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991.
992.
We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible--in one experiment--to screen more than 10 of these cells for growth under selective media containing 10(-4) M 5-fluoroorotic acid and 10(-4) M uridine. Cells capable of sustained growth in such media were eventually recovered. Clones of these cells, unlike clones of the parental population, required uridine for growth and contained only 0.39% as much orotate phosphoribosyltransferase, less than 0.5% as much 5-fluoroorotate phosphoribosyltransferase, and 0.013% as much orotidine-5'-monophosphate decarboxylase activity as the parental clones. The parental and variant clones had similar levels of activity for six other enzymes that participate in pyrimidine metabolism. 相似文献
993.
Elena Ingerman Jennifer Ying Hsiao R. Dyche Mullins 《The Journal of cell biology》2013,200(5):619-633
We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly. 相似文献
994.
Chia-Jung Chang Cheng-Chung Lee Yueh-Te Chan Devin L. Trudeau Mei-Huey Wu Chih-Hsuan Tsai Su-May Yu Tuan-Hua David Ho Andrew H.-J. Wang Chwan-Deng Hsiao Frances H. Arnold Yu-Chan Chao 《PloS one》2016,11(3)
Cellulases from Bacillus and Geobacillus bacteria are potentially useful in the biofuel and animal feed industries. One of the unique characteristics of these enzymes is that they are usually quite thermostable. We previously identified a cellulase, GsCelA, from thermophilic Geobacillus sp. 70PC53, which is much more thermostable than its Bacillus homolog, BsCel5A. Thus, these two cellulases provide a pair of structures ideal for investigating the mechanism regarding how these cellulases can retain activity at high temperature. In the present study, we applied the SCHEMA non-contiguous recombination algorithm as a novel tool, which assigns protein sequences into blocks for domain swapping in a way that lessens structural disruption, to generate a set of chimeric proteins derived from the recombination of GsCelA and BsCel5A. Analyzing the activity and thermostability of this designed library set, which requires only a limited number of chimeras by SCHEMA calculations, revealed that one of the blocks may contribute to the higher thermostability of GsCelA. When tested against swollen Avicel, the highly thermostable chimeric cellulase C10 containing this block showed significantly higher activity (22%-43%) and higher thermostability compared to the parental enzymes. With further structural determinations and mutagenesis analyses, a 310 helix was identified as being responsible for the improved thermostability of this block. Furthermore, in the presence of ionic calcium and crown ether (CR), the chimeric C10 was found to retain 40% residual activity even after heat treatment at 90°C. Combining crystal structure determinations and structure-guided SCHEMA recombination, we have determined the mechanism responsible for the high thermostability of GsCelA, and generated a novel recombinant enzyme with significantly higher activity. 相似文献
995.
A-C Tsai C-Y Wang J-P Liou H-C Pai C-J Hsiao J-Y Chang J-C Wang C-M Teng S-L Pan 《Cell death & disease》2014,5(4):e1162
Microtubule-binding agents, such as taxanes and vinca alkaloids, are used in the treatment of cancer. The limitations of these treatments, such as resistance to therapy and the need for intravenous administration, have encouraged the development of new agents. MPT0B271 (N-[1-(4-Methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-1-oxy-isonicotinamide), an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. Tubulin polymerization assay and immunofluorescence experiment showed that MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels. MPT0B271 reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of P-glycoprotein (P-gp), as determined by flow cytometric analysis of rhodamine-123 (Rh-123) dye efflux and the calcein acetoxymethyl ester (calcein AM) assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by the up-regulation of cyclin B1, p-Thr161 Cdc2/p34, serine/threonine kinases polo-like kinase 1, aurora kinase A and B and the downregulation of Cdc25C and p-Tyr15 Cdc2/p34 protein levels. The appearance of MPM2 and the nuclear translocation of cyclin B1 denoted M phase arrest in MPT0B271-treated cells. Moreover, MPT0B271 induced cell apoptosis in a concentration-dependent manner; it also reduced the expression of Bcl-2, Bcl-xL, and Mcl-1 and increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP). Finally, this study demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the human non-small cell lung cancer A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers. 相似文献
996.
