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)

Primary murine keratinocytes can be maintained in culture for extended periods in a proliferative, basal cell state under conditions of reduced extracellular Ca2+. In response to increased Ca2+ concentrations, the cells undergo a well-defined program of terminal differentiation, thus serving as a convenient model in which to study the genes involved in regulating this and possibly other differentiation cascades by DNA-mediated gene transfer. However, because of their sensitivity to increased Ca2+ concentrations, the introduction of exogenous genomic DNA into primary keratinocytes by conventional methods is problematic. We have optimized the calcium phosphate DNA transfection procedure by introducing conditions that reduce the potency of Ca2+ as a differentiation signal. Primary epidermal cells were transfected with pSV2CAT, a plasmid that codes for the enzyme chloramphenicol acetyltransferase CAT. Enzyme activity was measured in cell extracts under varying transfection conditions. When the K+ concentration of the medium used for transfection by calcium phosphate precipitation is reduced from 6.5 to 0.01 mM, CAT activity following transfection increases 2-3 times. Exposure to the DNA precipitate for 2-4 h is optimal. By the use of fibroblast conditioned medium following transfection, enzyme activity can be detected in cell extracts for at least 21 d, suggesting that the exogenous gene is integrated. The low K+/Ca2+ transfection method is more effective than SrCl2 used as an alternative for CaCl2 in Ca2+ sensitive cells. Low K+ medium enhances cell survival for Ca2+ mediated transfection but also appears to have a beneficial effect on DNA uptake or expression.
J Invest Dermatol 1988 Aug
PMID:Expression of transfected DNA by primary murine keratinocytes. 245 57

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.
J Invest Dermatol 1989 Feb
PMID:Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies. 246 54

The biologic activities of retinoic acid and 3,4-didehydroretinoic acid, two endogenous vitamin A derivatives in various tissues, were compared to their affinities for the nuclear retinoic acid receptors and their ability to induce transcriptional activation. Both retinoids were equipotent inducers of differentiation of F9 teratocarcinoma cells. In a morphologic assay, using reconstructed skin, retinoic acid and 3,4-didehydroretinoic acid inhibited keratinization at a concentration of 100 nM. In cultured keratinocytes, a 50% inhibition of the production of the keratinocyte transglutaminase enzyme was achieved with about 20 nM for both retinoids. The in vitro binding to the nuclear retinoic acid receptors alpha, beta, and gamma showed that retinoic acid and 3,4-didehydroretinoic acid had almost equal affinities for the receptors with Kds ranging from 3 to 47 nM. The transcriptional activation resulting from the addition of the two retinoids to cells co-transfected with alpha, beta, or gamma retinoic acid receptor expression vectors and a retinoic acid responsive element linked to the chloramphenicol acetyltransferase reporter gene was similar. Finally, it was demonstrated that retinoic acid did not metabolize to 3,4-didehydroretinoic acid, and a slow conversion of 3,4-didehydroretinoic acid into retinoic acid was not sufficient to explain the biologic effects produced by the former compound. In conclusion, the present study demonstrates that retinoic acid and 3,4-didehydroretinoic acid have the same activity in several different test systems, but their metabolism differs depending on the cell type used.
J Invest Dermatol 1994 Jan
PMID:Biologic activities of retinoic acid and 3,4-didehydroretinoic acid in human keratinocytes are similar and correlate with receptor affinities and transactivation properties. 750 53

The major alteration in photoaged skin is the deposition of massive amounts of abnormal elastic material, termed solar elastosis. In previous work, it has been shown that solar elastosis is accompanied by increased abundance of elastin and fibrillin mRNAs and upregulation of elastin promoter activity. Using a transgenic mouse line, which expresses the human elastin promoter, linked to a chloramphenicol acetyltransferase reporter gene, in a tissue-specific and developmentally regulated manner, we investigated the effects of ultraviolet A radiation and ultraviolet B radiation on human elastin promoter activity in vivo and in vitro. Irradiation of mice with a single dose of ultraviolet B radiation (491.4 mJ/cm2) resulted in an increase up to 8.5-fold in promoter activity, whereas a more modest increase of 1.8-fold was measured with ultraviolet A radiation (38.2 J/cm2). In addition, in vitro studies revealed over a thirtyfold increase in elastin promoter activity in response to ultraviolet B radiation (5.5 mJ/cm2), whereas no change was measured in response to ultraviolet A radiation (2.2 J/cm2). These results confirm the role of ultraviolet B radiation in elastin promoter activation in photoaging, and identify ultraviolet A radiation as a contributing factor. This system should serve as a useful in vivo and in vitro model to study cutaneous photoaging, and for testing compounds that may protect against cutaneous photodamage.
J Invest Dermatol 1995 Aug
PMID:Ultraviolet radiation activates the human elastin promoter in transgenic mice: a novel in vivo and in vitro model of cutaneous photoaging. 763 12

