Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.8.1.4 (diaphorase)
2,754 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the role of nitric oxide in the cerebellar degeneration during methylmercury intoxication, interaction of the change in nitric oxide synthase activity and degeneration of the granular layer neurons was examined in rats after methylmercury administration. Both reduced nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and anti-nitric oxide synthase antibody staining, and measurement of glutamate, and nitrite and nitrate levels in the cerebrospinal fluid were performed after oral administration of 5 mg/kg of methylmercury for 12 days. Nitric oxide synthase activity in the cerebellum was also assayed by monitoring the conversion of arginine to citrulline. Methylmercury levels in the blood and the cerebellum gradually increased up to day 13 after the initial methylmercury administration, and neurological disturbances, such as hindleg crossing and abnormal gait, were observed from day 17 after administration. Although a significant decrease in the number of granular layer neurons was recognized at day 84, no such decrease either in NADPH-diaphorase or anti-nitric oxide synthase antibody positive neurons was seen. Glutamate levels in the cerebrospinal fluid transiently increased at day 9 and finally decreased at day 84. Also a transient increase in both nitrite and nitrate levels in the cerebrospinal fluid and nitric oxide synthase activity in the cerebellum was seen prior to the start of degeneration of the granular layer neurons. These results suggest that nitric oxide may play an important role in the degeneration process of the granular layer neurons during methylmercury intoxication.
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PMID:Role of nitric oxide in the cerebellar degeneration during methylmercury intoxication. 910 26

Nitric oxide may serve as a retrograde messenger to refine or stabilize synapses in the developing nervous system. Whether this action is dependent upon glutamate and the N-methyl-D-aspartate receptor is not yet established. We have used the patch-cluster system in the intermediate gray layer (IGL) of the rat superior colliculus (SC), a system receiving both glutamatergic and cholinergic input, to study this question. The normal distribution and development of nitric oxide synthase (NOS) in SC was examined using nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry in Sprague-Dawley rats aged P4 to adulthood. Fibers containing acetylcholine (ACh) were identified using choline acetyltransferase (ChAT) immunocytochemistry. In addition, N omega-nitro-L-arginine, an inhibitor of NOS, was injected intraperitoneally from birth until P10, P14, P18, or P21-22 to determine if NOS inhibition would disrupt the formation of the ACh patches. Control animals were studied from the same age groups. Our results show NADPH-d-labeled cells within the periaqueductal gray and the deep gray layer of SC by P4, the earliest age examined. By P8-P9, cells in the IGL were well labeled by NADPH-d, while few in the superficial layers (SL) were labeled. SL cells were visible by P10 and were intensely labeled by P14. IGL cells transiently expressed NADPH-d in that the number of labeled cells increased from P8 to P35, then decreased in the adult. ChAT-labeled fibers first appeared in the IGL at P10, formed a characteristic two-tier pattern by P14, and established obvious patches by P21. Inhibition of NOS from birth produced no qualitative differences in the distribution or density of either ChAT-labeled fibers or NADPH-d-labeled cells and fibers at any of the ages examined. We therefore conclude that NO does not contribute to the refinement of cholinergic fiber patches in the rat SC, probably because the fiber system is not glutamatergic.
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PMID:Inhibition of nitric oxide synthase fails to disrupt the development of cholinergic fiber patches in the rat superior colliculus. 920 10

To reveal neurones in the cat medulla oblongata involved in carotid baroreceptor/chemoreceptor reflexes, the distribution of c-Fos oncoprotein immunoreactivity was studied following electrical stimulation of the right carotid sinus nerve. The neurochemistry of the activated neurones was investigated using antisera to tyrosine hydroxylase, neuropeptide Y, somatostatin, and glutamate. Nitric oxide containing neurones were identified using antiserum to nitric oxide synthase (NOS) and by the histochemical localization of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase. Following sinus nerve stimulation numerous c-Fos-IR cells were detected both ipsilaterally and contralaterally in the nucleus tractus solitarii, the area postrema and throughout the ventrolateral medulla. Dual labelling studies revealed that 3.3% of c-Fos-immunoreactive cells in the nucleus tractus solitarii were also immunoreactive for tyrosine hydroxylase. The double labelled cells were scattered within the medial and ventrolateral subnuclei, predominantly rostral to obex. A higher proportion (10.3%) of c-Fos-IR cells in the ventrolateral medulla also showed tyrosine hydroxylase immunoreactivity. Caudal to obex, these were scattered in the reticular formation between the spinal trigeminal nucleus and the lateral reticular nucleus, while more rostrally they were found within the lateral reticular nucleus, the nucleus ambiguus and the lateral tegmental field. Cells expressing c-fos and reactive for glutamate, neuropeptide Y or NADPH-diaphorase (or NOS) were only rarely seen, and co-localization of c-Fos and somatostatin immunoreactivities was not seen. These results suggest that of the neurones forming pathways within the medulla activated on carotid sinus nerve stimulation, presumably mediating baro- and chemoreceptor reflexes, relatively few utilize catecholamines, glutamate, neuropeptide Y or nitric oxide as their transmitter substance.
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PMID:Co-localization of c-Fos and neurotransmitter immunoreactivities in the cat brain stem after carotid sinus nerve stimulation. 931 68

