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Low-density lipoprotein preparation by combined diafiltration and ultracentrifugation 总被引:1,自引:0,他引:1
K A Pritchard J A Holland N J Rogers C C Crean T E Britton P Onigman M B Stemerman 《Analytical biochemistry》1988,174(1):121-127
A method for isolating low-density lipoprotein by combining diafiltration and ultracentrifugation is described. Diafiltration separates plasma components by use of an ultrafiltration membrane that excludes particles of molecular weight greater than 300,000. The retentate is concentrated three- to fourfold by ultrafiltration, allowing large-scale preparation of low-density lipoprotein. Low-density lipoprotein prepared in this manner is similar in physical, chemical, and biologic properties to low-density lipoprotein isolated by sequential density ultracentrifugation alone. When low-density lipoprotein, prepared by either method, was added to human umbilical vein endothelial cell cultures, no cytotoxicity was observed. The techniques described reduce the demand on multiple rotors and ultracentrifuges for large-scale preparation of low-density lipoprotein suitable and often needed for tissue culture studies. 相似文献
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Candida utilis was grown with glucose as growth-limiting carbon source in batch culture, steady-state continuous culture, and non-steady-state continuous culture. High cytochrome concentrations were routinely recorded in cells harvested in the latter stages of batch culture. They were occasionally recorded in cells from imprecisely controlled steady-state cultures, but precise control of the steady-state dissolved oxygen tension stabilized the cytochromes at relatively low levels. Controlled non-steady-state continuous cultures, imposed by pulse additions of ethanol, routinely produced cells with high cytochrome concentrations. A mechanism is proposed whereby maintenance of cytochrome derepression in continuous culture is dependent upon indefinitely prolonging an “overshoot” response in gene expression. 相似文献
36.
The recovery of radioactivity in the urine of guineapigs following a bolus intravenous dose of chromatographically pure 14C-Ntau-methylhistidine was measured in order to test whether the excretion of Ntau-methylhistidine (Ntau-MH) is a valid index of myofibrillar protein breakdown in these animals. Four male and four female guineapigs were dosed and after 7 days, 91.65+/-2.82% and 3.58+/-0.91% of injected radioactivity was recovered in the excreta and tissues, respectively. The average total recovery of 95.2+/-3.0% was not significantly different from 100%. Male guineapigs excreted the radioactivity more slowly than females (70% of the dose excreted within 74 h vs 39 h, respectively) but cumulative excretion at 7 days was the same for each sex. Chromatographic analysis of the urine showed almost all of the radioactivity to be associated with a single peak corresponding to Ntau-MH, indicating a lack of significant metabolism. These data show that although the clearance of 14C-Ntau-MH is slower than in rats or humans the urinary excretion of Ntau-MH is a valid index for myofibrillar protein degradation in the guineapig. 相似文献
37.
S R Compton D B Rogers K V Holmes D Fertsch J Remenick J J McGowan 《Journal of virology》1987,61(6):1814-1820
An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication. 相似文献
38.
Harry J. Klee Maria B. Hayford Stephen G. Rogers 《Molecular & general genetics : MGG》1987,210(2):282-287
Summary Cloning of genes by transformation with genomic banks and rescue of a phenotype has been extensively used in bacterial systems. This approach has not been possible in plant systems because of the large genome sizes and poor transformation frequencies of most plant species. Recent advances in plant transformation permit the generation of large numbers of transformants in petunia. We have used this system to rescue a model gene encoding resistance to kanamycin by shotgun cloning. The gene encoding neomycin phosphotransferase (NPTII) was introduced into the genome of Arabidopsis thaliana by Agrobacterium tumefaciens-mediated transformation. A genomic bank of DNA from this tissue was constructed in a cosmid vector containing features which would allow its use in plant transformation. The unselected genomic bank was mobilized from Escherichia coli to A. tumefaciens and used to retransform petunia leaf discs. The rescued gene was identified by its ability to confer a kanamycin-resistant phenotype in petunia tissue. The presence of the NPTII gene was confirmed by nopaline assay and Southern blot analysis. This experiment demonstrates the feasibility of gene rescue, in certain circumstances, in plants. 相似文献
39.
Ecology of bdelloids: how to be successful 总被引:7,自引:4,他引:3
Claudia N. Ricci 《Hydrobiologia》1987,147(1):117-127
Bdelloids inhabit many different environments. The entire taxon is an order belonging to the phylum Rotifera. In spite of its wide distribution, it has a very uniform morphology, suggesting that natural selection has had almost no effect on its morphological characteristics.This paper reviews the geographical and ecological distributions of bdelloids and their ability to tolerate different conditions is discussed. Two characteristics account for the wide distribution of bdelloids, parthenogenetic reproduction and their ability to withstand unfavorable conditions through anhydrobiosis. The former is an apomictic thelytoky which may ensure genetic homogeneity within clones. However, evidence which suggests variability among and within parthenogenetic clones is discussed. Some recent experimental evidence indicates that anhydrobiosis does not affect the life history of the surviving individuals. The effects of anhydrobiosis on bdelloid populations are considered. 相似文献
40.
Clostridium acetobutylicum Mutants That Produce Butyraldehyde and Altered Quantities of Solvents 总被引:5,自引:2,他引:3 下载免费PDF全文
Spontaneous mutants of Clostridium acetobutylicum NRRL B643 that were resistant to allyl alcohol (AA) were selected and characterized. These mutants contained 10- to 100-fold reduced activities of butanol and ethanol alcohol dehydrogenase. The AA mutants formed two groups and produced no ethanol. Type 1 AA mutants produced significant amounts of a new solvent, butyraldehyde, and contained normal levels of the coenzyme A-dependent butyraldehyde dehydrogenase (BAD). Type 2 AA mutants produced no significant butyraldehyde and lower levels of all solvents, and they contained 45- to 100-fold lower activity levels of BAD. Following ethyl methanesulfonate mutagenesis, low-acid-producing (Acid−) mutants were selected and characterized as superinduced solvent producers, yielding more than 99% of theoretical glucose carbon as solvents and only small amounts of acetate and butyrate. Following ethyl methanesulfonate mutagenesis, 13 sporulation-negative (Spo−) mutants were characterized; and 3 were found to produce only butyrate and acetate, a minor amount of acetone, and no alcohols. These Spo− mutants contained reduced butanol dehydrogenase activity and no BAD enzyme activity. The data support the view that the type 2 AA, the Acid−, and the Spo− mutants somehow alter normal regulated expression of the solvent pathway in C. acetobutylicum. 相似文献