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Find video protocols related to scientific articles indexed in Pubmed.
A Splice Variant of the Human Ion Channel TRPM2 Modulates Neuroblastoma Tumor Growth Through HIF-1/2?
J. Biol. Chem.
PUBLISHED: 11-14-2014
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The calcium permeable ion channel TRPM2 is highly expressed in a number of cancers. In neuroblastoma, full length TRPM2 (TRPM2-L) protected cells from moderate oxidative stress through increased levels of Forkhead transcription factor 3a (FOXO3a) and superoxide dismutase 2 (MnSOD). Cells expressing dominant negative short isoform (TRPM2-S) had reduced FOXO3a and MnSOD levels, reduced calcium influx in response to oxidative stress, and enhanced ROS, leading to decreased cell viability. Here, in xenografts generated with SH-SY5Y neuroblastoma cells stably expressing TRPM2 isoforms, growth of tumors expressing TRPM2-S was significantly reduced compared to tumors expressing TRPM2-L. Expression of HIF-1/2? was significantly reduced in TRPM2-S expressing tumor cells, as was expression of target proteins regulated by HIF-1/2? including those involved in glycolysis (LDHA, ENO2), oxidant stress (FOXO3a), angiogenesis (VEGF), mitophagy and mitochondrial function (BNIP3 and NDUFA4L2), and mitochondrial electron transport chain activity (complex IV, cytochrome oxidase 4.1/4.2). The reduction in HIF-1/2? was mediated both through significantly reduced HIF-1/2? mRNA levels and increased levels of von Hippel-Lindau in TRPM2-S expressing cells. Inhibition of TRPM2-L by pretreatment with clotrimazole or expression of TRPM2-S significantly increased sensitivity of cells to doxorubicin. Reduced survival of TRPM2-S expressing cells after doxorubicin was rescued by gain of HIF-1 or 2? function. These data suggest that TRPM2 activity is important for tumor growth, and cell viability and survival following doxorubicin, and that interference with TRPM2-L function may be a novel approach to reduce tumor growth through modulation of HIF-1/2?, mitochondrial function and mitophagy.
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Recombinant Clostridium difficile toxin B induces endoplasmic reticulum stress in mouse colonal carcinoma cells.
Acta Biochim. Biophys. Sin. (Shanghai)
PUBLISHED: 09-30-2014
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Clostridium difficile is the main cause of antibiotic-associated diarrhea and pseudomembranous colitis in humans and animals. Its pathogenicity is primarily linked to the secretion of two exotoxins (TcdA and TcdB). Although great progress in the toxic mechanism of TcdA and TcdB has been achieved, there are many conflicting reports about the apoptotic mechanism. More importantly, apoptotic endoplasmic reticulum (ER) stress has been reported in cells treated with Shiga toxins-another kind of cytotoxins that can cause diarrhea and colitis. Herein we checked whether TcdB can induce ER stress. The results showed that recombinant TcdB (rTcdB) activated molecular markers of unfolded protein response, suggesting that rTcdB induced ER stress in CT26 cells. However, rTcdB did not induce the up-regulation of C/EBP homologous protein (CHOP), a classic mediator of apoptotic ER stress, but it activated the precursor of cysteine aspartic acid-specific protease 12 (caspase-12), a controversial mediator of apoptotic ER stress. Besides, glucosyltransferase activity-deficient mutant recombinant TcdB induced ER stress, though it has no cytotoxic or cytopathic effect on CT26 cells. Altogether, these data demonstrated that ER stress induced by rTcdB is glucosyltransferase-independent, indicating that ER stress induced by rTcdB is non-apoptotic. This work also offers us a new insight into the molecular mechanism of CHOP protein expression regulation and the role of CHOP expression in ER stress.
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MiR-663 inhibits radiation-induced bystander effects by targeting TGFB1 in a feedback mode.
RNA Biol
PUBLISHED: 08-22-2014
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The mechanisms of radiation-induced bystander effects (RIBE) have been investigated intensively over the past two decades. Although quite a few reports demonstrated that cytokines such as TGF-?1 are induced within the directly irradiated cells and play critical roles in mediating the bystander effects, little is known about the signaling pathways that occur in bystander cells. The crucial question as to why RIBE signals cannot be infinitely transmitted, therefore, remains unclear. In the present study, we showed that miR-663, a radiosensitive microRNA, participates in the regulation of biological effects in both directly irradiated and bystander cells via its targeting of TGF-?1. MiR-663 was downregulated, while TGFB1 was upregulated in directly irradiated cells. The regulation profile of miR-663 and TGFB1, on the other hand, was reversed in bystander cells, in which an elevated miR-663 expression was exhibited and led to downregulation of TGF-?1. Further studies revealed that miR-663 interacts with TGFB1 directly and that through its binding to the core regulation sequence, miR-663 suppresses the expression of TGFB1. Based on the results, we propose that miR-663 inhibits the propagation of RIBE in a feedback mode, in which the induction of TGF-?1 by reduced miR-663 in directly irradiated cells leads to increased level of miR-663 in bystander cells. The upregulation of miR-663 in turn suppresses the expression of TGF-?1 and limited further transmission of the bystander signals.
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Optimization of key factors affecting hydrogen production from sugarcane bagasse by a thermophilic anaerobic pure culture.
