The production of coagulation factor VII by adipocytes is enhanced by tumor necrosis factor-α or isoproterenol.

BACKGROUND: A relationship has been reported between blood concentrations of coagulation factor VII (FVII) and obesity. In addition to its role in coagulation, FVII has been shown to inhibit insulin signals in adipocytes. However, the production of FVII by adipocytes remains unclear. OBJECTIVE: We herein investigated the production and secretion of FVII by adipocytes, especially in relation to obesity-related conditions including adipose inflammation and sympathetic nerve activation. METHODS: C57Bl/6J mice were fed a low- or high-fat diet and the expression of FVII messenger RNA (mRNA) was then examined in adipose tissue. 3T3-L1 cells were used as an adipocyte model for in vitro experiments in which these cells were treated with tumor necrosis factor-α (TNF-α) or isoproterenol. The expression and secretion of FVII were assessed by quantitative real-time PCR, Western blotting and enzyme-linked immunosorbent assays. RESULTS: The expression of FVII mRNA in the adipose tissue of mice fed with high-fat diet was significantly higher than that in mice fed with low-fat diet. Expression of the FVII gene and protein was induced during adipogenesis and maintained in mature adipocytes. The expression and secretion of FVII mRNA were increased in the culture medium of 3T3-L1 adipocytes treated with TNF-α, and these effects were blocked when these cells were exposed to inhibitors of mitogen-activated kinases or NF-κB activation. The ß-adrenoceptor agonist isoproterenol stimulated the secretion of FVII from mature adipocytes via the cyclic AMP/protein kinase A pathway. Blockade of secreted FVII with the anti-FVII antibody did not affect the phosphorylation of Akt in the isoproterenol-stimulated adipocytes. CONCLUSION: Obese adipose tissue produced FVII. The production and secretion of FVII by adipocytes was enhanced by TNF-α or isoproterenol via different mechanisms. These results indicate that FVII is an adipokine that plays an important role in the pathogenesis of obesity.

Promotion of DNA repair by nuclear IKKß phosphorylation of ATM in response to genotoxic stimuli.

Ataxia-telangiectasia mutated (ATM) is one of the key molecules involved in the cellular response to DNA damage. A portion of activated ATM is exported from the nucleus into the cytoplasm, where it activates the I kappa B kinase/nuclear factor kappa B (IKK/NF-κB) signaling pathway. It has been thought that activated IKKß, which is a critical kinase for NF-κB activation, generally resides in the cytoplasm and phosphorylates cytoplasmic downstream molecules, such as IκBα. Here, we identified a new role for IKKß during the response to DNA damage. ATM phosphorylation in response to alkylating agents consisted of two phases: the early phase (up to 3 h) and late phase (after 6 h). A portion of the activated IKKß generated during the DNA damage response was found to translocate into the nucleus and directly phosphorylate ATM in the late phase. Furthermore, the phosphorylation of ATM by nuclear IKKß was suggested to promote DNA repair. In parallel, activated IKKß induced classical NF-κB activation and was involved in anti-apoptosis. Our findings define the function of IKKß during the response to DNA damage, which promotes cell survival and DNA repair, and maintains cellular homeostasis.

Distinct mechanism of Helicobacter pylori-mediated NF-kappa B activation between gastric cancer cells and monocytic cells.

