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M John

Publications and source records attributed to M John.

At least 19 recordsLinked to original sources
PubMed1992

Ca2+/calmodulin-dependent cytochrome c reductase activity of brain nitric oxide synthase.

Nitric oxide acts as a widespread signal molecule and represents the endogenous activator of soluble guanylyl cyclase. In endothelial cells and brain tissue, NO is enzymatically formed from L-arginine by Ca2+/calmodulin-regulated NO synthases which require NADPH, tetrahydrobiopterin, and molecular oxygen as cofactors. Here we show that purified brain NO synthase binds to cytochrome c-agarose and exhibits superoxide dismutase-insensitive cytochrome c reductase activity with a Vmax of 10.2 mumol x mg-1 x min-1 and a Km of 34.1 microM. Cytochrome c reduction was largely dependent on Ca2+/calmodulin and cochromatographed with L-citrulline formation during gel filtration. When reconstituted with cytochrome P450, NO synthase induced a moderate Ca(2+)-independent hydroxylation of N-ethylmorphine. NO synthase also reduced the artificial electron acceptors nitro blue tetrazolium and 2,6-dichlorophenolindophenol. Cytochrome c, 2,6-dichlorophenolindophenol, and nitro blue tetrazolium inhibited NO synthase activity determined as formation of L-citrulline from 0.1 mM L-arginine in a concentration-dependent manner with half-maximal effects at 166, 41, and 7.3 microM, respectively. These results suggest that NO synthase may participate in cellular electron transfer processes and that a variety of electron-acceptors may interfere with NO formation due to the broad substrate specificity of the reductase domain of NO synthase.

2,6-Dichloroindophenol↗
PubMed1992

Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase.

L-Arginine-derived nitric oxide (NO) acts as an inter- and intra-cellular signal molecule in many mammalian tissues including brain, where it is formed by a flavin-containing Ca2+/calmodulin-requiring NO synthase with NADPH, tetrahydrobiopterin (H4biopterin) and molecular oxygen as cofactors. We found that purified brain NO synthase acted as a Ca2+/calmodulin-dependent NADPH:oxygen oxidoreductase, catalysing the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin, which inhibited the hydrogen peroxide formation with half-maximal effects at 11 microM and 0.3 microM respectively. Half-maximal rates of L-citrulline formation were observed at closely similar concentrations of these compounds, indicating that the NO synthase-catalysed oxygen activation was coupled to the synthesis of L-citrulline and NO in the presence of L-arginine and H4biopterin. N omega-Nitro-L-arginine, its methyl ester and N omega-monomethyl-L-arginine inhibited the synthesis of L-citrulline from L-arginine (100 microM) with half-maximal effects at 0.74 microM, 2.8 microM and 15 microM respectively. The N omega-nitro compounds also blocked the substrate-independent generation of hydrogen peroxide, whereas N omega-monomethyl-L-arginine did not affect this reaction. According to these results, activation of brain NO synthase by Ca2+ at subphysiological levels of intracellular L-arginine or H4biopterin may result in the formation of reactive oxygen species instead of NO, and N omega-nitro-substituted L-arginine analogues represent useful tools to effectively block NO synthase-catalysed oxygen activation.

Amino Acid Oxidoreductases↗
PubMed1992

Nitric oxide synthase-catalyzed activation of oxygen and reduction of cytochromes: reaction mechanisms and possible physiological implications.

Purified cerebellar nitric oxide (NO) synthase was found to reduce molecular oxygen to hydrogen peroxide at low concentrations of its substrate L-arginine or its cofactor tetrahydrobiopterin. The characteristics of oxygen reduction appeared to be similar to NO synthesis, as both reactions required reduced nicotinamide adenine dinucleotide phosphate (NADPH), were dependent on Ca2+/calmodulin, and showed optimal reaction rates at slightly acidic conditions. The electron transport from NADPH to molecular oxygen is probably mediated by the reduced flavins, flavine adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which are bound in stoichiometrical amounts to the enzyme. NO synthase shows similarities to cytochrome P450 (cytochrome c) reductase, another FAD- and FMN-containing enzyme, and we found that NO synthase reduced cytochromes and artificial, low molecular mass electron acceptors in a superoxide dismutase-insensitive manner. Thus, NO synthase apparently represents a Ca(2+)-regulated, soluble isoform of cytochrome P450 reductase.

