Biomedical subjects

T Yoshimura

Publications and source records attributed to T Yoshimura.

At least 181 records · Page 10Linked to original sources

PubMed1998

Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.

D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-decarboxylation [Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. M., Ringe, D., and Soda, K. (1992) Biochemistry 31, 6018-6023; Bhatia, M. B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K., and Manning, J. M. (1993) J. Biol. Chem. 268, 17687-17694]. beta-Decarboxylation of d-aspartate to d-alanine leads also to this inactivation [Jones, W. M., van Ophem, P. W., Pospischil, M. A., Ringe, D., Petsko, G., Soda, K., and Manning, J. M. (1996) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by d-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2 fragment derived from the substrate attached to the C'-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of d-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.

Alanine↗

PubMed1998

Isolation and characterization of a bovine neural specific protein (CRMP-2) cDNA homologous to unc-33, a C. elegans gene implicated in axonal outgrowth and guidance.

We have cloned the cDNA encoding bovine CRMP-2 from bovine brains. A full length cDNA encoding bovine CRMP-2 was isolated and sequenced. The deduced amino acid sequence reveals that the gene encodes a protein of 572 amino acids and is highly homologous to Caenorhabditis elegans unc-33, which controls the guidance and outgrowth of neuronal axons. The CRMP-2 transcript was present in bovine brains but not non-neural tissues, and its protein product existed in both soluble and membrane-bound forms. The expression of CRMP-2 protein and mRNA was upregulated during neuronal differentiation of rat PC12 cells. Immunoprecipitation of PC12 cell extracts shows that CRMP-2 was co-immunoprecipitated with a 190 kDa protein (p190). Both CRMP-2 and p190 were phosphorylated on serine residues in vivo and in vitro in a kinase assay of CRMP-2 immune complexes. Immunocytochemistry shows that CRMP-2 was exclusively localized in both the central and peripheral nervous systems in mouse embryos and detectable in the adult brain although the level of CRMP-2 decreased. The protein was expressed in the axon, dendrite, and cytoplasm of postmitotic neurons and in the cytoplasm of oligodendrocytes and astrocytes. The CRMP-2 gene maps to the region of mouse chromosome 14 syntenic with human chromosome 8p21. Taken together, these data suggest multi-functional roles for CRMP-2 in developing and adult nervous systems, and the biological activity of CRMP-2 could be regulated by phosphorylation reaction.

Amino Acid Sequence↗

PubMed1998

Identification of histidine 45 as the axial heme iron ligand of heme oxygenase-2.

A truncated, soluble, and enzymatically active form of human heme oxygenase-2 (DeltaHHO2) was expressed in Escherichia coli. To identify the axial heme ligand of HO-2, His-45 to Ala (DeltaH45A) and His-152 to Ala (DeltaH152A) mutants have been prepared using this expression system. DeltaH45A could form a 1:1 complex with hemin but was completely devoid of the heme degradation activity. A 5-coordinate-type ferrous NO EPR spectrum was observed for the heme-DeltaH45A complex. The DeltaH152A mutant was expressed as an inclusion body and was recovered from the lysis pellet by dissolution in urea followed by dialysis. The solubilized fraction obtained, however, was composed of a mixture of a functional enzyme and an inactive fraction. The inactive fraction was removed by Sephadex G-75 column chromatography since it eluted out of the column at the void volume. The gel filtration-purified DeltaH152A exhibited spectroscopic and enzymatic properties identical to those of wild-type. We conclude, in contrast to the previous reports (McCoubrey and Maines (1993) Arch. Biochem. Biophys. 302, 402-408; McCoubrey, W. K., Jr., Huang, T. J., and Maines, M. (1997) J. Biol. Chem. 272, 12568-12574), that His-45, but not His-152, in heme oxygenase isoform-2 is the proximal ligand of the heme and is essential for the heme degradation activity of the enzyme. His-152 appears to play a structural role in stabilization of the heme oxygenase protein.

