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)

Ferritin is the major intracellular iron-storage protein in eucaryotic cells and plays a prominent role in maintaining intracellular iron homeostasis. We observed that transfection of NIH-3T3 mouse fibroblasts with the adenovirus E1A oncogene specifically repressed the mRNA for one of the subunits of ferritin, ferritin H. This occurred in the absence of any effect of E1A on the mRNA for the L subunit of ferritin. The repression of ferritin H was not a general feature of oncogene expression since transfection of NIH-3T3 cells with H-ras did not affect ferritin composition. Deletion of the conserved regions of E1A responsible for immortalization and transcriptional repression impaired the ability of E1A to repress ferritin H. Immunoprecipitation of ferritin in E1A transfectants demonstrated that the decrease in the ferritin H/L ratio observed at the mRNA level was also exhibited at the protein level. The E1A-dependent inhibition of ferritin H was also observed in a chimeric gene containing the ferritin H promoter ligated to the chloramphenicol acetyltransferase reporter gene, but was not observed in control genes in which chloramphenicol acetyltransferase activity was dependent on promoters derived from SV40 or the interleukin-3 gene. This suggests that E1A may repress ferritin H at the transcriptional level. These results demonstrate that the adenovirus E1A oncogene specifically modulates ferritin H expression. They also suggest that alterations in cellular iron metabolism may be among the diverse array of cellular responses induced by E1A.
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PMID:Preferential repression of the H subunit of ferritin by adenovirus E1A in NIH-3T3 mouse fibroblasts. 846 62

The locus glc (min 64.5), associated with the glycolate utilization trait in Escherichia coli, is known to contain glcB, encoding malate synthase G, and the gene(s) needed for glycolate oxidase activity. Subcloning, sequencing, insertion mutagenesis, and expression studies showed five additional genes: glcC and in the other direction glcD, glcE, glcF, and glcG followed by glcB. The gene glcC may encode the glc regulator protein. Consistently a chloramphenicol acetyltransferase insertion mutation abolished both glycolate oxidase and malate synthase G activities. The proteins encoded from glcD and glcE displayed similarity to several flavoenzymes, the one from glcF was found to be similar to iron-sulfur proteins, and that from glcG had no significant similarity to any group of proteins. The insertional mutation by a chloramphenicol acetyltransferase cassette in either glcD, glcE, or glcF abolished glycolate oxidase activity, indicating that presumably these proteins are subunits of this enzyme. No effect on glycolate metabolism was detected by insertional mutation in glcG. Northern (RNA) blot experiments showed constitutive expression of glcC but induced expression for the structural genes and provided no evidence for a single polycistronic transcript.
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PMID:glc locus of Escherichia coli: characterization of genes encoding the subunits of glycolate oxidase and the glc regulator protein. 860 83

The cytoplasmic iron regulatory protein (IRP) modulates iron homeostasis by binding to iron-responsive elements (IREs) in the transferrin receptor and ferritin mRNAs to coordinately regulate transferrin receptor mRNA stability and ferritin mRNA translational efficiency, respectively. These studies demonstrate that thyroid hormone (T3) can modulate the binding activity of the IRP to an IRE in vitro and in vivo. T3 augmented an iron-induced reduction in IRP binding activity to a ferritin IRE in RNA electrophoretic mobility shift assays using cytoplasmic extracts from human liver hepatoma (HepG2) cells. Hepatic IRP binding to the ferritin IRE also diminished after in vivo administration of T3 with iron to rats. In transient transfection studies using HepG2 cells and a human ferritin IRE-chloramphenicol acetyltransferase (H-IRE-CAT) construct, T3 augmented an iron-induced increase in CAT activity by approximately 45%. RNase protection analysis showed that this increase in CAT activity was not due to a change in the steady state level of CAT mRNA. Nuclear T3-receptors may be necessary for this T3-induced response, because the effect could not be reproduced by the addition of T3 directly to cytoplasmic extracts and was absent in CV-1 cells which lack T3-receptors. We conclude that T3 can functionally regulate the IRE binding activity of the IRP. These observations provide evidence of a novel mechanism for T3 to up-regulate hepatic ferritin expression, which may in part contribute to the elevated serum ferritin levels seen in hyperthyroidism.
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PMID:Thyroid hormone modulates the interaction between iron regulatory proteins and the ferritin mRNA iron-responsive element. 866 26

