Association between dexamethasone treatment and alterations in serum concentrations of trace metals.

Previously, a significant elevation in the serum levels of iron (Fe) was observed within a few days after the initiation of cisplatin (CDDP)-based chemotherapy. To clarify the underlying mechanisms, the serum concentration of hepcidin, a negative regulator of Fe release, was determined in the clinical samples obtained from six patients with cancer. The result showed that the serum concentration of hepcidin in patients receiving CDDP-based chemotherapy was significantly increased after 4-6 days of treatment, in comparison to the baseline level, suggesting that aforementioned excessive systemic Fe was not explained by the change of serum hepcidin level. All these patients received antiemetic premedication. We next evaluated of the effects of Pt-containing drugs and prophylactic antiemetic dexamethasone medication on the serum concentration of trace metals in mice, and on the hepatic and renal concentration of trace metals. The serum concentrations of Fe, Cu, and Zn in the CDDP-treated and oxaliplatin-treated mice were not significantly altered in comparison to those of the vehicle-treated control group. The serum concentrations of Fe, Cu, and Zn were increased after 24 h of dexamethasone treatment, compared to those of the control group (P < 0.05). The hepatic concentration of Mn was significantly reduced, whereas those of Fe and Cu inclined to diminish. The present findings suggest that dexamethasone can partly contribute to the changes in the serum concentrations of trace metals during anticancer chemotherapy.

Determinants of hyperleptinaemia in an African population.

OBJECTIVE: To examine the determinants for elevated plasma leptin concentration in normal weight (NW), obese (OB), and morbidly obese (MO) individuals in Tanzania. DESIGN: Cross-sectional epidemiological study, the CARDIAC study. SETTING: Three areas in Tanzania; Dar es Salaam, urban (U), Handeni, rural (R) and Monduli, pastoralists (P), in August 1998. SUBJECTS: Five hundred and forty five participants from a random sample of 600 people aged 46-58 years. MAIN OUTCOME MEASURES: Plasma leptin concentrations, height, weight, body mass index (BMI), lipid profiles, haemoglobin A1c (HBA1c), and blood pressure (BP). RESULTS: Plasma leptin concentrations were higher in women than in men (women; 16.0 ng/mL, men; 3.1 ng/mL; p<0.0001). Women showed a higher mean body mass index (BMI), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) than men. In both genders, plasma leptin concentration, total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), triglycerides (TG), systolic BP (SBP) and diastolic BP (DBP) were significantly higher in OB than in NW participants. MO women had significantly higher leptin concentration, SBP and DBP compared with the other two groups. In NW men, log leptin concentrations showed a direct correlation with weight, BMI, HBA1c, TC, LDL-C, TG, SBP and DBP (all p<0.0001 except TG; p<0.001), while among NW women and OB men, weight and BMI correlated positively with log leptin (all p<0.05). OB women observed a positive correlation between log leptin and weight, BMI and LDL-C. Regression analysis indicated that among NW subjects, gender, BMI and TC explained 53.9% of the variation in log leptin. In OB subjects, gender, BMI and LDL-C explained 51.7% of the variability in leptin levels. No relationship was found between log leptin and CVD risk factors among MO subjects. CONCLUSION: The most important determinants for hyperleptinaemia in NW participants were gender, BMI, TC, while in addition to these LDL-C, was an important determinant of leptin concentration in OB individuals. In MO women, the high leptin concentrations did not reflect the amount of adipose stores.

Plasma soluble fas and soluble fas ligand in chronic glomerulonephritis.

