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21.
ATP-dependent drug transport by human P-glycoprotein (Pgp, ABCB1) involves a coordinated communication between its drug-binding site (substrate site) and the nucleotide binding/hydrolysis domain (ATP sites). It has been demonstrated that the two ATP sites of Pgp play distinct roles within a single catalytic turnover; whereas ATP binding or/and hydrolysis by one drives substrate translocation and dissociation, the hydrolytic activity of the other resets the transporter for the subsequent cycle (Sauna, Z. E., and Ambudkar, S. V. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 2515-2520; Sauna, Z. E., and Ambudkar, S. V. (2001) J. Biol. Chem. 276, 11653-11661). Trapping of ADP (or 8-azido-ADP) and vanadate (ADP.Vi or 8-azido-ADP.Vi) at the catalytic site, following nucleotide hydrolysis, markedly reduces the affinity of Pgp for its transport substrate [125I]iodoarylazidoprazosin ([125I]IAAP), resulting in dissociation of the latter. Regeneration of the [125I]IAAP site requires an additional round of nucleotide hydrolysis. In this study, we demonstrate that certain thioxanthene-based allosteric modulators, such as cis-(Z)-flupentixol and its closely related analogs, induce regeneration of [125I]IAAP binding to vanadate-trapped (or fluoroaluminate-trapped) Pgp without any further nucleotide hydrolysis. Regeneration was facilitated by dissociation of the trapped nucleotide and vanadate. Once regenerated, the substrate site remains accessible to [125I]IAAP even after removal of the modulator from the medium, suggesting a modulator-induced relaxation of a constrained transition state conformation. Consistent with this, limited trypsin digestion of vanadate-trapped Pgp shows protection by cis-(Z)-flupentixol of two Pgp fragments (approximately 60 kDa) recognizable by a polyclonal antiserum specific for the NH2-terminal half. No regeneration was observed in the Pgp mutant F983A that is impaired in modulation by flupentixols, indicating involvement of the allosteric modulator site in the phenomenon. In summary, the data demonstrate that in the nucleotide-trapped low affinity state of Pgp, the allosteric site remains accessible and responsive to modulation by flupentixol (and its closely related analogs), which can reset the high affinity state for [125I]IAAP binding without any further nucleotide hydrolysis. 相似文献
22.
Dey D Shepherd A Pachuau J Martin-Caraballo M 《American journal of physiology. Cell physiology》2011,300(3):C576-C587
Neuropoietic cytokines such as ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) stimulate the functional expression of T-type Ca(2+) channels in developing sensory neurons. However, the molecular and cellular mechanisms involved in the cytokine-evoked membrane expression of T-type Ca(2+) channels are not fully understood. In this study we investigated the role of LIF in promoting the trafficking of T-type Ca(2+) channels in a heterologous expression system. Our results demonstrate that transfection of HEK-293 cells with the rat green fluorescent protein (GFP)-tagged T-type Ca(2+) channel α(1H)-subunit resulted in the generation of transient Ca(2+) currents. Overnight treatment of α(1H)-GFP-transfected cells with LIF caused a significant increase in the functional expression of T-type Ca(2+) channels as indicated by changes in current density. LIF also evoked a significant increase in membrane fluorescence compared with untreated cells. Disruption of the Golgi apparatus with brefeldin A inhibited the stimulatory effect of LIF, indicating that protein trafficking regulates the functional expression of T-type Ca(2+) channels. Trafficking of α(1H)-GFP was also disrupted by cotransfection of HEK-293 cells with the dominant-negative form of ADP-ribosylation factor (ARF)1 but not ARF6, suggesting that ARF1 regulates the LIF-evoked membrane trafficking of α(1H)-GFP subunits. Trafficking of T-type Ca(2+) channels required transient activation of the JAK and ERK signaling pathways since stimulation of HEK-293 cells with LIF evoked a considerable increase in the phosphorylation of the downstream JAK targets STAT3 and ERK. Pretreatment of HEK-293 cells with the JAK inhibitor P6 or the ERK inhibitor U0126 blocked ERK phosphorylation. Both P6 and U0126 also inhibited the stimulatory effect of LIF on T-type Ca(2+) channel expression. These findings demonstrate that cytokines like LIF promote the trafficking of T-type Ca(2+) channels. 相似文献
23.
