Biomedical subjects

Y Doi

Publications and source records attributed to Y Doi.

At least 19 recordsLinked to original sources

PubMed1992

Interaction of gelsolin with covalently cross-linked actin dimer.

One of the two actin molecules in the ternary actin-gelsolin complex was selectively cross-linked to gelsolin when benzophenonemaleimide-actin (BPM-actin) was used [Doi, Y., Banba, M., & Vertut-Doi (1991a) Biochemistry 30, 5769-5777]. Here, we examine the interaction between gelsolin and BPM-actin dimer in which BPM-actin is covalently conjugated to unlabeled actin by p-phenylenedimaleimide (pPDM). BPM-actin dimer having an apparent molecular mass of 115 kDa is photo-cross-linked to gelsolin (90 kDa) more effectively than BPM-actin monomer in the presence of Ca2+, forming a cross-linked actin dimer-gelsolin (1:1) complex with a molecular mass of 210 kDa. The tight direct association of the dimer to gelsolin is shown by the titration of gelsolin with the fluorescently labeled dimer and by the higher concentration of phosphatidylinositol 4,5-bisphosphate required to inhibit the formation of BPM-dimer complex with gelsolin than that of BPM-monomer complex. However, an attempt to cross-link the two actin molecules in the ternary actin-gelsolin (2:1) complex by pPDM fails. The results argue that the topography of the two actin molecules in the actin-gelsolin (2:1) complex is similar, but not identical, to that of the barbed end of an actin filament.

4-Chloro-7-nitrobenzofurazan↗

PubMed1992

Effects of insulin and tyrosine kinase inhibitor on ion transport in the alveolar cell of the fetal lung.

We studied the effect of insulin and lavendustin-A (a tyrosine kinase inhibitor) on the short-circuit current (ISC) of primary cultures of fetal distal rat lung epithelium (FDLE). Insulin (2 microM) on the basolateral side of the monolayer increased ISC from 5.76 +/- 0.83 microA/cm2 (SEM, n = 7) to 7.23 +/- 1.00 microA/cm2 (p less than 0.01) under control conditions, and from 1.00 +/- 0.31 microA/cm1 to 1.53 +/- 0.34 microA/cm2 (p less than 0.05, n = 4) when amiloride (10 microM) was present on the apical side of the monolayer. Thus insulin increased both the amiloride-sensitive and insensitive ISC with the insulin-induced increase in ISC in the absence of amiloride (1.47 +/- 0.22 microA/cm2, n = 7) being significantly larger than that in the presence of 10 microM amiloride (0.53 +/- 0.14 microA/cm2, n = 4; p less than 0.025). Insulin's effect reached steady state in 1 hr. Lavendustin-A (10 microM), a tyrosine kinase inhibitor, applied to the apical side of the monolayer attenuated but did not completely block insulin's ability to increase in ISC; i.e., insulin increased ISC in lavendustin-A treated monolayers (0.63 +/- 0.09 microA/cm2, n = 5; p less than 0.0025) but the increase was significantly smaller than that without the pretreatment of lavendustin-A (p less than 0.05). In the presence of amiloride (10 microM) and lavendustin-A (10 microM) insulin was no longer able to increase ISC (change in ISC = 0.04 +/- 0.03 microA/cm2, n = 6), suggesting that lavendustin-A had blocked the insulin's effect on the amiloride-insensitive ISC. Lavendustin-A (10 microM) had no significant effect on the basal ISC in control and amiloride treated monolayers. Our studies demonstrate that insulin increases amiloride-insensitive ISC in FDLE via lavendustin-A sensitive tyrosine kinase and that insulin's action on the amiloride-sensitive ISC of FDLE is mediated through a lavendustin-A insensitive (and presumably tyrosine kinase-independent) pathway.

Amiloride↗

PubMed1992

Isolation of a novel protein from the outer layer of the vitelline membrane.

The outer layer of the vitelline membrane from hen egg yolk consists of ovomucin, vitelline membrane outer layer protein I (VMOI) and lysozyme. Here we report the occurrence of a further basic protein (pI 11.5) in the outer layer, which was designated as vitelline membrane outer layer protein II (VMOII). It was dissociated from the outer layer in a 10% (w/v) NaCl solution and purified to homogeneity by ion-exchange chromatography. VMOII is a simple protein with a molecular mass of 6000 Da, as determined by sedimentation equilibrium analysis. The amino acid composition of VMOII was characterized by the absence of Met and high contents of cystine (half) (14%) and basic amino acids (6% Arg, 6% Lys and 3% His). Analysis of carboxymethylated VMOII indicated that all cysteine residues were involved in disulphide bonding, which appears to facilitate the binding of SDS to the protein. Sequence comparison of the N-terminal 20 residues revealed no identity with other known proteins. VMOII contained a small amount of alpha-helix and was quite resistant to heat denaturation.