It has been reported that glomerular cells with apoptosis and positive Fas immunoreactivity are seen in proliferative glomerulonephritis (PGN). Fas induces apoptosis when it binds to Fas ligand (Fas-L) or soluble Fas-L (sFas-L). However, soluble Fas (sFas) blocks apoptosis by inhibiting binding between Fas and Fas-L or sFas-L. That is, Fas, Fas-L, and sFas-L are inducers of apoptosis, but sFas is an inhibitor of apoptosis. We studied the relationship between the plasma levels of sFas and sFas-L in 32 patients with various types of adult chronic glomerulonephritis. Patients with serum creatinine levels >1.5 mg/dl (132.6 micromol/l) were excluded. The plasma levels of sFas-L were within the normal limits in all patients. The plasma levels of sFas in the patients with minimal-change (n = 8) and membranous nephropathy (n = 7) were similar to the age- and sex-matched controls. However, the plasma sFas levels were significantly elevated in patients with mesangial PGN (n = 10) and membranoproliferative glomerulonephritis (n = 7)(3. 4 +/- 0.9 and 3.9 +/- 1.5 ng/ml, respectively) as compared with the age- and sex-matched controls (controls: 2.1 +/- 0.4 and 2.2 +/- 0.6 ng/ml, respectively). In PGN, according to increase of histological grade and decrease of creatinine clearance, the number of TUNEL-positive cells in glomeruli is decreased in spite of an increase of the Fas positivity, and plasma sFas is increased. The degree of proliferative change is determined by the balance between proliferation and apoptosis and/or necrosis. Therefore, increased plasma sFas in PGN may inhibit apoptosis in glomeruli and may be one of the progressing factors in PGN. Thus, we conclude that an increase in plasma sFas levels is important to the protection of apoptosis in PGN.

[Participation of apoptosis in renal amyloidosis].

Renal amyloidosis shows symptoms of renal dysfunction due to the deposition of amyloid protein in the kidney. Recently, it was reported that apoptosis plays an important role in the pathogenesis of type-2 diabetes mellitus and Alzheimer's disease of which amyloid deposition is seen in the tissue. We investigated whether or not apoptosis and related factors are observed in renal amyloidosis. In situ nick end labeling (TUNEL) was performed in seven autopsied renal tissues with primary and secondary amyloidosis and 10 autopsied renal tissues without renal disease as the control. The number of TUNEL-positive cells was significantly increased in both the glomeruli and tubulus of the kidney with amyloidosis than in the control. Electron microscopic analysis was performed on one biopsied renal tissue with amyloidosis and six biopsied renal tissues with minor abnormalities as the control. Typical apoptotic cells were observed only in the former. Bax product, an inducer of apoptosis, and Bcl-2 protein, an inhibitor of apoptosis, were examined immunohistochemically in the seven autopsied renal tissues with amyloidosis and 10 autopsied control tissues. Bax was overexpressed in the tubulus and glomeruli of subjects with renal amyloidosis, compared to the normal controls. However, Bcl-2 protein was not detected in the glomeruli in any of the subjects examined. These results indicate that apoptotic cells are increased in number in renal amyloidosis and Bax overexpression may play an important role in this increase.

Identification of cis-elements of the human endothelin-A receptor gene and inhibition of the gene expression by the decoy strategy.

Previously, we succeeded in molecular cloning of the cDNA and the gene for human endothelin-A receptor (ET-AR). In the present study, we define cis-elements in the 5'-flanking region of the ET-AR gene. Deletion analyses were performed in A7r5 cells, rat vascular smooth muscle cell line, and Chinese hamster ovary cells using ET-AR promoter-luciferase gene constructs including 5 kilobases of the 5'-flanking region. These analyses demonstrated the existence of one negative regulatory element (-2.0 kilobases to -857 bases) and two positive regulatory elements (-137 to -53 and -53 to +251). Gel mobility shift assay revealed a nuclear protein binding to the region (-104 to -78) (R1). DNase I footprinting analysis showed a footprint spanning from -91 to -83 whose sequence is CCCCACCTT (ETA-P1). When a plasmid including R1 fragments (R1 decoy) was co-transfected into A7r5 cells with ET-AR (-137 to +251)-luciferase gene construct, it significantly reduced the luciferase activity in a dose-dependent manner. Moreover, R1 decoy down-regulated the endogenous ET-AR mRNA in A7r5 cells by a maximum of 75%. Thus, we identified cis-elements that regulate basal transcriptional activity of the ET-AR gene and proved the feasibility to suppress the expression of the ET-AR gene by the DNA decoy strategy using the positive regulatory element we identified.

A comparative epidemiological study of the effects of regular exercise on health level.

