Michio Yamakido

The integrin alpha(9)beta(1) binds to a novel recognition sequence (SVVYGLR) in the thrombin-cleaved amino-terminal fragment of osteopontin.

The integrin α9β1 mediates cell adhesion to tenascin-C and VCAM-1 by binding to sequences distinct from the common integrin-recognition sequence, arginine-glycine-aspartic acid (RGD). A thrombin-cleaved NH2-terminal fragment of osteopontin containing the RGD sequence has recently been shown to also be a ligand for α9β1. In this report, we used site-directed mutagenesis and synthetic peptides to identify the α9β1 recognition sequence in osteopontin. α9-transfected SW480, Chinese hamster ovary, and L-cells adhered to a recombinant NH2-terminal osteopontin fragment in which the RGD site was mutated to RAA (nOPN-RAA). Adhesion was completely inhibited by anti-α9 monoclonal antibody Y9A2, indicating the presence of a non-RGD α9β1recognition sequence within this fragment. Alanine substitution mutagenesis of 13 additional conserved negatively charged amino acid residues in this fragment had no effect on α9β1-mediated adhesion, but adhesion was dramatically inhibited by either alanine substitution or deletion of tyrosine 165. A synthetic peptide, SVVYGLR, corresponding to the sequence surrounding Tyr165, blocked α9β1-mediated adhesion to nOPN-RAA and exposed a ligand-binding-dependent epitope on the integrin β1 subunit on α9-transfected, but not on mock-transfected cells. These results demonstrate that the linear sequence SVVYGLR directly binds to α9β1 and is responsible for α9β1-mediated cell adhesion to the NH2-terminal fragment of osteopontin.

Prediction of short- and intermediate-term prognoses of patients with acute myocardial infarction using myocardial contrast echocardiography one day after recanalization

OBJECTIVES This study sought to determine whether microvascular integrity in the risk area (RA) for myocardial infarction (MI) one day after recanalization predicts the outcome in patients with first acute MI. BACKGROUND Immediately after recanalization, microcirculation in the RA is modified by both hyperemic response and microvascular impairment. METHODS Fifty consecutive patients who underwent serial myocardial contrast echocardiography before and one day after recanalization (day 2) were studied. All patients had a completely occluded lesion in the left anterior descending coronary artery alone, and underwent successful reperfusion therapy. The relative size of the initial RA (RA ratio) and peak gray scale ratio (PGSR) within the RA on day 2 were determined. Patients were followed for a median of 22 months to evaluate clinical outcome. RESULTS On day 2, PGSR was a median of 0.46. Study patients were subdivided into two groups, group A of 24 patients with acceptable opacification (PGSR > 0.46 on day 2) and group B of 26 patients without it. Major cardiac events (cardiac death, nonfatal MI and repeat admission for congestive heart failure) were more frequently observed in group B (28% vs. 4%, Cox hazard ratio=8.5, p=0.05, 95% confidence interval [CI] 1.03 to 69.9). The median value of the RA ratio was 0.45. Patients (n=15) with RA ratio > 0.45 on day 1 and PGSR on day 2 < or = 0.46 exhibited a 10.7-fold relative risk for major cardiac events (p=0.005, 95% CI 2.06 to 55.8) and a 3.69-fold relative risk for composite cardiac events (major cardiac events and target lesion revascularizations) after the initial intervention (p=0.004, 95% CI 1.51 to 9.04). CONCLUSIONS The assessment of both the size of the initial RA and microvascular integrity on day 2 enables precise determination of the efficacy of reperfusion therapy and prediction of the short- and intermediate-term prognoses of patients with recanalized MI.

Diabetes Mellitus and Its Vascular Complications in Japanese Migrants on the Island of Hawaii

