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We conducted a survey of 111 healthy middle-aged and older women, aged 40 to 80 years, to elucidate the relationships between blood leptin and adiponectin levels and bone mineral density (BMD) in September 2008. We examined the associations between the blood levels of these adipocytokines and the lumbar spine (L2-L4), left femoral, and distal forearm BMD after adjustment for the age and body fat percentage. No correlation was observed between leptin and the BMD. This is presumably due to the fact that the influence of menopause, age, duration of postmenopausal period, and body fat percentage on the BMD is greater than that of leptin levels in healthy women. The adiponectin levels were significantly correlated negatively with the lumbar spine and femoral BMD in premenopausal women, and with forearm BMD in postmenopausal women, regardless of adjustment for age and body fat percentage. It seems likely that adiponectin influences the BMD at different skeletal sites in pre- and postmenopausal women.

Background: Phytosterolemia is one of the genetic disorders causing hypercholesterolemia and atherosclerosis together with the accumulation of plant sterol in plasma and tissues. The mutations in ABCG5 and ABCG8 genes, encoding sterolin-1 and -2, respectively, are responsible for phytosterolemia., Methods: We performed genetic analyses on 2 Japanese phytosterolemia patients., Results: We identified 2 mutations in the ABCG5 gene in these patients. The first patient was homozygous for a novel mutation, which was a 19-base pair tandem repeat insertion in exon 7, leading to a premature termination at codon 288. The second patient was a compound heterozygote; one of the mutations was the same as that found in the first patient, while the other mutation was a C to T substitution in exon 10, resulting in a premature termination at codon 446 (R446X). No other mutation was found in the ABCG5 and ABCG8 genes., Conclusions: This result was concordant with previous observations that found most Asian phytosterolemia patients possessed mutations in the ABCG5 gene, and the site of the novel mutation was completely different from these previous reports, necessitating the extensive analyses for phytosterolemia.

The purpose of this report was to describe the profile of esogastroduodenal disease diagnosed by upper digestive tract endoscopy (UDTE) in a rural area of Togo. This prospective study combines data collected during two two-week screening campaigns carried out in the Kara region. Patients were informed of the presence of the endoscopy team by means of a bulletin on a rural radio station. All male and female patients 15 years or older were included. A total of 220 UDTE procedure reports were recorded and analyzed including 107 men and 113 women with a mean age of 37.7 years (range: 15-84 years). Disease was detected in 72 procedures mainly in the 21 to 41 year age group (47.2 %) with a higher proportion of men than women: 38% versus 27% respectively. The most frequent indications for UDTE were epigastralgia (47.7 %) including 39% of procedures leading to the discovery of disease and diffuse abdominal pain (21.8 %). The procedure was carried out for follow-up purposes in 19.1% of cases. The most common lesions were peptic ulcer (34.2%), inflammatory disease including esophagitis, gastritis, and bulboduodenitis (32.4%), gastroduodenal bile reflux (9.3%), pylorobulbar stenosis (5.5%), tumoral disease (3.7%), and esophageal varicosities (3.7%). This study based on UDTE diagnostic procedures provided insight into the profile of esogastroduodenal disease in rural Africa. These screening campaigns required special organization using appropriate equipment and personnel.

Objective: To study the incidence and clinical features of acute juvenile female non-herpetic encephalitis (AJFNHE) in Japan., Methods: A nationwide questionnaire on patients with severe non-herpetic encephalitis of unknown etiology with a prolonged clinical course or death was sent to the departments of Internal Medicine, Neurology, Pediatrics, and Emergency and Critical Care at all hospitals with 200 beds or more in Japan., Results: The recovery rate was 25% (1,279 out of 5,030 departments) and 90 patients were enrolled in this study. The annual incidence was 0.33/10(6) population. 85% of patients were female. The means and standard deviations of age at onset and hospital stay were 26+/-10 years and 180+/-228 days. As first symptoms, fever and psychosis were presented in 90%. Among the neurological symptoms, disturbance of consciousness was presented in 92%, convulsions in 65%, and involuntary movements in 55%. Respiratory failure during hospitalization was observed in 71% and required care with mechanical ventilation. The detection rate of anti-GluR epsilon2 and/or delta1 antibodies was 67% of patients. Anti-N-methyl-D-aspartate receptor NR1/NR2 antibody was detected in all four examined patients with anti-GluR epsilon2 antibody, and also detected in both of the two examined patients without anti-GluR epsilon2 antibody. As for outcome, 46% returned to work and 37% returned home, but 7% died. Associated tumors were demonstrated in 39%. All reported patients had ovarian tumors, among which teratoma was the most frequent., Conclusion: A nationwide survey provided data for the annual incidence and clinical features of AJFNHE in Japan.

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor-deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.

Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their recognizable duplexes are limited to homopurine/homopyrimidine sequences because of interruption of pyrimidine base in the purine strand. This problem has not been fully solved despite numerous studies. We have previously reported that the novel nucleoside analogues (WNA: W-shaped nucleoside analogues), WNA-betaT and WNA-betaC, can recognize a TA and a CG interrupting sites to form triplexes with high stability and selectively, respectively. However, further investigations have shown that the triplex formation using the WNA derivatives is dependent on its neighbouring bases of the TFOs. In this study, the new WNA analogues having a variety aromatic ring were synthesized to evaluate effects on sequence dependency. And it was found that changes in the substituent of aromatic ring of WNA derivatives altered the binding selectivity and stability in the triplex formation.

