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A novel culture system where cancer cells are grown in the presence of nanoclay in adherent plates allows preparation of malignant and dispersed spheroids compared with those formed under ultralow-adherent conditions.

Hemoglobin vesicle (HbV), which is also called liposome-encapsulated hemoglobin, functions as a hemoglobin-based oxygen carrier and is expected to be utilized in emergency situations including hemorrhagic shock and several kinds of ischemic diseases. In the present study, we evaluated the efficacy of HbV for improving stroke-related symptoms induced by middle cerebral artery (MCA) occlusion/reperfusion and an intra-internal carotid arterial injection of arachidonic acid (AA) in rats. When HbV (10 mL/kg, i.v.) was administered to rats immediately after the MCA occlusion, it reduced the cerebral infarct volumes of the cortex and total of the cortex plus sub-cortex significantly as compared with saline as a vehicle. In AA-induced stroke model, HbV (10 mL/kg, i.v.) improved the motor dysfunction score and inhibited the increase in cerebral water content suggesting it could suppress cerebral edema. These results strongly suggest that HbV would provide a novel beneficial option for the treatment of stroke, especially acute ischemic stroke.

Two inter-related challenges must be overcome to develop a recombinant human serum albumin process. One is purity; the other is cost. Regarding cost, our goal is to produce recombinant human serum albumin at least as economically as plasma derived human serum albumin. To control production costs, maximum quantities of albumin must be produced from minimum volumes of cell culture, followed by high efficiency, high-yield purification methods. By introducing STREAMLINE technology, we have improved productivity by roughly 50% in terms of processing time and 45% in terms of yield. Furthermore, the life time of the gel and column are very long. After more than 1000 process cycles, performance remained unchanged.

The structure of recombinant human serum albumin derived from Pichia pastoris (rHSA) was analyzed in detail. Complete amino acid analysis was performed by the phenyl isothiocyanate precolumn labeling method. The amino terminal sequence was determined by the Edman degradation. The carboxyl terminal amino acid was determined by digestion with carboxypeptidase, and the carboxyl terminal peptide fragment was analyzed by electrospray mass spectrometry. The peptide fragments of rHSA digested with Lysylendopeptidase, Endoproteinase Glu-C, or Endoproteinase Asp-N were analyzed by electrospray mass spectrometry. The complete amino acid composition, the terminal sequences and the complete amino acid sequence of rHSA agreed with the primary structure deduced from its cDNA. The elution pattern of reduced and carboxymethylated rHSA digested with Lysylendopeptidase and the elution pattern of intact rHSA digested with pepsin were respectively similar to those of plasma-derived human serum albumin (pHSA). The pattern of CD spectrum of rHSA was identical in both shape and magnitude to that of pHSA. 1H-NMR spectra of rHSA and pHSA in deuterium oxide showed the same signal patterns in the observed region (delta 10.5-0.5 ppm). Cross peaks assigned to the alpha proton-beta proton of Asp-1 (delta 4.2/2.8 ppm) and the delta proton-epsilon proton of lysine residues (delta 2.8-3.2/1.4-2.0) showed the same cross peak patterns and chemical shifts in two-dimensional phase sensitive double-quantum filtered 1H-1H correlation spectra of rHSA and pHSA.

We developed enzyme immunoassays for human anti‐Pichia pastoris components (PPC) IgG and anti‐PPC IgE antibody titers. Anti‐PPC IgG antibody assay were performed using antigen‐coated plate and antihuman IgG peroxidase conjugate. The intra‐ and interassay coefficients of variation (CV) of anti‐PPC IgG antibody were 1.83–2.51% and 1.97–2.76%, respectively. The anti‐PPC IgE antibody assay was performed using an anti‐IgE monoclonal antibody‐coated plate, biotin‐labeled PPC and avidin‐labeled peroxidase, which was not subject to interference by the high titer of anti‐PPC IgG antibody. The intra‐ and interassay CV were 3.83–5.34% and 3.56–5.84%, respectively. We determined and compared anti‐PPC IgG antibody titers in the 40 normal individuals. We confirmed that a high titer of anti‐PPC IgG antibody is contained in all normal human sera and that these antibodies are directed primarily to mannan by immunoblotting analysis. The ratio of the maximum to minimum anti‐PPC IgG antibody titers in normal individuals was > 8,000. Anti‐PPC IgG antibody titers did not correlate with the age. However, we did not detect anti‐PPC IgE antibody in normal individuals. J. Clin. Lab. Anal. 11:196–201, 1997. © 1997 Wiley‐Liss, Inc.

