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M Nakane

Publications and source records attributed to M Nakane.

At least 91 records · Page 5Linked to original sources
PubMed1992

Degeneration of the ipsilateral substantia nigra following cerebral infarction in the striatum.

BACKGROUND AND PURPOSE: In rats, degeneration of the ipsilateral substantia nigra occurs a few weeks after occlusion of the middle cerebral artery. The aim of this study was to clarify whether similar change is observed in stroke patients. METHODS: Eighteen patients with striatal infarction and six patients with cortical infarction in the territory of the middle cerebral artery were examined by means of sequential magnetic resonance imaging. RESULTS: In all patients with striatal infarction, T2-weighted images revealed a high-signal-intensity spot in the ipsilateral substantia nigra. Changes in the ipsilateral substantial nigra appeared at day 14 after stroke on average and then became less intense and smaller a few months after the stroke. By contrast, we observed no nigral changes in any patient with cortical infarction. CONCLUSIONS: The degenerative change in the ipsilateral substantia nigra initially found in the rat model similarly occurred in patients with striatal infarction. This remote change in the substantia nigra may represent magnetic resonance imaging detection of neuropathologic changes in this region through the striatonigral pathway.

Aged↗
PubMed1992

Mapping of neural nitric oxide synthase in the rat suggests frequent co-localization with NADPH diaphorase but not with soluble guanylyl cyclase, and novel paraneural functions for nitrinergic signal transduction.

Nitric oxide synthases (NOS Types I-III) generate nitric oxide (NO), which in turn activates soluble guanylyl cyclase (GC-S). The distribution of this NO-mediated (nitrinergic) signal transduction pathway in the body is unclear. A polyclonal monospecific antibody to rat cerebellum NOS-I and a monoclonal antibody to rat lung GC-S were employed to localize the protein components of this pathway in different rat organs and tissues. We confirmed the localization of NOS-I in neurons of the central and peripheral nervous system, where NO may regulate cerebral blood flow and mediate long-term potentiation. GC-S was located in NOS-negative neurons, indicating that NO acts as an intercellular signal molecule or neurotransmitter. However, NOS-I was not confined to neurons but was widely distributed over several non-neural cell types and tissues. These included glia cells, macula densa of kidney, epithelial cells of lung, uterus, and stomach, and islets of Langerhans. Our findings suggest that NOS-I is the most widely distributed isoform of NOS and, in addition to its neural functions, regulates secretion and non-vascular smooth muscle function. With the exception of bone tissue, NADPH-diaphorase (NADPH-d) activity was generally co-localized with NOS-I immunoreactivity in both neural and non-neural cells, and is a suitable histochemical marker for NOS-I but not a selective neuronal marker.

Amino Acid Oxidoreductases↗
PubMed1992

Regulation and subcellular location of nitrogen oxide synthases in RAW264.7 macrophages.

In nitrinergic signal transduction, nitrogen oxide (NO) synthases (NOS) (EC 1.14.23) catalyze the conversion of L-arginine to L-citrulline and NO, which in turn activates soluble guanylyl cyclase. Macrophages were reported to contain a single isoform of NOS (type II, soluble, Ca(2+)-independent, 130-kDa) and only upon activation of the cells by interferon-gamma (INF) and lipopolysaccharides (LPS). By a mechanism involving L-type Ca2+ channels, calmodulin, and serine proteases, INF/LPS also induce a cytotoxic activation of macrophages. In RAW264.7 macrophages, NO release was detected upon activation of the cells by INF/LPS but also, although at a 20-fold lower level, in control cells. The latter constitutive NOS activity and NO release were Ca2+ dependent and were decreased in INF/LPS-activated RAW264.7 cells or with increasing passage number. RAW264.7 cells did not express soluble guanylyl cyclase, suggesting other target molecules for NO. In INF/LPS-activated cells, NOS activities and NO release were Ca2+ independent (type II) and coinduced with NADPH-diaphorase activities both in the soluble and in the particulate fractions. The NOS-II activities corresponded to a 130-kDa protein, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was not recognized in a protein immunoblot with anti-NOS-I antibody. The serine protease inhibitor tosyl-lysyl chloromethyl ketone abolished the induction of NOS-II by INF/LPS, by depleting intracellular thiol pools and interfering with protein synthesis. Induction of NOS-II by INF/LPS was transcriptionally based and, for maximal enzyme activity, required increased intracellular tetrahydrobiopterin levels, intracellular Ca2+ mobilization, and activation of non-L-type Ca2+ channels but, unlike the induction of macrophage-mediated cytotoxicity, neither L-type-Ca2+ channels nor calmodulin.

