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Biomedical subjects

S Tsuboi

Publications and source records attributed to S Tsuboi.

At least 109 records · Page 6Linked to original sources
PubMed1991

Fluorimetric and high-performance liquid chromatographic determination of D-lactate in biological samples.

D-Lactate in biological samples was converted into a strongly fluorescent substance in a one-vial reaction. It was first converted into the pyruvate hydrazone in the presence of D-lactate dehydrogenase, an NADH-reoxidation system using diaphorase, D,L-6,8-thioctamide and hydrazine. This hydrazone was then converted into 2-hydroxy-6,7-dimethoxy-3-methylquinoxaline by 1,2-diamino-4,5-dimethoxybenzene in 1 M hydrochloric acid, and the quinoxaline was extracted and measured fluorimetrically at 432 nm (excitation at 365 nm). The calibration curve for D-lactate was linear up to at least 100 nmol/ml of the assay mixture, with a determination limit of 2 nmol/ml. The quinoxaline was also analysed by high-performance liquid chromatography with fluorimetric detection. The calibration curve for D-lactate was linear from 500 fmol to 75 nmol in the reaction mixture. This method was 4000 times more sensitive than the fluorimetric method, and could determine D-lactate in blood plasma volumes of less than 1 microliter.

Animals↗
PubMed1991

Organ-specific occurrence and expression of the isoforms of nonspecific lipid transfer protein in castor bean seedlings, and molecular cloning of a full-length cDNA for a cotyledon-specific isoform.

Four kinds of nonspecific lipid transfer proteins (nsLTP) were purified from different organs of castor bean (Ricinus communis L.) seedlings. Amino acid compositions and amino-terminal sequences of the four nsLTPs were determined and compared with those of castor bean isoforms, nsLTP-A, -B, and -C, previously reported [Takishima et al. (1986) Biochim. Biophys. Acta 870, 248-255; Takishima et al. (1988) Eur. J. Biochem. 177, 241-249]. Two isoforms from the cotyledons were identified as nsLTP-A and -C, one isoform from the endosperms as nsLTP-B, and the other was a new isoform from the axes. This new isoform was named nsLTP-D and its amino acid sequence was determined. These results demonstrated organ-specific occurrence of the nsLTP isoforms in castor bean seedlings. The isoforms nsLTP-A, -B, -C, and -D showed similar transfer activity not only for phosphatidylcholine and phosphatidylethanolamine but also for monogalactosyldiacylglycerol, although the homology among their amino acid sequences ranged from 70 to 30%. Two cDNA clones (pnsLTP-C and pnsLTP-D) for nsLTPs of castor bean seedlings were isolated and sequenced. pnsLTP-C was the cDNA clone for nsLTP-C expressed in the cotyledons, and pnsLTP-D was that for nsLTP-D in the axis. A coupled in vitro transcription-translation analysis of both cDNA clones revealed that pnsLTP-C encodes the full-length of nsLTP-C precursor (pro-nsLTP-C), while pnsLTP-D encodes a part of nsLTP-D precursor. PronsLTP-C contained a 24-amino acid pre-sequence preceding the mature nsLTP-C (92 amino acids).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

An observation of ruptured annulus fibrosus in lumbar discs.

To observe anatomical or pathological changes in lumbar intervertebral discs, discography and computed tomography-discography (CTD) were performed on fresh human cadavers. The results of discograms and CTD were compared with histological findings of cross sections of discs. Preoperative CTD of lumbar disc herniation was investigated based on these results. Ruptures of the annulus fibrosus were divided into two categories: circumferential rupture and radial rupture. In CTD images of fresh human cadavers, most images of rupture of the annulus fibrosus showed anterior to lateral circumferential rupture. As disc degeneration progressed, circumferential rupture tended to coexist with radial rupture in many cases. In CTD cases of lumbar disc herniation, most images of ruptures of the annulus fibrosus showed a posterior radial rupture, which was the route for herniated nucleus. The greater the degree of degeneration, the more the images tended to show radial ruptures coexisting with circumferential ruptures.

Adult↗
PubMed1991

Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin.

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.

