Search PubMed⌕ Search

Biomedical subjects

M Nakane

Publications and source records attributed to M Nakane.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.

Amino Acid Oxidoreductases↗
PubMed1994

Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions.

Three isozymes of nitric oxide (NO) synthase (EC 1.14.13.39) have been identified and the cDNAs for these enzymes isolated. In humans, isozymes I (in neuronal and epithelial cells), II (in cytokine-induced cells), and III (in endothelial cells) are encoded for by three different genes located on chromosomes 12, 17, and 7, respectively. The deduced amino acid sequences of the human isozymes show less than 59% identity. Across species, amino acid sequences for each isoform are well conserved (> 90% for isoforms I and III, > 80% for isoform II). All isoforms use L-arginine and molecular oxygen as substrates and require the cofactors NADPH, 6(R)-5,6,7,8-tetrahydrobiopterin, flavin adenine dinucleotide, and flavin mononucleotide. They all bind calmodulin and contain heme. Isoform I is constitutively present in central and peripheral neuronal cells and certain epithelial cells. Its activity is regulated by Ca2+ and calmodulin. Its functions include long-term regulation of synaptic transmission in the central nervous system, central regulation of blood pressure, smooth muscle relaxation, and vasodilation via peripheral nitrergic nerves. It has also been implicated in neuronal death in cerebrovascular stroke. Expression of isoform II of NO synthase can be induced with lipopolysaccharide and cytokines in a multitude of different cells. Based on sequencing data there is no evidence for more than one inducible isozyme at this time. NO synthase II is not regulated by Ca2+; it produces large amounts of NO that has cytostatic effects on parasitic target cells by inhibiting iron-containing enzymes and causing DNA fragmentation. Induced NO synthase II is involved in the pathophysiology of autoimmune diseases and septic shock. Isoform III of NO synthase has been found mostly in endothelial cells. It is constitutively expressed, but expression can be enhanced, eg, by shear stress. Its activity is regulated by Ca2+ and calmodulin. NO from endothelial cells keeps blood vessels dilated, prevents the adhesion of platelets and white cells, and probably inhibits vascular smooth muscle proliferation.

Amino Acid Oxidoreductases↗
PubMed1994

Intracranial vertebral artery dissections: clinical, radiological features, and surgical considerations.

We reviewed 24 patients with intracranial vertebral artery dissections treated during the last 12 years. Sixteen patients were admitted with subarachnoid hemorrhage (SAH) and 8 did not have SAH. The mean age at the time of onset was 50.0 years. Male preponderance was noted. Among 21 patients with acute onset, 6 (29%) experienced prodromal neck pain and 3 (60%) of 5 SAH patients showed nuchal stiffness when examined within 6 hours of onset. The preoperative angiographical findings were uniform in SAH cases in contrast to the varied angiographical findings seen in non-SAH cases. So-called pearl and string sign was observed in most SAH cases, but the "string" was often so short and wide that the term "constriction" appeared more suitable. From intraoperative observations, the angiographical point of constriction seemed to represent the proximal or distal end of dissection. As for treatment, 19 patients underwent 20 surgeries. Trapping was performed in eight surgeries, base clipping was performed in five, and proximal clipping was performed in seven. Both trapping and base clipping prevented further bleeding, but trapping was associated with a high rate of postoperative lower cranial nerve palsy. Postoperative neurological complications were less frequent after proximal clipping, but subsequent postoperative bleeding occurred in one patient treated by this technique. The overall long-term outcome in the surgically treated cases in our series was favorable, but most patients suffered from various degrees of uncomfortable dysphagia or hoarseness for some period after surgery. It was also noted that, in half of the disabled cases, the major disability was attributable to lower cranial nerve palsy and respiratory troubles that developed postoperatively.

Adult↗
PubMed1993

Nitric oxide synthases in neuronal cells, macrophages and endothelium are NADPH diaphorases, but represent only a fraction of total cellular NADPH diaphorase activity.