Amino Acid Sequence↗

PubMed1992

A novel NADPH-dependent carbonyl reductase of Candida macedoniensis: purification and characterization.

A novel NADPH-dependent carbonyl reductase was purified to homogeneity from the soluble fraction of a cell extract of Candida macedoniensis AKU 4588. The enzyme catalyzes not only the reduction of quinones, but also the reduction of aromatic aldehydes, conjugated polyketones, 2'-ketopantothenate esters, and 4-chloro-3-oxobutanoate esters. The enzyme shows absolute specificity for NADPH as a coenzyme and also shows quite high affinity toward NADPH (Km less than 5 microM). The apparent Km values for menadione and p-toluquinone are 167 and 180 microM, respectively. The enzyme is not a flavoprotein and is a monomer protein with a relative molecular mass of 45,000. Dicoumarol, quercetin, and some sulfhydryl reagents inhibit the enzyme activity.

Alcohol Oxidoreductases↗

PubMed1992

The amino-terminal fragment of gelsolin is cross-linked to Cys-374 of actin in the EGTA-resistant actin-gelsolin complex.

It has been shown that the EGTA-resistant actin, one of the two actin molecules associated to gelsolin, can be predominantly cross-linked to gelsolin by benzophenone-4-maleimide (BPM), a photoaffinity-labeling reagent, which was conjugated to Cys-374 of actin prior to cross-linking (Doi, Y., Banba, M. and Vertut-Doï, A. (1991) Biochemistry 30, 5769-5777). When a chymotryptic digest of gelsolin containing the amino-terminal 15-kDa fragment was mixed with BPM-actin (42 kDa) and irradiated for cross-linking, a band of 58 kDa appeared on SDS-PAGE which was shown to contain actin molecule by using fluorescently labeled actin. The amino-terminal sequence of the 58-kDa complex was identical to that of gelsolin, confirming that the amino-terminal segment (residues 1-133) of pig plasma gelsolin lies closely to Cys-374 of actin in the EGTA-resistant complex.

Actins↗

PubMed1992

Simultaneous localization of histamine and factor VIII-related antigen in the endothelium of the human umbilical vein.

Simultaneous immunoelectron microscopic localization of histamine and factor VIII-related antigen was examined on the same ultrathin section of the endothelium of the human umbilical vein from full-term deliveries by means of the double-immunolabeling technique. Small gold particles demonstrating antibody reaction with histamine are preferentially located in the cytoplasmic matrix and organelles, especially in mitochondria and on the luminal membrane surface of the endothelial cells. The gold particles representing histamine immunoreactivity also located on some of Weibel-Palade (WP) bodies. In contrast, large gold particles demonstrating factor VIII-related antigen are concentrated preferentially on most WP bodies. Single labeling of either histamine or factor VIII-related antigen shows similar results to those of the double labeling. The present study indicates that some WP bodies are involved in storage of both factor VIII-related antigen and histamine, but others store factor VIII-related antigen only. This difference in contents of WP bodies may be induced during the development and maturation process of this inclusion. At any rate, it is reasonable to consider that WP bodies have important roles in both vascular tonus and hemostasis during the vascular obliteration.

Cytoplasm↗

PubMed1992

Studies on the hepatotoxicity induced by bis (tributyltin) oxide.

The toxic effects of bis (tributyltin) oxide (TBTO) on the rat liver were studied with an electron microscope and the accumulation sites of tin were determined with an X-ray microanalyzer. The activities of serum enzymes and the concentration of serum bilirubin were also analyzed. Male Wistar rats received an intramuscular injection of 0.5 ml/kg of TBTO. Marked swelling of the mitochondria appeared in the hepatocytes 4 h after injection of TBTO. Cytoplasmic vacuoles, which contained degenerated mitochondria, gradually increased in number in these hepatocytes. This in turn may have caused a decrease in the volume of hepatic cell cords and an enlargement of sinusoids in the entire hepatic lobule. However, fine structures of intrahepatic bile ducts were not altered. By X-ray microanalysis, tin peaks were preferentially obtained from swollen mitochondria of the hepatocytes. By polarographic analysis of the respiratory responses of mitochondria, it was demonstrated that rates of state 4 respiration and respiratory control ratio were significantly disturbed in TBTO-treated rats in comparison with those of controls. The activities of AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were significantly increased after TBTO treatment, but those of ALP (alkaline phosphatase), LAP (leucine aminopeptidase) and total bilirubin were not changed. These results indicated that parenterally administered TBTO accumulated in the liver cell mitochondria and disturbed oxidative phosphorylation. Mitochondrial dysfunction might induce severe damage of the hepatocytes. Four days after injection of TBTO, hepatic structures and chemical indices were almost restored by the regeneration of hepatocytes.