We have investigated the effect of Fab oligomerization on imaging efficacy in a pancreatic-carcinoma xenograft model in mice. Recombinant mouse/human chimeric Fab of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A10, which has been shown to react specifically with gastrointestinal cancers, was used in this study. Fab homo-oligomers (dimers and trimers) chemically linked through disulfide bonds (S-S Fab oligomers) were prepared by linkage of chimeric Fab with N-succinimidyl-3-(2-pyridyldithio)-propionate. Oligomers with S-S bonds showed 10-fold higher binding activity against human CEA than Fab, while the binding activity of oligomers was similar to that of F(ab′)2. In mice bearing pancreatic-carcinoma xenografts, tumor uptake of S-S oligomers was significantly greater than that of monomeric Fab, while there was no difference in tumor uptake between S-S Fab trimers and F(ab′)2. S-S oligomers showed more rapid clearance rates and uniform percolation in the tumor nodules than F(ab′)2. At 24 hr after injection, S-S Fab oligomers exhibited higher tumor-to-normal-tissue specificity ratios than did F(ab′)2. At 18 hr after injection, clear scintigraphic detection of the pancreatic-carcinoma tumors was obtained with 123I-labeled S-S Fab dimers. At 24 hr, improved tumor imaging was shown for 123I-labeled S-S Fab oligomers with slightly visible uptake in normal tissues, similar to that of F(ab′)2. S-S oligomers of chimeric A10 Fab may be useful as rapid diagnostic tools of pancreatic carcinomas. © 1996 Wiley-Liss, Inc.

To elucidate the risk factors for initiating glucose intolerance, the relevant factors were explored in a cross-sectional survey conducted in a sample population aged 40–79 years old selected from a Japanese community, Hisayama, Japan in 1988. A 75-g oral glucose tolerance test was used to classify 1,073 men (72.5% of the entire population in the same age range) and 1,407 women (80.5%) into normal, impaired glucose tolerance and diabetes mellitus groups. In all age and sex groups with normal glucose tolerance, the sum of fasting and 2-h post-load insulin values varied widely and demonstrated significant positive correlations with triglycerides, body mass index, waist-hip ratio, systolic and diastolic blood pressure, while it negatively correlated to HDL cholesterol (p

In an attempt to reduce the immunogenicity of two different murine anti‐carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs), KM10 and A10, we produced recombinant mouse/human chimeric MAbs and the respective Fab fragments carrying the variable regions of the murine MAbs. Chimeric A10 Fab fragment was expressed in Escherichia coli, and produced in large quantities in a mini‐jar fermentation system. In competitive binding assays, chimeric MAbs and their Fab fragments showed identical specificity to human CEA epitopes, as compared to the parental MAbs or Fab fragments. Both chimeric Fab fragments exhibited strong immunohistochemical reactivity with various gastrointestinal carcinomas and no reactivity with CEA‐related antigens, such as NCA (nonspecific cross‐reacting antigen) or BGPI (biliary glycoprotein I). Furthermore, chimeric KM10 MAb elicited substantially higher antibody‐dependent cellular cytotoxicity than the murine MAb. Complement‐dependent cytotoxicity in vitro was much weaker with chimeric KM10 MAb. These results indicate that chimeric MAbs or Fab fragments could potentially replace the parental murine antibodies or their Fab fragments in therapy or diagnosis of human gastrointestinal carcinomas.

