New mutants of apolipoprotein E associated with atherosclerotic diseases but not to type III hyperlipoproteinemia.

We analyzed the heterogeneity of apo E in very low density lipoprotein from 58 hyperlipidemic subjects with or without atherosclerosis, 69 patients with ischemic heart disease, and 100 apparently healthy individuals. Apo E gene frequencies in the group of healthy individuals were comparable with those in German and American populations. The distribution of six common apo E phenotypes in the groups of hyperlipidemia and ischemic heart disease was similar to that in the healthy group. In addition to the three major isoforms of apolipoprotein E (apo E-4, E-3, and E-2) and the new one (apo E-5) which was recently found in this laboratory, we have discovered an additional series of components, which showed themselves as at least three bands on an isoelectric focusing gel in the region of E-VII through E-V, in four patients with hyperlipidemia and atherosclerosis. The new series of apo E components, named apo E-Suita, was identical with the ordinary apo E in its interaction with heparin-Sepharose gel and with anti-apo E antibody. The most basic component of apo E-Suita (E-VII) was the unsialylated form and other components (E-VI and E-V), the sialylated forms. Family studies revealed that apo E-Suita was determined by inheritance of a new apo E allele located at the same locus as the hitherto known apo E components. Apo E-5 and apo E-Suita isoproteins had isoelectric points more basic than apo E-3, the parent type, by two and four units of charge, respectively. While the apo E-Suita isoprotein had the same molecular weight as ordinary major apo E isoproteins, the molecular weight of the apo E-5 isoprotein was approximately 1,500-2,000 lower than the other apo E isoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incidence of abnormal apo E components, including apo E-5 and apo E-Suita, was high among patients with hyperlipidemia and ischemic heart disease (7:127), while we could not find such components among 100 healthy individuals. Moreover, five of seven patients with the abnormal apo E had overt atherosclerotic disease. The findings suggest that these kinds of apolipoprotein mutation are closely related to the development of atherosclerosis.

Improvement of glucose tolerance in NIDDM by clofibrate. Randomized double-blind study.

A randomized double-blind study was performed to examine the effect of clofibrate on glucose tolerance in subjects with non-insulin-dependent diabetes mellitus (NIDDM). Clofibrate (1.5 g/day) or placebo was administered to 70 patients and an oral glucose tolerance test (OGTT) was performed before and 12 wk after treatment. Blood glucose levels were significantly improved in clofibrate-treated groups at all time points during OGTT, whereas there was no change in insulin levels. Improvement of fasting glucose levels required 8 wk of clofibrate treatment. Insulin binding to erythrocytes demonstrated no significant change in the clofibrate-treated subjects. These results suggest that clofibrate improves glucose tolerance in NIDDM subjects without a change in insulin receptors and that clofibrate increases insulin sensitivity through an unknown postreceptor mechanism.

Comparative analysis of developmentally regulated expressions of Gadd45a, Gadd45b, and Gadd45g in the mouse and marmoset cerebral cortex.

The cerebral cortex is an indispensable region that is involved in higher cognitive function in the mammalian brain, and is particularly evolved in the primate brain. It has been demonstrated that cortical areas are formed by both innate and activity-dependent mechanisms. However, it remains unknown what molecular changes induce cortical expansion and complexity during primate evolution. Active DNA methylation/demethylation is one of the epigenetic mechanisms that can modify gene expression via the methylation/demethylation of promoter regions. Three growth arrest and DNA damage-inducible small nuclear proteins, Gadd45 alpha, beta, and gamma, have been identified as regulators of methylation status. To understand the involvement of epigenetic factors in primate cortical evolution, we started by analyzing expression of these demethylation genes in the developing common marmoset (Callithrix jacchus) and mouse (Mus musculus) brain. In the marmoset brain, we found that cortical expression levels of Gadd45 alpha and gamma were reduced during development, whereas there was high expression of Gadd45 beta in some areas of the adult brain, including the prefrontal, temporal, posterior parietal and insula cortices, which are particularly expanded in greater primates and humans. Compared to the marmoset brain, there were no clear regional differences and constant or reduced Gadd45 expression was seen between juvenile and adult mouse brain. Double staining with a neuronal marker revealed that most Gadd45-expressing cells were NeuN-positive neurons. Thus, these results suggest the possibility that differential Gadd45 expression affects neurons, contributing cortical evolution and diversity.