Electron Spin Resonance Spectroscopy↗

PubMed1998

Nonstereospecific transamination catalyzed by pyridoxal phosphate-dependent amino acid racemases of broad substrate specificity.

Pyridoxal 5'-phosphate-dependent amino acid racemases of broad substrate specificity catalyze transamination as a side reaction. We studied the stereospecificities for hydrogen abstraction from C-4' of the bound pyridoxamine 5'-phosphate during transamination from pyridoxamine 5'-phosphate to pyruvate catalyzed by three amino acid racemases of broad substrate specificity. When the enzymes were incubated with (4'S)- or (4'R)-[4'-3H]pyridoxamine 5'-phosphate in the presence of pyruvate, tritium was released into the solvent from both pyridoxamine 5'-phosphates. Thus, these enzymes abstract a hydrogen nonstereospecifically from C-4' of the coenzyme in contrast to the other pyridoxal 5'-phosphate-dependent enzymes so far studied, which catalyze the stereospecific hydrogen removal. Amino acid racemase of broad substrate specificity from Pseudomonas putida produced D- and L-glutamate from alpha-ketoglutarate through the transamination with L-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the nonstereospecific overall transamination between L-ornithine and alpha-ketoglutarate. The cleavage and formation of the C-H bond at C-4' of the coenzyme and C-2 of the substrate thus occurs nonstereospecifically on both sides of the plane of the coenzyme-substrate complex intermediate. Amino acid racemase of broad substrate specificity is the first example of a pyridoxal enzyme catalyzing nonstereospecific transamination.

Amino Acid Isomerases↗

PubMed1998

Mutations of connexin32 in Charcot-Marie-Tooth disease type X interfere with cell-to-cell communication but not cell proliferation and myelin-specific gene expression.

Connexin32 (Cx32) is a gap junction protein and its mutations are responsible for X-linked Charcot-Marie-Tooth disease. We examined the functional abnormality of C6 glioma cells transfected with mutant (C53S and P172R) Cx32 genes. Nontransfected C6 did not express Cx32. Northern and Western blot analyses showed Cx32 mRNA and protein in cells with the wild-type gene as well as with the mutant Cx32 genes. An immunocytochemical study of cells with the wild-type gene showed the immunoreactive spots in the cell membrane. In cells with C53S or P172R mutant gene, however, the immunoreactivity was found in the cytoplasm. The scrape-loading method produced effective dye transfer in cells with the wild-type gene but not in those with mutant genes. A cell proliferation assay showed no differences in nontransfected cells, cells with the wild-type gene and those with the mutant genes. Messenger RNA expression for proteolipid protein did not change. These findings suggest that Cx32 gene mutation results in loss of cell-to-cell communication because of failure to incorporate Cx32 protein in the cell membrane. The mutations do not, however, interfere with cell proliferation or myelin-specific gene expression, at least myelin proteolipid protein expression in C6 glioma cells.

Blotting, Northern↗

PubMed1998

Acute glucosuria after continuous glucocorticoid loading in the rat in vivo.

We investigated the effects of the continuous infusion of various steroids in rats on renal tubular reabsorption of glucose in vivo to elucidate the pathogenesis of steroid-induced glucosuria. Urinary glucose excretion increased 60 min after administration of dexamethasone (2.38 mM). By 120 min, urinary excretion of glucose was three times higher in the dexamethasone group than in the control group (24.1 +/- 4.6 versus 72.4 +/- 16.7 micromol); the plasma level of glucose did not increase. Dexamethasone had no effect on the resorption of 1,5-anhydro-D-glucitol, which is a glucose-resembling polyol that is actively absorbed by the renal tubules as glucose. Neither estradiol nor progesterone increased urinary excretion of glucose. These findings suggest that continuous administration of a high-dose glucocorticoid selectively influences the glucose reabsorption system in the kidney.

Animals↗

PubMed1998

The oxygen and carbon monoxide reactions of heme oxygenase.

