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Summary Background & aims The associations between dietary magnesium intake and stroke and coronary heart disease (CHD) incidences are inconsistent and not established in Asian. We aimed to determine the association between dietary magnesium intake and the risk of stroke and CHD in a Japanese population. Subjects/Methods We studied 85,293 Japanese subjects by questionnaire at baseline (age 45–74 years, without cardiovascular disease or cancer in 1995 and 1998 for Cohorts I and II, respectively). The participants were followed until the end of 2009 and 2010 in Cohorts I and II, respectively. Dietary magnesium intake was estimated from a self-administered 138-item food-frequency questionnaire. Results After 1,305,738 person-years of follow-up, 4110 strokes and 1283 cases of CHD were documented. The multivariable-adjusted hazard ratios (HRs, 95% confidence intervals, 95%CIs) of CHD for the fourth and fifth quintiles of dietary magnesium intake were 0.70 (0.50–0.99) and 0.66 (0.44–0.97) in men (P for trend = 0.036), respectively, and third quintile of dietary magnesium intake was 0.61 (0.39–0.96) in women (P for trend = 0.241), compared with the lowest quintile in men and women. We observed no decreased risks of incident stroke in men or women with higher dietary magnesium intakes. Conclusions Higher dietary magnesium intake was associated with a reduced risk of CHD in Japanese men.

Aims Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. Methods and results The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000–04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50–79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality. The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73–1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55–0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64–0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01–4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07–11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03–1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40–0.99), respectively. Conclusion An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Background and aims Higher intake of fish or n-3 polyunsaturated fatty acids (PUFAs) has been associated with reduced risk of coronary heart disease (CHD). However, it is unclear whether increased blood levels of n-3 PUFAs are associated with reduced risk of CHD in the Japanese population. Methods The relationship between circulating levels of n-3 PUFAs (eicosapentaenoic acid + docosapentaenoic acid + docosahexaenoic acid) and risk of CHD was examined in a nested case-control study among participants in the Japan Public Health Center (JPHC)-based Study Cohort. Plasma n-3 PUFA phospholipid levels were measured at baseline by gas chromatography in 209 cases with CHD and 418 controls matched for sex, age, date of blood draw, time elapsed since last meal before blood collection, and study location. The CHD cases (n = 209) comprised 168 cases of myocardial infarction and 41 of sudden cardiac death, otherwise classified as 157 non-fatal and 52 fatal coronary events, respectively. Mean duration of follow-up was 13.5 years. Results Multivariate conditional logistic analysis showed no significant association between n-3 PUFAs and risk of total CHD. The odds ratio (OR) for the highest versus lowest quartiles of plasma n-3 PUFAs was 0.79 (95% confidence interval [95% CI]: 0.41–1.51, p for trend = 0.51). Subtype analysis of CHD revealed that the multivariate ORs for the highest versus lowest quartiles for n-3 PUFAs were 0.91 (95% CI: 0.43–1.89, p for trend = 0.90) for myocardial infarction, 0.08 (95% CI: 0.01–0.88, p for trend = 0.04) for sudden cardiac death, 0.89 (95% CI: 0.42–1.89, p for trend = 0.97) for nonfatal coronary events, and 0.12 (95% CI: 0.02–0.75, p for trend = 0.03) for fatal coronary events. Conclusions Plasma n-3 PUFA levels were not associated with risk of total CHD but were inversely associated with risks of sudden cardiac death and fatal coronary events among middle-aged Japanese individuals.

