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)

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 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

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

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

The experiments in this paper were designed to examine the therapeutic effects of adenoviral-mediated gene transfer of IFN-gamma into a mouse model of an established metastatic brain tumor. Temperature-sensitive replication-defective adenovirus was generated for gene transfer of IFN-gamma (AdIFN) and beta-galactosidase (AdBGAL) cDNAs in vivo. In this model, treatment with AdIFN elicits prolonged survival times and brain tumor rejection. Evidence against an immune-mediated response accounting for this result include: 1) absence of a memory immune response upon challenge, 2) lack of antitumor effects at sites distal to inoculation of AdIFN, and 3) preservation of the therapeutic effects of AdIFN in scid and beige mice and in inducible NO synthase (iNOS) knockouts. High concentrations of IFN-gamma do not inhibit tumor growth in vitro making it unlikely that the antitumor effect of this treatment acts directly on the growth of the tumor cells. However, gene transfer of IFN-gamma inhibits neovascularization of the tumor in a 3LL-Matrigel assay in vivo, and AdIFN induces apoptosis of endothelial cells in vivo, supporting the idea that AdIFN represses tumor growth by inhibiting angiogenesis. The substantial non-immune-mediated therapeutic benefits of AdIFN in animals paves the way for devising novel strategies for treating human brain tumors.
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PMID:Gene transfer of IFN-gamma into established brain tumors represses growth by antiangiogenesis. 1060 14

Vascular endothelial growth factor (VEGF) induces endothelial cell proliferation, migration, and actin reorganization, all necessary components of an angiogenic response. However, the distinct signal transduction mechanisms leading to each angiogenic phenotype are not known. In this study, we examined the ability of VEGF to stimulate cell migration and actin rearrangement in microvascular endothelial cells infected with adenoviruses encoding beta-galactosidase (beta-gal), activation-deficient Akt (AA-Akt), or constitutively active Akt (myr-Akt). VEGF increased cell migration in cells transduced with beta-gal, whereas AA-Akt blocked VEGF-induced cell locomotion. Interestingly, myr-Akt transduction of bovine lung microvascular endothelial cells stimulated cytokinesis in the absence of VEGF, suggesting that constitutively active Akt, per se, can initiate the process of cell migration. Treatment of beta-gal-infected endothelial cells with an inhibitor of NO synthesis blocked VEGF-induced migration but did not influence migration initiated by myr-Akt. In addition, VEGF stimulated remodeling of the actin cytoskeleton into stress fibers, a response abrogated by infection with dominant-negative Akt, whereas transduction with myr-Akt alone caused profound reorganization of F-actin. Collectively, these data demonstrate that Akt is critically involved in endothelial cell signal transduction mechanisms leading to migration and that the Akt/endothelial NO synthase pathway is necessary for VEGF-stimulated cell migration.
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PMID:Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt. 1078 12

In a transgenic model of ischemic cardiomyopathy in which monocytes are attracted to the myocardium by the targeted overexpression of monocyte chemoattractant protein-1 (MCP-1), we have observed the presence of endothelial NO synthase and platelet endothelial cell adhesion molecule-1-negative tunnels, occasionally containing blood-derived cells, that probe the cardiac tissue. Immunohistochemical data show that monocytes/macrophages (MCs/Mphs) drill tunnels using the broad-spectrum mouse macrophage metalloelastase. 5-Bromo-2'-deoxyuridine incorporation and neo-endothelial markers present in the microvasculature of MCP-1 mouse hearts suggest an active angiogenic process. Further studies will be required to establish that the MC-/Mph-drilled tunnels evolve to become capillaries, connected to the existing vessels and colonized by circulating endothelial cell progenitors. This possibility is supported by the availability of these cells, which is demonstrated by cell tagging with beta-galactosidase placed under an active endothelial Tie-2 promoter. This phenomenon might represent another mechanism, in addition to the secretion of the angiogenic factors, by which MCs/MPhs may participate in the elaboration of new blood vessels in adult tissues.
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PMID:Contribution of monocytes/macrophages to compensatory neovascularization: the drilling of metalloelastase-positive tunnels in ischemic myocardium. 1096 28

