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排序方式: 共有201条查询结果,搜索用时 265 毫秒
81.
Geoghegan JC Valdes PA Orem NR Deleault NR Williamson RA Harris BT Supattapone S 《The Journal of biological chemistry》2007,282(50):36341-36353
The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro.Ina PMCA reaction lacking PrPSc template seed, synthetic polyA RNA molecules induce hamster HaPrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an approximately 1-2.5-kb subset of [32P]polyA RNA molecules from a heterogeneous mixture ranging in size from approximately 0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions. 相似文献
82.
Kim EJ Kramer SF Hebert CG Valdes JJ Bentley WE 《Biotechnology and bioengineering》2007,98(3):645-654
RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system. 相似文献
83.
Lisa A. Gurski Xian Xu Lyana N. Labrada Ngoc T. Nguyen Longxi Xiao Kenneth L. van Golen Xinqiao Jia Mary C. Farach-Carson 《PloS one》2012,7(11)
To study the individual functions of hyaluronan interacting proteins in prostate cancer (PCa) motility through connective tissues, we developed a novel three-dimensional (3D) hyaluronic acid (HA) hydrogel assay that provides a flexible, quantifiable, and physiologically relevant alternative to current methods. Invasion in this system reflects the prevalence of HA in connective tissues and its role in the promotion of cancer cell motility and tissue invasion, making the system ideal to study invasion through bone marrow or other HA-rich connective tissues. The bio-compatible cross-linking process we used allows for direct encapsulation of cancer cells within the gel where they adopt a distinct, cluster-like morphology. Metastatic PCa cells in these hydrogels develop fingerlike structures, “invadopodia”, consistent with their invasive properties. The number of invadopodia, as well as cluster size, shape, and convergence, can provide a quantifiable measure of invasive potential. Among candidate hyaluronan interacting proteins that could be responsible for the behavior we observed, we found that culture in the HA hydrogel triggers invasive PCa cells to differentially express and localize receptor for hyaluronan mediated motility (RHAMM)/CD168 which, in the absence of CD44, appears to contribute to PCa motility and invasion by interacting with the HA hydrogel components. PCa cell invasion through the HA hydrogel also was found to depend on the activity of hyaluronidases. Studies shown here reveal that while hyaluronidase activity is necessary for invadopodia and inter-connecting cluster formation, activity alone is not sufficient for acquisition of invasiveness to occur. We therefore suggest that development of invasive behavior in 3D HA-based systems requires development of additional cellular features, such as activation of motility associated pathways that regulate formation of invadopodia. Thus, we report development of a 3D system amenable to dissection of biological processes associated with cancer cell motility through HA-rich connective tissues. 相似文献
84.
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86.
Aliya Sarmanova Michelle Hall Gwen S. Fernandes Archan Bhattacharya Ana M. Valdes David A. Walsh Michael Doherty Weiya Zhang 《Arthritis research & therapy》2017,19(1):281
Background
An important role for synovial pathology in the initiation and progression of knee osteoarthritis has been emphasised recently. This study aimed to examine whether ultrasonography-detected synovial changes associate with knee pain (KP) in a community population.Methods
A case–control study was conducted to compare people with early KP (n?=?298), established KP (n?=?100) or no KP (n?=?94) at baseline. Multinomial logistic regression was used to estimate the odds ratio (OR) and 95% confidence interval (CI) between groups adjusted for radiographic osteoarthritis (ROA) severity and other confounding factors. After 1 year, 255 participants with early and established KP completed the follow-up questionnaire for changes in KP. Logistic regression with adjustment was used to determine predictors of KP worsening.Results
At baseline, effusion was associated with early KP (OR 2.64, 95% CI 1.57–4.45) and established KP (OR 5.07, 95% CI 2.74–9.38). Synovial hypertrophy was also associated with early KP (OR 5.43, 95% CI 2.12–13.92) and established KP (OR 13.27, 95% CI 4.97–35.43). The association with effusion diminished when adjusted for ROA. Power Doppler signal was uncommon (early KP 3%, established KP 2%, controls 0%). Baseline effusion predicted worsening of KP at 1 year (OR 1.95, 95% CI 1.05–3.64). However, after adjusting for ROA, the prediction was insignificant (adjusted OR 0.95, 95% CI 0.44–2.02).Conclusions
Ultrasound effusion and synovial hypertrophy are associated with KP, but only effusion predicts KP worsening. However, the association/prediction is not independent from ROA. Power Doppler signal is uncommon in people with KP. Further study is needed to understand whether synovitis is directly involved in different types of KP.87.