NF-kappaB is a critical regulator of genes involved in inflammation. Gastric epithelial cells and macrophages are considered the main sources of pro-inflammatory cytokines. We investigated NF-kappaB activation by Helicobacter pylori in MKN45 gastric epithelial cells and THP-1 monocytic cells. Although, cag pathogenicity island (PAI)-positive H. pylori (wild type) activated NF-kappaB in both cells, isogenic mutant of cagE (DeltacagE) activated it only in THP-1 cells. Supernatant from the wild type culture could activate NF-kappaB in THP-1 cells but not in MKN45 cells. High density cDNA array analysis revealed that mRNA expression of NF-kappaB-regulated genes such as interleukin (IL)-8, tumor necrosis factor-alpha (TNFalpha), and IL-1beta was significantly up-regulated by the wild type in both cells, whereas it was up-regulated by DeltacagE only in THP-1 cells. Experiments using CD14-neutralizing antibody and IL-1 receptor-associated kinase (IRAK) assay showed that both wild type and DeltacagE H. pylori activated NF-kappaB through CD14 and IRAK in THP-1 cells but not in MKN45 cells. Macrophages from C3H/HeJ mice carrying point mutation in the Toll-like receptor 4 (TLR4) gene showed decreased NF-kappaB activation and TNFalpha secretion compared with C3H/HeN mouse macrophage when treated with H. pylori. In conclusion, H. pylori-induced NF-kappaB activation in epithelial cells is dependent on cag PAI and contact but does not involve CD14 and IRAK, whereas in macrophage/monocytic cells it is independent of cag PAI or contact but involves CD14 and TLR4.

Helicobacter pylori activates the cyclin D1 gene through mitogen-activated protein kinase pathway in gastric cancer cells.

Helicobacter pylori induces cellular proliferation in host cells, but the mechanism remains unclear. Thus, we examined the effect of H. pylori on cyclin D1, an important regulator of the cell cycle, especially in relation to intracellular signaling pathways. In a Northern blot analysis, cyclin D1 transcription in gastric cancer (AGS) cells was enhanced by coculture with H. pylori strain TN2 in a time-dependent and multiplicity-of-infection-dependent manner. An isogenic mutant form of vacA also increased cyclin D1 transcription, but mutant forms of cagE or the entire cag pathogenicity island did not enhance cyclin D1 transcription. These effects were confirmed with a luciferase assay of the cyclin D1 promoter (pD1luc). Cyclin D1 promoter activation by H. pylori was inhibited by MEK inhibitors (U0126 and PD98059), indicating that the mitogen-activated protein kinase pathway may be involved in intracellular signal transduction. In contrast, transfection of a reporter plasmid having any point mutations of the NF-kappaB binding sites in the promoter (pD1-kappaB1M, pD1-kappaB2M, or pD1-kappaB1/2M) or cotransfection of dominant negative IkappaBalpha did not affect cyclin D1 activation by H. pylori. In conclusion, H. pylori activates cyclin D1 through the mitogen-activated protein kinase pathway and not through NF-kappaB activation in AGS cells. This activation of cyclin D1 is partly dependent on the cag pathogenicity island but not on vacA.

Coronary remodeling of proximal and distal stenotic atherosclerotic plaques within the same artery by intravascular ultrasound study.

The aim of this intravascular ultrasound study was to compare the type and the degree of vessel remodeling in proximal and distal de novo lesions within the same coronary artery in patients with stable angina pectoris. Seventy-six de novo coronary artery lesions in 38 coronary arteries of 38 patients were imaged by intravascular ultrasound. The vessel area (VA) within the external elastic lamina and the lumen area (LA) were measured, and the wall area (VA-LA) was calculated at the lesion site, and the proximal and distal reference sites. The VA ratio was defined as (lesion VA/average of the proximal and distal reference VAs) to represent the degree of vessel remodeling. The proximal coronary segments showed compensatory enlargement more often (68% vs 29%, p < 0.01) than the distal segments, and the VA ratio at the lesion site was significantly larger (1.1 +/- 0.3 vs 1.0 +/- 0.2, p <0 .01) in proximal segments than in distal segments. The type of coronary remodeling was discordant in 61% and concordant in only 39% of coronary arteries between the proximal and distal segments. The type of coronary remodeling of proximal and distal coronary lesions was inhomogeneous, even within the same vessel. Proximal coronary segments showed more prominent compensatory enlargement than distal segments, which have a similar degree of luminal narrowings.

Determination of Helicobacter pylori virulence by simple gene analysis of the cag pathogenicity island.