While pre-treating a fat transplant recipient site with negative pressure has shown promise for increasing the fat survival rate, the underlying mechanisms have not been investigated, partly due to challenges related to immobilization of vacuum domes on large animal subjects. The aim of this study was to examine the effect of negative pressure treatment by External Tissue Expansion Device (ETED) on fat grating recipient sites in a porcine model. The ETED was designed to provide negative pressure on the dorsum of swine. Pressure treatment (-70 mmHg) was applied for 1 or 3 hours every other day for 10 and 20 treatments. The treated areas (3.5 cm in diameter) were harvested and examined for histological changes, vessel density, cell proliferation (Ki67) and growth factor expression (FGF-1, VEGF and PDGB-bb). The application of the ETED increased epidermis thickness even after 1-hour treatments repeated 10 times. The results of Ki67 analysis suggested that the increasing thickness was due to cell proliferation in the epidermis. There was a more than two-fold increase in the vessel density, indicating that the ETED promotes vascularization. Unexpectedly, the treatment also increased the number of hair follicles. Negative pressure provided by the ETED increases the thickness of epidermis section of tissue, cell proliferation and vessel density. The porcine model provides a better representation of the effect of the ETED on skin tissue compared to small animal models and provides an environment for studying the mechanisms underlying the clinical benefits of negative pressure treatment. 相似文献
997.
998.
Hyphae of light-grown V. agaricinum (Link) Corda contain many lipid bodies. The hypae were disrupted and the membranes immediately pelleted by a 48 000 g spin for 30 min. The major part (90%) of the carotenoids was found in a lipid layer on top of the supernatant. Carotenoids extracted from the lipid layer in diethyl ether had an absorption maximum at 488 nm. On the basis of spectra in different organic solvents the main component was tentatively. Thin layer chromatography of extracted and saponified carotenoids revealed the presence of one major and one minor component.
The 48 000 g pellet contained 80% of the NAD+ -dependent malate dehydrogenase (marker for mitrochondria), 25% of the antimycin-resistant NADH-cytochrome c reductase (marker for mitochodria and endoplasmic reticulum) and 10% of the proteins and the carotenoids. The latter were different from the carotenoids in the lipid layer in that the absorption maximum was at 471 nm in diethyl ether.
Further fractionation of the 48 000 g pellet showed the presence of carotenoids in both mitochondria and endoplasmic reticulum (ER) all with an absorption maximum at 471 nm. On a thin layer plate these carotenoids migrated in one band coinciding with the minor band of the lipid layer. On a continous Percoll gradient two main areas of mitochodrial activity (1.068 and 1.063 g ml−1 ) were clearly separated from the ER (1.059 g ml−1 ). The carotenoid content was about 0.6 and 0.7–1.0 μg (mg protein)−1 for mitochondria and ER, respectively, equivalent to 1–2 carotenoid molecules per 1000 lipid molecules. The possible role of these membrane-bound carotenoid molecules is discussed. 相似文献
The 48 000 g pellet contained 80% of the NAD
Further fractionation of the 48 000 g pellet showed the presence of carotenoids in both mitochondria and endoplasmic reticulum (ER) all with an absorption maximum at 471 nm. On a thin layer plate these carotenoids migrated in one band coinciding with the minor band of the lipid layer. On a continous Percoll gradient two main areas of mitochodrial activity (1.068 and 1.063 g ml
999.
Shian-Ying Sung Chia-Hui Liao Hsun-Pai Wu Wan-Chi Hsiao I-Hui Wu Jinpu Yu Sue-Hwa Lin Chia-Ling Hsieh 《PloS one》2013,8(8)
Bone marrow-derived mesenchymal stem cells (MSCs) are able to migrate to tumors, where they promote tumorigenesis and cancer metastasis. However, the molecular phenotype of the recruited MSCs at the tumor microenvironment and the genetic programs underlying their role in cancer progression remains largely unknown. By using a three-dimensional rotary wall vessel coculture system in which human MSCs were grown alone or in close contact with LNCaP, C4-2 or PC3 prostate cancer cell lines, we established in
vitro matched pairs of normal and cancer-associated MSC derivatives to study the stromal response of MSCs to prostate cancer. We observed that prostate cancer-associated MSCs acquired a higher potential for adipogenic differentiation and exhibited a stronger ability to promote prostate cancer cell migration and invasion compared with normal MSCs both in vitro and in experimental animal models. The enhanced adipogenesis and the pro-metastatic properties were conferred by the high levels of IL-6 secretion by cancer-associated MSCs and were reversible by functionally inhibiting of IL-6. We also found that IL-6 is a direct target gene for the let-7 microRNA, which was downregulated in cancer-associated MSCs. The overexpression of let-7 via the transfection of let-7 precursors decreased IL-6 expression and repressed the adipogenic potential and metastasis-promoting activity of cancer-associated MSCs, which was consistent with the inhibition of IL-6 3′UTR luciferase activity. Conversely, the treatment of normal MSCs with let-7 inhibitors resulted in effects similar to those seen with IL-6. Taken together, our data demonstrated that MSCs co-evolve with prostate cancer cells in the tumor microenvironment, and the downregulation of let-7 by cancer-associated MSCs upregulates IL-6 expression. This upregulation triggers adipogenesis and facilitates prostate cancer progression. These findings not only provide key insights into the molecular basis of tumor-stroma interactions but also pave the way for new treatments for metastatic prostate cancer. 相似文献
1000.