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)

Recent studies have demonstrated that sphingosine 1-phosphate (SPP) plays a functional role as a signaling molecule in gene expression in several kinds of cells. The present study demonstrates selective expression by ceramide of retinoic acid receptor-alpha (RAR-alpha) and retinoic X receptor-alpha (RXR-alpha) in osteoblastic MC3T3-E1 cells and a functional role of SPP-mediated AP-1 in the signaling mechanism of ligand-dependent transcriptional activity of heterodimers of these receptors in the cells. C(2)- and C(6)-ceramides selectively stimulated the expression of RAR-alpha and RXR-alpha genes, but not that of beta and gamma isoform genes of RAR and RXR, in the cells. The C(2)-ceramide-induced stimulation was clearly inhibited by dl-threo-dihydrosphingosine, an inhibitor of sphingosine kinase. SPP also selectively stimulated the expression of both receptors and increased the specific binding of the nuclear proteins to direct repeat 5 (DR-5), a consensus sequence of RAR-RXR. In addition, SPP markedly stimulated transient chloramphenicol acetyltransferase (CAT) activity of retinoic acid-dependent transcriptional activity in the cells transfected with a DR-5-CAT reporter gene. The SPP stimulation was activation protein-1 (AP-1)-dependent, because the SPP stimulatory action toward these nuclear gene expressions and the transient CAT activity were inhibited by antisense c-fos and c-jun oligonucleotides. We observed that SPP actually stimulated AP-1 transcriptional activity in the cells. This study suggests an important role of SPP-mediated AP-1 in the selective expression of RAR-alpha and RXR-alpha in osteoblastic cells via the sphingosine pathway.
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PMID:Selective stimulation by ceramide of the expression of the alpha isoform of retinoic acid and retinoid X receptors in osteoblastic cells. A role of sphingosine 1-phosphate-mediated AP-1 in the ligand-dependent transcriptional activity of these receptors. 1091 83

We have shown that the Escherichia coli phosphate-starvation-inducible psiE gene is regulated by both phosphate and the carbon source by using both lacZ and chloramphenicol acetyltransferase gene (cat) fusions. Yet, under all conditions tested, a single transcriptional start site lying 7 bp downstream of a predicted -10 region was revealed by primer extension analysis. DNase I footprinting showed that the PhoB transcriptional-activator protein protects two predicted pho boxes lying upstream of and near the -35 promoter region. Similar analysis showed that the cyclic AMP (cAMP)-cAMP receptor protein (cAMP-CRP) complex binds a region that overlaps with the downstream pho box. These results, together with measurements of the in vivo psiE promoter activity under various conditions, show that expression of the psiE gene is under direct positive and negative control by PhoB and cAMP-CRP, respectively.
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PMID:Dual transcriptional regulation of the Escherichia coli phosphate-starvation-inducible psiE gene of the phosphate regulon by PhoB and the cyclic AMP (cAMP)-cAMP receptor protein complex. 1098 67

Glucose-6-phosphatase (G6Pase) is a multicomponent system located in the endoplasmic reticulum comprising a catalytic subunit and transporters for glucose-6-phosphate, inorganic phosphate, and glucose. We have recently cloned a novel gene that encodes an islet-specific G6Pase catalytic subunit-related protein (IGRP) (Ebert et al., Diabetes 48:543-551, 1999). To begin to investigate the molecular basis for the islet-specific expression of the IGRP gene, a series of truncated IGRP-chloramphenicol acetyltransferase (CAT) fusion genes were transiently transfected into the islet-derived mouse betaTC-3 and hamster insulinoma tumor cell lines. In both cell lines, basal fusion gene expression decreased upon progressive deletion of the IGRP promoter sequence between -306 and -66, indicating that multiple promoter regions are required for maximal IGRP-CAT expression. The ligation-mediated polymerase chain reaction footprinting technique was then used to compare trans-acting factor binding to the IGRP promoter in situ in betaTC-3 cells, which express the endogenous IGRP gene, and adrenocortical Y1 cells, which do not. Multiple trans-acting factor binding sites were selectively identified in betaTC-3 cells that correlate with regions of the IGRP promoter identified as being required for basal IGRP-CAT fusion gene expression. The data suggest that hepatocyte nuclear factor 3 may be important for basal IGRP gene expression, as it is for glucagon, GLUT2, and Pdx-1 gene expression. In addition, binding sites for several trans-acting factors not previously associated with islet gene expression, as well as binding sites for potentially novel proteins, were identified.
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PMID:Characterization of the mouse islet-specific glucose-6-phosphatase catalytic subunit-related protein gene promoter by in situ footprinting: correlation with fusion gene expression in the islet-derived betaTC-3 and hamster insulinoma tumor cell lines. 1124 69

