Biomedical subjects

V Riveros-Moreno

Publications and source records attributed to V Riveros-Moreno.

At least 19 recordsLinked to original sources

PubMed2001

Impaired response to interferon-gamma in activated macrophages due to tyrosine nitration of STAT1 by endogenous nitric oxide.

1. Inducible NO synthase (iNOS) expression and activity were measured in the mouse macrophage cell line J774 after exposure to bacterial lipopolysaccharide (LPS) with or without interferon-gamma (IFN-gamma). 2. Inhibition of NOS activity by concomitant N(G)-monomethyl-L-arginine (L-NMMA) treatment further increased iNOS protein levels, with a substantial increase in iNOS half-life. 3. Western blotting and ELISA demonstrated that several cell proteins were tyrosine-nitrated when iNOS activity was high. 4. Rapid IFN-gamma-induced phosphorylation of STAT1 was reduced by about 40% when cells were pretreated to induce iNOS, unless L-NMMA was present during the pretreatment period. 2D gel electrophoresis demonstrated the presence of nitrotyrosine in STAT1 after iNOS induction, and confirmed the reduction in phospho-STAT1 on subsequent stimulation with IFN-gamma for 15 min and its partial restoration when L-NMMA was present during the pretreatment period. 5. We did not detect tyrosine nitration of the upstream kinase JAK2 in LPS+IFN-gamma pretreated cells, but JAK2 activity was also impaired, and was partially restored by concomitant L-NMMA pretreatment. 6. We conclude that endogenous production of NO induces feedback inhibition of signalling pathways activated by IFN-gamma, at least in part by nitrating tyrosine residues in STAT1 which prevents phosphorylation.

Animals↗

PubMed1999

Nitrotyrosine formation after activation of murine macrophages with mycobacteria and mycobacterial lipoarabinomannan.

Murine peritoneal macrophages, elicited with thioglycollate, were stimulated in vitro with lipopolysaccharide (LPS). The production of nitrite, superoxide anion (SOA), and the accumulation of nitrotyrosine in the cells increased after treatment, and all were inhibitable by the NO synthase inhibitor NG-monomethyl-L-arginine monoacetate (L-NMMA). This effect suggests a direct correlation between the accumulation of those metabolites and NO synthase activity. Lipoarabinomannan (LAM) purified from Mycobacterium tuberculosis was added to peritoneal macrophages in the presence of interferon-gamma (IFN-gamma); the cells produced nitrite and SOA, both inhibitable by L-NMMA. There was, as well, accumulation of nitrotyrosine in the macrophage proteins. Strikingly, the amount of nitrotyrosine measured after LAM plus IFN-gamma, or LAM plus the low molecular weight adjuvant glutamylmuramyl dipeptide (GMDP), increased significantly in the presence of L-NMMA. These results suggest that murine macrophages, upon LAM stimulation, might generate reactive nitrogen metabolites by a route other than NO synthase. Nitrotyrosine accumulation after infection of macrophages in vitro, with either live bacille Calmette-Guérin (BCG) or live M. tuberculosis, in the presence or absence of IFN-gamma, showed no correlation with nitrite production, suggesting a low superoxide production.

Animals↗

PubMed1998

Regulation by endogenous INTERLEUKIN-10 of the expression of nitric oxide synthase induced after ligation of CD23 in human macrophages.

The possible role of interleukin 10 (IL-10) as an endogenous inhibitor of CD23-driven inducible nitric oxide synthase (iNOS) expression in human macrophages was investigated. Cross-linking of CD23 by a monoclonal antibody induced iNOS mRNA, as detected by RT-PCR, and the production of NO measured as the stable derivative, nitrite. A linear correlation was observed between CD23 expression and iNOS activity or NO2- production. The iNOS activity reached a maximum 48 h after ligation of CD23, then declined rapidly until 72 h. In parallel, nitrite production was detected after 24 h and reached a maximum after 48 h. In addition, ligation of the CD23 molecule induced, in a time-dependent manner, the production of IL-10. As this cytokine is known to regulate iNOS induction and activity, we evaluated the effect of a neutralizing mAb to IL-10 on CD23-induced iNOS activity and nitrite production by CD23-bearing macrophages and found that both were significantly enhanced. Furthermore, the addition of exogenous IL-10 suppressed CD23-driven iNOS mRNA expression, iNOS activity and production of nitrite. These data suggest that, after CD23-ligation at the cell surface of human phagocytes, the secretion of IL-10 downregulates the CD23-induced NO production at the transcriptional level, thus providing an efficient feed-back mechanism.

