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Biomedical subjects

I Ohsawa

Publications and source records attributed to I Ohsawa.

At least 55 records · Page 3Linked to original sources
PubMed1997

The amino-terminal region of amyloid precursor protein is responsible for neurite outgrowth in rat neocortical explant culture.

We have previously shown that secreted forms of beta-amyloid precursor protein (APP(s)s) promote neurite outgrowth in embryonic rat neocortical explant culture. To determine the region of APP(s) responsible for its biological activity, we produced both amino- and carboxyl-terminal regions of APP(s) using a yeast expression system. The purified fragment corresponding to the amino-terminal region (NAPP) enhanced neurite outgrowth of neocortical explants, but the carboxyl-terminal region fragment did not. The neurite-promoting activity of full length APP(s) and NAPP was blocked by the antibody, 22C11, specific for the amino-terminal region, and the 16-mer peptide of epitope for 22C11 also enhanced neurite outgrowth. However, the 17-mer peptide which contains RERMS sequence did not enhance the neurite outgrowth, but promoted the survival of neocortical neurons in dissociated culture. These findings suggested that the amino-terminal region is responsible for the neurite-promoting activity of APP(s)s.

Amyloid beta-Protein Precursor↗
PubMed1997

Specific inhibitor for prolyl endopeptidase suppresses the generation of amyloid beta protein in NG108-15 cells.

A potent and specific prolyl endopeptidase inhibitor, JTP-4819, was used to investigate the role of prolyl endopeptidase in the generation of amyloid beta protein (A beta) from APP by NG108-15 cells. Synthetic substrates, 7-(succinyl-Ile-Ala)-4-methylcoumarinamide and Z(Val-Lys-Met)-4-methylcoumarinamide, respectively, corresponding to the C-terminal and N-terminal portions of A beta, were cleaved by NG108-15 cell lysates. Cleavage of the C-terminal portion, but not the N-terminal, was inhibited by JTP-4819 (IC50 = 0.6 nM). Western blot analysis showed that the A beta level in the culture medium of NG108-15 cells was increased by serum deprivation. JTP-4819 caused concentration (>10(-9) M)- and time-dependent inhibition of this serum deprivation-induced increase of A beta without having any effect on the level of the secretory form of APP. Using both specific anti-A beta (1-40) and anti-A beta (1-42) antisera, the A beta that increased with serum deprivation was confirmed to be A beta (1-40), suggesting that it might be produced by conversion of A beta (1-42) to A beta (1-40). These findings indicate that prolyl endopeptidase may be a key enzyme in the production of A beta by NG108-15 cells and that A beta secretion can be modulated by a prolyl endopeptidase inhibitor.

Amyloid beta-Peptides↗
PubMed1996

Effect of C-peptide administration on whole body glucose utilization in STZ-induced diabetic rats.

Recent studies suggest that C-peptide stimulates glucose transport in isolated skeletal muscle. In order to determine the effect of C-peptide on whole body glucose utilization, streptozotocin (60 mg kg-1) (STZ)-induced diabetic and normal rats were studied using the euglycaemic clamp procedure and continuous infusion of somatostatin (1.0 micrograms kg-1 min-1) in pentobarbital-anaesthetized rats. Plasma insulin levels during the 6.0- and 30.0-mU kg-1 min-1 insulin infusions rose to 70-90 microU mL-1 and 500-700 microU mL-1, respectively. Blood glucose concentrations were clamped at 7.5-7.9 mmol L-1 in the diabetic rats and at basal levels or 7.7 mmol L-1 in the non-diabetic (normal) rats. Biosynthetic human C-peptide (0.5 nmol kg-1 min-1) was infused in 12 diabetic and 11 normal rats, resulting in concentrations of 26-41 nmol L-1. The metabolic clearance rate of glucose (MCR) for the diabetic rats receiving C-peptide (12.0 +/- 1.0 mL kg-1 min-1) was significantly (P < 0.01) higher than that in the diabetic rats given saline (6.3 +/- 0.7 mL kg-1 min-1) or a randomly scrambled C-peptide (7.8 +/- 1.3 mL kg-1 min-1) at low-dose insulin infusion but not at the high-dose insulin infusion. In normal rats C-peptide did not significantly increase the MCR for glucose. These results thus demonstrate that C-peptide has the capacity to increase glucose utilization in STZ-induced diabetic rats.

Animals↗
PubMed1996

Exercise training prevents maturation-induced decrease in insulin sensitivity.

