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241.
242.
Gangnuss S Sutton-McDowall ML Robertson SA Armstrong DT 《Biology of reproduction》2004,71(4):1135-1141
In mice, exposure of the uterus to seminal plasma at mating initiates an inflammatory response within the endometrium, which is characterized by production of cytokines that recruit and activate leukocytes. We hypothesized that this seminal plasma-induced inflammatory response would extend to the ovary, increasing leukocyte abundance within corpora lutea and potentially enhancing progesterone synthesis. Female mice mated to males with their seminal vesicles surgically removed exhibited fewer macrophages within corpora lutea on the day after mating, compared with females mated to vasectomized or normal, intact males. The mean number of F4/80-positive macrophages and major histocompatibility complex (MHC) class II-positive activated macrophages was approximately 2-fold fewer in the absence of seminal vesicle fluid. The effects of seminal plasma on macrophage abundance subsided by Day 4 and were not accompanied by a change in serum progesterone levels during luteinization (Days 1, 2, or 4 after mating) or luteolysis (Days 6 or 9). In vitro secretion of progesterone from corpora lutea cultured with or without LH also did not differ between treatment groups. There was no effect of seminal plasma deficiency in males on the number of ovulated ova or corpora lutea in females. These results imply that seminal plasma exposure of the female reproductive tract at mating augments the macrophage population of newly formed corpora lutea, although these additional macrophages seem not to play a role in steroidogenesis and may instead be involved in tissue remodeling within corpora lutea. 相似文献
243.
Adenovirus type-5 entry and disassembly followed in living cells by FRET, fluorescence anisotropy, and FLIM
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Martin-Fernandez M Longshaw SV Kirby I Santis G Tobin MJ Clarke DT Jones GR 《Biophysical journal》2004,87(2):1316-1327
We have used fluorescence resonance energy transfer (FRET) to follow the process of capsid disassembly for adenovirus (Ad) serotype 5 (Ad5) in living CHO-CAR cells. Ad5 were weakly labeled on their capsid proteins with FRET donor and acceptor fluorophores. A progressive decrease in FRET efficiency recorded during Ad5 uptake revealed that the time course of Ad5 capsid disassembly has two sequential protein dissociation rates with half-times of 3 and 60 min. Fluorescence anisotropy measurements of the segmental motions of fluorophores on Ad5 indicate that the first rate is linked to the detachment from the capsid of the protruding, flexible fiber proteins. The second rate was shown to report on the combined dissociation of protein IX, penton base, and hexons, which form the rigid icosahedral capsid shell. Fluorescence lifetime imaging microscopy measurements using a pH-sensitive probe provided information on the pH of the microenvironment of Ad5 particles during intracellular trafficking, and confirmed that the fast fiber dissociation step occurred at the onset of endocytosis. The slower dissociation phase was shown to coincide with the escape of Ad5 from endocytic compartments into the cytosol, and its arrival at the nuclear membrane. These results demonstrate a rapid, quantitative live-cell assay for the investigation of virus-cell interactions and capsid disassembly. 相似文献
244.
245.
Distribution, genetic diversity, and variable expression of the gene encoding hyaluronate lyase within the Streptococcus suis population 总被引:4,自引:0,他引:4
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Although Streptococcus suis is an economically important pathogen of pigs and an occasional cause of zoonotic infections of humans knowledge of crucial virulence factors, and as a consequence targets for therapeutic or prophylactic intervention, remains limited. Here we describe a detailed study of the distribution, diversity, and in vitro expression of hyaluronate lyase, a protein implicated as a virulence factor of many mucosal pathogens. The gene encoding hyaluronate lyase, hyl, was present in all 309 bona fide S. suis isolates examined representing diverse serotypes, geographic sources, and clinical backgrounds. Examination of the genetic diversity of hyl by RFLP and sequence analysis indicated a pattern of diversity shared by many gram-positive surface proteins with a variable 5' region encoding the most distal cell surface-exposed regions of the protein and a much more conserved 3' region encoding domains more closely associated with the bacterial cell. Variation occurs by several mechanisms, including the accumulation of point mutations and deletion and insertion events, and there is clear evidence that genetic recombination has contributed to molecular variation in this gene. Despite the ubiquitous presence of hyl, the corresponding enzyme activity was detected in fewer than 30% of the 309 isolates. In several cases this lack of activity correlates with the presence of mutations (either sequence duplications or point mutations) within hyl that result in a truncated polypeptide. There is a striking absence of hyaluronate lyase activity in a large majority of isolates from classic S. suis invasive disease, indicating that this protein is probably not a crucial virulence factor, although activity is present in significantly higher numbers of isolates associated with pneumonia. 相似文献
246.
247.