STUDY OBJECT: To reveal the relationship between regular exercise and physical fitness, life style, mental health, and other cardiovascular risk factors among the Japanese. DESIGN: cross-sectional study. SUBJECTS: Three thousands, one hundred and thirty-two individuals (1,795 men and 1,336 women) over 40 years old who were examinees at one of seven centers for health promotion. METHODS: A questionnaire survey on the life style, a physical fitness tests and clinical laboratory tests were performed. RESULTS: Continuing regular exercise was associated with high levels of physical strength and their odds ratio (OR) was 2.09 (95% confidence interval(CI) 1.74-2.50). Many individuals in this group had good mental health: the OR for their sensation of fatigue (swift recovery) was 1.40(95% CI; 1.17-1.68) and that for mood recovery (fast) was 1.44(95% CI; 1.20-1.72). Thinness and obesity were scarce in this group. CONCLUSIONS: High physical fitness level, good mental health, standard body weight are more common in regular exercise group.

Glucocorticoid regulation of leptin synthesis and secretion in humans: elevated plasma leptin levels in Cushing's syndrome.

Leptin, the obese (ob) gene product, is an adipocyte-derived satiety factor that is involved in the regulation of food ingestion and body weight. To investigate glucocorticoid regulation of leptin synthesis and secretion in humans, we measured plasma leptin levels in patients with Cushing's syndrome with adrenal or pituitary adenoma and in patients with iatrogenic Cushing's syndrome. Plasma leptin levels in patients with Cushing's syndrome were significantly elevated compared to those in nonobese healthy subjects and obese subjects without any metabolic or endocrine diseases at a given percentage of body fat by analysis of covariance. In patients with adrenal or pituitary adenoma, after the tumor resection, plasma leptin levels were reduced, with a concurrent decrease in plasma cortisol levels. With no significant changes in body weight, plasma leptin levels were also elevated significantly in lean healthy volunteers 24 h after the administration of 1 mg dexamethasone. Dexamethasone potently induced ob gene expression and leptin secretion in the organ culture of human adipose tissue. The data demonstrate that glucocorticoids act, at least in part, directly on the adipose tissue and increase leptin synthesis and secretion in humans.

Factor VII and fibrinogen levels examined by age, sex, and other atherosclerotic risk factors in a Japanese population. The Jichi Medical School Cohort Study.

Factor VII coagulant activity (FVIIc) and fibrinogen (Fbg) levels have been investigated as cardiovascular risk factors. We studied these two factors with stratification for age, sex and blood pressure, and the relation with other atherosclerotic risk factors in a Japanese general population. The subjects were 3,139 Japanese (1,315 males and 1,824 females) aged 30 to 89 in 1992 and 1993. A linear increase with age was observed in the levels of Fbg in both men and women, but no differences were observed between men and women in each age group. A linear increase with age was also seen in the levels of FVIIc in women, but the levels of FVIIc in men were significantly higher for the age group 40-49 years than for any other age group. The levels of FVIIc in women were significantly higher than in men at age > or = 60 years. As concerning the effect of alcohol intake status, Fbg had a tendency to decrease with alcohol intake. Fbg and FVIIc levels were associated with an increase in smoking status in men, but no association was seen in women in either Fbg or FVIIc. FVIIc was positively correlated with age, body mass index, total cholesterol, triglycerides and fasting insulin level. Fbg was positively correlated with age, systolic blood pressure, diastolic blood pressure, total cholesterol, LDL-cholesterol and triglycerides in women, but Fbg had few positive correlations with risk factors in men. A comparison with previous Western studies showed that the Fbg levels of our Japanese population were lower than those of the Caucasians studied, but the present FVIIc levels were nearly the same level or slightly higher than theirs. The association of Fbg and FVIIc and with other cardiovascular risk factors in Japanese was similar to those observed in Caucasians.

Pathophysiological significance of the obese gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats: evidence for loss of its satiety effect in VMH-lesioned rats.