Oxidative stress has been proposed to play a crucial role in glomerulosclerosis, although its in vivo demonstration has proved taxing given the difficulty of inducing gene expression in specific renal cells. In this study, we examined whether the liver-directed expression of plasma platelet-activating factor acetylhydrolase (PAF-AH) would affect the glomerular pathophysiology in Imai rats, an animal model for glomerulosclerosis. Adenovirus-mediated liver-directed gene delivery of human PAF-AH resulted in a significant increase in plasma PAF-AH activity, which was detected almost exclusively on HDL. Histological examination of rats overexpressing PAF-AH showed not only the deposition of PAF-AH in mesangial cells, but also a reduction in hydroxynonenal and matrix protein content in the glomeruli. In situ hybridization analysis was negative for human PAF-AH mRNA in the kidney, while injection of HDL abundant in PAF-AH resulted in the deposition of PAF-AH in mesangial cells. Urine protein levels did not increase in rats overexpressing PAF-AH, while those of control rats increased significantly with age. This study provides direct evidence of the in vivo role of an enzyme that degrades lipid peroxides during the progression of glomerulosclerosis. Adenovirus-mediated extrarenal gene expression and lipoprotein-mediated glomeruli-targeted protein delivery promise to be a novel therapeutic approach to glomerulosclerosis.

This study aimed at assessing envenomations caused by snakebite in the Gabriel Touré hospital between January 1998 and December 1999. We included in total 112 victims of snakebites proved by the identification of the snake, the fangs traces and envenomation signs. The bites concerned 34 women (30.36%) versus 78 men (69.64%) from the Bamako district, while 66.7% were from a rural environment. The farmers were the most affected category (35.1%). The bite concerned the lower limb in 83.3% of the cases. Snakes such as Echis ocellatus, Bitis arietans, Naja nigricollis and N. katiensis caused the envenomations. The viperine syndrome dominated the clinical picture; its severity depended on the bleeding stage and the delay of administration of the antivenom serum (AVS) after the envenomation: 19 hours for Echis sp. and 2 hours for Naja sp. This study shows that the envenomation accidents caused by snakes are frequent with a lethality rate of 9.8%.

Probucol, an antioxidative and hypolipidemic agent, has been postulated to increase reverse cholesterol transport by enhancing cholesteryl ester transfer protein (CETP) activity. However, its clinical implication in CETP deficient patients has not been fully defined. To characterize the effects of probucol in the absence of CETP, we evaluated the changes in lipid profile, lipid peroxidation, and paraoxonase 1 (PON1) activity in two complete CETP deficient patients, caused by treatment with probucol. When the patients were not receiving probucol, low-density lipoprotein (LDL) particles were smaller and high-density lipoprotein (HDL) particles were larger in these patients than in controls. Treatment with probucol (500 mg/day) resulted in the decrease in the levels of HDL-C and apolipoprotein (apo) A-I up to 22%. The size of HDL particles became smaller. LDL cholesterol concentration did not change in one patient, while it decreased by 47% in the other. PON1 activity/HDL-C, which was about 40% lower in the patients before treatment than in controls with the matching PON1 genotype, increased by 30% during the treatment. Lag time for LDL and HDL in both cases became prolonged more than 1.8 times after administration of probucol. This study demonstrated for the first time that probucol reduces HDL-C even in humans with complete CETP deficiency. Probucol treatment in these patients was also associated with protection of lipoproteins against oxidative stress, suggesting a clinical benefit of this drug even in such a state.

Objective: Plasma platelet-activating factor (PAF) acetylhydrolase (AH) is an enzyme bound with lipoproteins that degrades not only PAF but also PAF-like oxidized phospholipids that are proposed to promote atherosclerosis. In this study, we investigated the distribution of PAF-AH protein among lipoprotein classes by using adenovirus-mediated gene transfer in mice, and we examined its effects on lipoprotein oxidation and foam cell formation of macrophages., Methods and Results: Adenovirus-mediated overexpression of PAF-AH in mice resulted in a 76- to 140-fold increase in plasma PAF-AH activity. Contrary to the previous report, overexpressed human PAF-AH protein was bound to very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein, and high density lipoprotein (HDL). All the lipoproteins with overexpressed human PAF-AH revealed more resistance against oxidative stress, which was associated with lower levels in autoantibody against oxidized low density lipoprotein in the plasma. In addition, HDL with human PAF-AH inhibited foam cell formation and facilitated cholesterol efflux in macrophages., Conclusions: These results suggest that human plasma PAF-AH exerts an antiatherogenic effect by binding to all the lipoproteins and thereby protecting them from oxidation, producing less proatherogenic lipoproteins and preserving HDL functions.

Objective: Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression., Methods and Results: When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-beta-D-xyloside (beta-DX) resulted in all 3 isoforms' reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with beta-DX resulted in all 3 exogenously derived apoE isoforms' reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4., Conclusions: The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.