A homology-modelled structure of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii, has been generated using the crystal structure of the enzyme from Pseudomonas fluorescens. Analysis of the halophilic enzyme structure identified a potential K+-binding site comprising four co-ordinated glutamate residues (E423 and E426 from each monomer) at the subunit interface of the dimeric protein. Whilst E426 is conserved throughout non-halophilic dihydrolipoamide dehydrogenases, E423 is only present in the halophilic enzyme. Four site-directed mutations of the Haloferax dihydrolipoamide dehydrogenase have been made (E423D, E423Q, E423S, and E423A) and the recombinant mutants expressed and characterised. From an analysis of their kinetic properties, salt-dependent activities and thermal stabilities, it is concluded that this site has an important influence on the halophilicity of the enzyme. The findings support the view that the arrangement and interaction of the negatively charged amino acids are as important as the total net charge in determining the adaptation of proteins to high salt concentrations.
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PMID:Site-directed mutagenesis and halophilicity of dihydrolipoamide dehydrogenase from the halophilic archaeon, Haloferax volcanii. 934 89

Glutamate excitocytotoxicity is implied in the cause of neuronal degeneration in the neostriatum, in which the toxicity may be mediated by different families of glutamate receptors. The precise cellular localization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA)-type glutamate receptor subunits (GluR1-4), one of the major family that involves in the mechanisms of glutamate excitocytotoxicity, in different populations of striatal neurons is therefore of special interest. Immunoreactivity for GluR2/3 subunits was detected in the medium-sized spiny neurons. By double labelling experiments, immunoreactivity for GluR1 and GluR4 was detected only in aspiny striatal neurons that display parvalbumin immunoreactivity, but not in the other neuron populations that display choline acetyltransferase or muscarinic m2 receptor immunoreactivity, nor neurons that display nitric oxide synthase immunoreactivity or nicotinamide adenine dinucleotide phosphate-diaphorase activity. These results indicate that GluR1 and GluR4 immunoreactivity is displayed only in the GABAergic interneurons in the neostriatum. In addition, almost all of the GluR1-immunoreactive neurons were found to display GluR4 immunoreactivity. This finding indicates for the first time that the striatal GABAergic interneurons co-express GluR1 and GluR4 subunits. The results of the present study indicate that there is a differential localization of AMPA-type glutamate receptor subunits in different populations of striatal neurons and they may have a different susceptibility to glutamate excitocytotoxicity.
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PMID:Cellular localization of GluR1, GluR2/3 and GluR4 glutamate receptor subunits in neurons of the rat neostriatum. 946 76

In the central nervous system (CNS), nitric oxide (NO) is thought to be involved in a variety of functions including synaptic plasticity, long term potentiation, and neurotoxicity. The aim of the present study was to investigate the expression of nitric oxide synthase (NOS) in the mouse CNS, following surgical injury to the hippocampus. NOS expression was assessed by histochemical detection of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity and immunohistochemistry of the inducible NOS (iNOS). Two days after injury to the CA1 hippocampal field, NADPH-diaphorase activity was detected in pyramidal and granular neurons and also in glial cells in the hippocampus, in contrast to the non-injured one where NADPH-diaphorase staining was observed only in a few interneurons. NADPH-diaphorase histochemistry combined with immunolabelling for GFAP and F4/80 demonstrated that these glial cells were astrocytes and microglia. This pattern of NOS expression is induced specifically after a hippocampal injury since lesion to the prefrontal or cerebellar cortex leads to NOS activity only in monocytes/macrophages like cells. Despite the large expression of NOS detected by NADPH-diaphorase histochemistry after lesioning the hippocampus, immunostaining for iNOS was confined to microglia. The fact that induction of high levels of NOS activity are detected in glial cells after a lesion to the hippocampus could be accounted for by the sensitivity of this structure to a high release of glutamate.
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PMID:Specific pattern of nitric oxide synthase expression in glial cells after hippocampal injury. 951 65