A homologue of the 'ferric uptake regulation' gene (fur) was isolated from the cyanobacterium Synechococcus sp. strain PCC 7942 by an Escherichia coli-based 'in vivo repression assay'. The assay uses a reporter-gene construct containing the promoter region of the iron-regulated cyanobacterial gene isiA, fused to the coding region for chloramphenicol acetyltransferase. The isolated gene codes for a protein that has 41% sequence similarity (36% identity) to Fur from E. coli and contains the putative iron-binding motif found in the Fur proteins of purple bacteria. No significant similarity was found to the DxtR repressor that regulates the expression of toxin and siderophore production in Gram-positive bacteria. Insertional mutagenesis of the cloned cyanobacterial fur gene led to the creation of heteroallelic mutants that showed iron-deficiency symptoms in iron-replete medium, including the constitutive production of flavodoxin and of hydroxamate siderophores. Failure to eliminate wild-type copies of the fur gene from the polyploid genome of Synechococcus 7942 implies that in this cyanobacterium Fur may have essential functions in addition to the regulation of genes involved in iron scavenging or photosynthetic electron transport.
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PMID:Fur regulates the expression of iron-stress genes in the cyanobacterium Synechococcus sp. strain PCC 7942. 870 86

The gene encoding Neisseria gonorrhoeae periplasmic binding protein FbpA contains two regions whose sequences exhibit homology with the Escherichia coli ferric uptake regulator protein (Fur) consensus binding sequence. In this study, DNase I footprinting experiments were employed to characterize the operator sequences within the fbpA promoter region to which E. coli Fur binds. A 160-bp fragment encompassing the promotor region and the putative iron boxes of the fbpA promoter was incubated with Fur, DNaseI was added, and the products of these reactions were sequenced to identify nucleotide peaks that were protected. At 50 nM Fur, a protected region that spanned 33 bp and extended 19 bp upstream and 8 bp downstream of the -35 region of the fbpA promoter was observed. At higher concentrations of Fur (75 and 100 nM), an extension of this protected region upstream of the -35 region was observed. Introduction of a plasmid carrying an fbpA-cat transcriptional fusion in E. coli H1717 (Fur+) resulted in an 88% induction of chloramphenicol acetyltransferase expression under conditions of iron restriction; however, chloramphenicol acetyltransferase expression was not responsive to iron in E. coli H1745 (Fur-), indicating that transcriptional regulation of fbpA in response to iron occurs via the negative regulator Fur. The extent of the fbpA operator sequence (42 bp), as defined by our footprinting analysis, would suggest the binding of two Fur repressor dimers.
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PMID:Analysis of Fur binding to operator sequences within the Neisseria gonorrhoeae fbpA promoter. 875 70

One of the primary endocrine hormones that influence the onset of Sertoli cell differentiation at puberty and help maintain differentiation in the adult testis is FSH. FSH can modulate the majority of Sertoli cell differentiated functions, including stimulation of the iron-binding protein transferrin. Previous studies have shown that FSH alters the levels of cAMP and the immediate early gene c-fos. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation by examining the actions of FSH on the promoter of the immediate early gene c-fos and the promoter of the downstream differentiated function gene transferrin. The regulation of c-fos by FSH was investigated with various chloramphenicol acetyltransferase (CAT) constructs containing segments of the c-fos promoter, such as the serum response element (SRE), cAMP response element (CRE), and AP1/phorbol ester/TPA response element (TRE), that were transfected into cultured Sertoli cells. Observations indicate that FSH can stimulate all three response elements, as well as a whole c-fos promoter construct. Interestingly, FSH was found to have a more dramatic effect on the SRE-CAT than a cAMP analog, suggesting a difference in the actions of the two agents. Gel mobility shift assays were performed to confirm the reporter gene results. Nuclear extracts of FSH-stimulated Sertoli cells caused a labeled AP1 oligonucleotide to form a DNA/protein complex (i.e., gel shift), indicating activation of the c-fos gene and binding of the c-fos/jun complex. Nuclear extracts from both FSH- and cAMP-stimulated Sertoli cells promoted similar gel shifts with SRE and CRE oligonucleotides. This observation supports the reporter gene data in indicating that FSH can influence both the SRE and CRE. A gel mobility shift assay was also performed with an oligonucleotide containing the 5'-flanking ETS domain of the SRE (ETS-SRE) that allows the formation of a ternary complex. FSH-stimulated Sertoli cell nuclear extracts were found to promote a unique ETS-SRE gel shift not present in cAMP-stimulated cells. The observations imply that FSH actions on the SRE are in part distinct from the actions of cAMP. Transferrin gene expression was examined to study the downstream regulation of Sertoli cell differentiation. CAT constructs containing deletion mutants of a 3-kb mouse transferrin promoter were used. When transfected into Sertoli cells, the 581-bp transferrin minimal promoter, previously shown to contain a CRE, had a significant response to cAMP and FSH. The 1.6-, 2.6-, and 3-kg transferrin promoter constructs also responded to FSH and cAMP to the same extent as, or to a lesser extent than, the 581-bp minimal promoter. Interestingly, the actions of FSH on the 581-bp minimal transferrin promoter were more dramatic than those of cAMP. The importance of FSH-induced c-fos in the regulation of transferrin expression was demonstrated in the current study when a c-fos antisense oligonucleotide was found to partially inhibit (50%) the ability of FSH to induce the expression of a transferrin promoter (CAT) construct. Therefore, FSH appears to act through multiple transcriptional activation pathways. The first involves cAMP and the CRE at both early-event genes (e.g., c-fos) and downstream genes (e.g., transferrin). It is likely that other pathways involve alternate signal transduction events (e.g., calcium mobilization) and promoter response elements (e.g., SRE). These multiple pathways may act in a compensatory manner to assure the ability of FSH to influence Sertoli cell differentiation and/or in a synergistic manner to amplify FSH actions.
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PMID:Transcriptional regulation of sertoli cell differentiation by follicle-stimulating hormone at the level of the c-fos and transferrin promoters. 883 93