Amino Acid Sequence↗

PubMed1997

Subcellular localization of nitric oxide synthase in the cerebral ventricular system, subfornical organ, area postrema, and blood vessels of the rat brain.

The distribution of neuronal nitric oxide synthase (nNOS) has been studied in the more rostral portion of the lateral ventricle, subfornical organ, area postrema and blood vessels of the rat central nervous system. nNOS was located by means of a specific polyclonal antibody, by using light and electron microscopy. Light microscopy showed immunoreactive varicose nerve fibers and terminal boutons-like structures in the lateral ventricle, positioned in supra- and subependimal areas. The spatial relationships between immunoreactive neuronal processes and the wall of the intracerebral blood vessels were studied. Electron microscopy showed numerous nerve fibers in the wall of the lateral ventricle; many were nNos-immunoreactive and established very close contact with ependymal cells. Immunoreactive neurons and processes were found in the subependymal plate of the ventricular wall, the subfornical organ, the area postrema, and the circularis nucleus of the hypothalamus. In these last three areas, the immunoreactive neurons were found close to the perivascular space of fenestrated and nonfenestrated blood vessels. The nNOS immunoreactivity was localized to the endoplasmic reticulum, cisterns, ribosomes, neurotubules, and in the inner part of the external membrane. In the terminal boutons, the reaction product was found surrounding the vesicle membranes. This distribution showed nNOS as a predominantly membrane-bound protein. The nitrergic nerve fibers present in the wall of the ventricular system might regulate metabolic functions as well as neurotransmission in the subfornical organ, area postrema and circularis nucleus of the hypothalamus.

Animals↗

PubMed1997

Interleukin-10 and interleukin-4 inhibit intracellular killing of Leishmania infantum and Leishmania major by human macrophages by decreasing nitric oxide generation.

The host response to Leishmania infection is regulated by a specific pattern of local cytokine production. We investigated the effect of interleukin (IL)-10 and IL-4 on the leishmanicidal activity of human macrophages (M phi). As with L. major, intracellular killing of L. infantum by human M phi was obtained following ligation of surface CD23 or cell treatment with interferon-gamma (IFN-gamma). This leishmanicidal activity required nitric oxide (NO) generation by activated M phi, and it was partially mimicked by cell treatment with chemical NO donors. Addition of recombinant human IL-10 or IL-4 to CD23 mAb or IFN-gamma decreased L. infantum and L. major killing by infected M phi. IL-10 was more potent than IL-4 in inhibiting the leishmanicidal activity of human M phi. Inhibition of Leishmania killing by IL-4 and IL-10 correlated with decreased NO generation from M phi, and was reversed when exogenous NO was added to cell cultures. Therefore, IL-10 and IL-4 down-regulate leishmanicidal activity of human M phi, in part by inhibiting NO generation by these cells.

Animals↗

PubMed1996

Identification of the domains of neuronal nitric oxide synthase by limited proteolysis.

