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:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cellular adherence is important for monocyte migration and function and is known to induce monocyte activation, leading to the production of mRNA for several proto-oncogenes and cytokines. In addition, since cellular adherence has important intracellular signalling function, it has the potential to enhance human immunodeficiency virus (HIV) replication in monocytic cells. We have investigated the effects of adhesion of the monocytic cell line THP-1 transfected with HIV1 or HIV2 long terminal repeat chloramphenicol acetyltransferase (LTR CAT) constructs. These studies have shown that adherence to tissue culture plastic or confluent endothelial cells is essential for enhanced HIV LTR CAT expression in lipopolysaccharide-stimulated cells. In addition, we have investigated the effects of engagement of specific adhesion molecules, using immobilized antibodies, on HIV replication in the promonocytic cell line OM101, which contains a single latent proviral copy of HIV. Such studies have demonstrated that engagement of CD18, the beta subunit of the lymphocyte function-related antigen-1 (LFA-1) and major histocompatibility complex class II (MHC II) enhanced HIV replication. LFA-1 is involved in both monocyte-endothelial cell interactions and monocyte-T-cell interactions, and MHC II is involved in monocyte interaction with antigen-specific T cells. These data suggest that such interactions of membrane adhesion molecules with their appropriate ligand enhance HIV replication in vivo. Thus, this study has demonstrated that cellular adherence is a key regulatory factor of HIV replication in monocytic cells.
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PMID:Cellular adherence enhances HIV replication in monocytic cells. 780 Sep 38

In vitro, HIV-1 infection of human fetal glial cells initiates a noncytopathic, productive infection that results in a long-term persistence during which the viral genome remains latent. The cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin 1 beta (IL-1 beta) reactivate HIV-1 gene expression in these cells, leading to production of infectious virus. Here we show that treatment of human fetal glial cells with TNF-alpha and IL-1 beta increase expression of the reporter gene chloramphenicol acetyltransferase (CAT) when placed under the control of the HIV-1 5' LTR. We also show that treatment of human fetal glial cells with TNF-alpha leads to increased binding of the nuclear transcription factor NF-kappa B (p50/p65) to a consensus kappa B-binding site present in the HIV-1 5'LTR. Our results suggest that TNF-alpha stimulation of HIV-1 gene expression in primary cultures of human fetal glial cells is mediated by an increase in binding of NF-kappa B (p50/p65) to the HIV-1 LTR. This is the first report documenting NF-kappa B-binding activity in primary cultures of human fetal glial cells.
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PMID:Stimulation of HIV type 1 gene expression and induction of NF-kappa B (p50/p65)-binding activity in tumor necrosis factor alpha-treated human fetal glial cells. 784 78

Human cytomegalovirus (HCMV) has been implicated as a potential cofactor in human immunodeficiency virus type 1 (HIV-1)-related disease. Previously, we reported that HCMV inhibits HIV-1 RNA and protein synthesis in cells productively infected with both viruses but, in transient assays, activates an HIV-1 long terminal repeat-chloramphenicol acetyltransferase (LTR-CAT) construct introduced into the cell by transfection (V. Koval, C. Clark, M. Vaishnav, S. A. Spector, and D. H. Spector, J. Virol. 65:6969-6978, 1991). We show here that HCMV can also activate an infectious proviral HIV-1 genome transiently transfected into a cell. To ascertain whether integration of the HIV-1 provirus plays a role in these differential effects, we generated monoclonal and polyclonal cell lines that each contain a single integrated copy of an HIV-1 LTR-CAT construct and compared the regulatory effects of HCMV and HIV-1 infection in these cells with those occurring in the same type of cell transiently transfected with the HIV-1 LTR-CAT construct. We find that HCMV activates the transfected HIV-1 promoter 230-fold but activates the integrated promoter only 2.8- to 54-fold. In contrast, HIV-1 stimulates the integrated HIV-1 promoter 2,700- to 6,000-fold but stimulates the transfected promoter only 80-fold. Thus, the relative response of the HIV-1 promoter to HCMV and HIV-1 regulatory proteins depends upon whether it is integrated. To determine if HIV-1 gene products are necessary for the HCMV-mediated repression, we constructed cell lines containing two different stably integrated HIV-1 proviruses: one is tat- and nef-minus and transcriptionally inactive, while the other is env- and nef-minus but actively expresses the other HIV-1 gene products. Upon infection with HCMV, HIV-1 antigen production was stimulated from the inactive HIV-1 genome but inhibited from the active genome. We propose that HCMV has two separate effects on HIV-1 replication during a coinfection. One is a slight stimulatory effect which would be undetectable during an active HIV-1 infection, while the other is a net inhibitory effect that is mediated by an interaction between HCMV and HIV-1 gene products.
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PMID:Differential effects of human cytomegalovirus on integrated and unintegrated human immunodeficiency virus sequences. 785