The O2 and CO reactions with the heme, alpha-hydroxyheme, and verdoheme complexes of heme oxygenase have been studied. The heme complexes of heme oxygenase isoforms-1 and -2 have similar O2 and CO binding properties. The O2 affinities are very high, KO2 = 30-80 microM-1, which is 30-90-fold greater than those of mammalian myoglobins. The O2 association rate constants are similar to those for myoglobins (kO2' = 7-20 microM-1 s-1), whereas the O2 dissociation rates are remarkably slow (kO2 = 0.25 s-1), implying the presence of very favorable interactions between bound O2 and protein residues in the heme pocket. The CO affinities estimated for both isoforms are only 1-6-fold higher than the corresponding O2 affinities. Thus, heme oxygenase discriminates much more strongly against CO binding than either myoglobin or hemoglobin. The CO binding reactions with the ferrous alpha-hydroxyheme complex are similar to those of the protoheme complex, and hydroxylation at the alpha-meso position does not appear to affect the reactivity of the iron atom. In contrast, the CO affinities of the verdoheme complexes are >10,000 times weaker than those of the heme complexes because of a 100-fold slower association rate constant (kCO' approximately 0. 004 microM-1 s-1) and a 300-fold greater dissociation rate constant (kCO approximately 3 s-1) compared with the corresponding rate constants of the protoheme and alpha-hydroxyheme complexes. The positive charge on the verdoporphyrin ring causes a large decrease in reactivity of the iron.

Animals↗

PubMed1998

Decline of circadian photosensitivity associated with retinal degeneration in CBA/J-rd/rd mice.

Although it is well known that visual image formation is mediated by rods and cones, photoreceptors implicated in entrainment of circadian rhythms remain unknown. In our previous study, 12-week-old CBA/J-rd/rd mice were found to show decreased sensitivity in circadian photoreception. Because rd gene induces different age-related degeneration of cones and rods, we have examined age-related changes in the sensitivity to a light pulse for phase-shifting the locomotor activity rhythms in CBA/J-rd/rd mice. Since mice have middlewave-sensitive cones (M-cone, 510 nm) and shortwave-sensitive cones (S-cone, 360 nm), a 15-min monochromatic light pulse (501.6 or 359 nm; 10 microW cm(-2) s(-1)) was given at circadian time (CT) 16, 2 weeks after being transferred to constant darkness (DD) and the magnitude of phase shift was determined. The amount of phase shift by a green light pulse (501.6 nm) gradually declined from 8 to 16 weeks after birth and the similar tendency was also observed on a UV pulse (359 nm). The similar pattern of age-related decline in the number of cones and circadian photosensitivity in CBA/J-rd/rd mice suggest that cones are involved in the circadian photoreception in mice.

Analysis of Variance↗

PubMed1998

Nonspecific and immune-specific up-regulation of cytokines in rabbit dermal tuberculous (BCG) lesions.

To our knowledge, this is the first sequential study of cytokines in tissue sections of developing and healing tuberculous (BCG) lesions. In situ hybridization, immunohistochemical, and RT-PCR techniques were used. Cytokine mRNAs showed a biphasic pattern. The percentage of mononuclear cells (MN) containing IL-1beta, TNF-alpha, MCP-1, and IL-8 mRNAs was highest in 1- to 3-day lesions, apparently because of the nonspecific inflammatory response caused by the tubercle bacilli in the BCG vaccine. At 5 days, this percentage was significantly reduced. With IFN-gamma, the peak and trough were delayed by 2 days. By 9 days, the percentage of MN containing the mRNAs of all five cytokines had again increased and the rabbits had become tuberculin-positive. In general, MCP-1 and TNF-alpha proteins and the vascular adhesion molecules, ICAM, VCAM, and perhaps ELAM, peaked at about 3 days. Many mononuclear cells surrounding the central areas of solid and liquefied caseous necrosis contained chemokine IL-8 mRNA. IL-8 is known to attract PMN, and PMN were present nearby. In contrast, MN containing chemokine MCP-1 mRNA were present more peripherally in areas rich in macrophages and lymphocytes. The early nonspecific cytokine response seems to be an adjuvant effect of the mycobacteria in BCG vaccine in that it causes a rapid entry of macrophages, lymphocytes, granulocytes, and probably dendritic cells into local sites of antigen deposition. This effect should be considered in developing improved vaccines for the prevention of tuberculosis, because BCG vaccines producing a strong early cytokine response should be more immunogenic than BCG vaccines with similar antigens producing a weak response.

