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1.
A C Porter 《Analytical biochemistry》1981,117(1):28-31
A liquid scintillation counting system is described that allows recovery of compounds for further study and analysis. For most classes of compounds tested (with the exception of steroids) the recovery was high (usually at least 90%) and in the case of nucleosides was accompanied by very little degradation of the sample. The counting method should be useful for the counting of samples where a high recovery of the compound is necessary. 相似文献
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D Racusen 《Analytical biochemistry》1979,99(2):474-476
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Purified liver microsomal NADPH-cytochrome P-450 reductase is able to catalyze the activation of [14C]ronidazole to metabolite(s) which bind covalently to protein. Like the reaction catalyzed by microsomes, protein alkylation catalyzed by the reductase is (1) sensitive to oxygen, (2) requires reducing equivalents, (3) is inhibited by sulfhydryl-containing compounds and (4) is stimulated several fold by either flavin mononucleotide (FMN) or methytlviologen. A cytochrome P-450 dependent pathway of ronidazole activation can be demonstrated as judged by the inhibition of the reaction by carbon monoxide, metyrapone and 2,4-dichloro-6-phenylphenoxyethylamine but the involvement of specific microsomal cytochrome P-450 isozymes has not been definitively established. Milk xanthine oxidase is also capable of catalyzing ronidazole activation. Polyacrylamide sodium dodecyl sulfate (SDS)-gel electrophoresis reveals that the reactive intermediate(s) of ronidazole does not alkylate proteins selectively. 相似文献
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The conditions used in some immunoblot procedures can fail to detect calmodulin, S100 proteins, and other proteins with similar physical properties. We describe here some of the basis of this difficulty, and provide an immunoblot protocol that allows the rapid and reproducible detection of calmodulin and S100 beta in crude biological samples. These proteins are rapidly transferred from sodium dodecyl sulfate-polyacrylamide gels to membrane matrices, and retention on the matrix is enhanced by a glutaraldehyde fixation step. Either nitrocellulose or a positively charged membrane filter (ZetaProbe) can be used as the immobilizing matrix. By combining microslab gel electrophoresis, 30 min electrophoretic transfer, and glutaraldehyde fixation of nitrocellulose paper, an immunoblot analysis can be done in an 8-hr day. 相似文献
6.
Fluorography--limitations on its use for the quantitative detection of 3H- and 14C-labeled proteins in polyacrylamide gels 总被引:1,自引:0,他引:1
The suitability of fluorography for the detection of 3H- and 14C-labeled proteins on polyacrylamide gradient gels has been investigated. It was found that the absorbance of the fluorographic film image produced by a given level of radioactivity decreased as the acrylamide concentration in the gel increased. The use of Coomassie brilliant blue protein dyes to stain the gel prior to fluorography reduced the absorbance of the fluorographic image. It is concluded that quantitative fluorography can only be applied to unstained gels of a uniform acrylamide concentration. 相似文献
7.
David E. Johnston Douglas M. Jefferson 《In vitro cellular & developmental biology. Animal》1994,30(7):464-470
Summary When primary cultures of hepatocytes are exposed to media containing fetal bovine serum (FBS) there is a rapid decrease in
levels of tissue-specific mRNAs such as albumin mRNA. We used Northern blot analysis to examine mRNA levels in cultured hepatocytes,
and characterized the factor in FBS that significantly reduces the steady state albumin mRNA level. Neonatal bovine serum
or serum derived from platelet-poor calf plasma proved as potent as did FBS, but commercial bovine serum albumin did not exhibit
this inhibitory activity. Inhibitory activity of FBS was not removed by moderate heat treatment, dialysis, or extraction with
organic solvents. However, incubation of FBS with a highly anionic detergent such as 0.1% sodium dodecyl sulfate orN-lauroyl sarcosine, followed by extensive dialysis, resulted in sera that did not inhibit expression of albumin mRNA. These
sera supported cell attachment and seemed non-toxic toward the cells. Ammonium sulfate fractionation of FBS showed the activity
was present in the 45 to 70% fraction, and trypsin digestion destroyed the inhibitory activity. Gel exclusion chromatography
gave a molecular weight 60 000 to 70 000. Fractionation of serum proteins by DEAE-Sephacel or Cibacron blue-agarose showed
enrichment for albumin in the most active fractions. Interestingly, metabolic labeling of secreted and cellular proteins with35S-methionine and cysteine showed no significant difference between hepatocytes maintained for 2 days beforehand in serum-free
or serum-supplemented media, and no difference between detergent-treated FBS and control FBS. Therefore, FBS contains a factor
that causes a significant decrease in steady state levels of mRNA for albumin and other mRNAs of tissue specific function,
but under these conditions albumin mRNA levels are not paralleled by synthesis of albumin or other proteins. 相似文献
8.
The use of a protein stain, [59Fe]ferrous bathophenanthroline, to radioactively label proteins in polyacrylamide gels after electrophoresis using simple staining and destaining procedures is described. 相似文献
9.
Cloning and sequence analysis of the Escherichia coli 4.5 S RNA gene 总被引:20,自引:0,他引:20
10.