Amino Acid Oxidoreductases↗
PubMed1991

Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.

The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.

Amino Acid Oxidoreductases↗
PubMed1991

Phosphorylation by calcium calmodulin-dependent protein kinase II and protein kinase C modulates the activity of nitric oxide synthase.

Nitric oxide synthase purified from rat brain, which is Ca2+ and calmodulin dependent, was phosphorylated by calcium calmodulin-dependent protein kinase II as well as protein kinase C. Phosphorylation by calcium calmodulin-dependent protein kinase II resulted in a marked decrease in enzyme activity (33% of control) without changing the co-factor requirements, whereas a moderate increase in enzyme activity (140% of control) was observed after phosphorylation by protein kinase C. These findings indicate that brain nitric oxide synthase activity may be regulated not only by Ca2+/calmodulin and several co-factors, but also by phosphorylation.

Adenosine Triphosphate↗
PubMed1991

Role of fibronectin in the inhibitory effect of TGF-beta on choline acetyltransferase activity in co-cultures of spinal cord neurons and myotubes.

We have recently shown that the enhanced expression of choline acetyltransferase (ChAT) activity in co-cultures of spinal cord motoneurons and muscle cells was blocked by transforming growth factor-beta (TGF-beta) (Dev. Brain Res., 57, 129-137, 1990). This study was performed to investigate the role of fibronectin in this effect. TGF-beta increased fibronectin level about 2-fold in extracellular matrix of spinal cord cells and skeletal myotubes in culture. Addition of a synthetic polypeptide that competitively inhibits fibronectin binding to its cell surface receptor recovered the TGF-beta-induced suppression of ChAT activity in co-cultures. The polypeptide did not affect ChAT activity in cultures of spinal cord cells alone or in co-cultures without TGF-beta. These results indicate that TGF-beta inhibits the stimulation of ChAT activity in spinal cord neurons in co-culture through a change in the composition and/or amount of fibronectin in the extracellular matrix at neuromuscular contacts.

Animals↗
PubMed1991

Expression of choline acetyltransferase activity in a co-culture of spinal cord and skeletal muscle cells is inhibited by myogenic differentiation inhibitors.

The effect of myogenic differentiation on the expression of choline acetyltransferase (ChAT) activity in co-cultured spinal cord neurons was studied. ChAT activity in spinal cord cells dissociated from 14-day mouse embryos was markedly increased when co-cultured with skeletal myotubes from 20-day embryos. This enhancement of ChAT activity was not observed in the presence of concanavalin A (ConA) or N-methyl-1-deoxynojirimycin (MDJN) which inhibits myoblast fusion, creatine phosphokinase and acetylcholinesterase activities in muscle cells. ChAT activity in spinal cord neurons cultured alone was unaffected by these agents. The inhibitory effect of ConA and MDJN was reversible, with an almost full recovery of ChAT activity following removal of the agents. Addition of ConA or MDJN after myotube formation exerted little inhibitory effect on ChAT activity. The effects of ConA and MDJN on ChAT activity in co-cultures were comparable to those on creatine phosphokinase and acetylcholinesterase. These observations indicate that the neurotrophic effects of skeletal muscle cells on spinal cord neurons are dependent on the differentiation state of the muscle cells.

