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排序方式: 共有250条查询结果,搜索用时 15 毫秒
1.
Kinetic measurements of fusion of phosphatidylserine-containing vesicles by electron microscopy and fluorometry 总被引:1,自引:0,他引:1
Large unilamellar vesicles (REV) containing phosphatidylserine and phosphatidylethanolamine at a ratio of 1:3 were induced to fuse by adding calcium (4 mM). The kinetics of fusion was monitored by fluorometry using terbium or dipicolinic acid-containing vesicles. The morphology and the states of vesicle aggregation and fusion were examined at approx. 2, 30, 60, 150 and 900 s after calcium addition, by rapid quenching and freeze-fracture electron microscopy. The size and the state of aggregation of vesicles are quantitated from 4000 randomly selected vesicles. The aggregation and fusion kinetics as assayed by fluorescence volume mixing is very well simulated and predicted by the mass action model. The model essentially predicts the time course of the distribution of the aggregates and the increase in size of fused particles as measured by electron microscopy, although in some cases the predicted fusion rate exceeds that by morphometric measurement. No morphological features can be defined as fusion intermediates, although bead-like and rim-like materials may be attributed to the remnants of broken diaphragms between fusion partners. 相似文献
2.
Summary Factors affecting the polyethylene glycol (PEG)-induced membrane fusion were examined. Human erythrocyte membrane ghosts, cytoskeleton-free vesicles budded from erythrocytes, mechanically disrupted erythrocyte vesicles, and recombinant vesicles from glycophorin and egg phosphatidylcholine were used as models. Fusion was monitored by darkfield light microscopy and by freeze-fracture electron microscopy. Osmotic swelling was found necessary for fusion between membrane ghosts following PEG treatment. The sample with the highest fusion percentage was sealed ghosts incubated in hypotonic media after at least 5 min of treatment in <25% PEG. At similar osmolarity, glycerol, dextran and PEG produced progressively more pronounced intramembranous particle (IMP) patching, correlating with their increasing fusion percentages. The patching of IMP preceded cell-cell contact, and occurred without direct PEG-protein interaction. The presence of cytoskeletal elements in small vesicles had no significant effect on fusion, nor on the aggregation of intramembranous particle (IMP) upon PEG treatment. Disrupting the membrane by lysolecithin, dimethylsulfoxide, retinol or mild sonication resulted in the fragmentation of ghosts without an increase in fusion percentage. The purity of the commercial PEG used had no apparent effect on fusion. We concluded that the key steps in PEG-induced fusion of cell membrane are the creation of IMP-free zones, and the osmotic swelling of cells after the formation of bilayer contacts during the PEG treatment. Cell cytoskeleton affects PEG-induced fusion only to the extent of affecting IMP patching. 相似文献
3.
4.
Linkage of tyrosine hydroxylase to four other markers on the short arm of chromosome 11. 总被引:5,自引:2,他引:3 下载免费PDF全文
Tyrosine hydroxylase is the rate-limiting enzyme in catecholamine synthesis; the gene has previously been cloned and localised to the short arm of chromosome 11. Because of the interest in tyrosine hydroxylase as a candidate gene for manic-depressive psychosis and other affective disorders, we carried out family studies to determine the linkage of tyrosine hydroxylase with insulin, beta-globin, D11S12 and Harvey-ras 1, members of a linkage group which has previously been localised to 11p. Using DNA from the Centre d'Etude du Polymorphisme Humain (CEPH) and from two large British pedigrees, we show that tyrosine hydroxylase is closely linked to these four loci (z = 7.36, theta = 0.04 for linkage to insulin) and suggest a gene order based on multipoint mapping. 相似文献
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6.