Seafood/fish intake has been regarded as a protective factor for coronary heart disease (CHD), while smoking is a strong risk factor. To examine whether associations between smoking and risk of CHD are modified by seafood/fish intake, we studied 72,012 Japanese men and women aged 45-74 years who completed 2 food frequency questionnaires, 5 years apart, during the period 1995-2009. After 878,163 person-years of follow-up, 584 incident cases of CHD (101 fatal and 483 nonfatal), including 516 myocardial infarctions, were documented. There was a clear dose-response association between smoking and CHD risk among subjects with a low seafood/fish intake (

We examined the association between green tea consumption and mortality due to all causes, cancer, heart disease, cerebrovascular disease, respiratory disease, injuries, and other causes of death in a large-scale population-based cohort study in Japan.We studied 90,914 Japanese (aged between 40 and 69 years) recruited between 1990 and 1994. After 18.7 years of follow-up, 12,874 deaths were reported. The association between green tea consumption and risk of all causes and major causes of mortality was assessed using the Cox proportional hazards regression model with adjustment for potential confounders.Hazard ratios for all-cause mortality among men who consumed green tea compared with those who drank less than 1 cup/day were 0.96 (0.89-1.03) for 1-2 cups/day, 0.88 (0.82-0.95) for 3-4 cups/day, and 0.87 (0.81-0.94) for more than 5 cups/day (P for trend.001). Corresponding hazard ratios for women were 0.90 (0.81-1.00), 0.87 (0.79-0.96), and 0.83 (0.75-0.91; P for trend.001). Green tea was inversely associated with mortality from heart disease in both men and women and mortality from cerebrovascular disease and respiratory disease in men. No association was found between green tea and total cancer mortality.This prospective study suggests that the consumption of green tea may reduce the risk of all-cause mortality and the three leading causes of death in Japan.

For Zn1-xMnx O/ZnO heterostructures pseudomorphically grown on ZnO (000) single-crystal substrates, spontaneous and piezoelectric polarization-induced sheet charges were investigated. The sheet carrier concentration of a Zn0.88Mn0.12O/ZnO heterostructure with a 10-nm-thick ZnMnO spacer layer was determined to be 5.6×1012 cm–2 by Hall measurement. For x = 0.12, the number of free electrons induced by spontaneous and piezoelectric polarizations was calculated to be 1.8×1012 cm–2, which is less than the experimental value. This discrepancy can be explained by considering an additional electric field generated by the ZnMnO/ZnO conduction band offset and ZnMnO spacer layer. Therefore, for ZnMnO/ZnO heterostructures, the effect of electric field should also be considered together with spontaneous and piezoelectric polarizations in carrier confinement. (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Expert system (ES)-assisted control has fully automated the operation of transportation facilities on the electrical steel finishing line, in the Mizushima Works of Kawasaki Steel Corporation. The system forms the optimum schedules for transferring slit coils and transfers them all on-line by 24 monorail-type coil carriers under its control. In order to obtain the necessary response for real-time use, control rules are divided into groups according to their functions, and only designated rule groups are made to run at one time, securing a response of less than 3 seconds. The adoption of ES has enabled the system successfully to handle the complex operations of the line, and reduced the man-months required for software development to one half compared with using conventional programming language. The system has been working smoothly, and contributing greatly to the high-efficiency operation of the line, since its commission in March 1990.

Several mAb (PG11, NG7, and ED4) against hamster complement C1s were obtained. PG11 and NG7 were shown to cross-react with human and rat C1s. By using an immunohistochemical method, we examined localization of C1s in tissues of hamsters and rats. Present results revealed a widespread yet specific staining of hamster C1s which is associated with endoderm-, mesoderm-, and neuroectoderm-derived cells. For example, chondrocyte of hyaline cartilage and surface epithelium of the stomach were strongly positive. Intestinal epithelium, muscle cells, pia mater and epithelium of the choroid plexus of the ventricle, and hepatocytes were also stained. The synthesis of hamster C1s in these organs was confirmed by RNA blot hybridization. Secretion of C1s into the culture medium was revealed by immunoblot analysis in cell lines of hepatocytes, kidney cells, and myoblasts of rat or hamster.

