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Diamine oxide and serum amine oxidase, which catalyse the oxidation of diamines and polyamines, respectively, were trapped within reconstituted Sendai virus envelopes. These loaded envelopes were incubated with cultured normal chick fibroblasts or with fibroblasts transformed by Rous sarcoma viruses. The binding of the reconstituted envelopes to the cultured cells was confirmed by scanning electron microscopy. It has been shown that the reconstituted envelopes (1-3 microns diameter) were attached to the eukaryotic cells. No significant changes in the morphology of the normal chick embryo fibroblasts were noted upon treatment with enzyme-loaded envelopes. On the other hand, chick embryo fibroblasts transformed by Rous sarcoma virus were affected by the microinjected amine oxidases. Scanning electron microscopy demonstrated the formation of holes in the microinjected cells. Similar morphological changes were also observed when diamine oxidase was microinjected into cultured glioma cells. These holes may be the result of the ejection of the nucleus. These findings are in line with the observed effect of the injected amine oxidases on macromolecular synthesis in normal and transformed chick embryo fibroblasts. 相似文献
3.
Amplification of sodium- and potassium-activated adenosinetriphosphatase in HeLa cells by ouabain step selection 总被引:8,自引:0,他引:8
A multistep selection for ouabain resistance was used to isolate a clone of HeLa S3 cells that overproduces the plasma membrane sodium, potassium activated adenosinetriphosphatase (Na+,K+-ATPase). Measurements of specific [3H]ouabain-binding to the resistant clone, C+, and parental HeLa cells indicated that C+ cells contain 8-10 X 10(6) ouabain binding sites per cell compared with 8 X 10(5) per HeLa cell. Plasma membranes isolated from C+ cells by a vesiculation procedure and analyzed for ouabain-dependent incorporation of [32P]phosphate into a 100,000-mol-wt peptide demonstrated a ten- to twelvefold increase in Na+,K+-ATPase catalytic subunit. The affinity of the enzyme for ouabain on the C+ cells was reduced and the time for half maximal ouabain binding was increased compared with the values for the parental cells. The population doubling time for cultures of C+ cells grown in dishes was increased and C+ cells were unable to grow in suspension. Growth of C+ cells in ouabain-free medium resulted in revertant cells, C-, with biochemical and growth properties identical with HeLa. Karyotype analysis revealed that the ouabain-resistant phenotype of the C+ cells was associated with the presence of minute chromosomes which are absent in HeLa and C- cells. This suggests that a gene amplification event is responsible for the Na+,K+-ATPase increase in C+ cells. 相似文献
4.
Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli. 总被引:11,自引:2,他引:9 下载免费PDF全文
D A Jans A L Fimmel L Langman L B James J A Downie A E Senior G R Ash F Gibson G B Cox 《The Biochemical journal》1983,211(3):717-726
The amino acid substitutions in the mutant c-subunits of Escherichia coli F1F0-ATPase coded for by the uncE429, uncE408 and uncE463 alleles affect the incorporation of these proteins into the cell membrane. The DNA sequence of the uncE429 allele differed from normal in that a G leads to A base change occurred at nucleotide 68 of the uncE gene, resulting in glycine being replaced by aspartic acid at position 23 in the c-subunit. The uncE408 and uncE463 mutant DNA sequences were identical and differed from normal in that a C leads to T base change occurred at nucleotide 91 of the uncE gene, resulting in leucine being replaced by phenylalanine at position 31 in the c-subunit. An increased gene dosage of the uncE408 or uncE463 alleles resulted in the incorporation into the membranes of the mutant c-subunits. The results are discussed in terms of the 'Helical Hairpin Hypothesis' of Engelman & Steitz [(1981) Cell 23,411-422]. 相似文献
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Introns are essential for growth-regulated expression of the mouse thymidylate synthase gene. 总被引:5,自引:1,他引:4 下载免费PDF全文
The thymidylate synthase (TS) gene is expressed at much higher levels in proliferating cells than in quiescent cells. We have been studying the sequences that are important for regulating the mouse TS gene. We previously showed that DNA sequences upstream of the essential promoter elements as well as downstream of the ATG codon are both necessary (but neither is sufficient) for normal regulation in growth-stimulated cells. In the present study, we examined the possible roles of the coding region, polyadenylation signal, and introns as downstream regulatory elements. Minigenes consisting of 1 kb of the TS 5'-flanking region, the coding region (with or without various introns at their normal locations), and polyadenylation signals from the TS gene, the human beta-globin gene, and the bovine growth hormone gene were stably transfected into wild-type mouse 3T6 cells. Minigenes that contained introns 5 and 6, 1 and 2, or 1 alone were regulated regardless of which polyadenylation signal was included. A minigene that contained an internally deleted version of intron 1 was also regulated in response to growth stimulation. However, when all introns were omitted, there was little if any change in the level of minigene expression as cells progressed from G1 through S phase. These observations indicate that TS introns contain sequences that are necessary for normal growth-regulated expression of the mouse TS gene. These sequences appear to be associated with sequences that are important for splicing and to function in cooperation with upstream regulatory elements to bring about normal S-phase-specific expression. 相似文献
8.