Objective: We examined the possible association between insulin resistance and carotid arteriosclerosis in subjects who had both normal fasting glucose and normal glucose tolerance after intake of a glucose load., Methods and Results: Our subjects were individuals who underwent general health screening at our institute, which included carotid ultrasound and oral glucose tolerance testing. Of the 1238 subjects enrolled in our study, 738 (60%) were classified as normal, defined as a normal fasting glucose level and normal glucose tolerance, and 334 (27%) and 166 (13%) were classified as borderline and diabetic, respectively, according to the criteria of the Japan Diabetes Society. The homeostasis model assessment of insulin resistance (HOMA-IR) was used as the index to measure insulin resistance. In normal-type subjects, univariate analysis showed that insulin resistance, but not insulin secretion, was associated with the presence of carotid plaque. Multivariate analysis showed that HOMA-IR was positively associated with carotid plaque in normal-type subjects, with an odds ratio of 1.19 (95% confidence interval, 1.00 to 1.41; P<0.05)., Conclusions: These data suggest the possibility that the presence of higher insulin resistance could be a risk factor for carotid arteriosclerosis in subjects with normal fasting glucose and normal glucose tolerance.

Objective: Heme oxygenase (HO) is important in the defense against oxidative stress and as a factor in an antiatherogenic mechanism. Compared with long (GT)(n) repeats, short (GT)(n) repeats in the human HO-1 gene promoter were shown to have higher transcriptional activity in response to oxidative stress. There is a strong link between oxidative stress and the pathogenesis of coronary artery disease (CAD)., Methods and Results: We screened the allelic frequencies of (GT)(n) repeats in the HO-1 gene promoter in 577 patients who underwent coronary angiography. Because the distribution of numbers of (GT)(n) repeats was bimodal, we divided the alleles into 2 subclasses: class S included shorter (<27) repeats, and class L included longer (> or =27) repeats. Multivariate logistic regression models including standard coronary risk factors revealed that the genotypes were significantly related to CAD status in hypercholesterolemic, diabetic patients or in smokers. In this study, the patients with shorter GT repeats were less likely to have CAD., Conclusions: Length polymorphism in the HO-1 gene promoter is related to CAD susceptibility in Japanese people who also have coronary risk factors such as hypercholesterolemia, diabetes, and smoking. HO-1 may play an antiatherogenic role in Japanese patients with these coronary risk factors.

We reported a 62-year-old man of late-onset familial amyloid polyneuropathy type I(transthyretin Met 30-associated familial amyloid polyneuropathy) from Ehime Prefecture. There was no family history related to endemic Japanese foci (Nagano and Kumamoto foci). He demonstrated paraesthesia in the legs and mild autonomic symptoms at the age of 52. These symptoms gradually developed. Analysis of the transthyretin gene from his leucocytes demonstrated he had Met 30 transthyretin mutation. Therefore, he was diagnosed with late-onset familial amyloid polyneuropathy type I(FAP 1). In some families, asymptomatic carriers with the mutant transthyretin gene were diagnosed. In early stage, this patient's polyneuropathy and autonomic nervous system dysfunction were less serious than those of FAP 1 patients from endemic Japanese foci. These symptoms of this patient was slowly progressive. He hoped liver transplantation (brain death or living-related) treatment if possible. Now he became 68-year-old and bed-ridden.

Paraoxonase1 (PON1) is a high-density lipoprotein (HDL)-associated protein which removes peroxidized lipids from lipoproteins. It has been proposed that apolipoprotein A-I (apoA-I) is an important determinant for its stabilization on HDL. However, little is known about its existence and activity in an apoA-I-deficient state in humans. To characterize the nature of PON1 in apoA-I deficiency, we investigated PON1 in an apoA-I-deficient patient. When serum was analyzed on fast protein liquid chromatography, PON1 protein was distributed almost exclusively on HDL despite the absence of apoA-I; on the other hand, 38.5% of PON1 protein was found in the lipoprotein-free fraction when the lipoproteins were fractionated through ultracentrifugation. The stability of PON1 activity in the patient serum was almost the same as in the normal control sera throughout incubation at 14 degrees C for 7 days. However, when the sera were incubated at 37 degrees C for 24 h, its activity declined more than those in the normal controls (19% versus 4% reduction of the initial values). Our results demonstrated that PON1 protein possesses a preferential association with HDL even in the absence of apoA-I, although apoA-I is a crucial factor for the maximal activity and stabilization of PON1.

A polymerase chain reaction (PCR) cloning procedure was developed for the resuscitation-promoting factor (Rpf) gene of Micrococcus luteus using strains NCIMB 13267, JCM 1464T, JCM 3347, and JCM 3348. A PCR product of the Rpf gene fragment was ligated into a cloning vector pBluescript II KS (+) with the restriction endonucleases Eco RI and Bam HI. The ligation mixture was used to transform Escherichia coli DH5alpha. The DNA sequence of the Rpf gene cloned from strain JCM 1464T was 84% homologous with that of NCIMB 13267, and from strains JCM 3347 and JCM 3348 it was 100% and 86% homologous, respectively. Recombinant Rpf proteins of M. luteus NCIMB 13267 and JCM 1464T after expression in E. coli BL21 harbouring the pET-19b-Rpf plasmid, and after purification, were approximately 16 kD for both strains.