We monitored the membrane fusion of liposomes to determine if the minimal components of soluble N-ethyl maleimide-sensitive factor attachment protein receptor (SNARE), which is involved in mast cell exocytosis, have fusogenic activity. Three core components of SNARE were reconstituted into liposomes. Membrane fusion between liposomes containing vesicle associated membrane protein (VAMP) -7 or -8 and liposomes containing synaptosomal-associated protein 23 kDa (SNAP23) and syntaxin-3 or -4 was monitored by fluorescence resonance energy transfer. The combination of SNAP23/syntaxin-3/VAMP-8 showed the most efficient liposome-liposome fusogenic activity. Liposomes with VAMP-7 exhibited poor fusogenic activity regardless of the syntaxin isoform. The core components of SNAP23, syntaxin-3, and VAMP-8 appear to be minimal machinery to induce membrane fusion, while VAMP-7 appears to be unessential for membrane fusion.

Regulated exocytosis is a very important process to secrete bioactive substances from cells, such as neurotransimitters, hormones, and inflammatory mediators from immune cells. Secretory cells have intracellular small vesicles called secretory vesicles or secretory granules and they contain bioactive substances. These contents are released by membrane fusion between secretory vesicles and the plasma membrane (exocytosis). Mast cells are typical non-neuronal secretory cells and secrete inflammatory mediators such as histamine. It has been shown that SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins play an essential role in exocytotic release in mast cells. In this study, we tried to develop an artificial system that mimics exocytotic release of mast cells, using liposomes in which purified SNARE proteins are reconstituted. To develop a such system, we try to prepare a cell-size giant liposome that contains smaller liposomes. A giant liposome and smaller liposomes correspond to the cell membrane and secretory vesicles, respectively. Preparing giant liposomes, LUVs that contain smaller liposomes inside like mast cells were formed. Diameter of inner liposomes ranged from 1 to 3 mum and the number of inner liposomes ranged from 1 to 20. We also succeeded preparing giant liposomes that contain calcein-labeled SUV by adding SUV at the process of rehydration. These system might be useful to construct an artificial exocytotic cells.

Exocytosis is a key process to secrete bioactive substances from cells, such as neurotransimitters, hormones, and inflammatory mediators from immune cells. Mast cells are typical non-neuronal secretory cells and secrete inflammatory mediators such as histamine and cause allergic responses. It has been shown that SNARE (soluble N-ethyl maleimide-sensitive factor attachment protein receptor) proteins play an essential role in exocytotic release in both neuronal cells and non-neuronal secretory cells. To mimic the exocytotic processes in mast cells, we developed an artificial membrane fusion system using liposomes that contains SNARE proteins. To prepare liposomes that contain SNARE proteins, SNARE proteins expressed in E. coli were purified and reconstituted into liposomes by solubilization-reconstitution methods using octylglucoside as a detergent. The membrane fusion is monitored by FRET (fluorescent resonance energy transfer) between fluorescence labeled phospholipids in liposomes (NBD- and rhodamine- labeled phospholipids). Using this assay system, we found that SNAP23, syntaxin 3 and VAMP8 are involved in exocytotic release in mast cells.

This paper explains the new run out table (ROT) cooling control system of Wakayama hot strip mill which has been in operation since December 1992. The proposed system has three main features, a precise cooling process model, a cooling profiles stabilizing method and feedforward and feedback control subsystems using the actual temperature of a running strip. The design of the model, method and control loops is outlined and the results of actual strip cooling are presented.

We have previously shown that non-transformed mouse A31 cells became tumorigenic when they were transfected with hamster C1s cDNA expression plasmid BCMGSNeoCS. In the present study, mutations were introduced into the cDNA at the activation cleavage site, Arg423(AGG) and the active center Ser617(AGC). These amino-acids were replaced by His423(CAC) and Thr617(ACC), respectively. The mutated cDNAs were inserted into BCMGSNeo and transfected to A31 and its polyoma-virus-transformed SEA7 cells. C1s produced from these transfectants lost their enzyme activity. Transfectants of these mutated C1s cDNA did not form tumors in nude mice, To distinguish between active and inactive C1s in situ, we have developed novel antibodies, one directed to the NH2-terminal neoepitope of the L chain and the other specific for uncleaved inactive C1s. These antibodies were used to characterize C1s produced by transfectants, so as to determine whether or not it was cleaved at the right position.

