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)

We developed and evaluated an in vivo athymic nude mouse model for tumor growth, angiogenesis, metastasis, and antineoplastic drug development. Melanoma cell lines expressing beta-galactosidase encoded by the Escherichia coli lac Z gene have been created by infecting an immortal murine melanocyte cell line with a recombinant retrovirus expressing the v-Ha-ras oncogene and lac Z to generate the MRB (melanoma, ras, beta-galactosidase) cell lines. The amelanotic, phorbol ester-independent, transformed melanoma cell lines developed tumors rapidly when injected subcutaneously into nude mice, as well as experimental lung metastases when injected i.v. into the tail vein. beta-galactosidase-expressing subcutaneous tumors and lung metastases stained blue with X-gal. The melanomas produced in nude mice have been characterized by using various histochemical and immunohistochemical staining methods to detect melanoma- and endothelial-cell-specific markers to determine the extent of neovascularization in MRB nude mouse tumors. Optimal staining of endothelial cells involved in tumor angiogenesis was observed by using ADPase activity and antiangiotensin-converting enzyme antibody staining. Attempts at indirect quantification of metastatic tumor cell number within the lung by either beta-galactosidase enzymatic activity or ELISA immunoreactivity were unsuccessful. However, the MRB cell lines should be useful in screening for and studying the mechanisms of action of antineoplastic, antimetastatic, and angiostatic drugs in vivo in athymic nude mice.
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PMID:Evaluation of a nude mouse tumor model using beta-galactosidase-expressing melanoma cells. 768 92

This study used reporter gene constructs containing regulatory regions of the c-fos, vasoactive intestinal peptide, and choline acetyltransferase genes to determine the role of p21ras and protein kinase C in the action of ciliary neurotrophic factor and leukemia inhibitory factor. Down-regulation of protein kinase C with phorbol ester did not affect the induction of either c-fos-beta-galactosidase or vasoactive intestinal peptide-luciferase by ciliary neurotrophic factor or leukemia inhibitory factor. In contrast, while leukemia inhibitory factor induction of choline acetyltransferase-luciferase expression was protein kinase C-independent, there appears to be both protein kinase C-dependent and -independent pathways for induction of choline acetyltransferase-luciferase by ciliary neurotrophic factor. Cotransfection of a dominant-negative mutant p21rasN17 blocked nerve growth factor-mediated induction of c-fos-beta-galactosidase, but did not affect induction of c-fos-beta-galactosidase, vasoactive intestinal peptide-luciferase, or choline acetyltransferase-luciferase by either ciliary neurotrophic factor or leukemia inhibitory factor. Thus, in contrast to the action of nerve growth factor, gene induction by ciliary neurotrophic factor, and leukemia inhibitory factor is ras-independent in IMR-32 neuroblastoma cells.
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PMID:Differential requirements for p21ras and protein kinase C in the regulation of neuronal gene expression by nerve growth factor and neurokines. 803 40

We have investigated the activity of CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase (EC 2.4.99.4) in FR3T3 cells transformed by the Ha-ras oncogene in which we have previously demonstrated the higher expression of the beta-galactosidase alpha-2,6-sialyltransferase (EC 2.4.99.1) [21]. We demonstrate that the presence of the activated ras gene decreases the activity of this specific alpha-2,3-sialyltransferase fourfold. According to the kinetic parameters and to mixing experiments, we can assume that this decreased enzymatic activity reflects a decrease in the number of active O-glycan alpha-2,3-sialyltransferase polypeptides in ras-transformed cells. However, no change in the binding of Peanut agglutinin was observed on the cell surface of ras-transformed FR3T3 suggesting that no change in the sialylation of O-glycan core 1 appeared in these cells, although the activity of the alpha-2,3-sialyltransferase was decreased.
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PMID:Sialyltransferase activity in FR3T3 cells transformed with ras oncogene: decreased CMP-Neu5Ac:Gal beta 1-3GalNAc alpha-2,3-sialyltransferase. 835 31

Raf-1 is a serine/threonine specific kinase that integrates signaling by a large number of mitogens to elicit a transcriptional response in the nucleus. Activated Raf-1 phosphorylates and activates MAPK/ERK kinase Mek), thus initiating the Mek--> MAP kinase cascade, which ultimately results in the phosphorylation and activation of transcription factors by MAP kinase. Here we have characterized the mechanism by which monoclonal antibody URP26K, which binds to an epitope in the Raf-1 kinase domain, inhibits intracellular signal transduction. This antibody preferentially immunoprecipitated the underphosphorylated, non-activated form of Raf-1 from quiescent cells. Baculovirus-expressed Raf-1 immunoprecipitated with URP26K was largely refractory to phosphorylation and activation mediated by protein kinase C (PKC)alpha or the tyrosine kinase Lck. In addition, URP26K reduced the binding of Raf-1 to its substrate Mek in vitro, but did not disturb the association of Raf-1 with Ras. Microinjection of URP26K into Rat-1 cells blocked DNA synthesis initiated by serum, insulin and various purified growth factors, but it did not block DNA synthesis initiated by v-ras. Microinjected URP26K also impaired the expression of stably transfected beta-galactosidase reporter genes regulated by minimal promoter elements. These results demonstrate, (i) that the URP26K monoclonal antibody inhibits Raf-1 by preventing activating Raf-1 phosphorylation and/or association with its substrate Mek, (ii) that inhibition of Raf-1 by URP26K does not interfere with Ras-induced DNA synthesis. In contrast to dominant negative Raf-1 mutants, which also block Ras signaling by binding to the Ras effector domain, antibody mediated Raf-1 inhibition thus reveals a branchpoint of mitogenic signaling at the level of Ras.
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PMID:Inhibition of Raf-1 signaling by a monoclonal antibody, which interferes with Raf-1 activation and with Mek substrate binding. 880 5