We characterized two Shiga toxin-producing Escherichia coli (STEC) O86:HNM isolates from a patient with hemolytic uremic syndrome (HUS) or bloody diarrhea. Both of them did not possess the eaeA gene. However, the isolate from a HUS patient carried genetic markers of enteroaggregative E. coli (EAEC) and showed aggregative adherence pattern to HEp-2 cells. The other isolate from bloody diarrhea, which was negative with EAEC markers, was diffusely adhered to HEp-2 cells. The stx2 gene in both E. coli O86:HNM strains was encoded in each infectious phage, which was partially homologous to that of strain EDL933, a STEC O157:H7. These results will help to explain the genotypic divergences of STEC.

Time series analysis was carried out on monthly data of body weight and blood pressure of one male subject from 29 to 65 years of age by the program Census X-11 and DECOMP. Body weight increased from 55 kg to 70 kg from 30 to 50 years, then decreased to 60 kg afterward. The trend factor of body weight obtained by X-11 fluctuated markedly even after the removal of seasonal and irregular components. The trend factor of systolic blood pressure increased from 120 mmHg to 140 mmHg from 40 to 55 years, while diastolic blood pressure increased from 70 mmHg to 90 mmHg around 40 years of age. These changes were also accompanied by many fluctuations. The program DECOMP suggested that the fluctuations observed in X-11 were expressed by an autoregressive model. From the viewpoint of the most appropriate autoregressive model, it is possible that at younger ages body weight and systolic blood pressure may be affected by the values of the previous several months. On the other hand, they may be affected by the values of the previous month or two at older ages. Spectral analysis was also applied to the autoregressive model. Peaks at about 20, 30, or 40 months were observed in body weight changes. The pattern of the seasonal factor changed with age in body weight and blood pressure. In body weight, the peak appeared in the summer at younger ages, but in the autumn or winter at older ages. In blood pressure, the peak did not always appear in the winter. Am. J. Hum. Biol. 12:526-541, 2000. Copyright 2000 Wiley-Liss, Inc.

Many specialists in various scientific fields are interested in human growth and development of body sizes of children as a physiological phenomenon. However, our knowledge on human growth has not yet reached a solid foundation and consensus, and at present gravity potential is known to be a lasting condition that influences human lives in various ways. Therefore, more basic research is needed by examining detailed processes of human growth on the earth, with the effect of gravity in prospect. Although modern human growth study is constructed mainly on the basis of observations at one-year intervals, whether such long-interval observations can reveal a true statistical model of human growth is questionable. Togo and Togo (1982, 1989) observed growth in five siblings at one-month intervals for more than 15 years and found fluctuations observed from the start to the end of growth observations consisted of trend, regular, and irregular factors. This indicates the importance of examining human growth by short-interval observation. Thus, our study focused on diurnal variations of height, measuring twice daily, and discusses the effects of gravity on growth of children.

We examined the effect of troglitazone on immunoreactive endothelin-1 (ET-1) secretion from cultured bovine vascular endothelial cells (bVECs). Insulin (10(-9)-10(-7) M) stimulated ET-1 secretion in a dose-dependent fashion without any kinetic change. Troglitazone (1-20 microM) dose-dependently inhibited both spontaneous and insulin-stimulated ET-1 secretion. This inhibitory effect of troglitazone was associated with reduced ET-1 mRNA levels. Addition of indomethacin (100 microM) or Nw-nitro-l-arginine methyl ester (1 mM) and downregulation of protein kinase C by prolonged pretreatment of the cells with a phorbol ester, 12-O-tetradecanoylphorbol 13-acetate, did not affect the inhibitory effect of troglitazone at concentrations up to 10 microM. Troglitazone did not change the intracellular Ca2+ concentration stimulated by angiotensin II (10 microM). Other PPARgamma ligands, pioglitazone (1-10 microM) and 15-deoxy-delta 12, 14-prostaglandin J2 (1-10 microM), but not a PPARalpha ligand, bezafibrate (1-10 microM), dose-dependently suppressed spontaneous ET-1 secretion from bVECs. These results, taken together, suggest that troglitazone inhibits ET-1 mRNA expression and secretion in bVECs possibly through activation of PPARgamma. This inhibition may contribute to the hypotensive effect of troglitazone in insulin-resistant subjects., (Copyright 1999 Academic Press.)

A total of 5,174 Japanese men were included in a cross-sectional study to examine the relationship between the glycated hemoglobin (HbA1C) level and the prevalence of proteinuria as determined using a reagent strip. The prevalence of proteinuria rose significantly at HbA1C levels above 5.9%, whereas no relationship was observed at HbA1C levels below 5.9%. Multiple logistic regression analysis showed that blood pressure and a family history of diabetes were independent factors associated with proteinuria in subjects with a HbA1C below 5.9% who were not under medication for diabetes. In contrast, HbA1C, obesity and smoking were associated with proteinuria in subjects who were under medication for diabetes and/or have a HbA1C above 5.9%. These findings suggest that maintaining a HbA1C level below 5.9%, non-smoking and a standard body weight may reduce the prevalence of proteinuria in Japanese men. Healthy life-style and standard body weight are especially important for subjects with a family history of diabetes.

