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)

A 1.3-kb segment of Escherichia coli DNA containing the regulatory gene, araC, and the promoter of the araBAD operon was amplified by the polymerase chain reaction (PCR) and cloned into pUC18, resulting in plasmid pKB130 that produced the alpha fragment of beta-galactosidase upon addition of L-arabinose (L-ara). A synthetic gene for human immunodeficiency virus (HIV)-1 preprotease was placed downstream of the ara-BAD promoter in pKB130 to create a translational fusion inducible by addition of L-ara. The fusion protein correctly autoprocessed in vivo to yield a mature 99-amino-acid HIV-1 protease, which was found predominantly in inclusion bodies. This material could be refolded to an active form, which was purified to homogeneity. A small fraction of the protease was expressed in vivo as a soluble active form, which allowed the monitoring of expression during fermentation by a rapid and simple whole cell assay employing an HIV-1 protease-specific fluorogenic substrate.
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PMID:High-level expression and purification of mature HIV-1 protease in Escherichia coli under control of the araBAD promoter. 136 41

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

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum. 165 5

Among 102 brains obtained from patients with acquired immune deficiency syndrome (AIDS), 34 cases with subacute AIDS encephalitis were characterized by immunohistochemistry using an antibody that binds to a human immunodeficiency virus-1 (HIV-1) envelope glycoprotein, gp41. This glycoprotein was detected in mononucleated and/or multinucleated cells in 90% of adult and 50% of pediatric brains with subacute AIDS encephalitis. In addition, many gp41-positive cells with bipolar or multipolar processes were found in 10 cases, and these cells occurred most frequently in the basal ganglia and internal capsule. The phenotype of the gp41-positive cells was determined using an improved double-labeling immunohistochemical technique that employed beta-galactosidase and peroxidase conjugated reagents. Cell-type specific markers for double-labeling included: Ricinus communis agglutinin-1 (RCA-1) for macrophages and microglia; Ulex europaeus agglutinin-1 for endothelium; anti-glial fibrillary acidic protein (GFAP) for astrocytes; anti-amyloid precursor protein for neurons; and anti-leukocyte common antigen for leukocytes. Results of double-immunostaining revealed that gp41-positive cells of all morphologic types, including cells with bipolar or multipolar processes, were double-labeled with RCA-1, but not with markers for astrocytes, neurons, or endothelia. These findings support the contention that HIV-1 infection of the CNS is predominantly restricted to cells of the macrophage/microglia lineage.
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PMID:Cellular localization of an HIV-1 antigen in subacute AIDS encephalitis using an improved double-labeling immunohistochemical method. 169 70

A strategy for the purification and cleavage of chimeric recombinant proteins based on a genetically engineered metal-binding peptide and a human renin cleavage site is described. Vectors were constructed to direct the synthesis of chimeric human immunodeficiency virus (HIV) reverse transcriptase (RT) or beta-galactosidase in Escherichia coli. As shown below, two control chimerics without the metal-binding peptide were also included: 1. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 2. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 3. Pro-Ile-Pro-Phe-His-Leu-Val-Ile-His-Ser-HIV RT 4. Pro-Ile-Pro-Phe-His-Leu-Leu-Tyr-Tyr-Ser-HIV RT 5. Pro-Ile-His-Asp-His-Asp-His-Pro-Phe-His-Leu-beta-galactosidase Both N-terminal sequencing and an enzyme-linked immunosorbent assay utilizing antibodies to the metal-binding peptide were used to characterize the purified chimeric proteins. The relative RT activity of the chimeric protein was indistinguishable from the HIV-1 RT without the fusion sequence, indicating that the metal-binding and renin-cleavage sequences have no effect on the polymerase function of HIV-1 RT. The cleavage by recombinant human renin occurred at the expected site. A future paper will describe results on the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography.
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PMID:Expression and characterization of chimeric rDNA proteins engineered for purification and enzymatic cleavage. 172 60

This report describes the vaccination of rhesus macaques with peptides selected from regions of the simian immunodeficiency virus (SIV) envelope that are hydrophilic, immunoreactive, and highly homologous with corresponding conserved envelope sequences of the human immunodeficiency virus (HIV). The peptides, produced as beta-galactosidase fusion proteins, induced virus-neutralizing and peptide-specific antibodies. After challenge with virulent virus, controls became virus positive and developed gradually rising antibody titers to SIV over 63 weeks. Immunized macaques developed a postchallenge anamnestic response to SIVenv antigens within 3-6 weeks followed by a gradual, fluctuating decline in SIV antibody titers and partial or total suppression of detectable SIV. Virus suppression correlated with prechallenge neutralizing antibody titers. Although the average CD4+ cell count in the blood of immunized macaques remained constant, the control macaques exhibited a progressive decrease developing about week 55 after challenge. The conserved nature of the HIV and SIV peptides and the similar humoral immunoreactivity in the respective hosts suggest that homologous HIV peptides may be important components of a successful immunization strategy.
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PMID:Protection of macaques with a simian immunodeficiency virus envelope peptide vaccine based on conserved human immunodeficiency virus type 1 sequences. 187 Nov 25

UV irradiation has been shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in cell culture; however, only limited studies have been described in vivo. UV light has been categorized as UV-A (400 to 315 nm), -B (315 to 280 nm), or -C (less than 280 nm); the longer wavelengths are less harmful but more penetrative. Highly penetrative UV-A radiation constitutes the vast majority of UV sunlight reaching the earth's surface but is normally harmless. UV-B irradiation is more harmful but less prevalent than UV-A. In this report, the HIV-1 LTR-luciferase gene in the skin of transgenic mice was markedly activated when exposed to UV-B irradiation. The LTR in the skin of transgenic mice pretreated topically with a photosensitizing agent (psoralen) was also activated to similar levels when exposed to UV-A light. A 2-h exposure to sunlight activated the LTR in skin treated with psoralen, whereas the LTR in skin not treated with psoralen was activated after 7 h of sunlight exposure. The HIV-1 LTR-beta-galactosidase reporter gene was preferentially activated by UV-B irradiation in a small population of epidermal cells. The transgenic mouse models carrying HIV-1 LTR-luciferase and LTR-beta-galactosidase reporter genes have been used to demonstrate the in vivo UV-induced activation of the LTR and might be used to evaluate other environmental factors or pharmacologic substances that might potentially activate the HIV-1 LTR in vivo.
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PMID:In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice. 190 29

The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.
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PMID:Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization. 210 12

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm. This effect is mediated by a specific RNA stretch (rev-responsive element = RRE) localized within a 3'-terminal segment of the mRNA encoding virion proteins. We present evidence that rev expressed as a beta-galactosidase fusion protein in E. coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or beta-galactosidase specificity. In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting.
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PMID:A rev/beta-galactosidase fusion protein binds in vitro transcripts spanning the rev-responsive element of human immunodeficiency virus type 1 (HIV-1). 211 May 33


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