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Biomedical subjects

Y Takata

Publications and source records attributed to Y Takata.

At least 199 records · Page 11Linked to original sources
PubMed1989

Insulin-like growth factor I receptors on erythrocytes in NIDDM.

We examined 125I-insulin-like growth factor I (125I-IGF-I) binding to erythrocytes from 24 normal and 21 non-insulin-dependent diabetic (NIDDM) subjects. 125I-IGF-I binding to human erythrocytes was specifically inhibited by unlabeled IGF-I, and Scatchard analysis indicated a curvilinear plot. There was a significant difference in IGF-I binding between normal and diabetic subjects (7.78 +/- 0.42 vs. 5.80 +/- 0.33%/2.4 x 10(9) cells/ml, P less than 0.001). Among diabetic patients, IGF-I binding to erythrocytes from those with retinopathy and those without retinopathy was comparable. These results suggested that decreased IGF-I binding might be a factor responsible for some pathological features such as delayed wound healing in diabetic patients.

Adult↗
PubMed1989

A biotin-avidin sandwich ELISA for quantification of intact complement component C9. The sera from hereditary C9 deficient individuals completely lack C9.

A two-site sandwich ELISA method was developed for quantitating intact C9 protein using MoAb P40 (anti-C9b antibody). This antibody reacted with monomeric C9 but not with polymerized C9. MoAb P40 was used as a capture antibody and MoAb X195 (anti-C9a antibody) as a detection antibody. This method is highly sensitive and can detect approximately 0.5 ng/ml of native C9. No cross-reactivities of either C6, C7, or C8 were observed even at concentrations of 10 micrograms/ml per component. In addition, this method allows for measurement of only intact C9 molecules, eliminating the interference of polymerized C9 or inactivated C9. Using this assay, no C9 at all was detected in sera from inherited C9 deficient individuals, including both healthy blood donors and patients with meningococcal meningitis; although by hemolytic assay, C9 levels were reported to be less than 0.2% those of NHS. Therefore, this two-site sandwich ELISA method can replace the hemolytic assay, and is especially useful for measuring small amounts of C9 in serum.

Animals↗
PubMed1989

Rat liver S-adenosylhomocysteinase. Spectrophotometric study of coenzyme binding.

Rat liver S-adenosylhomocysteinase, a homotetramer, was resolved by treatment with acid ammonium sulfate into apoenzyme and NAD. The apoenzyme thus prepared retained a tetrameric structure but differed in the mobility on nondenaturing polyacrylamide gel electrophoresis. The inactive apoenzyme was reactivated upon incubation with NAD. The restoration of activity paralleled with the tight binding of NAD to apoenzyme, and full activity was obtained when 4 mol of NAD were bound per mol of apoenzyme. The kinetics of reconstitution were apparently biphasic and suggest the existence of two conformers in a slow equilibrium, one of which binds the coenzyme rapidly while the other does so very slowly, if at all. In addition to NAD, apoadenosylhomocysteinase tightly bound nicotinamide hypoxanthine dinucleotide, 3-acetylpyridine adenine dinucleotide and nicotinic acid-adenine dinucleotide. NADP was not bound. Catalytic activity was found only with the enzyme reconstituted with NAD or nicotinamide hypoxanthine dinucleotide. The spectral change observed on interaction of apoadenosylhomocysteinase with NAD was similar to those seen with adenine nucleotides, and was largely approximated by the addition of dioxane to aqueous solutions of adenine nucleotides. By comparison of the difference spectra, it is suggested that the adenine portion of the coenzyme is bound in the hydrophobic pocket of the protein, and that the binding is accompanied by perturbation of tryptophan residue of the protein.

Adenosylhomocysteinase↗
PubMed1989

Binding specificity and intramolecular signal transmission of uncleaved insulin proreceptor in transformed lymphocytes from a patient with extreme insulin resistance.

An alteration of an amino acid sequence in the processing site of the insulin proreceptor by a point mutation of the insulin receptor gene produced extreme insulin resistance. We characterized functional properties of the unprocessed insulin receptor in transformed lymphocytes from a patient. Insulin binding to intact cells and to a partially purified insulin receptor preparation was radically decreased to 20% and 18% of the control values, respectively. In competitive insulin binding to intact cells, [LeuA3]-, [LeuB24]-, [SerB24]-insulin, and mini-proinsulin ([ B(1-29)-Ala-Ala-Lys-A(1-21)]-insulin) had the same relative binding activity in both the patient's and the control cells, but proinsulin and IGF-I were markedly less able to displace 125I-insulin in the patient's cells. In contrast to the study in intact cells, proinsulin and IGF-I as well as other insulin analogues had the same relative binding activity to bind to the partially lectin-purified insulin receptor preparations from both the patient's and the control cells. As regards the signal transduction after receptor binding, insulin-stimulated autophosphorylation of the unprocessed insulin proreceptor occurred proportionally to the amount of decreased insulin binding. With 0.025% trypsin treatment, the abnormal binding characteristics and autophosphorylation were normalized through conversion to functionally normal receptors. In spite of the abnormal processing, self-association of receptors into oligomeric structures was observed in the proreceptor. These results suggest that the unprocessed insulin proreceptor in the plasma membranes has an altered conformation which affects its binding characteristics but not its intramolecular signal transmission.