Advanced glyco-oxidation end products (AGEs) generate oxygen free radicals that potentiate the development of atherosclerosis. Thus, AGEs may potentiate the aggregation of human platelets through oxidative stress. AGE-bovine serum albumin (BSA) and AGE-poly-L-lysine were evaluated for aggregation of human platelets. Superoxide in platelet-rich plasma (PRP) was measured using lucigenin-derived chemiluminescence. The platelet aggregation induced by ADP or U46619 was potentiated by preincubation with AGE-BSA, by 40% and by 59%, P < .05, respectively, vs BSA. Aggregation was increased by AGEs in a dose-dependent manner. The production of superoxide was significantly greater in PRP incubated with AGE-BSA vs BSA. The other Maillard reaction products, such as Amadori-, pentosidine-, and carboxymethyl lysine (CML)-BSA had no effect. Superoxide dismutase or indomethacin abolished the enhancing effect of AGEs on the platelet aggregation. AGEs potentiate platelet aggregation possibly with superoxide anions and prostanoids. AGE-induced potentiation of platelet aggregation may be involved in the development of atherosclerosis., (Copyright 1998 Academic Press.)

Advanced glycation end products of bovine serum albumin (AGEs-BSA) exhibited biphasic effects on the proliferation of cultured rabbit vascular smooth muscle cells (VSMCs) in terms of [3H]thymidine incorporation and cell number count; a stimulatory effect was observed at 1-10 micrograms/ml and an inhibitory effect at more than 20 micrograms/ml, while it inhibited [3H]thymidine incorporation even at 1-10 micrograms/ml in cultured bovine vascular endothelial cells (VECs). Transient activation of p42 mitogen-activated protein kinase (MAPK) with a peak at around 5 min and a subsequent sustained phase was induced by AGEs-BSA in VSMCs, but not in VECs. The dependence of MAPK activation on AGEs-BSA dose was correlated with that of VSMCs proliferation.

In a previous study, we characterized prostanoid and thrombin receptors expressed on a megakaryoblastic cell line, MEG-01s (Blood 78, 2328-2336, 1991). In this study, we examines the mechanism of cross-talk between intracellular Ca2+ ([Ca2+]i) and cAMP signalings through prostanoid and thrombin receptors. Addition of a thromboxane (TX)A2 mimetic (U46619 or STA2) or thrombin stimulated the formation of inositol phosphates and dose-dependently augmented a prostaglandin (PG)I2 mimetic (iloprost)- or forskolin-induced cAMP formation. 12-O-tetradecanoylphorbol-13-acetate (TPA) and ionomycin, to lesser extent, also augmented iloprost-induced cAMP formation. The enhancing effect of U46619 or TPA on cAMP formation was inhibited by prolonged pretreatment of the cells with TPA (2.5 microM, 24 h), but not with calmodulin-antagonists; W-7, W-5, or KN-62. The elevation of [Ca2+]i induced by thrombin, STA2 or PGE2 was significantly suppressed by pretreatment of the cells with TPA (100 nM) as well as cAMP mimetics such as dibutyryl cAMP (5 mM), forskolin (5 microM) and iloprost (1 microM). These results suggest the key role of PKC on the cross-talk between [Ca2+]i and cAMP signalings through prostanoid and thrombin receptors; PKC, which is activated with TXA2 or thrombin, concomitantly suppress further [Ca2+]i elevation and enhances the PGI2 receptor-mediated cAMP formation, which, in turn, suppress [Ca2+]i elevation.

We examined the roles of myosin light-chain kinase in platelet responses to ADP using wortmannin, which almost completely inhibited myosin light-chain kinase at 3-6 microM. This concentration of wortmannin did not affect ADP-induced changes in the shape of the platelets, but it markedly inhibited aggregation in platelet-rich plasma and washed platelets. ML-9, another inhibitor of myosin light chain kinase, elicited similar effects on the platelet responses to wortmannin. Electron microscopic studies showed that there was no wortmannin effect on the ADP-induced spheration of discoid platelets, pseudopod formation, or granule centralization. Wortmannin at concentrations which prevented myosin light-chain kinase also inhibited platelet aggregation induced by ADP in the presence of U46619, an analogue of thromboxane A2, which is a prerequisite for ADP-induced irreversible aggregation. Although wortmannin partially inhibited protein kinase C, the protein kinase C inhibitor Ro-31-7549 (5 microM) prevented neither ADP- or ADP/U46619-induced changes in the shape of the platelets nor aggregation. These results suggest that myosin light-chain kinase activation is a prerequisite for ADP-induced platelet aggregation, but not for changes in their shape.

We examined the mechanisms of ATP release by human platelets using Ro-31-7549, a specific inhibitor of protein kinase C. Ro-31-7549 almost completely inhibited TPA-induced platelet aggregation and ATP release at 5-10 microM in washed platelets and in platelet-rich plasma. However, it suppressed thrombin- and U46619-induced ATP release by only 48% and 21%, respectively, and had little effect on aggregation in washed platelet suspensions containing serum or in platelet-rich plasma. The addition of GRGDS to prevent aggregation inhibited this residual thrombin-induced release by 53% and the residual U46619 release by 100% in the presence of Ro-31-7549. In washed platelet suspensions free of serum or plasma, Ro-31-7549 almost completely inhibited the ATP release and partially suppressed the aggregation induced by these agonists. These results suggested that there are protein kinase C-dependent and -independent mechanisms for ATP release by human platelets and that activation of the latter mechanism may depend on aggregation and plasma factors.

