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)

A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation. The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene. The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase. pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker. The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer. The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region. Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.
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PMID:A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. 146 57

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

Five constructions containing deletions of the promoter from an auxin-inducible gene of Arabidopsis thaliana, AtAux2-11, were fused to the coding region of the reporter gene LacZ, which encodes beta-galactosidase, and a polyadenylation 3'-untranslated nopaline synthase sequence from Agrobacterium. These chimeric genes were introduced into Arabidopsis by Agrobacterium tumefaciens-mediated transformation, and expression of the gene was examined by spectrophotometric and histochemical analyses. A 600 bp fragment from the AtAux2-11 promoter conferred histochemical patterns of staining similar to the longest 5' promoter tested, a 3.0 kb fragment. Localization of AtAux2-11/LacZ activity in the transgenic plants revealed spatial and temporal expression patterns that correlated with tissues and cells undergoing physiological processes modulated by auxin. LacZ activity was expressed in the elongating region of roots, etiolated hypocotyls, and anther filaments. Expression was detected in the vascular cylinder of the root and the vascular tissue, epidermis, and cortex of the hypocotyl, and filament. The AtAux2-11/LacZ gene was preferentially expressed in cells on the elongating side of hypocotyls undergoing gravitropic curvature. Expression of the chimeric gene in the hypocotyls of light-grown seedlings was less than that in etiolated seedling hypocotyls. The AtAux2-11/LacZ gene was active in the root cap, and expression in the root stele increased at sites of lateral root initiation. Staining was evident in cell types that develop lignified cell walls, e.g. trichomes, anther endothecial cells, and especially developing xylem. The chimeric gene was not expressed in primary meristems. While the magnitude of expression increased after application of exogenous auxin (2,4-D), the histochemical localization of AtAux2-11/LacZ remained unchanged. Transgenic plants with a 600 bp promoter construct (-0.6 kb AtAux2-11/LacZ) had higher levels of basal and auxin-inducible expression than plants with a 3.0 kb promoter construct. Transgenic plants with a -500 bp promoter had levels of expression similar to the -3.0 kb construct. The -0.6 kb AtAux2-11/LacZ gene responded maximally to a concentration of 5 x 10(-6) to 5 x 10(-5) M 2,4-D and was responsive to as little as 5 x 10(-8) M. The evidence presented here suggests that this gene may play a role in several auxin-mediated developmental and physiological processes.
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PMID:Expression of the Arabidopsis AtAux2-11 auxin-responsive gene in transgenic plants. 835 26

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

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

The current study was designed to determine the effect of recombinant endothelial nitric oxide synthase (eNOS) gene expression on endothelium-dependent relaxations to bradykinin in isolated canine basilar, coronary, or femoral arteries. Arterial rings were exposed ex vivo (30 minutes at 37 degrees C) to an adenoviral vector encoding either the eNOS gene (AdCMVeNOS) or the beta-galactosidase reporter gene (AdCMVbeta-Gal). Twenty-four hours after transduction, transgene expression was evident mainly in the adventitia. Expression of recombinant proteins was much higher in basilar arteries than in coronary or femoral arteries. Rings of control, AdCMVbeta-Gal, and AdCMVeNOS arteries with and without endothelium were suspended for isometric tension recording. Levels of cGMP were measured by radioimmunoassay. In AdCMVeNOS basilar arteries with endothelium, relaxations to low concentrations of bradykinin (3 x 10(-11) to 10(-9) mol/L) were significantly augmented. In contrast, in coronary and femoral arteries with endothelium, AdCMVeNOS transduction did not affect relaxations to bradykinin. Removal of the endothelium abolished bradykinin-induced relaxations in control and AdCMVbeta-Gal basilar arteries. However, in basilar arteries transduced with AdCMVeNOS even when the endothelium was removed, stimulation with bradykinin (3 x 10(-11) to 10(-9) mol/L) caused relaxations as well as increases in cGMP production. The relaxations to bradykinin were completely blocked by an NOS inhibitor, NG-nitro-L-arginine methyl ester. Electron microscopic analysis revealed that recombinant eNOS protein was expressed in fibroblasts of the basilar artery adventitia. These results suggest that genetically modified adventitial fibroblasts may restore production of NO in cerebral arteries without endothelium. Our findings support a novel concept in vascular biology that fibroblasts in the adventitia may play a role in the regulation of vascular tone after successful transfer and expression of recombinant eNOS gene.
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PMID:Adventitial expression of recombinant eNOS gene restores NO production in arteries without endothelium. 971 29

