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)

The nucleus tractus solitarius (NTS) of the brainstem is an important site for the regulation of sympathetic nerve activity. In the brain, nitric oxide (NO) has been shown to reduce blood pressure by inhibiting sympathetic nerve activity. However, most studies were performed in an acute state of anesthesia. Therefore, we developed a technique to increase the local production of NO in vivo by the transfer of endothelial NO synthase (eNOS) gene into the NTS. Adenovirus vectors encoding either the beta-galactosidase gene (Adbetagal) or the eNOS gene (AdeNOS) were infected into the NTS. In the Adbetagal-infected rats, the local expression of beta-galactosidase was confirmed by X-Gal staining, and beta-galactosidase activity was quantified using a colorimetric assay. In the AdeNOS-infected rats, the local expression of eNOS was confirmed by immunohistochemistry and Western blot analysis. Production of NO was measured by in vivo microdialysis. Blood pressure and heart rate were monitored by a radiotelemetry system in a conscious state. In the AdeNOS-infected rats, blood pressure and heart rate significantly decreased from d 5 to d 10, and then gradually recovered over time. These methods should be useful in examining the local effect on cardiovascular function of a particular substance produced by a specific gene in the brain.
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PMID:Adenovirus-mediated nitric oxide synthase gene transfer into the nucleus tractus solitarius in conscious rats. 1519 46

Vascular gene therapy is currently limited by low and transient levels of gene transfection. The objectives of this study were to determine whether peri-adventitial delivery of adenovirus coupled to nanoparticles could improve transfection efficiency and duration. Adenovirus was absorbed to the surface of nanoparticles that were made from poly(methylidene malonate)2.1.2 (PMM2.1.2). These complexes were found to have good adhesive properties to both cultured vascular smooth muscle cells and to the luminal and adventitial layers of excised rabbit carotid arteries. Adenovirus encoding to beta-galactosidase coupled to PMM2.1.2 nanoparticles or adenovirus alone were delivered locally to the adventitia of rabbit carotid arteries. Transfection rate was assessed histologically by the percentage of beta-galactosidase positive cells in the vessel wall at 1 and at 2 weeks. There was significantly higher transfection rate when adenovirus was complexed with nanoparticles as compared to free adenovirus (At 1 week: 10+/-3.9% beta-gal positive cells vs. 2.4+/-0.3% and at 2 weeks: 4.3+/-4.1% vs. 0%, P<0.005 for all). This difference was present in both the medial and adventitial layers. In conclusion, adenoviral mediated gene therapy was significantly enhanced by adsorbing the virus to PMM2.1.2 nanoparticles. This delivery method may be a good therapeutic strategy for the treatment of various vascular diseases.
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PMID:Poly(methylidene malonate 2.1.2) nanoparticles: a biocompatible polymer that enhances peri-adventitial adenoviral gene delivery. 1531

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.
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PMID:Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus. 1532 95

The Rho/Rho-kinase pathway in the nucleus tractus solitarii (NTS) of the brain stem contributes to blood pressure regulation. Activation of this pathway might be involved in the central nervous system mechanisms of hypertension. The aim of the present study was to determine whether baroreflex control of heart rate is altered by inhibition of Rho-kinase in the NTS. Adenovirus vectors encoding dominant-negative Rho-kinase or beta-galactosidase were transfected into the nucleus tractus solitarii of Wistar Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Baroreflex control of heart rate was examined by changing arterial pressure with an intravenous infusion of phenylephrine or sodium nitroprusside. The maximum gain of baroreflex control of heart rate was attenuated in SHR compared with WKY before the gene transfer. Transfection of adenovirus vectors encoding dominant-negative Rho-kinase significantly augmented the maximum gain in both WKY and SHR. The extent of this augmentation, however, was greater in SHR than in WKY. After treatment with metoprolol, the maximum gain was significantly decreased in rats transfected with adenovirus vectors encoding dominant-negative Rho-kinase, but not in nontransfected rats. In contrast, after treatment with atropine, the maximum gain was greater in rats transfected with adenovirus vectors encoding dominant-negative Rho-kinase compared with nontransfected rats, although it was decreased in both groups. These results suggest that inhibition of Rho-kinase in the NTS augments baroreflex control of heart rate, in both WKY and SHR, probably because of a cardiac sympathoinhibitory effect.
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PMID:Inhibition of Rho-kinase in the brainstem augments baroreflex control of heart rate in rats. 1535 14

Nerve injury leads to the induction of a large number of genes to repair the damage and to restore synaptic transmission. We have attempted to identify molecules whose mRNA expression is altered in response to facial nerve axotomy. Here we report that facial nerve axotomy upregulates Sonic hedgehog (Shh) and its receptor Smoothened (Smo) in facial motor neurons of adult rats, whereas facial nerve axotomy does not upregulate mRNA of Shh or Smo in neonatal rats. We tested whether overexpression of Shh in facial motor neurons of axotomized neonatal rats may promote neuronal survival. Adenovirus-mediated overexpression of Shh, but not that of beta-galactosidase, transiently rescues axotomy-induced neuronal cell death for 3-5 d after axotomy. Finally, the pharmacological inhibitor of Shh signaling, cyclopamine, induces motor neuron death in adult rats after axotomy. These results suggest that Shh plays a regulatory role in nerve injury.
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PMID:The upregulated expression of sonic hedgehog in motor neurons after rat facial nerve axotomy. 1535 5

