6R-[3H]tetrahydrobiopterin binding activities in rat brain.
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Amino Acid Oxidoreductases↗
Biomedical subjects
B Mayer
Publications and source records attributed to B Mayer.
Distribution and morphological features of nitrergic neurons in the porcine large intestine.
The distribution of nitric oxide synthase (NOS), an enzyme involved in the synthesis of the presumed non-adrenergic noncholinergic inhibitory neurotransmitter nitric oxide (NO), was demonstrated in the enteric nervous system of the porcine caecum, colon and rectum. Techniques used were NOS-immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd)-histochemistry. Throughout the entire large intestine, NOS-immunoreactive (IR) and NADPHd-positive neurons were abundant in the myenteric and outer submucous plexus. In the inner submucous plexus, only a small number of positive neurons were found in the caecum and colon, while a moderate number was observed in the rectum. The nitrergic neurons in the porcine enteric nerve plexuses were of a range of sizes and shapes, with a small proportion showing immunostaining for vasoactive intestinal polypeptide. Varicose and non-varicose NOS-IR and NADPHd-positive nerve fibres were present in the ganglia and connecting strands of all three plexuses. Nerve fibres were also numerous in the circular muscle layer, scarce in the longitudinal muscle coat and negligible in the mucosal region. The abundance of NOS/NADPHd in the intrinsic innervation of the caecum, colon and rectum of the pig implicates NO as an important neuronal messenger in these regions of the gastrointestinal tract.
Amino Acid Oxidoreductases↗
Nitric oxide synthase-immunoreactive axons innervating the guinea-pig lingual artery: an ultrastructural immunohistochemical study using elastic brightfield imaging.
The ultrastructure of nitric oxide synthase-immunoreactive (NOS-IR) axons innervating the guinea-pig lingual artery was investigated by means of pre-embedding immunohistochemistry using an indirect peroxidase technique and diaminobenzidine. Sections ranging in thickness from 60 to 500 nm were ultrastructurally evaluated in elastic brightfield imaging mode. Thick sections (optimum at 300 nm) were advantageous for enhancement of the labelling intensity, whilst some subcellular details were better revealed by thin sections. NOS-IR axon terminals often contained aggregations of large, dense-cored vesicles, consistent with a previous light microscopical report on colocalization of NOS and vasoactive intestinal peptide-immunoreactivity in these fibres. NOS-IR axons formed direct neuro-muscular junctions (width less than 50 nm) at the outer surface of the tunica media, thus providing a structural basis for "nitrergic" vasodilation. In addition, NOS-IR axons made direct contacts with non-varicose and varicose segments of non-reactive axons, suggesting interneuronal communication between these elements.
Amino Acid Oxidoreductases↗
Colocalization of vasoactive intestinal peptide and nitric oxide synthase in neurons of the ferret trachea.
Neurally-mediated relaxation of smooth muscle in human, guinea-pig, cat, and pig airways is largely attributed to a nonadrenergic, noncholinergic mechanism. While the specific transmitter(s) of this relaxant system have not been conclusively identified, vasoactive intestinal peptide and nitric oxide have emerged as likely mediators in airway smooth muscle. Both vasoactive intestinal peptide and nitric oxide relax guinea-pig, pig and human smooth muscle. Vasoactive intestinal peptide is present in nerve fibers associated with airway smooth muscle in humans and several animal species. In guinea-pigs, vasoactive intestinal peptide is released during electrical field stimulation of airway strips and the release correlates with the nonadrenergic relaxation. This relaxation is markedly reduced after incubation of tracheal tissue with a specific VIP antibody and by immunization to vasoactive intestinal peptide. Similarly, nonadrenergic relaxations induced by electrical field stimulation are reduced in human, pig, guinea-pig and bovine airways by nitric oxide synthesis inhibitors. Vasoactive intestinal peptide is present in nerve cell bodies of airway ganglia, suggesting that these nerves in airway smooth muscle originate from intrinsic neurons. It is stored in dense-core vesicles of nerve terminals near airway smooth muscle, suggesting that preformed vasoactive intestinal peptide is released by fusion of the vesicles with the cell membrane of the nerve terminal. Nitric oxide is probably generated by a novel mechanism involving de novo synthesis at the nerve terminal during neural activation by the action of the enzyme nitric oxide synthase.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Oxidoreductases↗
Distribution of nitric oxide synthase-immunoreactive neurons in the submucosal plexus of the porcine small intestine.
