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971.
Taru Sharma G Dubey PK Verma OP Pratheesh MD Nath A Sai Kumar G 《Biochemical and biophysical research communications》2012,424(3):378-384
Embryoid bodies (EBs) are used as in vitro model to study early extraembryonic tissue formation and differentiation. In this study, a novel method using three dimensional extracellular matrices for in vitro generation of EBs from buffalo embryonic stem (ES) cells and its differentiation potential by teratoma formation was successfully established. In vitro derived inner cell masses (ICMs) of hatched buffalo blastocyst were cultured on buffalo fetal fibroblast feeder layer for primary cell colony formation. For generation of EBs, pluripotent ES cells were seeded onto four different types of extracellular matrices viz; collagen-IV, laminin, fibronectin and matrigel using undifferentiating ES cell culture medium. After 5days of culture, ESCs gradually grew into aggregates and formed simple EBs having circular structures. Twenty-six days later, they formed cystic EBs over collagen matrix with higher EBs formation and greater proliferation rate as compared to other extracellular matrices. Studies involving histological observations, fluorescence microscopy and RT-PCR analysis of the in vivo developed teratoma revealed that presence of all the three germ layer derivatives viz. ectoderm (NCAM), mesoderm (Flk-1) and endoderm (AFP). In conclusion, the method described here demonstrates a simple and cost-effective way of generating EBs from buffalo ES cells. Collagen-IV matrix was found cytocompatible as it supported buffalo EBs formation, their subsequent differentiation could prove to be useful as promising candidate for ES cells based therapeutic applications. 相似文献
972.
973.
JS Yadav J Bezawada S Yan RD Tyagi RY Surampalli 《Canadian journal of microbiology》2012,58(8):937-952
Yeasts have a tradition in biotechnological applications, and Saccharomyces species are the most dominating representatives. Among the yeast species, Candida krusei has been isolated from different habitats, and in recent years, it has gained increased interest because of its diverse biotechnological role. It is found in many fermented food items and dairy products and has also been exploited for production of biochemicals and enzymes. However, because of its opportunistic pathogenic nature, it draws scientific attention regarding the safety of its industrial exploitation. Candida?krusei generally causes infections in immunocompromised patients, such as those suffering from Human immunodeficiency virus - acquired immune deficiency syndrome, and also in cancer patients. The recent increase in the use of immunosuppressive drugs has increased the chances of C.?krusei infections. Candida?krusei possesses an intrinsic resistance to many triazole antifungal drugs, especially fluconazole, which is a main drug used in antifungal therapy; therefore, there is serious concern regarding its safe industrial use. 相似文献
974.
Michael T. Lipari Wei Li Paul Moran Monica Kong-Beltran Tao Sai Joyce Lai S. Jack Lin Ganesh Kolumam Jose Zavala-Solorio Anita Izrael-Tomasevic David Arnott Jianyong Wang Andrew S. Peterson Daniel Kirchhofer 《The Journal of biological chemistry》2012,287(52):43482-43491
Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg218-Gln219 in the surface-exposed “218 loop.” This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg218-Gln219 more efficiently than furin but also cleaves at Arg215-Phe216. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser153–Arg218), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol. 相似文献
975.
Churamani D Geach TJ Ramakrishnan L Prideaux N Patel S Dale L 《The Journal of biological chemistry》2012,287(10):6974-6978
CD38 is a multifunctional protein possessing ADP-ribosyl cyclase activity responsible for both the synthesis and the degradation of several Ca(2+)-mobilizing second messengers. Although a variety of functions have been ascribed to CD38, such as immune responses, insulin secretion, and social behavior in adults, nothing is known of its role during embryonic development when Ca(2+) signals feature prominently. Here, we report the identification and functional expression of CD38 from Xenopus laevis, a key model organism for the study of vertebrate development. We show that CD38 expression and endogenous ADP-ribosyl cyclase activity are developmentally regulated during cellular differentiation. Chemical or molecular inhibition of CD38 abolished ADP-ribosyl cyclase activity and disrupted elongation of the anterior-posterior axis and differentiation of skeletal muscle, culminating in embryonic death. Our data uncover a previously unknown role for CD38 as an essential regulator of embryonic development. 相似文献
976.