Japanese migrants and their offspring on the island of Hawaii and Japanese living in Hiroshima were examined for diabetes mellitus and its vascular complications. the same methods and investigators were used in both locations. Death certificates of Japanese and Caucasians dying on the island during the past 26 yr were analyzed. Diabetes, defined as a venous serum glucose concentration of at least 200 mg/dl 2 h after a 50-g oral glucose load, was significantly more common in the Hawaiian Japanese than in the Hiroshima Japanese subjects. This suggests that diabetes is more prevalent in Japanese in Hawaii than in Japan, although lack of knowledge about the total population of Japanese migrants in Hawaii makes this generalization uncertain. The proportion of deaths attributed to diabetes was much higher in Japanese migrants and their offspring in Hawaii than in Japan. During the 1950s, the proportional death rate from diabetes was about half as large in Japanese Hawaiians as in Caucasian Hawaiians, but it increased to become 1.6 times the Caucasian rate during the 1970s. A nutritional study revealed that the total caloric intake was similar in Japanese in Hawaii and Hiroshima, although the estimated level of physical activity was less in the Hawaiian subjects. Consumption of animal fat and simple carbohydrates (sucrose and fructose) were at least twice as high in Hawaiian as in Hiroshima Japanese. Conversely, Hiroshima Japanese consumed about twice the amount of complex carbohydrate as the Hawaiian Japanese. These observations support the hypothesis that a high fat, high simple carbohydrate, low complex carbohydrate diet and/or reduced levels of physical activity increase risk of diabetes. The proportion of deaths attributed to ischemic heart disease was higher in both diabetic and nondiabetic Japanese Hawaiians than in diabetic subjects in Japan. The rates were similar for Japanese and Caucasians in Hawaii. There was no evidence of an environmental influence on the development of microangiopathy (retinopathy) in diabetes, as the prevalence of diabetic retinopathy (stratified for diabetes duration) was similar in Japanese subjects in Hawaii and in Japan, and it was similar to previous reports from England. On the other hand, diabetes alone did not appear to account for the greater prevalence of macroangiopathy in Hawaiian Japanese than in Hiroshima. Thus environmental factors, possibly including diet, appear to be involved in the development of macrovascular complications of diabetes.

Increased somatic cell mutant frequency in atomic bomb survivors.

Frequencies of mutant T-cells in peripheral blood, which are deficient in hypoxanthine guanine phosphoribosyltransferase (HPRT) activity, were determined for atomic bomb survivors by direct clonal assay using a previously reported method (Hakoda et al., 1987). Results from 30 exposed survivors (more than 1 rad exposed) and 17 age- and sex-matched controls (less than 1 rad exposed) were analyzed. The mean mutant frequency (Mf) in the exposed (5.2 X 10(-6); range 0.8-14.4 X 10(-6)) was significantly higher than in controls (3.4 X 10(-6); range 1.3-9.3 X 10(-6)), which was not attributable to a difference in non-mutant cell-cloning efficiencies between the 2 groups, which were virtually identical. An initial analysis of the data did not reveal a significant correlation between individual Mfs and individual radiation dose estimates when the latter were defined by the original, tentative estimates (T65D), even though there was a significant positive correlation of Mfs with individual frequency of lymphocytes bearing chromosome aberrations. However, reanalysis using the newer revised individual dose estimates (DS86) for 27 exposed survivors and 17 controls did reveal a significant but shallow positive correlation between T-cell Mf values and individual exposure doses. These results indicate that HPRT mutation in vivo in human T-cells could be detected in these survivors 40 years after the presumed mutational event.

Evaluation of coronary artery stenoses using electron-beam CT and multiplanar reformation.

PURPOSE We assessed the diagnostic value of electron-beam CT with multiplanar reformation for coronary artery stenoses. METHOD Thirty-seven patients who underwent conventional coronary angiography were evaluated with ECG-triggered thin section electron-beam CT with intravenous contrast enhancement. Multiplanar reformation of a stack of the images was performed to visualize coronary arteries. Two observers blind to the results of conventional coronary angiography independently evaluated the reformatted images. RESULTS The sensitivity and specificity for the detection of significant lesions were 100 and 100% in the left main coronary artery, 83 and 84% in the left anterior descending artery, 67 and 96% in left circumflex artery, 63 and 79% in the right coronary artery, and 74 and 94% for total results, respectively. All false-positive results in the left anterior descending artery were caused by wall calcification, and in the right coronary artery, 83% of the false-positive results were caused by small slice gaps in noncalcified segments. CONCLUSION Electron-beam CT was feasible for the detection of coronary artery stenoses. For interpretation of reformatted images, calcification and slice gaps should be taken into consideration.

Allelotype frequency of the thiopurine methyltransferase (TPMT) gene in Japanese.

Polymorphisms at three loci in the thiopurine methyltransferase (TPMT) gene are known to be responsible for azathioprine and 6-mercaptopurine (6MP) toxicity. Among them, only TPMT*3C variant allele with A719G mutation was found in 15/522 (2.9%; 17/1044 alleles; 1.6%) Japanese individuals including two homozygotes. The allele frequency was different from that in Caucasians, and investigation of TPMT polymorphisms with consideration of ethnic differences before administration of azathioprine or 6MP may provide clinically useful information.

Westernized food habits and concentrations of serum lipids in the Japanese.

