine-Dalgarno sequence is sequestered by base pairing. Sequence substitution experiments demonstrated that this stem-loop not only inhibits translation but induces premature termination.

The Dictyostelium developmental cDNA project: generation and analysis of expressed sequence tags from the first-finger stage of development.

In an effort to identify and characterize genes expressed during multicellular development ill Dictyostelium, we have undertaken a cDNA sequencing project. Using size-fractionated subsets of cDNA from the first finger stage, two sets of gridded libraries were constructed for cDNA sequencing. One, library S, consisting of 9984 clones, carries relatively short inserts, and the other, library L, which consists of 8448 clones, has longer inserts. We sequenced all the selected clones in library S from their 3'-ends, and this generated 3093 non-redundant, expressed sequence tags (ESTs). Among them, 246 ESTs hit known Dictyostelium genes and 910 showed significant similarity to genes of Dictyostelium and other organisms. For library L, 1132 clones were randomly sequenced and 471 non-redundant ESTs were obtained. In combination, the ESTs from the two libraries represent approximately 40% of genes expressed in late development, assuming that the non-redundant ESTs correspond to independent genes. They will provide a useful resource for investigating the genetic networks that regulate multicellular development of this organism.

Purification and DNA-binding properties of the integrase protein Int encoded by Lactobacillus plantarum phage.

The Lactobacillus plantarum temperate phage phi g1e (42,259 bp) encodes an integrase gene int linked to a phage attachment site attP (Kakikawa et al., 1997). To investigate phi g1e recombination, the integrase protein Int was overproduced in Escherichia coli under the T7 promoter, and purified. The Int protein had an apparent molecular mass of 42.0 kDa, corresponding well with that (45.5 kDa) predicted from the DNA sequence. Amino-acid sequencing revealed that the N-terminal 20 amino-acids of the purified Int protein completely coincided with those deduced from the DNA sequence, although deficient in the first methionine. Gel mobility-shift assays demonstrated that Int bound specifically to the attP region. In addition, footprinting analysis showed that Int protected about 35 bases, containing the 24-bp core domain at attP, from DNase I attack. These results are indicative of site-specific interaction of Int with the attP site, the reaction prerequisite for integration and excision of the phi g1e genome into and/or out of the host chromosome.

Characterization of the genes encoding integrative and excisive functions of Lactobacillus phage øg1e: cloning, sequence analysis, and expression in Escherichia coli.

øg1e is a temperate phage of the Lactobacillus strain G1e. The phage-host junctions attR and attL cloned from the lysogen have a 24-bp common (core) sequence implicated in recombination. DNA sequencing analysis of a 5.2-kbp SacI fragment of the øg1e phage genome (42.5 kbp) revealed two possible open reading frames (ORF), xis and int, and the phage attachment (recombination) site (attP), whose 24-bp sequence is identical to the core sequence detected in attR and attL. The deduced int product (Int) is a basic protein of 391 amino acids with an estimated pI of 9.70, and significantly resembles other presumed integrases encoded by the Lactobacillus and Lactococcus phages including øadh and øLC3, as well as the Escherichia coli phages such as lambda. The predicted øg1e xis protein (Xis) is small and very acidic (66 amino acids; pI 4.55), and shows a resemblance (32% overall identity) with a putative excisionase encoded by the Staphylococcus phage ø11. The øg1e Int with a deduced molecular mass of 45.5 kDa was overproduced in E. coli cells, and electrophoretically analyzed.

Promoter/repressor system of Lactobacillus plantarum phage og1e: characterization of the promoters pR49-pR-pL and overproduction of the cro-like protein cng in Escherichia coli.

The Lactobacillus plantarum phage og1e (42259bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M.

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in beta-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease (Staphylococcus aureus strain V8) and SPase (S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene (proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Expression of stem cell factor in human aortic endothelial and smooth muscle cells.

It has been confirmed that the receptor protein encoded by the c-kit proto-oncogene is expressed by cells of the hematopoietic, gonadal, pigment, and mast cell lineages and that its ligand, stem cell factor (SCF), is mainly expressed in their microenvironment. In a previous study we investigated the expression of the c-kit gene in human aortic endothelial cells (EC). In the present study we investigated the expression of SCF in human aortic EC and smooth muscle cells (SMC). Reverse transcription (RT)-PCR and Northern blot analyses showed that both human arterial EC and SMC expressed mRNA specific for the SCF gene. In addition, tissue-specific expression of the SCF gene was confirmed by in situ hybridization in the EC and the SMC. Western blot analysis and immunocytochemistry showed evidence of production of SCF protein in both the EC and the SMC. These results indicate the existence of mast cell-SMC interaction and of an autocrine loop of c-kit and its ligand on the surface of EC, suggesting that the interaction between c-kit protein and SCF may play an important role in metabolism of arterial wall and in the pathogenesis of atherosclerosis in the arterial intima.

Cloning and nucleotide sequences of the BanI restriction-modification genes in Bacillus aneurinolyticus.

The genes of the BanI restriction-modification system specific for GGPyPuCC were cloned from the chromosomal DNA of Bacillus aneurinolyticus IAM1077, and the coding regions were assigned on the nucleotide sequence on the basis of the N-terminal amino acid sequences and molecular weights of the enzymes. The restriction and modification genes coded for polypeptides with calculated molecular weights of 39,841 and 42,637, respectively. Both the enzymes were coded by the same DNA strand. The restriction gene was located upstream of the methylase gene, separated by 21 bp. The cloned genes were significantly expressed in E. coli cells, so that the respective enzymes could be purified to homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration indicated that the catalytically active form of the endonuclease was dimeric and that of the methylase was monomeric. Comparison of the amino acid sequences revealed no significant homology between the endonuclease and methylase, though both enzymes recognize the same target sequence. Sequence comparison with other related enzymes indicated that BanI methylase contains sequences common to cytosine-specific methylases.

Neural cell adhesion molecule expression in thyroid cancer.

Neural cell adhesion molecule (NCAM) is an integral membrane glycoprotein that participates in cell-cell interactions. We examined the expression of neural cell adhesion molecule (NCAM) by immunohistochemistry in 65 cases of thyroid disease. In the majority of thyroid tumors, NCAM expression was detected in tumor cells. In chronic thyroiditis, Hurthle cells demonstrated expression of NCAM. In normal thyroid gland, neither follicular nor parafollicular cells expressed NCAM at detectable levels. All papillary carcinomas and most poorly differentiated follicular carcinomas and some follicular adenomas demonstrated NCAM expression. In thyroid carcinoma, clinical stage and recurrent laryngeal nerve paralysis did not correlate significantly with expression of NCAM. Papillary carcinomas with strong expression of NCAM were more likely to have extra capsular invasion. It is supposed that immunohistochemical detection of NCAM expression supports histologic diagnosis and offers prognostic information.