The authors studied mortality from major causes of death and risk factors in the elderly in a long-term prospective survey conducted in a Japanese suburban community, Hisayama. In the baseline survey in 1961, we scrutinized 1658 residents of the town aged 40 years or older accounting for 92% of the total population in this age range. Of those, 591 residents (245 men and 346 women) aged 60 years or older, who were free from major cardiovascular disease, were selected for the present study. They were followed-up for 26 years from 1961 to 1987. The average age was 67 years for men and 70 years for women, being significantly higher for women than for men. During the follow-up period, 529 subjects (89.5%) died, and 448 were autopsied (autopsy rate 84.7%). The all-cause mortality (per 1,000 person-years) after adjustment for age was 89.9 for men and 56.7 for women, the former being significantly higher than the latter (p0.01). The age-adjusted mortality from cerebrovascular disease was estimated to be 21.4 for men and 9.9 for women, i.e. 8.9 and 8.8 from heart disease, and 19.9 and 10.6 from neoplasm, and 18.1 and 12.2 from-pneumonia, respectively. There was significant sex difference in mortality from cerebrovascular disease, neoplasm and pneumonia (p0.01) but not from heart disease (p0.1). Multiple Cox's proportional hazards regression analysis showed systolic blood pressure and male sex to be significant risk factors for death by cerebrovascular disease. Systolic blood pressure was also a predictor for death by heart disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperinsulinemia has been closely associated with hypertension in several epidemiological studies, but little is known about this condition in the elderly. The authors studied the relationship between serum insulin and blood pressure levels, and the prevalence of hypertension according to insulin levels in the elderly in a Japanese rural community, Hisayama. In 1988, 75 g oral glucose tolerance test was performed on 426 male and 567 female Hisayama residents aged 60 to 79 years. Fasting and 2 hr serum insulin values were measured by radioimmunoassay. In the subjects, excluding those receiving antihypertensive drugs, the sum of fasting and 2 hr postload insulin (sigma IRI) significantly correlated with systolic (r = 0.15 and 0.25 for males and females, respectively) and diastolic blood pressure (r = 0.20, 0.16). In multiple regression analyses, the correlation with systolic blood pressure remained significant in females after controlling for age, body mass index, alcohol intake, smoking habits, serum total cholesterol, HDL-cholesterol, triglycerides, and fasting plasma glucose. In contrast, sigma IRI did not remain as an independent variable relevant to blood pressure among males. The age-adjusted prevalence rates of hypertension (> or = 160/95 mmHg or receiving drug treatment) significantly increased with increasing quartiles of sigma IRI in females. The prevalence of hypertension which was not treated with drugs also increased significantly with increasing sigma IRI in females. However, no significant association was observed in males. In conclusion, the present study suggests that hyperinsulinemia can be related to hypertension in the female elderly in the general population of Japan.

We have fused a cDNA gene encoding mature human serum albumin (HSA) to several secretory leader-encoding sequences. The hybrid genes were cloned into an episomal vector under the control of several yeast promoters and then introduced into yeast cells. The GAL1 promoter in combination with either the native HSA pre-sequence or a modified HSA pre-sequence gave the highest production of immunoreactive HSA, 90 mg/liter being reached in a shake flask culture. The invertase pre-sequence, the mating factor alpha 1 prepro-sequence, and the modified HSA pre-sequence directed accurate processing. In contrast, the chicken lysozyme pre-sequence and the native HSA pre-sequence directed incorrect processing. Episomal vectors were unstable within the host cells under non-selective culture conditions. To improve the plasmid stability, the hybrid genes were incorporated into an integrative vector. Transformants carrying multicopies of the plasmid integrated at the LEU2 locus stably secreted HSA. The highest yield of 65 mg/liter in a shake flask culture was obtained with the combination of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter and the modified HSA pre-sequence. By constructing transformed strains containing multicopies of plasmids integrated at both the chromosome LEU2 and HIS4 loci, we have obtained a stable strain that continuously secretes as much as 85 mg HSA per liter of culture medium.

To elucidate the effect of glucose intolerance on cardiovascular disease in the current Japanese population, we performed a 75-g oral glucose tolerance test in 2,427 Hisayama residents aged 40–79 years in 1988, who were free from a previous history of stroke or myocardial infarction, and followed them prospectively for 5 years. The prevalence of diabetes (NIDDM) among men was 13% and that of impaired glucose tolerance (IGT) was 20%; the corresponding values for women were 9 and 19%, respectively. The age- and sex-adjusted incidence of cerebral infarction (6.5 per 1,000 person-years, P 0.01) and coronary heart disease (5.0 per 1,000 person-years, P 0.05) was significantly higher in subjects with NIDDM than in those with normal glucose tolerance (1.9 and 1.6 per 1,000 person-years, respectively). In addition, subjects with IGT and NIDDM had a higher risk of cardiovascular disease including stroke and coronary heart disease than did those with normal glucose tolerance after adjustment for age and sex, namely the relative risk for IGT was 1.9 (95% CI 1.2–3.2), and the relative risk for NIDDM was 3.0 (95% CI 1.8–5.2). These associations remained significant even after controlling for six other risk factors including hypertension in multivariate analysis. Our data suggest that NIDDM is a significant risk factor for both cerebral infarction and coronary heart disease and also that IGT itself is a risk factor for cardiovascular disease in the general Japanese population today.