Because arginase hydrolyzes arginine to produce ornithine and urea, it has the potential to regulate nitric oxide (NO) and polyamine synthesis. We tested whether expression of the cytosolic isoform of arginase (arginase I) was limiting for NO or polyamine production by activated RAW 264.7 macrophage cells. RAW 264.7 cells, stably transfected to overexpress arginase I or beta-galactosidase, were treated with interferon-gamma to induce type 2 NO synthase or with lipopolysaccharide or 8-bromo-cAMP (8-BrcAMP) to induce ornithine decarboxylase. Overexpression of arginase I had no effect on NO synthesis. In contrast, cells overexpressing arginase I produced twice as much putrescine after activation than did cells expressing beta-galactosidase. Cells overexpressing arginase I also produced more spermidine after treatment with 8-BrcAMP than did cells expressing beta-galactosidase. Thus endogenous levels of arginase I are limiting for polyamine synthesis, but not for NO synthesis, by activated macrophage cells. This study also demonstrates that it is possible to alter arginase I levels sufficiently to affect polyamine synthesis without affecting induced NO synthesis.
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PMID:Arginase I: a limiting factor for nitric oxide and polyamine synthesis by activated macrophages? 1108 91

Endothelial dysfunction is frequently involved in the pathogenesis of vascular disease. While nitric oxide (NO) inhibits smooth muscle cell proliferation, its effect on endothelial cell proliferation is unclear. The aim of this study was to determine if adenoviral-mediated gene transfer of endothelial NO synthase (eNOS) to human umbilical vein endothelial cells (HUVECs) would result in increased generation of NO and affect endothelial cell proliferation. HUVECs were transduced with adenoviral vectors encoding eNOS (AdeNOS) or beta-galactosidase (Ad beta gal) or exposed to diluent (control). AdeNOS-transduced cells showed increased eNOS expression as detected by Western blot analysis, and increased concentrations of cGMP (control 0.7 +/- 0.1; Ad beta gal 0.9 +/- 0.2; AdeNOS 3.1 +/- 0.5 pmol/mg protein; p < 0.001) and nitrite (control 11.8 +/- 1.2; Ad beta gal 13.3 +/- 1.7; AdeNOS 21.1 +/- 2.2 nmol/mg protein/hour; p < 0.01). DNA synthesis as assessed by [(3)H]thymidine incorporation and cell counts were significantly reduced (by approximately 30%) in AdeNOS-transduced HUVECs. Expression of mitogen-activated protein kinase was also decreased in AdeNOS-transduced cells. This study shows that adenoviral-mediated gene transfer of eNOS to HUVECs inhibits endothelial cell proliferation.
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PMID:Expression and function of recombinant endothelial nitric oxide synthase in human endothelial cells. 1114 98

The clinical outcome of vascular stenting is limited by in-stent stenosis. Increased nitric oxide (NO)/cGMP signaling by L-arginine (L-Arg) supplementation, the substrate for NO synthase (NOS), or NOS gene transfer may reduce in-stent neointima formation. After stenting, vascular cell proliferation in rat carotid arteries, as measured by 5'-bromodeoxyuridine (5'-BrdU) incorporation, indicated 15+/-8%, 28+/-5%, and 33+/-7% 5'-BrdU-positive vascular cells at 4, 7, and 14 days, respectively. Reporter beta-galactosidase gene transfer efficacy was evidenced by 30% beta-galactosidase-expressing medial smooth muscle cells at 14 days. The intima-to-media ratio (I/M) progressively increased to 2.32+/-0.24 at 14 days. To target in-stent neointima formation, animals were infected with adenoviral vectors (4x10(10) plaque-forming units per mL) expressing NOS2 (AdNOS2) or no transgene (AdRR5), or they received daily doses of L-Arg (500 mg. kg(-1). (d-1) IP). The neointima at 14 days was smaller in L-Arg-treated than in untreated rats (I/M 1.25+/-0.35 vs 2.32+/-0.24, P<0.05, n=7 each) or in AdRR5- and AdNOS2-infected rats (I/M 2.57+/-0.43, n=7 and 1.82+/-0.75, n=8, respectively; P<0.05 for both). The effect of L-Arg was abolished by simultaneous administration of N(G)-nitro L-arginine methyl ester, an NOS inhibitor (2.03+/-0.39, P<0.05, vs L-Arg). Inflammation was markedly less in L-Arg- and AdNOS2-treated than in AdRR5-infected rats. Supplemental L-Arg reduces neointima formation after stenting by way of an NOS-dependent mechanism and may be a valuable strategy to target in-stent stenosis.
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PMID:L-arginine administration reduces neointima formation after stent injury in rats by a nitric oxide-mediated mechanism. 1159 33


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