We report a novel technique that combines high-resolution scanning electron microscopy (SEM) of intracellular structures with backscattered electron imaging (BEI) of colloidal gold-labeled intracellular ligands. Murine dorsal root ganglia were immersion-fixed, freeze-cleaved, labeled with gold complexes, and critical point-dried. Specimens were carbon-coated and viewed by BEI. They were then minimally sputter-coated with gold and previously identified cells relocated by secondary electron imaging (SEI). This permitted increased resolution of intracellular detail while gold particles remained detectable by BEI. Incubation with RNAse-gold and DNAse-gold complexes resulted in specific labeling of cytoplasm and nucleus, respectively. Immunolabeling of neurofilament (NF) and small nuclear ribonucleoproteins (snRNP) resulted in selective labeling of intracellular antigens. Nonspecific binding was abolished by use of 1% skin milk. Specifically, incubation with monoclonal anti-NF68 resulted in labeling of cytoplasm in 66% of neurons, notably of the large cells known to contain large amounts of NF. Satellite cells, which lack NF, showed low levels of background label. Human autoimmune anti-Sm serum recognizes snRNP particles, with the exception of the nucleolar U3 snRNP. Labeling with this serum resulted in specific labeling of 92% of nuclei, with only background labeling over nucleoli and cytoplasm. The results show that it is feasible to employ high-resolution SEM in conjunction with colloidal gold labeling to localize intracellular ligands in situ. 相似文献
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P Deberghes A M Betbeder F Boisard R Blanc J F Delaby S Krivobok R Steiman F Seigle-Murandi EE Creppy 《Mycotoxin Research》1995,11(1):37-47
The toxicity of ochratoxin A (OTA), a mycotoxin produced by fungi ofAspergillus orPenicillium genera is now well documented. Its nephrotoxicity, immunosuppression, teratogenicity, and carcinogenicity have been widely studied. Physical and biochemical methods have been studied to prevent these toxinogenicAspergillus andPenicillium from producing OTA, and/or to destroy the mycotoxin when already produced in a liquid or a solid medium. Repeated freezing at ? 20?C and thawing at + 26?C aleatory reduce OTA production in a liquid medium. Exposure to UV B for different periods of time is efficient in preventing OTA production in a liquid medium. Gamma-irradiation from 2 to 5 kGy gives good results in preventing the production of OTA or destroying it when already produced. Carboxypeptidase is very efficient at 5 units/50 ml in a liquid medium for cleaving the OTA already produced. 相似文献
9.
Ergosta-5,7,9,22-tetraen-3-β-ol (dehydroergosterol) was synthesized and employed as a probe of cholesterol behavior in phospholipid bilayers. Circular dichroism (CD) spectra were obtained. The CD of dehydroergosterol in sonicated egg phosphatidylcholine vesicles was dependent on cholesterol concentration, while in unsonicated egg phosphatidylcholine liposomes and in vesicles obtained by oxctylglucoside dialysis, the CD observed was independent of cholesterol content. The CD of dehydroergosterol in sonicated sphingomyelin vesicles exhibited a different dependence on cholesterol content than seen in sonicated egg phosphatidylcholine vesicles. These data are interpreted in terms of differences between the packing of cholesterol in systems of large and small radii of curvature and in different interactions between dehydroergosterol and phosphatidylcholine and sphingomyelin. 相似文献
10.
L. T. Boni T. P. Stewart J. L. Alderfer S. W. Hui 《The Journal of membrane biology》1981,62(1-2):65-70
Summary Fusion between unilamellar vesicles of both egg phosphatidylcholine and bovine phosphatidylserine was induced by polyethylene glycol. Aggregation and fusion events were monitored by electron microscopy and turbidity measurements. The threshold concentration of polyethylene glycol for aggregation and fusion is found to be independent of lipid concentration. Typically, aggregation of phosphatidylcholine vesicles starts at 2.5% (wt/wt) polyethylene glycol, but fusion is not significant until the polyethylene glycol concentration reaches 35%. Multilamellar vesicles were formed as a result of fusion.Abbreviations PEG
Polyethylene glycol
- IMP
Intramembranous particle
- PC
Phosphatidylcholine
- PS
Phosphatidylserine
- SUV
Small unilamellar vesicles
- MLV
Multilamellar vesicles
- DPPC
Dipalmitoyl phosphatidylcholine
- DSC
Differential scanning calorimetry 相似文献