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 target gene expression to malignant hepatic cells, we have constructed recombinant retroviral vectors containing a reporter gene encoding nuclear beta-galactosidase (nls-LacZ) under transcriptional control of regulatory sequences from the rat alpha-fetoprotein (AFP) or human insulinlike growth factor II (IGFII) genes. The AFP and IGFII P3 promoters activate transcription during fetal development and are often reactivated in hepatocellular carcinoma (HCC). Infection of several cultured cell types with the retroviral vector containing the IGFII P3 sequence resulted in expression of the reporter gene in all cell lines tested, including those that do not produce IGFII. In contrast, selective expression was achieved by vectors containing the AFP transcriptional regulatory sequence. Nuclear beta-galactosidase activity was detectable in cells from lines that produce AFP, and not in cells that do not express the AFP gene. In most infected cell lines, retroviral RNA synthesis from the 5' LTR was inhibited, and deletion of the retroviral LTR enhancer did not change expression from either the IGFII P3-nls-LacZ or the AFP-nls-LacZ cassettes. After treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor (EGF), the decrease in concentrations of endogenous AFP messenger RNA (mRNA) and nls-LacZ mRNA transcribed from the transferred AFP regulatory sequence were similar. In the context of an integrated provirus, the AFP transcriptional regulatory sequence is therefore subject to similar regulatory control as that of the endogenous gene. These data show that the AFP sequence, and not the IGFII P3 promoter we used, is suitable for targeting gene expression to malignant hepatic cells.
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PMID:Hepatoma cell-specific expression of a retrovirally transferred gene is achieved by alpha-fetoprotein but not insulinlike growth factor II regulatory sequences. 748 90

Complementary DNA clones for exogastrula-inducing peptides (EGIPs) of the sea urchin Anthocidaris crassispina, which are related to epidermal growth factor (EGF), were obtained from a cDNA library of late gastrula embryos using, as probe, the partial cDNA for one of the EGIP (EGIP-D) obtained by the reverse-transcription PCR method. The longest cDNA was composed of 1662 bp, and encoded a protein of approximately 36 kDa with a region that resembled a signal sequence. The deduced protein contains the sequences of EGIP-C, EGIP-D, and EGIP-A in that order, followed by the sequence for an unidentified EGIP-like polypeptide. When expressed in Escherichia coli as a fusion protein with beta-galactosidase, the product for the cDNA was specifically recognized by a rabbit antibody raised against EGIP-D that had been purified from embryos. Characteristic amino acid residues were found around the N-terminus and the C-terminus of each EGIP sequence, suggesting a specific processing mechanism for the generation of the individual EGIPs from the precursor. RNA-blot analysis revealed the presence of EGIP mRNA in unfertilized eggs. The level of this mRNA decreased gradually after fertilization, began to increase dramatically after the onset of gastrulation, and continued to increase through the pluteus stage. Genomic Southern-blot analysis suggested that this gene is present as a single copy. A homology search showed that the EGIP cDNA has a similarity to the cDNA for SpEGF2 which was cloned as a gastrula-specific gene in another sea urchin, Strongylocentrotus purpuratus.
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PMID:Molecular cloning of a cDNA that encodes the precursor to several exogastrula-inducing peptides, epidermal-growth-factor-related polypeptides of the sea urchin Anthocidaris crassispina. 770 69

The effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on mature marmoset (Callithrix jacchus) Sertoli cells in vitro were investigated using light and electron microscopic and histochemical means. The morphological data were substantiated by morphometric analysis at the electron microscopic level. In a bicameral chamber system, cultured Sertoli cells displayed a high degree of ultrastructural differentiation and exhibited a polarized appearance. Basally located tight junctions joined adjacent cells. Germ cells of early stages of development were regularly seen. Under the influence of IGF-I, cells developed extensive cell-cell contacts. The surface density of smooth endoplasmic reticulum was increased. In contrast, the volume density of lipid inclusions was decreased. The morphological integrity of enclosed germ cells was maintained for a longer period. With EGF, cells were arranged in loose aggregates. Intercellular spaces were widened. The volume density of lipid inclusions was increased. Germ cells exhibited profound signs of degeneration early in culture, paralleled by increased development of phagolysosomes and high acid-beta-galactosidase activity. Under the influence of either growth factor, mitochondria displayed a shift from the crista to the tubulo-vesicular type. Mitochondrial dimensions and the volume density of mitochondrial compartment were increased. In comparison with control cultures all documented changes were statistically significant. Our findings indicate that marmoset Sertoli cells are target cells for EGF and IGF-I. Moreover, the dynamics of intercellular contacts, germ cell survival, and morphological indices of lipid and/or steroid metabolism seem to be differentially modulated.
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PMID:Growth factors (EGF, IGF-I) modulate the morphological differentiation of adult marmoset (Callithrix jacchus) Sertoli cells in vitro. 786 Apr 20