Nitric oxide synthase (EC 1.14.13.39) binds arginine and NADPH as substrates, and FAD, FMN, tetrahydrobiopterin, haem and calmodulin as cofactors. The protein consists of a central calmodulin-binding sequence flanked on the N-terminal side by a haem-binding region, analogous to cytochrome P-450, and on the C-terminal side by a region homologous with NADPH:cytochrome P-450 reductase. The structure of recombinant rat brain nitric oxide synthase was analysed by limited proteolyis. The products were identified by using antibodies to defined sequences, and by N-terminal sequencing. Low concentrations of trypsin produced three fragments, similar to those in a previous report [Sheta, McMillan and Masters (1994) J. Biol. Chem. 269, 15147-15153]: that of Mr approx. 135000 (N-terminus Gly-221) resulted from loss of the N-terminal extension (residues 1-220) unique to neuronal nitric oxide synthase. The fragments of Mr 90000 (haem region) and 80000 (reductase region, N-terminus Ala-728) were produced by cleavage within the calmodulin-binding region. With more extensive trypsin treatment, these species were shown to be transient, and three smaller, highly stable fragments of Mr 14000 (N-terminus Leu-744 within the calmodulin region), 60000 (N-terminus Gly-221) and 63000 (N-terminus Lys-856 within the FMN domain) were formed. The species of Mr approx. 60000 represents a domain retaining haem and nitroarginine binding. The two species of Mr 63000 and 14000 remain associated as a complex. This complex retains cytochrome c reductase activity, and thus is the complete reductase region, yet cleaved at Lys-856. This cleavage occurs within a sequence insertion relative to the FMN domain present in inducible nitric oxide synthase. Prolonged proteolysis treatment led to the production of a protein of Mr approx. 53000 (N-terminus Ala-953), corresponding to a cleavage between the FMN and FAD domains. The major products after chymotryptic digestion were similar to those with trypsin, although the pathway of intermediates differed. The haem domain was smaller, starting at residue 275, yet still retained the arginine binding site. These data have allowed us to identify stable domains representing both the arginine/haem-binding and the reductase regions.

Amino Acid Sequence↗

PubMed1996

Characteristics of nitric oxide synthase type I of rat cerebellar astrocytes.

We have previously reported that stimulation of astrocyte cultures by particular agonists and calcium ionophores induces cyclic GMP formation through activation of a constitutive nitric oxide synthase (NOS) and that astrocytes from cerebellum show the largest response. In the present work we have used rat cerebellar astrocyteenriched primary cultures to identify and characterise the isoform of NOS expressed in these cells. The specific NOS activity in astrocyte homogenates, determined by conversion of [3H]arginine to [3H]citrulline, was ten times lower than in homogenates from cerebellar granule neurons. Upon centrifugation at 100,000 g, the astroglial activity was recovered in the supernatant, whereas in neurons around 30% of the activity remained particulate. The cytosolic NOS activities of both astrocytes and granule neurons displayed the same Km for L-arginine, dependency of calcium, and sensitivity to NOS inhibitors. Expression of NOS-I in astrocyte cytosolic fractions was revealed by Western blot with a specific polyclonal antiserum against recombinant NOS-I. Double immunofluorescence labelling using anti-glial fibrillary acidic protein (GFAP) and anti-NOS-I antibodies revealed that a minor population of the GFAP-positive cells, usually in clusters, presented a strong NOS-I immunostaining that was predominantly located around the nuclei and had a granular appearance, indicating association with the endoplasmic reticulum-Golgi system. Astrocytes of stellate morphology also showed immunoreactivity in the processes. Similar staining was observed with the avidin-biotin-peroxidase complex using different anti-NOS-I antisera. With this method the majority of cells showed a weak NOS-I immunoreactivity around the nuclei and cytosol. A similar pattern was observed with the NADPH-diaphorase reaction. These results demonstrate that the NOS-I expressed in astrocytes presents the same biochemical characteristics as the predominant neuronal isoform but may differ in intracellular location.

Animals↗

PubMed1996

Prevention by lamotrigine, MK-801 and N omega-nitro-L-arginine methyl ester of motoneuron cell death after neonatal axotomy.

Motoneuron cell death was analysed in the rat facial motor nucleus after neonatal facial nerve transection. In situ DNA fragmentation labelling showed that axotomized motoneurons die by an apoptotic mechanism. In order to investigate the existence of excitotoxic mechanisms in this type of neuronal death, rats were treated with several agents known to possess neuroprotective action through a variety of mechanisms. The Na+ channel inhibitor lamotrigine and the antagonist for the N-methyl-D-aspartate-type glutamate receptor, dizocilpine maleate (MK-801) were found to be able to rescue motoneurons from cell death induced by axotomy. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester was also able to protect motoneurons from death, but to a lesser extent. The distribution of constitutive and inducible isoforms of nitric oxide synthase was investigated by immunocytochemistry in the facial motor nucleus. No changes were detected in constitutive nitric oxide synthase immunoreactivity in the facial motor nucleus after axotomy. However, in the axotomized facial motor nucleus, inducible nitric oxide synthase showed a positive immunolabelling specifically located in activated astrocytes, but not in microglia. Nitric oxide derived from activated astrocytes may have a role in promoting excitotoxic mechanisms in axotomized motoneurons. We conclude that excitotoxic mechanisms involving apoptotic cell death are present when immature motoneurons die as a consequence of target disconnection.