G Protein-coupled receptor kinase 5 is localized to centrosomes and regulates cell cycle progression
Michal AM So CH Beeharry N Shankar H Mashayekhi R Yen TJ Benovic JL 《The Journal of biological chemistry》2012,287(9):6928-6940
G protein-coupled receptor kinases (GRKs) are important regulators of G protein-coupled receptor function and mediate receptor desensitization, internalization, and signaling. While GRKs also interact with and/or phosphorylate many other proteins and modify their function, relatively little is known about the cellular localization of endogenous GRKs. Here we report that GRK5 co-localizes with γ-tubulin, centrin, and pericentrin in centrosomes. The centrosomal localization of GRK5 is observed predominantly at interphase and although its localization is not dependent on microtubules, it can mediate microtubule nucleation of centrosomes. Knockdown of GRK5 expression leads to G2/M arrest, characterized by a prolonged G2 phase, which can be rescued by expression of wild type but not catalytically inactive GRK5. This G2/M arrest appears to be due to increased expression of p53, reduced activity of aurora A kinase and a subsequent delay in the activation of polo-like kinase 1. Overall, these studies demonstrate that GRK5 is localized in the centrosome and regulates microtubule nucleation and normal cell cycle progression. 相似文献
977.
978.
We report the use of surface plasmon-coupled emission (SPCE) as an analytical tool to study the photophysics of surface-adsorbed
fluorescently labeled proteins. The study uses plasma etching of PMMA surface followed by deposition of poly(diallyldimethylammonium
chloride) (PDDA) for surface protein detection. PDDA increases the overall amount of the captured protein and also promotes
dye aggregation. The photon-sorting properties of the SPCE process allows for wavelength separation of the individual components
from the protein–dye aggregates. This has been exploited to study the fluorescence emissions from casein labeled with fluorescein
isothiocyanate and concanavalin A labeled with tetramethylrhodamine. Based on the current findings, the proteins can be used
to measure background fluorescence or to monitor the microenvironments in fluoroimmunoassays on SPCE substrates. 相似文献
979.
Su-Ni Tang Chandan Singh Dara Nall Daniel Meeker Sharmila Shankar Rakesh K Srivastava 《Journal of molecular signaling》2010,5(1):1-15
Background
Much attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables.Results
Our data indicate that human prostate cancer cell lines contain a small population of CD44+CD133+ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44+α2β1+CD133+ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSC's migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.Conclusion
Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment. 相似文献980.
Sai K. Buddai Juliana M. Layzer Genmin Lu Christopher P. Rusconi Bruce A. Sullenger Dougald M. Monroe Sriram Krishnaswamy 《The Journal of biological chemistry》2010,285(8):5212-5223
The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA11F7t) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA11F7t inhibits thrombin formation catalyzed by prothrombinase without obscuring the active site of Xa within the enzyme complex. Selective inhibition of protein substrate cleavage arises from the ability of the aptamer to bind to factor Xa and exclude interactions between the proteinase and cofactor within prothrombinase. Competition for enzyme complex assembly results from the binding of RNA11F7t to factor Xa with nanomolar affinity in a Ca2+-dependent interaction. RNA11F7t binds equivalently to the zymogen factor X as well as derivatives lacking γ-carboxyglutamic acid residues. We suggest that the ability of RNA11F7t to compete for the Xa-Va interaction with surprisingly high affinity likely reflects a significant contribution from its ability to indirectly impact regions of Xa that participate in the proteinase-cofactor interaction. Thus, despite the complexity of the macromolecular interactions that underlie the assembly of prothrombinase, efficient inhibition of enzyme complex assembly and thrombin formation can be achieved by tight binding ligands that target factor Xa in a discrete manner. 相似文献