Alanine Transaminase↗

PubMed1992

Effects of non-collagenous proteins on the formation of apatite in calcium beta-glycerophosphate solutions.

The effects of the non-collagenous proteins; osteonectin, bone Gla protein and dentine phosphoprotein, on the formation of apatite were studied in calcium beta-glycerophosphate solutions containing catalytic amounts of alkaline phosphatase under physiological conditions. In the system used, calcium phosphate precipitates de novo at levels of supersaturation precisely determined through the enzymatic hydrolysis of beta-glycerophosphate. At 1.7 mM of calcium beta-glycerophosphate, calcium phosphate precipitated when inorganic phosphate accumulated to about 1.4 mM. In the presence of the proteins, however, a greater accumulation of inorganic phosphate was needed for calcium phosphate to precipitate, suggesting that a higher degree of supersaturation, though still a slight undersaturation with respect to dicalcium phosphate dihydrate, is required for calcium phosphate to precipitate in the presence of the proteins. At the same protein (micrograms/ml) concentration, dentine phosphoprotein was approximately four times as effective as bone Gla protein, which was about twice as effective as osteonectin in delaying precipitation. The proteins also retarded subsequent crystal growth, with apatite formed in the presence of the more inhibitory proteins having the smallest crystals, especially in width.

Alkaline Phosphatase↗

PubMed1992

Changes in Na concentration in cerebrospinal fluid during acute hypernatremia and their effect on drinking in juvenile rats.

To clarify if there are age-related differences in osmoreception, we measured changes in Na concentration in cerebrospinal fluid ([Na]csf) during and after acute hypernatremia in juvenile (JR) and adult rats (AR). The Na concentrations in plasma ([Na]pl) and [Na]csf were measured during intravenous infusion of 1 M NaCl solution (INF, 0.2 ml/100 g body wt. for 10 min) and for 20 min thereafter in anesthetized rats. To measure [Na]pl, a flow-through Na-sensitive electrode was placed in an extracorporeal shunt from a carotid artery to a jugular vein, and to measure [Na]csf, a Na-sensitive electrode was placed in the right lateral ventricle. There was a linear relationship between delta[Na]pl and delta[Na]csf during INF. The slope of delta[Na]pl vs. delta[Na]csf was 0.42 +/- 0.03 (n = 7) in JR, which was significantly higher (p less than 0.01) than in AR (0.23 +/- 0.04, n = 6). We also measured water intake during and after INF of the same amount of 1 M NaCl solution in conscious JR (n = 6) and AR (n = 6). The JR drank 1.93 +/- 0.22 ml/100 g body wt. of water within 30 min of the start of INF, which was significantly more (p less than 0.05) than AR drank (1.20 +/- 0.13 ml/100 g body wt.). These results indicate that the movement of Na or water, or both, between the blood and cerebrospinal fluid in acute hypernatremia is greater in JR, and that the osmoreceptors in JR are more sensitive than those in AR.

Animals↗

PubMed1992

The role of the rat submandibular gland in the excretion of bis (tributyltin) oxide: electron microscopy, X-ray microanalysis and atomic absorption analysis.

The role of the submandibular glands in the excretion of parenterally administered bis (tributyltin) oxide (TBTO) was studied. Fine structural alterations of the submandibular glands were observed with an electron microscope. Accumulation sites of TBTO were determined with an X-ray microanalyzer and tin concentrations in saliva and blood were measured by atomic absorption spectrophotometry.

Animals↗

PubMed1992

Biosynthesis and n.m.r. studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16.

Alcaligenes eutrophus H16 was grown on mixtures of 1H- and 2H-acetate as carbon sources. The accumulation of deuterated poly(3-hydroxybutyrate) (P(3HB)) was observed. The deuterium distributions in the isolated P(3HB)s were determined from 1H and 2H-n.m.r. spectra and confirmed by 13C-n.m.r. spectra. Although one would expect to synthesize P([2,2,4,4,4-2H5]3HB) when the cells were grown on 2H-acetate as the sole carbon source, the methyl, methylene and methine groups of the P(3HB) contained both deuterium and proton. This observation indicates some substitution from 2H to 1H during the P(3HB) synthesis. The 2H content in the methyl groups was larger than that in the methylene groups, which suggests a kinetic isotope effect in the P(3HB) synthesizing process. The deuterium distributions in the two magnetically non-equivalent methylene protons were determined to be different, which indicates stereoselectivity at the C2 site.