Nucleic acid amplification was performed for five loci in the cag pathogenicity island (PAI) of Helicobacter pylori (comprising cagA, the cagA promoter region, cagE, cagT, and the left end of cagII [LEC]), and gastric inflammation in patients was evaluated. Of 204 H. pylori isolates from Japanese patients (53 with peptic ulcer, 55 with gastric cancer, and 96 with chronic gastritis), 197 (96.6%) were positive for all five loci. Two isolates (1%) were negative for all five loci, and five isolates (2.4%) were positive for only cagA and LEC. These latter seven isolates were all from patients with mild chronic gastritis. Neutrophil infiltration in gastric mucosa was significantly milder in patients infected with partially or totally deleted-PAI strains than in those with intact-PAI strains. The cagE gene was a more accurate marker of an intact cag PAI than the cagA gene, and cagE seemed to be more useful in discriminating between H. pylori strains causing different rates of disease progression.

A comparison of mutation spectra detected by the Escherichia coli lac(+) reversion assay and the Salmonella typhimurium his(+) reversion assay.

Each of the Escherichia coli tester strains in the WP3101P-WP3106P series contains an F' plasmid with a different base substitution mutation within the lacZ gene. Each of the six possible base substitution mutations, therefore, can be assayed with these strains by Lac(+) reversion. We used the strains to characterize the mutational profiles of 21 chemical mutagens, including alkylating agents, base analogs and oxidative compounds. We also assayed the mutagens with Salmonella typhimurium tester strains TA7002, TA7004 and TA7005, which detect A.T-->T.A, G.C-->A.T and G.C-->T.A mutations, respectively, and we compared the sensitivity and specificity of the two systems. Escherichia coli strain WP3102P was more sensitive than the S.TYPHIMURIUM: strains to G.C-->A.T transitions induced by N(4)-aminocytidine, 5-azacytidine, cumene hydroperoxide (CHP), t-butyl hydroperoxide (BHP), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), methyl methane sulfonate and N-ethyl-N-nitrosourea (ENU), while the reverse was true for G.C-->A.T transitions induced by 2-aminopurine and phosmet. Escherichia coli strain WP3104P, which detects G.C-->T.A transversions, was superior to the S.TYPHIMURIUM: strains in detecting transversions induced by N(4)-aminocytidine, 5-azacytidine, 5-diazouracil, CHP, BHP, ENNG, ENU, 4-nitroquinoline 1-oxide (4-NQO) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). Escherichia coli WP3105P was also more sensitive than S. TYPHIMURIUM: to A.T-->T.A transversions induced by N-methyl-N- nitrosourea (MNU), CHP and 4-NQO, but it was less sensitive to those induced by ENNG, ENU and 2-aminopurine. The present results indicate that the E.COLI: Lac(+) reversion system with tester strains WP3101P-WP3106P is as sensitive as the S.TYPHIMURIUM: His(+) reversion system for the detection of specific mutations induced by a variety of direct mutagens.

H. pylori activates NF-kappaB through a signaling pathway involving IkappaB kinases, NF-kappaB-inducing kinase, TRAF2, and TRAF6 in gastric cancer cells.

BACKGROUND & AIMS: H. pylori infection on gastric epithelial cells has been shown to induce NF-kappaB activation, but the mechanism of intracellular signal conduction that leads to NF-kappaB activation is not clear. The aim of this study was to analyze the molecular mechanism responsible for H. pylori-mediated NF-kappaB activation on gastric cancer cells. METHODS: NF-kappaB activation by H. pylori was tested by using luciferase reporter assay. IkappaBalpha degradation by H. pylori infection was assessed by immunoblotting. IKKalpha and IKKbeta activation was analyzed by kinase assay. In transfection experiments, effects of dominant negative IkappaBalpha, IKKalpha, IKKbeta, NF-kappaB-inducing kinase (NIK), TRAF2, and TRAF6 mutants were investigated. The effects of an IKKbeta-specific inhibitor, aspirin, on NF-kappaB activation and IL-8 secretion were also analyzed. RESULTS: H. pylori promotes degradation of IkappaBalpha, a cytoplasmic inhibitor of NF-kappaB. In kinase assay, H. pylori induced IKKalpha and IKKbeta catalytic activity in gastric cancer cells. Transfection of kinase-deficient mutant of either IKK inhibited H. pylori-mediated NF-kappaB activation dose-dependently. Aspirin inhibited both NF-kappaB activation and IL-8 secretion induced by H. pylori. NF-kappaB activation was also inhibited by transfection of kinase-deficient NIK or a dominant negative mutant of upstream adapter protein TRAF2 or TRAF6. CONCLUSIONS: H. pylori induces NF-kappaB activation through an intracellular signaling pathway that involves IKKalpha, IKKbeta, NIK, TRAF2, and TRAF6.