This study analyses the occurrence of NOS-immunoreactive neurons in the enteric nervous system of the porcine small intestine, and particularly their distribution pattern in the ganglia of the two submucosal plexuses, the external submucosal plexus and the internal submucosal plexus. Numerous NOS-immunoreactive nerve cell bodies were found in the external submucosal plexus, as well as in the myenteric plexus. In 1 cm2 of the surface of the submucosal whole-mount preparation, we counted 186 NOS-immunoreactive nerve cell bodies in the external submucosal plexus, which is about 27% of the total number of nerve cells in that plexus. As against this, we found only very few NOS-immunoreactive nerve cells in the internal submucosal plexus. In the external submucosal plexus, these cells are often located together, forming aggregates within the ganglia. NOS-immunoreactive nerve cells are of different sizes and shapes. Some of them have a multidendritic uniaxonal appearance. NOS-immunoreactive nerve fibres can be observed in all three ganglionic networks, surrounding NOS-immunoreactive as well as nonimmunoreactive nerve cell bodies and forming varicosities in close proximity to the nerve cell bodies.
Amino Acid Oxidoreductases↗
[Tarsal flap technique. A technically simple procedure for correction of lower lid flaccidity, senile ectropion and ptosis of the lateral canthus].
Lower lid flaccidity, senile extropion and canthal malposition are the principal indications for the tarsal flap technique. This is a technically simple procedure avoiding sutures on the border of the lashes and avoiding the disadvantages of the technique of Kuhnt - Szymanowski, which cause a shortening of palpebral length due to tarsal resection.
Aged↗
Nitric oxide synthase neurons in rat brain express more NMDA receptor mRNA than non-NOS neurons.
Nitric oxide, the gaseous neuronal messenger, is produced by nitric oxide synthase (NOS) in a Ca2+ dependent manner. Indirect evidence suggests that glutamate, acting through the NMDA subtype of excitatory amino acid receptor, is the principle activator signal for NOS in the brain. To date there is a lack of direct evidence demonstrating NOS and NMDA receptors, or receptor message, in the same neurons. A double labeling technique was developed which uses NOS immunocytochemistry in combination with in situ hybridization for NMDA NR1 receptor mRNA. Quantitative analysis of the silver grain labeling resulting from the hybridization procedure revealed that the majority of NOS positive cells in the cerebral cortex, striatum and midbrain contained a significantly greater amount of NR1 receptor mRNA than non-NOS neurons in the same regions. The amount of NR1 mRNA per cell varies in the cortical and striatal NOS cells, with some cells showing no NR1 expression. These results indicate that NOS containing neurons do not reflect a single phenotype of mRNA expression and further suggest that some NOS cells may be activated by a non-NMDA receptor mechanism.
Amino Acid Oxidoreductases↗
Inhibition of Ca2+ transport pathways in thymic lymphocytes by econazole, miconazole, and SKF 96365.
