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.

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.

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.

Multiplicity in type V adenylylcyclase: type V-a and type V-b.

Type V mammalian adenylylcyclase cDNA was originally isolated from two animal species, the dog and rat. The amino acid sequences from the two species are highly homologous, but completely different in the putative N-terminal, cytoplasmic region. Northern blot analysis using oligonucleotide probes unique to either of the two clones has revealed that the two forms of type V adenylylcyclase mRNA, canine form (= type V-a) and rat form (= type V-b), are co-expressed as splicing variants in both species. Genomic Southern blot analysis has suggested that the two forms are the products of a single gene. When overexpressed, however, deletion of the N-terminal domain did not alter any biochemical properties. Thus multiple splicing variants with unique N-terminal amino acid sequences of type V adenylylcyclase can be generated from a single gene, however, biochemical properties of these variants may not be different.

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.

Effects of DNA repair deficiency on the mutational specificity in the lacZ gene of Escherichia coli.

The mutational specificities of various chemical mutagens were compared in isogenic E. coli strains with different DNA repair capabilities (wild-type, uvrA, umuC, and uvrA umuC) in a reversion assay employing a set of mutant lacZ genes that can detect two types of transitions, four types of transversions, and five kinds of specific frameshift events. A uvrA derivative was more sensitive than the wild-type strain to 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone for +1G, -1G, -2(C-G), +1A and -1A frameshifts, G.C-->A.T transitions, and G.C-->T.A transversions. In a uvrA background, G.C-->T.A transversions and +1G, +1A, and -1A frameshifts appeared to be umuC-dependent, while G.C-->A.T transitions were not. N-Ethyl-N'-nitro-N-nitrosoguanidine was more mutagenic in a uvrA background for five kinds of frameshifts and G.C-->A.T transitions, but not for G.C-->T.A, A.T-->C.G, and A.T-->G.C base substitutions. A.T-->C.G transversions were totally dependent on umuC gene function. For the investigation of mutational specificities induced by frameshift mutagens, an rfa mutation was additionally introduced. The rfa strain responded to 2-nitrofluorene, which induced primarily -2(C-G) frameshift mutations. In an rfa uvrA background, benzo[a]pyrene induced +1G, -1G, +1A, and -1A frameshifts. 2-Aminoanthracene induced +1G, -1G, and +1A, but not -1A, frameshifts, with -1G frameshifts predominating. Ethidium bromide induced only two types of frameshifts, -1G and +1A. Frameshifts induced by ICR-170 were independent of umuC gene function, while those by induced 1-nitropyrene were partly umuC-dependent.

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.

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.

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.

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.

[Assessment of denervated but viable myocardium in patients with myocardial infarction--by myocardial imaging with 201Tl and 123I-MIBG].

The present study assessed sympathetic function in viable infarcted areas of the myocardium following the onset of myocardial infarction. The subjects were 19 patients with myocardial infarction. After exercise on a bicycle ergometer, simultaneous SPECT with Tl-201 and I-123 MIBG was performed. The behavior of MIBG following exercise remains to be clarified, but in the present study MIBG provided images different from those obtained with Tl. While the redistribution of Tl in the infarcted area was observed in 8 of 19 patients, MIBG was absent in the infarcted area in both the initial and delayed scans. In the patient undergoing CABG, the infarcted area showed no MIBG despite the normal perfusion of Tl. The previous proposal that the redistribution of Tl indicates myocardial viability to some extent suggests that the sympathetic function of the area of the myocardium showing the redistribution of Tl decreases even though the area is viable. These findings indicate that after the onset of myocardial infarction, there is a denervated but viable area in the myocardium, despite the viability demonstrated by Tl imaging. They also indicate that concurrent myocardial imaging with MIBG is useful for the detection of such a myocardium.

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.

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.