Animals↗

PubMed1996

Specific cleavage of immunoglobulin G by copper ions.

The hinge region of a recombinant-DNA-produced human IgG1 (Campath 1H) is specifically cleavable at a single copper-sensitive peptide bond, yielding a distinct fragment resolved by size-exclusion high-performance liquid chromatography. This novel metal ion-catalysed cleavage at slightly alkaline pH is inhibited by EDTA and its rate is reduced at slightly acidic conditions (pH 5-6) and accelerated by increasing concentrations of cupric ion and higher temperature. Complete cleavage was observed after incubation at pH 8 for 24 h with 1 mM CuCl2. Sequence analysis determined the cleavage site to be the Lys226-Thr227 bond in the hinge-region sequence DKTHT. Cleavage of other IgGs was observed to varying degrees, and specific cleavage of synthetic peptides containing this pentapeptide sequence was also observed.

Alemtuzumab↗

PubMed1996

Depolymerization of macrophage microfilaments prevents induction and inhibits activity of nitric oxide synthase.

We have investigated the relationship between peritoneal murine macrophage cytoskeleton and nitric oxide (NO) synthase (NOS). Activation of the cells with lipopolysaccharide plus interferon-gamma (LI) induced iNOS, detected by nitrite or by labeled L-citrulline production and by a specific antibody against macrophage iNOS. Addition of cytochalasin B (a microfilament-depolymerizing agent) caused a dose-dependent inhibition in NO production by macrophages, whereas colchicine (a microtubule depolymerizing agent) inhibited it only by 20% and not dose-dependently. Addition of cytochalasin B together with LI abolished nitrite and L-citrulline accumulation as well as the amount of iNOS antigen in activated macrophage. Moreover, addition of cytochalasin B 6 or 12 h after stimulus, also decreased the nitrite and L-citrulline production by macrophages although iNOS antigen content by Western blot was the same in the presence or in the absence of cytochalasin B added 12 h after activation. Since cytochalasin B failed to inhibit iNOS activity directly, its inhibitory effects on NO production by macrophages is likely to be indirect, through microfilament network in central regions of cells, but not in filaments seen at pseudopodia or edging processes. Our findings demonstrate that disruption of microfilaments but not of microtubules prevents the iNOS induction process and inhibits its enzymatic activity in activated macrophages.

Actin Cytoskeleton↗

PubMed1995

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

Amino Acid Oxidoreductases↗

PubMed1995

Purification to homogeneity and characterisation of rat brain recombinant nitric oxide synthase.

We have previously demonstrated high expression of rat neuronal nitric oxide synthase (NO synthase) in a baculovirus system [Charles, I. G., Chubb, A., Gill, R., Clare, J., Lowe, P. N., Holmes, L. S., Page, M., Keeling, J. G., Moncada, S. & Riveros-Moreno, V. (1993) Biochem. Biophys. Res. Commun. 196, 1481-1489], where a small proportion of the expressed enzyme was soluble and active, but the majority was insoluble (approximately 15% of the total insoluble proteins). NO synthase is a complex enzyme, requiring several cofactors for full activity. These include tightly bound FAD, FMN, heme and tetrahydrobiopterin, in addition to calmodulin and NADPH. Here, we report that a substantial proportion of the total NO synthase produced becomes soluble following addition of hemin (2.5 micrograms/ml) to the culture medium. However, the enzyme purified under these conditions had very low specific activity, 50 nmol.min-1.mg-1, after ADP-Sepharose affinity purification. Full activity (approximately 800 nmol.min-1.mg-1) could, however, be obtained by including precursors for the cofactors, nicotinic acid, riboflavin, and sepiapterin in the culture medium. We demonstrate that the enzyme activity is exclusively associated with the dimeric form of the enzyme, which had the following molar ratios for the cofactors: heme, 0.92; FAD, 0.57; FMN, 0.34; H4biopterin, 0.32, with a specific activity of 1500 nmol.min-1.mg-1. The provision of substantial quantities of good quality enzyme, as described here, will facilitate the studies on the relationship between enzyme structure and its mechanism of catalysis.