We investigated the existence and the function of 5-hydroxytryptamine(4) (5-HT4) receptors and the effect of TKS159, a novel agonist of 5-HT4 receptor, on guinea pig stomach. The mechanical activity and the release of [3H]ACh were measured using preparations of muscle layers attached to intramural plexus from guinea pig stomach. 5-HT in the presence of 1 microM methysergide, 1 microM ketanserin and 1 microM granisetron, 5-methoxytryptamine or TKS159 enhanced the electrical transmural stimulation-evoked contraction and [3H]ACh release in strips of the stomach in a concentration-dependent manner. This enhancement by 5-HT, 5-methoxytryptamine or TKS159 was antagonized by SDZ 205-557 or atropine. Cisapride, metoclopramide and TKS159 enhanced the electrical transmural stimulation-evoked contraction and release of [3H]ACh in a concentration-dependent manner. We conclude that the pharmacological characteristics of the receptor, which mediates contraction of the guinea pig stomach by the activation of cholinergic nerves, are consistent with its being of the putative 5-HT4 receptor type and that TKS159 is an agonist at 5-HT4 receptors.

While the mortality rate of cerebral hemorrhage of Okinawa Prefecture is the lowest in Japan, the presence of a high incidence rate for cerebral hemorrhage has been pointed out in Hirara-city, Okinawa Prefecture. In order to develop an effective counter-measure, a case-control study of risk factors for cerebral hemorrhage was performed. Cases were 62 residents in Hirara city aged 40-69 years who had experienced cerebral hemorrhage between April 1987 through March 1992. From the 62 cases interviewed, responses were obtained from 49 cases. Controls were 147 residents without a history of stroke who were matched to cases individually by sex and age (control/case ratio = 3). From the 49 cases, 28 cases who had participated in residential mass health examinations before experiencing the cerebral hemorrhage, were matched individually sex and age with different randomly selected controls (control/case ratio = 10). From conditional logistic regression analysis of the interview survey data, history of hypertension, interruption of hypertension therapy, family history of stroke, long working hours, overwork and difficulty of taking a day-off each week were strongly related to increased risk of cerebral hemorrhage. Smoking, preference for salty taste and habitual drinking, also, elevated risk slightly. From analysis of the residential mass health examination data, high blood pressure was found to be strongly associated with cerebral hemorrhage, while serum cholesterol was inversely related.

Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of C1s.

The activated first component of human complement, C1-s, was shown to cleave type I and II collagen and gelatin. The proteolytic activity was heat labile and was inhibited by a monoclonal antibody (M241) which recognized light chain of active human C1-s or by a serine protease inhibitor, DFP, but not by a chelating agent.

Transfer of radioactive materials to fixed cells from an overlying layer of living cells has been examined to determine whether fixed cells can act as acceptors of glycosyltransferases of living cells. After the incubation of living cells were removed by EDTA treatment, and the radioactivity associated with the fixed cells was determined. Lipids, proteins and carbohydrates were found to be transfered from the living cells to the fixed cells. The amount of radioactivity transferred to the fixed cells was dependent on the number of both fixed and living cells and increased with the time of incubation. When fixed cells were treated with chloroform-methanol before the addition of living cells, the transfer of both lipids and proteins to the fixed cells decreased drastically, but only a slight decrease incarbohydrate transfer was observed. Most of the radioactive materials transferred from living cells labeled with glucosamine or fucose to chloroform-methanol-treated fixed cells were solubilized by trypsin but not by the detergents tested. Approximately 55% of the materials transferred from the cells labeled with glucosamine could be solubilized by hyaluronidase and chondroitinase, and the rest was solubilized by neuraminidase and a glycosidase mixture. The treatment of chloroform-methanol-extracted fixed cells with trypsin caused a significant decrease in the transfer from cells labeled with glucosamine. When nucleotide sugars were used as the radioactive precursor, no significant amount of radioactivity was transferred to the fixed cells.

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca 2+ concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N -ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca 2+ dependent, the target molecule that interacts with Ca 2+ is not clear. Synaptotagmin is a Ca 2+ sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca 2+ , low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca 2+ , and phosphatidylserine.