Cytochrome P-450 has been proposed to underlie the mechanism of regulation of the plasma membrane Ca2+ permeability by the Ca2+ content of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. We have investigated the effects on divalent cation uptake in rat thymic lymphocytes of three structurally related imidazole reagents reported to inhibit redox mechanisms. Changes in intracellular Ca2+ concentration and intracellular Mn2+ concentration were measured fluorimetrically with indo-1 and/or quin-2. Econazole, miconazole, and SKF 96365 were found to be potent blockers of Ca2+ and Mn2+ uptake activated by release of Ca2+ from intracellular stores induced by thapsigargin. Additionally, we found that concentrations of these agents required to abolish divalent cation uptake also released Ca2+ from the thapsigargin-sensitive intracellular stores, consistent with inhibition of the endosomal Ca(2+)-ATPase. In agreement with this suggestion, we have found that all three of these agents are potent inhibitors of isolated sarcoplasmic reticulum Ca(2+)-ATPase. We conclude that econazole, miconazole, and SKF 96365 inhibit cytochrome P-450-independent filling of intracellular Ca2+ pools, as well as store-regulated Ca2+ entry, and caution against the use of these compounds as selective inhibitors of cytochrome P-450.
Animals↗
Hypercholesterolemia is associated with a reduced response of smooth muscle guanylyl cyclase to nitrovasodilators.
A diminished relaxant response of atherosclerotic arteries to nitrovasodilators has been frequently observed in advanced stages of hypercholesterolemia. In the present study, we investigated whether this effect might be a result of reduced activity of smooth muscle guanylyl cyclase. Experimental atherosclerosis was induced by feeding rabbits a cholesterol-rich diet (1%) over a period of 4 months. Aortas were removed and homogenized, and guanylyl cyclase activity was measured in the 100,000 g supernatants. Sodium nitroprusside, which stimulated cyclic GMP (cGMP) formation in control tissues almost 200-fold (from 3 to 585 pmol cGMP.mg-1 x min-1), increased enzyme activities in atherosclerotic aortas only approximately 90-fold (from 3 to 257 pmol cGMP.mg-1 x min-1). Similarly, the maximal stimulatory effects of S-nitroso-glutathione were reduced from 200-fold (controls) to 114-fold in atherosclerotic tissues. Basal guanylyl cyclase activities were identical in both atherosclerotic and control vessels. Hypercholesterolemia also reduced the activity of smooth muscle adenylyl cyclase. In control aortas, basal and NaF-stimulated enzyme activities were 24 and 349 pmol cAMP.mg-1 x min-1, respectively, whereas cAMP formation was reduced in atherosclerotic aortas to 7 (basal) and 96 (NaF) pmol cAMP.mg-1.min-1. The stimulatory effect of NaF (approximately 14-fold) remained unchanged. Since adenylyl and guanylyl cyclase have important functions in regulating vascular tone, reduced activities of both enzymes may contribute to the diminished relaxant and/or enhanced vasoconstricting effects of vasoactive compounds in atherosclerotic blood vessels.
Adenylyl Cyclases↗
New dimensions for the vital storage of microsurgical free flaps: an experimental approach.
A revolutionary circulatory system has been developed to nourish big, free osteomusculocutaneous flaps extracorporally. Thus we will be able to transplant the free flaps to defect areas that have no sufficient vascular situation. In respect to the cold storage of microsurgical free flaps, to date maximal periods of ischaemic tolerance have been considerably exceeded; the maximal period is currently 168 hours. The vitality of the flap is monitored through parameters setting forth the consumption of oxygen together with histology and electron microscopy. The oxygenation of the nutritive medium is achieved through an aqueous phase gas exchange.
Animals↗
Novel actions of methylene blue.
Methylene blue has been frequently used as an inhibitor of soluble guanylyl cyclase. We found that endothelium-dependent relaxations of isolated blood vessels were considerably more sensitive to inhibition by methylene blue than relaxation induced by direct activators of soluble guanylyl cyclase. Similar data were obtained in the presence of superoxide dismutase, indicating that the diverse potencies of methylene blue were not due to superoxide-induced inactivation of nitric oxide (NO). Subsequent experiments revealed that methylene blue is an inhibitor of purified NO synthase. Conversion of L-arginine to L-citrulline was inhibited by the dye in a concentration-dependent fashion with half-maximal effects observed at 5.3 microM and 9.2 microM in the absence and presence of superoxide dismutase, respectively. Purified soluble guanylyl cyclase, however, was far less sensitive to methylene blue. When the enzyme was maximally stimulated with S-nitroso-glutathione, cyclic guanosine monophosphate, (cGMP) formation was reduced by 50% at approximately 60 microM methylene blue; 1 mM produced maximal inhibitions of about 70%. Our data indicate that methylene blue is only a poor inhibitor of soluble guanylyl cyclase. The dye seems to act primarily via inhibition of NO synthase, with enzyme-bound heme being a possible target in its inhibitory action.
