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51.
A mutant affected in the b subunit (coded by the uncF gene) of the F1F0-ATPase in Escherichia coli was isolated by a localized mutagenesis procedure in which a plasmid carrying the unc genes was mutagenized in vivo. The biochemical properties of cells carrying the uncF515 allele were examined in a strain carrying the allele on a multicopy plasmid and a mutator-induced polar unc mutation on the chromosome. The strain carrying the mutant unc allele was uncoupled with respect to oxidative phosphorylation. Membrane-bound ATPase activity was very low or absent, and membranes were somewhat proton permeable. It was concluded that the F0 sector was assembled. Determination of the DNA sequence of the uncF515 allele showed it differed from wild type in that a G----A substitution occurred at position 392, resulting in glycine being replaced by aspartate at position 131. Genetic complementation tests indicated that the uncF515 allele complemented the uncF476 allele (Gly 9----Asp). Two-dimensional gel electrophoresis of membrane preparations indicated that the uncF515 and uncF476 alleles interrupted assembly of the F1F0-ATPase at different stages.  相似文献   
52.
The interaction of calmodulin with amphiphilic peptides   总被引:16,自引:0,他引:16  
Calmodulin has recently been shown to form exceptionally tight, calcium-dependent complexes with several natural peptides (Kdiss greater than 10(-7) M). These peptides were demonstrated to be capable of forming basic, amphiphilic alpha-helices. To further illustrate the importance of this structural feature for calmodulin binding, several other amphiphilic alpha-helical peptides were tested for their ability to bind calmodulin. To monitor complexes of high affinity (greater than 10(8) M-1), a new competition assay was devised with Sepharose 4B-conjugated melittin. Stoichiometries were assessed by electrophoresis and equilibrium size exclusion chromatography. Three peptides, which were designed to form idealized amphiphilic alpha-helices were tested. The basic peptides, N alpha-9-fluorenylmethoxycarboxyl-(FMOC)-(Leu-Lys-Lys-Leu-Leu-Lys-L eu)1 and FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 bind calmodulin in a 1:1 complex with dissociation constants of 150 and 3 nM, respectively. The acidic peptide, FMOC-(Leu-Glu-Glu-Leu-Leu-Glu-Leu)2 failed to bind calmodulin, even at micromolar concentrations. Complex formation between calmodulin and the 14-residue basic peptide leads to an increase in the helicity of the complex which is attributed to an increase of about 50% in the helicity of the peptide. Calmodulin also interacts with the neutral alpha-helical peptide toxin delta-hemolysin. Concomitant with binding, the fluorescence maximum of the unique Trp residue increases 2-fold and is blue-shifted. A dissociation constant could not be unambiguously estimated though, since delta-hemolysin has a strong tendency to self-aggregate. The above data support our hypothesis that a basic, amphiphilic alpha-helix is a structural feature which underlies the calmodulin-binding properties common to a variety of peptides.  相似文献   
53.
A retrospective study of 2,912 cryptorchid dogs identified 14 breeds with significantly high risk. Among six distinct closely interrelated breed groups (e.g., toy, miniature, and standard poodles), the risk in the smaller breed was always greater than that in the larger relative, suggesting that genetically influenced maldescent could be, in part, related to physical size or the rate of growth of the involved structures. Testicular tumors were diagnosed in 5.7% of the cryptorchid dogs; half had only Sertoli cell tumors, one-third had only seminomas. The relative risk for Sertoli cell tumor or seminoma was not directly related to a familial risk for cryptorchism. Using the health experience of a control population composed of male dogs with anal sac disease (N = 4,184), there is an estimated relative risk of 9.2 in cryptorchid dogs to develop a testis tumor (95% confidence interval, 5.9-14.3) and 4.2 in dogs with inguinal hernia (95% confidence interval, 1.8-9.5). Considering that the anatomical development of the genital tract, testis descent, and tunic relationships in dog are very similar to that in man, and that the associations of cryptorchism and inguinal hernia with testis neoplasms are also similar, the dog should be an excellent model system to further investigate the causes of human cryptorchism.  相似文献   
54.
