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)

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

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

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coli beta-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37 degrees C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.
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PMID:Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries. 1040 17

L-arginine (L-Arg) may be limiting for inducible nitric oxide synthase (NOS) activity and under certain circumstances, such as increased concentrations of a NOS inhibitor, may also be limiting for endothelial NOS activity. It is unknown if L-Arg is limiting for recombinant eNOS activity in the vascular wall after adenoviral mediated gene transfer. Our aim was to examine, if L-Arg is limiting for recombinant eNOS activity in the normal or atherosclerotic vessel wall. Rings of rabbit aorta from chow or cholesterol fed animals were transduced with adenovirus vector encoding eNOS (AdeNOS) or beta-galactosidase (AdbetaGal). After 24 h, transgene expression was confirmed and vasomotor studies were performed in the absence or presence of L-Arg. During maximal contractions to phenylephrine (10(-5) M), L-Arg (3 mM) was added to the organ chamber for 30 min. Subsequently, relaxations to acetylcholine during half-maximal contractions were obtained. In the chow- and cholesterol-fed animals, relaxations were significantly enhanced in the NOS and NOS + L-Arg groups compared to the betaGal and betaGal + L-Arg groups. There was no difference between NOS and NOS + L-Arg or betaGal and betaGal + L-Arg rings from chow- or cholesterol-fed animals. While gene transfer of eNOS enhances endothelium-dependent vasorelaxation in the normal and atherosclerotic vessel wall, L-arginine is not limiting for recombinant eNOS activity.
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PMID:L-arginine availability is not limiting for nitric oxide generation from recombinant endothelial nitric oxide synthase. 1062 19

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

Our previous ex vivo and in vivo studies reported that expression of the recombinant endothelial nitric oxide (NO) synthase (eNOS) gene in adventitial fibroblasts recovers NO production in arteries without endothelium in response to bradykinin. The present study was designed to characterize subtypes of bradykinin receptors on adventitial fibroblasts coupled to the activation of recombinant eNOS. Endothelium-denuded segments of canine basilar arteries were transduced with beta-galactosidase (beta-Gal) gene or eNOS gene ex vivo, using a replication-defective adenoviral vector (10(10) plaque-forming units/ml) for 30 min at 37 degrees C. Twenty-four hours later, isometric force recording or cGMP measurement was carried out. B(1) bradykinin receptor agonist (des-Arg(9)-bradykinin, 10(-10)-10(-8) mol/l) did not significantly affect vascular tone in control or beta-Gal gene-transduced canine basilar arteries without endothelium. In contrast, this agonist caused concentration-dependent relaxations in recombinant eNOS gene-transduced arteries without endothelium. Relaxations to B(1) receptor agonist in the eNOS arteries were abolished by B(1) receptor antagonist (des-Arg(9)-[Leu(8)]bradykinin, 6 x 10(-9) mol/l) but not by B(2) receptor antagonist (Hoe-140, 5 x 10(-8) mol/l). Bradykinin did not significantly alter vascular tone in control or beta-gal arteries without endothelium, whereas this peptide (10(-11)-10(-8) mol/l) induced concentration-dependent relaxations, as well as an increase in cGMP formation in endothelium-denuded eNOS-transduced arteries. Stimulatory effects of bradykinin were prevented in the presence of a B(2) receptor antagonist but not in the presence of a B(1) receptor antagonist. B(1) and B(2) receptor antagonists had no effect on relaxations to substance P, confirming the selectivity of the compounds. Our results suggest that B(1) and B(2) bradykinin receptors are coupled to activation of recombinant eNOS expressed in adventitial fibroblasts.
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PMID:B(1) and B(2) bradykinin receptors on adventitial fibroblasts of cerebral arteries are coupled to recombinant eNOS. 1066 66

Resistance arteries are an important target for vascular gene therapy because they play a key role in the regulation of tissue blood flow. The present study was designed to determine the effects of recombinant endothelial (e) nitric oxide synthase (NOS) gene expression on vasomotor reactivity of small brain stem arteries (internal diameter, 253 +/- 2.5 microm). Arterial rings were exposed ex vivo to an adenoviral vector (10(9) and 10(10) plaque-forming units/ml) encoding eNOS gene or beta-galactosidase gene. Twenty-four hours after transduction, vascular function was examined by isometric force studies. Transgene expression was evident mainly in adventitia. In arteries with endothelium transduced with eNOS gene but not with control beta-galactosidase gene, relaxations to bradykinin and substance P were significantly augmented. Removal of endothelium abolished relaxations to bradykinin and substance P in control and beta-galactosidase arteries. However, in endothelium-denuded arteries transduced with recombinant eNOS, bradykinin and substance P caused relaxations that were abolished in the presence of the NOS inhibitor N(G)-nitro-L-arginine methyl ester. In control arteries, endothelium removal augmented relaxations to the nitric oxide donors sodium nitroprusside and diethylamine NONOate. This augmentation was absent in eNOS gene-transduced arteries without endothelium. Our results suggest that, in small brain stem arteries, expression of recombinant eNOS increases biosynthesis of nitric oxide. Adventitia of small arteries is a good target for expression of recombinant eNOS. Genetically engineered adventitial cells may serve as a substitute source of nitric oxide in cerebral arteries with dysfunctional endothelium.
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PMID:Effects of recombinant eNOS gene expression on reactivity of small cerebral arteries. 1066 71

