Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It is postulated that vascular disease involves a disturbance in the homeostatic balance of factors regulating vascular tone and structure. Recent developments in gene transfer techniques have emerged as an exciting therapeutic option to treat vascular disease. Several studies have established the feasibility of direct in vivo gene transfer into the vasculature by using reporter genes such as beta-galactosidase or luciferase. To date no study has documented therapeutic effects with in vivo gene transfer of a cDNA encoding a functional enzyme. This study tests the hypothesis that endothelium-derived nitric oxide is an endogenous inhibitor of vascular lesion formation. After denudation by balloon injury of the endothelium of rat carotid arteries, we restored endothelial cell nitric oxide synthase (ec-NOS) expression in the vessel wall by using the highly efficient Sendai virus/liposome in vivo gene transfer technique. ec-NOS gene transfection not only restored NO production to levels seen in normal untreated vessels but also increased vascular reactivity of the injured vessels. Neointima formation at day 14 after balloon injury was inhibited by 70%. These findings provide direct evidence that NO is an endogenous inhibitor of vascular lesion formation in vivo (by inhibiting smooth muscle cell proliferation and migration) and suggest the possibility of ec-NOS transfection as a potential therapeutic approach to treat neointimal hyperplasia.
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PMID:Gene therapy inhibiting neointimal vascular lesion: in vivo transfer of endothelial cell nitric oxide synthase gene. 753 5

To examine whether nitric oxide (NO) has a protective effect against Ca2+ overdose or a beneficial action on myocardial cells, we employed direct gene-transfer of endothelial (type III) nitric oxide synthase (eNOS), using HVJ (Sendai virus) coated liposomes and beta-galactosidase (lac-z) as a marker for the transfection. The transfection efficiency of the lac-z gene was comparable with adenovirus as a vector, though the subsequent inflammation was much improved. The lac-z gene transfection was restricted to myoplasm between two intercalated discs, indicating that the transfected gene dose not permeate the disc. Co-transfection with human eNOS gene revealed degraded myoplasm of not only transfected cells but adjacent myocytes, fibrotic changes and infiltration of mononuclear cells seven days after the transfection. Electron microscopy of the lesions revealed a huge accumulation of mitochondria and loss of myofilaments, though fragmentation of nucleus or cytoplasm was not obvious. We conclude that an expression of human eNOS gene in cardiomyocytes causes a degenerative process, incompatible with typical apoptosis.
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PMID:Toxic action of nitric oxide on myocardial cells: direct evidence from gene transfer in vivo. 895 73

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.
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PMID:Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery. 904 52

We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that overproduction of .NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.
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PMID:Modulation of adenovirus-mediated gene transfer by nitric oxide. 916 Aug 30

We have designed a system in which to test gene transfer into gut neurons consisting of an organ culture of neonatal rat small intestine. The tissue was exposed to herpes simplex- and adenovirus-derived vectors: (1) a temperature-sensitive herpes simplex virus-1 (HSV1) vector (tsK-beta gal) containing the lacZ gene encoding beta-galactosidase (beta-gal), under the transcriptional control of the HSV1 immediate-early 3 (IE3) promoter; (2) RAd35, an E1-/E3- replication-deficient adenovirus expressing lacZ under the control of a truncated HCMV major IE promoter; and (3) RAd122, an E1-/E3- replication-deficient adenovirus expressing the lacZ under the control of the RSV LTR. Forty-eight hours after the vector was added to the organ culture, we detected beta-gal using immunohistochemistry or X-gal histochemistry in tissue sections examined by light microscopy. We encountered a distinctive staining of cells arranged in two concentric circles corresponding in location to the myenteric and submucosal plexuses. Cells in these areas were of similar size and morphology to neonatal enteric neurons, as visualized by NADPH-diaphorase histochemistry and immunocytochemical staining with antibodies to the neuronally expressed proteins PGP 9.5, or neurofilaments. Double labelling with antibodies recognizing neurofilaments and beta-galactosidase revealed that most cells infected by tsK were neurons, while the RAd35 and 122 vectors only infected non-neuronal cells. We thus demonstrate that both HSV1- and adenovirus-derived vectors can be used to transfer genes to the gut in vitro, but they transduce different populations of target cells.
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PMID:Gene transfer into enteric neurons of the rat small intestine in organ culture using a replication defective recombinant herpes simplex virus type 1 (HSV1) vector, but not recombinant adenovirus vectors. 917 19

Gene transfer with replication-deficient adenovirus is a potentially useful tool to study vascular biology. We have constructed a replication-deficient adenovirus (AdRSVeNOS) that carries cDNA for endothelial nitric oxide synthase (eNOS). Transfection of COS-1 cells with AdRSVeNOS increased nitric oxide synthase activity (measured as production of L-citrulline from L-arginine) that was calcium dependent and inhibited by N omega-nitro-L-arginine methyl ester. To investigate effects of overexpression of eNOS on vascular function, we incubated common carotid arteries from rabbits in organ culture with AdRSVeNOS or AdRSV beta gal encoding beta-galactosidase. Transgene expression and responses to vasoactive agents were examined 1 day after transduction. Histochemical staining of beta-galactosidase and immunohistochemistry for eNOS indicated transgene expression in endothelium and adventitial cells. After precontraction with phenylephrine, vessels treated with AdRSVeNOS demonstrated greater relaxation to acetylcholine than vessels treated with vehicle or AdRSV beta gal. Relaxation to calcium ionophore A-23187 was much greater in vessels treated with AdRSVeNOS than in vessels treated with vehicle or AdRSV beta gal. Augmented relaxation in response to A-23187 was also observed after denudation of endothelium in vessels treated with AdRSVeNOS and was inhibited by N omega-nitro-L-arginine. Thus vasorelaxation in response to stimuli that release nitric oxide is augmented after adenovirus-mediated overexpression of eNOS. Transgene expression in adventitial cells appears to be sufficient to alter vasomotor function.
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PMID:Altered vascular function after adenovirus-mediated overexpression of endothelial nitric oxide synthase. 924 99