NADPH diaphorase activity is used as a histochemical marker for neuronal nitric oxide (NO) synthase; however, it remains unclear whether these activities are directly correlated in all tissues. In N1E-115 neuroblastoma cells, NADPH diaphorase activity was found primarily in the particulate fraction, whereas NO synthase activity was mostly soluble. Non-induced macrophages expressed significant NADPH diaphorase activity (which was mostly particulate) but virtually no NO synthase activity. Induction of macrophages produced marked increases in both NO synthase and NADPH diaphorase activities in the soluble and particulate fractions. In endothelial cells, both NO synthase and NADPH diaphorase activities were found mostly in the particulate fraction. Purified NO synthases from brain (type I), macrophages (type II), and endothelium (type III) all showed NADPH diaphorase activity; relative activities were: macrophage > endothelium > brain. These data indicate that all known NO synthases are NADPH diaphorases; however, NO synthases represent only a fraction of total cellular NADPH diaphorase activity and these activities are not always co-localized.

Amino Acid Oxidoreductases↗
PubMed1993

Neurons in rat cerebral cortex that synthesize nitric oxide: NADPH diaphorase histochemistry, NOS immunocytochemistry, and colocalization with GABA.

Neurons that stain for NADPH diaphorase, which colocalizes with nitric oxide synthase (NOS), are scattered uniformly across neocortex, and denser in entorhinal cortex. In the primary sensorimotor cortex, 0.5-2% of neurons contain NOS. These are most numerous in layers II-III, whereas NOS-positive fibers are concentrated in layers IV and VI. Most stained neurons are aspiny bipolar cells. Some in deep layers are multipolar; very few are pyramidal-shaped. In layer IV, NOS-positive neurons and their dendrites are confined to the septa between barrels. Retrograde tracing experiments demonstrate that NOS-positive cells are local circuit neurons. Double staining demonstrates that NOS-positive neurons also contain GABA.

Amino Acid Oxidoreductases↗
PubMed1993

Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain.

In the presence of added flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both cytosolic and particulate nitric oxide synthase (NOS) activities can be detected in rat brain. Developmental changes of the cytosolic and particulate NOS in rat cerebellum and cerebrum were determined biochemically and immunochemically. Particulate NOS activity in the cerebrum increased during the first week of development, but then decreased and became almost undetectable in adult rats. In contrast, the cytosolic NOS in cerebellum showed low activity in newborns, but then constantly increased reaching an 8-fold higher level in adult rats. The activities of cerebellar particulate and cerebral cytosolic NOS also increased slightly during maturation. Western blot analysis using a polyclonal antibody raised against rat cerebellar cytosolic NOS revealed that the particulate and cytosolic fractions of 1-week-old and adult rat brains contained the same 160 kDa NOS protein. The relative content of the NOS protein correlated well with the relative amount of NOS activity in all brain fractions. These results indicate that distribution of NOS in the rat brain changes during maturation, but the same NOS protein is likely to be responsible for activities in immature and mature brains, cytosolic and particulate fractions. We suggest that nitric oxide might play a role in functional differentiation of the brain.

Amino Acid Oxidoreductases↗
PubMed1993

A correlation between soluble brain nitric oxide synthase and NADPH-diaphorase activity is only seen after exposure of the tissue to fixative.

In histochemical studies using fixed brain tissue, NADPH-diaphorase has been found to be colocalized with soluble nitric oxide synthase. In the present study, using fresh tissues from eight different regions of rat brain, NADPH-diaphorase activity was found mostly in the particulate fraction, whereas most of the nitric oxide synthase activity was located to the cytosolic fraction. Also, the distribution of NADPH-diaphorase activity among brain regions was different from that of nitric oxide synthase. Pretreatment of the fractions with paraformaldehyde virtually abolished the NADPH-diaphorase activity in the particulate fraction, whereas 40-60% of the NADPH-diaphorase activity remained intact in the cytosolic fraction. These results suggest that during fixation most NADPH-diaphorase activity is inactivated and only some of the NADPH-diaphorase activity associated with soluble nitric oxide synthase remains intact.

Amino Acid Oxidoreductases↗
PubMed1993

Neurons in rat hippocampus that synthesize nitric oxide.

We studied the distribution and light- and electron-microscopic morphology of neurons in the hippocampal formation containing nitric oxide synthase (NOS), and thus likely to release nitric oxide, a freely diffusible neuromediator implicated in long-term potentiation. Only a small fraction of hippocampal neurons contained NOS or its marker, NADPH diaphorase. Most of the positive neurons were in the pyramidal layer of the subiculum, stratum radiatum of Ammon's horn, and subgranular zone of the dentate gyrus. Positive neurons were also conspicuous in the molecular layer of the dentate gyrus and in the pyramidal layer of CA3, sparse in the pyramidal layer of CA2 and CA1, and almost absent from presubiculum and parasubiculum. Numerous positive fibers were seen, especially in stratum radiatum and stratum lacunosum-moleculare of Ammon's horn. Double staining experiments demonstrated that nearly all NADPH diaphorase-positive neurons in the hippocampus also contained gamma-aminobutyric acid. On the basis of their morphology, distribution, and inhibitory neurotransmitter content, most NOS-positive cells in the hippocampus are probably local circuit neurons. These data suggest that nitric oxide in CA1 may function as a paracrine agent, rather than a spatially precise messenger, in long-term potentiation.