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbetagal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbetagal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbetagal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbetagal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were significantly increased (P<0. 05), while iNOS protein levels remained unchanged (P>0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector significantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.
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PMID:Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat. 1100 94

Neuronal nitric oxide synthase (nNOS) plays a modulatory role in the biology of a variety of neuroendocrine tissues and is especially relevant to gonadal function. We have previously reported the cloning and characterization of a variant of the nNOS protein, termed testis nNOS (TnNOS), the mRNA for which was restricted in expression to male gonadal tissues. To examine the cell-specificity of the testis-specific NOS regulatory regions we defined patterns of beta-galactosidase expression of an insertional transgene in which the reporter gene lacZ was under the transcriptional control of the human TnNOS promoter. beta-galactosidase activity was detected exclusively in the interstitial cells of the testis in transgenic mice. These cells also evidenced positive staining for nNOS protein and were identified as androgen-producing Leydig cells by staining with the Leydig cell marker, P(450)scc. Expression of the promoter was absent in cells of the seminiferous tubules, specifically germline cells of different stages and Sertoli cells. In contrast to the male gonad, beta-galactosidase activity was not detected in ovaries of adult female mice. Activity was also not evident in organs known to express full-length nNOS, such as skeletal muscle, kidney, or cerebellum. The same pattern of beta-galactosidase staining was observed in independent transgenic founders and was distinct from that observed for an endothelial NOS promoter/reporter transgene. In the testis of male adult eNOS promoter-reporter transgenic mice, beta-galactosidase activity was expressed only in endothelial cells of large- and medium-sized arterial blood vessels. Transcriptional activity of the human TnNOS promoter could not be detected in a variety of cell types, including Leydig cells, using episomal promoter-reporter constructs suggesting that a nuclear environment and higher order genomic complexity are required for appropriate promoter function. The restricted expression pattern of an nNOS variant in Leydig cells of the male gonad suggests an important role in the regulation of testosterone release and represents an intriguing model with which to dissect the molecular basis of Leydig cell-specific gene expression.
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PMID:An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. 1178 30

Diabetic vasculopathy is central to the development of diverse cardiovascular, renal, retinal, and neurological complications of diabetes. We previously demonstrated that growth of endothelial cells on glycated extracellular matrix proteins (collagen and matrigel) results in a significant decrease in cell proliferation. In the present study, we show that early-passage human umbilical vein endothelial cells (HUVECs) grown on glycated collagen (GC) express hallmarks of premature cell senescence, ie, increase in the proportion of cells expressing senescence-associated beta-galactosidase activity, apoptotic rate, and p53 and p14(AFR) expression, but in contrast to replicative senescence, display neither attrition of telomeres nor decrease in telomerase activity. An increased frequency of prematurely senescent cells was similarly observed in vivo in aortae of young Zucker diabetic rats, compared with lean controls. NO production by HUVECs grown on GC was decreased, despite a 3-fold increase in eNOS expression and was associated with the increased nitrotyrosine-modified proteins. Development of premature senescence of HUVECs on GC could be prevented and reversed by treatments with the peroxynitrite scavenger, ebselen, eNOS intermediate N(omega)-hydroxy-L-arginine (NOHA), or superoxide dismutase mimetic Mn-TBAP. Concomitant with the reversal of senescence, ebselen, and NOHA each restored NO production to levels observed with HUVECs grown on unmodified collagen. Our findings indicate that diabetes mellitus in vivo and GC exposure in vitro elicit premature senescence of the vascular endothelium, a process with distinct pathogenetic mechanisms. Premature senescence of the vascular endothelium is hypothesized to be an important contributor to diabetic vasculopathy and a consequence of reduced NO availability, peroxynitrite, and/or superoxide excess.
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PMID:Glycated collagen I induces premature senescence-like phenotypic changes in endothelial cells. 1208 67


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