A new isoform of apolipoprotein E--apo E-5--associated with hyperlipidemia and atherosclerosis.

Heterogeneity of apolipoprotein E (apo E) was analyzed by isoelectric focusing of apo VLDL in patients with hyperlipidemia and/or atherosclerosis. Six major apo E phenotypes were shown, in agreement with the current genetic model which is composed of 3 major apo E isoproteins, apo E-4, apo E-3 and apo E-2, resulting from three apo E alleles, epsilon 4, epsilon 3 and epsilon 2, at a single genetic locus. We recognized an additional apolipoprotein band, which is located basic to apo E-4 on an isoelectric focusing gel, in 3 patients with hyperlipidemia. The new apolipoprotein component, named apo E-5, was identical with ordinary apo E in apparent molecular weight by SDS-polyacrylamide gel electrophoresis and in its interactions with heparin-Sepharose gel and with anti-apo E antibody. This mutant apo E isoprotein had an isoelectric point more basic by one unit of charge than apo E-4. Two of 3 patients had the phenotype E5/3, and the other the phenotype E5/4. Genetic analysis of the apo E phenotypes in family members of the patients indicated the presence of a new apo E allele (epsilon 5) at the same genetic locus as hitherto known alleles. Since most of the subjects above 50 years old with apo E-5 had ischemic heart disease or cerebral infarction, it was suggested that the mutant apo E-5 may possibly be related to the development of atherosclerosis.

Coronary artery disease in heterozygous familial hypercholesterolemia.

Serum lipids, lipoproteins and Achilles tendon thickness in 52 patients with heterozygous familial hypercholesterolemia (FH) were investigated in order to clarify what are the important factors for the development of coronary artery disease (CAD) in heterozygous FH patients. There were no significant differences in the average concentration of total cholesterol and triglyceride between the patients with and those without CAD. The HDL cholesterol (HDL-C) level was significantly lower in patients with CAD than in those without, and the HDL-C value was within the normal range in most of the patients with heterozygous FH, if not associated with CAD. Although most of the males aged over 50 years had CAD and a decreased level of HDL-C, many of the aged females were without signs of CAD. The HDL-C value of heterozygous FH patients with CAD was significantly lower compared with the age-matched group without CAD. The Achilles tendon was thicker in patients with CAD than in those without CAD, both for males and females, although it was less closely correlated with the incidence of CAD than HDL-C or the atherogenic index. A forecast concerning the development of CAD in heterozygous FH may be possible if we consider multiple parameters, such as HDL-C, atherogenic index, Achilles tendon thickness, etc.

Reproducibility of major diagnoses in a binational study of lung cancer in uranium miners and atomic bomb survivors.

A binational panel of four Japanese and four American pathologists examined 208 pulmonary neoplasms, according to the World Health Organization (WHO) recommendations, second edition, for the histologic typing of lung tumors. The study design included independent evaluations by pathologists working alone, followed by group reviews. The individual evaluations, and their implications for reproducibility of the WHO recommendations, are reported. Consensus (agreement by six or more pathologists) with respect to major (ie, first digit) diagnosis was obtained for 76.4% of the cases. Consensus was obtained for 72.5% of the cases with any major diagnosis of small cell cancer; the comparable figures for adenocarcinoma and squamous cell carcinoma were 56% and 48%, respectively. American pathologists were twice as likely as Japanese pathologists to diagnose large-cell cancer, the only significant national difference. Consensus was far less frequent with the minor (ie, second digit) diagnosis categories. This study shows that lung cancers continue to be difficult to classify reproducibly.