We recently reported that expression of recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts restores NO formation in canine cerebral arteries without endothelium in response to bradykinin ex vivo and in vivo. The present study was designed to further characterize the stimuli that can activate recombinant eNOS enzyme expressed in the adventitia of cerebral arteries. To stimulate recombinant eNOS, we used serum (0. 1-10%), substance P (10(-11)-3 x 10(-9) M), and ANG II (10(-7)-10(-5) M) because they increase intracellular calcium concentrations in fibroblasts. Endothelium-denuded segments of canine basilar arteries were incubated with an adenoviral vector encoding beta-galactosidase gene or eNOS gene for 30 min at 37 degrees C. After 24 h, vasomotor activity and cGMP formation in eNOS or beta-galactosidase arteries were examined by isometric force recording and by radioimmunoassay, respectively. In control arteries and beta-galactosidase gene-transduced arteries, serum caused concentration-dependent contractions, whereas in recombinant eNOS gene-transduced arteries, serum produced concentration-dependent relaxations. Substance P and ANG II had no effect on vascular tone in control and beta-galactosidase arteries but caused concentration-dependent relaxations as well as a significant increase in cGMP levels in eNOS arteries. These relaxations were blocked by the NOS inhibitor NG-nitro-L-arginine methyl ester. Chemical treatment or mechanical inactivation of adventitial function significantly attenuated substance P-induced relaxations and ANG II-induced relaxations. These findings demonstrate that serum, substance P, and ANG II cause adventitia-dependent relaxations in cerebral arteries expressing the recombinant eNOS gene. This mechanism of vasodilatation may have beneficial effects in the prevention and treatment of vascular disorders characterized by the diminished bioavailability of NO, such as cerebral vasospasm.
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PMID:Adventitia-dependent relaxations of canine basilar arteries transduced with recombinant eNOS gene. 1051 60

Introducing recombinant genes into donor hearts may offer a therapeutic intervention that could potentially attenuate the complications of heart transplantation, including rejection, infection and accelerated atherosclerosis. In the cardiovascular system, reduced bioactivity of endothelial nitric oxide is a feature of atherosclerosis and vascular injury. Nitric oxide is an arterial vasodilator that also inhibits proliferation of vascular smooth muscle cells and platelet aggregation. Experiments were designed to determine the distribution of adenoviral-mediated transfer of recombinant endothelial nitric oxide synthase gene (eNOS) and the effect of recombinant gene expression on the function of transplanted hearts. Adenoviral vectors for (a) bovine eNOS (AdeNOS) or (b) beta-galactosidase (AdLacZ; control) were infused into two groups (n = 12, per group) of explanted rat hearts. The transduced hearts were then implanted heterotopically into the abdomen of syngeneic recipient rats. After four days, the hearts were excised and examined for distribution and function of the recombinant genes. Polymerase chain reaction (PCR) verified the presence of the recombinant eNOS gene in eNOS-transduced but not in beta-galactosidase-transduced hearts; reverse transcriptase-PCR identified mRNA for eNOS in AdeNOS-transduced hearts. NOS activity (conversion of tritiated L-arginine to citrulline) was greater in homogenates of AdeNOS-compared to AdLacZ-transduced hearts. Positive immunoreactivity for eNOS was present in cardiomyocytes predominantly in eNOS-transduced hearts. Myocardial contractility and coronary blood flow, as determined using a Langendorff preparation, were not different between hearts transduced with AdeNOS or AdLacZ. These results suggest that, up to four days post transplantation, adenoviral-mediated transfer of eNOS into transplanted hearts is possible. However, expression of the recombinant protein did not result in measurable changes in myocardial contractility or coronary perfusion.
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PMID:Distribution and function of recombinant endothelial nitric oxide synthase in transplanted hearts. 1053 12

Adrenal zona glomerulosa (ZG) cells do not contain nitric oxide (NO) synthase (NOS). We conferred endothelial NOS activity onto adrenal ZG cells through transduction with a recombinant adenovirus encoding the endothelial NOS gene (AdeNOS) to determine the effect of endogenous NO on aldosterone synthesis. A 135-kDa protein band immunoreactive to anti-endothelial NOS antibody was observed in Western blots of AdeNOS-transduced ZG cells but not in control cells or cells transduced with adenovirus encoding the beta-galactosidase gene (AdbetaGal). Nitrate/nitrite production in AdeNOS-transduced ZG cells increased from 0.15+/-0.01 to 0.27+/-0.01 micromol/L after stimulation with 1 nmol/L angiotensin II. The treatment of AdeNOS-transduced cells with 30 micromol/L L-nitro-arginine decreased angiotensin II-stimulated nitrite production from 0.27+/-0. 01 to 0.17+/-0.01 micromol/L. Basal and angiotensin II-stimulated nitrite production was not increased in AdbetaGal-transduced or control cells. AdeNOS-transduced cells demonstrated diaminofluorescein-2 diacetate fluorescence, which was blocked by pretreatment with L-nitro-arginine. Angiotensin II-stimulated aldosterone synthesis decreased from 5123+/-177 pg/mL in AdbetaGal-transduced ZG cells to 72+/-27 pg/mL in AdeNOS-transduced cells. Treatment with the NOS inhibitor thiocitrulline (30 micromol/L) increased angiotensin II-stimulated aldosterone synthesis to 2158+/-45 pg/mL after AdeNOS transduction. These data demonstrate that adenovirus-mediated gene transfer of eNOS in ZG cells results in the expression of active endothelial NOS enzyme and that this endogenous NO production by ZG cells decreases aldosterone synthesis.
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PMID:Inhibition of adrenal cell aldosterone synthesis by endogenous nitric oxide release. 1064 19

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.
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PMID:Adenovirus-mediated gene transfer to human cerebral arteries. 1099 58


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