To investigate the association of westernized food habits and concentrations of serum lipids in the Japanese, we studied 1200 healthy Japanese living in Hiroshima prefecture and 1483 ethnic Japanese from Hiroshima prefecture living in the Hawaii Islands and Los Angeles. The nutritional assessments were made by the same dietitians. No major difference was observed in the total energy intake between the Japanese and the Japanese-Americans in both males and females. However, the intake of animal fat and simple carbohydrates (especially fructose) were markedly greater, and that of complex carbohydrates lower, in the Japanese-Americans compared with the Japanese. The mean serum cholesterol (CH), LDL-CH and serum triglyceride (TG) levels were significantly higher in the Japanese-Americans in both sexes. The mean HDL-CH concentration was similar between the two groups in males, but it was approximately 7 mg/dl higher in Japanese-American females. Using the 75 percentile values of CH and TG in the Japanese in Hiroshima, the frequency of WHO types IIa and IIb hyperlipidemia was about twice as high in the Japanese-Americans. These results suggest that westernized food habits in the Japanese include qualitative changes in animal fat, simple carbohydrate and complex carbohydrate diet rather than an increase in the total energy intake. These changes are associated with marked increases in the concentrations of serum CH and TG and increased prevalence of types IIa and IIb hyperlipidemia.

The high prevalence of diabetes mellitus and hyperinsulinemia among the Japanese-Americans living in Hawaii and Los Angeles.

A comparative epidemiologic investigations was conducted among Japanese immigrants and their offspring living in Hawaii and the Los Angeles area, and among Japanese living in Hiroshima. All subjects received an oral glucose tolerance test and the diagnosis of diabetes was made on the basis of WHO criteria. In the subjects aged 40 years or older, the age, sex, and obesity-specific prevalence of diabetes was 2-3 times higher among Japanese living in America than those in Hiroshima. The fasting and post-glucose load serum immunoreactive insulin concentrations were higher in the Japanese-Americans compared to the Hiroshima inhabitants with the same degree of obesity and glucose tolerance. We suggest that a westernized lifestyle induces peripheral insulin resistance and promotes the development of diabetes among Japanese.

Cyclooxygenase‐2 (COX‐2) mRNA Expression Levels in Normal Lung Tissues and Non‐small Cell Lung Cancers

One of the cyclooxygenase (COX) isoforms, COX‐2, is over expressed in various human cancers. In this study, we examined the gene expression levels of COX‐2 in primary non‐small cell lung cancers (NSCLC), metastatic lymph nodes, and normal lung tissues. The expression levels of the COX‐2 gene were assessed by means of the reverse transcription polymerase chain reaction in 76 autopsy samples (29 primary NSCLC, 29 corresponding normal lung tissues, and 9 metastatic lymph nodes). The expression levels in NSCLC (both adenocarcinomas and squamous cell carcinomas) were significantly higher than in normal lung tissues and were significantly higher in adenocarcinomas than in squamous cell carcinomas. Differences between the levels of expression of COX‐2 in primary tumors and their corresponding metastatic lymph nodes in 9 adenocarcinoma patients were not significant. Our results indicate that COX‐2 may be associated with carcinogenesis of NSCLC, and that it may be a target for the treatment of NSCLC.

Prognostic significance of p53 and ras gene abnormalities in lung adenocarcinoma patients with stage I disease after curative resection.

We investigated the prognostic significance of p53 gene abnormalities and ras gene mutations in patients with curatively resected stage I lung adenocarciiioma. Formalin‐fixed and paraffin‐embedded tissues were obtained from 30 patients who had undergone curative resection for stage I lung adenocarciiioma. Abnormalities of the p53 gene were detected using polymcrasc chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE) analysis and immunohistochemistry and ras mutations were detected using PCR‐restriction fragment length polymorphism (RFLP) analysis. Both univariate and multivariate analyses were performed to assess the relationship between the presence of abnormalities of these genes and the patients’disease‐free survival. Eleven tumors (37%) had mutated p53 sequences and 11 (37%) showed p53 overexpression. A total of 15 tumors (50%) had p53 gene abnormalities and the concordance rate was 73%. Seven tumors (23%) showed mutated ras sequences. The univariate analysis revealed that the disease‐free survival of patients with any p53 abnormality was shorter than that of those without abnormalities (P=0.02, generalized Wilcoxon test), and survival of those with p53 protein overexpression was more significantly shorter (P=0.003, generalized Wilcoxon test). Multivariate analysis using the Cox proportional hazards model indicated that the presence of p53 abnormalities was a significantly (P=0.01) unfavorable prognostic factor. There was no significant correlation between the presence of ras mutation and survival. These results suggest that analysis of the p53 gene may be helpful for the selection of high‐risk patients for clinical trials of adjuvant therapy for stage I lung adenocarcinoma.