The methylotrophic yeast, Pichia pastoris, is widely used as a host strain for the production of a variety of heterologous proteins. We used P. pastoris for the production of recombinant human serum albumin (rHSA). In several runs of fed-batch fermentation, rapid degradation of rHSA was observed, coinciding with a sudden increase of protease activity in the culture broth. Monitoring the changes in the concentration of the medium components during fermentation suggested that this phenomenon was caused by nitrogen starvation. Increased initial concentrations of ammonia and phosphoric acid in the medium prevented the protease production during fermentation. Using this improved medium, stable production of rHSA of around 1.4 g/l was achieved. Although protease activity in the culture broth of the improved medium was not detected by the casein plate method at the end of fermentation, potential protease activity remained and could be activated by decreasing the pH of the culture broth, a high degradation rate of 660 mg HSA/l/h was observed at pH 4.3, but degradation did not occur above pH 5.9.

We have developed a fermentation process for recombinant human serum albumin (rHSA) production using an expression strain of the methylotrophic yeast Pi-chia pastoris. The high productivity of the process enables it to compete with the production of plasma derived HSA. After purification of the rHSA, the content of yeast derived contaminants was less than 1 ng/250 mg of rHSA. The results from structural analyses suggested that purified rHSA possessed an identical conformation to plasma derived HSA. Furthermore, no neoantigenicity different from that of plasma derived HSA was observed. The efficacy and safety of rHSA were tested in clinical studies, and it was shown that there was no difference between rHSA and plasma derived HSA in a comparison study. The high efficacy of rHSA with little or no adverse reaction was confirmed in these studies.

We analyzed and compared the physicochemical and immunochemical properties of recombinant human serum albumin (rHSA) from Pichia pastoris with those of plasma-derived human serum albumin (pHSA). The second virial coefficient of rHSA, obtained from colloid osmotic pressure measurements at pH 6.7 +/- 0.1 was not significantly different from that of pHSA (P > 0.05). A 25% rHSA solution exhibited Newtonian flow, and the viscosity of 25% rHSA at 20 +/- 0.02 degrees C was not significantly different from that of 25% pHSA (P > 0.05). We analyzed the long- and medium-chain fatty acid composition of rHSA by reverse-phase HPLC using 9-anthryldiazomethane as the fluorescent labeling reagent. The total amount of fatty acid was higher for pHSA than for rHSA. The fatty acid composition of the rHSA preparation was the same as that of the pHSA preparation. However, the amounts of palmitic acid (C16:0) and stearic acid (C18:0) in rHSA were much lower than those in pHSA. Interestingly, we found that P. pastoris produced linolenic acid (C18:3) because it was detected in rHSA. The immunochemical properties of rHSA were analyzed by a parallel line assay method using anti-pHSA polyclonal antibody, and were identical to those of pHSA (P > 0.05).

We have developed a recombinant human serum albumin (rHSA) from Pichia pastoris which expresses high levels of heterologous proteins. rHSA is used clinically in high concentration (approximately 250 mg/ml in a 50 mL vial). We had to consider not only proteins from host cells as impurities but also mannan, which exhibits harmful effects on humans. However, the analysis of mannan in biopharmaceuticals produced from yeast has not been reported. Contaminating mannans in the final product were one important index to assess the clinical safety of rHSA. We have developed a highly sensitive enzyme immunoassay (EIA), utilizing an avidin-biotin system, for the detection of either the protein or mannan polysaccharide components from P. pastoris components (PPC) in rHSA. In addition, we used anion exchange chromatography with pulsed amperometric detection (AE-PAD) for monosaccharide analysis of glycoconjugates for the detection of mannan from PPC in rHSA. The detection limits of the EIA for PPC (PPC EIA) and the AE-PAD were 1 ng of protein/250 mg of rHSA and 180 ng of mannose/mg of rHSA, respectively. The mannan content in partially purified rHSA as determined by the AE-PAD was about same as the PPC content as determined by the PPC EIA. We showed that the PPC EIA and the AE-PAD are useful methods for the purity analysis of biopharmaceuticals produced from yeast.

In the case where the supply of material is limited from natural resources and/or risks of infection are to be avoided, recombinant proteins are an important substitute. Consequently, the physicochemical characterization of the primary and tertiary structures of such materials that are to be used clinically is indispensable. In this context, disulfide linkages play a significant structural role and their determination is of paramount importance. As the demand for human serum albumin (HSA), which contains 35 cysteine residues, is continually increasing, its industrial-scale production from the genetically engineered yeast Pichia pastoris is of interest. The present paper describes a methodology that allows the characterization of the multi-disulfide linkages including exact positions in purified recombinant HSA by use of gas-phase protein sequencing. Mild Edman degradation followed by isocratic analysis of the phenylthiohydantoin amino acids in combination with multienzymatic digestions in acidic conditions allowed the exact positions of the 17 disulfide bridges and 1 sulfhydryl group to be rigorously determined. The sulfhydryl content of the present recombinant HSA was the same as plasma HSA.