Alcaligenes↗

PubMed1992

Effects of hormonal supplements on the maintenance of cardiac function in potential donor patients after cerebral death.

It is well-known that cardiac function in cerebrally dead patients rapidly deteriorates, leaving the organ unfit for donation. This study investigated whether or not cardiac function in patients with cerebral death can be maintained in a desirable condition with hormonal supplementation. In studies of changes in hormones before and after cerebral death, insulin, glucagon, triiodothyronine, thyroxine, cortisol, vasopressin, epinephrine, and norepinephrine values were measured with a lapse of time after cerebral death. Among them, triiodothyronine and cortisol levels were markedly reduced after cerebral death; therefore, these two hormones were selected as hormonal supplements. The average period from the judgment of cerebral death to cardiac arrest was 4.3 days in 12 patients with no hormonal supplement (group I) and more than 11.5 days in 4 patients with hormonal supplement (group II). This period for patients in group II was significantly longer (p less than 0.05). In 2 of the group II patients the hormonal supplementation was discontinued at the family's request, and in the other 2 patients, it was discontinued because of proposed renal donation. Hemodynamic comparisons between the two groups showed that the mean arterial pressure and the left ventricular maximum dp/dt were significantly higher (p less than 0.01) as was the cardiac index (p less than 0.05) on the 3rd day after cerebral death in members of group II. Thereafter, in group II, an excellent hemodynamic state was maintained until hormonal supplements were discontinued. We conclude that the triiodothyronine and cortisol supplements were effective in the maintenance of cardiac function in patients after cerebral death.

Adolescent↗

PubMed1992

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.

Carboxylic Ester Hydrolases↗

PubMed1992

Continuous measurement of Na concentration in CSF during gastric water infusion in dehydrated rats.

To assess the differential stimulus to central and intravascular osmoreceptors during recovery from thermal dehydration, we measured Na concentrations in cerebrospinal fluid ([Na]CSF) and plasma ([Na]p) continuously and compared these during simulated drinking by gastric water infusion (INF) in euhydrated and thermally dehydrated rats under anesthesia. Continuous measurement of [Na]CSF was obtained with a double-barreled Na electrode placed in the lateral ventricle. Continuous measurement of [Na]p was obtained from a flow cell Na electrode in an extracorporeal shunt. Measurements were made during 10 min of INF (2.5 ml/100 g body wt) into the stomach and during 20 min of recovery. Changes in [Na]CSF always lagged behind those in [Na]p and were quantitatively smaller after INF. The decrease in [Na]CSF occurred sooner in dehydrated than in euhydrated rats in response to the decrease in [Na]p (P < 0.01). These results suggest that water and/or Na movement between blood and CSF is accelerated during restitution from thermal dehydration, acting to prevent overhydration during the early phase of rehydration.

Animals↗

PubMed1992

Sequential appearance of fibronectin, collagen and elastin during fatty streak initiation and maturation in hypercholesterolemic fat-fed rabbits.

To elucidate whether tissue fibronectin increases in the early stages of atherogenesis induced by hypercholesterolemia without mechanical trauma, we investigated sequential changes in the distribution of tissue fibronectin during fatty streak initiation and maturation in the aortas of hypercholesterolemic fat-fed rabbits. The presence of fibronectin was examined on immunoperoxidase stained tissue specimens with the aid of a microscope-photometric technique. Twenty male albino rabbits were used. Cholesterol supplemented chow (1%) was given for 4 weeks (n = 6), 8 (n = 5) or 14 weeks (n = 5). A membrane-like layer positive for fibronectin was observed along the endothelium in the normal aorta. After 4 weeks of the cholesterol-feeding, fatty streaks were initiated in the intima, where fibronectin was more densely accumulated than the normal intima. After 8 weeks of the cholesterol-feeding, fatty streaks were expanding, associated with the dense staining for fibronectin. After 14 weeks, fibronectin was still concentrated in the endothelial layer and also in the superficial areas of the thickened intima, but decreased in the deep areas of the thickened intima where collagen and elastin appeared as bundles. The photometric data of fibronectin supported these visual observations. Thus, fibronectin appeared early and disappeared later in the intima during the process of fatty streak initiation and maturation. These findings suggest that in hypercholesterolemia without mechanical endothelial injury, fibronectin may play an important role in an early process of atherogenesis.