Amino Acid Sequence↗
PubMed1991

Amino acids and peptides. XXX. Synthesis of eglin c (41-49) and eglin c (60-63) and examination of their inhibitory activity towards human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin.

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OH and H-Thr-Asn-Val-Val-OH, which correspond to the sequences 41-49 and 60-63 of eglin c, respectively, were synthesized by a conventional solution approach using the newly developed 6-chloro-2-pyridyl ester method. The inhibitory activities of the above two peptides against human leukocyte elastase, cathepsin G, porcine pancreatic elastase and alpha-chymotrypsin were examined in comparison with those of the corresponding methyl esters.

Amino Acid Sequence↗
PubMed1991

Effects of antioxidants on survival of adult rat hepatocytes under various oxygen tensions in serum-free primary culture.

Effects of antioxidants, such as superoxide dismutase, vitamin C, vitamin E, 4-(0-benzylphenoxy)-N-methylbutylamine hydrochloride (bifemelane), and selenite on survival of adult rat hepatocytes were examined under normoxic and hyperoxic conditions in serum-free primary culture. The tested antioxidants, except for vitamin C, significantly increased the survival rate of hepatocytes under the normoxic condition (under air). Thus, even the normoxic culture condition is hyperoxic for hepatocytes. Elevation of oxygen tension (40% O2) caused severe morphologic degeneration of hepatocytes and remarkable decrease in the survival rate of the cells. Addition of the antioxidants effectively protected hepatocytes from the morphologic degeneration, and significantly improved the survival of the cells under the hyperoxic condition. These findings indicate that the antioxidants can maintain the long-term survival of hepatocytes in serum-free primary culture.

Animals↗
PubMed1991

[Immunotherapy using high concentration purified antigen showed remarkable effect in all cases].

Hoya (sea-squirt) asthma is a typical type I occupational asthma. A total of 22 females with this disease whose age ranged from 22 to 69 years were treated for two years with high concentration purified sea-squirt antigen named Ei-M having a molecular weight of 22,800. One case who developed ordinary bronchial asthma abandoned the immunotherapy during the early stage of the therapy, but in the remaining 21 cases remarkable effects were rapidly observed from the early stage of treatment. These cases did not develop asthmatic attacks even through they engaged in oyster shucking work and no symptomatic therapy was indicated. Serum anti-Ei-M IgG antibody was significantly elevated after the therapy. None of the treated cases developed any side effects.

Adult↗
PubMed1990

Synthesis of a trihexacontapeptide corresponding to the sequence 8-70 of eglin c and studies on the relationship between the structure and the inhibitory activity against human leukocyte elastase, cathepsin G and alpha-chymotrypsin.

A trihexacontapeptide corresponding to the sequence 8-70 of eglin c and its related peptides were synthesized by the conventional solution method and their inhibitory activity against human leukocyte elastase, cathepsin G and alpha-chymotrypsin was examined. Although synthetic eglin c (41-49) inhibited cathepsin G and alpha-chymotrypsin (Ki = 4.0 x 10(-5) M and 2.0 x 10(-5) M, respectively) but not leukocyte elastase, the synthetic trihexacontapeptide potently inhibited cathepsin G, alpha-chymotrypsin and leukocyte elastase (Ki = 1.8 x 10(-9) M, 1.4 x 10(-9) M and 2.2 x 10(-9) M, respectively). The relationship between the structure of eglin c and the inhibitory activity against the above enzymes is also described.

Amino Acid Sequence↗
PubMed1990

Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens.

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker's yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.

Adenosine Diphosphate↗
PubMed1990

Enhancing effect of S-(1,2-dicarboxyethyl)glutathione on epidermal growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes.

A tripeptide S-(1,2-dicarboxyethyl)glutathione (DCE-GS) has been reported to be present in the lens, liver, and heart. Effects of DCE-GS and its derivatives and analogues on hepatocyte deoxyribonucleic acid (DNA) synthesis were examined using primary cultures of adult-rat hepatocytes. DCE-GS alone had no effect on DNA synthesis of hepatocytes. However, when DCE-GS was added with epidermal growth factor (EGF), the tripeptide effectively enhanced EGF-stimulated DNA synthesis of hepatocytes under the culture conditions of low cell density, but not high cell density. On the other hand, some esters and amides of DCE-GS and DCE-GS analogues showed a suppressive effect on DNA synthesis of hepatocytes in the absence of EGF. The derivatives and analogues together with EGF had no effect or rather a suppressive effect on stimulation of hepatocyte DNA synthesis by EGF. Therefore, the two carboxy groups in the substituent probably play an important role in the stimulative activity of DCE-GS. In addition, it seems likely that one of in vivo physiological roles of DCE-GS is related to liver regeneration.