Amino Acid Oxidoreductases↗
PubMed1991

Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase.

Brain nitric oxide synthase is a Ca2+/calmodulin-regulated enzyme which converts L-arginine into NO. Enzymatic activity of this enzyme essentially depends on NADPH and is stimulated by tetrahydrobiopterin (H4biopterin). We found that purified NO synthase contains enzyme-bound H4biopterin, explaining the enzymatic activity observed in the absence of added cofactor. Together with the finding that H4biopterin was effective at substoichiometrical concentrations, these results indicate that NO synthase essentially depends on H4biopterin as a cofactor which is recycled during enzymatic NO formation. We found that the purified enzyme also contains FAD, FMN and non-heme iron in equimolar amounts and exhibits striking activities, including a Ca2+/calmodulin-dependent NADPH oxidase activity, leading to the formation of hydrogen peroxide at suboptimal concentrations of L-arginine or H4biopterin.

Amino Acid Oxidoreductases↗
PubMed1991

Free radicals in children with rheumatic diseases in serum and synovial fluid.

Free radicals, demonstrable by thiobarbituric acid, are also significant in the pathogenic concept of juvenile chronic arthritis. 41 patients without therapy demonstrate significantly increased values in serum, compared with the same patients after a treatment of 4 weeks. There are relations between ESR and the concentration of thiobarbituric acid reactive substances in serum, but without statistic significance. In the synovial fluid the radicals are increasingly demonstrable, but there is no relation to the number of cells or granulocytes.

Arthritis, Juvenile↗
PubMed1990

Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin.

L-Arginine-derived nitric oxide acts as an inter- and intracellular signal molecule with cytosolic guanylyl cyclase as the effector system. Two NO synthase isoenzymes are postulated: a cytokine-inducible enzyme in macrophages and a constitutive, Ca2(+)-regulated enzyme in various other cells. An NO synthase was isolated from porcine cerebellum by ammonium sulfate precipitation and affinity chromatography on 2',5'-ADP-Sepharose. The enzyme was identified as an NO synthase with a specific NO-chemiluminescence method and with purified cytosolic guanylyl cyclase as an NO-sensitive detection system. The purified NO synthase was, besides Ca2+/calmodulin and NADPH, largely dependent on tetrahydrobiopterin as a cofactor.

Amino Acid Oxidoreductases↗
PubMed1990

Bronchiolitis in tropical south India.

In a prospective hospital-based study of 328 children under 5 years of age with acute lower respiratory infections, 114 (35%) were diagnosed to have acute bronchiolitis. Of them, 87 (76%) were less than 1 year and 107 (94%) were less than 2 years of age. Signs of severe lower respiratory infections, namely tachypnea (respiratory rate greater than 50/min) and subcostal retraction, were present in 95% and 93%, respectively. Of 88 children of whom roentgenographs were taken, 30 (34%) had evidence of pneumonia. No clinical signs discriminated between those with and without pneumonia. By culture or immunofluorescence antigen detection, viruses were found in 81 (71%) children with bronchiolitis; respiratory syncytial virus was the most common agent, found in 65 (57%). Parainfluenza viruses were the next most common, found in 12 (11%). Most cases of bronchiolitis occurred in outbreaks during the rainy months of August through November, coinciding with respiratory syncytial virus outbreaks. Although bacterial culture of blood was done in 56 children, no respiratory pathogen was isolated. In one child with bronchiolitis and consolidation, postmortem lung aspirate yielded Staphylococcus aureus. Thus, bronchiolitis is primarily a viral syndrome in this tropical region, just as it is in temperate regions. Eight (7%) children died (all were infants); 5 had roentgenographic pneumonia and the remaining had other abnormalities contributing to death; all had been treated with antibiotics. Since one third of lower respiratory infections are bronchiolitis, and among infants under 1 year of age bronchiolitis comprises 47% of all lower respiratory infection cases, criteria for antibiotic management must take into account the availability of roentgenographic investigation.

