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Biomedical subjects

M Sugimoto

Publications and source records attributed to M Sugimoto.

At least 217 records · Page 12Linked to original sources
PubMed1994

Total synthesis of a modified ganglioside, de-N-acetyl GM2.

Regio- and stereo-selective glycosylation of a sialyl donor 6 that carries a N-phthaloyl protecting group at C-5 with a lactosyl acceptor 7 armed with a pivaloyl group at O-2a was performed to give the expected glycotrioside 5. Subsequent glycosylation of 5 with 2-azido galactosyl donor 4 gave glycotetraosyl derivatives 18 and 19. After conversion of 18 into imidates 25 and 26, coupling with (2S,3R,4E)-3-O-benzoyl-2-N-tetracosanoylsphingenine (2) was executed to afford completely protected ganglioside analogues 27 and 28. Selective cleavage of the methyl ester and N,O-deprotection gave the target de-N-acetyl GM2 (1).

Carbohydrate Conformation↗
PubMed1994

A morphometric study of spermatogenesis in the testes of mice of a senescence accelerated strain.

The morphometric parameters of spermatogenic cells in a mouse strain prone to accelerated senescence (SAM-P), a novel murine model of spontaneously promoted aging, were compared with those of a SAM resistant strain (SAM-R) after birth until 40 weeks (mean life span of SAM-P). A mixture of gonocytes and spermatogonia were present in the testis in 1-week-old mice, and no gonocytes were observed in 2-week-old mice. At 6 weeks of age, the absolute number of spermatogonia in SAM-P was 27% greater than that in SAM-R, whereas the cell number in 40-week-old SAM-P was 17% less than in SAM-R. Primary spermatocytes were first observed in 3-week-old animals, and the cell numbers in SAM-P at 3, 5 and 6 weeks were 78%, 31% and 25%, respectively, greater than in SAM-R, whereas the cell number in SAM-P at 40 weeks was 30% less than SAM-R. Round spermatids were first observed in all SAM-P at 4 weeks old, but 20% of SAM-R had no spermatids and the rest had only a few. At 5 and 6 weeks old, the absolute numbers of round spermatids in SAM-P were 38% and 41%, respectively, greater than in SAM-R, whereas the cell number in 40-week-old SAM-P was about 34% less than SAM-R. These results indicate that testicular maturation begins at an earlier age in SAM-P than SAM-R. Furthermore, at the age of 40 weeks signs of testicular deterioration are evident in SAM-P mice only.

Aging↗
PubMed1994

Structural elements influencing von Willebrand factor (vWF) binding affinity for platelet glycoprotein Ib within a dispase-digested vWF fragment.

We investigated the structural elements in human von Willebrand factor (vWF) that influence binding affinity for platelet glycoprotein (GP) Ib using a dispase-digested vWF fragment as a prototype (residues Leu480/Val481-Gly718 of the vWF subunit; Andrews et al, Biochemistry 28:8326, 1989). The major structural features of this fragment are a large A1-loop formed by an intrachain disulfide bond between Cys509 and Cys695 and six O-linked sugar chains. The fragment was chemically modified by (1) reduction and S-carboxyamido-methylation (R/A), (2) desialylation (DS), or (3) a combination of both (R/A-DS). The GPIb binding affinity of these fragments was basically evaluated by competitive binding assay with anti-GPIb monoclonal antibody (LJ-Ib1), a receptor blocker for vWF (Sugimoto et al, Biochemistry 30:5202, 1991). Both the prototype and the R/A fragments were also assessed for their function in shear-induced platelet aggregation. Results unambiguously demonstrated that the presence of a disulfide bridge (Cys509-Cys695) within this domain downregulates the affinity of vWF to GPIb. In addition, it was also demonstrated that the terminal sialic acids attached to six o-linked sugar chains within this domain contribute to optimal functional modulation by the antibiotic ristocetin, but not by snake venom botrocetin.

Animals↗
PubMed1994

Identification of a new collagen IV chain, alpha 6(IV), by cDNA isolation and assignment of the gene to chromosome Xq22, which is the same locus for COL4A5.