Transaldolase regulates redox-dependent apoptosis through controlling NADPH and ribose 5-phosphate production via the pentose phosphate pathway. The minimal promoter sufficient to drive chloramphenicol acetyltransferase reporter gene activity was mapped to nucleotides -49 to -1 relative to the transcription start site of the human transaldolase gene. DNase I footprinting with nuclear extracts of transaldolase-expressing cell lines unveiled protection of nucleotides -29 to -16. Electrophoretic mobility shift assays identified a single dominant DNA-protein complex that was abolished by consensus sequence for transcription factor ZNF143/76 or mutation of the ZNF76/143 motif within the transaldolase promoter. Mutation of an AP-2alpha recognition sequence, partially overlapping the ZNF143 motif, increased TAL-H promoter activity in HeLa cells, without significant impact on HepG2 cells, which do not express AP-2alpha. Cooperativity of ZNF143 with AP-2alpha was supported by supershift analysis of HeLa cells where AP-2 may act as cell type-specific repressor of TAL promoter activity. However, overexpression of full-length ZNF143, ZNF76, or dominant-negative DNA-binding domain of ZNF143 enhanced, maintained, or abolished transaldolase promoter activity, respectively, in HepG2 and HeLa cells, suggesting that ZNF143 initiates transcription from the transaldolase core promoter. ZNF143 overexpression also increased transaldolase enzyme activity. ZNF143 and transaldolase expression correlated in 21 different human tissues and were coordinately upregulated 14- and 34-fold, respectively, in lactating mammary glands compared with nonlactating ones. Chromatin immunoprecipitation studies confirm that ZNF143/73 associates with the transaldolase promoter in vivo. Thus, ZNF143 plays a key role in basal and tissue-specific expression of transaldolase and regulation of the metabolic network controlling cell survival and differentiation.
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PMID:ZNF143 mediates basal and tissue-specific expression of human transaldolase. 1470 49

We recently compared the regulation of glucose-6-phosphatase (G-6-Pase) catalytic subunit and glucose 6-phosphate (G-6-P) transporter gene expression by insulin in conscious dogs in vivo (Hornbuckle LA, Edgerton DS, Ayala JE, Svitek CA, Neal DW, Cardin S, Cherrington AD, and O'Brien RM. Am J Physiol Endocrinol Metab 281: E713-E725, 2001). In pancreatic-clamped, euglycemic conscious dogs, a 5-h period of hypoinsulinemia led to a marked increase in hepatic G-6-Pase catalytic subunit mRNA; however, G-6-P transporter mRNA was unchanged. Here, we demonstrate, again using pancreatic-clamped, conscious dogs, that glucagon is a candidate for the factor responsible for this selective induction. Thus glucagon stimulated G-6-Pase catalytic subunit but not G-6-P transporter gene expression in vivo. Furthermore, cAMP stimulated endogenous G-6-Pase catalytic subunit gene expression in HepG2 cells but had no effect on G-6-P transporter gene expression. The cAMP response element (CRE) that mediates this induction was identified through transient transfection of HepG2 cells with G-6-Pase catalytic subunit-chloramphenicol acetyltransferase fusion genes. Gel retardation assays demonstrate that this CRE binds several transcription factors including CRE-binding protein and CCAAT enhancer-binding protein.
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PMID:Selective stimulation of G-6-Pase catalytic subunit but not G-6-P transporter gene expression by glucagon in vivo and cAMP in situ. 1472 27

Cell-free translation systems generally utilize high-energy phosphate compounds to regenerate the adenosine triphosphate (ATP) necessary to drive protein synthesis. This hampers the widespread use and practical implementation of this technology in a batch format due to expensive reagent costs; the accumulation of inhibitory byproducts, such as phosphate; and pH change. To address these problems, a cell-free protein synthesis system has been engineered that is capable of using pyruvate as an energy source to produce high yields of protein. The "Cytomim" system, synthesizes chloramphenicol acetyltransferase (CAT) for up to 6 h in a batch reaction to yield 700 microg/mL of protein. By more closely replicating the physiological conditions of the cytoplasm of Escherichia coli, the Cytomim system provides a stable energy supply for protein expression without phosphate accumulation, pH change, exogenous enzyme addition, or the need for expensive high-energy phosphate compounds.
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PMID:Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. 1500 37

The antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin, sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing of this plasmid revealed that it is 120 592 bp in size and has a G+C content of 53.1 mol%. The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are almost identical to corresponding regions located on the F-plasmid and on R100. The three replicons encode replication initiation (rep) and replication control, multimer resolution (mrs), post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra) module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly mosaic 35 991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/sulfonamide-resistance module, remnants of the beta-lactam-resistance transposon Tn1, a macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402 integrating a class 1 integron and the Tn21-specific transposition module. A truncated version of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107 encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition siderophore system (iuc/iut); (ii) a putative high-affinity Fe(2+) uptake system which was previously identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating that pRSB107 was gradually assembled by integration of different horizontally acquired DNA segments via transposition or homologous recombination.
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PMID:The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions. 1581 78

GnRH peptide analogs are widely used to treat diverse clinical conditions. However, they have poor oral activity and exhibit rapid metabolic clearance, thus requiring injection and depot formulation. Because steroid hormones are bound to plasma proteins, we explored the possibility of conjugating hydroxylated progesterones to GnRH analogs to reduce metabolic clearance of the peptides. Conjugation of [D-Lys6]GnRH agonist to the alpha11-hydroxyl of alpha11-hydroxyl progesterone via a hemi-succinate bridge increased the plasma half-life after iv injection in rabbits by 3.6-fold while retaining high binding affinity, thus providing proof of concept. Five GnRH antagonists were then synthesized with 21-hydroxyprogesterone conjugated via C21-hydroxyl to positions six (conjugates A and B) and position seven (conjugates C and D) of GnRH antagonists. In the fifth compound the NH2 terminus of a GnRH antagonist lacking the first two amino acids was conjugated via the C21-hydroxyl to 21-hydroxyprogesterone (conjugate E). All five analogs bound to guinea pig progesterone binding globulin with relatively high affinities (264-1020 nM). Moreover, all five conjugates retained high progestogenic activity in stimulating a progesterone-response-element-driven chloramphenicol acetyltransferase reporter gene in the T47D breast cancer cell line. Conjugation via the epsilon-amino function of D-Lys6 (conjugates A and B) produced compounds with high binding affinity for the human GnRH receptor (15 and 7 nM) comparable to that of the unconjugated GnRH antagonists (4 and 26 nM). Conjugation via the epsilon-amino function of Lys7 (conjugates C and D) or the NH2 terminus of an N-terminally truncated antagonist (conjugate E) produced compounds of low binding affinity. Conjugates A and B also exhibited high functional antagonism of GnRH stimulation of inositol phosphate production in COS-7 cells expressing the human GnRH receptor (2.6 and 16 nM) compared with the unconjugated antagonists (1.3 and 122 nM). In accordance with their poor receptor binding affinity, conjugates C, D, and E had poor functional antagonism. Preliminary dose-finding studies in female marmosets showed transitory progesterone inhibition by 0.25 mg and prolonged suppression of 12 and 17 d by 0.5- and 1.0-mg doses. Injection of conjugate A in adult male marmosets (0.5 mg sc) rapidly suppressed plasma testosterone levels, which remained suppressed for at least 3 d. In contrast, the unconjugated parent antagonist alone or with progesterone suppressed testosterone for only 8 h to 1 d. The findings demonstrate that conjugation of progesterone to GnRH antagonists conveys plasma binding and progestogenic properties and increases their efficacy and duration of action in vivo. These new GnRH antagonists show promise as therapeutic agents for hormone-dependent diseases and as contraceptives.
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PMID:Bifunctional gonadotropin-releasing hormone antagonist-progesterone analogs with increased efficacy and duration of action. 1622 68

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

When a transient or stable transfection assay is developed for a promoter, a primary objective is to quantify promoter strength. Because transfection efficiency in such assays can be low, promoters are commonly fused to heterologous reporter genes that encode enzymes that can be quantified using highly sensitive assays. The reporter protein's activity or fluorescence within a transfected cell population is approximately proportional to the steady-state mRNA level. Although the Escherichia coli lacZ gene, encoding beta-galactosidase (beta-gal), can be used as a standard reporter for monitoring the strength of a promoter or enhancer in a transient or stable transfection assay, it is predominantly used as an internal control during transient transfection experiments. When used in this manner, cells are usually transfected with the control plasmid (containing a ubiquitously active viral promoter fused to the E. coli lacZ gene) and an experimental plasmid containing another reporter gene (e.g., luciferase or chloramphenicol acetyltransferase [CAT]) under the control of the promoter or enhancer of interest. The basic colorimetric assay described here is the simplest and least expensive assay for quantifying beta-gal activity. The cells are lysed and, after determining the total protein concentration in the extracts, an aliquot of the extract is mixed with the reaction substrate, O-nitrophenyl-beta-D-galactopyranoside (ONPG), in a buffer containing sodium phosphate and magnesium chloride. When the yellow product becomes visible, the optical densities of the samples are determined spectrophotometrically.
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PMID:Beta-galactosidase assay. 2043 10


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