Animals↗

PubMed1998

IL-8 is an essential mediator of the increased delayed-phase vascular permeability in LPS-induced rabbit pleurisy.

We investigated the involvement of IL-8 in the delayed vascular permeability (VP) in rabbit lipopolysaccharide (LPS)-pleurisy. Maximal level of interleukin-8 (IL-8) was detected in pleural fluid at 2 h after LPS injection and anti-IL-8 inhibited the delayed VP by 90%. Injection of homologous IL-8 induced VP, the time-course of which preceded that of LPS-induced delayed VP. Production of IL-8 in LPS-pleurisy was inhibited with anti-tumor necrosis factor alpha (TNF-alpha), whereas the production of TNF-alpha was not affected with anti-IL-8. Injection of IL-8 did not induce TNF-alpha production and anti-TNF-alpha had no effect on IL-8-induced VP. Injection of homologous TNF-alpha induced IL-8 production and VP, and TNF-alpha-induced delayed VP was blocked with anti-IL-8. These results indicate important roles of IL-8 in LPS-induced delayed VP and that TNF-alpha causes the delayed VP through the production of IL-8.

Animals↗

PubMed1998

Interleukin 8 and monocyte chemoattractant protein 1 production by cultured human airway smooth muscle cells.

Leukocyte accumulation and activation are key events in the pathogenesis of inflammatory lung disease. The ability of human airway smooth muscle cells (HASM) to contribute to the inflammatory process by its ability to produce the chemokines interleukin (IL) 8, monocyte chemotactic protein (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) was investigated. Cultured HASM, when stimulated with the pro-inflammatory cytokines IL-1 alpha (0.01-1 ng/ml) or tumour necrosis factor alpha (TNF-alpha, 0.3-30 ng/ml), synthesize and release substantial amounts of IL-8, as assessed by specific immunoassay, bioasssay (elevation of intracellular free calcium in human neutrophils), and upregulation of mRNA. These stimuli also increased MCP-1 production and mRNA expression, but RANTES mRNA expression was not detected at 24 h. The smooth muscle spasmogen endothelin 1 (1 microM) was unable to stimulate IL-8 or MCP-1 release or mRNA expression. These data indicate that HASM may constitute an important source of leukocyte attractants in the inflamed lung, where the inducing stimuli, IL-1 alpha and TNF-alpha, are also likely to be present.

Bronchi↗

PubMed1998

Immunohistochemical demonstration of aberrant glycosylation and epidermal growth factor receptor in tubal metaplasia of the uterine cervix.

Tubal metaplasia (TM) of the uterine cervix may be confused with endocervical glandular dysplasia or adenocarcinoma of the uterine cervix in histologic specimens. TM is characterized histologically by architecturally normal endocervical glands containing ciliated or clear cells, nonciliated cells, and intercalated or peg cells, resembling normal tubal mucosa. Although several characteristics of TM are still undetermined, TM is considered a benign lesion. In this study, the expressions of epithelial-specific antigen (ESA), epidermal growth factor receptor (EGFR), and fucosyl SSEA1 (Le(y)) antigens were examined by an immunohistochemical method in 14 cases of TM in the uterine cervix (5 with normal endocervix, 5 with endocervical glandular dysplasia, 3 with adenocarcinoma in situ, and 1 with microinvasive adenocarcinoma). In the normal endocervical cells, expression of the EGFR antigen was rarely observed in the cytoplasm, and ESA staining was generally restricted to the basolateral membrane. Le(y) expression was occasionally observed in the cytoplasm of subcolumnar reserve cells. On the other hand, expression of EGFR, ESA, and Le(y) was observed in the cytoplasm of TM as well as in adenocarcinomas of the uterine cervix. These results suggest that TM in the uterine cervix might possess a neoplastic character.