We examined the effects of exercise training initiated before maturation or after maturation on insulin sensitivity and glucose transporter GLUT-4 content in membrane fractions of skeletal muscle. Female Wistar rats (4 wk of age) were divided into sedentary and exercise-trained groups. At 12 wk of age, a subset of the trained animals (Tr) was killed along with a subset of sedentary controls (Sed). One-half of the remaining sedentary animals remained sedentary (Sed-Sed) while the other half began exercise training (Sed-Tr). The remaining rats in the original trained group continued to train (Tr-Tr). Euglycemic clamp (insulin infusion rate at 6 mU.kg body wt-1. min-1) was performed at 4, 12, and 27 wk. After euglycemic clamp in all animals except the 4-wk-old, hindlimb (gastrocnemius and part of quadriceps) muscles were removed for preparation of membrane fractions. In sedentary rats, glucose infusion rate (GIR) during euglycemic clamp was decreased from 15.9 mg.kg-1.min-1 at 4 wk of age to 9.8 mg.kg-1.min-1 at 12 wk of age and 9.1 mg.kg-1.min-1 at 27 wk of age. In exercise-trained rats, the GIR was not significantly decreased by maturation (at 12 wk) and further aging (at 27 wk). Initiation of exercise after maturation restored the GIR at 27 wk of age to the same levels as these for the corresponding exercise-trained rats. GLUT-4 content in plasma and intracellular membrane fractions of hindlimb muscle obtained just after euglycemic clamp showed the same trend as the results of GIR. These results suggest that exercise training prevented the maturation-induced decrease in insulin sensitivity. Improvement of insulin sensitivity caused by exercise training was attributed, at least in part, to the increase in insulin-sensitive GLUT-4 on the plasma membrane in skeletal muscle.

Age Factors↗
PubMed1996

Elevation of plasma thrombomodulin level in primary glomerulonephritis with heavy proteinuria.

Thrombomodulin (TM) is a thrombin receptor found on endothelial cells. TM acts as a cofactor for the thrombin-catabolized activation of protein C and protects these cells from the formation of thrombi. We hypothesized that the soluble form of TM which reflects the damage of the endothelial cells and that simultaneously, soluble TM will be affected by renal excretion because a significant positive correlation has been found between soluble TM and serum creatinine (sCr) in the renal failure state. However, there have been no reports on the relationship between plasma TM and clinical parameters except for sCr in primary glomerulonephritis (PGN). Plasma TM (pTM) and urinary TM (uTM) were measured in 107 patients with PGN using a one-step sandwich enzyme immunoassay. These values were assessed together with other laboratory and histological findings. We were able to divide all subjects into two groups: an sCr-dependent group whose sCr level was over 1.2 mg/dl and an sCr-independent group whose sCr level was under 1.2 mg/dl. In the sCr-independent group, patients who suffered from nephrotic syndrome (NS) had a much higher pTM level than patients who did not suffer from NS. Histological findings and other parameters were not correlated with pTM or uTM. Therefore, the two patients who suffered from NS with very high pTM levels and normal sCr level at the time of admission exhibited a decrease in their pTM value as their condition improved. We concluded that in our patients, pTM was affected not only by renal excretion of TM, but also by renal damage with heavy proteinuria and may have been associated with an ongoing disease process in PGN.

Adolescent↗
PubMed1995

Expression, purification, and neurotrophic activity of amyloid precursor protein-secreted forms produced by yeast.

The secreted form of amyloid precursor protein (APPs) including most of the extracellular domain of APP is released from the cell surface, suggesting physiological significance of APPs in vivo. We used the methylotrophic yeast Pichia pastoris as a host system for the production of recombinant APPs (rAPPs). Two rAPPss derived from isoforms of APP (APP695 and APP770) were secreted into the culture medium from the yeast, which carried cDNA encoding the N-terminal portion of APP under the control of a P. pastoris alcohol oxidase promoter. Like APPss produced by the transfected COS-1 cells, the purified rAPPss from yeast were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants. These rAPPss could be valuable tools for investigating the biological functions of APPss.

Amino Acid Sequence↗
PubMed1995

A clinical survey on the compliance of exercise therapy for diabetic outpatients.

