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41.
Human β-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/β-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input. pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5′ or 3′ end, a 3′ poly A tail, or a 5′-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%–40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain β-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection. 相似文献
42.
Radioimmunoassay of neuropeptide Y 总被引:5,自引:0,他引:5
The development of a radioimmunoassay to the newly isolated peptide, neuropeptide Y is described. Four separate antisera have been developed using different immunisation schedules. Two of these antisera (YNI and YNIO) are directed to the C-terminal region of the peptide and cross-react with the related peptide PYY, whereas YN7 is specific being directed to the N-terminal region of NPY, YN6 is similarly specific for NPY, but is unable to bind the available fragments. These four antisera provide similar results for determination of NPY immunoreactivity within porcine brain extracts, however YN6 consistently undervalues all extracts from the other species examined (human, rat, guinea pig, cat and mouse). Chromatographic analysis by means of reverse phase high pressure liquid chromatography (HPLC) shows that NPY immunoreactivity of human extracts elutes in an earlier position than the porcine standard. It seems likely therefore that human and porcine NPY differ in their amino acid sequences. 相似文献
43.
Peptide YY (PYY) immunoreactivity is co-stored with glucagon-related immunoreactants in endocrine cells of the gut and pancreas 总被引:2,自引:0,他引:2
A. Ali-Rachedi I. M. Varndell T. E. Adrian D. A. Gapp S. Van Noorden S. R. Bloom J. M. Polak 《Histochemistry and cell biology》1984,80(5):487-491
Summary In this study we report the localisation of PYY immunoreactivity in intestinal mucosa endocrine (EG) cells containing glucagon-related peptides and also in foetal pancreatic A cells of rat and man. Radioimmunoassay of human foetal pancreatic extracts revealed the presence of PYY immunoreactivity, the concentration of which declined with age (from 65.42 pmol/g at week 20 to 17.0 pmol at week 40; correlation coefficient=–0.893), in contrast to the amount of glucagon which remained statistically constant throughout the same foctal period. The identity of this PYY immunoreactive material with the original 36 amino acid porcine peptide has been shown by high pressure liquid chromatography (HPLC). 相似文献
44.
We compare a recombinant cDNA plasmid (pAF81) complementary to a fetal skeletal muscle actin mRNA with a plasmid (pAM91) complementary to the actin mRNA expressed in adult skeletal muscle. The two mRNAs are significantly diverged in silent nucleotide positions; they are coexpressed in fetal skeletal muscle, and in differentiating muscle cell cultures their accumulation begins coordinately. The sequence of pAF81 shows that the amino acid sequence of mouse fetal skeletal muscle actin is almost identical to that of adult bovine cardiac actin. Hybridization of pAF81 to RNA from different mouse tissues shows that fetal skeletal muscle actin mRNA is very homologous or identical to fetal and adult cardiac actin mRNA. Only one gene homologous to pAF81 is detected on blots of restricted mouse DNA. We conclude that this gene must be expressed both in fetal skeletal muscle and in fetal heart. Whereas mRNA transcribed from this gene is the major actin mRNA species in adult heart, it is present in low amounts, if at all, in adult skeletal muscle. 相似文献
45.
A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound. 相似文献
46.
Adrian P. Sorrentino Jorge Zorzópulos Héctor F. Terenzi 《Archives of biochemistry and biophysics》1977,180(2):232-238
Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed. 相似文献
47.
Bernard Junod Adrian Griffiths Roland Lüthi Hannes B. St?helin Heikki Savolainen et al. 《International journal of biometeorology》1992,36(3):179-189
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Our Planet, Our Health Report of the who commission on health and environment 相似文献48.
We describe a generally applicable method of adapting Dictyostelium from growth on a bacterial food source to axenic growth. Cells are initially selected by growth on a plastic substratum but subsequently acquire the ability to grow in suspension culture. These strains can be transformed efficiently by DNA-mediated gene transfer. 相似文献
49.
Plasma and tissue alterations of peptide YY and enteroglucagon in rats after colectomy. 总被引:4,自引:0,他引:4
A P Vukasin G H Ballantyne O Nilsson A J Bilchik T E Adrian I M Modlin 《The Yale journal of biology and medicine》1992,65(1):1-15
Peptide YY (PYY) and enteroglucagon are produced by endocrine cells of the colonic mucosa. PYY inhibits upper gastrointestinal motility, and enteroglucagon is trophic for small bowel mucosa. Adaptive increase in the production and release of these peptides may improve functional results after colorectal resections. We hypothesized that if segments of the colon were resected, then production and release of PYY and enteroglucagon would increase in the remaining segments of bowel. Animals which underwent colonic transections and partial resections had transient elevations of PYY up to 250 +/- 80 pmol/L, which dropped to control group levels in the second week following surgery. Rats with an abdominal colectomy had significantly greater PYY levels than all other groups from the third (208 +/- 30 pmol/L) to the thirty-eighth (100 +/- 16 pmol/L) week of the study. Circulating levels of enteroglucagon were elevated to 156 +/- 35 pmol/L in rats with a right hemicolectomy during the first week following surgery. Enteroglucagon levels did not significantly vary in the other groups studied. Both tissue PYY (413 +/- 33 pmol/gram) and tissue enteroglucagon (171 +/- 17 pmol/gram) were significantly elevated in the rectums of the rats with an abdominal colectomy, as compared to all other groups. The elevated tissue levels may thus account for the ability to maintain elevated plasma PYY. Double immunogold labeling of endocrine cells in the colorectal tissue for PYY and enteroglucagon revealed both peptides within the same endocrine cells and secretory granules. These studies support the hypothesis that circulating levels of PYY are elevated after major colonic resections and suggest that L-type endocrine cells may participate in adaptive responses which improve intestinal function following colonic surgery. 相似文献
50.
Antigen-coated plastic tubes were used with 125I- or enzyme-labeled staphylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G (IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive adsorption. Haptens with free carboxyl groups were bound covalently to poly-l-lysine and these conjugates passively adsorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using 125I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, IgM, and IgE in serum in volumes up to 1 ml. 相似文献