The present study examined the pattern and amplitude of seasonality of growth in body weight of Japanese children living in Tokyo, and analyzed the relationship between physique and seasonal variation. The body weights of 125 school children were measured monthly for 6 years (excluding August). The data were analyzed by time-series analysis. Significant seasonal variations were observed in about 80% of the children. Although individual seasonal patterns and amplitudes varied among children, minimum seasonal factors generally appeared in the Summer. Seasonal variations of group means of body weight were significant in both sexes. The seasonal pattern in boys was low in the Summer and high in the Fall and Winter. Deceleration of weight growth in the Summer was observed in both sexes, even though there were no major stresses in the Summer. The amplitudes of seasonal variation were 1.2% for body weight in boys and 0.7% in girls. In addition, children who were high on the BMI or Rohrer's index had larger amplitudes of seasonal variation, even when body weight of each child was taken into consideration. © 1994 Wiley-Liss, Inc., (Copyright © 1994 Wiley-Liss, Inc., A Wiley Company.)

Thapsigargin-activated Ca2+ entry into platelets was examined in the presence of S-145, a thromboxane A2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H2/thromboxane A2. With external Ca2+ present, 0.2 microM thapsigargin caused a prompt increase in intracellular Ca2+ concentration ([Ca2+]i) followed by a gradual increase. Pretreatment with 6 microM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca2+]i. In Ca(2+)-free EGTA buffer, thapsigargin induced a smaller increase in [Ca2+]i, and subsequent addition of Ca2+ to the buffer caused a further prompt increase in [Ca2+]i, demonstrating external Ca2+ entry. Wortmannin only partly inhibited this entry of external Ca2+. The wortmannin-insensitive Ca2+ entry pathway remained open for more than 6 min in Ca(2+)-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078-17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca2+ entry by two mechanisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.

Stature and body weight of two children, 12.5 and 9.3 years, and their mother were measured twice daily for 488 days, immediately after rising and just before bed. The time of the measurements was also recorded and diurnal changes during daytime and nighttime were estimated. Stature began to decrease instantaneously after rising. Therefore, morning measurements were made immediately after rising. Stature decreased during the day, and the mean daytime loss was 1.78 cm in the older child, 1.61 cm in the younger child, and 1.43 cm in the adult. Stature increased during sleep hours at night, and the mean nighttime gain was 1.79 cm, 1.63 cm, and 1.43 cm in each subject, respectively. Stature also increased after naps and a bath. Body weight decreased during sleep hours at night, and the mean nighttime loss was 0.46 kg, and 0.38 kg, and 0.36 kg in each subject, respectively. Diurnal changes were largest in the older child, while the ratios of changes (ΔST/ST, ΔWT/WT) were largest in the younger child whose growth rate was the greatest. In the children, the nighttime gain of stature was not as highly correlated with hours of sleep as in the adult. Diurnal change varies depending upon sleep hours and stage of growth. Rapid growth in stature of 1 cm in only three weeks was observed once in the younger child. During this period, diurnal change was smaller than the mean value over 488 days. In the older child, the growth rate decreased in both stature and weight about three months after menarche. © 1993 Wiley-Liss, Inc., (Copyright © 1993 Wiley-Liss, Inc., A Wiley Company.)

Anthropometric data, nutrient intake data, and blood biochemical parameters were analyzed for 66 elementary school children living in Tokyo, Japan, and their nutritional status was evaluated focusing on three problems: (1) zinc nutriture and growth, (2) anemia with iron deficiency, and (3) lipid nutriture and obesity. The subjects' mean energy and protein intakes met the recommended levels for Japanese children. However, their zinc intake levels were inadequate at 7.2, 8.3, and 8.5 mg in grades 2 (mean age: 8 yr), 4 (10 yr), and 6 (12 yr), respectively. Mean serum zinc concentration was 0.82 +/- 0.15 microgram/ml; the percentages of subjects who showed serum zinc concentration lower than 0.68 microgram/ml, the lower limit of the normal serum zinc concentration, were 28.6, 15.4, and 5.0% in grades 2, 4, and 6, respectively. These serum zinc concentrations indicated the existence of marginal zinc deficiency in some children, particularly in grade 2, though it was not severe enough to retard growth. Their iron intake levels (8.2, 10.2, and 10.2 mg for grades 2, 4, and 6, respectively) in combination with the proportion of iron intake from animal foods (37%) were judged to be adequate because no children showed serum ferritin, serum iron, or transferrin saturation levels lower than the criteria levels recommended for iron deficiency. Moreover, no definitely anemic children were found. Daily lipid intakes were 65.7, 74.5, and 78.3 g in grades 2, 4, and 6, respectively, and the mean percentage of energy intake from lipid to total energy intake, 32%, exceeded the level recommended. Mean serum total cholesterol concentrations and the percentage of subjects with elevated cholesterol levels (greater than or equal to 200 mg/dl) were high compared with the reported values. Means of the body mass index (BMI) and Rohrer Index (RI) for the subjects were slightly higher than Japanese standards. With these parameters for obesity, triglycerides and atherogenic index were positively correlated and HDL cholesterol and HDL cholesterol percentage to total cholesterol were negatively correlated.