The ligand binding properties and esterase-like activity of recombinant human serum albumin (rHSA) expressed by Pichia pastoris were compared with those of plasma derived human albumin (pHSA). The binding of long fatty acid ions was determined by the equilibrium partition method using radiolabeled palmitate. The association constants and the number of binding sites of diazepam, salicylate and warfarin were determined by specific and nonspecific binding models. The high affinity binding of bilirubin was kinetically determined from the oxidation rate of free bililubin in the binding mixture. The binding parameters of these five ligands obtained with rHSA were within the same range observed with pHSA preparations. The kinetic parameters for hydrolytic activity of rHSA toward p-nitrophenyl acetate was also similar to pHSA. These results indicate that rHSA and pHSA have the same functional property.

To obtain a relatively true mortality from malignant neoplasms, we studied the frequency of cancers in the different sites and the changing patterns of the frequency and sites over time among residents of the community of Hisayama, where an autopsy-based population survey (autopsy rate, 80%) has been conducted since 1961. During the 30-year period from 1962 to 1991, we found 438 malignant neoplasms in 407 cases among 1,250 consecutive autopsies. Stomach cancer was most frequent in type of cancer, with 123 cases (9.8%), followed by lung cancer in 62 (5.0%), colorectal cancer in 42 (3.4%), liver cancer in 37 (3.0%), and pancreatic cancer in 30 (2.4%). We compared the mortality from cancers for both autopsy and nonautopsy cases (the proportional mortality) among three 10-year periods, which included early (1962 to 1971), middle (1972 to 1981), and late (1982 to 1991) periods. The proportional mortality from all cancers, as well as for lung, colorectal, and liver cancers, showed an increase in recent years, while stomach and pancreatic cancer showed a decrease. These figures were nearly similar to the mortality statistics for the Japanese population as a whole except for the observed decreasing trend in mortality from pancreatic cancer.

We purified a 58 kDa serine protease from culture-supernatant of Pichia pastoris and found that the NH2-terminal amino acid sequence of this protease is closely homologous to that of mature protein of Saccharomyces cerevisiae carboxypeptidase Y (CPY), which is encoded by the PRC1 gene. Using the S. cerevisiae PRC1 gene as a hybridization probe, a cross-hybridizing fragment of P. pastoris genomic DNA was identified and the gene, PRC1, encoding CPY, was cloned. The open reading frame of the P. pastoris PRC1 gene consists of 1569 bp encoding a protein of 523 amino acids. The molecular mass of the protein is calculated to be 59.44 kDa without sugar chains. The protein comprises 20 amino acids of pre (signal)-peptide, 87 amino acids of pro-peptide and 416 amino acids of mature peptide, and has four N-glycosylation sites. The NH2-terminal amino acid sequence of mature peptide is completely identical with that of the protease purified from the culture-supernatant. There is 61% identity between the amino acid sequences of P. pastoris Prc1p and S. cerevisiae Prc1p. Chromosomal disruption of the PRC1 gene resulted in the loss of CPY activity. Over-expression of the PRC1 gene under regulation of the P. pastoris AOX1 promoter resulted in accumulation of a large amount of active CPY in the intracellular fraction, and secretion of a slightly larger molecule that is thought to be pro-CPY. The nucleotide sequence data reported in this paper will appear in the EMBL Nucleotide Sequence Databases under the Accession Number X87987.

