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31.
Inhibition of clinical human immunodeficiency virus (HIV) type 1 isolates in primary CD4+ T lymphocytes by retroviral vectors expressing anti-HIV genes. 总被引:4,自引:3,他引:1
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T Vandendriessche M K Chuah L Chiang H K Chang B Ensoli R A Morgan 《Journal of virology》1995,69(7):4045-4052
Gene therapy may be of benefit in human immunodeficiency virus type 1 (HIV-1)-infected individuals by virtue of its ability to inhibit virus replication and prevent viral gene expression. It is not known whether anti-HIV-1 gene therapy strategies based on antisense or transdominant HIV-1 mutant proteins can inhibit the replication and expression of clinical HIV-1 isolates in primary CD4+ T lymphocytes. We therefore transduced CD4+ T lymphocytes from uninfected individuals with retroviral vectors expressing either HIV-1-specific antisense-TAR or antisense-Tat/Rev RNA, transdominant HIV-1 Rev protein, and a combination of antisense-TAR and transdominant Rev. The engineered CD4+ T lymphocytes were then infected with four different clinical HIV-1 isolates. We found that replication of all HIV-1 isolates was inhibited by all the anti-HIV vectors tested. Greater inhibition of HIV-1 was observed with transdominant Rev than with antisense RNA. We hereby demonstrated effective protection by antisense RNA or transdominant mutant proteins against HIV-1 infection in primary CD4+ T lymphocytes using clinical HIV-1 isolates, and this represents an essential step toward clinical anti-HIV-1 gene therapy. 相似文献
32.
33.
Summary Intermolecular transposition of Tn2660 into pCR1 was measured at 30°C in recA
– and recA
+ hosts as between 2.6 and 5.5x10–3, a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 104 transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10–3 and 10–4 when a mutant Tn2660 (resulting in the synthesis of a temperaturesensitive -lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition. 相似文献
34.
Summary Inverse repeats of the transposon Tn2660 in either a ColEl or an R6K replicon, with or without inversions of the parental DNA sequences between the repeats, show no detectable (<2%) evidence of recombination between the repeats after 60 generations of growth in either recA or recA
+ hosts. In contrast, attempts made to construct plasmids which carry two direct repeats by in vitro cleavage and ligation in a recA host were unsuccessful, although homologous plasmids with inverse repeats could be constructed, and other plasmids were found consistent with products of recombination between the direct repeats of a transient intermediate structure. It is concluded that in recA or recA
+ hosts recombination between direct repeats of a transposon is frequent, whereas recombination between inverse repeats of a homologous structure has not been observed. A model to explain this difference depends upon a mechanism that produces a nick in only one of the pair of strands at the internal resolution site (IRS) sequence of the transposon. 相似文献
35.
The two major forms of rabbit liver microsomal cytochrome P-450, P-450LM2 and P-450LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450LM4 has a much higher tryptophan content than P-450LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450LM2 gives a much stronger Type I difference spectrum than does P-450LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450LM2 and P-450LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in α-helical content, particularly with P-450LM4, in the presence of the phospholipid. 相似文献
36.
Summary Using gel filtration chromatography, we find a single peak of deoxythymidine phosphorylating activity in Chlamydomonas reinhardti. This activity has characteristics of a thymidine kinase, in that (1) it will utilize ATP (or dATP) or CTP (or dCTP) as phosphoryl donor, but not AMP or phenyl phosphate, and (2) it is inhibited by dTTP (and less so by dTDP, dUTP, and dUDP) but is unaffected by 3–5 cyclic AMP.Partially purified Chlamydomonas thymidine kinase has a pH optimum near 8.5, and a molecular weight of 80,000 to 85,000 daltons. Kinetic studies indicate a ping-pong mechanism with a Km for thymidine of 1.5x10-7 moles per liter. 5-Bromo-and 5-fluorodeoxyuridine, and to a lesser degree deoxyuridine, are competitive inhibitors, but significant phosphorylation of these nucleosides could not be demonstrated in vitro by thymidine kinase.While thymidine is phosphorylated to dTMP by crude Chlamydomonas extracts, greater than 80% of the product formed by the partially purified enzyme is dTTP. Further, the gel filtration elution position of the single deoxythymidylate kinase activity present in cell extracts coincides with that of thymidine kinase. These results suggest that a multifunctional enzyme, rather than three separate phosphorylating activities, may be responsible for dTTP formation.Abbreviations MES
2(N-morpholino) ethanesulfonic acid
- TES
N-tris (hydroxymethyl) methyl-2-amino ethanesulfonic acid
- tris
tris-hydroxyamino methane
- NEM
N-ethyl maleimide
- PEI
polyethyleneimine
- TLC
thin-layer chromatography; nucleotides abbreviated by CBN rules 相似文献
37.
Gong CS Chen LF Flickinger MC Chiang LC Tsao GT 《Applied and environmental microbiology》1981,41(2):430-436
d-Xylulose, an intermediate of d-xylose catabolism, was observed to be fermentable to ethanol and carbon dioxide in a yield of greater than 80% by yeasts (including industrial bakers' yeast) under fermentative conditions. This conversion appears to be carried out by many yeasts known for d-glucose fermentation. In some yeasts, xylitol, in addition to ethanol, was produced from d-xylulose. Fermenting yeasts are also able to produce ethanol from d-xylose when d-xylose isomerizing enzyme is present. The results indicate that ethanol could be produced from d-xylose in a yield of greater than 80% by a two-step process. First, d-xylose is converted to d-xylulose by xylose isomerase. d-Xylulose is then fermented to ethanol by yeasts. 相似文献
38.
A high-d-xylulose mixture (d-xylose-d-xylulose = 33:67) was prepared from the cold ethanol extract of preisomerized d-xylose solution (d-xylose-d-xylulose = 77:23). Fusarium oxysporum f. sp. lini and Aspergillus niger were demonstrated to preferentially utilize d-xylose in the mixture of d-xylose and d-xylulose. Chromatographically pure d-xylulose was thus obtained in 90% yield. A high-d-xylulose mixture was also incubated with Rhodotorula toruloides, Klebsiella pneumoniae, Candida utilis, or Mucor rouxii.d-Xylose and d-xylulose were simultaneously consumed. When borate was added to the mixture, a d-xylulose-borate complex was formed, and it could be used to protect d-xylulose from being utilized. 相似文献
39.
A radioactive assay for acetylcholinesterase is described. The assay is based on the separation of [14C]acetate from [14C]acetylcholine by differential adsorption of the former on DEAE anion-exchange disks. The procedure is simple and sensitive and eliminates the use of ion-exchange resin columns or organic extractions. Moreover, when unpurified enzyme preparations are assayed, linear steady-state kinetics can be observed with this method as contrasted to the nonlinear colorimetric method using acetylthiocholine and dithiobisnitrobenzoate. This method also permits the detection in biological samples of low levels of acetylcholinesterase activity, which is not detectable by the colorimetric method. Using the present radioactive method, cellular levels of acetylcholinesterase have been surveyed in N4TG1 neuroblastoma cells, NG108-15 neuroblastoma x glioma hybrid cells, H9c2 myoblasts, and 3T3-L1 and 3T3-C2 fibroblasts. 相似文献