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 new procedure for a sialidase assay, by bioluminescence, has been developed. The substrate, N- acetylneuraminyllactose (sialyllactose), hydrolysed by the sialidase activity, releases lactose. This lactose is hydrolysed with beta-galactosidase. The released galactose is oxidized with galactose dehydrogenase and NAD. The NADH produced in the last step is measured by a luminescence system, coupling two enzymes, NAD(P)H dehydrogenase (FMN) and luciferase. This microassay, which is specific, rapid, simple and ultra-sensitive, is a measure for amounts as little as (at least) 5 pmol of N-acetylneuraminic acid (corresponding to 0.15 ng of the released sialic acid). It uses commercialized reagents (non-radioisotopic) and avoids interferences common in other procedures. This method has been used for measuring sialidase activity directly on intact virus, avoiding inconvenient modifications produced in the extraction of the enzyme. The specific activity of sialidase of influenza virus X31 (H3N2), determined by this procedure, is 0.65 U/mg of total virus protein.
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PMID:Sialidase assay by luminescence in the low picomole-range of sialic acid. Its application to the measurement of this activity in influenza virus. 673 52

Replication-deficient recombinant adenovirus vectors have been used to transfer foreign genes effectively to a wide variety of cell types in vivo and in vitro. We have now used adenovirus containing either the Escherichia coli beta-galactosidase (beta-gal) gene (AdHCMVsp1LacZ) or the firefly luciferase gene (Ad5-luc3) to test the hypothesis that efficiencies of adenovirus-mediated gene delivery into organ cultures of smooth muscle differ according to the anatomic origin of the muscle. Thoracic aorta and renal artery were isolated from 9-week-old male Sprague-Dawley rats and exposed to adenovirus after 16 hours of incubation with serum-free medium (Dulbecco's modified Eagle's medium). With the use of histochemical methods, beta-gal staining was noted in both endothelial and adventitial cells but not in the muscular media of thoracic aorta and renal artery exposed to AdHCMVsp1LacZ. The efficiency of the transfection, assessed either by counting of beta-gal-stained cells in intact vessels or by measurement of beta-gal activity in tissue extracts, was higher in renal artery than thoracic aorta (P < .05). Consistent with this result, luciferase activity in renal artery exposed to Ad5-luc3 (15.9 +/- 2.1 x 10(6) relative light units per milligram protein) was higher than that in thoracic aorta (8.3 +/- 2.0 x 10(6), P < .05). To determine whether increased efficiency of adenovirus-mediated gene transfer into renal artery is a function of the replication status of vessels, we assessed [3H]thymidine incorporation. [3H]Thymidine uptake by thoracic aorta was only 63% of that in renal artery (P < .05), indicating that more proliferating cells are present in renal artery. We conclude that the efficiency of adenovirus-mediated gene transfer into cultured renal artery is enhanced compared with that into thoracic aorta and propose that the increase in efficiency is related to the higher proliferative activity of renal artery.
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PMID:Heterogeneity of adenovirus-mediated gene transfer in cultured thoracic aorta and renal artery of rats. 749 65