To examine whether the epidermal growth factor (EGF)-like domain Pro47-Asp87 is involved in the interaction of tissue plasminogen activator (t-PA) with platelets, we have expressed this domain in E. coli. The peptide fragment was produced from a plasmid expression vector as a fusion protein with beta-galactosidase Met1-Val444 at high yield in eight clones of E. coli. The fusion protein was purified and subjected to mild acid hydrolysis with formic acid, then the peptide Pro47-Asp87, identified by immunoblotting using specific antibodies to t-PA, was isolated by HPLC. After incubation with blood platelets spin labelled with 16-doxylstearic acid or 5-doxylstearic acid, the Pro47-Asp87 peptide fragment reduced fluidity of the membrane lipid bilayer to the same extent as did intact t-PA as indicated by ESR measurements. Our data suggest that the EGF-like domain of t-PA can directly interact with blood platelets and thus it seems to contain those sites of the t-PA molecule that bind the platelet membrane components.
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PMID:The epidermal growth factor-like domain from tissue plasminogen activator. Cloning in E. coli, purification and ESR studies of its interaction with human blood platelets. 803 Mar 71

Trophic factors may function as one of the epigenic signals responsible for the proliferation, growth, migration, and differentiation of neurons and glia during embryogenesis. The present study reports that basic fibroblast growth factor (bFGF) at high concentrations (10-100 ng/ml) is a mitogen for embryonic spinal cord cells that have already committed to a neuronal pathway and are expressing neuronal phenotypes (neuroblasts). Neuroblasts proliferate with a doubling time of 2.5 d. To characterize the nature of cells proliferating in response to bFGF, we have established long-term cultures of neuroblasts that can be passaged, freeze thawed, and recultured. In cultures the proportion of astrocytes remained the same, indicating limited survival and proliferation of these cells in response to bFGF. These results indicate that bFGF has mitogenic effects preferably on neuroblasts. The morphological and biochemical characterizations of the neuronal populations present in the long-term neuroblast cultures are presented here. The presence of cholinergic and GABAergic neurons in the cultures was established by immunocytochemical analysis. The cultures contain a small number of motoneurons as judged by their immunostaining with ChAT, low-affinity NGF receptor (LNGFR), and large size. Among all other growth factors tested for their mitogenic effects on embryonic spinal cord cells in culture, only epidermal growth factor (EGF) showed such effects, but to a lesser degree. The proliferative nature of neuroblasts has made it possible to transduce the Escherichia coli beta-galactosidase (LacZ) gene stably into these cells in vitro using a retroviral vector. The transfected cells expressing the foreign gene can be passaged, freeze thawed, and recultured without the loss of transgenes. The ability to transduce foreign genes stably into these cells permits implantation of these cells in the spinal cord to study cellular and biochemical behaviors and gene expression in defined neuronal populations in in vivo environments.
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PMID:Spinal cord neuroblasts proliferate in response to basic fibroblast growth factor. 820 71

Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer using asialofetuin-labeled liposomes (AF-liposomes) were examined with HepG2 cells and rat hepatocytes in primary culture. AF-liposomes were sufficiently taken up by both types of cells through the asialoglycoprotein receptor-mediated endocytosis. In HepG2 cells, bacterial beta-galactosidase (beta-Gal) gene expression was observed by transfection using AF-liposomes encapsulating plasmid pCMV beta DNA (AF-liposome-pCMV beta). By addition of dioleoylphosphatidylethanolamine (DOPE) to the liposomal lipid composition (AF-liposome(DOPE)-pCMV beta), the transfection efficiency was clearly increased. The effects of DOPE were more conspicuous in the presence of chloroquine in the medium throughout the transfection. When pCMV beta complexed with gramicidin S (pCMV beta (GrS)) was encapsulated (AF-liposome(DOPE)-pCMV beta (GrS) and was transfected to HepG2 cells, an significantly high beta-Gal activity in the cells was observed as compared with that in the cells transfected with AF-liposome(DOPE)-pCMV beta. No effects of GrS were found in the transfection using AF-non-labeled control liposomes. In primary culture of rat hepatocytes, no beta-Gal gene expression was observed even though AF-liposome(DOPE)-pCMV beta was introduced into the cells prepared from adult rats. However, following the transfection with AF-liposome(DOPE)-pCMV beta, the beta-Gal activity was expressed in the cells from immature rats cultured in the medium supplemented with epidermal growth factor and insulin, and the transfection efficiency was 2-fold higher than that transfected with pCMV beta encapsulated in AF-non-labeled control liposomes. By the complex formation of pCMV beta with GrS, the transfection efficiency of AF-liposome(DOPE)-pCMV beta (GrS) increased according to the increase of GrS in the complex. It was shown that AF-liposome(DOPE)-pCMV beta (GrS) did efficiently introduce and express beta-Gal gene in both HepG2 cells and primary hepatocytes in the receptor mediated manner.
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PMID:Effects of fusogenic and DNA-binding amphiphilic compounds on the receptor-mediated gene transfer into hepatic cells by asialofetuin-labeled liposomes. 861 7