The substantia gelatinosa of the spinal cord (lamina II) is the major site of integration for nociceptive information. Activation of NMDA glutamate receptor, production of nitric oxide (NO), and enhanced release of substance P and calcitonin gene-related peptide (CGRP) from primary afferents are key events in pain perception and central hyperexcitability. By combining reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry for NO-producing neurons with immunogold labeling for substance P, CGRP, and glutamate, we show that (1) NO-producing neurons in lamina IIi are islet cells; (2) these neurons rarely form synapses onto peptide-immunoreactive profiles; and (3) NADPH diaphorase-positive dendrites are often in close spatial relationship with peptide-containing terminals and are observed at the periphery of type II glomeruli showing glutamate-immunoreactive central endings. By means of confocal fluorescent microscopy in acute spinal cord slices loaded with the Ca2+ indicator Indo-1, we also demonstrate that (1) NMDA evokes a substantial [Ca2+]i increase in a subpopulation of neurons in laminae I-II, with morphological features similar to those of islet cells; (2) a different neuronal population in laminae I-IIo, unresponsive to NMDA, displays a significant [Ca2+]i increase after slice perfusion with either substance P and the NO donor 3morpholinosydnonimine (SIN-1); and (3) the responses to both substance P and SIN-1 are either abolished or significantly inhibited by the NK1 receptor antagonist sendide. These results provide compelling evidence that glutamate released at type II glomeruli triggers the production of NO in islet cells within lamina IIi after NMDA receptor activation. The release of substance P from primary afferents triggered by newly synthesized NO may play a crucial role in the cellular mechanism leading to spinal hyperexcitability and increased pain perception.
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PMID:Nitric oxide-producing islet cells modulate the release of sensory neuropeptides in the rat substantia gelatinosa. 985 75

The roles of lysine-54 (K54) and glutamate-192 (E192) of human dihydrolipoamide dehydrogenase (E3) in stabilizing the thiolate-FAD intermediate during electron transfer were investigated by site-directed mutagenesis. Recombinant human E3s, wild-type, K54E, S53K54-K53S54 (SK-KS), and E192Q, were overexpressed, purified, and characterized. Only K54E and SK-KS E3s had about 25% less bound FAD compared to wild-type, implicating that K54 is crucial for the protein-FAD interaction. The specific activities of all mutant E3s were markedly decreased (<5% wild-type). In the case of K54E E3, the Km for lipoamide in the reverse reaction was increased by about twofold. Surprisingly, for both SK-KS and E192Q E3s, the Kms for both dihydrolipoamide (forward reaction) and lipoamide (reverse reaction) were markedly reduced. The catalytic rate constants (kcat/Km) for both reactions for SK-KS E3 were significantly lower than wild-type, indicating that K54 is crucial for the catalytic efficiency of the enzyme. Fluorescence spectral analyses showed that the FAD in E3s were reduced by the addition of dihydrolipoamide, and that its reoxidation by NAD+ in the mutant E3s was slower than wild-type E3. Interestingly, in K54E E3 dihydrolipoamide reduced FAD efficiently only when NAD+ was present, indicating that K54 stabilizes the thiolate-FAD interaction. The lack of the formation of thiolate-FAD intermediate in the absence of NAD+ in K54E E3 was also confirmed by CD spectra. The SK-KS mutation demonstrates that the correct sequence of residues is as critical as the nature of the amino acid residues. These results suggest that K54 plays an important role in stabilizing the thiolate-FAD intermediate during the electron transfer in the reaction, and E192 is involved in maintaining correct orientation of K54 during catalysis.
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PMID:Site-directed mutagenesis of human dihydrolipoamide dehydrogenase: role of lysine-54 and glutamate-192 in stabilizing the thiolate-FAD intermediate. 1033 57

