Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

HIV-associated dementia results from neuronal loss and an alteration of neuronal function due to a loss of synapses. While HIV infection in astrocytes is limited, astrocytes exhibit a chronic nonproductive infection that can lead to the release of neurotoxic proteins. Additionally, infection can disrupt the normal neurotrophic role of astrocytes that results in neuronal death. Gonadal steroid hormones are known to act as trophic and protective factors in the brain under a variety of normal and pathological conditions. In the present study, to determine if estrogen plays a role in the ability of Tat to function as a transcriptional activator within astrocytes, we examined the effect of estrogen on regulation of viral transcription. We utilized an immortalized human astrocyte cell line (SVGA) stably transfected with a reporter plasmid containing the HIV-1IIIB LTR driving the chloramphenicol acetyltransferase (CAT) gene. The amount of transcriptional activity was measured by quantifying the amount of CAT produced. We determined that 17beta-estradiol treatment (1 nM) had no effect on basal LTR activity. Following transfection with a Tat-expressing plasmid, there was a 100-fold increase in CAT production. This induction was reduced by 40% in cells pretreated with 17beta-estradiol. 17beta- Estradiol only suppressed transcription stimulated by Tat. Furthermore, we determined that this effect was specific to 17beta-estradiol and estrogen receptor agonists. This activity was limited to astrocytes as no effect was observed in a monocytic cell line. Finally, the mechanism of action did not involve an alteration in levels of Cdk9 or Cyclin T1 proteins necessary for Tat activation of the HIV-1 LTR. This study demonstrates a novel activity of 17beta-estradiol in glial cells that could play a role in the maintenance of neuronal health during HIV infection of the central nervous system.
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PMID:Estradiol negatively regulates HIV-LTR promoter activity in glial cells. 1662 39

It has been suggested that the common (betaalpha)(8)-barrel enzyme fold has evolved by the duplication and fusion of identical (betaalpha)(4)-half barrels, followed by the optimisation of their interface. In our attempts to reconstruct these events in vitro we have previously linked in tandem two copies of the C-terminal half barrel HisF-C of imidazole glycerol phosphate synthase from Thermotoga maritima and subsequently reconstituted in the fusion construct HisF-CC a salt bridge cluster present in wild-type HisF. The resulting recombinant protein HisF-C*C, which was produced in an insoluble form and unfolded with low cooperativity at moderate urea concentrations has now been stabilised and solubilised by a combination of random mutagenesis and selection in vivo. For this purpose, Escherichia coli cells were transformed with a plasmid-based gene library encoding HisF-C*C variants fused to chloramphenicol acetyltransferase (CAT). Stable and soluble variants were identified by the survival of host cells on solid medium containing high concentrations of the antibiotic. The selected HisF-C*C proteins, which were characterised in vitro in the absence of CAT, contained eight different amino acid substitutions. One of the exchanges (Y143C) stabilised HisF-C*C by the formation of an intermolecular disulfide bond. Three of the substitutions (G245R, V248M, L250Q) were located in the long loop connecting the two HisF-C copies, whose subsequent truncation from 13 to 5 residues yielded the stabilised variant HisF-C*C Delta. From the remaining substitutions, Y143H and V234M were most beneficial, and molecular dynamics simulations suggest that they strengthen the interactions between the half barrels by establishing a hydrogen-bonding network and an extensive hydrophobic cluster, respectively. By combining the loop deletion of HisF-C*C Delta with the Y143H and V234M substitutions, the variant HisF-C**C was generated. Recombinant HisF-C**C is produced in soluble form, forms a pure monomer with its tryptophan residues shielded from solvent and unfolds with similar cooperativity as HisF. Our results show that, starting from two identical and fused half barrels, few amino acid exchanges are sufficient to generate a highly stable and compact (betaalpha)(8)-barrel protein with wild-type like structural properties.
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PMID:Stabilisation of a (betaalpha)8-barrel protein designed from identical half barrels. 1763 94

This unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV-1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin-layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV-1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or "down-regulate") HIV transcription.
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PMID:Regulation of the HIV promoter/enhancer. 1843 98


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