Cytoplasmic isoactins of mouse fibroblast L-cells were examined by two-dimensional gel electrophoresis. In contrast to other cultured cell lines, which contain both beta- and gamma-actin, L-cells contained only beta-actin species. This unique phenomenon was due neither to the transformed status of this cell line nor to the characteristic nature of adipose tissue fibroblasts of C3H mice. When RNA of L-cells was translated in a nuclease-treated reticulocyte lysate system, actin synthesized in vitro also appeared as only one species, beta-actin. Therefore, the genetic information for gamma-actin is absent at the level of translatable mRNA in L-cells.

The amount of Forssman glycolipid (GL-5) was investigated in two clones of Nil hamster cells grown either in monolayer or in spinner culture. GL-5 assayed by the complement-fixation inhibition test increased with increasing cell density in the monolayer. However, cells grown in spinner cultures failed to show the density-dependent response in both clones examined. Cells transformed by hamster sarcoma virus did not show the density-dependent increase of GL-5, even in cells grown in monolayer. The effect of transfer from confluent to sparse cultures on the amount and the synthesis of GL-5 was also examined. It is suggested that the GL-5 that accumulates in cells during confluency is diluted into the daughter cells and that the decrease of the Forssman lipid does not precede cell division.

We have characterized properties of a melanoma antigen with a mouse-specific melanoma epitope expressed on B16 melanoma by using syngeneic monoclonal antibodies with antimetastatic ability. The molecule recognized by the antibody is a membrane glycoprotein with a molecular weight of 80,000. Studies on tunicamycin treatment indicated that the core size of the molecule appeared to have a molecular weight of 69,000 and also suggested that the carbohydrate moiety was greatly responsible for the conformation of the mouse melanoma epitope. The antigen was released or shed into the culture medium from the cell surface, and the turnover rate of the antigen was within 1.5 h.

Oncogenic transformation of mouse 10T 1/2 fibroblasts induced upon exposure to X-ray or N-methyl-N'-nitro-N-nitrosoguanidine was suppressed if lipopolysaccharide (LPS) was present in the culture medium. The suppressive effect of LPS was exerted within 24 h after irradiation. Suppression was dependent on the concentration of LPS added and LPS (2 micrograms/ml) derived from Salmonella minnesota R595 reduced the number of transformed type III foci per dish from 0.39 to 0.15. Indomethacin (1 to 30 microM) further enhanced the effect of LPS in a dose-dependent manner.

Glycolipid synthesis was investigated in secondary hamster embryo cells and in two Nil (hamster) clones at 24-hr intervals following infection with the hamster sarcoma virus. In all cases lower levels of complex glycolipids were being synthesized by 14 days postinfection. The time courses for these changes, however, were complex and characteristic for each cell type. Ahigher rate of Forssman antigen synthesis was found in infected secondary hamster fibroblasts as compared to uninfected cells at one point during the transition to transformation. In some cases the changes in glycolipid pattern were found to precede changes in morphology and glucose uptake and in other cases they were found to follow these changes.

When Nil2C2, a metastatic clone derived from hamster embryo fibroblasts (Nil), was inoculated over [3H]leucine-labeled fixed cells, Nil2C2 cells solubilized and phagocytosed fixed cells, and the radioactivity was released into the culture medium as trichloroacetic acid-soluble fragments. The solubilization of fixed cells was dependent on both the time of incubation of living cells with fixed cells and the number of living cells inoculated. Nil2C2 cells were shown by autoradiographic and electron microscopic studies to peel off fixed cells and ingest them as large fragments. The solubilization of fixed cells was significantly decreased when plasminogen was depleted from the culture medium. Protease inhibitors such as leupeptin, epsilon-aminocaproic acid, and soybean trypsin inhibitor partially inhibited the proteolysis and phagocytosis of Nil2C2 cells. Mouse peritoneal macrophages activated by Salmonella typhimurium solubilized fixed cells after the addition of 12-O-tetradecanoylphorbol-13-acetate. However, they did not phagocytose fixed cells as large fragments.