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)

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.
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PMID:Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells. 255 44

The transcriptional and translational signals required for efficient expression of the chloramphenicol acetyltransferase, beta-galactosidase, and tissue plasminogen activator genes, under the control of the polyhedrin promoter in Spodoptera frugiperda cells infected with Autographa californica nuclear polyhedrosis virus, were investigated by SDS-PAGE and RNA dot blot analysis. The recombinant baculoviruses all contained alterations in the leader sequence or 5' proximal coding region of the polyhedrin gene. Highest levels of foreign proteins and polyhedrin-linked mRNAs were observed when portions of the coding sequence of the polyhedrin gene were fused in phase with the foreign gene. Recombinant viruses in which the foreign gene was inserted upstream from the polyhedrin ATG start codon expressed nonfused products but at lower levels than contructs which produced fusion proteins. A corresponding decrease in the levels of mRNAs produced by such constructs was also observed. Some constructs in which the foreign gene was inserted out of phase downstream from the polyhedrin start codon expressed nonfused protein products at low levels but produced polyhedrin-linked mRNA at levels comparable to vectors which produced protein fusions. These data suggest that reinitiation of translation can take place at AUG start codons a short distance downstream from the primary polyhedrin start codon. These results indicate that sequences immediately upstream from the polyhedrin start codon are important for regulation of transcription and that additional sequences near the AUG start codon can have a dramatic influence on the levels of translation observed.
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PMID:Signals important for high-level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors. 314 47

The death of cultured insect cells after baculovirus infection is a time-dependent event. Without a quantitative model, it is difficult to characterize its kinetics. Our group has shown that the cell survival rate can be characterized by use of the n-target theory, which involves only two parameters: the number of hypothetical inactivation targets (n) and the first-order death rate (k). In this study, we used different recombinant viruses to examine the effect of heterologous protein expression on the cell survival rate. The proteins expressed were beta-galactosidase, human T-cell leukemia virus type I p40x, human interleukin-2, and human tissue plasminogen activator (tPA). The survival rate was affected by protein expression, but the n value remained constant if the protein expression level was high (above 30 mg/L). Low-level expression of secreted, glycosylated tPA resulted in a reduced n value, which was restored to the normal value when the tPA signal peptide and prosequence were deleted. In addition, if the n value was normal (10-11), the level of protein expression correlated negatively with the death rate. However, if the n value was reduced by unfavorable culture conditions or foreign protein expression, the expression level correlated positively with the death rate. A dimensionless plot with kt as the dimensionless time shows that alteration of the k value while retaining constant n is equivalent to a rescaling of time. Therefore, the survival curves with constant n reduce to a single curve on the dimensionless plot.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Heterologous protein expression affects the death kinetics of baculovirus-infected insect cell cultures: a quantitative study by use of n-target theory. 776 27

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

Previously, we produced transformed insect cell lines that can express a selected foreign protein constitutively, in the absence of baculovirus infection (D. L. Jarvis, J. G. W. Fleming, G. R. Kovacs, M. D. Summers, and L. A. Guarino, Bio/Technology 8:950-955, 1990). These cells contain stably integrated copies of chimeric genes consisting of the promoter from an immediate-early baculovirus gene, IE1, and the sequences encoding either human tissue plasminogen activator or Escherichia coli beta-galactosidase. Transcription of the integrated genes in these cells is specifically controlled by the IE1 promoter. The purpose of this study was to determine how baculovirus infection influences IE1-mediated foreign protein production by these stably transformed insect cell lines. The results showed that viral infection transiently stimulated and then strongly inhibited the production of both tissue plasminogen activator, a secreted protein, and beta-galactosidase, an intracellular protein. These effects reflected virus-induced changes in the steady-state levels of RNA produced by the integrated genes. Transient assays showed that expression of the viral IEN gene alone could account for the increased levels of RNA observed early in infection. The precise mechanism accounting for the decreased levels of RNA observed later in infection was not determined. However, we obtained evidence that the native IE1 promoter remains active throughout infection, which suggested indirectly that the integrated IE1 promoter is transcriptionally inactivated at late times of baculovirus infection. Thus, the same promoter behaved quite differently late in infection, depending on its local environment. Neither methylation nor degradation appeared to be responsible for inactivating IE1-mediated expression of the integrated genes. The significance of these results with respect to the baculovirus-host interaction and the practical applications of stably transformed insect cell lines are discussed.
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PMID:Effects of baculovirus infection on IE1-mediated foreign gene expression in stably transformed insect cells. 847 63