Animals↗

PubMed1992

Negative washout rate of myocardial thallium-201--a specific marker for high grade coronary artery narrowing.

A decline is usually observed in the myocardial uptake of thallium-201 in delayed imaging compared with initial imaging. In patients with severe coronary artery disease (CAD), the uptake is sometimes higher in the delayed than in the initial imaging, which is expressed as negative washout rate. To evaluate the diagnostic implications of this negative washout rate the findings of dipyridamole thallium scintigraphy in 582 patients with coronary artery disease were evaluated. The negative washout rate was present in 201 of 582 patients (35%). It had a significant association with high grade coronary artery narrowing of > or = 90%. Sensitivity in detecting patients with this high grade narrowing by negative washout rate was 48%, its specificity was 93%, and its positive predictive value was 94%. Sensitivity to detect individual coronary artery narrowing of > or = 90% did not decrease according to the extent of CAD, with the highest detection in the left anterior descending coronary artery and the lowest in the left circumflex coronary artery. Since patients with high grade coronary narrowing often require coronary intervention, the results of this study suggest the diagnostic importance of negative washout rate in the identification of the particular subset of patients with CAD.

Adult↗

PubMed1992

Production of active and inactive renin by adrenal explant cultures and the existence of a prorenin activating enzyme in the adrenal gland.

We previously demonstrated high levels of inactive renin (IR) in nephrectomized rat plasma which provided evidence for extra-renal sources of IR. However, the sources of IR were unknown. This study examined whether the adrenal gland is capable of producing active renin (AR) and IR, using explants of adrenal capsules (glomerulosa portions) from control and nephrectomized (Nepex) rats. Explants from both control and Nepex rats produced large quantities of IR and small quantities of AR. The productions of both IR and AR in Nepex rats were significantly greater than in control rats. High potassium culture medium markedly increased IR and slightly, but significantly, enhanced AR in both control and Nepex rats. On the other hand, sodium had little effect on either IR or AR. A renin-angiotensin system has been reported in the adrenal gland. Recently, a prorenin activating enzyme was demonstrated in the kidney and the aortic wall. The next study was performed to demonstrate the existence of a prorenin activating enzyme in the adrenal gland, using inactive renin from culture medium from Nepex rat explants as the substrate. A 26 KD component in adrenal zona glomerulosa tissues isolated by Sephacryl S-200 column chromatography, converted IR to AR in IR rich medium. The pH optimum for prorenin activation by the enzyme was 6.5. Inhibitor studies indicate that this enzyme is a serine protease. These data suggest that (1) the adrenal gland is an extra-renal source of inactive renin and (2) a prorenin activating enzyme exists in the adrenal gland.

Adrenal Glands↗

PubMed1992

[Prognosis of asymptomatic elderly patients with aortic stenosis].

To elucidate the prognosis of elderly patients with asymptomatic aortic stenosis (AS) and to assess the timing of aortic valve replacement (AVR), 21 asymptomatic patients (8 men, 13 women, mean age: 75 +/- 8 years (54-89 years)), who had Doppler echocardiographic evidence of a significant aortic pressure gradient of greater than 40 mmHg (mean gradient: 75 +/- 31 mmHg), were followed for 33 +/- 10 months. During the follow-up, there were 4 cardiac events (2 cardiac deaths, 2 late AVRs), and 2 non-cardiac deaths (cerebro-vascular accidents). Among 15 survivors, 13 patients were in NYHA class I--II, and the remaining 2 patients were found to have malignant disease. Compared to the 17 patients without cardiac events, those with cardiac events had significantly larger CTR (58 +/- 6% vs. 53 +/- 3%; p less than 0.01), although there were no significant difference in electrocardiographic LVH, echocardiographic LV mass, and Doppler pressure gradient between the two groups. The prevalence of the development of cardiac symptoms during the follow-up was not high (12%) in patients without cardiac events. Among 4 patients with cardiac events, one patient who was 89 years-old at diagnosis died of heart failure, one patient had fatal myocardial infarction which seemed to be unrelated to AS, and two patients had successful late AVR because of new heart failure. The low incidence of fatal cardiac events in asymptomatic patients with aortic stenosis and the relatively high possibility of developing non-cardiac events in elderly patients indicate that the decision-making for AVR should not be solely based upon the pressure gradient detected by Doppler echocardiography.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