By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue sample...By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.展开更多
Nitric oxide is an important neuromodulator in the brain and is involved in the development of visual system. But it is not clear how nitric oxide and nitric oxide synthase （NOS） are involved in the developing visua...Nitric oxide is an important neuromodulator in the brain and is involved in the development of visual system. But it is not clear how nitric oxide and nitric oxide synthase （NOS） are involved in the developing visual cortex of rodents. Thus we examined the expression of NOS activity in the postnatal developing visual cortex of the golden hamster by using histochemical technique for NADPH-diaphorase （NADPH-d）. A heavily stained NADPH-d band was observed in the neuropil of the visual cortex. This NADPH-d band initially appeared in the cortical plate from the day of birth （P0） to postnatal day 4 （P4）. From P7 to P21, this band was confined to area 17 and migrated to the deeper layers Ill IV and V VI before it eventually disappeared at P28. Such developmental trends of the band correlated well with the process of formation and establishment of the geniculo-cortical projection patterns. Thus, the areal specific development of the band suggests that NOS is closely related to the cortical differentiation and synaptic formation of the primary visual cortex. On the other hand, monocular eye enucleation on P1 could not alter the appearance of this NADPH-d positive band, indicating a non-activity dependant role of NOS. In addition, differences in the laminar distributions and developmental sequence between the heavily and lightly stained NADPH-d positive neurons during development suggest that they play different roles in the development.展开更多
We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohis...We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohistochemical staining and histochemical staining. The periventricular region cells exhibited the morphology of neurons and glia including radial glia-like cells and contained several neurochemical substances. Heterogeneous populations of tyrosine hydroxylase-, gamma aminobutyric acid-immunoreactive, as well as nicotinamide adenine dinucleotide phosphate diaphorase-positive cells were observed in proliferating cell nuclear antigen-immunoreactive proliferative zones in periventricular area of diencephalon, central grey layer of dorsomedial tegmentum, medulla and spinal cord. Immunolocalization of Pax6 in the cherry salmon brain revealed a neuromeric construction of the brain at various stages of postnatal ontogenesis, and this was confirmed by tyrosine hydroxylase and gamma aminobutyric acid labeling.展开更多
The data of our investigations contribute to understanding of cellular mechanisms of the teleost fishes CNS forming in postembryonic development. The revealed peculiarities of structural and neurochemical organization...The data of our investigations contribute to understanding of cellular mechanisms of the teleost fishes CNS forming in postembryonic development. The revealed peculiarities of structural and neurochemical organization and description of basic histogenetic processes (proliferation, migration and neuronal cell differentiation) during the brain forming in fish, which have signs of fetal organization, widen the existing knowledge about histogenesis of these structures in postembryonic development. It seems conceivable, that during postembrional development in teleost fishes some neurotransmitters and gaseous mediators (NO and H2S) act as factors, which initiate and regulate the cellular and the tissues processes of genetic program during the brain development. Materials of this investigation define a new experimental model for studying of postembrional neurogenesis processes.展开更多
Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was us...Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was used in our experiment. Twenty SD rats were randomly divided into KA 30, 60, 90, 200min and control groups. The brain sections were stained by NADPh (nicotinamide adenine dinucleotide phosphate ) diaphorase (Nd ) histochemically, and were further stained by GABA immunohistochemically.Results: Histological and immunohistochemical study revealed that in KA groups the number of Nd and GABA-positive double labelled neurons in CA3 region, CA3 region and dentate gyms was significantly reduced,compared with the control group. Conclusion: Nd coexisted with GABA in the brain. Reduction of GABA release led to relief of GABA-ergic inhibition and in the same way, reduction of NO release weakened its negative feedback modulation. Therefore neuronal synchronous paroxysmal discharges increased. GABA and NO,both having antiepileptic action, acted through different ways or different link in the same way. NO may involve in the effect of GABA-ergic neurons and play cooperative antiepileptic action with GABA.展开更多
The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids ...The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,展开更多
文摘By combining native polyacrylamide gel electrophoresis (PAGE) and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining, a simple method for detecting NADPH-cytochrome P450 reductase in tissue samples was established. When rat liver microsomal fractions were examined by this method, several bands with different mobility were visualized. Western blot analysis indicated that the band which appeared in the most anodal position among them represented NADPH-cytochrome P450 reductase. SDS-PAGE/Western blot analysis revealed that the molecular weight of the protein forming the band was around 80 kDa, which was identical to that of rat NADPH-cytochrome P450 reductase. The intensity level of NADPH-diaphorase staining assigned to this enzyme estimated by this method increased four times in microsomal fractions prepared from rat fed ethanol chronically compared to that from controls. When a dilution series of a rat liver microsomal fraction was examined by this method and SDS-PAGE/Western blot analysis, their staining intensities representing this enzyme were significantly correlated with each other. Using the naive PAGE/NADPH-diaphorase staining method, NADPH-cytochrome P450 reductase is detected in rat liver microsomes. This method is beneficial because compared with the conventional SDS-PAGE/Western blot analysis, the quantification of NADPH-cytochrome P450 reductase in tissue samples is allowed to be more easily done.