55.
Restriction of bacteriophage plaque formation in Streptomyces spp.   总被引:15,自引:11,他引:4  
Several Streptomyces species that produce restriction endonucleases were characterized for their ability to propagate 10 different broad host range bacteriophages. Each species displayed a different pattern of plaque formation. A restrictionless mutant of S. albus G allowed plaque formation by all 10 phages, whereas the wild-type strain showed plaques with only 2 phages. DNA isolated from three of the phages was analyzed for the presence of restriction sites for Streptomyces species-encoded enzymes, and a very strong correlation was established between the failure to form plaques on Streptomyces species that produced particular restriction enzymes and the presence of the corresponding restriction sites in the phage DNA. Also, the phages that lacked restriction sites in their DNA generally formed plaques on the corresponding restriction endonuclease-producing hosts at high efficiency. The DNAs from the three phages analyzed also generally contained either many or no restriction sites for the Streptomyces species-produced enzymes, suggesting a strong evolutionary trend to either eliminate all or tolerate many restriction sites. The data indicate that restriction plays a major role in host range determination for Streptomyces phages. Analysis of bacteriophage host ranges of many other uncharacterized Streptomyces hosts has identified four relatively nonrestricting hosts, at least two of which may be suitable hosts for gene cloning. The data also suggest that several restriction systems remain to be identified in the genus Streptomyces.  相似文献   
56.
Recycling of platelet phosphorylation and cytoskeletal assembly   总被引:9,自引:2,他引:7  
The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.  相似文献   
57.
This prospective study attempted to determine if nonreversible occlusive vascular changes in the skin contribute to skin slough after rhytidectomy. The dermal microvasculature from 83 consecutive rhytidectomies was evaluated for intimal proliferation and/or hyalin sclerosis. Occlusive vascular disease increased progressively with age in all patients, but smokers and ex-smokers had significantly greater involvement than nonsmokers at any given age (p = 0.03). Severe occlusive vascular disease and skin slough were associated (p = 0.02), and there was a strong trend toward an association between active smoking and skin slough (p = 0.06). Among smokers, there was a significant relationship between skin slough and failure to abstain from smoking postoperatively (p = 0.006). We conclude that with aging, nonreversible occlusive changes develop in the dermal microvasculature. These changes appear to be accelerated by cigarette smoking. Our data, however, show that these nonreversible occlusive vascular changes by themselves do not completely account for the occurrence of skin slough after rhytidectomy.  相似文献   
58.
Pseudomonas aeruginosa strain PAO1, growing in low-iron medium, produces two siderophores, pyochelin and pyoverdin, in massive bursts as the culture shifts from logarithmic phase to stationary phase. Two medium components, oxygen and iron, prolonged the logarithmic phase when they were added to the medium. Oxygen and iron appeared to be in demand during this period because, as heme synthesis increased in response to the low oxygen concentration in the medium, a situation resulting from the high density of bacteria present in the medium during late log phase, the iron content of the bacteria decreased. These phenomena resulted in the production of massive amounts of siderophores late in the log phase to supply iron for the increased heme synthesis.  相似文献   
59.
60.
Properties of human anticentromere autoantibodies were analyzed. In intact cells or isolated cell fractions, these sera stain the centromeres of mitotic chromosomes and discrete speckles (prekinetochores) in nuclei. Staining is also retained in matrix preparations from nuclei or chromosomes. Immunoprecipitation or immunoblotting demonstrates protein antigens of 14, 20, 23, and 34 kd in HeLa nuclei and chromosomes; immunoprecipitates of nuclei also contain a protein of 15.5 kd. Matrix preparations contain only the 20, 23, and 34 kd species. Absorption of the anticentromere serum with any one of the four nuclear antigens immobilized on nitrocellulose is sufficient to eliminate centromere staining. Using a lysed cell model for microtubule nucleation, anticentromere sera are shown to inhibit specifically the organization of microtubules at the kinetochore.  相似文献   
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