Cross-sectional study on the relationship between smoking or smoking cessation and trait anxiety.

OBJECTIVE: The relationships between trait anxiety, or anxiety proneness, and smoking and between trait anxiety and smoking cessation, among an adult population were investigated. METHODS: The subjects were 2,669 male Japanese personnel working for a Japanese government agency. Participants completed a self-administered questionnaire on smoking and smoking cessation status and other habits. Trait anxiety was evaluated with the trait anxiety part of the standardized Japanese version of the Spielberger State-Trait Anxiety Inventory. Trait anxiety is regarded as the long-term, more endogenous general type of anxiety. Odds ratios of the single 2 x 2 table were calculated and a logistic regression analysis was used to adjust for age. RESULTS: After adjusting for age, high trait anxiety did not increase the risk of smoking and was not related to success in abstaining from smoking. More subjects with high trait anxiety had planned to stop smoking (adjusted odds ratio: 1.39, P = 0.01) but did not actually succeed in doing so. CONCLUSION: The present study did not support the hypothesis that high trait anxiety increased the risk of having a smoking habit and that high trait anxiety increased the chance of abstaining from smoking. However, the study did show that high trait anxiety was related to the planning of smoking cessation, but not to actually giving up the smoking habit.

Mutation spectra of chemical mutagens determined by Lac+ reversion assay with Escherichia coli WP3101P-WP3106P tester strains.

We previously reported the development of mutation-specific Escherichia coli B tester strains WP3101 to WP3106 from strain WP2uvrA. In this study we constructed their pKM101-containing derivatives WP3101P to WP3106P, and further isolated their rfa derivatives WP4101-WP4106 and WP4101P-WP4106P. The six kinds of F' plasmids (lacI-, lacZ-, proAB+), each of which carries a different lacZ allele, contained in the above strains were originally derived from E. coli K-12 strains CC101-CC106. All the tester strains show Lac- and Trp- phenotype. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid. The trpE65(ochre) allele in the same strains enables them to be used for Trp+ reversion assays as well. In the present paper, we evaluated the sensitivity, specificity, and usefulness of the newly developed tester strains. Strains WP3101P-WP3106P were highly sensitive to determine mutational profile of heterocyclic amines with S9 mix-mediated metabolic activation and most of the oxidative mutagens and free radical generators tested. Every type of base-pair substitutions induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) or 5-diazouracil were detected in strains WP3101P-WP3106P, while A:T-->C:G and G:C-->A:T mutations induced by MeIQ, and A:T-->C:G, G:C-->A:T, and G:C-->C:G by 5-diazouracil were not detected in pKM101-free tester strains. In pKM101-carrying strains, cumene hydroperoxide induced all types of base substitutions, while formaldehyde preferentially induced G:C-->T:A transversions. Phenazine methosulfate induced predominantly G:C-->A:T transitions and G:C-->T:A transversions, while H2O2 induced predominantly G:C-->T:A and A:T-->T:A transversions. Introduction of the rfa mutation considerably enhanced sensitivity to bulky mutagens such as polycyclic aromatic compounds. All six possible base substitutions induced by 9, 10-dimethyl-1,2-benzanthracene (DMBA) were detected in tester strains WP4101P-WP4106P. In conclusion, our tester strains WP3101P-WP3106P and WP4101P-WP4106P permitted rapid and simple detection of specific mutations induced by variety of mutagens.

Development of new tester strains derived from E. coli WP2uvrA for the determination of mutational specificity.