ErbB Receptors↗

PubMed1998

Vascular leiomyosarcoma arising from the inferior vena cava diagnosed by intraluminal biopsy.

A 61-year-old woman developed pain in the right thigh, paraplagia of the lower extremities and lumbago in November 1996. A lumbar spine roentgenogram showed lytic change in L2, and magnetic resonance imaging showed a patchy destructive lesion and compression of the dural sac from the right by a tumour. Computed tomography (CT) myelography showed a motheaten destructive lesion in L2 and projection of the tumour into the spinal canal. Abdominal ultrasound, CT and cavography showed dilatation of the inferior vena cava (IVC) and an intraluminal tumour about 2x2.8x4 cm in size in the IVC. The tumour arose from the IVC just beneath the renal vein and extended to just short of the right atrium. Both vertebral and intraluminal biopsy materials showed the same morphology, in which atypical spindle cells admixed with multinucleated giant cells proliferated in a fascicular growth pattern. Neoplastic cells were strongly positive for alpha-smooth muscle actin. We diagnosed vascular leiomyosarcoma arising from the IVC with metastasis to the lumbar vertebrae. Cases of vascular leiomyosarcoma diagnosed by intraluminal biopsy are rare.

Biopsy↗

PubMed1998

Impact of retrograde cerebral perfusion with posterolateral thoracotomy on distal arch aneurysm repair.

BACKGROUND: Repair of distal aortic arch aneurysms is difficult to accomplish through a median sternotomy or left thoracotomy, and stroke and respiratory disorders often become lethal complications with the use of circulatory arrest. We investigated the use of retrograde cerebral perfusion with a posterolateral thoracotomy in the repair of distal arch aneurysms. METHODS: Thirty-eight patients underwent repair of a distal arch aneurysm. They were divided into three groups according to the method of surgical repair used. Sixteen patients (group I) underwent proximal anastomosis of the graft with the use of an aortic cross-clamp. Eight patients (group II) underwent open proximal anastomosis with the use of retrograde cerebral perfusion (oxygenated blood perfusion through a superior vena cava cannula) and a median sternotomy and anterolateral thoracotomy. Fourteen patients (group III) also underwent open anastomosis with the use of retrograde cerebral perfusion (cerebral perfusion through blood returned to the right atrium with the patient in the Trendelenburg position) and a posterolateral thoracotomy. RESULTS: The operative mortality rate in group I was 25.0%; 4 of 16 patients died of stroke, myocardial infarction, and intestinal necrosis. In group II, 3 of 8 patients (37.5%) died of respiratory failure and aortic dissection. In group III, only 1 of 14 patients (7.1%) died, as a result of heart failure. CONCLUSIONS: The use of retrograde cerebral perfusion with a posterolateral thoracotomy is an alternative method that minimizes the risk of stroke and respiratory failure during distal aortic arch operations.

Adult↗

PubMed1998

Cholesterol 7-hydroxylase knockout mouse: a model for monohydroxy bile acid-related neonatal cholestasis.

BACKGROUND & AIMS: Cyp 7-/- mice lack a functional cholesterol 7alpha-hydroxylase enzyme and develop cholestasis before up-regulation of 27-hydroxycholesterol 7alpha-hydroxylase activity. Because 7alpha-hydroxylation is not the initial step in this metabolic pathway, we tested the hypothesis that cholesterol 27-hydroxylase is expressed at an earlier step and leads to the production of monohydroxy bile acids. METHODS: Polymerase chain reaction with specific oligonucleotides was used to detect messenger RNA (mRNA) coding for cholesterol 27-hydroxylase in 5-day-old normal and Cyp 7-/- mice. Gas-liquid chromatography-mass spectrometry and reverse isotope dilution were used to identify intermediates in the cholesterol 27-hydroxylase metabolic pathway. Light and electron microscopy were used to evaluate the morphological appearance of the liver. RESULTS: mRNA for cholesterol 27-hydroxylase was identified in the liver and spleen. The monohydroxy bile acids 3beta-hydroxy-5-cholenoate and 3alpha-hydroxy-5beta-cholanoate together with their precursor, 27-hydroxycholesterol, were identified in liver homogenates. Cholestasis, present focally, was manifested as dilated bile canaliculi, partial loss of microvilli, and retention of electron-dense biliary material. CONCLUSIONS: The cholesterol 27-hydroxylase metabolic pathway of bile acid synthesis is expressed in neonatal life. The absence of 7alpha-hydroxylase activities unmasks the cholestatic potential of monohydroxy bile acids. The Cyp 7-/- knockout mouse mimics cholestatic events known to occur in humans and provides a unique opportunity for studying regulatory determinants.