To clarify the present state of exercise therapy for diabetes mellitus, we conducted a survey of 570 diabetic outpatients by written questionnaires. The results revealed that approximately 30% of the patients did not implement the prescribed exercise regimen. The principal reasons for low compliance were 'lack of time to do' and 'lack of mind to do'. 'Lack of time to do' was particularly numerous in male patients. Other reasons were 'lack of guidance by physician', 'lack of interest', 'lack of understanding of procedure'. These findings indicated that the patients should be motivated adequately when they are diagnosed as having diabetes and subsequently encouraged to reinforce their intentions by the physician and co-medical staff. Furthermore, they showed that specific approaches suitable for each individual patient and not standardized guidelines should be devised. As regards the daily environment of patients, the cooperation of the patient's family would be effective and the exercise facilities and the exercise guidance personnel should be augmented.

Diabetes Mellitus↗
PubMed1995

Effect of daily voluntary running on in vivo insulin action in rat skeletal muscle and adipose tissue as determined by the microdialysis technique.

The effect of physical training on in vivo insulin-stimulated glucose utilization in relation to glycolysis (lactate formation) in rat peripheral tissues was investigated in 8 sedentary controls (SC) and 7 voluntary running rats (VR). We used a sequential euglycemic clamp procedure (insulin infusion rate; 6.0, 30.0 mU/kg.min) in combination with a microdialysis technique in M. quadriceps femoris, vastus lateralis, and inguinal adipose tissue. In the clamp study, glucose infusion rate (GIR) averaged over 45-75 min during the 6.0-mU/kg.min insulin infusion was significantly (p < 0.01) higher in VR (15.36 +/- 0.83 mg/kg.min, mean +/- SE) than in SC (10.41 +/- 0.88 mg/kg.min), and the lack of a significant difference in GIR between VR and SC was found during the 30.0-mU/kg.min insulin infusion. In these tissues, there was no significant difference in dialysate lactate levels between VR and SC in the basal state without insulin or glucose infusion, or at an insulin infusion rate of 30.0 mU/kg.min. However, dialysate lactate concentrations in muscle averaged over 45-75 min during the 6.0-mU/kg.min insulin clamp procedure in VR (8.51 +/- 0.71 mg/dl) were significantly (p < 0.05) higher than in SC (6.18 +/- 0.48 mg/dl). These results indicated that insulin action in skeletal muscle and adipose tissue could be evaluated in vivo by using the microdialysis technique, and that an increase in GIR in VR was, in part, explained by an increase in lactate formation in skeletal muscle.

Adipose Tissue↗
PubMed1993

Effects of adrenodemedullation on in vivo insulin-stimulated glucose utilization in relation to glycolysis in rat peripheral tissue.

The effect of adrenodemedullation (ADMX) on insulin action was examined in anesthetized rats by means of a three-step euglycemic clamp procedure (insulin infusion rate: 0, 6.0 and 30.0 mU.kgBW-1.min-1) combined with a microdialysis technique in skeletal muscle and adipose tissue. The dialysate lactate levels in the above tissues increased in parallel with the plasma lactate levels during the sequential euglycemic clamp. In the euglycemic clamp, the glucose infusion rate (GIR) was significantly (P < 0.01) higher in ADMX rats (13.41 +/- 0.82 mg.kgBW-1.min-1) than in SHAM rats (10.21 +/- 0.87 mg.kgBW-1.min-1) during the 6.0-mU.kgBW-1.min-1 insulin infusion, and the lack of a significant difference between ADMX and SHAM rats was observed during the 30-mU.kgBW-1.min-1 insulin infusion. In skeletal muscle, the concentration of lactate in dialysate was significantly higher in ADMX rats (9.29 +/- 1.01 mg/dl) than in SHAM rats (6.22 +/- 0.47 mg/dl) (P < 0.05) at an insulin infusion rate of 6.0mU.kgBW-1.min-1. In adipose tissue, no significant difference in dialysate lactate levels was found between ADMX and SHAM rats at any insulin infusion rate. These results suggest that 1) it is possible to determine insulin action in skeletal muscle and adipose tissue in vivo by using the microdialysis technique, that 2) ADMX appears to result in a significant increase in insulin sensitivity, and that 3) lactate formation increased in skeletal muscle, but not in adipose tissue.

Adipose Tissue↗
PubMed1991

Isolation of Bacillus sphaericus biotin synthesis control mutants: evidence for transcriptional regulation of bio genes.