To date, five distinct alpha chains have been identified in basement membrane collagen IV. We have cloned a gene encoding a new alpha chain belonging to basement membrane collagen IV by cDNA isolation under low stringency conditions. Isolation of overlapping clones and the technique of 5' rapid amplification of cDNA ends enabled us to derive the primary structure of the entire polypeptide. The deduced collagen polypeptide contained 1678 amino acid residues, including a 21-residue signal peptide, a 24-residue amino-terminal NC domain, a central 1405-residue collagenous (COL1) domain, and a 228-residue carboxyl-terminal NC1 domain. We have designated this newly found alpha chain as the alpha 6(IV) chain. The gene was mapped to chromosome Xq22 by in situ hybridization of metaphase lymphocytes. This is the same region of chromosome X where the gene coding for the alpha 5(IV) collagen chain (COL4A5) resides. Mutations in COL4A5 have been characterized in more than 50 patients with Alport syndrome. However, some of the X-linked cases of Alport syndrome are not apparently caused by COL4A5 mutations. The gene we describe in this paper therefore seems to be a candidate for mutations in this group of Alport syndrome patients.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca.

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Human alpha-galactosidase gene expression: significance of two peptide regions encoded by exons 1-2 and 6.

Two proteins with alpha-galactosidase activity, alpha-galactosidase A (alpha-GalA) and alpha-galactosidase B (alpha-GalB, or alpha-N-acetylgalactosaminidase; alpha-NAGA) have a high homology of amino-acid sequence. Point mutations of the alpha-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1-2 and/or 6 sequences of alpha-GalA cDNA were partly substituted by the corresponding regions of alpha-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1-2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved alpha-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl alpha-galactopyranoside were compared with those of alpha-GalA. The catalytic activity was slightly low in CMB12, decreased to 1/10 in CMB6, and restored to a significant degree in CMB126. Km was more than 4-fold higher for CMB6 and CMB126 than for alpha-GalA. The pH optimum was 4.0 for both CMB12 and alpha-GalA, 4.8 for CMB6, and 4.6 for CMB126 and alpha-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in alpha-GalB, CMB126, alpha-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Gal alpha 1-6Glc) and methyl alpha-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for alpha-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal alpha-linked galactose. We conclude that (1) the two regions encoded by exons 1-2 and 6 contribute to the alpha-galactosidic cleavage, and (2) an increase in Km of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal alpha-linked galactose.

Amidohydrolases↗
PubMed1994

Distribution of 3 alpha-hydroxysteroid dehydrogenase (bile acid binder) in rat small intestine: comparison with glutathione S-transferase subunits.

We identified and quantitated the Y' bile acid binder, i.e., 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), in rat small intestinal mucosa, and compared its longitudinal distribution with that of glutathione S-transferases (GST). The enzyme activity of 3 alpha-HSD in intestinal mucosa was approximately one-third of that in liver, and it had similar activity in the proximal, middle, and distal portions of the intestine. Immunoreactive protein corresponding to hepatic bile acid binder was detected in all segments of rat small intestine mucosal cytosol by Western blot analysis. There was no significant difference in the concentration of bile acid binder, assayed by enzyme-linked immunosorbent assay (ELISA), between the proximal and the distal intestine, this being 2.93 +/- 0.03 and 3.29 +/- 0.95 nmol/g tissue, respectively (mean +/- SD of four animals). On the other hand, the concentration of GST 1-1 showed sharp longitudinal decline and that of GST 3-4 was negligible in the small intestine, as we previously reported, indicating that bile acid binder was a prominent cytosol binding protein in the distal intestine. These results suggested the possible role of bile acid binder in the intracellular transport of bile acids in the ileum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Characteristics of an attenuated vaccinia virus strain, LC16m0, and its recombinant virus vaccines.

This article reviews studies concerning the characteristics of a vaccinia virus strain, LC16m0, and its recombinant virus vaccines. The LC16m0 strain is one of several temperature-sensitive and further attenuated variants derived from the Lister (Elstree) strain of vaccinia virus, which has a proven safety record in human populations. Several types of recombinant vaccinia viruses expressing a foreign antigen gene from a pathogenic virus have been constructed using the LC16m0 strain as a vector, and their immunological and virological characteristics have been investigated extensively. These studies indicate that the LC16m0 strain has potential as a vector in a live recombinant vaccine. The advantages, disadvantages and future prospects of vector are discussed.

Hepatitis B Vaccines↗
PubMed1994

An improved method of determining free and acetylated polyamines by HPLC involving an enzyme reactor and an electrochemical detector.