Animals↗
PubMed1990

S-(1,2-dicarboxyethyl)glutathione and activity for its synthesis in rat tissues.

The contents of S-(1,2-dicarboxyethyl)glutathione (DCE-GS) in several tissues of rat were determined by HPLC. The peptide was present at concentrations (nmol/g tissue) of 119 in lens, 71.6 in liver, and 27.4 in heart. It was, however, not detected in spleen, kidney, cerebrum, or cerebellum. In rat liver, DCE-GS was located primarily in the cytosolic fraction. The substrates for the enzymic synthesis of DCE-GS were GSH and L-malate. In rats, the DCE-GS-synthesizing activity was found to be highest in the liver and in the cytosol of rat liver subcellular fractions. The DCE-GS-synthesizing enzyme was partially purified from rat liver cytosolic fraction by ammonium sulfate fractionation, Phenyl Superose chromatography, hydroxyapatite chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be 53 kDa by gel filtration and SDS-PAGE, showing it to be a monomeric protein. The Km values for GSH and L-malate were 2.3 and 4.0 mM at 37 degrees C, respectively. The enzyme did not utilize 1-chloro-2,4-dinitrobenzene, 1,2-dichloro-4-nitrobenzene, p-nitrophenyl bromide, trans-4-phenyl-3-buten-2-one, or p-nitrobenzyl chloride, which were substrates for previously characterized glutathione S-transferases. The isolated enzyme preparation showed no fumarase activity, which supported the conclusion that the formation of DCE-GS was not the result of a nonenzymic reaction following the synthesis of fumarate from L-malate by the isolated enzyme. The N-terminal amino acid of this polypeptide was presumably blocked since no sequence was obtained by automatic sequencing after electro-blotting onto a siliconized-glass fiber (SGF) sheet.

Amino Acid Sequence↗
PubMed1990

Amino acids and peptides. XXVIII. Synthesis of peptide fragments related to eglin c and studies on the relationship between their structure and effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin.

Various peptide fragments related to eglin c, which consists of 70 amino acid residues, were synthesized by a conventional solution method and their inhibitory effects on leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Among them, H-Arg-Glu-Tyr-Phe-OMe (eglin c 22-25) and H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe (eglin c 41-49) inhibited cathepsin G and alpha-chymotrypsin but not leukocyte elastase, while H-Thr-Asn-Val-Val-OMe (eglin c 60-63) inhibited leukocyte elastase but not cathepsin G or alpha-chymotrypsin, although eglin c potently inhibited leukocyte elastase, cathepsin G and alpha-chymotrypsin. These results indicated that the interaction sites of eglin c with leukocyte elastase, cathepsin G and alpha-chymotrypsin might be different.

Amino Acid Sequence↗
PubMed1990

Synthesis of a heptacontapeptide corresponding to the entire amino acid sequence of eglin C.

A heptacontapeptide corresponding to the entire amino acid sequence of eglin c was synthesized by the conventional solution method using a minimal protecting method. The synthetic eglin c exhibited a symmetrical single peak on HPLC at the same retention time as an authentic eglin c, and had the same inhibitory activity against human leukocyte elastase, cathepsin G and alpha-chymotrypsin (Ki = 6.0 x 10(-9) M, 5.5 x 10(-9) M and 2.5 x 10(-9) M, respectively) as N alpha-acetyl-eglin c synthesized genetically (Ki = 5.1 x 10(-9) M, 1.5 x 10(-8) M and 2.2 x 10(-9) M, respectively).

Amino Acid Sequence↗
PubMed1990

Amino acids and peptides. XXIX. Synthesis of peptide fragments related to active center of eglin c and studies on the relationship between their structure and their inhibitory activity against cathepsin G and alpha-chymotrypsin.