Joydip Karmakar Rajib Roychowdhury Rup Kumar Kar Debal Deb Narottam Dey 《Physiology and Molecular Biology of Plants》2012,18(2):125-132
A total of ten rare indigenous rice landraces of West Bengal were screened for germination potential and seedling growth under varying concentrations of sodium chloride (NaCl) and polyethylene glycol (PEG) solutions as osmotic stress inducing agents. Among the studied rice landraces Kelas and Bhut Moori showed highest degree of tolerance to induced osmotic stresses. Proline content of the studied lines was also determined. Genetic relationship among the studied rice landraces was assessed with 22 previously reported osmotic stress tolerance linked Simple Sequence Repeat (SSR) markers. The identified allelic variants in form of amplified products size (molecular weight) for each SSR marker were documented to find out allele mining set for the linked markers of the studied genotypes in relation to osmotic stress tolerance. A Microsatellite Panel was constructed for the different allelic forms (size of amplified products) of each used marker. Among 22 SSR markers, ten showed unique alleles in form of single specific amplified product for the studied four genotypes which can be used for varietal identification. Genetic relationship among the studied rice lines was determined and a dendrogram was constructed to reveal their genetic inter-relationship. Polymorphism Information Content (PIC) for each used marker was also calculated for the studied rice lines. 相似文献
24.
Mitali Ray Niladri Sekhar Bhunia Anindya Sundar Bhunia Sajal Ray 《Fish & shellfish immunology》2013,34(1):244-253
A comparative analyses of hemocytes of molluscs, Pila globosa (Gastropoda: Prosobranchia), Bellamya bengalensis (Gastropoda: Prosobranchia) and Lamellidens marginalis (Bivalvia: Eulamellibranchiata) were carried out for morphotype and subpopulation identification, analyses of phagocytosis and generation of cytotoxic agents. Flow cytometry and microscopic analyses of hemocytes revealed the existence of agranulocytes (blast like cells, round hyalinocytes and spindle hyalinocytes), semigranulocytes (semigranular asterocytes and round semigranulocytes) and granulocytes (round granulocytes, spindle granulocytes and granular asterocytes) as three morphotypes. In P. globosa, granulocytes and semigranulocytes and in B. bengalensis granulocytes and agranulocytes are the chief phagocytes and major producers of superoxide anion and nitric oxide. In L. marginalis, granulocytes were identified as principal phagocytes with prominent activity of superoxide anion and nitric oxide. Highest activity of phenoloxidase was recorded in the agranulocytes of P. globosa with moderate activities among other morphotypes of all three species. Differential result may be due to species specific response, non-identical habitat preference and related adaptation of the species to their different ecological niches. 相似文献
25.
26.
Dey Chyi Sheu Jan Yi Chang Chung Yih Wang Chang Ta Wu Chen Ji Huang 《Bioprocess and biosystems engineering》2013,36(11):1745-1751
High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L?1 yeast extract and 300 g L?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days. 相似文献
27.
Scott J Diede Zizhen Yao C Chip Keyes Ashlee E Tyler Joyoti Dey Christopher S Hackett Katrina Elsaesser Christopher J Kemp Paul E Neiman William A Weiss James M Olson Stephen J Tapscott 《Epigenetics》2013,8(12):1254-1260
Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development. 相似文献
28.
Pei Fan Zixuan Chen Peng Tian Wen Liu Yan Jiao Yi Xue Anindya Bhattacharya Jianmin Wu Meifen Lu Yuqi Guo Yan Cui Weikuan Gu Weiwang Gu Junming Yue 《PloS one》2013,8(4)
miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including αSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways. 相似文献
29.
30.
Isothermal calorimetry (ITC) is efficient in characterizing and recognizing both high affinity and low affinity intermolecular interactions quickly and accurately. Adriamycin (ADR) and daunomycin (DNM) are the two anticancer drugs whose activity is achieved mainly by intercalation with DNA. During chemotherapy, normal human genomic DNA and mutated DNA from K562 leukemic cells show different thermodynamic properties and binding affinities on interaction with ADR and DNM when followed by ITC. Normal DNA shows more than one step in kinetic analysis, which could be attributed to outside binding, intercalation and reshuffling as suggested by Chaires et al. (1985); whereas K562 DNA fits a different binding pattern with higher binding affinities (by one order or more) compared to normal DNA. Structural properties of the interaction were followed by laser Raman spectroscopy, where difference in structure was apparent from the shifts in marker B DNA Raman bands (Ling et al., 2005). A correlation of thermodynamic contribution and structural data reveals step wise changes in normal genomic DNA conformation on drug binding. The overall structural change is higher in normal DNA–DNM interaction suggesting a partial B to A transition on drug binding. Such large changes were not observed for K562 DNA–DNM interaction which showed B to A transition properties in native from itself corroborating with our earlier findings (Ghosh et al., 2012). 相似文献