The effect of bcl-2 expression on cell viability and recombinant protein synthesis was investigated in the Spodoptera frugiperda Sf-9 and Trichoplusia ni BTI-Tn-5B1-4 (High Fivetrade mark) insect cell lines. It was found that coinfection with a baculovirus expressing bcl-2 [Autographa californica nuclear polyhedrosis virus (AcNPV)-bcl2] extended the life span of High Fivetrade mark cells but not Sf-9 cells when compared to infection with recombinant baculoviruses expressing either human tissue plasminogen activator (AcNPV-tPA) or Escherichia coli beta-galactosidase (AcNPV-betagal). Similar results were obtained in coinfection experiments; i.e., AcNPV-bcl2 coinfection increased the life span of High Fivetrade mark cells over that of cells infected with either AcNPV-tPA or AcNPV-betagal alone, but they did not affect the life span of coinfected Sf-9 cells. Coinfection of Sf-9 cells with AcNPV-bcl2 and AcNPV-betagal resulted in a decrease in the maximum beta-gal expression levels of over 90% when compared to infection with AcNPV-betagal alone. A similar trend was found in the beta-gal mRNA levels. Coinfection also resulted in a reduced beta-gal expression level in High Fivetrade mark cells, but the reduction was consistent with what would be expected when two recombinant viruses compete for use of the cellular machinery. In contrast to the inhibitory effect of AcNPV-bcl2 coinfection on betagal expression, t-PA expression levels were either not affected (Sf-9 cells) or were increased 50% (High Fivetrade mark cells) over those obtained by infection with AcNPV-tPA alone. These results support the hypotheses that bcl-2 can inhibit transcription of genes under polyhedrin promoter control and that beta-gal expression levels, but not t-PA expression levels, are controlled at the transcriptional level. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 380-390, 1997.
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PMID:bcl-2 expression in Spodoptera Frugiperda Sf-9 and Trichoplusia Ni BTI-Tn-5B1-4 insect cells: effect on recombinant protein expression and cell viability. 1864 41

Vein grafts are used to bypass coronary arterial stenosis, but many grafts thrombose soon after surgery. A model was developed in the pig to allow continuous measurement of blood flow and production of flow-restricting thrombi (cyclic flow reductions; CFRs). Saphenous vein lumen was exposed to adenovirus ex vivo, to over-express human tissue plasminogen activator (h-tPA), with beta-galactosidase adenovirus as a control. The vein segments were engrafted into carotid arteries and examined 0, 1 or 3 days later (4-7 animals/group). Untransduced grafts examined on the day of surgery developed repeated CFRs at both normal and restricted flow, but their frequency declined in grafts examined after 3 days. Adenovirus transduction was evident as beta-galactosidase or h-tPA expression 1 day after engraftment. Blood flow was increased 1.4-fold in h-tPA transduced grafts after 1 day [control 390 (280-510), h-tPA 550 (450-660) ml/min; p=0.02 (expressed as mean (95% confidence intervals)]. CFRs were less severe (p=0.002) in the h-tPA transduced grafts than beta-galactosidase-transduced grafts. CFRs were also less frequent in unstenosed undamaged h-tPA grafts [control 17 (6.1-29), h-tPA 7.6 (1.7-14) CFR/hr; p=0.02], but this difference was reduced after damage or stenosis. CFRs formed faster in h-tPA than in beta-galactosidase-transduced grafts [control 14 (11-17), h-tPA 23 (19-27) ml/min(2); p<0.001], and resolved twofold faster [control 25 (22-30), h-tPA 48 (39-60) ml/min(2); p<0.001]. Hence, in this model, local gene therapy with h-tPA increased graft blood flow and decreased measures of early graft thrombosis, namely quicker CFR resolution and decreased frequency and severity.
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PMID:Reduction of early vein graft thrombosis by tissue plasminogen activator gene transfer. 1957 59

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