Multifrequency phase fluorometry, in conjunction with site-directed mutagenesis, has allowed the determination of the fluorescence lifetimes of each of the three tryptophan residues of the type III variant of chloramphenicol acetyltransferase (CATIII). The mutant proteins retaining a single tryptophan yield lifetimes of 1.36, 2.00, and 1.17 ns for Trp-16, -86, and -152, respectively. Binding of chloramphenicol shortens the fluorescence lifetimes of all three tryptophans to some extent, in particular those of Trp-86 and Trp-152 (decreases of 51% and 39%, respectively). The mechanism of fluorescence quenching is believed to be radiationless energy transfer. Estimates of Trp-chloramphenicol distances by energy-transfer calculations are in good agreement with those determined from the crystal structure of CATIII. Despite binding at the same site in wild-type CATIII, CoA and ethyl-S-CoA produce different responses in global lifetime measurements--increases of 8% and 31%, respectively. Examination of each of the one-Trp CATIII variants, generated by site-directed mutagenesis, yields a variety of responses. Trp-152, located within the CoA binding site, responds to both CoA and its thioalkyl derivative with a 27-30% increase in fluorescence lifetime. Trp-16, distant from the CoA site, does not differentiate between the two ligands (7% increase in lifetime). However, Trp-86 shows a striking difference in binding responses, only a 4% decrease with CoA but a 14% reduction with ethyl-S-CoA. Each of the two-Trp CAT variants shows little change in global fluorescence lifetime on association with CoA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tryptophan fluorescence of chloramphenicol acetyltransferase: resolution of individual excited-state lifetimes by site-directed mutagenesis and multifrequency phase fluorometry. 789 46

Two distinct species of caprine arthritis encephalitis virus (CAEV) tat cDNAs were isolated early after infection of a Himalayan tahr cell line. Sequence analyses predicted that one cDNA (pCEV/e1) represented a polycistronic transcript that encodes Tat and Rev as well as an N-terminally truncated transmembrane protein and a protein, designated X, whose function is unknown; whereas the other cDNA (pCEV/f1) encodes Tat and the env gene products. pCEV/e1 trans-activated a CAEV LTR-chloramphenicol acetyltransferase reporter gene in goat synovial membrane cells. This activity was shown to be encoded by the Tat open reading frame by analysis of a deletion mutant. Because the pCAEV/f1 insert was unstable in plasmid form, its Tat activity could not be convincingly demonstrated. The target sequences for Tat within the CAEV LTR were localized to the U3 region which, when placed in either orientation upstream of heterologous promoters, was able to confer responsiveness to Tat.
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PMID:Characterization of cDNAs species encoding the Tat protein of caprine arthritis encephalitis virus. 794 54

We have previously isolated a HeLa cell cDNA encoding a 21-kDa polypeptide that is 48% similar to transcription factor IIS. To explore the possibility that p21 plays a role in transcriptional regulation in vivo, we tested the effect of p21 expression on the synthesis of reporter chloramphenicol acetyltransferase (CAT) in transfected COS-1 cells. CAT formation under control of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter was decreased nearly 20-fold in cells coexpressing p21. In contrast, CAT production under control of other sequence elements was only slightly reduced (human immunodeficiency virus type 1 LTR, simian virus 40 early promoter), unaffected (human heat shock protein of 70-kDa promoter, adenovirus major late promoter TATA box), or increased (terminal deoxynucleotidyltransferase initiator element, c-fos promoter) by p21 coexpression as compared to cells cotransfected with the parental vector. The abundance of steady-state CAT transcripts from RSV LTR was also decreased by p21 expression in a dose-dependent manner, suggesting that transcription of RSV LTR/CAT is under negative control by p21. Consistent with an effect on transcription, p21 was localized in nuclei of transfected cells. Deletion analysis of p21 indicated that the sequences essential for inhibition of RSV LTR function include the previously identified ARg/Ser-rich region and zinc finger-like motif. Proliferation of chicken embryo fibroblasts transfected with an infectious molecular clone of RSV was diminished by p21 expression, which also resulted in fewer transformed foci.
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PMID:Down-regulation of Rous sarcoma virus long terminal repeat promoter activity by a HeLa cell basic protein. 797 97

Interference with T cell activation signals by Human immunodeficiency virus (HIV) gene products is suggested to contribute to the impairment of immune functions observed in AIDS. Interleukin-2 (IL-2) and HIV share common stimulatory signals triggered during T cell activation. The role of HIV tat, which is the main enhancing factor for viral LTR, in the regulation of IL-2 gene transcription has been studied following transient expression of the tat gene in phorbol ester and calcium ionophore-activated Jurkat cells transfected with IL-2 promoter-chloramphenicol acetyltransferase reporter constructs. We observed that tat increased the IL-2 promoter transcriptional activity in response to phorbol ester and ionomycin. This tat-dependent synergism mapped to the (-279 to -263 bp) NFAT motif of the IL-2 enhancer, which was sufficient to be transactivated by tat. Our data suggest that tat links T cell activating signals to the shared IL-2 and HIV regulation. This may play a role in the early phase of HIV infection.
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PMID:Human immunodeficiency virus type-1 tat enhances interleukin-2 promoter activity through synergism with phorbol ester and calcium-mediated activation of the NF-AT cis-regulatory motif. 799 66