We show that a new rat chondrosarcoma (RCS) cell line established in long-term culture from the Swarm tumor displayed a stable differentiated chondrocyte-like phenotype. Indeed, these cells produced the collagen types II, IX, and XI and alcian blue-stainable cartilage-specific proteoglycans, but no type I or type III collagen. To functionally characterize their chondrocytic nature, the cells were stably transfected with a type II collagen/beta geo chimeric gene which confers essentially perfect chondrocyte-specific expression in transgenic mice. RCS cells expressed both beta-galactosidase and G418 resistance, in comparison with similarly transfected 10T1/2 and NIH/3T3 fibroblasts which did not. These cells were then used to perform a systematic deletion analysis of the first intron of the mouse type II collagen gene (Col2a1) using transient expression experiments to determine which segments stimulated expression of a luciferase reporter gene in RCS cells but not in 10T1/2 fibroblasts. Cloning of two tandem copies of a 156-base pair (bp) intron 1 fragment (+2188 to +2343) in a construction containing a 314-bp Col2a1 promoter caused an almost 200-fold increase in promoter activity in RCS cells but no increase in 10T1/2 cells. DNase I footprint analysis over this 156-bp fragment revealed two adjacent protected regions, FP1 and FP2, located in the 3'-half of this segment, but no differences were seen with nuclear extracts of RCS cells and 10T1/2 fibroblasts. Deletion of FP2 to leave a 119-bp segment decreased enhancer activity by severalfold, but RCS cell specificity was maintained. Further deletions indicated that sequences both in the 5' part of the 119-bp fragment and in FP1 were needed simultaneously for RCS cell-specific enhancer activity. A series of deletions in the promoter region of the mouse Col2a1 gene progressively reduced activity when these promoters were tested by themselves in transient expression experiments. However, these promoter deletions were all activated to a similar level in RCS cells by a 231-bp intron 1 fragment that included the 156-bp enhancer. The RCS cell-specific activity persisted even if the Col2a1 promoter was replaced by a minimal adenovirus major late promoter. This 231-bp intron 1 fragment also had strong enhancing activity in transiently transfected mouse primary chondrocytes. Our experiments establish the usefulness of RCS cells as an experimental system for studies of the control of chondrocyte-specific genes, provide an extensive delineation of segments in the Col2a1 first intron involved in chondrocyte-specific activity, and show that promoter sequences are dispensable for chondrocyte specificity.
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PMID:Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene. 749 38

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

Intramuscular injection of naked plasmid DNA provides a means for gene transfer and expression in striated muscle. In this study, the effects of treating muscle with normal saline, etidocaine, mepivacaine, acetic anhydride, sodium bicarbonate, Notechis scutatus venom, cardiotoxin and bupivacaine before plasmid DNA injection on foreign gene expression were evaluated. Dose dependence, strain and species specificity, the time interval between pharmacological agent and plasmid DNA injection, the stability of gene expression and the fate of the injected plasmid DNA were studied using reporter gene expression, by histological examination and semi-quantitative polymerase chain reaction. Of the various agents tested, the best enhancement of foreign gene expression occurred in muscle treated with 0.75% bupivacaine five to seven days before plasmid DNA injection. Rat and mouse quadriceps muscle treated with 0.75% bupivacaine had levels of luciferase activity four- to 40-times greater than non-bupivacaine-treated muscle. Also, beta-galactosidase expressing myofibers were observed throughout the length of the muscle in samples treated with 0.75% bupivacaine before reporter gene injection. Muscle treated with 0.75% bupivacaine fully recovered from the degeneration caused by its injection with no long-term effects histologically. The heightened level of reporter gene expression persisted in 0.75% bupivacaine-treated muscle for one month, but decreased to that of non-bupivacaine-treated muscle by two months after plasmid DNA injection. Enhancement of foreign gene expression may be particularly advantageous in vaccination protocols employing intramuscular plasmid injection.
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PMID:Pharmacological enhancement of in vivo foreign gene expression in muscle. 758 66

The tie gene encodes a receptor tyrosine kinase that is expressed in the endothelium of blood vessels, particularly during embryonic development and angiogenesis in adults. We have cloned and characterized the mouse tie gene and isolated the human and mouse tie promoters. The promoter activities of human and mouse tie were analyzed using luciferase reporter gene constructs in transfected cell lines and beta-galactosidase constructs in transgenic mice. In transfection assays of cultured cells, both human and mouse promoter DNA fragments showed activity that was not restricted to endothelial cells. In contrast, in transgenic mice both promoters directed expression of the reporter gene to endothelial cells undergoing vasculogenesis and angiogenesis. In adult mice, tie promoter activity in lung and many vessels of the kidney was as high as in the vessels of the corresponding embryonic tissues, whereas in the heart, brain and liver, tie promoter activity was downregulated and restricted to coronaries, cusps, capillaries, and arteries. Our results show that the endothelial cell-type specificity of the tie promoter in vivo can be transferred to heterologous genes by using relatively short promoter fragments. The tie promoter, thus, has useful properties for potential gene therapy.
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PMID:Endothelial-specific gene expression directed by the tie gene promoter in vivo. 765 12