Anti-Bacterial Agents↗
PubMed1990

Urokinase and tissue type plasminogen activators in human keratinocyte culture.

Using immunocytochemical and biochemical techniques, we have demonstrated that cultured human epidermal keratinocytes contain both urokinase and tissue type plasminogen activators. In subconfluent colonies the distribution of the two enzymes differed. Tissue type plasminogen activator (tPA) was distributed evenly throughout the colony, while, as we have demonstrated previously, urokinase type plasminogen activator (uPA) was preferentially localized at the migrating edges of the colony. Using zymographic analyses, both tPA and uPA activities were detected in cell extracts. Depending on the procedure used to prepare cell extracts, tPA was detected either as free enzyme or in complex with PA inhibitor type 1. PA inhibitor type 1 was deposited onto the extracellular matrix of the keratinocyte cultures and formed a complex with cell-associated tPA when cells and matrix were extracted together. The most differentiated keratinocytes in the culture, which were spontaneously shed from the culture surface, also contained both tPA and uPA. However, these spontaneously shed cells had a higher ratio of tPA:uPA than did the less differentiated cells from the same culture. In conjunction with our previous studies, these results demonstrate the complex nature of the plasminogen activator system, including enzymes and inhibitors, that is present in human keratinocytes. In addition, our data suggest that the relative amounts of uPA and tPA in epidermal cells vary with differentiation state.

Cell Adhesion↗
PubMed1990

Preliminary results of concurrent radiotherapy and chemotherapy in advanced cervical carcinoma: a phase I-II prospective intergroup NCOG-RTOG Study.

Thirty-eight patients with advanced carcinoma of the cervix were prospectively treated with a concurrent combination of radiotherapy (RT) and chemotherapy (CT) using the drugs 5-fluorouracil (5FU), mitomycin C and cis-platinum as part of a Northern California Oncology Group (NCOG) and Radiation Therapy Oncology Group (RTOG) intergroup study. RT consisted of 36.00 Gy to the pelvis in 4 weeks followed by a 9.00-Gy parametrial boost. This was followed by two intracavitary applications for a total of 4000 mg hr of radium equivalent when possible. 5FU (1000 mg/m2/24 hr for 96 hr by iv infusion) and mitomycin C (10 mg/m2/iv bolus) were given during the second week of external RT. 5FU (dose as above) and cis-platinum (75 mg/m2/iv over 6 hr) were given during the first intracavitary application. Of 36 patients evaluable for toxicity, 11% had grade 3 nonhematological toxicity and 11% had reversible grade 4 hematological toxicity. There were no toxic deaths. A complete response rate of 62.5% was obtained overall (median survival not reached). This study suggests that this particular combination of RT and CT in advanced cervical carcinoma is effective and well tolerated.

Adult↗
PubMed1990

Prophylactic irradiation of the para-aortic lymph node chain in stage IIB and bulky stage IB carcinoma of the cervix, initial treatment results of RTOG 7920.

From November 1979 to October 1986, 367 patients were entered onto RTOG 7920 and randomized to receive either pelvic irradiation alone or pelvic plus para-aortic radiation. Patients with Stage IIB cervical carcinoma who had not undergone curative surgery and patients with Stages IB and IIA cervical carcinoma who were determined by digital exam to have primary tumors measuring 4 cm or greater in lateral dimension were eligible for this study. Clinically apparent or surgically involved para-aortic nodes were reason for exclusion from the study. Pelvic irradiation consisted of 1.6-1.8 Gy per day for 5 days per week to a total of 40-50 Gy. Para-aortic irradiation delivered 44 to 45 Gy in 1.6-1.8 Gy per day, 5 days per week. Pelvic irradiation was to be completed in 4 1/2 to 6 1/2 weeks and para-aortic irradiation in 4 1/2 to 5 1/2 weeks. Intracavitary brachytherapy delivered a total of 4000-5000 mg hr of radium-equivalents or 30-40 Gy to point A. Patients were stratified prior to random treatment assignment by histology, para-aortic nodal status (negative vs. unevaluated), and FIGO stage. As of June 1, 1989, 30 cases were excluded, including five patients who were inevaluable. Two patients who refused the assigned treatment were also excluded. Therefore, a total of 330 cases were analyzable. At 5 years the estimates of survival, the primary endpoint, for the pelvic only and pelvic plus para-aortic irradiation arms are 55% and 65%, respectively (p = 0.043). Several secondary endpoints were also analyzed. Estimates for loco-regional control at 5 years are, for pelvic irradiation only, 66%, and for pelvic plus para-aortic irradiation, 75% (p = 0.21). Distant metastases are estimated in 32% of pelvic irradiation only patients and 25% of pelvic plus para-aortic irradiation patients at 5 years (p = 0.17). When the first disease failure patterns are examined, more patients fail distally when treated only with pelvic radiation than when using pelvic plus para-aortic fields (p = .04). In analysis of patients with grade 3 (severe), grade 4 (life-threatening), and grade 5 (fatal complications), 8% of the patients in both groups had grade 3 severe complications. In the pelvic plus para-aortic group, 11 patients had grade 4 and 2 had grade 5 complications, whereas 6 had grade 4 and none had grade 5 in the pelvic only treatment group.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗
PubMed1990