We followed 828 nondemented residents of Hisayama Town, Kyushu, Japan, aged 65 years or older (88.3% of the elderly population) for 7 years starting in 1985 in order to determine the type-specific incidence of dementia and its risk factors in the general Japanese population. Only two subjects were lost to the follow-up, during which period 103 subjects developed dementia. Morphologic examination of the brains of 89 subjects (86.4%) was made by autopsy or CT. We made the initial diagnosis of dementia based on the DSM-III-R criteria, with the diagnoses of vascular dementia (VD) being based on the NINDS-AIREN criteria and Alzheimer's disease (AD) on the NINCDS-ADRDA criteria. The incidence of VD and AD increased with age for both sexes. The age-adjusted total incidence (per 1,000 person-years) of dementia was 19.3 for men and 20.9 for women. The corresponding rates for VD were 12.2 for men and 9.0 for women, and for AD, 5.1 for men and 10.9 for women. Among the VD subjects whose brain morphology we examined, the most frequent type of stroke was multiple lacunar infarcts (42%), but half these subjects lacked a stroke episode in their histories. Multivariate analysis showed that age, prior stroke episodes, systolic blood pressure, and alcohol consumption were significant independent risk factors for the occurrence of VD. In contrast, age and a low score on Hasegawa's dementia scale were significant risk factors for AD, and physical activity was a significant preventive factor for AD.(ABSTRACT TRUNCATED AT 250 WORDS)

The methylotrophic yeast Pichia pastoris has two alcohol oxidase genes, AOX1 and AOX2. The AOX2 gene is transcribed at a much lower level than the AOX1 gene. Apart from this difference in expression levels, the two genes are regulated similarly. To study the role of cis-acting elements in the promoter region of the AOX2 gene, we constructed expression plasmids in which the human serum albumin (HSA) gene was placed under the control of various deleted or mutated AOX2 promoter derivatives. By analyzing the expression of HSA in P. pastoris transformants, we have identified three cis-acting regulatory elements in the AOX2 promoter. The positive cis-acting element AOX2-UAS, located between positions −337 and −313 (relative to the transcription initiation codon), is required for response to transcriptional induction by methanol in an orientation-independent manner, and artificial amplification of the AOX2-UAS resulted in an increase in the transcriptional activity of the promoter. A sequence homologous to AOX2-UAS was also found in the AOX1 promoter, and in methanol-regulated promoters in other methylotrophic yeast. Two negative cis-acting elements, AOX2-URS1 and AOX2-URS2 play a role in repressing transcription from the AOX2 promoter. The function of AOX2-UAS is completely repressed by this unique repression system when both the AOX2-URS1 and AOX2-URS2 are functional.

We compared type-specific prevalences of dementia between two surveys of Hisayama residentsor = 65 years old conducted in 1985 and 1992. The overall age-adjusted prevalence was greatly decreased from 5.4% in 1985 to 3.3% in 1992 for men, while it was changed little from 7.5 to 6.3% for women. The prevalence of vascular dementia (VD) decreased during the intervening 7 years in men but not in women. The prevalence of Alzheimer's type (SDAT) was the same in either sex. The ratio of VD to SDAT prevalence was 1.8 in 1985 but fell to 1.1 in 1992. The background factors responsible for this change are discussed.

We determined the population-based prevalence of diabetes mellitus in members of the Japanese community, Hisayama aged 40–79 years old by a 75-g oral glucose tolerance test. The basic population used to calculate diabetic prevalence was 1,077 men (72.8% of the whole population in the same age range) and 1,413 women (80.8%) including ten diabetic patients on insulin therapy. In addition, we compared the prevalence of history of diabetes which was acquired by interview or questionnaire, between participants and non-participants in the 75-g oral glucose tolerance test, but they were not statistically different. The age-adjusted prevalence of diabetes to world population was 12.7% for men and 8.4% for women, and that of impaired glucose tolerance was 19.6% for men and 18.4% for women. These figures were much higher than those previously reported from several Japanese communities. The results obtained from the present study could reveal true prevalence of diabetes among the Japanese population. In addition, the reasons for the increasing prevalence of diabetes among the recent Japanese population are also discussed.

We tested radiolocalization of pancreatic carcinoma xenografts in nude mice with recombinant mouse/human chimeric Fab fragment (chimeric Fab) of A10 anti-CEA monoclonal antibody (MAb). When compared to the parental A10 MAb, we obtained higher tumor to normal tissue labeling ratios and clearer tumor detection using chimeric Fab. No significant difference in tumor to normal tissue ratio was observed between the chimeric Fab and murine Fab fragment prepared from the A10 MAb. Our results indicate that chimeric A10 Fab may be a potential candidate for localization of pancreatic carcinoma studies.