Four cloned cell lines, MT-7, MT-8, MT-9 and MT-10, were established from a transplantable malignant fibrous histiocytoma (MFH) of F344 rats to investigate the histogenesis of the tumor. Cells of MT-7, MT-9 and MT-10 had fine structures characteristic of histiocytes, such as numerous cell processes, many lysosomes and well-developed cytoplasmic organelles. They stained positively for histiocytic lysosomal and antigenic markers. In addition, MT-9 cells contained microfilaments and well-developed RER in their cytoplasm, suggesting that they may be facultative fibroblasts. MT-8 cells stained weakly for histiocytic markers and had scant cytoplasmic organelles. They were identified as undifferentiated mesenchymal cells. The tumors induced in syngeneic rats by inoculating MT-7 or MT-10 consisted of a mixture of the pleomorphic, myxoid and storiform types of MFH, and those by MT-9 were of the storiform type. Cells forming these tumors stained positively for histiocytic markers. Tumors induced by MT-8 consisted of undifferentiated cells negative to these stainings. The histogenesis of MFH is surmised to be related to various differentiation stages shifting from undifferentiated cells to histiocytic cells capable of acting as facultative fibroblasts.

A cis-diamminedichloroplatinum (CDDP)-selected cell line (MT-R10) was induced by continuous exposure of an in vitro passaged cell line (MT-P) established from a transplantable rat malignant fibrous histiocytoma (MFH-MT) to CDDP. MT-R10, capable of proliferating in the presence of 1.0 microgram CDDP/ml, was passaged in CDDP-free medium. The doubling time of MT-R10 at passage 10 (MT-R10/10) was almost the same as that of MT-P, being 22.3 and 25.5 h, respectively. The concentration of CDDP required for 50% inhibition of MT-R10/10 proliferation was two-fold higher than that of MT-P. MT-R10 consisted of round, epithelial-type cells arranged in compact sheets. Ultrastructurally, MT-R10 had numerous free ribosomes, some mitochondria, and other poorly developed cytoplasmic organelles suggesting its undifferentiated nature. MT-R10 showed no reaction for acid phosphatase or non-specific esterase. Tumors induced in syngeneic rats by inoculation with MT-R10 consisted of small, round, undifferentiated cells with scanty cytoplasm. They showed organoid and trabecular patterns, and were often arranged in compact sheets. The neoplastic cells showed no reaction for any of the histiocytic lysosomal and antigenic markers tested, but exhibited a strong reaction for alkaline phosphatase. Bone formation was often observed in the tumors. These observations suggest that CDDP-selected, undifferentiated cells may have osteogenic potential and may be one of the progenitor cells of MFH-MT.

The purpose of the present study is to examine the distribution pattern of acridine orange (AO) binding to DNA in rat adrenal pheochromocytoma PC12 cells that respond to nerve growth factor (NGF). PC12 cells were incubated in a medium containing 100 ng/ml of NGF for 1-14 days for AO ultracytochemistry. Electron microscopic studies revealed that AO binds to DNA exclusively within the euchromatin portion of the cell nucleus. About 35% of the untreated PC12 cells showed characteristic electron-dense interaction products within the nuclei. The average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area were 45 and 1.6, respectively. In the presence of NGF, cell proliferation was suppressed. The cells gradually extend neurite-like cell processes. Mean 3H-labeling index was 40.0 +/- 2.2% in untreated cells and 13.0 +/- 2.3% in PC12 cells with NGF for 6 days after incubation for 30 min. With increasing the cellular differentiation percentages of AO positive cells and average numbers of AO chromatin interaction products per cell nucleus and per micron 2 nuclear area showed a progressive decrease. The results of the present and previous studies suggest that AO chromatin interaction products may be indicative of cell proliferation and differentiation.

The relationship of fat mass, lean body mass (LBM), and fat distribution to blood pressure was examined in Japanese adult men and women. Percent body fat was estimated using two skinfold thicknesses, and fat mass and LBM were then calculated. Correlation coefficients showed that fat mass, fat distribution, and age were weakly associated with blood pressure levels, and that the correlation coefficients of LBM to blood pressure levels were nearly zero. Stepwise regression analysis revealed that fat mass and age contributed significantly to the variations in blood pressure levels. In addition, only in the case of men, trunk-extremity skinfolds ratio was also entered into the model for diastolic and mean arterial blood pressure. It thus has been concluded that fat mass may be the more predictive determinant of blood pressure level than LBM in the Japanese population, and that the independent correlation of fat distribution to blood pressure is rather small.

Cefminox (CMNX, MT-141), a new cephamycin antibiotic, was studied in the field of obstetrics and gynecology, and the following results were obtained. The absorption and the penetration of CMNX into pelvic dead space exudate were good. The mean peak serum level after single intravenous injection was 190.8 micrograms/ml. The level in pelvic dead space exudate reached a peak of 36.6 micrograms/ml 4 hours after an intravenous injection of 1 g and 6.5 micrograms/ml after 12 hours, thus the level over MIC against main pathogenic organisms was maintained for a long time. CMNX was administered against gyneco-obstetrical infections such as intrauterine, intrapelvic, adnexal infections and postoperative wound infections with daily dose of 2 g and was effective in 11 cases out of 12 cases (91.7%), and 81.8% of bacteriological effect was obtained. No side effect was observed. From the above results the usefulness of CMNX in the field of obstetrics and gynecology was suggested.