Oxidation products of caffeic acid as model substances for the antigonadotropic activity of plant extracts.

Phenolic plant constituents exert antigonadotropic activity following an oxidation. The resulting complex mixture of mostly instable products impedes the elucidation of the various oxidation steps as well as the mode of antigonadotropic action. Thus caffeic acid was chosen as a single model phenolic to facilitate the interpretation. The oxidation of caffeic acid with KMnO4 as well as with polyphenoloxidase leads to the instable caffeic acid o-quinone as the first oxidation product. Following the initial oxidation, a number of products was indicated via HPLC. Two of them were isolated and characterized as oligomers of caffeic acid, one of them with phenolic, acid and, quinoic structural components and a relative molecular mass similar to caffeic acid tetramer. It was shown that caffeic acid quinone cannot be the antigonadotropically active principle. Correspondingly, the isolated oxidation products exhibit pronounced antigonadotropic activity. It could be proved that oxidation products of caffeic acid bind to PMSG, forming PMSG-inhibitor-complexes. In such complexes the gonadotropic activity of PMSG is completely abolished.

Animals↗
PubMed1990

Structures of compounds with antigonadotropic activity obtained by in vitro oxidation of caffeic acid.

Two new cyclolignan derivatives were isolated by HPLC from the mixture of substances obtained after oxidation of caffeic acid with KMnO4. Their structures were elucidated by spectroscopic methods as 2,3-dicarboxy-6,7-dihydroxy-1-(3', 4'-dihydroxy)-phenyl-1, 2-dihydronaphthalene (1) and 3-carboxy-6,7-dihydroxy-1-(3', 4'-dihydroxy)-phenylnaphthalene (2). Compounds 1 and 2 exhibit antigonadotropic activity as do the extracts of crude drugs of Lycopus europaeus and Lithospermum officinale after oxidation by plant enzymes.

Animals↗
PubMed1990

Cerebrospinal fluid C-reactive protein measurement--a bedside test in the rapid diagnosis of bacterial meningitis.

C-reactive protein (C-RP) determinations were performed by the Latex agglutination method on the cerebrospinal fluid (CSF) samples of 212 patients with clinical features suggestive of meningitis. Patients were grouped as follows Group I: bacterial meningitis and partially treated bacterial meningitis (n = 22). Group II: viral encephalitis (n = 11). Group III: tuberculous meningitis (n = 18). Group IV: (i) febrile convulsions (n = 87); (ii) epileptic seizures (n = 70); (iii) intracranial haemorrhage (n = 4). C-RP was a better indicator of bacterial meningitis (sensitivity 91 per cent) than the Gram's stain (sensitivity 46 per cent). C-RP was positive in 91 per cent of patients in Group I, none in Groups II and III and 0.6 per cent in Group IV. C-RP determination in CSF proved to be a useful indicator of bacterial meningitis and served to distinguish it from viral encephalitis, tuberculous meningitis, febrile convulsions and other central nervous system disorders.

Bacterial Infections↗