1-Deoxynojirimycin↗
PubMed1991

Expression of soluble guanylate cyclase activity requires both enzyme subunits.

Soluble guanylate cyclase purified from rat lung exists as a heterodimer of two subunits (70 kDa and 82 kDa). Recent cloning and sequencing of both subunit entities have revealed their primary structures. Transient expression in COS-7 cells by transfection with expression vectors containing the coding regions of the 70 kDa or the 82 kDa subunit cDNA showed no guanylate cyclase activity when cells were transfected with either subunit cDNA alone. However, a marked enzymatic activity was found after transfection with both subunits that was activated by sodium nitroprusside. The combination of separately expressed guanylate cyclase subunits could not reconstitute enzymatic activity in vitro. Furthermore, cotransfection with antisense oligonucleotides against the 70 kDa subunit or the 82 kDa subunit mRNA inhibited the guanylate cyclase activity. These data indicate that both the 70 kDa and the 82 kDa subunits must be present and interactive with each other in order to see basal guanylate cyclase activity and activation with sodium nitroprusside.

Animals↗
PubMed1991

Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.

The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.

Amino Acid Oxidoreductases↗
PubMed1991

Simultaneous establishment of monoclonal antibodies specific for either cyclobutane pyrimidine dimer or (6-4)photoproduct from the same mouse immunized with ultraviolet-irradiated DNA.

Six new monoclonal antibodies (TDM-2, TDM-3, 64M-2, 64M-3, 64M-4 and 64M-5) specific for ultraviolet (UV) induced DNA damage have been established. In the antibody characterization experiments, two TDM antibodies were found to show a dose-dependent binding to UV-irradiated DNA (UV-DNA), decrease of binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, binding to DNA containing cyclobutane thymine dimers, and unchanged binding to UV-DNA after photoisomerization of (6-4)photoproducts to Dewar photoproducts. These results indicated that the epitope of TDM monoclonal antibodies was the cyclobutane pyrimidine dimer in DNA. On the other hand, four 64M antibodies were found to show a dose-dependent binding to UV-DNA, unchanged binding to UV-DNA after cyclobutane pyrimidine dimer photoreactivation, undetectable binding to DNA containing thymine dimers, and decrease of binding to UV-DNA after photoisomerization of (6-4)photoproducts. These results indicated that the epitope of 64M antibodies was the (6-4)photoproduct in DNA. This is the first report of the simultaneous establishment of monoclonal antibodies against the two different types of photolesions from the same mouse. By using these monoclonal antibodies, we have succeeded in measuring both cyclobutane pyrimidine dimers and (6-4)photoproducts in the DNA from human primary cells irradiated with physiological UV doses.

Animals↗
PubMed1990

Inhibition by transforming growth factor beta of choline acetyltransferase stimulation in a co-culture of spinal cord and muscle cells from mice.

Choline acetyltransferase (CAT) activity increased 11-fold in co-cultures of spinal cord and muscle cells from fetal mice relative to cultures of spinal cord cells alone. The addition of transforming growth factor-beta (TGF-beta) to the medium at 30 pM throughout the culture period inhibited the increase of CAT activity in the co-cultures, but did not affect the activity in cultures of spinal cord cells alone. TGF-beta did not inhibit glutamic acid decarboxylase activity in the co-cultures. Other growth factors such as epidermal growth factor, fibroblast growth factor and beta-NGF had little or no effect on CAT activity. TGF-beta markedly inhibited the fusion of myoblasts to myotubes and the expression of marker enzymes for muscle differentiation. When TGF-beta was included during muscle culture and removed before inoculation with spinal cord cells, myoblasts did not subsequently form myotubes. CAT activity in the spinal cord cells, however, markedly increased in co-cultures with the undifferentiated myoblasts. When TGF-beta was added to the co-cultures after myotube formation was complete, the increase in CAT activity was inhibited according to the length of TGF-beta treatment. These results suggest that TGF-beta inhibits the muscle-induced stimulation of CAT activity by inhibiting the production, secretion and/or action of trophic factors from muscle.