88.
Minicircle variable region probes for characterization of Leishmania (Viannia) species. 总被引:1,自引:0,他引:1
O Fernandes M P Catanho I Segura L A Labrada R Derré N Saravia W Degrave 《The Journal of parasitology》1999,85(3):563-568
The minicircle molecules present in the kinetoplast DNA (kDNA) network constitute a particularly useful molecular tool because they are a multicopy target and present a variable region that differs among minicircle classes in the same network. Using the polymerase chain reaction (PCR) and a set of primers directed outwardly from the minicircle conserved region, it is possible to prepare molecular probes representing the pool of variable regions from the different minicircle classes in the kDNA. In order to examine the specificity of the minicircle variable region as hybridization probes in Leishmania (Viannia) species, such fragments were amplified from reference strains and from a panel of isolates representing the zymodeme diversity of Leishmania (Viannia) in Colombia. The size of the amplified products was conserved in Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, and Leishmania (Viannia) panamensis (650 bp) and diverged in Leishmania (Viannia) equatorensis and Leishmania (Viannia) colombiensis (850 bp). The amplified products were further hybridized to variable region pools of Leishmania braziliensis, Leishmania panamensis, Leishmania guyanensis, and Leishmania equatorensis reference strains. The results obtained from the hybridization experiments support this approach as a means of defining relationships among strains. Hybridization allowed homologies to be perceived, whereas restriction fragment length analysis of the amplified products yielded strain-specific profiles. Apparently, L. (V.) equatorensis and L. (V.) colombiensis minicircle variable regions have no or only low homology with those of other Leishmania (Viannia) species, showing the divergence of those species within the subgenus. 相似文献
89.
Lynn F Cherkas Elizabeth C Oelsner Y T Mak Anna Valdes Tim D Spector 《Twin research》2004,7(6):649-658
In humans, in contrast to animals, the genetic influences on infidelity are unclear. We report here a large study of over 1600 unselected United Kingdom female twin pairs who confidentially reported previous episodes of infidelity and total lifetime number of sexual partners, as well as attitudes towards infidelity. Our findings demonstrate that infidelity and number of sexual partners are both under moderate genetic influence (41% and 38% heritable, respectively) and the genetic correlation between these two traits is strong (47%). Conversely, attitudes towards infidelity are driven by shared and unique environmental, but not genetic, influences. A genome-wide linkage scan identified three suggestive but nonsignificant linkage areas associated with infidelity and number of sexual partners on chromosomes 3, 7 and 20 with a maximum LOD score of 2.46. We were unsuccessful in associating infidelity or number of sexual partners with a locus implicated in other mammals' sexual behavior, the vasopressin receptor gene. Nonetheless, our findings on the heritability of sexual infidelity and number of sexual partners provide support for certain evolutionary theories of human sexual behavior, as well as justifying further genetic and molecular research in this domain. 相似文献
90.
Effects of receptor concentration, media pH and storage on nicotinic receptor-transmitted signal in a fiber-optic biosensor 总被引:3,自引:0,他引:3
The optical signal generated by a nicotinic acetylcholine receptor (nAChR)-based optical biosensor was dependent on the density of toxin binding sites (i.e. receptors) immobilized on the surface of the fiber. The maximum density of nAChR receptors absorbed on the optic fibers, measured using [125I]-alpha-bungarotoxin (the alpha-neurotoxin of the Bungarus snake venom) binding, was 6.2 pmol of receptor sites per fiber. Incubation time, that was required for maximal noncovalent immobilization of the receptor protein on the fiber, was less than 10 min. Immobilization of the nAChR protein on the quartz fiber was affected by pH of the medium, with pH 3.5 as the optimal. Stored in phosphate buffered saline (PBS) or in a dry nitrogen atmosphere, the nAChR optical sensor showed no loss of activity over the first 3 days, then showed a slow but gradual loss in activity (45% over the next 30 days). 相似文献