We have developed a set of multipurpose tester strains (WP3101 to WP3106) derived from E. coli WP2uvrA for the detection and classification of mutagens. Six kinds of F' plasmid (lacI, lacZ, proAB+) in strains CC101-CC106, each of which carried a different lacZ allele, were transferred to a delta(lac-pro) derivative of WP2uvrA. Assays for transitions and transversions are based upon Lac+ reversion of a specific mutation located within the lacZ gene on an F' plasmid in strains WP3101-WP3106. In addition, the trpE65(ochre) allele in the same strains is available for Trp+ reversion assays. Using the new tester strains, we investigated the mutational specificities of various chemical mutagens. Base analog mutagens and alkylating mutagens induced specific types of base substitutions. G:C-->A:T transitions and G:C-->T:A transversions predominated in mutagenesis induced by 4-nitroquinoline 1-oxide. Only a slight increase in G:C-->T:A transversions was observed in cells treated with 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), although the potent mutagenicity of AF-2 was detected in a concurrent Trp+ reversion assay in the same strain. Sodium azide, on the other hand, was negative in the Trp+ reversion assay but specifically induced G:C-->A:T transitions. Present finding suggested that target sites for AF-2- and azide-induced lesions may largely depend on sequence context.

Detection of in vivo genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) by the alkaline single cell gel electrophoresis (Comet) assay in multiple mouse organs.

We tested the genotoxicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX) in the mouse in 6 organs (liver, lung, kidney, brain, spleen, and bone marrow) and in the mucosa of stomach, jejunum, ileum, colon, and bladder using the alkaline single-cell gel electrophoresis (SCG) (Comet) assay modified by us. Mice were sacrificed 1, 3, 6, and 24 h after oral administration of the mutagen at 100 mg/kg. MX yielded statistically significant DNA damage in the liver, kidney, lung, and brain and in all the mucosa samples. While DNA damage persisted in the gastrointestinal and urinary tract for 6-24 h after a single oral dosing, it peaked in the liver at 1 h and returned to almost the control level at 3 h. Our present results suggest that MX is genotoxic for various mouse organs, but not for the hematopoietic system, and that the alkaline SCG assay with a homogenization technique can be used to predict genotoxicity in the gastrointestinal and urinary tracts.

Food-derived heterocyclic amines potentiate the mutagenicity of a drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

We investigated the enhancing effect of heterocyclic amines on base-substitution mutations with 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 2-amino-3,4-dimethyl-imidazo[4,5-f]quinoline (MeIQ). We compared the mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the presence and absence of the heterocyclic amines in E. coli WP2 (trpE) and in excision repair-deficient strains WP2s (uvrA, trpE) and ZA500 (uvrA, rfa, trpE). Since the assay was performed without microsomal metabolic activation, Trp-P-1 and MeIQ alone were not mutagenic. In WP2, trp+ reversions induced by MX were greatly potentiated by Trp-P-1 and slightly potentiated by MeIQ. Mutation enhancement was not observed in strains WP2s and ZA500, suggesting that a functional DNA excision repair system is necessary for the combined action of MX and heterocyclic amines. Our finding implies that the combined effect of mutagens as well as the effect of individual mutagens, should be considered in risk evaluation.

Enhanced generation of A:T-->T:A transversions in a recA730 lexA51(Def) mutant of Escherichia coli.

RecA730 belongs to a class of mutant RecA protein that is often referred to as RecA*, since it is constitutively activated for coprotease functions in the absence of exogenous DNA-damage. Escherichia coli strains carrying recA730 (or other recA* alleles) exhibit dramatic increases in SOS-dependent spontaneous mutator activity. We have analyzed the specificity of this mutator phenotype by employing F'-plasmids carrying a set of mutant lacZ genes that can individually detect two types of transitions, four types of transversions, and four kinds of specific frameshift events. Analysis revealed that most of the spontaneous mutagenesis in a recA730 lexA51(Def) strain (which expresses derepressed levels of all LexA-regulated proteins) can be attributed to a specific increase in A:T-->T:A, A:T-->C:G and G:C-->T:A transversions, with the A:T-->T:A transversions occurring most frequently. These transversion events were completely abolished in a delta umuDC strain, indicating that the functionally active UmuD'C proteins are normally required for their generation. The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-->5' proofreading) subunit of DNA polymerase III. Such an observation raises the possibility that the wild-type epsilon protein is in activated in strains expressing the RecA730 and UmuD'C proteins.