R G Wolfe R Nakayama D Holten 《Biochemical and biophysical research communications》1979,89(1):108-115
The relative rate of synthesis of glucose-6-P dehydrogenase increases up to 8-fold when fasted rats are fed a 60% carbohydrate, fat-free diet for 3 days but the specific activity of the enzyme only increases 2 to 3 fold. This suggests that the high carbohydrate diet also causes a 2 to 3 fold increase in the rate of glucose-6-P dehydrogenase degradation. The nutritional induction of this enzyme in adipose tissue is primarily due to a large increase in the rate of its synthesis. 相似文献
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A Strauss C Bennett A M Donohue J Rodkey A W Alberts 《Biochemical and biophysical research communications》1977,77(4):1224-1230
Effect of hemin, mild periodate oxidation and concanavalin A (Con A) on biosynthesis of membrane proteins and hemoglobin, in the rabbit reticulocyte, was examined. Whereas addition of hemin to the incubation medium stimulates synthesis of both hemoglobin and membrane proteins, addition of Con A, at concentrations which agglutinate cells, selectively stimulates membrane protein biosynthesis. Mild periodate treatment of cells inhibits synthesis of hemoglobin and membrane proteins; this inhibition is not related to oxidation of a membrane component since hemoglobin synthesis in a cell free lysate of treated cells is similarily inhibited. 相似文献
12.
G M Ward S Clark L C Harrison 《Biochemical and biophysical research communications》1984,123(2):849-853
We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects. 相似文献
13.
Identification in rat atrial tissue of multiple forms of natriuretic polypeptides of about 3,000 daltons 总被引:7,自引:0,他引:7
K Kangawa A Fukuda I Kubota Y Hayashi H Matsuo 《Biochemical and biophysical research communications》1984,121(2):585-591
Rat atrial natriuretic peptides of relatively low molecular weight have been isolated from the alpha-component of rectum relaxant activity corresponding to about 3,000 daltons, which was obtained as a side fraction in our previous isolation of beta-rat atrial natriuretic polypeptide (beta- rANP ). In contrast to the same fraction from human atria, the rat atrial alpha-component was found to contain six or more distinct but related peptides, eliciting a potent natriuretic activity. Six of them (B-II, C, D, E, B-I and A), containing 35, 33, 32, 31, 28 and 25 amino acid residues, respectively, have been purified to homogeneity and sequenced. All these peptides were found to correspond to the C-terminal sequence of beta- rANP composed of 48 residues, with varying N-terminal elongations. This indicates that these peptides are derived from beta- rANP . Peptide B-I, composed of 28 residues, is identical to alpha-human atrial polypeptide(alpha- hANP ), with a single replacement of Ile for Met at position 12. 相似文献
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F Peillon A M Brandi D Bression M Le Dafniet J Racadot 《Biochemical and biophysical research communications》1983,112(1):42-46
The ability of 2-Hydroxyestradiol, a catecholestrogen, and 17 beta Estradiol to interact with the dopamine inhibition of prolactin and with dopamine receptors has been tested on dispersed human prolactin-secreting cells obtained from ten pituitary adenomas. There is a 80% inhibition of prolactin secretion obtained by addition of dopamine in a superfusion system. This inhibition is not affected by preexposure to the steroids, or by their introduction into the perifusion medium. Moreover 2 Hydroxyestradiol and 17 beta Estradiol do not interact with the binding of 3H Domperidone to DA receptors. 相似文献
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Jian-bo Fan Wei LiuKun Yuan Xin-hui ZhuDa-wei Xu Jia-jia ChenZhi-ming Cui 《Biochemical and biophysical research communications》2014
Pleiotrophin (Ptn) plays an important role in bone growth through regulating osteoblasts’ functions. The underlying signaling mechanisms are not fully understood. In the current study, we found that Ptn induced heparin-binding epidermal growth factor (HB-EGF) release to trans-activate EGF-receptor (EGFR) in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, Ptn activated Akt and Erk signalings in cultured osteoblasts. The EGFR inhibitor AG1478 as well as the monoclonal antibody against HB-EGF (anti-HB-EGF) significantly inhibited Ptn-induced EGFR activation and Akt and Erk phosphorylations in MC3T3-E1 cells and primary osteoblasts. Further, EGFR siRNA depletion or dominant negative mutation suppressed also Akt and Erk activation in MC3T3-E1 cells. Finally, we observed that Ptn increased alkaline phosphatase (ALP) activity and inhibited dexamethasone (Dex)-induced cell death in both MC3T3-E1 cells and primary osteoblasts, such effects were alleviated by AG1478 or anti-HB-EGF. Together, these results suggest that Ptn-induced Akt/Erk activation and some of its pleiotropic functions are mediated by EGFR trans-activation in cultured osteoblasts. 相似文献
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HeLa cell polysomes were oxidized with sodium periodate and reduced with sodium borohydride to induce covalent crosslinks between ribosomal RNA and nearby proteins. We proved that RNA was tryly crosslinked to protein in oxidized, and not in control, samples using denaturing cesium trichloroacetate density gradients and phenol extraction. By both one- and two-dimensional gel analysis, we found that protein S3a can be crosslinked to 18S RNA, protein L3 to 28S RNA, and proteins L7′ and L23′ to 5.8S RNA. Because of the specificity of the periodate reaction, and since we were able to crosslink protein S1 to 16S RNA in , 30S ribosomal subunits, it is likely that we have crosslinked proteins to the 3′OH ends of HeLa polysomal RNAs. 相似文献
20.
M. P. Lopez M. J. Gomez-Lechon J. V. Castell 《In vitro cellular & developmental biology. Plant》1984,20(12):923-931
Summary Liver parenchymal cells cultured in serum-free medium may retain their ability to synthesize glycogen in response to insulin.
Specific hormone requirements are needed by hepatocytesto retain the biochemical pattern of mature cells. Insulin supplementation
of culture medium seems to be essential to maintain the glycogen synthesis rate of cultured hepatocytes. The continuous presence
of dexamethasone amplified the insulin-induced glucogen synthesis. Cytophotometric analysis showed differences in the way
that individual cells accumulate glycogen in response to insulin stimulus, which indicates that liver parenchymal cells in
culture are functionally heterogeneous.
The financial support for this work was from the Fondo de Investigationes Sanitarias de la Seguridad Social, grants 41/82
and 48/82. 相似文献