Biotechnol Biofuels
PUBLISHED: 08-20-2014
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Hydrogen is regarded as an attractive future energy carrier for its high energy content and zero CO2 emission. Currently, the majority of hydrogen is generated from fossil fuels. However, from an environmental perspective, sustainable hydrogen production from low-cost lignocellulosic biomass should be considered. Thermophilic hydrogen production is attractive, since it can potentially convert a variety of biomass-based substrates into hydrogen at high yields.
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Down-regulation of BTG1 by miR-454-3p enhances cellular radiosensitivity in renal carcinoma cells.
Radiat Oncol
PUBLISHED: 08-12-2014
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B cell translocation gene 1 (BTG1) has long been recognized as a tumor suppressor gene. Recent reports demonstrated that BTG1 plays an important role in progression of cell cycle and is involved in cellular response to stressors. However, the microRNAs mediated regulatory mechanism of BTG1 expression has not been reported so far. MicroRNAs can effectively influence tumor radiosensitivity by preventing cell cycle progression, resulting in enhancement of the cytotoxicity of radiotherapy efficacy. This study aimed to demonstrating the effects of microRNAs on the BTG1 expression and cellular radiosensitivity.
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Modulation of microRNAs by ionizing radiation in human gastric cancer.
Oncol. Rep.
PUBLISHED: 04-08-2014
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Gastric cancer is one of the most common cancers in China. Although surgery is the primary therapeutic method, radiotherapy has become an integral part, particularly in the early and intermediate stages of gastric cancer. microRNAs (miRNAs) are involved in the regulation of diverse cellular processes in response to intrinsic and extrinsic stress. A change in miRNA expression profile has been identified in various types of tumor cells in response to radiation; however, there is no relevant information concerning gastric cancer. In the present study, we investigated the miRNA profiles of two clinical gastric cancer samples exposed to X?rays using miRNA microarray. We found that 16 miRNAs were downregulated and 2 miRNAs were upregulated significantly in both irradiated samples when compared with the unirradiated samples. Decreases in the levels of miR?300 and miR?642 expression were confirmed by qRT?PCR in more clinical samples and in cultured cell lines. We predicted the targets of the two miRNAs with TargetScan and classified all the candidate targets with Gene Ontology, which indicated that both miR?300 and miR?642 potentially regulate cellular radiation response by modulating apoptosis, cell cycle regulation and DNA damage and repair pathway-related genes. Cell cycle assay and immunofluorescence assay demonstrated that miR?300 regulates radiation?induced G2 cell cycle arrest and DNA damage repair. In conclusion, our findings indicate that ionizing radiation modulates the miRNA expression profile, and the changes in several specific miRNAs such as miR?300 have the potential to be used in the treatment, diagnosis and prognosis of gastric cancer.
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The combined effects of X-ray radiation and hindlimb suspension on bone loss.
J. Radiat. Res.
PUBLISHED: 04-03-2014
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Outer space is a complex environment with various phenomena that negatively affect bone metabolism, including microgravity and highly energized ionizing radiation. In the present study, we used four groups of male Wistar rats treated with or without four-week hindlimb suspension after 4 Gy of X-rays to test whether there is a combined effect for hindlimb suspension and X-ray radiation. We tested trabecular parameters and some cytokines of the bone as leading indicators of bone metabolism. The results showed that hindlimb suspension and X-ray radiation could cause a significant increase in bone loss. Hindlimb suspension caused a 56.6% bone loss (P = 0.036), while X-ray radiation caused a 30.7% (P = 0.041) bone loss when compared with the control group. The combined factors of hindlimb suspension and X-rays exerted a combined effect on bone mass, with a reduction of 64.8% (P = 0.003).
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Comparative performance of aldolase and lactate dehydrogenase rapid diagnostic tests in Plasmodium vivax detection.
Malar. J.
PUBLISHED: 03-10-2014
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Misdiagnosis of malaria by commercial rapid diagnostic tests (RDTs) is a major cause of concern in the diagnosis of malaria. This retrospective study was aimed at assessing the relative performance of four RDTs with emphasis on the detection of two Plasmodium vivax antigens: aldolase and lactate dehydrogenase (LDH).
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Draft Genome Sequence of an Anaerobic, Thermophilic Bacterium, Thermoanaerobacterium aotearoense SCUT27, Isolated from a Hot Spring in China.
Genome Announc
PUBLISHED: 02-15-2014
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Thermoanaerobacterium aotearoense SCUT27, isolated from a hot spring in China, is a strictly anaerobic, thermophilic bacterium capable of degrading xylan and converting both pentose and hexose to ethanol with high yields. Here, we report the draft genome sequence of SCUT27, which reveals insights into the mechanisms of carbon source coutilization and xylan degradation in this thermophilic microorganism.
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TRPM2 channels protect against cardiac ischemia-reperfusion injury: role of mitochondria.