To explore the pathophysiological significance of the obese (ob) gene product, leptin, in ventromedial hypothalamus (VMH)-lesioned rats, we examined the synthesis and secretion of leptin and its satiety effect in VMH-lesioned rats compared with those in sham-operated rats. Northern blot analysis revealed that ob gene expression is markedly augmented in the mesenteric and sc white adipose tissue, but remained unchanged in the epididymal white adipose tissue during the development of obesity in VMH-lesioned rats. Plasma leptin levels were relatively constant in sham-operated rats, but were elevated during the development of obesity in VMH-lesioned rats. In sham-operated rats, a single i.v. (1.0 mg/rat) or intracerebroventricular (2.0 micrograms/rat) injection of recombinant human leptin reduced food intake and body weight gain in sham-operated rats. By contrast, no significant effect on food intake or body weight gain was observed in VMH-lesioned rats. The present study provides evidence that VMH-lesioned rats overproduce leptin and increase its release but cannot respond to it and suggests that the loss of its satiety effect contributes to the development of obesity and the obesity-related phenotypes in VMH-lesioned rats.

Augmentation of obese (ob) gene expression and leptin secretion in obese spontaneously hypertensive rats (obese SHR or Koletsky rats).

To explore the pathophysiologic roles of the obese (ob) gene product, leptin, in the development of obesity and hypertension, we examined ob gene expression and leptin secretion in obese spontaneously hypertensive rats (obese SHR or Koletsky rats) at the stage of established obesity and hypertension. Expression of the ob gene was augmented in the epididymal, mesenteric, subcutaneous, and retroperitoneal white adipose tissue (WAT) from 20-week-old male obese SHR compared to their lean littermates (lean SHR). Using a radioimmunoassay for rat leptin, we also measured plasma leptin levels in 20-week-old lean and obese SHR. Plasma leptin levels in obese SHR (292.5 +/- 37.1 ng/ml) were more than 100-fold higher than those in lean SHR (2.8 +/- 1.0 ng/ml). The present study demonstrates that ob gene expression and leptin secretion are markedly augmented in obese SHR.

Re-evaluation of the SA gene in spontaneously hypertensive and Wistar-Kyoto rats.

1. To investigate whether the difference in the SA gene expression in the kidneys is causally related to the pathogenesis of hypertension, we reassessed the expression of the SA gene in the kidneys of the spontaneously hypertensive rat (SHR), its stroke-prone substrain (SHRSP) and Wistar-Kyoto (WKY) rat from different sources (SHR/Izm, SHRSP/Izm and WKY/ Izm from Izumo colony; SHR/Crj and WKY/Crj from Charles River Laboratories). 2. At the age of 5 weeks, high levels of the SA mRNA were expressed in the kidneys of SHRSP/Izm, SHR/Izm, SHR/Crj and WKY/Izm, while very low levels of the SA mRNA were observed in those of WKY/Crj. At the age of 8 weeks, the expression of the SA mRNA in the kidneys of WKY/Izm was at the same level as in those of SHRSP/Izm and two SHR strains. 3. Four genetic markers at the SA locus, an StuI restriction fragment length polymorphism and three microsatellite markers, were not polymorphic among Izumo strains of SHR, SHRSP and WKY rats. 4. In situ hybridization showed strong signals of the SA mRNA in the renal proximal tubules, while no positive signals were detected in the glomeruli. 5. Because WKY/Izm has normal blood pressure, our observations indicate that a simple difference of the SA gene expression in the kidney cannot be an explanation for the difference of blood pressure between SHR(SP)/Izm and WKY/Izm.

Self-association of LIM-kinase 1 mediated by the interaction between an N-terminal LIM domain and a C-terminal kinase domain.

LIM-kinase 1 (LIMK1) and 2 (LIMK2) are members of a novel class of protein kinases containing two LIM motifs at the N-terminus. The LIM motif is thought to be involved in protein-protein interactions. We report here evidence that LIMK1 self-associates and also associates with LIMK2. In vivo and in vitro binding analyses using variously deleted mutants of LIMKI revealed that the self-association of LIMK1 was caused by interaction between the N-terminal LIM domain and the C-terminal kinase domain. The association of LIMK1 with itself and with LIMK2 is important for understanding how activities and functions of LIMK family kinases are regulated.