We developed an improved system for the simultaneous measurement of free and acetylated polyamines, which comprised a HPLC pump, a separation column, an enzyme reactor, and an electrochemical detector, connected in series. Polyamines were separated with an isocratic elution system, and the separated polyamines were introduced into the enzyme reactor, in which they were deacetylated and oxidized to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with the electrochemical detector. Analysis of a mixture of nine standard polyamines including both free forms and acetylated derivatives with this method revealed that the analytical variables were satisfactory. For the analysis of polyamines in urine, pretreatment of samples with a weakly acidic ion exchange resin was necessary to reduce the interfering substances present in the urine. On successive determinations of polyamines in a urine sample, the coefficients of variation obtained were below 5.4%, except that for spermine (27.6%), and the analytical recovery rates were above 90%, except that for acetylputrescine (78.5%). The correlation coefficient between the total polyamine content in urine estimated by our method and that obtained by means of a commercially available enzymatic assay system was calculated to be 0.98, and the regression equation was expressed as y = 0.81 x + 0.89.

Acetylation↗
PubMed1994

Laser high performance liquid chromatography determination of prostaglandins in nasal lavage fluid in allergic rhinitis.

This study was designed to analyse prostaglandins (PGs) in human nasal lavage fluid using the combination of microcolumn high performance liquid chromatography and a He/Cd laser induced fluorescence detection system. Forty-seven patients with allergic rhinitis and 12 healthy volunteers were investigated. Four species of PG, i.e. PGD2, PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were detected in the nasal lavage fluid. Concentrations of PGD2 (1.33 +/- 0.17 nmol/ml) and PGE2 (0.87 +/- 0.11 nmol/ml) in nasal lavage fluid from patients with allergic rhinitis (the allergy group) were significantly increased compared with those of volunteers (the control group, 0.23 +/- 0.16 nmol/ml, 0.29 +/- 0.19 nmol/ml, respectively). On the other hand, no significant differences were observed in concentrations of either PGF2 alpha or 6-keto-PGF1 alpha between the control group and the allergy group. Histamine concentration in nasal lavage fluid was significantly increased in the allergy group (53 +/- 7.6 nmol/l) compared with the control group (3.4 +/- 1.0 nmol/l). No significant correlation was observed between PGD2 and histamine concentration (r = 0.24), or between PGE2 and histamine concentration (r = 0.08) in nasal lavage fluid from patients with allergic rhinitis. Treatment with oxatomide, an anti-histamine and anti-allergic drug, significantly improved symptom scores, but did not alleviate them completely. Concentrations of each PG detected in nasal lavage fluid did not change significantly after oxatomide treatment. It is concluded that, not only histamine but also PGs, particularly PGD2 and PGE2, might be involved in the genesis of allergic rhinitis.

Adolescent↗
PubMed1994

Basement membrane and carcinogenesis: ultrastructural observations in the basement membrane of the bladder epithelium in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN).

This study investigated the structural alterations in the basement membrane (BM) of the bladder epithelium in rats treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) using transmission electron microscopy. Following administration of BBN, thickening of the BM of the bladder epithelium was observed and remained almost constant from 4 to 20 weeks, although the pathological changes in the rat bladder epithelium induced by BBN altered over the same period of 20 weeks. The reason for this phenomenon can be explained by the increased interfacial area between the basal epithelial cells and the BM of the rat bladder epithelium due to an increase in the number and size of the microvilli on the basal cell surfaces adjacent to the BM. Our results also showed that the frequency of hemidesmosomes increased progressively during the period of carcinogenesis, especially in the lesions of noninvasive transitional cell carcinoma (TCC) in the rat bladder. It is suggested that the neosynthesis of BM components can be carried out both by benign hyperplastic cells and by noninvasive TCC cells of rat bladder. The alterations in the BM thickness may be affected by the changes in the number and size of the microvilli occurring on the basal cell surfaces adjacent to the BM. Both an increased frequency of hemidesmosomes and the neosynthesis of BM are closely related to cell proliferation during carcinogenesis.

Animals↗
PubMed1994

Adult Chédiak-Higashi syndrome presenting as parkinsonism and dementia.

Chédiak-Higashi syndrome (CHS) in children can be a fatal disease. We describe the oldest known CHS patient first seen with a neurologic disorder in early adult life. From the age of 22, this 39-year-old woman developed mental deterioration, parkinsonism including resting tremor at the tongue, mandible, and hands, oculogyric crisis, muscular atrophy of limbs, and loss of tendon reflexes. MRI showed marked temporal dominant brain atrophy and diffuse spinal cord atrophy. Partial albinism, pan-leukocytic giant granules, and profoundly decreased NK-cell activity were compatible with childhood CHS, but apparently normal neutrophil function prolonged her survival. Stimulated proliferation of lymphocytes was less than 40% that of normal controls.