Radiation-associated lung cancer: a comparison of the histology of lung cancers in uranium miners and survivors of the atomic bombings of Hiroshima and Nagasaki.

A binational panel of Japanese and American pulmonary pathologists reviewed tissue slides of lung cancer cases diagnosed among Japanese A-bomb survivors and American uranium miners and classified the cases according to histological subtype. Blind reviews were completed on slides from 92 uranium miners and 108 A-bomb survivors, without knowledge of population, sex, age, smoking history, or level of radiation exposure. Consensus diagnoses were obtained with respect to principal subtype, including squamous-cell cancer, small-cell cancer, adenocarcinoma, and less frequent subtypes. The results were analyzed in terms of population, radiation dose, and smoking history. As expected, the proportion of squamous-cell cancer was positively related to smoking history in both populations. The relative frequencies of small-cell cancer and adenocarcinoma were very different in the two populations, but this difference was accounted for adequately by differences in radiation dose or, more specifically, dose-based relative risk estimates based on published data. Radiation-induced cancers appeared more likely to be of the small-cell subtype, and less likely to be adenocarcinomas, in both populations. The data appeared to require no additional explanation in terms of radiation quality (alpha particles vs gamma rays), uniform or local irradiation, inhaled vs external radiation source, or other population difference.

Effect of atenolol on serum beta 2-microglobulin level in hypertensive diabetic patients.

The effects of atenolol (50 mg once daily) on serum beta 2-microglobulin levels in 11 hypertensive diabetic patients uncomplicated by renal dysfunction were studied. Atenolol significantly decreased serum beta 2-microglobulin levels (micrograms/mL) at four weeks (1.5 +/- 0.13) and at eight weeks (1.4 +/- 0.09) from pretreatment level (1.8 +/- 0.17) (P less than 0.05, respectively), along with statistically significant antihypertensive effects. Blood urea nitrogen, serum creatinine, fasting plasma glucose, HbA1C levels, and body weight remained unchanged. The results suggest that atenolol provides a favorable effect on renal function in hypertensive diabetic patients uncomplicated by renal dysfunction.

Evaluation of transcatheter arterial embolization with epirubicin-lipiodol emulsion for hepatocellular carcinoma.

A total of 18 patients with hepatocellular carcinoma (HCC) were treated by transcatheter arterial embolization (TAE) with a 4'-epi-doxorubicin (EDX)-lipiodol emulsion. Infusion of the EDX-lipiodol emulsion (EDX-L) via the hepatic artery was followed by the injection of gelatin sponge in 12 cases. The response and survival of these 12 patients following EDX-L treatment were compared with those of 42 subjects treated with a doxorubicin-lipiodol emulsion (DX-L) and those of 23 patients treated by TAE with gelatin sponge (GS) only. In the group treated with EDX-L, nine cases were AFP-positive in sera and four showed a decrease in serum AFP values to less than 10% of the pretreatment level. Seven cases showed a partial response, and nine cases showed no change in the size of the tumor. In the group treated with EDX-L, nine cases are alive, and the oldest has survived for more than 431 days since the treatment. The half-year survival value was 57%, and the 1-year survival value was 49%. These values did not differ significantly from those calculated for the group treated with DX-L. The 1-year survival value determined for patients treated with a lipiodol emulsion (EDX-L or DX-L) followed by GS was 65%, and the 2-year survival value was 39%. These results rates are significantly better than those obtained in patients treated with GS only (1-year survival, 39%; 2-year survival, 13%.

Relationships between serum cholesterol and obesity: a field study on nutritional background of hypercholesterolemia.

1. Serum cholesterol levels in Japan were unrelated to total fat intake and its quality. 2. A significant positive correlation was found between serum cholesterol level and BMI, and the total energy intake was significantly higher in hypercholesterolemic group than in normocholesterolemic group. 3. Hypercholesterolemia seems to occur in the background of accumulation of body fat due to relative excess of energy intake in Japan.