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P450 play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and beta-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of beta-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism.
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PMID:The other kind of biological NMR--studies of enzyme-substrate interactions. 889 75

Sertoli cells are critical for testicular function and maintenance of the spermatogenic process. The induction of Sertoli cell differentiation in the embryo promotes testicular development and male sex determination. The progression of Sertoli cell differentiation during puberty promotes the onset of spermatogenesis. The maintenance of optimal Sertoli cell differentiation in the adult is required for spermatogenesis to proceed. The current study was designed to investigate the transcriptional regulation of Sertoli cell differentiation through the analysis of a previously identified marker of differentiation, transferrin gene expression. Sertoli cells produce transferrin to transport iron to developing spermatogenic cells sequestered within the blood-testis barrier. The transferrin promoter was characterized and found to contain two critical response elements, designated Sertoli element 1 (SE1) and Sertoli element 2 (SE2). Through sequence analysis, SE2 was found to contain an E-box response element, which has been shown to respond to basic-helix-loop-helix (bHLH) transcription factors. The bHLH proteins are a class of transcription factors associated with the induction and progression of cell differentiation. bHLH proteins dimerize through the conserved helix-loop-helix region and bind DNA through the basic region. Nuclear extracts from Sertoli cells were found to cause an E-box gel shift when the cells were stimulated to differentiate in culture, but not under basal conditions. The SE2 gel shift of Sertoli nuclear extracts was competed with excess unlabeled SE2 or E-box DNA fragments. Several Sertoli nuclear proteins associate with the SE2 gel shifts, including 70-, 42-, and 25-kDa proteins. Therefore, the critical SE2 element in the transferrin promoter is an E-box element capable of binding bHLH transcription factors. The ubiquitously expressed E12 bHLH protein dimerizes with numerous cell-specific bHLH factors. A Western blot analysis demonstrated that E12 was present in Sertoli cell nuclear extracts and associated with the SE2 gel shift. A ligand blot of Sertoli cell nuclear extracts with radiolabeled E12 had apparent bHLH proteins when the cells were stimulated to differentiate. The E-box sequence in the SE2 fragment of the transferrin promoter was CATCTG and was similar in gel shifts to the consensus E-box elements (CANNTG) previously characterized. A bHLH inhibitory factor (Id) competed and inhibited formation of the Sertoli cell nuclear extract E-box gel shift. To extend this observation, Id protein was overexpressed in cultured Sertoli cells. A transferrin promoter chloramphenicol acetyltransferase construct was used to monitor Sertoli cell function. The presence of Id suppressed the activation of the promoter induced by Sertoli differentiation factors. Therefore, the inhibition of Sertoli bHLH factors by Id suppressed Sertoli cell differentiated function, as measured by transferrin expression. An E-box-chloramphenicol acetyltransferase construct was also found to be active in Sertoli cells when cells were induced to differentiate. Screening the computerized nucleotide data bases demonstrated that putative E-box response elements are present in the promoters of a large number of Sertoli cell differentiated genes. In summary, a critical E-box response element has been identified in the transferrin promoter that can be activated by bHLH factors (e.g. E12) present in Sertoli cells. Inhibition of Sertoli bHLH factors by Id suppresses Sertoli cell differentiated function (i.e. transferrin expression), suggesting that bHLH transcription factors may be important in regulating Sertoli cell differentiated functions.
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PMID:Role of basic-helix-loop-helix transcription factors in Sertoli cell differentiation: identification of an E-box response element in the transferrin promoter. 900 1

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.
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PMID:A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide. 907 50

The control of the expression of the pilin gene (pilE) in Neisseria gonorrhoeae under a wide variety of growth conditions has been studied. The expression of pilE was measured using transcriptional fusions between pilE and the gene encoding chloramphenicol acetyltransferase (CAT), and the level of pilin production was measured by Western blot analysis. Many of the conditions tested affected both growth rate and pilin gene expression (e.g. isoleucine, high osmolarity, high temperature, anaerobic growth, pH 6, urea and iron depletion). Changes in the level of many other proteins were also observed, depending on the conditions, indicating that gonococci undergo an adaptive response to environmental variations. Moreover, environment-induced changes in the level of many proteins, including pilin, seem to involve the pilA/pilB regulatory system, which has been previously proposed to modulate the expression of the gonococcal pilin gene.
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PMID:Control of Neisseria gonorrhoeae pilin gene expression by environmental factors: involvement of the pilA/pilB regulatory genes. 916 25


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