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21.
Jaime F. Angulo Jaime Schwencke Patrice L. Moreau Ethel Moustacchi Raymond Devoret 《Molecular & general genetics : MGG》1985,201(1):20-24
Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD
+) strains carrying mitochondrial DNA or not (rho
0). This was also true for the following DNA-repair deficient (rad
-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents. 相似文献
22.
23.
In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model 总被引:1,自引:0,他引:1
Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction. 相似文献
24.
Summary Induction of prophage occurs in recA441 mutant lysogens after a shift to 42° C in the presence of adenine. If the synthesis of RecA441 protein is maintained at a low basal level by the presence of a second mutation in the recA441 gene, recA453, induction of prophage is prevented. The ability to induce prophage is restored by the introduction, on a transducing phage, of a second recA gene carrying the recA430 mutation; by itself, the RecA430 protein is devoid of activity against the repressor (Rebollo et al. 1984). In order to explain how the RecA430 protein might complement the RecA441 protein to provide repressor cleavage in a recA453-441 (recA430) diploid lysogen, we characterized the cleavage reaction catalysed by a mixture of these proteins in vitro. Our results suggest that, in the presence of dATP, the RecA441 and RecA430 proteins form mixed multimers on single-stranded DNA, in which the RecA441 protein molecules enhance the DNA binding affinity of RecA430 protein molecules, but RecA430 protein molecules support no cleavage of the repressor.Although the effects of the RecA430 and single-strand binding (SSB) proteins are similar in vitro, we show that the SSB protein cannot substitute for the RecA430 protein in restoring repressor cleavage in a recA453-441 lysogen. Comparison of the stimulatory effect of long single-stranded DNA with that of (dA)14 oligonucleotides on the RecA441 protein-directed cleavage of the repressor in the presence of various nucleoside triphosphates (NTPs) indicates that the cooperative binding of the RecA441 protein to single-stranded DNA stabilizes the RecA protein-DNA complexes so that they remain intact long enough to support cleavage of the repressor. We conclude that the low basal level of the RecA441 protein in a recA453-441 cell is sufficient to cleave the repressor, under conditions where a normal basal level of RecA430 protein is also present allowing the formation of mixed multimers on single-stranded DNA regions normally present in the cell. 相似文献
25.
Molecular cloning and analysis of Staphylococcus aureus chromosomal aminoglycoside resistance genes 总被引:5,自引:0,他引:5
Most of the aminoglycoside resistant Staphylococcus aureus strains isolated in France are resistant to all the antibiotics belonging to this family. Two aminoglycoside-modifying enzymes were detected in the wild-type strains studied: an APH3'III and an AAC6'-APH2". These strains also carry two types of streptomycin resistance: high-level resistance due to chromosomal mutation(s) affecting ribosome affinity and low-level resistance, the mechanism of which was not characterized. All the aminoglycoside resistance genes were located on the chromosome. DNA fragments of 1.5 and 1.95 kb carrying the aphA and aacA genes, respectively, were isolated, by cloning, from the cellular DNA of a clinical isolate. When these genes were introduced into Escherichia coli and Bacillus subtilis strains, the enzymes synthesized were indistinguishable from those produced by the S. aureus strains. When the cellular DNAs of wild-type and resistant strains were hybridized with the cloned fragments, sequences homologous to the fragment carrying the aphA gene were found to be located at the same chromosomal site, while those hybridizing with the fragment carrying the aacA gene were at different chromosomal sites. 相似文献
26.
Identification of distinct accumulation sites of 4-aminoquinoline in chloroquine sensitive and resistant Plasmodium berghei strains 总被引:1,自引:0,他引:1
We report the synthesis of an analogue of chloroquine (CQA) which can be used as a probe to visualize accumulation of 4-aminoquinoline by electron microscopy. A mouse monoclonal antibody against CQA was raised and used for immunodetection by the protein-A gold method on ultrathin cryosections, of CQA treated parasites. We demonstrate that in a P. berghei chloroquine(CQ)-sensitive strain (N strain) the chloroquine analogue used accumulates in the endocytic vacuoles where hemoglobin (Hb) degradation is occurring. In contrast, in a P. berghei CQ-resistant strain (RC strain) the probe was found scattered all over the cytoplasm of the parasite. This result suggests that endocytic vacuoles of the parasite could constitute the site of antimalarial action of CQ. 相似文献
27.
In the Staphylococcus aureus strain harbouring the plasmid RPAL, the resistance to aminoglycoside antibiotics results from two inactivating reactions catalyzed by a 6'-N-aminoglycoside acetyltransferase and a 2"-O-amino-glycoside phosphotransferase. These enzymes are copurified with a constant ratio between the two activities, the purification process consisting in affinity chromatography, native electrophoresis and gel exclusion chromatography. The kinetic mechanisms of each activity have been determined from studies of initial velocities, as well as product and dead-end inhibitions. Both activities follow a random rapid equilibrium mechanism. The substrates and cofactors of one reaction have been tested as effectors of the other reaction. No interaction between the two activities has been observed. However, the GTP cofactor of phosphotransferase protects, at weak concentrations, the acetyltransferase against thermal inactivation, which suggests that the two activities may be associated. 相似文献
28.
29.
H. Moreau A. Bernadac Y. Gargouri F. Benkouka R. Laugier R. Verger 《Histochemistry and cell biology》1989,91(5):419-423
Summary The presence in human gastric juice of a lipase secreted by the gastric mucosae has been reported previously, but its exact cellular origin has not yet been established. Polyclonal antibodies specific to human gastric lipase (HGL) were prepared, and used by an immunofluorescence technique to label cells producing HGL. This immunocytolocalization was correlated with that of pepsin (chief cells) and parietal cells using specific polyclonal or monoclonal antibodies.Our results clearly establish that HGL is exclusively located in the chief cells of fundic mucosa; furthermore, it was found to be always co-located with pepsin. No HGL was observed in the parietal or mucus cells. HGL was always detected intracellularly, either in secretory granules of the apical region of the chief cells, or revealed by more diffuse cytoplasmic labelling.Abbreviations HGL
Human gastric lipase
- SDS PAGE
Sodium dodecyl sulfate-polyAcrylamid gel electrophoresis
- PBS
Phosphate buffer saline 相似文献
30.
Lisette Waits Stephanie Dunkle F. E. Wilkinson P. Moreau Keri Safranski T. Reust Dorothy M. Morré D. J. Morré 《Protoplasma》1990,154(1):8-15
Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [14C]acetate. Time- and temperature-dependent transfer of [14C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [14C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [3H]squalene which was converted into sterol or with [14C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS
dictyosome-like structure(s)
- PBS
phosphatebuffered saline
- HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BSA
bovine serum albumin 相似文献