RESUMO
The gene coding for the human estrogen receptor protein was expressed as a ubiquitin fusion under the control of the lambda PL promoter in Escherichia coli. Analysis of extracts by Western blot showed that intact receptor protein was produced only when PL promoter was depressed by nalidixic acid at 30 degrees C. To ascertain the intactness of the expressed protein, estrogen receptor in bacterial extracts was titrated with either 17 beta-[3H]estradiol or 17 beta-[125I]iodoestradiol, and the mass was quantified by enzyme immunoassay. Cell extracts contained 484 fmol of estrogen receptor per mg of soluble protein by titration with a Kd of 3.2 x 10(-10) M. The simultaneous analysis by enzyme immunoassay resulted in 487 fmol/mg of extract protein indicating that most of the expressed protein bound ligand with wild type properties. Ligand competition analysis demonstrated that expressed receptor contained a binding domain preferentially recognizing estrogenic substances identical with that of wild-type estrogen receptor. The E. coli-expressed estrogen receptor also associated specifically with estrogen response DNA elements. Full length receptor protein was detected by ligand binding and immunorecognition using size exclusion chromatography. Hydrophobic interaction chromatography showed a major isoform which exhibited both ligand binding and immuno-recognition identical with the estrogen receptor from human breast tumor tissues. These data indicate that estrogen receptor expressed in E. coli retained characteristics of the native protein found in human tissues.
Assuntos
Proteínas de Ligação a DNA/genética , Receptores de Estrogênio/genética , Western Blotting , Clonagem Molecular , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Estradiol/metabolismo , Expressão Gênica , Humanos , Receptores de Estrogênio/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitinas/genéticaRESUMO
Cellular changes in hepatocytes of green sunfish (Lepomis cyanellus) exposed to arsenic-contaminated or control lake water were compared with the level of arsenic in the liver. Standard stereological procedures involved conversion of two-dimensional data (i.e. fractional measurements of morphological changes) to three-dimensional data for interpretation. Both the volume and numbers of nuclei increased slightly with increasing concentration of arsenic in the liver. Significant increases (p less than 0.01) were observed in the volumes occupied by necrotic and fibrous bodies as arsenic levels in the liver increased; linear regression analysis of these data resulted in 0.9066 and 0.9359 correlation coefficients for necrotic and fibrous bodies, respectively, when volume changes were considered on a unit body weight basis. The volume occupied by necrotic areas, abnormal lysosomes, and autophagic vacuoles increased with increased arsenic concentration. The surface density of rough endoplasmic reticulum increased with increasing arsenic concentration; linear regression resulted in a correlation coefficient of 0.8367 when data were based on unit body weight.