S. Smith J. Robinson J. Hollyer R. Bhatt S. Ash S. Shaunak 《BMJ (Clinical research ed.)》1996,312(7028):416-420
OBJECTIVE: To develop and evaluate a model of health care for HIV positive patients involving specialist, hospital based teams and primary health care teams. DESIGN: One year retrospective and a 2 1/2 year prospective study. SETTING: Two hospitals in West London and 88 general practitioners in 72 general hospitals. SUBJECTS: 209 adults with HIV infection. INTERVENTION: General practitioners enrolled in the project were faxed structured outpatient clinic summaries. When hospital inpatients were discharged, a brief discharge summary was faxed. General practitioners had access to consultant physicians skilled in HIV medicine through a 24 hour mobile telephone service. An HIV/AIDS management and treatment guide containing relevant local information was produced. Quarterly discussion forums for general practitioners were held, and a regular newsletter was produced. MAIN OUTCOME MEASURES: Hospital attendance and general practitioner consultations; perceived benefits and problems of patients and general practitioners. RESULTS: The average length of a hospital inpatient stay was halved for those patients who had participated in the project for two years, and the average number of visits to the outpatient clinic per month fell for patients with AIDS. There was a substantial increase in the number of visits to general practitioners by patients with AIDS and symptomatic HIV infection. Patients and general practitioners both felt that the standard of health care provided had improved. CONCLUSIONS: This model of health care efficiently and effectively utilised existing teams of hospital and primary health care professionals to provide care for HIV positive patients. Simple, prompt, and regular communication systems which provided information relevant to the needs of general practitioners were central to its success. 相似文献
9.
Ronald J. Ash Gary A. Flynn Rob M.J. Liskamp Harry C.J. Ottenheijm 《Biochemical and biophysical research communications》1984,125(2):784-789
Sparsomycin, ScRs configuration, was the most potent of the four possible stereoisomers as a competitive inhibitor of peptide bond formation. In addition, the configuration of the two chiral centers dictated whether the compound exhibited time- and temperature-dependent inhibition of peptidyl transferase when incubated with polysomes prior to enzyme assay. The data corroborate the thesis that a peptidyl transferase-mediated acylation of the pivotal sulfoxide moiety and subsequent Pummerer rearrangement play a significant role in the inhibitory properties of sparsomycin. 相似文献
10.
Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy 总被引:13,自引:0,他引:13 下载免费PDF全文
A new indirect method for fluorescence localization of proteins making use of the avidin-biotin complex is described. We have prepared both a biotin-modified rabbit heavy meromyosin (BHMM) and a biotin-modified antibody to a smooth muscle myosin. After fixation, cells can be treated with either BHMM, which binds to actin, or the biotinyl antibody, which binds to myosin. In a second step the cell are treated with a fluorescent derivative of avidin (Fl-avidin) which binds to the biotinyl proteins and thus indirectly reveals the location of the cellular action or myosin. 相似文献