Adult↗
PubMed1989

Unprocessed insulin proreceptor in cultured fibroblasts from a patient with extreme insulin resistance.

Insulin receptors and IGF-I receptors in cultured fibroblasts were investigated in a patient with extreme insulin resistance due to unprocessed insulin receptors. Insulin binding to cultured fibroblast monolayers and partially purified insulin receptors was extremely decreased to 27% and 18% of control value, respectively. Affinity cross-linking study revealed that molecular weight of the insulin receptor was 210 kDa and that it could not be dissociated to alpha- and beta-subunit with dithiothreitol treatment. Because IGF-I binding to the fibroblasts from the patient was normal and alpha-subunit of IGF-I receptor was 135 KDa, the defect was specific to the insulin receptor. Autophosphorylation of the 210 kDa unprocessed insulin proreceptor was stimulated by insulin in a dose-dependent manner. In the fibroblasts from the patient, insulin-stimulated alpha-aminoisobutyric acid uptake was fivefold shifted to the right in the dose-response curve (ED50 20 ng/mL for the patient v 3.5 ng/mL for the control subjects), but the maximally stimulated uptake was normal. With 0.025% trypsin treatment, insulin binding and alpha-aminoisobutyric acid uptake were normalized. These results suggested that (1) abnormal processing of insulin proreceptor also occurred in the cultured fibroblasts, (2) the postreceptor steps of insulin action were totally intact, and (3) IGF-I receptors were normally processed in this patient.

Adult↗
PubMed1989

Glucose tolerance and insulin secretion in conscious and unrestrained normotensive and spontaneously hypertensive rats.

We compared the glucose tolerance and insulin responses to intravenous (IV) glucose administration of a dose of 1 g/kg body weight in a conscious and unrestrained state of spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) with catheters chronically indwelled into artery and vein. Both plasma glucose levels at two minutes and ten minutes following IV glucose load as well as the incremental and total areas of plasma glucose were slightly but significantly lower in SHR than in WKY. Glucose disappearance rate (K value) was 7.7 +/- 0.3%/min in SHR, being slightly but significantly higher than that of 6.8 +/- 0.3%/min in WKY. On the other hand, insulin responses to the glucose load at ten minutes and 30 minutes as well as incremental and total insulin areas were significantly lower in SHR than in WKY. There was no significant difference in insulinogenic index between SHR and WKY. Our observations suggest that in a conscious and unrestrained state, SHR have the greater glucose tolerance associated with reduced insulin secretion than do WKY.

Animals↗
PubMed1989

Unprocessed insulin proreceptors due to point mutation at the cleavage site.

Two sisters presented with severe insulin resistance and markedly decreased insulin binding to erythrocytes, cultured fibroblasts and transformed lymphocytes. The dose-response curve of insulin-stimulated amino acid uptake in the fibroblasts was shifted to the right. The molecular weight of the insulin receptor on the transformed lymphocytes from the patients was 210,000 and could not be dissociated to alpha- and beta-subunits by dithiothreitol treatment. However, the proreceptor was cleaved by trypsin and this led to the production of alpha-subunit with normal insulin binding. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patients. The polymerase chain reaction was used to obtain a large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough cDNA for the region to be sequenced. The results showed an AGG (Arg) to AGT (Ser) point mutation, resulting in the change of the interconnecting sequence of the two subunits from -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser-. These results suggest that the tertiary structure change of the cleavage site leads to production of unprocessed insulin proreceptors.

Adult↗
PubMed1989

Age and hypertension promote endothelium-dependent contractions to acetylcholine in the aorta of the rat.

