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)

Adult rat chromaffin cells in vitro show a large proliferative response to NGF, followed by neuronal differentiation. Infection of replicating chromaffin cells with a retrovirus carrying the Escherichia coli beta-galactosidase (beta-gal) gene demonstrates beta-gal expression in cells that continue to multiply, that differentiate into neurons, and that become static. The effects of NGF on proliferation and differentiation are abolished by the protein kinase inhibitors K252a and staurosporine, and by cholera toxin, an activator of adenylate cyclase. They are diminished, but not abolished, by high concentrations of dexamethasone. Both cholera toxin alone and phorbol myristate acetate (PMA), an activator of protein kinase C, elicit small and inconsistent mitogenic responses. The responses to PMA cannot be shown to be additive with the effects of NGF. NGF is a known mitogen and neuritogen for chromaffin cells from neonatal rats, but has not previously been believed to affect similarly chromaffin cells from adults. The present findings suggest that portions of NGF signaling pathways might continue to be involved in regulating proliferation of adult rat chromaffin cells in vivo, and might be constitutively activated in PC12 cells and other adrenal medullary tumors. They further suggest that rat chromaffin cells might be propagated in vitro to obtain large numbers of sympathetic neurons expressing normal or exogenous genes.
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PMID:Nerve growth factor is a potent inducer of proliferation and neuronal differentiation for adult rat chromaffin cells in vitro. 846 33

In vivo gene transfer of angiogenic growth factors represents a potential approach to the treatment of ischemic diseases. The present study examined the in vitro and in vivo effects of two replication-deficient recombinant adenovirus (Ad) vectors coding for human acidic fibroblast growth factor (aFGF1-154). One vector codes for the nonsecreted form of the peptide (AdCMV.aFGF1-154), and the other vector codes for a recombinant, secreted form (AdCMV.sp+aFGF1-154). AdCMV.NLS beta gal, an adenovirus vector coding for beta-galactosidase (beta-Gal), was used as a control. Assessment of proliferation of starved human umbilical vein endothelial cells infected with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154 (20 pfu/cell) showed approximately 6- and 10-fold increase in cell number over control, respectively. Infection with AdCMV.sp+aFGF1-154 and with AdCMV.aFGF1-154 enhanced endothelial cell differentiation into capillary-like structures in vitro. However, this effect was significantly more pronounced with AdCMV.sp+aFGF1-154 than with AdCMV.aFGF1-154. Angiogenesis in vivo was assessed by injecting subcutaneously into mice 750 microliters of reconstituted basement membrane proteins (Matrigel) and the Ad vectors (2 x 10(8) pfu). After 14 days, there was histologic evidence of neovascularization in the animal's tissue surrounding the Matrigel plugs with AdCMV.aFGF1-154 and AdCMV.sp+aFGF1-154. Further, the hemoglobin content of the Matrigel plugs with AdCMV.aFGF1-154 and with AdCMV.sp+aFGF1-154 was, respectively, 2.3- and 2.6-fold higher than with AdCMV.NLS beta gal. Together, these observations support the concept that adenovirus vectors coding for various forms of acidic FGF1-154 may be used to induce angiogenesis in vivo and may provide a new therapeutic approach to ischemic diseases.
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PMID:In vivo angiogenesis induced by recombinant adenovirus vectors coding either for secreted or nonsecreted forms of acidic fibroblast growth factor. 857 18

A pantropic pseudotyped retroviral vector containing the envelope protein of vesicular stomatitis virus was used as a gene transfer vector in the dwarf surfclam, Mulinia lateralis. These pantropic retroviral vectors have an extremely broad host cell range and can infect many nonmammalian species. Newly fertilized dwarf surfclam eggs were electroporated at 700 V in the presence of 1 x 10(4) colony-forming units of pantropic pseudotyped retroviral particles. Infection was well tolerated and did not affect the survival rate of the embryos. Gametes collected from P1 presumptive transgenic animals were analyzed for the presence of provirus by PCR, and in different experiments 13-33% of the gamete pools were positive for the transgene. Dot blot hybridization of DNA samples from the F1 offspring of two different crosses between infected P1 and wild-type individuals revealed that 28% and 31% of F1 offspring were transgenic, respectively. Southern blot analysis of DNA isolated from PCR-positive F1 animals confirmed integration of a single copy of the provirus into the host genome. Thus, the germ lines of these two P1 transgenic animals were mosaic for the transgene. Expression of beta-galactosidase encoded by the provirus was detected in transgenic but not control surfclam embryos. Pantropic pseudotyped retroviral vectors provide a useful method for the stable introduction of foreign genetic information into surfclams and may facilitate the introduction of desirable genetic traits into commercially important shellfish and crustaceans.
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PMID:Production of transgenic dwarf surfclams, Mulinia lateralis, with pantropic retroviral vectors. 862 62

CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to be important regulators of the hepatocyte phenotype. However, the specific physiological roles of different isoforms are poorly understood because hepatocytes express multiple C/EBPs, and various isoforms have overlapping functions. To identify the functions of C/EBPalpha in mature hepatocytes, replication-defective adenovirus vectors were used to efficiently and homogeneously overexpress the mouse C/EBPalpha gene in a SV40 virus-conditionally transformed rat hepatocyte line that can be induced to express C/EBPbeta and C/EBPdelta but that has little endogenous C/EBPalpha expression. Hepatocytes were infected with a recombinant adenovirus vector carrying the cDNA for C/EBPalpha driven by Rous sarcoma virus promoter elements (AdCEBPalpha) or a similar vector carrying the Escherichia coli lacZ gene (Adbetagal). Staining for beta-galactosidase demonstrated an infection efficiency of 100% at a multiplicity of infection of 25 plaque-forming units/cell and persistence of foreign gene expression for at least 9 days. Cultures infected with AdCEBPalpha had 50-fold higher levels of C/EBPalpha mRNA and protein than those infected with Ad-beta-gal, but similar expression of C/EBP-beta. Infection with AdCEBPalpha inhibited proliferation in cells expressing little C/EBPbeta, even when proliferation was driven by the SV40 transforming antigen, and also blunted mitogenic induction of the c-myc proto-oncogene in nontransformed cells with high levels of C/EBPbeta. Although overexpression of C/EBPalpha consistently increased C/EBPalpha DNA binding activity, it was not sufficient for albumin expression. Infection with AdCEBPalpha only increased albumin mRNA levels in nontransformed cells that also expressed relatively high levels of C/EBPbeta. Thus, in hepatocytes, C/EBPalpha has a dominant antiproliferative function, but must interact with other factors to regulate hepatocyte-specific gene expression.
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PMID:Adenovirus-mediated transfer of CCAAT/enhancer-binding protein-alpha identifies a dominant antiproliferative role for this isoform in hepatocytes. 863 55

To examine the potential use of adenovirus vectors in cancer gene therapy as a mechanism for purging bone marrow cells of possible breast cancer contaminants, we compared the infection efficiency of adenovirus and the transfection efficiency of plasmid DNA in the presence of adenovirus in human breast cancer and bone marrow cells. Following infection of breast cancer cells with an adenovirus expressing beta-galactosidase gene, high levels of beta-galactoside activity were observed. No beta-galactosidase activity was observed in low-density human bone marrow cells. A replication-deficient adenovirus mutant dl312 enhanced the transfection efficiency of a plasmid DNA-expressing beta-galactosidase gene into breast cancer cells, and addition of a liposome, lipofectamine, further enhanced the transfection efficiency. In contrast, human bone marrow cells treated under the same conditions expressed very low levels of transfected beta-galactosidase DNA. Transfection of cells with plasmid DNA expressing a truncated but fully active Pseudomonas exotoxin gene in the presence of dl312 and lipofectamine resulted in marked breast cancer cell killing, whereas colony-forming unit granulocyte-macrophage (CFU-GM) were relatively resistant to these treatments. A recombinant adenovirus expressing human wild-type p53 protein (AdWTp53) was also highly cytotoxic to breast tumor cells. Infection of breast cancer cells with AdWTp53 (100 plaque-forming units/cell) resulted in 100% loss of the clonogenicity of breast tumor cells. However, colony formation from CFU-GM was relatively resistant to the cytotoxic effects of AdWTp53 alone or in the presence of pULI100 plasmid and lipofectamine. On the basis of these results, it is proposed that human adenoviruses are potentially useful for cancer gene therapy and bone marrow purging.
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PMID:Adenovirus-mediated gene transfer to human breast tumor cells: an approach for cancer gene therapy and bone marrow purging. 864 Aug 24

A defective herpes simplex virus-1 (HSV-1) vector system was used to study cell type-specific expression of the tyrosine hydroxylase (TH) gene. HSV-1 particles containing 663 bp (pTHlac 663), 278 bp (pTHlac 278), or 181 bp (pTHlac 181) of the rat TH promoter driving E. coli LacZ were used to infect superior cervical ganglia (SCG: TH-expressing tissue) and dorsal root ganglia (DRG:non-TH-expressing tissue) cultures. One day after infection, expression of beta-galactosidase was visualized by X-gal cytochemistry. Following viral transduction with pTHlac 663 at a multiplicity of infection of 0.2, 14.4% of the SCG neurons were X-gal positive whereas only about 0.9% of DRG neurons were X-gal positive. Infection with either pTHlac278 or 181 resulted in 3-fold more X-gal-positive DRG neurons. These results suggest that (i) the defective HSV-1 vector system may be useful in defining regulatory promoter motifs; (ii) 663 bp of the rat TH promoter contains sufficient information for cell type-specific expression in peripheral nervous system neurons; and (iii) sequences between -278 and -663 contain an element(s) that represses gene expression in non-catecholamingeric neurons.
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PMID:A herpes simplex virus-1 vector containing the rat tyrosine hydroxylase promoter directs cell type-specific expression of beta-galactosidase in cultured rat peripheral neurons. 871 59