Acetylcholinesterase↗
PubMed1990

Molecular cloning and expression of cDNAs coding for soluble guanylate cyclase from rat lung.

Complementary DNA clones corresponding to the 70- and 82-kDa subunits of soluble guanylate cyclase of rat lung have been isolated. Blot hybridization of total poly(A)+ RNA from rat tissues detected mRNA of about 3.4 kilobases for the 70-kDa subunit and about 5.5 kilobases for the 82-kDa subunit. Messenger RNA levels of both subunits were abundant in lung and cerebrum, moderate in cerebellum, heart, and kidney, and low in liver and muscle, consistent with previously described enzyme activities in these tissues. Southern blot analysis of high molecular weight genomic DNA from rat liver indicated that the genes for the 70- and 82-kDa subunits are different. The carboxyl-terminal region of the 70- and 82-kDa subunits showed a high degree of homology and also had a partial homology with the putative catalytic domain of particulate guanylate cyclase and adenylate cyclase, indicating that both the 70- and 82-kDa subunits have catalytic domains. The cDNAs were subcloned to an expression vector and transfected to L cells. The cells transfected with cDNA of the 70-kDa subunit or the 82-kDa subunit showed no guanylate cyclase activity, whereas the cells transfected with both the 70- and 82-kDa subunit cDNAs showed significant guanylate cyclase activity that was activated markedly by sodium nitroprusside. These data suggest that both subunits are required for both the basal catalytic and regulatory activity of soluble guanylate cyclase. Presumably both catalytic subunits must be present and interactive to permit synthesis of cyclic GMP and nitrovasodilator activation.

Amino Acid Sequence↗
PubMed1990

9,11-epoxy-9-homoprosta-5-enoic acid analogues as thromboxane A2 receptor antagonists.

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha, 2 alpha(Z),3 alpha(1E,3S*,4R*),4 alpha]-7-[3-(3-hydroxy-4-phenyl-1-pentenyl)-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (4), was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Alcohol 4 was the only member in a series of allylic alcohols which did not display direct contractile activity in the rat stomach strip model. Alcohol 4 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.65 +/- 0.1 microM); (b) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (pA2 = 8.0 +/- 0.2) or rat aorta (pA2 = 8.1 +/- 0.2); and (c) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (1 mg/kg iv). A radioiodinated analogue of 4 bound in a specific and saturable manner to human platelet membranes with a Kd = 2.3 +/- 0.9 nM. Modification of the alpha-chain, in an attempt to minimize in vivo metabolism, resulted in TxA2 receptor antagonists of reduced in vitro potency.

Animals↗
PubMed1990

7-Oxabicyclo[2.2.1]heptyl carboxylic acids as thromboxane A2 antagonists: aza omega-chain analogues.

A novel bicyclic prostaglandin analogue, [1S-[1 alpha, 2 alpha (Z), 3 alpha, 4 alpha]]-7-[3-[[[[(1- Oxoheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hept-2- yl]-5-heptenoic acid [-)-7) was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Unlike the related series of omega-chain allylic alcohols, amide 7 and its congeners were uniformly free of direct contractile activity in vitro (bovine coronary) and in vivo (anesthetized guinea pig). Amide 7 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.18 +/- 0.006 microM), (b) 11,9-epoxymethano-PGH2 induced platelet aggregation of human platelet-rich plasma (I50 = 0.24 microM), (c) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (Kb = 3.0 +/- 0.3 nM) or rat aorta (Kb = 8.8 +/- 1.1 nM), and (d) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (0.1-1.0 mg/kg iv). Amide 7 inhibited the binding of [5,6-3H2]-[1S- (1 alpha, 2 alpha (Z), 3 alpha, 4 alpha)]-7-[3-[[2-[(Phenyl- amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid to human platelet membranes in a specific and saturable manner with a Kd = 49.6 +/- 1.4 nM.

Animals↗