This study was undertaken to compare age-related changes in endothelium-dependent vascular responses in both hypertensive and normotensive rats. Aorta from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) aged 4-6 weeks (young), 3-6 months (adult), and 12-25 months (old) were examined for relaxation to acetylcholine, adenosine 5'-triphosphate (ATP), and sodium nitroprusside. Rubbed (endothelium denuded) aorta from all groups displayed neither relaxation nor contraction to acetylcholine. Maximal relaxation responses to acetylcholine were reduced progressively with increasing age in unrubbed aorta of both SHR and WKY rats. In addition, acetylcholine caused not only dose-dependent relaxations at lower concentrations but also increases in tension at higher concentrations in unrubbed aorta of old WKY rats as well as adult and old SHR. However, indomethacin completely inhibited the tension development. As a result, aorta treated with indomethacin demonstrated similar acetylcholine-induced, endothelium-dependent relaxations in all groups. The thromboxane A2 synthetase inhibitor (E)-7-phenyl-7-(3-pyridyl)-6-heptanoic acid (CV-4151) partially but significantly depressed the increases in tension in aorta of old WKY rats. The degrees of endothelium-dependent relaxations to ATP and endothelium-independent relaxations to sodium nitroprusside were almost similar in all groups. These findings suggest that the release of or vascular responsiveness to endothelium-derived relaxing factor in the aorta is well maintained through senescence in both strains and that, in the aorta of not only SHR but also old normotensive WKY rats, the endothelium releases contracting factors that may be thromboxane A2 and other vasoconstrictor prostanoids.

Acetylcholine↗
PubMed1989

Hypotensive effects of nifedipine in patients with primary hyperparathyroidism: case reports.

Nifedipine was administered orally to 2 patients with primary hyperparathyroidism before and after parathyroidectomy. The operation lowered serum calcium concentration and parathyroid hormone but did not alter plasma renin activity, plasma aldosterone concentration, and serum magnesium. The hypotensive effects of nifedipine were markedly enhanced with the decrease in serum calcium concentration following parathyroidectomy. Thus, the level of serum calcium concentration may modulate the hypotensive effect of nifedipine in humans.

Aged↗
PubMed1988

C5 convertase of the alternative complement pathway: covalent linkage between two C3b molecules within the trimolecular complex enzyme.

C5 convertase of the alternative C pathway is a complex enzyme consisting of three C fragments--one molecule of a major fragment of factor B (Bb) and two molecules of a major fragment of C3 (C3b). Within this C3bBbC3b complex, the first C3b binds covalently to the target surface, and Bb, which bears a catalytic site, binds noncovalently to the first C3b. In the present investigation, we studied the nature of the convertase that is assembled on E surfaces and obtained evidence that the second C3b binds directly to the alpha'-chain of the first through an ester bond rather than to the target surface. Thus, the alternative pathway C5 convertase could be described as a trimolecular complex in which Bb binds noncovalently to a covalently linked C3b dimer. We also obtained evidence that not only the second C3b but also the first C3b participates in binding C5, that is, covalently-linked C3b dimer acts as a substrate-binding site. Because of this two-site binding, the convertase has a much higher affinity for C5 than the surrounding monomeric C3b molecules. Based on this evidence, a new model of the alternative pathway C5 convertase is proposed. Covalent association of two subunits and the bivalent binding of the substrate are then common properties of the alternative and classical pathway C5 convertases.

Animals↗
PubMed1988

Recombinant rat liver guanidinoacetate methyltransferase: reactivity and function of sulfhydryl groups.

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo[14C]acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5'-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine [2,2'-dithiobis(ethylamine)] causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.

Animals↗
PubMed1988

Insulin resistance by unprocessed insulin proreceptors point mutation at the cleavage site.

Failure to cleave the interconnecting site between alpha- and beta-subunit produced insulin proreceptors in the plasma membranes which had markedly low affinity to insulin, leading to extreme insulin resistance in a patient. We performed cDNA sequence analysis of the cleavage site of the insulin proreceptor from the patient. Polymerase chain reaction was used to obtain large amount of cDNA coding for the region including the interconnecting site. A thermostable DNA polymerase, Taq polymerase, successfully produced enough amount of cDNA of the region to be sequenced. The results showed AGG (Arg) to AGT (Ser) point mutation, resulting in the change of interconnecting sequence of the two subunits from -Arg-Lys-Arg-Arg- to -Arg-Lys-Arg-Ser-. These results suggest that the tertial structure change of the cleavage site leads to production of unprocessed insulin proreceptors.

Base Sequence↗
PubMed1988

Lack of increase in concentrations of cerebrospinal fluid sodium in rats with various stages of DOCA-salt hypertension.

Experiments were conducted in conscious rats to determine whether DOCA-salt treatment could cause an elevation of sodium concentration of cerebrospinal fluid (CSF), which may be responsible for the enhanced activity of sympathetic nervous system (SNS) and increased secretion of vasopressin (AVP). Systolic blood pressure (SBP) and mean arterial pressure (MAP) were gradually but consistently increased by DOCA-salt treatment. Serum Na concentration was similarly increased with time by DOCA-salt, and significantly higher than control in the 4th treatment week. In contrast, DOCA-salt did not alter the CSF Na levels at any time during treatment. A relationship between SBP and CSF Na was never evident at any stage of the DOCA-salt hypertension. The decrease in MAP following administration of the vasopressin V1-receptor antagonist, d(CH2)5Tyr(Me)AVP (30 micrograms/kg), or hexamethonium (30 mg/kg) was enhanced in the DOCA-treated rats, as compared to findings in the controls. These hypotensive effects were gradually, but progressively enhanced with the development of hypertension by DOCA-salt treatment. We tentatively conclude that mechanisms accounting for the enhanced activity of SNS and AVP in DOCA-salt hypertensive rats are independent of an increased Na concentration in the CSF.