H-Ser-Pro-Val-Thr-Leu-Asp-Leu-Arg-Tyr-OMe, corresponding to the sequence 41-49 of eglin c, inhibited human leukocyte cathepsin G and alpha-chymotrypsin. In order to gain further insight into the relationship between the structure and the inhibitory activity against cathepsin G and alpha-chymotrypsin, peptide fragments related to the above nonapeptide were synthesized by a conventional solution method and their inhibitory activities were examined. The smallest peptide which exhibited inhibitory effects on the above enzymes was H-Pro-Val-Thr-Leu-OMe, corresponding to the sequence 42-45 of eglin c.

Amino Acid Sequence↗
PubMed1990

Fluid movement across the blood-retinal barrier: a review of studies by vitreous fluorophotometry.

Vitreous fluorophotometry was used to study the fluid movement across the blood-retinal barrier. The effect of plasma osmolality and intraocular pressure on the movement of the fluorescent tracer confirmed the interaction between the tracers and the fluid flow across the blood-retinal barrier. The measurement of outward and inward permeabilities to carboxyfluorescein revealed the existence of a posteriorly directed fluid flow in the vitreous cavity of cynomolgus monkey eyes with retinal detachment. Fluid movement across the blood-retinal barrier was shown to be inhibited by furosemide and enhanced by acetazolamide. Furthermore, vitreous fluorophotometry proved to be useful in estimating the in vivo fluid dynamics in the vitreous and across the human retina.

Animals↗
PubMed1990

Extravascular albumin concentration of the uvea.

The hypothesis that uveal vessels absorb fluid was tested by measuring the albumin in extravascular uveal tissues and in plasma. From these results the effective albumin concentration was calculated in both rabbits and monkeys. Three separate methods were used to measure uveal albumin, and the results of these were compared. In method 1, the intravenous fluorescein isothiocyanate (FITC)-albumin concentration found in the uvea 5 min after injection (intravascular tracer) was subtracted from that found 2 hr after injection (intravascular plus extravascular tracer) to determine the extravascular albumin concentration. In method 2, intravenous FITC-albumin was followed by vascular washout after a 2-hr equilibration period to determine extravascular uveal albumin. In method 3, the endogenous extravascular albumin concentration of uveal tissues was measured with an enzyme-linked immunosorbent assay (ELISA) after vascular washout. The effective albumin concentration was determined by dividing the data in methods 1, 2, and 3 by the extravascular albumin space volume. The effective albumin concentration in monkey (as percentage of plasma) was, for methods 1, 2, and 3: iris 2, 3, and 4%; pars plicata 14, 12, and 7%; pars plana 2, 10, and 12%; and choroid 2, 12, and 10%, respectively. In rabbit, the extravascular albumin concentrations were: iris 10, 21, and 7%; pars plicata 69, 26, and 39%; pars plana 41, 46, and 10%; and choroid 88, 30, and 26%, respectively. These findings are lower than previously reported in rabbits, yet are consistent with previous estimates in monkeys. These results support the hypothesis that uveal vessels are capable of fluid absorption, since a large colloid osmotic gradient exists across the vessel wall.

Albumins↗
PubMed1989

Synthesis of peptide fragments related to eglin c and examination of their inhibitory effect on human leukocyte elastase, cathepsin G and alpha-chymotrypsin.

Various kinds of peptide fragments related to eglin c were prepared by the conventional solution method and their inhibitory effects on human leukocyte elastase, cathepsin G and alpha-chymotrypsin were examined. Peptide (31-40) inhibited cathepsin G (Ki = 2.3 x 10(-4) M), peptide (41-49) potently inhibited cathepsin G and alpha-chymotrypsin (Ki = 4.2 x 10(-5) M and 2.0 x 10(-5) M, respectively), and peptide (60-63) inhibited leukocyte elastase (Ki = 1.6 x 10(-4) M), whereas, peptide (31-35) weakly inhibited both elastase and cathepsin G (Ki = 2.1 x 10(-3) M and 7.3 x 10(-4) M, respectively).

Amino Acid Sequence↗