Cutaneous aging consists of chronologic aging as well as actinic damage, referred to as photoaging. Most of the morphologic changes associated with an aged appearance result from actinic damage to the skin. The morphologic changes in sun-damaged skin are associated with accumulation of material having the staining characteristics of elastin, known as solar elastosis, in the superficial dermis. Previous studies have demonstrated the presence of elastin within areas of solar elastosis; however, little is known about the mechanisms leading to elastin accumulation in photoaged skin. In addition, fibrillin, the fibrillar component of elastic fibers, has been found in small amounts in solar elastosis. In this study we demonstrate increased elastin mRNA levels in photoaged skin, as well as increased elastin and fibrillin mRNAs in skin explant-derived fibroblasts using Northern hybridizations, compared with controls from sun-protected sites of the same individual. Increased elastin mRNA levels result from transcriptional upregulation of the gene, as demonstrated by transient transfections with a human elastin promoter/chloramphenicol acetyltransferase construct. Elevated mRNA levels were also correlated with increased elastin and fibrillin deposition in paired biopsy specimens from photodamaged and non-sun-exposed skin, as demonstrated by immunohistochemical staining. Thus, approaches to counteract transcriptional activation of elastin gene expression may be useful in preventing the changes associated with cutaneous photoaging.
J Invest Dermatol 1994 Aug
PMID:Enhanced elastin and fibrillin gene expression in chronically photodamaged skin. 804 Jun 8

We evaluated SR11237, a retinoid X receptor (RXR)-specific compound, for its pharmacologic effects on cell differentiation in F9 embryonal carcinoma cells and rhino mouse epidermis. SR11237 can cause RXR/RXR homodimers to form and transactivate a reporter gene containing a RXR-response element. We confirmed, using nuclear receptor co-transfection assays in COS-1 cells, that SR11237 is effective at transactivating a chloramphenicol acetyltransferase reporter gene through RXRs but not retinoic acid receptors. When SR11237 was tested for its ability to modulate cell differentiation, it was inactive on F9 embryonal carcinoma cells and rhino mouse skin. Because differentiation in these systems is known to be regulated by RAR-specific compounds, such as all-trans-retinoic acid and (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-prope nyl benzoic acid], our results with SR11237 are compatible with the concept that classical retinoid pleiotropic responses are mediated by RXR/RAR heterodimeric nuclear receptors rather than through RXR/RXR homodimers.
J Invest Dermatol 1994 May
PMID:A pleiotropic response is induced in F9 embryonal carcinoma cells and rhino mouse skin by All-trans-retinoic acid, a RAR agonist but not by SR11237, a RXR-selective agonist. 817 47

Tyrosinase is the principal enzyme in the biosynthesis of melanin. The expression of tyrosinase is tissue-specific and appears to be regulated by various hormonal and environmental factors. Elucidation of the genomic structure and molecular basis of control of tyrosinase gene expression will greatly enhance our understanding of the regulation of human pigmentation. To this end, we have isolated and performed restriction mapping of recombinant cosmid and lambda phage clones containing the human tyrosinase gene, sequenced a 2.2-kilobase (kb) region of its promoter, and determined the potential regions regulating the tyrosinase gene expression in transient-expression system. The human tyrosinase gene is comprised of five exons and four introns. Based on our restriction mapping studies, the gene spans a distance of over 65-kb on chromosome 11 (q14-->q21). We constructed a series of plasmids (pHTY-CAT) that contain 5' sequential deletions of the human tyrosinase 5' flanking sequence fused to the reporter gene, chloramphenicol acetyltransferase (CAT). The plasmids were used to locate promoter regions that are potential regulators of tyrosinase gene expression in a transient expression system using melanoma cell lines. In human melanoma cells, the plasmid construct with a -2020 base pair (bp) promoter yielded the highest CAT activity. When the deletions reached -1739 bp, the CAT activity was dramatically reduced, indicating that important enhancer elements for transcription control are present between -1739 and -2020 bp. Further deletions up to -550 bp also resulted in dramatic decreases of CAT activity. However, when the deletion included -550 bp of the 5' flanking sequence, there was 26 percent of the CAT activity compared to that of the -2020 bp promoter. Deletions beyond -550 bp also showed markedly decreased CAT activity. Based on our data, we suggest that human tyrosinase gene expression is governed by both tissue-specific and multiple regulatory elements.
J Invest Dermatol 1994 May
PMID:Structural organization of the human tyrosinase gene and sequence analysis and characterization of its promoter region. 817 57