Smooth muscle cells (SMCs) play a key role in the pathogenesis of vascular diseases. The objectives of this study were to determine whether transfer of recombinant endothelial nitric oxide synthase (eNOS) gene to porcine coronary artery smooth muscle cell (CSMCs) would result in expression of a functional enzyme and to assess the effect of expression of eNOS on cell proliferation. CSMCs were transduced in vitro with adenoviral vectors encoding cDNA for eNOS (AdeNOS) and beta-galactosidase (Ad beta Gal). In contrast to Ad beta Gal- or sham-transduced cells, CSMCs transduced with AdeNOS stained positive with the NADPH-diaphorase stain, acquired calcium-dependent NOS activity (measured by the conversion of [3H]L-arginine to [3H]L-citrulline), had increasing cyclic 3',5' cGMP levels with increasing concentrations of the vector, and produced increased amounts of nitrite. cGMP production by AdeNOS-transduced cells was augmented by increasing intracellular levels of the eNOS cofactor tetrahydrobiopterin. CSMCs transduced with AdeNOS showed diminished serum-stimulated DNA synthesis as measured by thymidine uptake. Cell proliferation was diminished in AdeNOS-transduced CSMCs as assessed by cell counts 3 and 6 days after serum stimulation of quiescent CSMCs. The present study demonstrates that adenovirus-mediated gene transfer of eNOS to CSMCs results in the expression of a functional enzyme whose activity can be augmented by increasing intracellular levels of tetrahydrobiopterin. Expression of recombinant eNOS in CSMCs results in inhibition of serum-stimulated DNA synthesis and cell proliferation. These findings imply that eNOS gene transfer to SMCs may be a unique mode of increasing local NO production in the arterial wall.
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PMID:Expression and function of recombinant endothelial NO synthase in coronary artery smooth muscle cells. 940 8

2-Methoxyestradiol (2ME) is an endogenous metabolite of estradiol (E2) and is known to inhibit tumor angiogenesis. In the present study, the direct effects of 2ME on the vascular endothelial cells were examined. 2ME enhanced apoptosis and beta-galactosidase expression in bovine vascular endothelial cells. A nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin (SNAP) also enhanced beta-galactosidase expression, suggesting a possible role of NO in mediating the action of 2ME. 2ME increased the cellular content of nitric oxide synthase (NOS) and the production of NO. In addition, 2ME altered the membrane localization pattern of NOS. These suggest that the effects of 2ME on apoptosis and senescence of vascular endothelial cells were mediated, at least partly, by NOS and NO.
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PMID:2-Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and beta-galactosidase expression in vascular endothelial cells. 967 76

TNP470, a derivative of fumagillin, suppressed in vivo growth of human PLC/PRF/5 hepatoma and ameliorated cachexia of hepatoma-bearing mice. These in vivo effects were associated with reductions in microvessel and macrophage counts. In in vitro experiments, TNP470 inhibited the growth and migration of human hepatoma and bovine vascular endothelial (VE) cells. TNP470 did not inhibit the production of VE growth factor by the hepatoma, which suggests that this compound acts directly on VE cells in vivo. In contrast, TNP470 inhibited the production of leukemia inhibitory factor, which may be related to the amelioration of cancer cachexia. TNP470 induced apoptosis and enhanced the expression of beta-galactosidase, a biomarker of senescence, which was partly mimicked by a nitric oxide (NO) donor S-nitroso-N-acetyl penicillamin. TNP470 inhibited myristoylation and membrane translocation of NO synthase and increased the cellular content of NO synthase and production of NO. Therefore, it is suggested that the actions of TNP470 are mediated, at least in part, through the inhibition of membrane translocation of biologically active proteins.
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PMID:Suppression of hepatoma growth and angiogenesis by a fumagillin derivative TNP470: possible involvement of nitric oxide synthase. 972 89

Nitric oxide (NO), a mediator involved in penile erection, is synthesized by the nitric oxide synthase (NOS) family of enzymes. It has been shown that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of endothelial NOS (eNOS) could enhance erectile responses, we administered a recombinant adenovirus containing the eNOS gene (AdCMVeNOS) into the corpora cavernosum of the aged rat. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue 1 day after intracavernosal administration of AdCMVbetagal; 1 day after administration of AdCMVeNOS, transgene expression was confirmed by immunoblot staining of eNOS protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with eNOS, and erectile responses to acetylcholine and zaprinast were enhanced at a time when the erectile response to the NO donor sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate was not altered. These results suggest that in vivo gene transfer of eNOS, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.
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PMID:Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. 1050 Feb 31


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