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1.
Colin K.W. Watts Robert L. Sutherland 《Biochemical and biophysical research communications》1984,120(1):109-115
Saturation and competitive binding analyses demonstrated the presence of a high affinity (KD = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver. 相似文献
2.
Jon G. Wilkes John B. Sutherland 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1998,717(1-2)
The chromatographic analysis of carboxyl-containing mycotoxins, such as fumonisin B1, ochratoxin A, and citrinin, presents a continual challenge. Toxins must first be extracted from foods or tissues and then cleaned up before chromatographic separation and detection. Liquid–liquid extraction efficiencies for some carboxylic mycotoxins are marginal for spiked samples and uncertain for incurred residues. Immunoaffinity columns may be useful for concentrating mycotoxins from samples before chromatography. In almost every case, more than one analytical method must be used to confirm the identification of the mycotoxin. The fumonisins are especially troublesome to analyze because they are relatively insoluble in organic solvents, they are not separated easily by gas chromatography, and they do not respond to the usual absorbance or fluorescence detectors used in liquid chromatography. Fluorescence derivatization and electrospray liquid chromatography–mass spectrometry have now made it possible to detect trace levels of mycotoxins. The purity of mycotoxin standards for toxicological studies can be determined by liquid chromatography with either an evaporative light scattering detector or electrospray mass spectrometer. New developments in capillary electrophoresis, nonporous microsphere liquid chromatography, and detection methods for low-volatility compounds show promise for improving the analysis of mycotoxins in the future. 相似文献
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Streptomyces setonii strain 75Vi2 was grown at 45 degrees C in liquid media containing yeast extract and trans-cinnamic acid, p-coumaric acid, ferulic acid, or vanillin. Gas chromatography, thin-layer chromatography, and mass spectrometry showed that cinnamic acid was catabolized via benzaldehyde, benzoic acid, and catechol; p-coumaric acid was catabolized via p-hydroxybenzaldehyde, p-hydroxybenzoic acid, and protocatechuic acid; ferulic acid was catabolized via vanillin, vanillic acid, and protocatechuic acid. When vanillin was used as the initial growth substrate, it was catabolized via vanillic acid, guaiacol, and catechol. The inducible ring-cleavage dioxygenases catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected with an oxygen electrode in cell-free extracts of cultures grown in media with aromatic growth substrates and yeast extract. 相似文献
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A human colon adenocarcinoma cell line, WiDr, has been grown in monolayer, as multicellular spheroids, and as xenografted tumors in immune-deprived mice. The growth and radiation responses of the cells under these different growth conditions were compared. The mean doubling time of monolayer cultures was 0.8 day and the initial volume doubling times of spheroids and xenografts averaged 1.2 and 6 days, respectively. The mean total viable cell plating efficiencies were 82, 63, and 7% for cells from monolayers, spheroids, and xenografted tumors, respectively. The radiation responses of single cell suspensions prepared from WiDr tumors (8-10 mm in diameter), exponentially growing monolayer cultures (5 days growth), and spheroids (1200 microns in diameter) irradiated in air at 4 degrees C were similar. Values for D0 were 1.5 Gy and for n between 3 and 5. Nitrogen curves were characterized by a D0 of 5 Gy and n between 3 and 6. Oxygen enhancement ratios were approximately 3.3. Both spheroids and tumors had radioresistant components to the 37 degrees C/air-breathing survival curves with estimated hypoxic fractions of 8 and 12%, respectively. The final portion of the survival curves for irradiations in nitrogen and under normal growth conditions were parallel for both tumors and spheroids. Thus WiDr spheroids appear to model accurately the radiation sensitivity of WiDr tumors. 相似文献
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Solubilization and characterization of a lactogenic receptor from human placental chorion membranes 总被引:2,自引:0,他引:2