J. Biol. Chem.
PUBLISHED: 02-03-2014
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Cardiac TRPM2 channels were activated by intracellular adenosine diphosphate-ribose and blocked by flufenamic acid. In adult cardiac myocytes the ratio of GCa to GNa of TRPM2 channels was 0.56 ± 0.02. To explore the cellular mechanisms by which TRPM2 channels protect against cardiac ischemia/reperfusion (I/R) injury, we analyzed proteomes from WT and TRPM2 KO hearts subjected to I/R. The canonical pathways that exhibited the largest difference between WT-I/R and KO-I/R hearts were mitochondrial dysfunction and the tricarboxylic acid cycle. Complexes I, III, and IV were down-regulated, whereas complexes II and V were up-regulated in KO-I/R compared with WT-I/R hearts. Western blots confirmed reduced expression of the Complex I subunit and other mitochondria-associated proteins in KO-I/R hearts. Bioenergetic analyses revealed that KO myocytes had a lower mitochondrial membrane potential, mitochondrial Ca(2+) uptake, ATP levels, and O2 consumption but higher mitochondrial superoxide levels. Additionally, mitochondrial Ca(2+) uniporter (MCU) currents were lower in KO myocytes, indicating reduced mitochondrial Ca(2+) uptake was likely due to both lower ?m and MCU activity. Similar to isolated myocytes, O2 consumption and ATP levels were also reduced in KO hearts. Under a simulated I/R model, aberrant mitochondrial bioenergetics was exacerbated in KO myocytes. Reactive oxygen species levels were also significantly higher in KO-I/R compared with WT-I/R heart slices, consistent with mitochondrial dysfunction in KO-I/R hearts. We conclude that TRPM2 channels protect the heart from I/R injury by ameliorating mitochondrial dysfunction and reducing reactive oxygen species levels.
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Induced overexpression of phospholemman S68E mutant improves cardiac contractility and mortality after ischemia-reperfusion.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 01-31-2014
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Phospholemman (PLM), when phosphorylated at Ser(68), inhibits cardiac Na+ / Ca2+ exchanger 1 (NCX1) and relieves its inhibition on Na+ -K+ -ATPase. We have engineered mice in which expression of the phosphomimetic PLM S68E mutant was induced when dietary doxycycline was removed at 5 wk. At 8-10 wk, compared with noninduced or wild-type hearts, S68E expression in induced hearts was ?35-75% that of endogenous PLM, but protein levels of sarco(endo)plasmic reticulum Ca2+ -ATPase, ?1- and ?2-subunits of Na+ -K+ -ATPase, ?1c-subunit of L-type Ca2+ channel, and phosphorylated ryanodine receptor were unchanged. The NCX1 protein level was increased by ?47% but the NCX1 current was depressed by ?34% in induced hearts. Isoproterenol had no effect on NCX1 currents but stimulated Na+ -K+ -ATPase currents equally in induced and noninduced myocytes. At baseline, systolic intracellular Ca2+ concentrations ([Ca2+]i), sarcoplasmic reticulum Ca2+ contents, and [Ca(2+)]i transient and contraction amplitudes were similar between induced and noninduced myocytes. Isoproterenol stimulation resulted in much higher systolic [Ca2+]i, sarcoplasmic reticulum Ca2+ content, and [Ca2+]i transient and contraction amplitudes in induced myocytes. Echocardiography and in vivo close-chest catheterization demonstrated similar baseline myocardial function, but isoproterenol induced a significantly higher +dP/dt in induced compared with noninduced hearts. In contrast to the 50% mortality observed in mice constitutively overexpressing the S68E mutant, induced mice had similar survival as wild-type and noninduced mice. After ischemia-reperfusion, despite similar areas at risk and left ventricular infarct sizes, induced mice had significantly higher +dP/dt and -dP/dt and lower perioperative mortality compared with noninduced mice. We propose that phosphorylated PLM may be a novel therapeutic target in ischemic heart disease.
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Preparation and characterization of double crosslinked hydrogel films from carboxymethylchitosan and carboxymethylcellulose.
Carbohydr Polym
PUBLISHED: 01-26-2014
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A novel crosslinked hydrogel film was prepared from carboxymethylchitosan (CMCS) and carboxymethylcellulose (CMC) by ionical and covalent crosslinking with CaSO4 and genipin, respectively. The swelling ratio of the crosslinked CMCS/CMC hydrogel films was investigated at different pH solutions (1-9), and the results indicated that the crosslinked hydrogels had the swelling-deswelling properties with two primary peaks of swelling ratio at pH 3 and 7. The surface morphologies of the crosslinked hydrogels at different pH values provided evidences of the swelling-deswelling properties. The mechanical properties of the hydrogel films were also examined. The ionical and covalent crosslinking were found to have the primary impact on the toughness and max load, respectively, of the crosslinked hydrogels. The cells comparatively cultured on the crosslinked hydrogels and the negative and positive controls suggested the biocompatibility of the crosslinked CMCS/CMC films. This kind of hydrogel films have potential application in drug delivery vehicles and skin tissue engineering.
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Utility of Clostridium difficile Toxin B for Inducing Anti-Tumor Immunity.
PLoS ONE
PUBLISHED: 01-01-2014
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Clostridium difficile toxin B (TcdB) is a key virulence factor of bacterium and induces intestinal inflammatory disease. Because of its potent cytotoxic and proinflammatory activities, we investigated the utility of TcdB in developing anti-tumor immunity. TcdB induced cell death in mouse colorectal cancer CT26 cells, and the intoxicated cells stimulated the activation of mouse bone marrow-derived dendritic cells and subsequent T cell activation in vitro. Immunization of BALB/c mice with toxin-treated CT26 cells elicited potent anti-tumor immunity that protected mice from a lethal challenge of the same tumor cells and rejected pre-injected tumors. The anti-tumor immunity generated was cell-mediated, long-term, and tumor-specific. Further experiments demonstrated that the intact cell bodies were important for the immunogenicity since lysing the toxin-treated tumor cells reduced their ability to induce antitumor immunity. Finally, we showed that TcdB is able to induce potent anti-tumor immunity in B16-F10 melanoma model. Taken together, these data demonstrate the utility of C. difficile toxin B for developing anti-tumor immunity.