The causes of death for the elderly were prospectively studied in Hisayama, Japan, a rural community. We compared 1,621 subjects, aged 40 years or over, recruited in 1961, and 2,053 subjects recruited in 1974. Each cohort was studied in a follow-up that lasted 10 years; they had autopsy rates of 82.1% and 86.1% during each 10-year period, respectively. The most common causes of death for those aged 70 years or over were cerebrovascular disease, malignant neoplasms, and pneumonia. Deaths due to cerebrovascular disease tended to decrease in the recent cohort, but the proportion of decline was more prominent in cases aged 40 to 69 years. There was a sex difference in the changing pattern of mortality from heart diseases including ischemic heart disease. Deaths by both heart diseases and ischemic heart disease increased in the more recent cohort of aged women, whereas they decreased in the aged men. Pneumonia was an important cause of death for the elderly in both cohorts. Deaths due to "senility" were rare, being only 1% of the deceased aged 70 or over. With prolonged lifespan, especially for women, the impact of atherosclerosis and its related disorders on the recent Japanese aged population appears to have increased.

OBJECTIVES To study the relationship between serum insulin and blood pressure, as well as the prevalence of hypertension according to the insulin level in a general Japanese population. DESIGN In 1988 a cross-sectional community survey was conducted among Hisayama residents aged 40-79 years. METHODS A total of 1073 males and 1407 females (72.5 and 80.5% of the total population, respectively) underwent comprehensive investigation, including a 75-g oral glucose-tolerance test. Fasting and 2-h serum insulin levels were measured by radioimmunoassay. RESULTS The sum of the fasting and 2-h postloading insulin levels was significantly correlated with the systolic blood pressure (SBP; r = 0.18 and 0.26 for males and females, respectively) and the diastolic blood pressure (DBP; r = 0.24 and 0.19, respectively) in the subjects not receiving antihypertensive drugs. In multiple regression analysis the correlation with blood pressure remained significant in both sexes even after controlling for age, body mass index, alcohol intake, smoking, a family history of hypertension, serum total cholesterol and fasting plasma glucose. The age- and sex-adjusted prevalence of hypertension (SBP > or = 160 mmHg or DBP > or = 95 mmHg, or both, or receiving drug treatment) increased significantly with an increase in the sum of fasting and 2-h postload insulin levels in both the non-obese subjects (body mass index or = 25 kg/m2). Multiple logistic regression showed that the sum of fasting and 2-h postload insulin levels was a significant factor with an independent relationship to hypertension, even after taking the other risk factors into account. CONCLUSION The present study suggests that hyperinsulinaemia is related to hypertension in a general Japanese population.

0.6%のD, L-メチオニンを添加した16%カゼイン食に5mg/100gのエリタデニンを投与すると, イ) 炭水化物源がショ糖であってもαデンプンであっても, ロ) コレステロール+コール酸ソーダの並食の有無にかかわらず, ハ) 飼育期間の長短にかかわらず, ラットの血漿脂質 (総および遊離コレステロール, 燐脂質) が有意に低下したが, 同時に肝グリセリドが著しく増大し, 19%を越える脂肪肝の生成が見られた。

The mechanism of the anti-allergic activity of carboxamidemethylated Fc (CM-Fc) was examined in vitro. CM-Fc had no effect on the proliferation of lymphocytes induced by concanavalin A (Con A), lipopolysaccharide (LPS), T cell-dependent mitogen or T cell-independent mitogen.CM-Fc inhibited the activation of glycogen-stimulated guinea pig macrophages by FMLP and A23187, but not by phorbol 12-myristate 13-acetate (PMA).

A large-scale purification method for hepatitis B surface antigen produced in a recombinant yeast (Saccharomyces cerevisiae) was established. The resulting HBsAg was greater than 99% pure and suitable for vaccine use. The yeast-derived HBsAg was structurally and biochemically similar to plasma-derived HBsAg. The anti-HBs antibody producing potency of the yeast-derived vaccine in mice was significantly higher than that of the plasma-derived vaccine. The yeast-derived vaccine induced protective antibody against hepatitis B virus of either adr or ayw subtype in a chimpanzee efficacy study. These observations demonstrate the usefulness of the yeast-derived vaccine as a second-generation hepatitis B vaccine.

Two series of bis [2- (E-2-alkenoylamino) ethyl] disulfides (compds. I) and 2- (E-alkenoyl-amino) ethyl carbamoylmethyl sulfides (compds. II) were synthesized from 2-trans unsaturated fatty acids (C6 : 1-C14 : 1) and cystamine, and their effects on carrageenin-induced paw edema were studied. Several of compds. I and II showed significant anti-edema activity at a dose of 25 mg/kg, i. p. These derivatives were much more potent than the starting materials. The alkene chain length influenced the activity. The compounds (compds. I-3, I-8, II-3 and II-8) with C8 : 1 and C13 : 1 fatty acids showed the highest activity in both series. Compounds I-3 and II-3 with the C8 : 1 fatty acid were also effective when administered orally at a dose of 10 mg/kg.