A comparative epidemiological study on the effects of physical fitness on health level.

Clinical tests, a questionnaire on life style and mental health, and physical fitness tests were conducted on 3,132 individuals (1,796 men and 1,336 women) to find the relationships between physical fitness level and life style, mental health, or cardiovascular risk factors among Japanese. Individuals with an advanced physical fitness level also exhibited optimum mental health: the odds ratio (OR) and 95% confidence interval (CI) for their sensation of fatigue (fast recovery) was 1.16 (95% CI = 1.00 to 1.35) and that for mood recovery (fast) was 1.29 (95% CI = 1.12 to 1.49). Among those with an advanced physical fitness level, the following were rare (OR): daily smoking [0.78]; obesity (over 120%) [0.73 (95% CI = 0.62 to 0.87)]; thick subcutaneous fat (over 20 mm on the back) [0.77 (95% CI = 0.66 to 0.90)]; high blood sugar level [0.64 (95% CI = 0.50 to 0.82)]; and hypertension [0.67 (95% CI = 0.49 to 0.91)]. These are risks for circulatory diseases. These data show that risk factors associated with cardiovascular disease are more common in those with a low physical fitness level.

Development of radioimmunoassay for human leptin.

Using recombinant human leptin, we have produced an antiserum for human leptin and developed a radioimmunoassay (RIA) specific and sensitive for human leptin. We detected leptin-like immunoreactivity (-LI) in culture media of adipose tissue from subcutaneous abdominal fat in human. The plasma leptin-LI concentration in nonobese subjects (17.6

Identification and characterization of a novel family of serine/threonine kinases containing two N-terminal LIM motifs.

We previously isolated human cDNA coding for LIMK1 (LIM motif-containing protein kinase-1), a putative protein kinase containing two LIM motifs at the N terminus and an unusual protein kinase domain at the C terminus. In the present study, we isolated human cDNA encoding LIMK2, a second member of a LIMK family, with a domain structure similar to LIMK1 and 50% overall amino acid identity with LIMK1. The protein kinase domains of LIMK1 and LIMK2 are unique in that they contain an unusual sequence motif Asp-Leu-Asn-Ser-His-Asn in subdomain VIB and a highly basic insert between subdomains VII and VIII. Expression patterns of LIMK1 and LIMK2 mRNAs in human tissues differ significantly. Chromosomal localization of human LIMK1 and LIMK2 genes was assigned to 7q11.23 and 22q12, respectively, by fluorescence in situ hybridization. The Myc epitope-tagged LIMK1 and LIMK2 proteins transiently expressed in COS cells exhibited serine/threonine-specific kinase activity toward myelin basic protein and histone in in vitro kinase assay. Immunofluorescence and subcellular fractionation analysis revealed that Myc-tagged LIMK1 and LIMK2 were localized mainly in the cytoplasm. The "native" LIMK1 protein endogenously expressed in A431 epidermoid carcinoma cells also exhibited serine/threonine kinase activity. The specific activity of native LIMK1 from A431 cells was apparently much higher than that of "recombinant" LIMK1 ectopically expressed in COS cells, hence, it is likely that there is a mechanism, by which native LIMK1 is activated. A 140-kDa tyrosine-phosphorylated protein (pp140) was co-immunoprecipitated with native LIMK1 form A431 cell lysates; therefore, pp140 may be a LIMK1-associated protein involved in the regulation of LIMK1 function.

Physical-strength tests and mortality among visitors to health-promotion centers in Japan.