Comparative genomic hybridization-array analysis enhances the detection of aneuploidies and submicroscopic imbalances in spontaneous miscarriages
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Schaeffer AJ Chung J Heretis K Wong A Ledbetter DH Lese Martin C 《American journal of human genetics》2004,74(6):1168-1174
Miscarriage is a condition that affects 10%-15% of all clinically recognized pregnancies, most of which occur in the first trimester. Approximately 50% of first-trimester miscarriages result from fetal chromosome abnormalities. Currently, G-banded chromosome analysis is used to determine if large-scale genetic imbalances are the cause of these pregnancy losses. This technique relies on the culture of cells derived from the fetus, a technique that has many limitations, including a high rate of culture failure, maternal overgrowth of fetal cells, and poor chromosome morphology. Comparative genomic hybridization (CGH)-array analysis is a powerful new molecular cytogenetic technique that allows genomewide analysis of DNA copy number. By hybridizing patient DNA and normal reference DNA to arrays of genomic clones, unbalanced gains or losses of genetic material across the genome can be detected. In this study, 41 product-of-conception (POC) samples, which were previously analyzed by G-banding, were tested using CGH arrays to determine not only if the array could identify all reported abnormalities, but also whether any previously undetected genomic imbalances would be discovered. The array methodology detected all abnormalities as reported by G-banding analysis and revealed new abnormalities in 4/41 (9.8%) cases. Of those, one trisomy 21 POC was also mosaic for trisomy 20, one had a duplication of the 10q telomere region, one had an interstitial deletion of chromosome 9p, and the fourth had an interstitial duplication of the Prader-Willi/Angelman syndrome region on chromosome 15q, which, if maternally inherited, has been implicated in autism. This retrospective study demonstrates that the DNA-based CGH-array technology overcomes many of the limitations of routine cytogenetic analysis of POC samples while enhancing the detection of fetal chromosome aberrations. 相似文献
248.
An immobilization procedure for protein on surface plasmon resonance sensor (SPR) chips is described. The target protein, cyclophilin D, is thereby genetically linked to a mutant of the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT). The procedure includes the immobilization of an alkylguanine derivative on the surface by amine coupling and contact of the surface with a solution of the fusion protein (TCypD-hAGT). TCypD-hAGT could be immobilized using buffer solutions of purified protein or cell extracts. High densities of covalently linked proteins were achieved by either procedure. Binding experiments performed with the ligand cyclosporin A indicate relative binding activities close to 100%. The KD value (12 nM) and the kinetic rate constants kon (3 × 105 M−1s−1) and koff (4 × 10−3s−1) are given and compared to values determined for cyclophilin D linked to the surface by amide coupling chemistry. The KD value is in excellent agreement with the KD value determined in solution by fluorescence titration. 相似文献
249.
Under anaerobiosis, the mitochondrion of Saccharomyces cerevisiae is restricted to unstructured promitochondria. These promitochondria provide unknown metabolic functions that are required for growth. Since high glucose concentrations are mainly fermented by S. cerevisiae during stationary phase (due to nitrogen starvation), an optimized promitochondria isolation procedure was investigated. Firstly, the unusual promitochondria ultrastructure was checked in intact cells by electron microscopy using a cryo-fixation and freeze-substitution method. The rapid response of anaerobic cells toward oxygen justified the adoption of several critical steps, especially during spheroplasting. Control of spheroplasting was accompanied by a systematic analysis of spheroplast integrity, which greatly influence the final quality of promitochondria. Despite the presence of remnant respiratory chain components under anaerobiosis, characterization of isolated promitochondria by high-resolution respirometry did not reveal any antimycin A- and myxothiazol-sensitive NADH and NADPH oxidase activities. Moreover, the existence of a cyanide-sensitive and non-phosphorylating NADH-dependent oxygen consumption in promitochondria was demonstrated. Nevertheless, promitochondria only slightly contribute to the overall oxygen consumption capacity observed in highly glucose-repressed anaerobic cells. 相似文献
250.
Schaeffer LM McCormack FX Wu H Weiss AA 《Journal of immunology (Baltimore, Md. : 1950)》2004,173(3):1959-1965
Surfactant protein A (SP-A) plays an important role in the innate immune defense of the respiratory tract. SP-A binds to lipid A of bacterial LPS, induces aggregation, destabilizes bacterial membranes, and promotes phagocytosis by neutrophils and macrophages. In this study, SP-A interaction with wild-type and mutant LPS of Bordetella pertussis, the causative agent of whooping cough, was examined. B. pertussis LPS has a branched core structure with a nonrepeating trisaccharide, rather than a long-chain repeating O-Ag. SP-A did not bind, aggregate, nor permeabilize wild-type B. pertussis. LPS mutants lacking even one of the sugars in the terminal trisaccharide were bound and aggregated by SP-A. SP-A enhanced phagocytosis by human monocytes of LPS mutants that were able to bind SP-A, but not wild-type bacteria. SP-A enhanced phagocytosis by human neutrophils of LPS-mutant strains, but only in the absence of functional adenylate cyclase toxin, a B. pertussis toxin that has been shown to depress neutrophil activity. We conclude that the LPS of wild-type B. pertussis shields the bacteria from SP-A-mediated clearance, possibly by sterically limiting access to the lipid A region. 相似文献