Amino Acid Oxidoreductases↗
Characterization of endothelial cell amino acid transport systems involved in the actions of nitric oxide synthase inhibitors.
N omega-Substituted analogues of L-arginine have proven useful as specific inhibitors of nitric oxide formation in various biological systems. In the present study we describe the characteristics of amino acid transporters that mediate uptake of N omega-methyl-L-arginine (L-NMA) and N omega-nitro-L-arginine (L-NNA) into cultured porcine aortic endothelial cells. The transport of L-[14C]NMA showed biphasic kinetics, with Km values of 4 and 368 microM, and was inhibited by L-arginine, L-homoarginine, L-lysine, and L-ornithine but not by L-leucine or L-isoleucine. Similar transport kinetics (Km values of 6 and 609 microM) and substrate specificities were obtained for L-[3H]arginine uptake, indicating that L-arginine and L-NMA are transported by the same system. In contrast to L-arginine and L-NMA transport, uptake of L-[3H]NNA was monophasic (Km = 617 microM) and was inhibited by L-leucine and L-isoleucine but not by L-arginine, L-homoarginine, L-NMA, L-lysine, or L-ornithine. Uptake studies with L-[3H]leucine revealed that the transport of this amino acid occurred in a manner very similar to that of L-[3H]NNA transport, suggesting that the uptake of both compounds may be mediated by the same system. In additional experiments, we determined the effects of L-NMA and L-NNA on the A23187-induced accumulation of intracellular cGMP, to establish to what extent these transport systems are involved in the actions of nitric oxide synthase inhibitors. L-Lysine and L-ornithine, which both inhibited L-NMA uptake, increased the IC50 of L-NMA from 7.8 microM to 57 microM but did not reduce the inhibitory effects of L-NNA. In the presence of L-leucine or L-isoleucine, however, which both inhibited L-NNA uptake, the IC50 of L-NNA was increased from 1.2 microM to 37 microM but the inhibitory actions of L-NMA remained unaffected. These data demonstrate that the endothelial transport systems for L-arginine and L-leucine mediate the biological effects of L-NMA and L-NNA, respectively.
Amino Acid Oxidoreductases↗
Characterization of nitric oxide synthase, soluble guanylyl cyclase, and Ca2+/calmodulin-stimulated cGMP phosphodiesterase as components of neuronal signal transduction.
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3',5'-Cyclic-GMP Phosphodiesterases↗
Long-lasting expression of JUN and KROX transcription factors and nitric oxide synthase in intrinsic neurons of the rat brain following axotomy.