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pH-responsive micelles based on (PCL)2(PDEA-b-PPEGMA)2 miktoarm polymer: controlled synthesis, characterization, and application as anticancer drug carrier.
Nanoscale Res Lett
PUBLISHED: 01-01-2014
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Amphiphilic A2(BC)2 miktoarm star polymers [poly(?-caprolactone)]2-[poly(2-(diethylamino)ethyl methacrylate)-b- poly(poly(ethylene glycol) methyl ether methacrylate)]2 [(PCL)2(PDEA-b-PPEGMA)2] were developed by a combination of ring opening polymerization (ROP) and continuous activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP). The critical micelle concentration (CMC) values were extremely low (0.0024 to 0.0043 mg/mL), depending on the architecture of the polymers. The self-assembled empty and doxorubicin (DOX)-loaded micelles were spherical in morphologies, and the average sizes were about 63 and 110 nm. The release of DOX at pH 5.0 was much faster than that at pH 6.5 and pH 7.4. Moreover, DOX-loaded micelles could effectively inhibit the growth of cancer cells HepG2 with IC50 of 2.0 ?g/mL. Intracellular uptake demonstrated that DOX was delivered into the cells effectively after the cells were incubated with DOX-loaded micelles. Therefore, the pH-sensitive (PCL)2(PDEA-b-PPEGMA)2 micelles could be a prospective candidate as anticancer drug carrier for hydrophobic drugs with sustained release behavior.
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Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.
PLoS ONE
PUBLISHED: 01-01-2014
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The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.
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Inoculation and alkali coeffect in volatile fatty acids production and microbial community shift in the anaerobic fermentation of waste activated sludge.
Bioresour. Technol.
PUBLISHED: 09-04-2013
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Batch fermentations of waste activated sludge (WAS) at alkaline pH with different inocula were performed. Paper mill anaerobic granular sludge (PAS) and dyeing mill anaerobic sludge (DAS) were used as inocula. At pH 10 the inoculation did not increase the volatile fatty acids (VFAs) production compared to the non-inoculated samples fermented in the same conditions, and the maximal VFAs yield of non-inoculated WAS was higher than inoculated WAS. However, at pH 9 the inoculation with PAS increased the sludge hydrolysis and VFAs production was 1.7-fold higher than that in non-inoculated WAS (yield 52.40mg/g of volatile solid). Denaturing gradient gel electrophoresis analysis revealed that 3 bacterial species, identified as Proteocatella, Tepidibacter, and Clostridium, disappeared when inoculated with PAS at pH 9 or at pH?10. The results showed that the inoculation with PAS can be helpful to achieve a relatively high VFAs production from WAS in a moderate alkaline environment.
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MicroRNAs regulate bone metabolism.
J. Bone Miner. Metab.
PUBLISHED: 08-20-2013
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Osteoporosis is caused by an unbalance between bone formation and bone resorption. Bone homeostasis is regulated by intricate mechanisms. Recently, a novel class of regulatory factors termed microRNAs (miRNAs) has been found to play a crucial role in cell cycle control, apoptosis and other cellular processes including metabolism and differentiation. Published data have shown that some miRNAs regulate bone homeostasis, including bone formation, resorption, remodeling, repair and bone-related disease, by regulating the expression of certain cytokines and transcription factors. This review highlights the current knowledge of miRNAs and their involvement in the regulation of bone formation, bone resorption and the pathways regulating the progression of osteoporosis.
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Characterization of the glutamate-specific endopeptidase from Bacillus licheniformis expressed in Escherichia coli.
J. Biotechnol.
PUBLISHED: 08-02-2013
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Glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL) is widely used in peptide recovery and synthesis because of its unique substrate specificity. However, the mechanism underlying its specificity is still not thoroughly understood. In this study, the roles of the prosegment and key amino acids involved in the proteolytic activity of GSE-BL were investigated. Loss of the GSE-BL prosegment severely restricted enzymatic activity toward Z-Phe-Leu-Glu-pNA. A homologous model of GSE-BL revealed that it contains the catalytic triad "His47, Asp96 and Ser 167", which was further confirmed by site-directed mutagenesis. In vitro mutagenesis further indicated that Val2, Arg89 and His190 are essential for enzymatic activity toward Z-Phe-Leu-Glu-pNA. Moreover, the catalytic efficiency of Phe57Ala GSE-BL toward Z-Phe-Leu-Glu-pNA was 50% higher than that of the native mature GSE-BL. This is the first study to fully elucidate the key amino acids for proteolytic activity of GSE-BL. Mature GSE-BL could be obtained through self-cleavage alone when Lys at -1 position was replaced by Glu, providing a new strategy for the preparation of mature GSE-BL. This study yielded some valuable insights into the substrate specificity of glutamate-specific endopeptidase, establishing a foundation for broadening the applications of GSE-BL.