Adult↗
PubMed1994

Amino acid sequence of a porcine zona pellucida glycoprotein ZP4 determined by peptide mapping and cDNA cloning.

Mammalian zona pellucida is an attractive target for developing a contraceptive vaccine. In this study, the amino acid sequence of a core protein of porcine zona pellucida glycoprotein ZP4 was determined by peptide mapping and cDNA cloning. Two kinds of ZP4 peptides were identified: one consisted of 128 amino acid residues and the other of 133 amino acid residues with an additional five amino acid sequence at the carboxy-terminal end of the 128 amino acid peptide. Both peptides had two potential N-linked glycosylation sites. The 128 amino acid peptide showed 39.1% similarity to the amino-terminal region of mouse ZP2 polypeptide. The positions of five cysteine residues were the same for porcine ZP4 and mouse ZP2. The cloned cDNA possessed an additional 195 nucleotides at the 3' end of the sequence corresponding to the 133 amino acid peptide. This additional sequence was found to encode the amino-terminal 10 amino acid sequence of porcine ZP2 polypeptide. These results suggest that porcine ZP4 and ZP2 are derived from a common parent polypeptide by proteolytic cleavage at the position between 133 and 134 residues.

Amino Acid Sequence↗
PubMed1994

Long-term changes in the haversian systems following high-dose irradiation. An ultrastructural and quantitative histomorphological study.

The effects of high-dose irradiation on the morphology of haversian bone were studied, over a fifty-two-week period, in seventy-seven adult rabbits, after the administration of a single dose of radiation (therapeutic x-ray; twenty-five, fifty, or 100 gray) to one knee joint. The specimens of bone were examined with microangiography, light and transmission electron microscopy, and histomorphometry. Analysis was performed on the haversian bone in the subchondral bone plate of weight-bearing portions of the femoral condyles. Microangiography demonstrated dilatation of the microvasculature four weeks after irradiation. Beginning at twelve weeks, there was a marked decrease in vascularity; no obvious recovery of the subchondral bone had occurred by fifty-two weeks. At four weeks, morphological analysis revealed two changes in the haversian canals: simple occlusion of the haversian vessels with loss of cells in the canal, and dilatation of the capillaries with abnormal resorption of the perivascular bone matrix by osteoclasts. The abnormal bone resorption was not coupled with subsequent new-bone formation, resulting in increased porosity. Beginning at four weeks, a progressive decrease in the number of haversian vessels and in cellularity became prominent. The decrease in cellularity involved all types of cells, including endothelial cells, pericytes, perivascular mesenchymal cells, osteoblasts, osteocytes, and osteoclasts. The loss of perivascular cells was often but not always associated with occlusion of the haversian vessels. Histomorphometry revealed both time-dependent and dose-dependent decreases in capillary density (the number of intraosseous capillaries per unit area) and in the number of osteocytes in the subchondral bone plate. The porosity of the same areas showed a significant increase by four weeks (p < 0.001 after administration of twenty-five gray and p < 0.01 after administration of both fifty and 100 gray), but between twelve and fifty-two weeks, there was only a slight additional increase. Statistical analysis revealed significant correlations between capillary density and osteocyte survival (p < 0.001) and between capillary density and porosity (p < 0.001). The portion of the subchondral bone plate that was located farthest from the non-irradiated normal bone showed progressive damage and no sign of recovery at fifty-two weeks.

Animals↗
PubMed1994

Role of cell-mediated immunity in bovine leukemia virus (BLV) infection in ruminants: its implication for the vaccination strategy against retroviruses.

Recent studies in the immunopathogenesis of bovine leukemia virus (BLV) infection in ruminants, reviewed herein, provide an insight into the vaccination strategy against retrovirus infection. It was shown that vaccination of naive sheep with a recombinant vaccinia virus (RVV) expressing BLV envelope glycoprotein protected the animals against BLV infection. The involvement of cell-mediated immunity (CMI) in this phenomenon was strongly suggested. The postinfection vaccination, that is, the vaccination of BLV-infected animals with RVV, also significantly suppressed BLV replication in the carrier animals. These findings support the idea that vaccination against retroviruses should put its emphasis on the induction of CMI and that such vaccine could be used not only for prophylactic but also for therapeutic purposes.

Animals↗