Tumorigenicity study of disodium glycyrrhizinate administered orally to mice.

Disodium glycyrrhizinate (DG) was administered at concentrations of 0.15 (maximum tolerated dose), 0.08, 0.04 or 0% in the drinking-water to groups of 50, 70, 60 and 60 male B6C3F1 mice, respectively. Female groups, each consisting of 50 mice, were given DG in the drinking-water at concentrations of 0.3 (maximum tolerated dose), 0.15, 0.08 or 0%. Treatment was continued for 96 wk and the experiment was terminated at wk 110. There was no difference between treated and control groups in tumour incidence, in the latent period before tumours appeared or in the distribution of different types of tumour. Thus the long-term oral administration of DG to mice did not yield any evidence of chronic toxicity or tumorigenicity.

Hormonal regulation of glandular kallikrein activity and its inhibitor in human plasma.

Plasma glandular kallikrein (GK) activity, serum 3,5,3'-triiodothyronine (T3), thyroxine (T4), reverse T3, thyroid-stimulating hormone, plasma alpha 2 macroglobulin (alpha 2M) and alpha 1 antitrypsin (alpha 1AT) levels were determined in twenty-nine non-diabetic female subjects given normal ad-lib diet or one of several kinds of caloric composition for more than 7 days after an overnight fast. Positive exponential correlation was found between serum T3 level and plasma GK activity (r = 0.54), and there was positive linear correlation between serum T3 level and plasma 2M level (r = 0.51). Positive natural logarithmic correlation was found between plasma GK activity and plasma alpha 2M level (r = 0.47). There was no correlation between plasma GK activity and plasma alpha 1AT level, and between serum T3 level and plasma alpha 1AT level. No correlation was found between serum T4 level and plasma GK activity, and between serum T4 level and plasma alpha 2M level. There was no correlation between serum T4 and T3 level, but positive exponential correlation was found between serum T4 and reverse T3 level (r = 0.82). These data suggest that T3 responds to dietary carbohydrate through Kallikrein-Kinin system, and T3 regulates both GK activity and its inhibitor (alpha 2M) in human plasma.

Evaluation of fasting serum insulin levels among Japanese school-age children.

We examined the physical constitution, serum lipids, fasting serum insulin (IRI), food intake, and physical activity in 1,330 children in three age groups (6-7, 9-10, and 12-13) in Nagao village for the period 1994-1996. Serum total cholesterol (TC) and high-density lipoprotein-cholesterol (HDL-C) levels were positively correlated, and HDL-C and TG were negatively correlated in all age groups. IRI was significantly higher among the elder children, and it was also higher in girls than in boys. IRI was positively correlated with the body mass index (BMI) and triceps skinfold thickness in ages 9-10 and 12-13, but no significant correlation was noted with waist/hip ratio (W/H ratio). In ages 12-13, IRI was positively correlated with TC and TG and negatively correlated with HDL-C. In high-IRI children (above 11.4 microU/mL) ages 12-13, the high-fat diet (fat-energy ratio above 30%) and low physical activity children (physical activity score below 10 points) were more frequent, and the mean levels of triceps skinfold were higher. The positive correlation between TC and HDL-C was not significant among high-IRI children. We concluded that a high-fat diet, low physical activity, and body fat accumulation influence the IRI level, which is closely related to serum lipid levels.

Relationship between protein intake and nitrogen balance in obese patients on low energy diet.

The effect of nitrogen intake on nitrogen balance was studied in six obese patients receiving low energy diets. They were given a control diet containing 2,000 kcal of energy and 80 g of protein for the first ten days. Then they were given Diet A with 1,100 kcal of energy and 70 g of protein for the next 2 weeks, followed by Diet B with 1,100 kcal of energy and 50 g of protein for 2 weeks. The relationship between nitrogen intake (X, mg/kg) and nitrogen balance (Y, mg/kg) during the low energy diet periods was statistically significant, with Y = 0.388X-60.32 (SD = 17.71, r = +0.67, n = 11, p less than 0.05). The nitrogen and protein requirements were estimated from this equation to be 201.1 mg/kg and 1.26 g/kg, respectively. In our experiment, the nitrogen balance in obese patients was well maintained although total energy was reduced to 1,100 kcal/day in Diet A. It is suggested that protein quantity in the diets should be taken into account when a low energy diet is used for the treatment of obesity.