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Malondialdehyde regulates glucose-stimulated insulin secretion in murine islets via TCF7L2-dependent Wnt signaling pathway.
Mol. Cell. Endocrinol.
PUBLISHED: 06-24-2013
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Evidence showed strong relations between malondialdehyde (MDA) levels and different pathological stages of diabetes. Here, an explicit system with freshly isolated islets and precisely controlled MDA gradient was employed to investigate the physiological effect of MDA on GSIS. Promoter analysis, pathway mapping, Q-PCR profiling, and siRNA verification were performed to clarify the intracellular signaling pathways. MDA at a moderately high level (5 and 10?M) promoted GSIS and accompanied with ATP/ADP ratio, cytosolic Ca(2+) level, and key regulators (GK, GLUT2, PDX1, and UCP2) changes in islets. Both upstream (PI3K and p-AKT) and downstream (TCF7L2 and TCF7) factors of Wnt pathway showed greatest changes. Knockdown of TCF7L2 blocked the MDA-induced GSIS elevation. These results indicated that MDA acted as a signaling messenger and regulated islet GSIS mainly through Wnt pathway. Therefore, the elevated MDA level and up-regulated Wnt signaling pathway could be an etiological factor in the development of hyperinsulinemia and type 2 diabetes.
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Efficient production of l-lactic acid by an engineered Thermoanaerobacterium aotearoense with broad substrate specificity.
Biotechnol Biofuels
PUBLISHED: 06-07-2013
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Efficient conversion of lignocellulosic biomass to optically pure lactic acid is a key challenge for the economical production of biodegradable poly-lactic acid. A recently isolated strain, Thermoanaerobacterium aotearoense SCUT27, is promising as an efficient lactic acid production bacterium from biomass due to its broad substrate specificity. Additionally, its strictly anaerobic and thermophilic characteristics suppress contamination from other microoragnisms. Herein, we report the significant improvements of concentration and yield in lactic acid production from various lignocellulosic derived sugars, achieved by the carbon flux redirection through homologous recombination in T. aotearoense SCUT27.
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Effects of shielding on the induction of 53BP1 foci and micronuclei after Fe ion exposures.
J. Radiat. Res.
PUBLISHED: 05-31-2013
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High atomic number and high-energy (HZE) particles in deep space are of low abundance but substantially contribute to the biological effects of space radiation. Shielding is so far the most effective way to partially protect astronauts from these highly penetrating particles. However, simulated calculations and measurements have predicted that secondary particles resulting from the shielding of cosmic rays produce a significant fraction of the total dose and dose equivalent. In this study, we investigated the biological effects of secondary radiation with two cell types, and with cells exposed in different phases of the cell cycle, by comparing the biological effects of a 200 MeV/u iron beam with a shielded beam in which the energy of the iron ion beam was decreased from 500 MeV/u to 200 MeV/u with PMMA, polyethylene (PE), or aluminum. We found that beam shielding resulted in increased induction of 53BP1 foci and micronuclei in a cell-type-dependent manner compared with the unshielded 200 MeV/u Fe ion beam. These findings provide experimental proof that the biological effects of secondary particles resulting from the interaction between HZE particles and shielding materials should be considered in shielding design.
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A new strategy for recovery of two peptides without Glu employing glutamate-specific endopeptidase from Bacillus licheniformis.
Enzyme Microb. Technol.
PUBLISHED: 05-08-2013
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The difficulty in the purification of bioactive peptide limited its application in food, drug and cosmetic industry. Here we report a new strategy for the recovery of two peptides employing glutamate-specific endopeptidase from Bacillus licheniformis (GSE-BL), which shows strong specificity for Glu residue. Human glucagon and human beta-defensin-2 (HBD-2) were peptides without Glu residue, and Glu residue was introduced between affinity tag and target peptide as recognition site of GSE-BL. Tagless human glucagon with the same HPLC retention time as native human glucagon and mature HBD-2 with antibacterial activity and cytotoxicity were obtained after GSE-BL treatment. This strategy has great potential in the recovery of bioactive peptide without Glu residue, thus facilitating large scale preparation of peptide and widening the application of bioactive peptide.
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Radiation-induced cellular senescence results from a slippage of long-term G2 arrested cells into G1 phase.
Cell Cycle
PUBLISHED: 04-09-2013
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Diploid cells undergoing senescence and mitotic slippage have been reported in the literature. However, the mechanisms triggering senescence in long-term G2-arrested cells are currently unclear. Previously, we reported that the cell cycle of the human uveal melanoma cell line, 92-1, is suspended for up to 6 d upon exposure to 10 Gy ionizing radiation (IR), followed by senescence. In the current study, we initially distinguished senescence in long-term blocked 92-1 cells from mitotic slippage by confirming the blockage of cells in the G2 phase. We subsequently showed that the genes essential for G2-M transition are prematurely downregulated at both the transcriptional and translational levels. Furthermore, levels of the G1-specific markers, Cyclin D1 and Caveolin-1, were distinctly increased, while S/G2-specific markers, Cyclin B1 and Aurora A, were significantly downregulated. These findings collectively imply that long-term G2-arrested cells undergo senescence via G2 slippage. To our knowledge, this is the first study to report that the cellular process of G2 slippage is the mechanism responsible for senescence of cells under long-term G2 arrest.