A metallothionein isoform metallothionein-II was isolated from the livers of zinc acetate-treated rats. Metallothionein-II, which showed a single band on polyacrylamide gel electrophoresis, was subjected to two kinds of anti-ulcer screening systems. It was shown that intravenously administered metallothionein-II suppressed the formation of rat water-immersion stress- and HCl-ethanol-induced gastric ulcer significantly. The effect may partly be derived from the zinc contained in the metallothionein-II. However, the effect of metallothionein-II was much stronger than that of an equivalent mole of zinc. Apparently, metallothionein-II had an anti-ulcerogenic activity not based on the effect of intrinsic zinc.

Immunoglobulin G from human serum was digested with papain to give an Fc fragment, which was carboxamidemethylated [carboxamidemethylated Fc (CM-Fc), M.W. about 25000].Types I, III and IV allergic reactions were significantly depressed by the administration of CM-Fc.

From spinach leaves a green particulate fraction was obtained which consumed oxygen and took up inorganic phosphate in the presence of the Krebs cycle intermediates in a similar manner as particles obtained from animal and etiolated plant tissues. This fraction was mainly composed of green particles similar in properties to the mitochondria from other sources. From these results, it was concluded that chloroplast fragments had the activity of oxidative phosphorylation.

The effect of eritadenine, a hypocholesterolemic factor ef Shiitake mushroom Lentinus edodes, and excess methionine on the lipid contents of rat liver was investigated and the following results were obtained.1) Addition of eritadenine and more than 0.45% methionine to the diet containing 18% casein decreased the plasma total cholesterol level, but total lipid in the liver exceeded 20%.2) Addition of 8mg% eritadenine and up to 2.4% methionine to CLEA CE-2 diet did not affect the liver lipid level, but the plasma total cholesterol level increased slightly but significantly.

Colored food extracts such as soy sauce were extracted with phenol saturated with water removing most of the colored materials. Nucleotides remained in the aqueous phase. The preliminary decolorization was proposed for the determination of 5'-IMP and 5'-GMP in colored foods.

The efficacy and safety of a recombinant yeast-derived hepatitis B vaccine were evaluated in 209 subjects after three administrations at 0, 4 and 20 weeks. Subjects were divided into four groups given 5 micrograms vaccine subcutaneously, 10 micrograms subcutaneously, 10 micrograms intramuscularly and 20 micrograms subcutaneously to define the effective dose and to compare the effect of administration. Seroconversion of the antibody to hepatitis B surface antigen after the third vaccination reached 96.6% in the group given 5 micrograms vaccine subcutaneously and 100% in the other groups. The final geometric mean antibody titres were 700 IU/l in subjects given 5 micrograms subcutaneously, 2004 IU/l in those given 10 micrograms subcutaneously, 4674 IU/l in those given 10 micrograms intramuscularly and 3342 IU/l in those given 20 micrograms subcutaneously. In the groups given 10 micrograms, the early seroconversion rate of the antibody to hepatitis B surface antigen and the geometric mean antibody titres after the third vaccination were significantly higher in subjects administered intramuscularly than subcutaneously (P less than 0.05). No major adverse effects were observed and minor reactions were the same as, or less than, those reported for the plasma-derived vaccine. Before and after administration, no significant fluctuation in the yeast antibody titre was observed. These results demonstrate the efficacy and safety of the yeast-derived vaccine, and show that 10 micrograms was the effective dose.

Twenty-six first episodes of subarachnoid hemorrhage occurred among 1,621 Hisayama residents aged greater than or equal to 40 years during the 22-year follow-up of a prospective study. Subarachnoid hemorrhage was confirmed by both clinical and autopsy findings. The average annual incidence (96.1/100,000 population) was 3-13 times higher than any previously reported and steeply increased with age in both sexes, being 2.3 times higher for women than for men after adjusting for age. Nine patients (35%) died less than or equal to 8 hours after the onset of subarachnoid hemorrhage. None was correctly diagnosed on the death certificates, and four of the nine (44%) were misdiagnosed as intracerebral hemorrhage. We found the survival rate of patients suffering subarachnoid hemorrhage to be much lower than previously reported because we detected a large number of sudden deaths due to subarachnoid hemorrhage through the high rate of autopsy in our cohort (81.4%).