Animals↗

PubMed1998

Chemokine production and adhesion molecule expression by neural cells exposed to IL-1, TNF alpha and interferon gamma.

We investigated the effect of TNF alpha, IL-1alpha and IFN gamma on two neuroblastoma (NB) cell lines (SK-N-SH and SK-N-MC). These lines responded differentially to IL-1alpha, TNF alpha and IFN gamma for MCP-1 and IL-8 production and expression of the ICAM-1 and VCAM-1 adhesion molecules. None of the cytokines induced MCP-1 or IL-8 on SK-N-MC cells. Both chemokines were produced in response to IL-1alpha by SK-N-SH cells, while TNF alpha induced mainly MCP-1 production. Addition of IFN gamma decreased IL-8, but not MCP-1 production. These responses correlated with monocyte and neutrophil chemotactic activity in NB culture supernatants. This activity was neutralized by antibodies to IL-8 and MCP-1. The expression of ICAM-1 on SK-N-MC was up-regulated by TNF alpha or IFN gamma, while IL-1alpha also upregulated ICAM-1 on SK-N-SH cells. VCAM-1 expression on SK-N-SH was induced by IL-1alpha and TNF alpha and IFN gamma synergized with TNF alpha in this respect on both NB cell lines. These results suggest that mechanisms for chemokine production and VCAM-1 and ICAM-1 upregulation by inflammatory cytokines differ and IFN gamma, in conjunction with TNF alpha, stimulate neural cell responses (high MCP-1 and VCAM-1 and decreased IL-8) favouring mononuclear cell recruitment.

Antibodies, Monoclonal↗

PubMed1998

Rare case of autoimmune hyperchylomicronemia during pregnancy.

A pregnant woman was found to have severe hypertriglyceridemia, fasting chylomicronemia, and low platelet count. The activities of serum lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) proved to be extremely low. The activities of the enzymes in normal plasma were completely inhibited by addition of the patient's plasma. We concluded that autoantibodies to lipases were responsible for this patient's hypertriglyceridemia.

Adult↗

PubMed1998

Modulation of Th1- and Th2-like cytokine production from mitogen-stimulated human peripheral blood mononuclear cells by phosphodiesterase inhibitors.

1. Effects of phosphodiesterase (PDE) inhibitors on the production of IFN-gamma, IL-2, IL-4 and IL-5 by phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) were investigated. In addition, we investigated the effects of dibutyrylcyclic AMP (dbcAMP) and a beta-adrenoceptor agonist on production of these cytokines. 2. Type IV, type III and nonselective PDE inhibitors were effective at inhibiting the production of IFN-gamma and IL-2 production in a dose-dependent manner. In contrast, IL-4 and IL-5 production was inhibited by only the highest concentration of type IV inhibitor, and other agents had no effect on the production. 3. Similarly, dbcAMP inhibited the production of IFN-gamma and IL-2 more potently than IL-4 and IL-5. 4. The addition of a beta-adrenoceptor agonist increased the inhibitory effect of PDE inhibitors tested on the production of IFN-gamma and IL-2. 5. These results indicate that PDE inhibitors or cAMP-elevating agents modulate Th1 cytokine more effectively than Th2 cytokine production.

Adrenergic beta-Agonists↗