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Coordinated regulation of cardiac Na(+)/Ca (2+) exchanger and Na (+)-K (+)-ATPase by phospholemman (FXYD1).
Adv. Exp. Med. Biol.
PUBLISHED: 03-15-2013
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Phospholemman (PLM) is the founding member of the FXYD family of regulators of ion transport. PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. The TM domain of PLM interacts with TM9 of the ?-subunit of Na(+)-K(+)-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of Na(+)/Ca(2+) exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of Na(+)-K(+)-ATPase by decreasing K(m) for Na(+) and increasing V(max), and simultaneous inhibition of Na(+)/Ca(2+) exchanger. Enhanced Na(+)-K(+)-ATPase activity lowers intracellular Na(+), thereby minimizing Ca(2+) overload and risks of arrhythmias. Inhibition of Na(+)/Ca(2+) exchanger reduces Ca(2+) efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy. In acute cardiac ischemia and chronic heart failure, either expression or phosphorylation of PLM or both are altered. PLM regulates important ion transporters in the heart and offers a tempting target for development of drugs to treat heart failure.
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Cell-based screening of traditional chinese medicines for proliferation enhancers of mouse embryonic stem cells.
Biotechnol. Prog.
PUBLISHED: 03-11-2013
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A high-throughput cell-based method was developed for screening traditional Chinese herbal medicines (TCHMs) for potential stem cell growth promoters. Mouse embryonic stem (mES) cells expressing enhanced green fluorescent protein (EGFP) were cultured in growth media supplemented with various TCHM extracts. The dosage-dependent effects of TCHM extracts on cell growth, including proliferation and cytotoxicity, were assessed via EGFP fluorescence measurement. Seven TCHMs were investigated, and among them Panax notoginseng (PN), Rhizoma Atractylodis macrocephalae, Rhizoma chuanxiong, and Ganoderma lucidum spores (GLS) showed potential to improve mES cell proliferation. Eleven mixtures of these four TCHMs were then studied, and the results showed that the mixture of PN and GLS had the strongest growth promoting effect, increasing the specific growth rate of mES cells by 29.5% at a low dosage of 0.01% (wt/vol) PN/GLS (P<0.01) and 34.2% at 0.1% (wt/vol) PN/GLS (P<0.05) compared to the control. The growth promoting effect of PN/GLS was further confirmed with ES cells cultured in spinner flasks. A 29.3-fold increase in the total cell number was achieved in the medium supplemented with 0.01% PN/GLS after 5 days, while the control culture only gave a 16.8-fold increase. This cell-based screening method thus can provide an efficient and high-throughput way to explore potential stem cell growth promoters from TCHMs.
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Fast and continuous preparation of high polymerization degree cellulose nanofibrils and their three-dimensional macroporous scaffold fabrication.
Nanoscale
PUBLISHED: 02-16-2013
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C6-carboxy-cellulose with a carboxylate content of 0.8 mmol g(-1) was obtained by oxidation of once-dried cellulose, using the 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO)/NaClO/NaClO2 system at pH 6.8 and 60 °C for 16 h. This method, with the addition of reagents in the order TEMPO, NaClO and NaClO2, was 38 h faster than a previously published method. Individualized cellulose nanofibrils with a width of 3-5 nm and a length of several hundred nanometers were prepared by homogenizing the C6-carboxy-cellulose-water suspension. Macroporous cellulose nanofibril/poly(vinyl alcohol) scaffolds with interconnected large pores of 20-100 ?m diameter and small pores of 2-10 ?m diameter were fabricated. The cellulose nanofilaments formed nanofibrous structures on the surface of the PVA wall, which was similar to that of the collagen skeleton of the extracellular matrix. NIH/3T3 cells were cultured in the scaffolds for 4 weeks, SEM observation showed that the cells were anchored and clustered on the cellulose nanofilaments, forming spherical colonies. The extracellular matrix (ECM) was filled with mineralized particles.
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TRIM5? polymorphism identification in cynomolgus macaques of Vietnamese origin and Chinese rhesus macaques.
Am. J. Primatol.
PUBLISHED: 02-10-2013
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TRIM5? is a retroviral restriction factor, in which the B30.2 (SPRY) and coiled-coil domains cooperate to determine the specificity of TRIM5?-mediated capture of retroviral capsids. Here, all exons of TRIM5? were analyzed in 39 Vietnamese cynomolgus macaques (VCE) and 29 Chinese rhesus macaques (CR). Our results revealed the presence of 22 alleles using the PHASE 2.0 software package (PHylogenetics And Sequence Evolution), including two novel species-specific alleles with a low frequency in VCE in exons 4 and 8, which encoded the coiled-coil and B30.2 (SPRY) domains, respectively. Nine alleles were detected in both VCE and CR, while four alleles were likely shared between the species. Of these alleles, the highest frequencies of 38% and 26% occurred in VCE and CR, respectively. Importantly, we found that some alleles encoded the same coiled-coil domain, but not the SPRY domain. In contrast, other alleles encoded the same SPRY domain, but not the coiled-coil domain. Our findings will contribute to the understanding of the interaction between the two domains and the determination of the specificity of TRIM5?-mediated capture of retroviral capsids. Our results from the phylogenetic trees constructed for VCE and CR suggested that the macaques ability to inhibit SIV replication became gradually stronger if they carried corresponding alleles in four clades (clades4-7). More interesting, in clade3, both novel allele pairs (4E100a, 10E147a) and allele pairs (7R17b and 13R11b), which had the strong ability to inhibit SIV replication, originated from the same ancestral allele, suggesting that the novel alleles might play a key role to determine an animals ability to inhibit SIV/HIV replication. However, further studies are needed to increase our understanding of the genetic background of TRIM5? in these two macaque species.