Stimulation of the nucleus magnocellularis (NMC) of the medulla produces changes in locomotion, muscle tone, heart rate, and blood pressure. Glutamatergic input has been found to modulate muscle tone, whereas cholinergic input has been found to mediate cardiovascular changes produced by stimulation of the NMC. The current study was designed to identify the brainstem afferents to NMC by using retrograde transport of wheat germ agglutinin and horseradish peroxidase (WGA-HRP) combined with glutamate and choline acetyltransferase (ChAT) immunohistochemical and nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemical techniques. Fifty nanoliters of 2.5% WGA-HRP were microinjected into the NMC in the cat. A heavy density of WGA-HRP-labeled neurons was found in the ipsilateral mesencephalic reticular formation (MRF), periaqueductal gray, Kolliker-Fuse nucleus, and pontis centralis caudalis (PoC), in the contralateral pontis centralis oralis (PoO), and bilaterally in the nucleus paragigantocellularis lateralis. A moderate density of retrogradely labeled neurons was found in the ipsilateral side of the nuclei parvocellularis, retrorubral (RRN), PoO, and vestibular complex, in the contralateral PoC and nucleus gigantocellularis, and bilaterally in the inferior vestibular nucleus. Retrograde HRP/glutamate-positive cells could be found throughout the brainstem, with a high percentage in RRN, PoO, PoC, and MRF. Double-labeled WGA-HRP/ChAT neurons were found in the pedunculopontine nucleus. Double-labeled WGA-HRP/NADPH-d-positive neurons could be seen in many nuclei of the brainstem, although the number of labeled neurons was small. The dense glutamatergic projections to the NMC support the hypothesis that rostral brainstem glutamatergic mechanisms regulate muscle activity and locomotor coordination via the NMC, whereas the pontine cholinergic projections to the NMC participate in cardiovascular regulation.
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PMID:Brainstem projections to the ventromedial medulla in cat: retrograde transport horseradish peroxidase and immunohistochemical studies. 1034 May 15

Experiments were carried out to investigate the functional and anatomical relationships between neuronal elements and cerebral microvessels in 300-350-microm thick coronal hippocampal slices maintained at 33-35 degrees C, obtained from 150-200 g male Wistar rats. Cerebral arterioles (9-22 microm in diameter) were visualized in situ and pre-constricted by 22.0+/-6.6% by the addition of the thromboxane A2 agonist U46619 (75 nM), to the bathing medium. The glutamate agonist N-methyl-D-aspartate (0.01-1 mM) produced a dose-related increase in luminal diameter of pre-constricted vessels. In the presence of 4 microM haemoglobin to scavenge nitric oxide from the extravascular environment of the slice, the increase in diameter evoked by 0.1 mM N-methyl-D-aspartate was significantly reduced from 17.5+/-4.6% to 4.8+/-1.7% indicating that N-methyl-D-aspartate-induced vasodilatation of cerebral microvessels is mediated via a mechanism which involves neuronally-derived nitric oxide. In a parallel anatomical study, beta-nicotinamide adenine dinucleotide phosphate-dependent diaphorase staining was used to reveal the enzyme nitric oxide synthase in vascular endothelium and neurons in slices. A small subpopulation (< 11 cells per slice) of darkly-stained multipolar neurons, 21-32 microm in diameter was observed to give rise to a dense network of fine diaphorase-reactive nerve fibres that ramified throughout the whole of the hippocampus and appeared to come into close apposition with arterioles. Morphometric analysis of the relationship between cerebral microvessels, beta-nicotinamide adenine dinucleotide phosphate, reduced form-dependent diaphorase-reactive neuronal elements and individual pyramidal layer neurons, identified by filling with biocytin, revealed that for a given point on a pyramidal layer neuron, the proximity of the nearest diaphorase-reactive nerve fibre was less than 10 microm, whilst the distance to the nearest arteriole (the smallest functional unit for controlling blood flow) was in excess of 70 microm. Such a distance would probably preclude diffusion of vasoactive metabolites in effective concentrations from the area of increased neuronal activity. We therefore propose that the diaphorase-reactive nerve network constitutes the functional link. It is possible that during periods of increased neuronal activity, spillover of glutamate from synapses may activate the diaphorase-reactive network. Release of nitric oxide from the network in the vicinity of local cerebral arterioles may then produce relaxation of the vascular smooth muscle, enabling increased blood flow into the capillary network supplying the region of increased metabolic activity. This study has shown that the process whereby increases in neuronal activity elicit a local change in cerebral blood flow remains functionally intact in hippocampal slice preparations. Nitric oxide of neuronal origin appears to be involved in mediating the coupling between neurons and cerebral arterioles. Stereological analysis of the relationship between neuronal and vascular elements within hippocampal slices suggested that a small subpopulation of nitric oxide synthase-containing neurons which give rise to a diffuse network of fine nitric oxide synthase-containing nerve fibres that lie in close apposition to cerebral arterioles may provide the anatomical substrate for coupling of blood flow to metabolism.
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PMID:Neurovascular relationships in hippocampal slices: physiological and anatomical studies of mechanisms underlying flow-metabolism coupling in intraparenchymal microvessels. 1039 29


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