The safety of replication-defective viruses used as vectors is based on the deletion of essential gene(s). Adenovirus vector safety relies on the deletion of the E1A/E1B region. This region encodes the immediate-early proteins that trans activate all other early regions, so DNA replication in these deletion mutants is dramatically reduced. We have previously shown that E1A deletion is efficient in vivo and significantly reduces the dissemination of adenovirus in mice and cotton rats. However, the pattern of dissemination of E1A-deleted and wild-type viruses showed that both could be localized in the same tissues, thus involving a theoretical risk of phenotypic complementation if a recipient of E1A-deleted adenovirus is infected after adenovirus-mediated gene therapy by a wild-type adenovirus. In this report, we show that complementation can be evidenced in vitro in Vero cells infected with E1A/E1B-defective adenovirus vectors expressing reporter genes (either beta-galactosidase or luciferase), passaged three times until no infectious virus can be recovered by plating on 293 cells, and then infected with wild-type adenovirus 5. A mixed virus population was maintained at a stable state for at least 10 passages. In contrast, no evidence of complementation was found in cotton rats inoculated intravenously or intramuscularly with Ad-beta-gal-nls and Ad-luc and infected 24 h later intranasally with wild-type adenovirus 5. No increase in the level of luciferase expression was found in these animals, compared with that in controls, nor was any viral population expressing beta-galactosidase or luciferase isolated from various organs or any animal excretion or secretion.
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PMID:Lack of evidence of phenotypic complementation of E1A/E1B-deleted adenovirus type 5 upon superinfection by wild-type virus in the cotton rat. 766 53

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.
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PMID:Gene expression following direct injection of DNA into liver. 771 Nov 40

Little is known of the relation between recovery of contraction and the regulation of contractile protein gene expression in ventricular myocytes after severe ATP depletion. We have examined alterations in activation of an MLC-2 luciferase fusion gene in cultured neonatal rat ventricular myocytes produced by exposure to 2 mM Na CN and 20 mM 2-deoxyglucose, and after recovery is serum or serum free medium. The effects of metabolic inhibition followed by recovery on expression on an RSV-luciferase activity were also investigated. Myocytes were co-transfected with a CMV beta-galactosidase fusion gene, and luciferase activities were normalized relative to beta-galactosidase activity to control for transfection efficiency. Two hours of metabolic inhibition produced significant cell injury, as documented by disorganization of myofilaments, and reduction in luciferase and beta-galactosidase activity within transfected cells. Cells allowed to recover for 48 h in serum free hormone supplemented medium showed a further decline in corrected luciferase activity, consistent with a marked reduction in MLC-2 gene transcription. Cells recovered from severe metabolic inhibition in serum free medium also showed failure to redevelop contractile activity, and failure of redevelopment of organized myofibrils. In contrast, myocytes exposed to serum during the 48 h recovery period had a marked increase in luciferase activity, resumed contractile activity and re-established organized myofilaments. There were no significant differences between RSV luciferase activities in cells recovered in serum versus serum free media. In ventricular myocytes in which contraction was inhibited by exposure to 10 microM verapamil, MLC-2 luciferase activity declined by 87%. However, even when contractile activity was inhibited by exposure to verapamil during recovery from metabolic inhibition, exposure to serum containing medium caused a significantly greater increase in MLC-2 luciferase activity than did serum free medium. Thus, the effects of serum on MLC-2 gene expression were not solely due to an effect of serum on recovery of contractile activity. Verapamil had no consistent effect on expression of RSV luciferase. These results suggest that expression of the MLC-2 gene is markedly reduced following recovery from severe metabolic inhibition, an effect largely due to cessation of myocyte contractile activity. Resupply of growth factors present in fetal calf serum reactivate expression of this gene, and this is associated with resumption of contractile activity and redevelopment of organized myofibrils. These results suggest that reactivation of contractile protein gene expression during recovery from metabolic inhibition may be beneficial in allowing cells to recover from this insult.
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PMID:Regulated expression of a contractile protein gene correlates with recovery of contractile function after reversible metabolic inhibition in cultured myocytes. 776 Mar 76

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.
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PMID:Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters. 779 76


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