Coronary artery stenosis in rats affects beta-adrenergic receptor signaling in myocytes.

OBJECTIVE: The purpose of this study was to determine whether the early chronic ischemic cardiomyopathy produced by non-occlusive coronary artery constriction was characterized by alterations in the regulation of beta-adrenoreceptor (beta-AR) signaling. METHODS: Coronary artery narrowing was surgically induced in rats and the animals sacrificed at 7 and 14 days. The changes in the biochemical properties of the multiple components of the beta-AR pathway were examined in enzymatically dissociated myocytes. RESULTS: Coronary stenosis, involving an average 55% reduction in luminal diameter, was associated with left ventricular failure and right ventricular dysfunction at both time intervals. A decrease in the quantity of beta-AR was detected at 7 days and preceded the loss of high-affinity binding sites. This regulatory modification was characterized by a reduction in beta 1 and beta 2 receptors and a shift in the isoproterenol dose response curve indicating a functional correlation between the decrease in beta-AR and attenuated inotropic support of the myocardium. The percentage of beta-AR binding agonist with high affinity decreased significantly at 14 days along with a further reduction in the density of beta 1 and beta 2 receptors. Reconstitution studies with cyc S49 lymphoma cells did not detect an impairment of Gs alpha functional activity, but the quantity of Gi alpha was increased at both intervals. Finally, activation of the catalytic unit of adenylyl cyclase by forskolin and GTP was not altered by coronary stenosis, however, basal cyclic AMP in myocytes was depressed at 14 days. CONCLUSIONS: Coronary stenosis induces distinct and progressive modifications in the beta-AR signaling cascade which may contribute to the impaired ventricular performance in this model of myocardial ischemia.

Hypertrophic obstructive cardiomyopathy due to a novel T-to-A transition at codon 624 in the beta-myosin heavy chain (beta-MHC) gene possibly related to the sudden death.

Many missense mutations in the beta-myosin heavy chain have been reported in patients with hypertrophic obstructive cardiomyopathy (HOCM). However, the controversy is present whether the mutation accompanying the change of electric charge is related with poorer prognosis. The proband, a 48-year-old female, of the family was diagnosed clinically as HOCM, and a structural analysis of the cardiac beta-MHC gene showed that the proband and her junior daughter had a novel mutation with T to A transition in codon 624 replacing tyrosine with asparagine, which was not present in her husband, elder daughter and son. The proband's husband, son and two daughters were healthy except that the ECG of junior daughter (15-year-old) showed complete right bundle branch block. Proband's mother died suddenly after the delivery of the proband and the proband also collapsed suddenly. The occurrence of sudden death in proband and her mother suggested that HOCM with this novel mutation might be associated with a high risk of sudden death irrespective of the absence of charge alteration.

In vivo genotoxicity of heterocyclic amines detected by a modified alkaline single cell gel electrophoresis assay in a multiple organ study in the mouse.

We used a modification of the alkaline single cell gel electrophoresis (SCG) (Comet) assay to test the in vivo genotoxicity of 6 heterocyclic amines, Trp-P-1 (25 mg/kg), Trp-P-2 (13 mg/kg), IQ (13 mg/kg), MeIQ (13 mg/kg), MeIQx (13 mg/kg) and PhIP (40 mg/kg), in mouse liver, lung, kidney, brain, spleen, bone marrow and stomach mucosa. Mice were sacrificed 1, 3, and 24 h after intraperitoneal injection. Trp-P-2, IQ, MeIQ, and MeIQx yielded statistically significant DNA damage in the stomach, liver, kidney, lung and brain; Trp-P-1 in the stomach, liver and lung; and PhIP in the liver, kidney and brain. None of the heterocyclic amines induced DNA damage in the spleen and bone marrow. Our results suggest that the alkaline SCG assay applied to multiple organs is a good way to detect organ-specific genotoxicity of heterocyclic amines in mammals.