Animals↗
PubMed1988

Lack of increase in sodium concentration of cerebrospinal fluid in DOCA-salt hypertensive rats.

The purpose of the present study is to determine whether DOCA-salt treatment could cause an elevation of Na concentration of cerebrospinal fluid (CSF), which may increase blood pressure (BP) as a result of enhanced activity of sympathetic nervous system (SNS) and vasopressin (AVP). Blood pressure was gradually increased by DOCA-salt treatment. Serum NA was also elevated with time by DOCA-salt, and significantly higher in DOCA rats on the 4th treatment week, as compared with controls. By contrast, DOCA-salt did not alter CSF Na at any time of treatment. No relationship was detected between BP and CSF Na at any stage of DOCA-salt hypertension. The precent fall in BP by AVP antagonist or hexamethonium was always greater in DOCA-treated rats than that in the controls. These hypotensive effects were gradually, but consistently enhanced with the development of hypertension in DOCA rats. It is concluded that the enhanced activity of both SNS and AVP system responsible for DOCA-salt hypertension may be attributed to the mechanism(s) unrelated to the increased CSF Na concentration.

18-Hydroxycorticosterone↗
PubMed1988

Ageing suppresses endothelium-dependent relaxation and generates contraction mediated by the muscarinic receptors in vascular smooth muscle of normotensive Wistar-Kyoto and spontaneously hypertensive rats.

We evaluated the effects of ageing and hypertension on endothelium-dependent relaxation and contraction of vascular smooth muscles. Aortic rings with and without endothelium from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR), divided into young, adult and old groups, were examined for relaxation in response to acetylcholine, adenosine triphosphate (ATP) and sodium nitroprusside. Relaxation responses to acetylcholine and ATP in SHR were either equal to or greater than those in WKY. Relaxations induced by ATP were not altered by ageing, while the degree of acetylcholine-induced endothelium-dependent relaxations was in an inverse order of age in both SHR and WKY. In old SHR and WKY and adult SHR, lower doses of acetylcholine caused relaxations, but increases in the dose of acetylcholine induced contraction. Acetylcholine-induced contractions were abolished by indomethacin or endothelium rubbing. We conclude that endothelium-dependent relaxations are not diminished by spontaneous hypertension but the relaxations mediated by the muscarinic receptors are reduced with ageing. Further, acetylcholine causes endothelium-dependent contractions by releasing cyclo-oxygenase products, not only in SHR but also in aged normotensive WKY.

Acetylcholine↗
PubMed1988

Differential effects of verapamil, nicardipine and diltiazem on Ca2+-dependent and Ca2+-independent noradrenaline release and contraction in isolated canine saphenous veins.

We reported that verapamil, nicardipine and diltiazem inhibited both 3H overflow and contraction induced by transmural nerve stimulation (TNS) in the canine saphenous vein preloaded with 3H-noradrenaline. In the present study, the effects of these Ca channel antagonists in the presence of tetraethylammonium (TEA) were investigated to evaluate whether the antagonists act by inhibiting Ca2+ influx into the nerve endings. The effects of the drugs on both responses to tyramine were also studied. In the 1 mmol/l-TEA-pretreated veins, verapamil and nicardipine inhibited only the contraction. Diltiazem inhibited the two parameters, the effects being nearly equipotent to those in the absence of TEA. The L-cis isomer of diltiazem inhibited the TNS-evoked responses to a similar degree as diltiazem, which was unaffected by 1 mmol/l TEA. Verapamil, nicardipine and diltiazem inhibited the TNS-evoked 3H overflow in 0.25 mmol/l Ca2+ and 20 mmol/l TEA medium, the inhibitions by the two former antagonists being greater than those seen in normal Krebs solution. TEA (1 mmol/l) added after the Ca channel antagonists, overcame the inhibition of the evoked overflow by diltiazem more poorly than that by verapamil or nicardipine. Only verapamil inhibited both responses to tyramine. These data together with previous results suggest that unlike verapamil and nicardipine, diltiazem may inhibit the TNS-evoked neurotransmitter release via a mechanism being unrelated to its Ca2+ influx-inhibiting action, resulting in an inhibition of the contraction, and that only verapamil inhibits the tyramine-evoked neurotransmitter release.

Animals↗