The present study examined the role of inducible nitric oxide synthase (iNOS) in the rostral ventrolateral medulla (RVLM) of the brain stem, where the vasomotor center is located, in the control of blood pressure and sympathetic nerve activity. Adenovirus vectors encoding iNOS (AdiNOS) or beta-galactosidase (Adbetagal) were transfected into the RVLM in Wistar-Kyoto (WKY) rats. Blood pressure and heart rate were monitored using a radiotelemetry system. iNOS expression in the RVLM was confirmed by immunohistochemical staining or Western blot analysis. Mean arterial pressure significantly increased from day 6 to day 11 after AdiNOS transfection, but did not change after Adbetagal transfection. Urinary norepinephrine excretion was significantly higher in AdiNOS-transfected rats than in Adbetagal-transfected rats. Microinjection of aminoguanidine or S-methylisothiourea, iNOS inhibitors, or tempol, an antioxidant, significantly attenuated the pressor response evoked by iNOS gene transfer. The levels of thiobarbituric acid-reactive substances, a marker of oxidative stress, were significantly greater in AdiNOS-transfected rats than in Adbetagal-transfected rats. Dihydroethidium fluorescence in the RVLM was increased in AdiNOS-transfected rats. In addition, nitrotyrosine-positive cells were observed in the RVLM only in AdiNOS-transfected rats. Intracisternal infusion of tempol significantly attenuated the pressor response and the increase in the levels of thiobarbituric acid-reactive substances induced by AdiNOS transfection. These results suggest that overexpression of iNOS in the RVLM increases blood pressure via activation of the sympathetic nervous system, which is mediated by an increase in oxidative stress.
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PMID:Overexpression of inducible nitric oxide synthase in rostral ventrolateral medulla causes hypertension and sympathoexcitation via an increase in oxidative stress. 1559 Dec 32

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BK(Ca)) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase BK(Ca) channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli beta-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10(-5) M) increased 14,15-EET release twofold and BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10(-5) M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BK(Ca) channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S),15(R)-EET from endogenous arachidonic acid to activate BK(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.
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PMID:Regulation of potassium channels in coronary smooth muscle by adenoviral expression of cytochrome P-450 epoxygenase. 1614 53

Recombinant adenoviral vectors encoding human HDL-cholesterol receptor SR-BI (Ad/hSR-BI) or beta-galactosidase (Ad/lacZ), respectively, were purified using a Source15 Q anion-exchange (AEX) column and quantified using two parallel Taqman Real Time PCR systems with different target sequences. Adenovirus concentrations were ascertained by 260 nm measurements, purity by 260/280 nm ratio and SDS-PAGE. Subsequently, adenoviruses were validated by experimental intravenous application into New Zealand White rabbits. Transgene expression was verified by functional assays determining plasma clearance rate of 3H-HDL-cholesterol, and was not affected by 21-months storage at -80 degrees C. No alterations of liver enzymes and C-reactive protein (CRP) upon Source15 Q adenovirus treatment could be detected, demonstrating biological safety of our protocol.
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PMID:In vivo application of adenoviral vectors purified by a Taqman Real Time PCR-supported chromatographic protocol. 1653 Feb 64

Activation of nuclear factor-kappaB (NF-kappaB) can promote or inhibit apoptosis. Oxidative stress is an important mechanism by which certain anticancer drugs kill cancer cells, and is also one of the mechanisms that activate NF-kappaB. We therefore examined hepatic expression of the NF-kappaB monomer p65 in human hepatocellular carcinoma (HCC) tissue samples from eight patients and compared it with their respective samples of surrounding liver tissues. We also studied the effect of NF-kappaB inhibition in human HCC cells exposed to oxidative stress, by infecting HuH7 cells with a recombinant adenovirus carrying mutant IkappaBalpha (mIkappaBalpha). Cultured HuH7 cells were infected with mIkappaBalpha or beta-galactosidase (beta-Gal) for 24 hr followed by treatment with increasing concentrations of H2O2. Cytotoxicity, NF-kappaB translocation, NF-kappaB DNA binding, cell proliferation, and apoptosis were determined. The monomer p65 was overexpressed in six of eight human HCC tissues. In HuH7 cells, introduction of mIkappaBalpha potently inhibited the translocation, activation, and DNA binding of NF- kappaB. In control (beta-Gal-infected) HuH7 cells, exposure to H2O2 produced a dose-dependent increase in apoptosis, regardless of NF-kappaB status. mIkappaBalpha-mediated inhibition of NF-kappaB activation sensitized HuH7 cells to H2O2-induced inhibition of cell growth, and further promoted cell death. Addition of H2O2 (200-500 microM) to control or mIkappaBalpha-infected HuH7 cells enhanced caspase-3 activity and cleavage. Adenovirus-mediated transfer of mIkappaBalpha potently inhibits NF-kappaB activity in HuH7 cells, and this enhances oxidative stress-induced cell killing.
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PMID:Constitutive activation of NF-kappaB in human hepatocellular carcinoma: evidence of a cytoprotective role. 1654 77

Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.
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PMID:Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6. 1664 80


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