基金the Fundamental Research Funds for the Central Universities,No.21609101the National Basic Research Program of China (973 Program),No.2011CB707501
文摘Nitric oxide is an important neuromodulator in the brain and is involved in the development of visual system. But it is not clear how nitric oxide and nitric oxide synthase （NOS） are involved in the developing visual cortex of rodents. Thus we examined the expression of NOS activity in the postnatal developing visual cortex of the golden hamster by using histochemical technique for NADPH-diaphorase （NADPH-d）. A heavily stained NADPH-d band was observed in the neuropil of the visual cortex. This NADPH-d band initially appeared in the cortical plate from the day of birth （P0） to postnatal day 4 （P4）. From P7 to P21, this band was confined to area 17 and migrated to the deeper layers Ill IV and V VI before it eventually disappeared at P28. Such developmental trends of the band correlated well with the process of formation and establishment of the geniculo-cortical projection patterns. Thus, the areal specific development of the band suggests that NOS is closely related to the cortical differentiation and synaptic formation of the primary visual cortex. On the other hand, monocular eye enucleation on P1 could not alter the appearance of this NADPH-d positive band, indicating a non-activity dependant role of NOS. In addition, differences in the laminar distributions and developmental sequence between the heavily and lightly stained NADPH-d positive neurons during development suggest that they play different roles in the development.
基金supported by a grant from Far Eastern Branch of Russian Academy of Sciences, No.12-Ⅲ-A-06-095
文摘We investigated the distribution of gamma aminobutyric acid, tyrosine hydroxylase and nitric oxide-producing elements in a cherry salmon Oncorhynchus masou brain at various stages of postnatal ontogenesis by immunohistochemical staining and histochemical staining. The periventricular region cells exhibited the morphology of neurons and glia including radial glia-like cells and contained several neurochemical substances. Heterogeneous populations of tyrosine hydroxylase-, gamma aminobutyric acid-immunoreactive, as well as nicotinamide adenine dinucleotide phosphate diaphorase-positive cells were observed in proliferating cell nuclear antigen-immunoreactive proliferative zones in periventricular area of diencephalon, central grey layer of dorsomedial tegmentum, medulla and spinal cord. Immunolocalization of Pax6 in the cherry salmon brain revealed a neuromeric construction of the brain at various stages of postnatal ontogenesis, and this was confirmed by tyrosine hydroxylase and gamma aminobutyric acid labeling.
文摘The data of our investigations contribute to understanding of cellular mechanisms of the teleost fishes CNS forming in postembryonic development. The revealed peculiarities of structural and neurochemical organization and description of basic histogenetic processes (proliferation, migration and neuronal cell differentiation) during the brain forming in fish, which have signs of fetal organization, widen the existing knowledge about histogenesis of these structures in postembryonic development. It seems conceivable, that during postembrional development in teleost fishes some neurotransmitters and gaseous mediators (NO and H2S) act as factors, which initiate and regulate the cellular and the tissues processes of genetic program during the brain development. Materials of this investigation define a new experimental model for studying of postembrional neurogenesis processes.
文摘Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was used in our experiment. Twenty SD rats were randomly divided into KA 30, 60, 90, 200min and control groups. The brain sections were stained by NADPh (nicotinamide adenine dinucleotide phosphate ) diaphorase (Nd ) histochemically, and were further stained by GABA immunohistochemically.Results: Histological and immunohistochemical study revealed that in KA groups the number of Nd and GABA-positive double labelled neurons in CA3 region, CA3 region and dentate gyms was significantly reduced,compared with the control group. Conclusion: Nd coexisted with GABA in the brain. Reduction of GABA release led to relief of GABA-ergic inhibition and in the same way, reduction of NO release weakened its negative feedback modulation. Therefore neuronal synchronous paroxysmal discharges increased. GABA and NO,both having antiepileptic action, acted through different ways or different link in the same way. NO may involve in the effect of GABA-ergic neurons and play cooperative antiepileptic action with GABA.
基金Project supported by the State Commission of Science and Technology of China,the National Institutes of Health (USA)(EY 08338) and the International Human Frontier Science Program Organization.
文摘The carp retina was examined by NADPH diaphorase histochemistry to determine if the staining pattern of retinal cells was changed depending on the adaptation state of the retina. When dark-adapted for 5 h, ellipsoids of inner segments of both rods and cones and some horizontal cells were heavily stained. Staining was also found in subpopulations of amacrine cells and ganglion cells. In addition, Muller cells were strongly positive for NADPH diaphorase. When light-adapted for 5h, ellipsoids of photoreceptors and ganglion cells were less intensely stained, whereas Muller cells and horizontal cells became negative for NADPH diaphorase. Furthermore, rod ON-center bipolar cells were clearly stained. The difference of staining of amacrine cells between dark- and light-adapted retinas was not significant. The differences in diaphorase-staining pattern between dark- and light-adapted retinas suggest that Muller cells, some horizontal cells and rod ON-center bipolar cells contain inducible nitric oxide synthase,