In adult rats, the medial forebrain bundle (MFB) and mammillothalamic tract (MT) were unilaterally transected, resulting in axotomy of neurons in numerous areas such as the substantia nigra (SN), ventral tegmental area (VTA), nucleus (ncl.) mammillaris (MnM), and ncl. parafascicularis of the thalamus (PF). In these areas, expression of the transcription factor proteins c-JUN, JUN B, JUN D, c-FOS, FOS B, KROX-20, KROX-24, and CREB was investigated by immunocytochemistry up to 150 d. In parallel, the expression of nitric oxide synthase (NOS) was investigated both immunocytochemically and by the NADPH-diaphorase reaction (NDP), and the antibody against NOS was further characterized. The colocalization of c-JUN with NDP or NOS was also studied in the axotomized neurons. c-JUN and JUN D became visible in nuclei of many neurons of the ipsilateral MnM, PF, VTA, and SN (predominantly in the pars compacta and those double labeled by tyrosine hydroxylase, TH) after 36 hr, not after 24 hr, following transection of MFB and MT. In MnM, c-JUN and JUN D persisted at a nearly maximal level for up to 150 d. In PF, these proteins returned to control levels after 75 d. Expression of c-JUN and JUN D declined in the VTA after 30 d, but in the SN, it already declined after only 10 d. KROX-24 had a later onset of expression, being visible after 3 d in all investigated areas, and its pattern was similar to that of JUN proteins, although labeling was visible in fewer nuclei and declined earlier. JUN B, c-FOS, FOS B, and KROX-20 were not expressed in these areas, and substantial alterations of CREB immunoreactivity (CREB-IR) could not be detected. A subset of SN neurons (predominantly in the pars reticularis and negative for TH) presented an early and transient expression of all studied JUN, FOS, and KROX-24 proteins within 3 hr of transection that declined between 24 hr and 48 hr to basal levels. This expression pattern is typical of that caused by transynaptic stimulation (probably due to excitation of descending striatal neurons running within the MFB) and was clearly distinct from that evoked by c-JUN, JUN D, and KROX-24 IRs after 36 hr (predominantly in the pars compacta). An ipsilateral increase in NOS and NDP became visible in many neurons of the MnM after 10 d, but not after 5 d, and this persisted up to 150 d. The temporospatial pattern of NDP was similar to the pattern of NOS-IR.(ABSTRACT TRUNCATED AT 400 WORDS)
Amino Acid Oxidoreductases↗
Intracavernous application of SIN-I in rabbit and man: functional and toxicological results.
The mode of action of the active metabolite SIN-I of the vasodilator prodrug molsidomine was studied in vitro and in vivo in corpus cavernosum of rabbit and man. SIN-I produces a dose-dependent relaxation of isolated human cavernous smooth muscle strips. In the rabbit, the intracavernous application of SIN-I increased the intracavernous pressure to a full erection (approximately 100 cm H2O). This response was highly reproductible. SIN-I was also injected intracavernously 6 times in five rabbits over 2 weeks; no inflammatory or fibrotic reactions were found on histology. SIN-I may be a reliable drug for the treatment of impotence without side-effects.
Animals↗
Secondary structure of Src homology 2 domain of c-Abl by heteronuclear NMR spectroscopy in solution.
The Src homology 2 (SH2) domain is a recognition motif thought to mediate the association of the cytoplasmic proteins involved in signal transduction by binding to phosphotyrosyl-containing sequences in proteins. Assignments of nearly all 1H and 15N resonances of the SH2 domain from the c-Abl protein-tyrosine kinase have been obtained from homonuclear and heteronuclear NMR experiments. The secondary structure has been elucidated from the pattern of nuclear Overhauser effects, from vicinal coupling constants, and from observation of slowly exchanging amino hydrogens. The secondary structure contains two alpha-helices and eight beta-strands, six of which are arranged in two contiguous, antiparallel beta-sheets. Residues believed to be involved in phosphotyrosyl ligand binding are on a face of one beta-sheet. The alignment of homologous sequences on the basis of secondary structure suggests a conserved global fold in a family of SH2 domains.
Amino Acid Sequence↗
Brain nitric oxide synthase is a haemoprotein.
Brain nitric oxide (NO) synthase showed pyridine haemochrome spectra typical of ferroprotoporphyrin IX-containing enzymes. The haem content of purified NO synthase was in the range 0.7-0.9 mol/mol of 160 kDa subunit. In the presence of CO, NO, KCN and miconazole, the L-citrulline-forming activity of NO synthase was markedly diminished, demonstrating that enzyme-bound haem is involved in enzymic NO synthesis.
Amino Acid Oxidoreductases↗
Dynamics of sine-Gordon solitons in the annular Josephson junction.
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Journal Article↗