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The second member of transient receptor potential-melastatin channel family protects hearts from ischemia-reperfusion injury.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 02-01-2013
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The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-3 days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the ?1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by 30 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (SODs) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.
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Plasmodium vivax aldolase-specific monoclonal antibodies and its application in clinical diagnosis of malaria infections in China.
Malar. J.
PUBLISHED: 01-17-2013
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Most rapid diagnostic tests (RDTs) currently used for malaria diagnosis cannot distinguish the various Plasmodium infections. The development of a Plasmodium vivax specific RDTs with high sensitivity to sufficiently differentiate the two most common Plasmodium infections would be very crucial for disease treatment and control.
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Enhanced lipid productivity and photosynthesis efficiency in a Desmodesmus sp. mutant induced by heavy carbon ions.
PLoS ONE
PUBLISHED: 01-01-2013
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The unicellular green microalga Desmodesmus sp. S1 can produce more than 50% total lipid of cell dry weight under high light and nitrogen-limitation conditions. After irradiation by heavy (12)C(6+) ion beam of 10, 30, 60, 90 or 120 Gy, followed by screening of resulting mutants on 24-well microplates, more than 500 mutants were obtained. One of those, named D90G-19, exhibited lipid productivity of 0.298 g L(-1)?d(-1), 20.6% higher than wild type, likely owing to an improved maximum quantum efficiency (Fv/Fm) of photosynthesis under stress. This work demonstrated that heavy-ion irradiation combined with high-throughput screening is an effective means for trait improvement. The resulting mutant D90G-19 may be used for enhanced lipid production.
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Induced overexpression of Na(+)/Ca(2+) exchanger does not aggravate myocardial dysfunction induced by transverse aortic constriction.
J. Card. Fail.
PUBLISHED: 01-01-2013
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Alterations in expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) have been implicated in the pathogenesis of heart failure.
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Constitutive overexpression of phosphomimetic phospholemman S68E mutant results in arrhythmias, early mortality, and heart failure: potential involvement of Na+/Ca2+ exchanger.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 11-11-2011
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Expression and activity of cardiac Na(+)/Ca(2+) exchanger (NCX1) are altered in many disease states. We engineered mice in which the phosphomimetic phospholemman S68E mutant (inhibits NCX1 but not Na(+)-K(+)-ATPase) was constitutively overexpressed in a cardiac-specific manner (conS68E). At 4-6 wk, conS68E mice exhibited severe bradycardia, ventricular arrhythmias, increased left ventricular (LV) mass, decreased cardiac output (CO), and ?50% mortality compared with wild-type (WT) littermates. Protein levels of NCX1, calsequestrin, ryanodine receptor, and ?(1)- and ?(2)-subunits of Na(+)-K(+)-ATPase were similar, but sarco(endo)plasmic reticulum Ca(2+)-ATPase was lower, whereas L-type Ca(2+) channels were higher in conS68E hearts. Resting membrane potential and action potential amplitude were similar, but action potential duration was dramatically prolonged in conS68E myocytes. Diastolic intracellular Ca(2+) ([Ca(2+)](i)) was higher, [Ca(2+)](i) transient and maximal contraction amplitudes were lower, and half-time of [Ca(2+)](i) transient decline was longer in conS68E myocytes. Intracellular Na(+) reached maximum within 3 min after isoproterenol addition, followed by decline in WT but not in conS68E myocytes. Na(+)/Ca(2+) exchange, L-type Ca(2+), Na(+)-K(+)-ATPase, and depolarization-activated K(+) currents were decreased in conS68E myocytes. At 22 wk, bradycardia and increased LV mass persisted in conS68E survivors. Despite comparable baseline CO, conS68E survivors at 22 wk exhibited decreased chronotropic, inotropic, and lusitropic responses to isoproterenol. We conclude that constitutive overexpression of S68E mutant was detrimental, both in terms of depressed cardiac function and increased arrhythmogenesis.
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Cloning, expression, purification, and characterization of a glutamate-specific endopeptidase from Bacillus licheniformis.
Protein Expr. Purif.
PUBLISHED: 10-14-2011
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A gene encoding a glutamate-specific endopeptidase (GSE) from Bacillus licheniformis (BL) has been cloned in Escherichia coli cells. The recombinant protein was expressed as cytoplasmic insoluble inclusion bodies. Immobilized metal affinity chromatography was employed to purify the protein, and then a 27-kDa GSE intermediate was obtained by gradient urea dialysis. The remaining pro-peptide was completely removed by treatment with trypsin to obtain mature GSE-BL with a molecular weight of 26 kD at a final yield of up to 140.9 mg/L. With Z (benzyloxycarbomyl)-Phe-Leu-Glu-pNA (p-nitroanilide) as the substrate, the optimal temperature and pH conditions for the enzyme were 37 °C and 8.5, respectively, K(m) was 1.495 ± 0.034 mM, and V(max) was 50.237 ?mol/mg min. The presence of calcium and ferrous ions enhanced the catalytic activity of GSE-BL. These results suggest that the recombinant protein is a relatively stable specific proteinase that could be effectively utilized in protein structure analysis, peptide synthesis, and the food industry.