Amino Acid Oxidoreductases↗

PubMed1995

Induction of nitric oxide synthase in colonic smooth muscle from patients with toxic megacolon.

BACKGROUND & AIMS: Colonic inflammation may lead to motility disturbances, including severe atony. Nitric oxide is released by inflamed tissue and induces smooth muscle relaxation. The aim of this study was to analyze NO generation pathways in colonic tissue from patients who had ulcerative colitis with or without toxic megacolon and in tumor-free samples from patients with colonic neoplasm. METHODS: Enzymatic activity was determined by transformation of [14C]arginine to [14C]citrulline in mucosa and muscular layer samples. Immunostaining of tissue sections with antibody against inducible NO synthase was investigated. The effects of endotoxin on NO synthase activity was tested in muscle strips from human colon. RESULTS: Ca(2+)-independent NO synthase was undetectable or very low in muscularis propria from tumor and colitis controls. In contrast, specimens from patients with toxic megacolon had high activity (P < 0.05). Positive immunostaining for inducible NO synthase was found in muscular layers from patients with megacolon but not in tumor and colitis controls. Finally, endotoxin induced Ca(2+)-independent NO synthase activity in colonic muscle. CONCLUSIONS: Toxic megacolon is associated with the appearance of inducible NO synthase in the colonic muscularis propria. Local generation of excessive amounts of NO may be responsible for the colonic dilatation that is the hallmark of this syndrome.

Adult↗

PubMed1995

Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human osteoblast-like cells and its functional role in osteoblast metabolic activity.

Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.

Alkaline Phosphatase↗

PubMed1995

Nitric oxide synthesis in locust olfactory interneurones

The brain of the locust Schistocerca gregaria contains a nitric oxide synthase (NOS) that has similar properties to mammalian neuronal NOS. It catalyses the production of equimolar quantities of nitric oxide (NO) and citrulline from l-arginine in a Ca2+/calmodulin- and NADPH-dependent manner and is inhibited by the Nomega-nitro and Nomega-monomethyl analogues of l-arginine. In Western blots, an antiserum to the 160 kDa rat cerebellar NOS subunit recognises a locust brain protein with a molecular mass of approximately 135 kDa. NOS is located in several parts of the locust brain, including the mushroom bodies, but it is particularly abundant in the olfactory processing centres, the antennal lobes. Here it is present in two groups of local interneurones (a pair and a cluster of about 50) that project into the neuropile of the antennal lobes. The processes of these neurones terminate in numerous glomerulus-like structures where the synapses between primary olfactory receptor neurones and central interneurones are formed. NOS-containing local interneurones have also been identified in the mammalian olfactory bulb, suggesting that NO performs analogous functions in locust and mammalian olfactory systems. As yet, nothing is known about the role of NO in olfaction, but it seems likely that it is involved in the processing of chemosensory input to the brain. The locust antennal lobe may be an ideal 'simple' system in which this aspect of NO function can be examined.

Journal Article↗

PubMed1995

Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria.

Physiological and histochemical studies have recently supported the notion that nitric oxide (NO) is the transduction signal responsible for the non-adrenergic, non-cholinergic relaxation of the vasculature as well as the airways of the mammalian lung. We report the presence of immunoreactivity to NO synthase (NOS) in nerve cell bodies and nerve fibres in the neural plexus of the buccal cavity and lungs of the frog, Rana temporaria, using the indirect immunocytochemical technique of avidin-biotin and the NADPH-diaphorase technique. The neural ganglia located next to the muscle layer and within the connective tissue of the buccal cavity were partially immunoreactive for NOS. In the lungs, NOS immunoreactivity occurred in nerve cell bodies, as well as in both myelinated and unmyelinated nerve fibres. Fine nerve fibres immunoreactive to NOS were observed within the muscle fibre bundles and next to the respiratory epithelium. Both the presence of NOS immunoreactivity and the positive histochemical reaction for NADPH-diaphorase in the neural plexus of amphibian respiratory tract suggests a broad evolutionary role for NO as a peripheral neurotransmitter.