Human immunodeficiency virus 1 (HIV1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin 6 (IL-6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL-6 secretion of HIV1-infected cells may include transactivation of the IL-6 gene by HIV1. To test this hypothesis, we used the pIL6Pr-chloramphenicol acetyltransferase (CAT) plasmid, an IL-6 promoter-CAT construct, as a target of the transactivating function of the HIV1 TAT protein. By cotransfecting the pIL6Pr-CAT and the tat-expressing pSVT8 plasmid in MC3 B-lymphoblastoid or in HeLa epithelial cells, we observed that TAT transactivates the human IL-6 promoter. These results were confirmed when pIL6Pr-CAT was transfected in MC3 or HeLa cells that constitutively expressed the tat gene in a sense (pSVT8 cells) or antisense (pSVT10 cells) orientation. 5' deletion plasmids of pIL6Pr-CAT, in which regions at -658, -287, and -172 were inserted 5' to the cat gene, were transiently transfected in pSVT10 and pSVT8 cells and showed that TAT-induced activation of the IL-6 promoter required a minimal region located between -287 and -54 bp. Moreover, experiments with plasmids carrying the -658, -287, and -172 bp regions of the IL-6 promoter inserted downstream to a TAR-deleted HIV1-LTR identified the sequence of -172 to -54 as the minimal region of the IL-6 promoter required for TAT to transactivate the TAR-deleted HIV1-LTR. By DNA-protein binding experiments, tat-transfected cells expressed a consistent increase in kappa B and nuclear factor (NF)-IL-6 binding activity. Accordingly, the pDRCAT and IL-1REK9CAT, carrying tandem repeats of NF-kappa B or NF-IL6 binding motifs, respectively, were activated in TAT-expressing cells. The biological relevance of the TAT-induced IL-6 secretion was addressed by generating 7TD1 cells, an IL-6-dependent mouse cell line, stably expressing the tat gene. These tat-positive cells expressed the endogenous IL-6 gene, secreted high amounts of murine IL-6, and grew efficiently in the absence of exogenous IL-6. Moreover, the tat-positive 7TD1 cells sustained the growth of parental 7TD1 cells and showed a dramatic increase in their tumorigenic potency. These results suggest that TAT protein may play a role in the pathogenesis of some HIV1-associated diseases by modulating the expression of host cellular genes.
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PMID:The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 TAT protein. 811 88

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus. Sequence analysis of the CAEV genome revealed the presence of a small open reading frame (ORF) which shares amino acid identity with the visna virus tat gene. Using an infectious molecular clone of CAEV the role of the tat ORF in viral replication was examined. Mutations were made in the tat ORF that introduced two in frame stop codons six amino acids downstream of the tat AUG; in addition, a deletion mutant was made that removed most of the tat ORF. Both of these mutants had greatly reduced virus titers (> 1000-fold less than the wild type infectious clone). Co-transfection of a tat expressing plasmid with these viruses containing the tat ORF mutations resulted in higher levels of virus production demonstrating that the effects of both mutants are tat specific. These mutants provide data that the CAEV tat gene is necessary for efficient virus replication. Analysis of the RNA in these transfected cells showed that complementation of the tat gene was in trans and not the result of recombination. Analysis of the gag and rev proteins in the transfected cells demonstrated that these proteins were not detectable in cells transfected with the tat mutants but could be readily detected when the mutations were complemented in trans with a tat expression vector. To test for tat mediated trans-activation a plasmid expressing the CAEV tat ORF was co-transfected with plasmids containing either the CAEV or visna virus LTR driving transcription of the bacterial chloramphenicol acetyltransferase gene (CAT). These experiments indicate that one function of the CAEV tat protein is to trans-activate gene expression from the viral promoter. RNase protection analysis of CAT mRNA from co-transfected cells demonstrated that CAEV Tat trans-activates gene expression by increasing steady-state levels of mRNA.
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PMID:The CAEV tat gene trans-activates the viral LTR and is necessary for efficient viral replication. 821 71

BTEB, a GC box-binding transcription factor, was tested for its ability to activate the human immunodeficiency virus type 1 long terminal repeat (HIV-1 LTR). An electrophoretic mobility shift assay demonstrated specific binding of BTEB to GC boxes of the HIV-1 LTR. When a BTEB expression vector was cotransfected into A3.01 cells with a fusion gene of HIV-1 LTR and chloramphenicol acetyltransferase (CAT) structural gene, the CAT activity was increased. This increase was accompanied by an increase in the content of CAT mRNA. Transcriptional activity of the HIV-1 LTR, stimulated by Tat, was further enhanced by the expression of BTEB. BTEB also activated the LTR activity in cooperation with phorbol 12-myristate 13-acetate. Northern blot analysis showed that various T cell and macrophage/monocyte cell lines expressed the BTEB mRNA to a level comparable with that of Sp1, another GC box-binding transcription factor. These results suggest that BTEB, like Sp1, is involved in transcriptional activation of the HIV-1 LTR.
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PMID:Activation of the human immunodeficiency virus type 1 long terminal repeat by BTEB, a GC box-binding transcription factor. 825 32


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