Clinical studies were done on cefoxitin (CFX), the first cephamycin antibiotic, in the field of obstetrics and gynecology and following results were obtained. CFX was administrated by intravenous drip infusion for average 6.9 days at a daily dose of 2--6 g to 58 patients; 15 with intrauterine infections, 5 with intrapelvic infections, 11 with adnexitis, 3 with mastitis, 5 with urinary tract infections, 2 with other infections and 17 for prophylaxis of postoperative infections. The clinical results were excellent in 8 cases, good in 39 cases, fair in 4 cases, poor in 6 cases and unknown in 1 case, with the efficacy of 82.5%. Total bacteriological effective rate was 88.9%. As to side effects, eruption was observed only in 1 case. From the results of the present study, the usefulness of CFX was demonstrated in the field of obstetrics and gynecology.

Although the reeler, an autosomal recessive mutant mouse with the abnormality of lamination in the central nervous system, died about 3 weeks of age when fed ordinary laboratory chow, this mouse could grow up normally and prolong its destined, short lifespan to 50 weeks and more when given assistance in taking paste food and water from the weaning period. Histopathological examinations of the brain of this mutant mouse revealed no significant age-related difference, suggesting that this mutation does not fundamentally obstruct the spontaneous growth despite the abnormality of the central nervous systems. This mutant mouse is advantageous for investigations on influences of the central nervous system on the aging process and longevity.

1. We performed light and electron microscopic autoradiography of GABA and opiate receptors using 3H-muscimol and 3H-DAMA. 2. Selection of development method is important for electron microscopic autoradiography. The authors obtained silver grains in a pattern precisely localized to the cytoplasmic membrane, i.e., the receptor site by means of EAA development. 3. We observed 3H-muscimol binding site in the neuropiles of the cerebellar molecular layer and the substantia nigra ultrastructurally. Receptor sites labeled with silver grains are located not only on the synaptic regions but also on the plasma membranes of the dendrites and axons not corresponding to synaptic sites. 3H-DAMA binding sites in the caudate nucleus and the amygdaloid nucleus are noticed on the plasma membranes of the axons, dendrites, and nerve cell somas, some corresponding to synapses, others not. There are still problems to be solved in the autoradiographical observation of neurotransmitter receptors. Nevertheless, this technique is very important in observing the morphological relationship between transmitters and their receptor sites.

Pharmacokinetic studies and clinical evaluations of latamoxef (LMOX) were carried out in perinatal mothers and infants. LMOX was promptly absorbed after intravenous injection and intravenous drip infusion in pregnant women, producing dose-related peak blood levels. Placental transference to the fetus was favorable. After intravenous injection and intravenous drip infusion of 1.0--2.0 g of LMOX, drug concentration of the cord blood, amniotic fluid and fetal blood exceeded MICs of the main pathogenic organisms. By administration of the dose of 1.0--2.0 g twice a day, it is possible to successfully prevent or treat uterine infections. LMOX was effective in the prophylaxis or therapy of perinatal infections. Moreover, newborn infants delivered from mothers receiving LMOX treatment having drug concentrations exceeded MICs of main pathogenic organisms however not remained more than 12 hours after birth, and did not exhibit any laboratory test abnormalities. The above results demonstrated that LMOX is a clinically useful antibiotic for the prophylaxis and treatment of perinatal infections.

Morphologic and functional characteristics were investigated on in vitro passaged cells (MT-P) derived from a rat transplantable malignant fibrous histiocytoma (MFH-MT). There were spindle, polygonal, and giant cell types in MT-P. Ultrastructurally, the polygonal and giant cells had the abundant cytoplasm with many lysosomes and processes, whereas the spindle cells possessed smooth cell surface and a small number of lysosomes in their cytoplasm. Immunorosette formation for Fc- and C3-surface receptors and phagocytic activity were demonstrated in 10-20% of MT-P. MT-P were positive for acid phosphatase, nonspecific esterase and alkaline phosphatase. Chromosomes counted in 100 MT-P ranged from 32 to 100 with two peaks of 64 and 76. Tumors induced in syngeneic rats by inoculating MT-P showed variable histologic patterns. They were composed partly of histiocytic cells arranged in a compact sheet. Fibroblastic cells often arranged in a storiform pattern or were supported by myxoid matrix. Osteosarcoma-like structures were occasionally found in the tumors. These results suggest that MFH-MT is heterogeneous, although some cells constituting the tumors have histiocytic markers.

This paper examines the differentiating patterns of subsistence activities, fish-eating habit, mercure level in human biological specimens, and the interrelation among these variables on several small Japanese islands. The catch and supply of fish vary according to patterns of division of labour on each island. The frequency of fish-eating is generally regulated by the availability of fish, and is well associated with the level of mercury in hair or red cells. Difference in variety of fish consumed has a modifying effect on the mercury level. The importance of understanding the total ecological setting relating to any environmental problem should be emphasized.