Animals↗

PubMed1994

Localization of inducible nitric oxide synthase in acute renal allograft rejection in the rat.

There is increasing evidence for a role for nitric oxide (NO) in the alloimmune response and induction of NO synthesis occurs during allograft rejection. The aim of this study was to investigate the source of NO synthesis in rejecting allografts. Localization of inducible nitric oxide synthase (iNOS) was studied by immunohistochemistry, in a rat model of acute renal allograft rejection, in unmodified Lewis recipients in which rejection is complete 7 days after transplantation of F1 hybrid Lewis-Brown Norway kidneys. High levels of iNOS expression were found in infiltrating mononuclear cells in glomeruli and interstitium of rejecting kidneys; there was no expression in parenchymal renal cells, or in control isografts of either rat strain. Expression of iNOS in the cortex was present from 4 to 6 days posttransplantation, and had declined by the 7th day, where expression was principally in the medulla. The pattern of iNOS staining was similar to ED1 staining, a marker for rat macrophages. These findings suggest that infiltrating macrophages in the graft reaction are a prominent source of NO; this iNOS expression supports a role for NO in the modulation of local allogeneic responses, and possibly as a mediator of cytotoxic graft damage.

Amino Acid Oxidoreductases↗

PubMed1994

Localization of nitric oxide synthase in the adult rat brain.

The distribution of the immunoreactivity to nitric oxide synthase has been examined from rostral to caudal areas of the rat central nervous system using light microscopy. Endogenous nitric oxide synthase was located using a specific polyclonal antiserum, produced against affinity purified nitric oxide synthase from whole rat brain, following the avidin-biotin peroxidase procedure. Immunoreactive cell bodies and processes showed a widespread distribution in the brain. In the telencephalon, immunoreactive structures were distributed in all areas of the cerebral cortex, the ventral endopiriform nucleus and claustrum, the main and accessory olfactory bulb, the anterior and posterior olfactory nuclei, the precommisural hippocampus, the taenia tecta, the nucleus accumbens, the stria terminalis, the caudate putamen, the olfactory tubercle and islands of Calleja, septum, globus pallidus and substantia innominata, hippocampus and amygdala. In the diencephalon, the immunoreactivity was largely found in both the hypothalamus and thalamus. In the hypothalamus, immunoreactive cell bodies were characteristically located in the perivascular-neurosecretory systems and mamillary bodies. In addition, immunoreactive nerve fibres were detected in the median eminence of the infundibular stem. The mesencephalon showed nitric oxide synthase immunoreactivity in the ventral tegmental area, the interpeduncular nucleus, the rostral linear nucleus of the raphe and the dorsal raphe nucleus. Immunoreactive structures were also found in the nuclei of the central grey, the peripeduncular nucleus and substantia nigra pars lateralis, the geniculate nucleus and in the superior and inferior colliculi. The pons displayed immunoreactive structures principally in the pedunculopontine and laterodorsal tegmental nuclei, the ventral tegmental nucleus, the reticulotegmental pontine nucleus, the parabrachial nucleus and locus coeruleus. In the medulla oblongata, immunoreactive neurons and processes were detected in the principal sensory trigeminal nucleus, the trapezoid body, the raphe magnus, the pontine reticular nuclei, the supragenual nucleus, the prepositus hypoglossal nucleus, the medial and spinal vestibular nuclei, the dorsal cochlear nucleus, the medullary reticular field, the nucleus of the solitary tract, the gracile and cuneate nuclei, the dorsal nucleus of the vagus nerve and the oral, interpolar and caudal parts of the spinal trigeminal nucleus. In the cerebellum, the stellate and basket cells showed immunoreactivity, which was also seen in the basket terminal fibres of the Purkinje cell layer. Isolated immunoreactive Purkinje cells were found in the vermis and parafloccular regions of the cerebellum. In the granular layer of the cerebellum, the granular cells and glomeruli were also immunoreactive. Numerous positive varicose nerve fibres and occasional neurons were also found in the lateral and interposed cerebellar nuclei.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Oxidoreductases↗