Effects of low energy diets on protein metabolism studies with [15N]glycine in obese patients.

The effects of low energy diets on protein metabolism in terms of the metabolic pool, active protein pool, and active and inactive protein synthesis rates were studied using [15N]glycine in five obese patients (percentage of ideal body weight, 120-190%). For 10 days, the patients were given a control diet containing 2,000 kcal of energy and 80 g of protein. For the next 2 weeks, they were given Diet A with 1,100 kcal of energy and 70 g of protein, and for the last 2 weeks given Diet B with 1,100 kcal of energy and 50 g of protein. During the Diet A period, the active protein pool and the active and inactive protein synthesis rates were about the same as during the control diet period, although the metabolic pool tended to be slightly smaller than during the control diet period. During the Diet B period, the metabolic pool, active protein pool, and active protein synthesis rate were all significantly different from the values during the control diet period. The results suggest that protein metabolism in obese patients is not maintained with less than 70 g of protein daily when energy intake was restricted to 1,100 kcal/day.

A 1-year follow-up study on the effect of atenolol on serum beta 2-microglobulin level in hypertensive diabetic patients.

Hypertensive diabetic patients are particularly prone to renal function impairment. A total of nine out-patients with diabetes and hypertension were, therefore, entered into this single-blind uncontrolled study on the effects of 50 mg/day atenolol on reducing blood pressure and preserving normal kidney functioning. Treatment and evaluations were continued for 12 months. Serum beta 2-microglobulin concentration was used as the index for measuring renal impairment. Atenolol significantly reduced heart rate, systolic and diastolic blood pressure, and serum beta 2-microglobulin concentrations compared with baseline. Plasma glucose and glycosylated haemoglobin levels were unchanged, and blood urea nitrogen levels were increased slightly (non-significant). Serum creatinine showed a tendency (non-significant) to reflect the changes in beta 2-microglobulin concentration. Ways in which atenolol may act to improve kidney functioning are suggested. It is concluded that atenolol is a favourable choice for the treatment of hypertension in diabetic patients with normally functioning kidneys since, even in long-term use, normal renal functioning is preserved.

Tumorigenicity study of sodium erythorbate administered orally to mice.

Sodium erythorbate (SE) was administered at concentrations of 0, 1.25, or 2.5% (maximum tolerated dose, MTD) in the drinking-water to groups of 50 male B6C3F1 mice respectively. Female groups, each consisting of 50 mice, received SE in the drinking-water at concentrations of 0, 2.5 or 5% (MTD). Treatment continued for 96 wks and the experiment was terminated during wk 110. Tumors were observed at various sites including the liver, hematopoietic system, lung and soft tissue. However, at any of the sites, the tumor incidence, the time to death with tumors or the histological distribution of tumors did not differ significantly from those in the untreated control group. Thus, the present study did not demonstrate a tumorigenic effect of SE on B6C3F1 mice by means of oral administration.

[Method for remnant lipoproteins--quantitative analysis of serum remnant-like particle cholesterol (RLP-C)].

The important role of remnant lipoprotein, which is linked up to serum insulin, in the development of atherosclerosis is well known. So, measurement of remnant has a benefit as the indicator of cardiovascular disease-risk. Recently, Nakajima et al have developed a simple, rapid assay method, using a immunoaffinity gel mixture of anti-apo B-100 and anti-apo A1 monoclonal antibodies coupled with Sepharose 4B. The apoprotein composition of RLP which is unbound with mixed gel is as similar to apo-E rich VLDL. Clinical significance of RLP-C has been already indicated by many reports.