Prolactin has a wide range of actions, including osmoregulation and the control of mammary gland development and lactation. These effects are mediated through a high-affinity cell surface receptor, which has been well characterized in a number of animal tissues. The molecular characteristics of the human receptor are unknown, however. The present studies were initiated, therefore, to determine the binding and molecular characteristics of the lactogenic receptor of human placental chorion membranes. Subcellular fractionation studies showed that the bulk of the receptor sedimented in the microsomal fraction at 45,000gav. Endogenous ligand was dissociated from the receptor with 3.5 M MgCl2 or 0.05 M acetate buffer (pH 4.8) with preservation of binding activity. The microsomal receptor bound human growth hormone (hGH), human prolactin (hPRL), ovine prolactin (oPRL), and human placental lactogen (hPL) but not non-primate growth hormones, indicating a narrow specificity for lactogenic hormones. The binding was only partially reversible in agreement with the known binding kinetics of animal lactogenic receptors. The receptor was solubilized with 45% yield from the microsomes using 16 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) detergent-250 mM NaCl, and the binding activity was fully restored by a two-fold dilution in the binding reaction to reveal a KD of 0.8 nM for hGH and a binding capacity of 200 fmol of specifically bound hGH per mg of microsomal protein. Gel filtration chromatography indicated the minimum molecular weight of the ligand-receptor complex was approximately 60,000 daltons, and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of covalently cross-linked 125I-hGH-receptor complexes revealed a molecular size of 58,000 daltons. When account was taken of the contribution of the ligand, a molecular weight of 36,000 for the receptor's binding domain was obtained. These data indicate that the chorion lactogenic receptor has very similar binding and molecular characteristics to the lactogenic receptors from other mammalian species. Chorion membranes are thus a convenient source of material for the further purification and characterization of the human lactogenic receptor. 相似文献
9.
The human metallothionein gene complex on chromosome 16 has been remapped to 16q13 using high-resolution in situ hybridization. The complex is not disrupted by the rearrangement breakpoint on the long arm of chromosome 16 in patients with myelomonocytic leukemia with abnormal eosinophils, as had been previously reported. The locus order on 16q is cen-MT-FRA16B-D16S4-inversion breakpoint-HP-tel. 相似文献
10.
Expression and function of cell surface extracellular matrix receptors in mouse blastocyst attachment and outgrowth 总被引:13,自引:7,他引:6 下载免费PDF全文
Mouse-hatched blastocysts cultured in vitro will attach and form outgrowths of trophoblast cells on appropriate substrates, providing a model for implantation. Immediately after hatching, the surfaces of blastocysts are quiescent and are not adhesive. Over the period 24-36 h post-hatching, blastocysts cultured in serum-free medium become adhesive and attach and spread on the extracellular matrix components fibronectin, laminin, and collagen type IV in a ligand specific manner. Attachment and trophoblast outgrowth on these substrates can be inhibited by addition to the culture medium of an antibody, anti-ECMr (anti-extracellular matrix receptor), that recognizes a group of 140-kD glycoproteins similar to those of the 140-kD extracellular matrix receptor complex (integrin) recognized in avian cells by CSAT and JG22 monoclonal antibodies. Addition to the culture medium of a synthetic peptide containing the Arg-Gly-Asp tripeptide cell recognition sequence of fibronectin inhibits trophoblast outgrowth on both laminin and fibronectin. However, the presence of the peptide does not affect attachment of the blastocysts to either ligand. Immunoprecipitation of 125I surface-labeled embryos using anti-ECMr reveals that antigens recognized by this antibody are exposed on the surfaces of embryos at a time when they are spreading on the substrate, but are not detectable immediately after hatching. Immunofluorescence experiments show that both the ECMr antigens and the cytoskeletal proteins vinculin and talin are enriched on the cell processes and ventral surfaces of trophectoderm cells in embryo outgrowths, in patterns similar to those seen in fibroblasts, and consistent with their role in adhesion of the trophoblast cells to the substratum. 相似文献