Involucrin is one of the precursor proteins of keratinocyte cornified envelope. Although the formation of the cornified envelope is induced by tumor-promoting phorbol esters, the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the involucrin gene expression remains unknown. We have isolated a 5'-upstream region of human involucrin gene and examined its TPA-dependent promoter activity. The involucrin upstream region with the untranslated first exon was connected to chloramphenicol acetyltransferase (CAT)-involucrin promoter expression vector (INV-CAT) and was transfected into fetal rat keratinizing epidermal (FRSK) cells. The INV-CAT-transfected FRSK cells showed considerable CAT activity that was significantly augmented by the treatment of cells with TPA. FRSK cells that were transfected with a reversely oriented 5'-upstream sequence revealed little CAT activity and did not respond to TPA. The effect of TPA was significantly inhibited by the protein kinase C inhibitor 1-(5-isoquinoline-sulfonyl)-2-methyl piperazine dihydrochloride (H-7). Other protein kinase C activators (1-oleoyl-2-acetylglycerol and mezerein) also induced the INV-CAT promoter activity, whereas 4-O-methyl phorbol myristate acetate, a very weak protein kinase C activator, had only a slight effect. Analysis of the nucleotide sequence of the 5'-upstream region detected several 5'-TGANTCAA-3' sequences that are highly conserved TPA-response elements (TRE). Cotransfection of both c-jun and c-fos expression vectors with the INV-CAT vector into FRSK cells resulted in increased CAT activity. Cotransfection of either the c-jun or c-fos vector singly with the INV-CAT vector into FRSK cells had negligible effects. Dexamethasone significantly inhibited the TPA-induced promoter activity in the INV-CAT-transfected FRSK cells. These results indicate that involucrin gene expression is positively controlled by TPA through the activation of the protein kinase C/TRE system.
J Invest Dermatol 1993 Jan
PMID:Analysis of the 5'-upstream promoter region of human involucrin gene: activation by 12-O-tetradecanoylphorbol-13-acetate. 838 Aug 29

Ultraviolet (UV) irradiation of human cells induced expression of a stably maintained fusion gene consisting of the human immunodeficiency virus long terminal repeat promoter controlling the bacterial chloramphenicol acetyltransferase gene. Two experiments demonstrated that DNA damage can initiate induction: UV induction was greater in DNA repair-deficient cells from a xeroderma pigmentosum patient than in repair-proficient cells, and transfection of UV-irradiated DNA into unirradiated cells activated gene expression. Increased repair of cyclobutane pyrimidine dimers by T4 endonuclease V abrogated viral gene activation, suggesting that dimers in DNA are one signal leading to increased gene expression. This signal was spread from UV-irradiated cells to unirradiated cells by co-cultivation, implicating the release of soluble factors. Irradiation of cells from DNA repair-deficiency diseases resulted in greater release of soluble factors than irradiation of cells from unaffected individuals. These results suggest that UV-induced cyclobutane pyrimidine dimers can activate the human immunodeficiency virus promoter at least in part by a signal-transduction pathway that includes secretion of soluble mediators.
J Invest Dermatol 1993 Jun
PMID:Cyclobutane pyrimidine dimers in UV-DNA induce release of soluble mediators that activate the human immunodeficiency virus promoter. 838 27

Involucrin is one of the precursor proteins of keratinocyte cornified envelope that is formed beneath the inner surface of the cell membrane during terminal differentiation. Although involucrin is specifically expressed in the upper squamous cells of the epidermis, the precise regulatory mechanism of involucrin gene expression remains unknown. Transcriptional enhancer factor 1 (TEF-1), which binds to SV40 enhancer, is a nuclear protein expressed in various types of cells including keratinocytes. Immunohistochemical study has revealed that TEF-1 protein is highly expressed on the basal cell layer of the epidermis. To examine the possible regulatory mechanism of involucrin gene expression by TEF-1 protein, we analysed involucrin promoter activity of the INV-CAT vector, which was constructed by connecting the 5' upstream region of the involucrin gene (-801 bp upstream from the transcription start site and downstream including the untranslated first exon) to the chloramphenicol acetyltransferase (CAT) reporter gene. The INV-CAT vector was transfected to SV40-transformed human keratinocytes (SVHK). Cotransfection of the TEF-1 expression vector significantly repressed INV-CAT promoter activity in a dose-dependent manner. The repression was also observed by transfection of the GAL4-TEF-1 vector, which was constructed by replacement of the TEF-1 DNA binding domain by the GAL4 activator domain. This suggests that TEF-1-induced repression is due to interference/squelching of a limiting transcriptional intermediary factor that is essential for involucrin expression.(ABSTRACT TRUNCATED AT 250 WORDS)
Arch Dermatol Res 1995
PMID:Repression of involucrin gene expression by transcriptional enhancer factor 1 (TEF-1). 855 86


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