A follow-up study was conducted to clarify the relationship between physical-strength level and risk of death from all causes and from cancer and cardiovascular disease. The 7286 persons who were examined at seven health-promotion centers throughout Japan between 1982 and 1987 were followed up. By January 1992, 6259 persons (85.9%) had been contacted by questionnaire. They included 3117 men (49.8% of all subjects studied) (average age 53.6 years at baseline, SD = 9.0 years, range 40-84 years), and 3142 women (50.2%) (average age 54.5 years at baseline, SD = 8.5 years, range 40-85 years). The follow-up period for each person averaged 6.1 years, for a total of 38,253 person-years. During this period, 155 deaths were reported. At baseline, five physical-strength tests (grip strength, side step, vertical jump, standing trunk flexion, and sit-ups) were performed. Five clinical laboratory tests (thickness of skinfold, blood sugar, total serum cholesterol, percent vital lung capacity, and blood pressure) were also conducted. The examinees were questioned about smoking status (current smoker, nonsmoker, and ex-smoker). Men with thicker skinfold [relative risk (RR) = 2.11] and higher levels of blood sugar (RR = 1.89) had an excess risk of death from all causes. Men with higher serum cholesterol (RR = 5.08), thicker skinfold (RR = 4.54), and elevated blood pressure (RR = 2.33) had an excess risk of death from cardiovascular disease. In women, no relationship was seen between clinical laboratory tests and an excess risk of death. Men exhibiting lower values for side step (RR = 2.43), vertical jump (RR = 2.37), sit-ups (RR = 1.93) and grip strength (RR = 1.92) also had an excess risk of death from all causes. Furthermore, men with lower heights for vertical jump (RR = 5.51) had an excess risk of death from cardiovascular disease. After adjustment for skinfold thickness, blood sugar, total serum cholesterol, blood pressure, percent vital lung capacity and smoking status, men with a lower level of side step, vertical jump, and grip strength had an excess risk of death from all causes. No such relationship was seen between physical-strength level and an excess risk of death in women. It is concluded that a low level of physical strength might be significantly correlated with subsequent health outcomes in men.

Lipoprotein(a) levels in the Japanese population: influence of age and sex, and relation to atherosclerotic risk factors. The Jichi Medical School Cohort Study.

The authors studied the distribution of lipoprotein(a) (Lp(a)) levels with stratification for age and sex, as well as the relation between Lp(a) and atherosclerotic risk factors in a large Japanese population between 1992 and 1993. The subjects were 1,235 males and 1,762 females over 30 years old. Lp(a) was measured by an enzyme-linked immunosorbent assay. Lp(a) levels were higher in females than in males. The increase in Lp(a) with age was statistically significant, and the proportion of subjects with Lp(a) levels > 30 mg/dl also increased with age. In the obese subjects (body mass index (BMI) (kg/m2) > 26), Lp(a) levels were lower than in the non-obese subjects (BMI < or = 26) (p < 0.01 in males; p < 0.05 in females). Male alcohol drinkers had lower Lp(a) levels than nondrinkers (p < 0.05). Age, low density lipoprotein subtracting Lp(a) cholesterol [Lp(a) x 0.3], and fibrinogen level were all positively correlated with Lp(a) in both sexes. Alcohol consumption (g/day) and triglycerides were inversely correlated with Lp(a) in males, while total cholesterol subtracting Lp(a) cholesterol [Lp(a) x 0.3], high density lipoprotein, and factor VII were positively correlated in females. Multiple logistic regression analysis showed that triglycerides in males and BMI and fibrinogen in females were significant independent variables. The authors conclude that Lp(a) level is affected by various factors, such as alcohol drinking, BMI, sex, and age, and is not only correlated with lipid levels but also with hemostatic factors such as fibrinogen and factor VII.

Cis elements for transcriptional regulation of the human endothelin-A receptor gene.

To elucidate the transcriptional regulation of the endothelin-A receptor (ET-AR) gene, we defined the cis elements in the 5'-flanking region by functional analysis. Chimeric gene constructs were prepared using serial deletion mutants of a 5-kilobase (kb) fragment of the human ET-AR 5'-flanking region and the luciferase gene and were transfected into a rat vascular smooth-muscle cell line, A7r5. Deletion of the region between -2.0 kb and -857 bases caused a twofold increase in luciferase activity, and deletion from -137 bases to -53 bases reduced the activity by 50%. These results indicate the existence of a negative regulatory element and a positive regulatory element, respectively. The gel shift assay revealed the existence of nuclear proteins in A7r5 cells that bind to the region containing the positive regulatory element. Thus, we identified cis regulatory elements and nuclear factors that regulate the expression of the ET-AR gene.