Amino Acid Oxidoreductases↗
PubMed1993

Cloned human brain nitric oxide synthase is highly expressed in skeletal muscle.

Complementary DNA clones corresponding to human brain nitric oxide (NO) synthase have been isolated. The deduced amino acid sequence revealed an overall identity with rat brain NO synthase of about 93% and contained all suggested consensus sites for binding of the co-factors. The cDNA transfected COS-1 cells showed significant NO synthase activity with the typical co-factor requirements. Unexpectedly, messenger RNA levels of this isoform of NO synthase was more abundant in human skeletal muscle than human brain. Moreover, we detected high NO synthase activity and the expressed protein in human skeletal muscle by Western blot analysis, indicating a possible novel function of NO in skeletal muscle.

Adolescent↗
PubMed1993

Characterization and localization of endothelial nitric oxide synthase using specific monoclonal antibodies.

We have produced specific monoclonal antibodies (MAb) against particulate bovine aortic endothelial nitric oxide synthase. In Western blots, native and cultured bovine aortic endothelial cells as well as cultured bovine microvascular endothelial cells possess immunoreactive NO synthase. In dot blots, MAb H210 and H32 detect 1 ng and 100 pg of purified endothelial NO synthase, respectively. Both antibodies are specific to the endothelial NO synthase and do not cross-react with other known isoforms of NO synthase, namely from the brain, from cytokine/endotoxin-induced macrophages, or from cytokine/endotoxin-induced vascular smooth muscle cells. Immunohistochemical studies demonstrated the specificity of endothelial NO synthase for endothelial cells in various bovine and human tissues. Many types of endothelial cells, macrovascular, microvascular, arterial, and venous were found to possess this specific isoform of NO synthase. Electron microscopy showed the enzyme to be associated with the plasma membrane, membranes of cytoplasmic vesicles, and in the cytoplasm in human umbilical vein endothelial cells. The results demonstrate that particulate endothelial NO synthase is present in a site to act rapidly to produce NO for release into the blood or toward the smooth muscle in many vascular beds.

Amino Acid Oxidoreductases↗
PubMed1993

Cerebral cysticercosis.

A 44-year-old female underwent a right frontal craniotomy under a diagnosis of metastatic brain tumor and a fluid-filled cystic lesion was extirpated. One rice grain sized nodule was identified in the cyst and histopathological examination of the nodule showed a scolex with hooks and suckers. These macro and microscopic findings indicated a diagnosis of cerebral cysticercosis, caused by infection of the larval stage of some type of Taenia species. A therapeutic course of praziquantel was given after craniotomy. The patient has been a resident of the Kanto area of Japan since birth and has never been outside Japan.

Adult↗
PubMed1993

Isoforms of nitric oxide synthase: functions in the cardiovascular system.

Various cell types, including endothelial cells, can synthesize nitric oxide (NO). Three different isoforms of NO synthase have been characterized, purified and cloned. Isozyme I is present in neuronal cells of the brain (where NO may mediate synaptic plasticity), in peripheral non-adrenergic non-cholinergic (NANC) neurons (where NO acts as an atypical neurotransmitter relaxing vascular and non-vascular smooth muscle), and in various specialized epithelial cells. Macrophages can be induced with bacterial endotoxin and/or cytokines to express isozyme II. The high concentrations of NO produced by this isoform have cytostatic effects on parasitic microorganisms and tumour cells. A similar isozyme can be induced in the vascular wall (presumably in smooth muscle cells) in sepsis and during cytokine therapy. The large amounts of NO produced by this enzyme contribute to the symptoms of septic shock, such as vasodilatation and microvascular endothelial damage. Endothelial cells contain isoform III of NO synthase which seems to be unique for this cell type. Endothelium-derived NO is a physiologically significant vasodilator and inhibitor of platelet aggregation and adhesion. In addition, vascular NO can prevent leukocyte adhesion to the endothelium by interfering with the adhesion molecule CD11/CD18, and NO has also been shown to inhibit the proliferation of vascular smooth muscle cells. Hence, NO represents a protective factor against vascular damage and probably atherogenesis.