The effects of mitogens and agents affecting tyrosine phosphorylation signaling on androgen-regulated transcription were investigated. CV-1 and HeLa cells were cotransfected with an androgen receptor (AR) expression vector and an androgen-responsive chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse mammary tumor virus promoter. Growth factors [epidermal growth factor (EGF) and insulin-like growth factor I] that activate receptor tyrosine kinases, an inhibitor of phosphotyrosine phosphatases (vanadate), or an inhibitor of tyrosine kinases (genistein) did not influence basal promoter activity or that of unliganded AR. However, EGF, insulin-like growth factor I, and vanadate enhanced AR-dependent transactivation by 1.5- to 2.5-fold, and genistein diminished it by two thirds in the presence of androgen. None of the treatments affected pRSV-CAT or pSV-beta-galactosidase expression, suggesting that gross activation of the transcription machinery was not involved. A reporter with two androgen response elements (AREs) in front of the thymidine kinase promoter (p delta ARE2tk-CAT) was used to examine promoter specificity. EGF activated this reporter even in the absence of androgen. However, when EGF was used concomitantly with testosterone, it augmented the action of androgen. Vanadate enhanced androgen-induced transactivation 2-fold without altering basal promoter activity. Neither EGF nor vanadate altered immunoreactive AR content or elicited changes in the receptor's DNA-binding properties. The intracellular content of hormone-binding AR was not influenced by EGF, but was decreased by vanadate and increased by genistein, as judged by [3H]mibolerone binding assays. An AR form lacking the hormone-binding domain (delta 641-902 mutant) transactivated p delta ARE2tk-CAT reporter similar to or better than the wild-type receptor in the presence of androgen. The transactivation by the delta 641-902 mutant was augmented by EGF and vanadate, but was attenuated by genistein, implying that the steroid-binding region is not critical for regulatory events initiated by tyrosine phosphorylation. Collectively, these data indicate that there is cross-talk between androgen-mediated signaling systems and growth factor/receptor tyrosine kinase pathways.
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PMID:Effects of mitogens on androgen receptor-mediated transactivation. 882 95

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

The aim of this work was to develop a procedure for the lipid-mediated transfection of DNA into normal adult human hepatocytes in culture. Cells were plated in a serum-free culture medium at various cell densities, on plastic or collagen-coated dishes, both in the absence and in the presence of epidermal growth factor (EGF). The cells were incubated for various periods of time with mixtures of DNA-lipofectin or DNA-3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-chol) liposomes, and the efficiency of transfection was assessed by measuring the activity of reporter genes, beta-galactosidase or chloramphenicol acetyl-transferase (CAT). For comparison, similar experiments were carried out with human cell lines including HepG2, Caco-2, and WRL68. The efficiency of transfection (in percentage of cells) was not significantly different after transfection with lipofectin or DC-chol and comprised between 0.04 and 1.7% (extreme values) for different cultures. The efficiency of transfection decreased as the age or density of the culture increased and increased in cultures treated with EGF. Direct measurement of the rate of DNA synthesis suggested that the efficiency of transfection was related to the number of cells entering the S phase. Under the same conditions, the efficiency of transfection was one to two orders of magnitude greater in the three cell lines. A plasmid harboring 660 bp of the 5'-flanking region of CYP1A1 (containing two xenobiotic enhancer elements) fused upstream of the promoter of thymidine kinase and the CAT reporter gene was constructed. When this plasmid was transfected in human hepatocytes, CAT activity was induced as expected. We conclude that normal adult human hepatocytes can be transfected with exogenous DNA and that the transfected construct is regulated in the manner expected from in vivo studies.
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PMID:Lipid-mediated transfection of normal adult human hepatocytes in primary culture. 912 68

Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron-glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for beta-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10-11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15-18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that > 62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.
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PMID:In vitro cell density-dependent clonal growth of EGF-responsive murine neural progenitor cells under serum-free conditions. 939 57


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