The genes involved in biotin synthesis have recently been isolated from Bacillus sphaericus [Gloeckler et al., Gene 87 (1990) 63-70]. Sequence analysis revealed that they are organized into two gene clusters, designated bioXWF and bioDAYB. The 5'-noncoding region of the bioD locus fused to the xylE reporter gene was inserted into the Gram-positive pUB110 replicon and the resulting plasmid was introduced into B. sphaericus IFO3525. Transformants expressed the xylE gene only if the biotin concentration in the growth medium remained below 50 ng/ml. After mutagenesis, colonies were screened for their ability to express the chromogenic marker in the presence of an excess of biotin. Most of these mutants escaped biotin repression of xylE gene expression. Classical genetic analysis showed they formed two main categories: chromosomal mutations, pleiotropically acting in trans on both bioXWF and bioDAYB 5'-noncoding regions, in which a 15-bp region common to both promoters represented a hot-spot for the second class of plasmid-associated mutations. These results, completed by the identification of transcription start points for the bioD and bioX genes, strongly suggest that this 15-bp sequence overlaps the site of a biotin-mediated negative regulation circuit controlling the transcription of the bio genes.

Amino Acid Sequence↗
PubMed1991

Effects of training and training cessation on insulin action.

Physical training has been shown to improve glucose tolerance and insulin action. In the present study, insulin action was determined using the euglycemic clamp technique in six trained male athletes compared with six untrained controls matched by age, sex, and weight at 14, 38, and 86 hours and at 6 days after cessation of exercise. The rate of insulin-mediated glucose uptake (glucose disposal) was 9.40 +/- 0.46 mg.kg-1.min-1 (mean +/- SEM) for the athletes at 14 h after the last exercise bout, compared with 6.80 +/- 0.86 mg.kg-1.min-1 obtained for the untrained controls (p less than 0.01). Glucose disposal was gradually decreased to 7.78 +/- 0.87 mg.kg-1.min-1 at 38 h, 6.82 +/- 0.49 mg.kg-1.min-1 at 86 h and to 7.11 +/- 1.00 mg.kg-1.min-1 at 6 days after cessation of physical training. At 38 h, 86 h, and 6 days of detraining, glucose disposal exhibited by training athletes did not differ significantly from untrained controls. These results suggest that physical training increases insulin action, and that this effect could be reversed to the control levels within 38 h after detraining.

Adolescent↗
PubMed1990

Cloning and characterization of the Bacillus sphaericus genes controlling the bioconversion of pimelate into dethiobiotin.

Using 8.8 kb of genetic information from Bacillus sphaericus, it was possible to confer to Escherichia coli bio- strains, including delta bioA-D, bioC-, bioH-, the ability to convert exogenous pimelate into biotin. The bio genes were borne on two recombinant plasmids with inserts of 4.3 kb and 4.5 kb, which had been isolated from a genomic bank of HindIII-digested B. sphaericus DNA, by phenotypic complementation of various E. coli bio mutants. The B. sphaericus bioD and bioA genes were unambiguously identified within the 4.3-kb insert and shown to be closely linked to bioY (coding for a protein with a presently unknown function) and to bioB [Ohsawa et al., Gene 80 (1989) 39-48]. These genes are clustered in the order bioDAYB. The 4.5-kb fragment contains genetic information for three different proteins, the products of bioX, bioW and bioF. Complementation studies using an E. coli bioF mutant and a B. subtilis bio112TG3 strain, revealed that the third ORF of this cluster encodes 7-keto-8-aminopelargonic acid synthetase. A combination of bioW and bioF allows an efficient complementation of E. coli bioC and bioH mutants, provided that pimelate is added to the biotin-depleted growth medium. No function could be identified for the product of bioX. The gene order of this cluster is bioXWF. By sequence analysis, the two cloned DNA fragments were shown to bear overlapping open reading frames and secondary structures at their 3' ends, typical of transcription terminators.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus↗
PubMed1989

Cloning of the biotin synthetase gene from Bacillus sphaericus and expression in Escherichia coli and Bacilli.

Biotin synthetase (BS) catalyses the biotransformation of dethiobiotin (DTB) to biotin. Here we report the cloning, characterization and expression of the gene encoding BS of Bacillus sphaericus. A recombinant plasmid pSB01, containing an 8.2-kb DNA fragment from B. sphaericus, was isolated by phenotypic complementation of an Escherichia coli bioB strain. Nucleotide sequence analysis of this fragment and N-terminal sequence determination of the recombinant protein product revealed that the bioB gene of B. sphaericus consists of a 996-bp open reading frame which is closely associated with at least one other gene. E. coli cells transformed with a bioB expression vector performed efficient bioconversion of DTB to biotin under defined culture conditions. Biotin production from transformed Bacillus subtilis and B. sphaericus recombinant strains was also demonstrated. Comparison of the amino acid sequences of BS from E. coli and B. sphaericus revealed extensive similarity.

Amino Acid Sequence↗