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 have constructed a HeLa cell line that both expresses high levels of CD4 and contains a single integrated copy of a beta-galactosidase gene that is under the control of a truncated human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). This cell line, called CD4-LTR/beta-gal, can be used to determine quantitatively the titer of laboratory-adapted HIV strains, and the method used to do so is as sensitive as the determination of viral titers in a T-cell line by end point dilution. Using this cell line as a titer system, we calculated that HIV-1 stocks contain only one infectious particle per 3,500 to 12,000 virions. Virus derived from a molecular clone of a macrophagetropic provirus will not infect this cell line. We have also cocultivated peripheral blood lymphocyte cultures from HIV-infected individuals with the CD4-LTR/beta-gal indicator cells. In a majority of primary isolates (five of eight), including isolates from asymptomatic patients, rare virus-infected cells that can activate the beta-galactosidase gene are present.
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PMID:Detection of replication-competent and pseudotyped human immunodeficiency virus with a sensitive cell line on the basis of activation of an integrated beta-galactosidase gene. 154 59

Chemical conjugates of recombinant soluble CD4 (sCD4) with toxins, or with antibodies that activate cytotoxic T cells, can be used to direct selective killing of human immunodeficiency virus (HIV)-infected cells. This approach takes advantage of the ability of sCD4 to bind with high affinity to gp120, the envelope protein of HIV-1, which is expressed on actively infected cells. However, conjugation of sCD4 via reagents that target amino groups may reduce its affinity for gp120, since at least one such group is important for gp120 binding. Here, we describe a novel cross-linking reagent which enables the conjugation of sCD4 via its carbohydrate moieties rather than its free amino groups. This heterobifunctional reagent, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), combines a nucleophilic hydrazide with an electrophilic maleimide, thereby allowing coupling of carbohydrate-derived aldehydes to free thiols. We describe conditions by which MPBH is coupled selectively to the sialic acid residues of sCD4, and exemplify the use of MPBH by conjugating sCD4 to hemoglobin and to beta-galactosidase. We show that, whereas conjugation of sCD4 via amino groups markedly reduces its gp120 binding affinity, conjugation via the carbohydrate chains using MPBH does not affect binding. Moreover, we demonstrate the ability of a sCD4-MPBH-fluorescein conjugate to label HIV-infected human CEM cells selectively. These results indicate that, by targeting its carbohydrate moieties, sCD4 can be cross-linked to other molecules without compromising its function. The approach described here can be useful for glycoproteins in which amino groups, but not carbohydrates, are important for function. More generally, this approach can be considered for use in cross-linking glycoconjugates to compounds which either contain thiols, or to which thiols can be added.
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PMID:Conjugation of soluble CD4 without loss of biological activity via a novel carbohydrate-directed cross-linking reagent. 163 20

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed.
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PMID:Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata. 197 17

The gene for the CD4-membrane glycoprotein-receptor for HIV has been cloned. The 179 amino acids fragment of the CD4-receptor responsible for binding of gp120 HIV glycoprotein has been fused with beta-galactosidase and shown to be expressed in Escherichia coli cells. The recombinant protein in ELISA and immunoblotting techniques reacts with the monoclonal antibodies OKT4A and Leu3A known to block the interaction between the CD4 and gp120 HIV glycoprotein. The recombinant protein can be used for different scientific and practical purposes including studying of the mechanisms for HIV interaction with the sensitive cells as well as for viral gp120 protein purification, etc.
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PMID:[Cloning and expression of the CD4 receptor gene from human T-lymphocytes in Escherichia coli cells]. 202 97

A quantitative bioassay for human immunodeficiency viruses has been developed on the basis of the ability of the tat gene to transactivate the expression of an integrated beta-galactosidase gene in a HeLa-CD4+ cell line. Infection by a single virion of HIV-1 or HIV-2 corresponds to a unique blue syncytium or a cell cluster detected after fixation and addition of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (a beta-galactosidase substrate). The number of infected lymphoid cells in a culture (stimulated human peripheral blood lymphocytes and cell lines) can also be quantified by cell-to-cell transmission of HIV into the HeLa-CD4(+)-beta-galactosidase monolayer. Infections by simian immunodeficiency viruses are similarly detected. This assay has been used to determine the dose response of drugs, the half-life of HIV at 37 degrees C, and the appearance of infectious particles after virus infection.
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PMID:Activation of a beta-galactosidase recombinant provirus: application to titration of human immunodeficiency virus (HIV) and HIV-infected cells. 211 May 96

Primate lentiviruses such as human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) are phenotypically diverse, and virus isolates vary in cytopathicity, replication rate, and cell tropism. While all virus isolates infect primary peripheral blood lymphocytes, only a subset of strains infect established CD4-expressing T-cell lines. Here, we describe the development and characterization of a macaque cell line that can be infected by all of the strains of SIV that we have tested, including macrophage- and T-cell-tropic strains, primary and cell-line adapted strains, and SIVmac, SIVMne, and SIVsm isolates. The cells can be infected by strains of HIV type 2 (HIV-2) to varying degrees, but not by either cloned or primary isolates of HIV type 1 (HIV-1). This cell line is a derivative of a rhesus macaque mammary tumor cell line (CMMT) engineered to express human CD4. For these studies, a CMMT-CD4 clone expressing an integrated copy of a truncated HIV-1 long terminal repeat fused to the beta-galactosidase gene (LTR-beta-gal) was established to allow detection of infectious SIV after a single round of replication. Here, we demonstrate the ability of the CMMT-CD4-LTR-beta-gal cell line to rapidly and quantitatively detect infectious SIV. Using these cells to assay virus, we could readily measure neutralizing antibody activity in animals infected with different SIV isolates. Neutralizing activity was detected against the homologous virus and lower, but detectable, activity was measured against heterologous virus. Thus, this system, which is highly sensitive and can detect infection by all of the SIV isolates we tested, is a rapid method for detecting infectious virus and quantitating neutralizing antibody activity.
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PMID:Characterization of a CD4-expressing macaque cell line that can detect virus after a single replication cycle and can be infected by diverse simian immunodeficiency virus isolates. 749 63

