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961.
Gustin DJ Sehon CA Wei J Cai H Meduna SP Khatuya H Sun S Gu Y Jiang W Thurmond RL Karlsson L Edwards JP 《Bioorganic & medicinal chemistry letters》2005,15(6):1687-1691
A novel series of competitive, reversible cathepsin S (CatS) inhibitors was discovered and optimized. The 4-(2-keto-1-benzimidazolinyl)-piperidin-1-yl moiety was found to be an effective replacement for the 4-arylpiperazin-1-yl group found in our earlier series of CatS inhibitors. This replacement imparted improved PK properties as well as decreased off-target activity. Optimization of the ketobenzimidazole moiety led to the discovery of the lead compound JNJ 10329670, which represents a novel class of selective, noncovalent, reversible, and orally bioavailable inhibitors of cathepsin S. 相似文献
962.
Three opposing pathways are proposed for the release of malaria parasites from infected erythrocytes: coordinated rupture of the two membranes surrounding mature parasites; fusion of erythrocyte and parasitophorus vacuolar membranes (PVM); and liberation of parasites enclosed within the vacuole from the erythrocyte followed by PVM disintegration. Rupture by cell swelling should yield erythrocyte ghosts; membrane fusion is inhibited by inner-leaflet amphiphiles of positive intrinsic curvature, which contrariwise promote membrane rupture; and without protease inhibitors, parasites would leave erythrocytes packed within the vacuole. Therefore, we visualized erythrocytes releasing P. falciparum using fluorescent microscopy of differentially labeled membranes. Release did not yield erythrocyte ghosts, positive-curvature amphiphiles did not inhibit release but promoted it, and release of packed merozoites was shown to be an artifact. Instead, two sequential morphological stages preceded a convulsive rupture of membranes and rapid radial discharge of separated merozoites, leaving segregated internal membrane fragments and plasma membrane vesicles or blebs at the sites of parasite egress. These results, together with the modulation of release by osmotic stress, suggest a pathway of parasite release that features a biochemically altered erythrocyte membrane that folds after pressure-driven rupture of membranes. 相似文献
963.
964.
965.
Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531 总被引:12,自引:0,他引:12
Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM)
cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative
and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed.
In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445
and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445
and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative
detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed,
which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits
of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were
10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter,
five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR
systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as
bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed
as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531.
The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established
event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for
GM cotton identification and quantification. 相似文献
966.
As an alternative to the mean regression model, the quantile regression model has been studied extensively with independent failure time data. However, due to natural or artificial clustering, it is common to encounter multivariate failure time data in biomedical research where the intracluster correlation needs to be accounted for appropriately. For right-censored correlated survival data, we investigate the quantile regression model and adapt an estimating equation approach for parameter estimation under the working independence assumption, as well as a weighted version for enhancing the efficiency. We show that the parameter estimates are consistent and asymptotically follow normal distributions. The variance estimation using asymptotic approximation involves nonparametric functional density estimation. We employ the bootstrap and perturbation resampling methods for the estimation of the variance-covariance matrix. We examine the proposed method for finite sample sizes through simulation studies, and illustrate it with data from a clinical trial on otitis media. 相似文献
967.
Production of biologically active human interleukin-4 in transgenic tobacco and potato 总被引:2,自引:1,他引:1
Ma S Huang Y Davis A Yin Z Mi Q Menassa R Brandle JE Jevnikar AM 《Plant biotechnology journal》2005,3(3):309-318
Interleukin-4 (IL-4) is a pleiotropic cytokine that plays a key regulatory role in the immune system. Recombinant human IL-4 (rhIL-4) offers great potential for the treatment of cancer, viral and autoimmune diseases. Unfortunately, the high production cost of IL-4 associated with conventional expression systems has, until now, limited broader clinical testing, particularly with regard to the more convenient and safer oral delivery of IL-4 as opposed to parenteral injection in patients. In this study, we investigated the feasibility of transgenic plants for the cost-effective production of rhIL-4. IL-4 expression vectors with different modifications under the control of a constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter were introduced into tobacco by Agrobacterium-mediated transformation. Transgenic tobaccos expressing various levels of rhIL-4 protein were generated. Higher expression was achieved through IL-4 retention in the endoplasmic reticulum (ER), with the maximal accumulation being approximately 0.1% of total soluble protein (TSP) in the leaves. No improvement in expression was further achieved by replacing the native signal peptide of IL-4 with the plant signal peptide. The best rhIL-4-expressing vector shown in tobacco was selected and further transferred into potato plants. The analysis of transgenic tubers also revealed various levels of rhIL-4, with the highest being 0.08% of TSP. Sensitive in vitro T-cell proliferation assays showed that plant-derived rhIL-4 retained full biological activity. These results suggest that plants can be used to produce biologically active rhIL-4 and probably many other mammalian proteins of medical significance. Moreover, the production of plants expressing rhIL-4 will enable the testing of plant rhIL-4 by oral delivery for the treatment of clinical diseases. 相似文献
968.
969.
Yurkovetskiy A Choi S Hiller A Yin M McCusker C Syed S Fischman AJ Papisov MI 《Biomacromolecules》2005,6(5):2648-2658
Modification of proteins with hydrophilic polymers is an effective strategy for regulation of protein pharmacokinetics. However, conjugates of slowly or non-biodegradable materials, such as poly(ethylene glycol), are known to cause long-lasting cell vacuolization, in particular in renal epithelium. Conjugates of more degradable polymers, e.g., polysaccharides, have a significant risk of immunotoxicity. Polymers that combine complete degradability, long circulation in vivo, and low immuno and chemical toxicity would be most beneficial as protein conjugate components. This study explores new fully biodegradable hydrophilic polymers, hydrophilic polyals. They are nontoxic, stable at physiological conditions, and undergo proton-catalyzed hydrolysis at lysosomal pH. The model enzyme-polyal conjugates were prepared with 61-98% yield using conventional and novel conjugation techniques and retained 90-95% of specific activity. The model conjugates showed a significant prolongation of protein circulation in rodents, with a 5-fold reduction in the renal accumulation. The data suggests that hydrophilic polyals may be useful in designing protein conjugates with improved properties. 相似文献
970.
Many genes are involved in mammalian cell apoptosis pathway. These apoptosis genes often contain characteristic functional domains, and can be classified into at least 15 functional groups, according to previous reports. Using an integrated bioinformatics platform for motif or domain search from three public mammalian proteomes (International Protein Index database for human, mouse, and rat), we systematically cataloged all of the proteins involved in mammalian apoptosis pathway. By localizing those proteins onto the genomes, we obtained a gene locus centric apoptosis gene catalog for human, mouse and rat.Further phylogenetic analysis showed that most of the apoptosis related gene loci are conserved among these three mammals. Interestingly, about one-third of apoptosis gene loci form gene clusters on mammal chromosomes, and exist in the three species, which indicated that mammalian apoptosis gene orders are also conserved. In addition, some tandem duplicated gene loci were revealed by comparing gene loci clusters in the three species. All data produced in this work were stored in a relational database and may be viewed at http://pcas.cbi.pku.edu.cn/database/apd.php. 相似文献