Organotypic cocultures of thalamic and cortical explants have recently been used to study the development of the thalamocortical axonal network in the mammalian neocortex. To explore the possibility of genetically manipulating organotypic explants, rat thalamocortical (TC) cocultures were infected with the recombinant adenovirus, Adv/RSV beta gal. Infection of the cortical explants resulted in long-term expression (2 weeks) of the reporter gene (beta-galactosidase) with no significant alterations to the structural integrity of the explants. By micro-injecting the adenoviruses into cortical explants a significant degree of spatial control over reporter gene expression was obtained. DiI-labeled axonal projections from thalamic explants into infected (n = 116) and control cortical (n = 120) explants were also analyzed. There was no significant difference in the extent or degree of TC ingrowth into infected or control cortical explants. Thalamic explants were also efficiently infected with the Adv/RSV beta gal virus. While the pattern and extent of TC ingrowth from infected thalamic explants was similar to controls, the percentage of viable, infected thalamic explants was decreased. These experiments were necessary precursors for future studies using recombinant adenoviruses and organotypic cocultures. Genetic manipulation of these cocultures should enable the dissection of proteins involved in the development of axonal networks in the mammalian neocortex, using a system amenable to direct manipulation and observation.
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PMID:Adenovirus-mediated expression of a reporter gene in thalamocortical cocultures. 871 24

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.
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PMID:Adenovirus-mediated overexpression of human transforming growth factor-beta 1 in rat cardiac fibroblasts, myocytes and smooth muscle cells. 873 1

The glycerol phosphate shuttle consists of FAD-linked mitochondrial glycerol 3-phosphate dehydrogenase (mGPDH) and its cytosolic NAD-linked isoform (cGPDH). Impaired mGPDH activity has recently been suggested to be one of the primary causes of insulin secretory defects in beta-cells. We found that mGPDH and cGPDH activities in MIN6 cells are comparable to those of isolated islets and higher than those in HIT cells by eightfold and threefold, respectively. Therefore, we selected the MIN6 cell line as a beta-cell model with normally regulated insulin secretion and normal shuttle enzyme activities and the HIT cell line as a beta-cell model with impaired insulin secretion and lower activities of these enzymes. The role of these dehydrogenases in glucose-stimulated insulin secretion was addressed by examining the effects of overexpression of mGPDH and/or cGPDH via recombinant adenoviruses in these cells. Infection with recombinant adenovirus with a cDNA encoding the Escherichia coli beta-galactosidase gene resulted in expression of its gene in 90% of MIN6 and HIT cells. Infection with a recombinant adenovirus with mGPDH cDNA (Adex1CAmGPDH) caused 2.1-fold and 5.7-fold increases in dehydrogenase activity as compared with those of control MIN6 and HIT cells, respectively. Infection with a recombinant adenovirus with cGPDH cDNA (Adex1CAcGPDH) caused a more than 50-fold increase in activity in both cell lines. Glycerol phosphate shuttle flux, as estimated by [2-3H]glycerol conversion to [3H]H2O, was increased to 120-130% by infection with Adex1CAmGPDH, but not with Adex1CAcGPDH infection, in both MIN6 and HIT cells. No further increase in flux through the glycerol phosphate shuttle was detected when the cells were infected with Adex1CAmGPDH together with Adex1CAcGPDH. Furthermore, neither [U-14C]glucose oxidation nor the insulin secretory response to glucose was affected in either cell line. Thus, mGPDH abundance in MIN6 and HIT cells is not directly related to their insulin secretory capacity in response to glucose, and reduced expression of mGPDH is not the primary cause of abnormal insulin secretory responses in HIT cells. The present data indicate that the emerging hypothesis pointing to mGPDH deficiency as a possible cause of NIDDM needs to be carefully evaluated.
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PMID:Effect of mitochondrial and/or cytosolic glycerol 3-phosphate dehydrogenase overexpression on glucose-stimulated insulin secretion from MIN6 and HIT cells. 877 29

Glycoprotein H (gH) of pseudorabies virus (PrV) is a structural component of the virion and forms a complex with another glycoprotein, gL. For a detailed analysis of the function of PrV gH, we isolated a gH-deficient mutant on trans-complementing gH-expressing cells after insertion of a beta-galactosidase expression cassette into a partially deleted gH gene. The absence of gH did not affect primary or secondary attachment of PrV but the mutant was not infectious. The defect in infectivity could partially be overcome by experimentally induced membrane fusion using PEG, which suggests that gH was necessary for fusion between virion and cellular membranes. After intranasal inoculation into mice, the LD50 of complemented gH- PrV was more than four orders of magnitude higher than that of wild-type PrV. Infection of the respiratory epithelium was much less efficient with complemented gH- PrV as compared with rescued PrV, reflecting the lack of direct cell-to-cell spread. Complemented gH- PrV was able to penetrate into a few trigeminal and sympathetic first order neurons accessible from the nasal cavity, whereas transneuronal transfer in the second order neurons was not observed. In summary, gH is essential for entry and cell-to-cell spread in cell culture, and for propagation in the nervous system of mice. This substantiates the hypothesis that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms.
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PMID:Glycoprotein gH of pseudorabies virus is essential for penetration and propagation in cell culture and in the nervous system of mice. 881 Oct 28


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