The haematopoietic development of embryonic stem (ES) cell injection chimaeras was analysed using beta-galactosidase expression from an X-linked transgene as a marker to distinguish the ES-derived cell population from the host cells. The number of cells in the different haematopoietic cell subpopulations was determined by flow cytometry. When the proportions of ES-derived cells in the antigen-positive lineages were compared to the ES cell contribution to all cells in teh organs, we found an unexpected bias in the haematopoietic differentiation of ES-derived cells. ES descendants were overrepresented in the bone marrow B lymphoid cell population and the splenic myeloid cells but were underrepresented in the CD4-positive T lymphoid cells in the spleen. These results were obtained by comparison with control female animals that were X chromosome mosaic for beta-galactosidase expression. These findings of uneven contribution to haematopoietic development by ES cells indicate that the commitment of ES cell descendants may be different from that of the host cells.
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PMID:The development of haematopoietic cells is biased in embryonic stem cell chimaeras. 764 91

The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.
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PMID:Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8 alpha in yeast. 766 3

The nef gene product of human immunodeficiency virus type 1 (HIV-1) promotes more-rapid kinetics of viral replication in primary peripheral blood mononuclear cells. We have previously shown that these enhancing effects of Nef on HIV-1 replication reflect an increase in viral infectivity detectable both in limiting dilution assays and through a single-cycle infection of the HeLa-CD4-long terminal repeat-beta-galactosidase indicator cell line. We now demonstrate that nef-defective HIV-1 can be rescued to near wild-type levels of infectivity by coexpressing Nef in trans in the cell line producing the virus. This observation indicates that HIV-1 virions produced in the presence of Nef are intrinsically different. However, we show that the major viral structural proteins are quantitatively similar in purified viral preparations. We also demonstrate the functional equivalence of the gp120-gp41 envelope glycoprotein complexes of Nef+ and Nef- HIV-1 through an assay for viral entry. Finally, we show that env-defective Nef+ HIV-1 pseudotyped with an amphotropic envelope is also more infectious than similarly pseudotyped Nef- HIV-1. Thus, the production of HIV-1 in the presence of Nef results in viral particles that are more infectious, and this increased infectivity is manifested at a stage after viral entry but prior to or coincident with HIV-1 gene expression.
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PMID:Expression of the human immunodeficiency virus type 1 (HIV-1) nef gene during HIV-1 production increases progeny particle infectivity independently of gp160 or viral entry. 798 59

The fusogenic activities of enveloped-virus glycoproteins were analyzed by using a quantitative, sensitive, rapid, and highly versatile recombinant vaccinia virus-based assay measuring activation of a reporter gene upon fusion of two distinct cell populations. One population uniformly expressed vaccinia virus-encoded viral glycoproteins mediating specific binding and fusion activities; the other expressed the corresponding cellular receptor(s). The cytoplasm of one population also contained vaccinia virus-encoded bacteriophage T7 RNA polymerase; the cytoplasm of the other contained a transfected plasmid with the Escherichia coli lacZ gene linked to the T7 promoter. When the two populations were mixed, cell fusion resulted in activation of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase activity was assessed by colorimetric assay of detergent cell lysates or by in situ staining. We applied this approach to study the human immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4 interaction. Beta-Galactosidase was detected within 1 h after cell mixing and accumulated over the next several hours. Cell fusion dependence was demonstrated by the strict requirement for both CD4 and functional Env expression and by the inhibitory effects of known fusion-blocking monoclonal antibodies and pharmacological agents. Quantitative measurements indicated much higher sensitivity compared with analysis of syncytium formation. The assay was used to probe mechanisms of the cell type specificity for Env-CD4-mediated fusion. In agreement with known restrictions, cell fusion occurred only when CD4 was expressed on a human cell type. Membrane vesicle transfer experiments indicated that CD4 initially produced in either human or nonhuman cells was functional when delivered to human cells, suggesting that the fusion deficiency with nonhuman cells was not associated with irreversible defects in CD4. We also demonstrated that the infectivity specificities of different human immunodeficiency virus type 1 isolates for peripheral blood lymphocytes versus continuous CD4+ cell lines were associated with corresponding fusion selectivities of the respective recombinant Env proteins. The assay enabled analysis of the fusogenic activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the paramyxovirus simian virus 5. This system provides a powerful tool to study fusion mechanisms mediated by enveloped-virus glycoproteins, as well as to screen fusion-blocking antibodies and pharmacological agents.
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PMID:Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a novel recombinant vaccinia virus-based assay quantitating cell fusion-dependent reporter gene activation. 805 23


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