Amino Acid Oxidoreductases↗
PubMed1992

Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities.

NO synthase (NOS; EC 1.14.23) catalyzes the conversion of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [32P]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near Km concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1992

Particulate and soluble bovine endothelial nitric oxide synthases are structurally similar proteins yet different from soluble brain nitric oxide synthase.

In cultured bovine aortic endothelial cells (BAECs), 95% of the total endothelial nitric oxide (NO) synthase (type III) activity was found in the particulate fraction and only 5% was found in the soluble fraction. The soluble and particulate endothelial NO synthase activities behaved similarly on anion-exchange and gel filtration chromatography, whereas the soluble brain NO synthase (type I) had chromatographic properties different from the type III endothelial NO synthases. We have purified the particulate endothelial NO synthase from cultured and native BAECs using affinity chromatography on 2',5'-ADP Sepharose followed by Superose 6 gel filtration chromatography. Subsequently, monoclonal antibodies were generated against the purified particulate endothelial NO synthase. In protein immunoblotting analyses, crude and partially purified samples of particulate and soluble type III endothelial NO synthase demonstrated a single band at a molecular mass of 135 kDa with monoclonal antibody (MAb) H32. Purified type I soluble brain NO synthase did not cross-react with MAb H32. These data indicate that the soluble and particulate endothelial NO synthase are structurally similar proteins and represent an isozyme that can be distinguished from the brain NO synthase.

Amino Acid Oxidoreductases↗
PubMed1992

Focus formation by the human immunodeficiency virus (HIV) in the immobilized MT-4 cell culture and its application to the evaluation of anti-HIV agents.

Immunofluorescence studies were performed on the infection of monolayer cultures of immobilized MT-4 cells with human immunodeficiency virus type 1 (HIV-1). By using the anti-viral p24 monoclonal antibody, we could observe formation of foci of p24 antigen-positive cells within 3 to 4 days when the infection was initiated with a relatively small amount of the virus. Frequency of the focus formation was in proportion to the dose of input virus (ranging from 0.001 to 0.1 PFU/cell), which allowed us to apply this phenomenon to the assay of anti-HIV agents as well as to the estimation of relative infectivity of the virus stocks. When antiviral agents were added to the infected cultures, number of foci as well as the size of each focus was reduced in a concentration-dependent manner. The dose required for reducing the number of foci by 50% was calculated to be 6 ng/ml and 8 ng/ml for tunicamycin (TM) and azidothymidine (AZT), respectively. These values are comparable to those obtained by other current assay methods. In addition, focus reduction assay is also useful in searching for such antiviral agents that would inhibit or block the early step of viral replication cycle.

Antibodies, Monoclonal↗
PubMed1992

Characterization and localization of nitric oxide synthase in non-adrenergic non-cholinergic nerves from bovine retractor penis muscles.

1. Partially purified soluble nitric oxide (NO) synthase was isolated from the bovine retractor penis muscle (BRP), a tissue in which the inhibitory response to non-adrenergic non-cholinergic nerve (NANC) stimulation appears to be mediated by NO or NO-like material. 2. NO synthase from BRP used L-arginine as a substrate, required NADPH, tetrahydrobiopterin, and FAD as co-factors and was Ca2+/calmodulin-dependent. The activity of NO synthase was inhibited by NG-methyl-L-arginine and NG-nitro-L-arginine, and haemoglobin blocked the effect of NO formed by the enzyme. 3. On reducing SDS polyacrylamide gel electrophoresis the apparent molecular mass of NO synthase from BRP was 160 +/- 2 kDa, which is similar to that of the cerebellar NO synthase. Protein immunoblot and immunoprecipitation showed that NO synthase from BRP cross-reacted with the selective antiserum to neuronal NO synthase from rat cerebellum. 4. Immunohistochemistry using the same antiserum demonstrated that NO synthase in BRP was located exclusively within nerve fibres. Thus, autonomic nerves synthesizing the NANC neurotransmitter seem to contain an isoform of NO synthase which is similar to that from rat cerebellum.

Amino Acid Oxidoreductases↗