An understanding of how oncogenes affect differentiated liver functions might lead to improved treatments for liver cancer or other disorders where liver-specific functions are compromised. A retroviral vector that coexpressed beta-galactosidase (beta-gal) and activated Ras genes (Ras-gal) was transduced into a small fraction of adult rat hepatocytes in vivo. Hepatocytes from Ras-gal-transduced diethylnitrosamine-untreated livers and hepatocellular carcinomas (HCC) from Ras-gal-transduced diethylnitrosamine-treated rats were analyzed for liver functions by performing histochemical assays on liver sections. Ras-gal-transduced hepatocytes failed to express gluconeogenic, ketogenic, and urea pathway enzymes. In contrast, several enzymes involved in fat synthesis were strongly activated, and microvesicular fat accumulated. These metabolic changes are induced in normal livers by insulin, a hormone that activates p21-ras. The deregulation of p21-ras may inhibit these liver-specific functions and may induce fat synthesis in both malignant and nonmalignant liver diseases. Furthermore, treatment with drugs that inhibit the attachment of p21-ras to the plasma membrane might reverse these changes. The alterations in enzymatic functions in the HCCs were similar to those observed in the hepatocytes, although each of the two cancers had a region that abruptly lost its expression of liver-specific enzymes and acquired the expression of genes that are more characteristic of oval or bile ductule cells. This suggests that a single genetic event in a malignant cell may dramatically alter its apparent phenotype. The identification of this putative gene might lead to insights into the regulation of the phenotype of normal cells in the liver.
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PMID:Alterations in enzymatic functions in hepatocytes and hepatocellular carcinomas from Ras-transduced livers resemble the effects of insulin. 885 86

Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in mediating the actions of insulin.
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PMID:Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport. 899 89

Products of ras oncogenes strongly stimulate the activity of the reporter gene, chloramphenicol acetyltransferase (CAT), driven by a 1.2 kb fragment of the murine cytomegalovirus (MCMV) immediate early (IE) gene enhancer (pCMVCAT). To define the role of proteins binding to the unique cAMP response element (CRE) present in the IE enhancer, NIH 3T3 cells were cotransfected with prasZip6 plasmid, a mammalian expression vector containing a v-Ha-ras cDNA, together with p(delta)ACMVCAT (pCMVCAT without the CRE sequence). Lower stimulation of CAT activity was indeed observed upon deletion of the CRE sequence. Decreased levels of p(delta)ACMVCAT were also observed in cell lines carrying stably transfected ras oncogenes. Further support for the role of the CRE sequence in MCMV enhancer activation comes from the finding that v-Ha-ras expression increases the activity of a reporter gene, beta-galactosidase, driven by three tandem copies of CRE sequence about six-fold. Moreover, this transactivation was prevented by cotransfection of the dominant inhibitor mutant Ha-ras (Leu-61; Ser-186) and was not suppressed by cotransfection of Ha-ras (Asn-17), suggesting that the effect is due to activated ras protein, rather than normal p21ras. Finally the transactivation observed is accompanied by an increase in nuclear proteins binding to a labelled oligonucleotide homologous to the CRE sequence, as shown in a gel retardation assay. These results suggest that the CRE element contributes to the transactivation of the MCMV IE gene enhancer by ras oncogenes.
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PMID:cAMP response element of murine cytomegalovirus immediate early gene enhancer is transactivated by ras oncogene products. 904 20

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.
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PMID:Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p. 926 16

The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.
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PMID:Lac/Tet dual-inducible system functions in mammalian cell lines. 956 36

Hepatitis C virus (HCV) is a major human pathogen causing mild to severe liver disease worldwide. This positive strand RNA virus is remarkably efficient at establishing chronic infections. Although a high rate of genetic variability may facilitate viral escape and persistence in the face of Ag-specific immune responses, HCV may also encode proteins that facilitate evasion of immunological surveillance. To address the latter possibility, we examined the influence of specific HCV gene products on the host immune response to vaccinia virus in a murine model. Various vaccinia/HCV recombinants expressing different regions of the HCV polyprotein were used for i.p. inoculation of BALB/c mice. Surprisingly, a recombinant expressing the N-terminal half of the polyprotein (including the structural proteins, p7, NS2, and a portion of NS3; vHCV-S) led to a dose-dependent increase in mortality. Increased mortality was not observed for a recombinant expressing the majority of the nonstructural region or for a negative control virus expressing the beta-galactosidase protein. Examination of T cell responses in these mice revealed a marked suppression of vaccinia-specific CTL responses and a depressed production of IFN-gamma and IL-2. By using a series of vaccinia/HCV recombinants, we found that the HCV core protein was sufficient for immunosuppression, prolonged viremia, and increased mortality. These results suggest that the HCV core protein plays an important role in the establishment and maintenance of HCV infection by suppressing host immune responses, in particular the generation of virus-specific CTLs.
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PMID:Suppression of host immune response by the core protein of hepatitis C virus: possible implications for hepatitis C virus persistence. 991 17


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