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Right ventricular outflow pacing induces less regional wall motion abnormalities in the left ventricle compared with apical pacing.
Europace
PUBLISHED: 09-22-2011
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This study aimed to explore if the right ventricular outflow tract (RVOT) pacing is superior to right ventricular apical (RVA) pacing on the overall left ventricular (LV) function and regional wall motion.
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Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes.
J. Biol. Chem.
PUBLISHED: 09-20-2011
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MraY translocase catalyzes the first committed membrane-bound step of bacterial peptidoglycan synthesis leading to the formation of lipid I. The essential membrane protein therefore has a high potential as target for drug screening approaches to develop antibiotics against gram-positive as well as gram-negative bacteria. However, the production of large integral membrane proteins in conventional cellular expression systems is still very challenging. Cell-free expression technologies have been optimized in recent times for the production of membrane proteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF). We report the development of preparative scale production protocols for the MraY homologues of Escherichia coli and Bacillus subtilis in all three cell-free expression modes followed by their subsequent quality evaluation. Although both proteins can be cell-free produced at comparable high levels, their requirements for optimal expression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF production followed by defined treatment with detergents. In contrast, the E. coli MraY appears to be unstable after post- or cotranslational solubilization in detergent micelles. Expression kinetics and reducing conditions were identified as optimization parameters for the quality improvement of E. coli MraY. Most remarkably, in contrast to B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only the production in the L-CF mode in the presence of preformed liposomes resulted in stable and translocase active protein samples.
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Detection of novel human MiRNAs responding to X-ray irradiation.
J. Radiat. Res.
PUBLISHED: 07-26-2011
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Up to now, more than 1048 human miRNAs have been identified. However, the recognition of new human miRNAs is becoming more and more difficult. Based on the hypothesis that the expression of some miRNAs can be induced by ionizing radiation, total RNAs of HeLa cells were isolated 1 h after exposure to 2 Gy of X-rays, and total small RNAs were enriched and sequenced by PAGE and Solexa technology, respectively. As a result, 421 kinds of known miRNAs and 337 kinds of unknown sequences were identified, among which 10 novel miRNAs were characterized by bioinformatic approach and verified by qRT-PCR. Finally, putative targets of these miRNAs were predicted by TargetScan software and compared with known proteins down-regulated by radiation. It was confirmed that some of the targets of these novel miRNAs were radiation-related proteins. These results imply that these 10 novel miRNAs are radiation-related miRNAs. This study reveals a new way to find novel miRNAs.
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Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method.
PLoS ONE
PUBLISHED: 07-11-2011
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The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.
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Residues 248-252 and 300-304 of the cardiac Na+/Ca2+ exchanger are involved in its regulation by phospholemman.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 07-06-2011
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Using split cardiac Na(+)/Ca(2+) exchangers (NCX1), we previously demonstrated that phospholemman (PLM) regulates NCX1 by interacting with the proximal linker domain (residues 218-358) of the intracellular loop of NCX1. With the use of overlapping loop deletion mutants, interaction sites are localized to two regions spanning residues 238-270 and residues 300-328 of NCX1. In this study, we used alanine (Ala) linker scanning to pinpoint the residues in the proximal linker domain involved in regulation of NCX1 by PLM. Transfection of human embryonic kidney (HEK)293 cells with wild-type (WT) NCX1 or its Ala mutants but not empty vector resulted in NCX1 current (I(NaCa)). Coexpression of PLM with WT NCX1 inhibited I(NaCa). Mutating residues 248-252 (PASKT) or 300-304 (QKHPD) in WT NCX1 to Ala resulted in loss of inhibition of I(NaCa) by PLM. By contrast, inhibition of I(NaCa) by PLM was preserved when residues 238-242, 243-247, 253-257, 258-262, 263-267, 305-309, 310-314, 315-319, 320-324, or 325-329 were mutated to Ala. While mutating residue 301 to alanine completely abolished PLM inhibition, mutation of any single residue 250-252, 300, or 302-304 resulted in partial reduction in inhibition. Mutating residues 248-252 to Ala resulted in significantly weaker association with PLM. The NCX1-G503P mutant that lacks Ca(2+)-dependent activation retained its sensitivity to PLM. We conclude that residues 248-252 and 300-304 in the proximal linker domain of NCX1 were involved in its inhibition by PLM.
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Cell cycle suspension: a novel process lurking in G? arrest.
Cell Cycle
PUBLISHED: 05-01-2011
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Cell cycle checkpoint is a self-protective mechanism for cells to monitor genome integrity and ensure the high-fidelity transmission of genetic information to daughter cells. Insufficient function of cell cycle checkpoints has been demonstrated to partially account for tumor initiation, promotion and progression. In the ten melanoma cell lines that we tested in preliminary experiments, two human uveal melanoma cell lines, 92-1 and OCM-1, were found to be significantly different in terms of radiosensitivity but similar in DNA repair ability. Evident G 2 arrest was induced in both cell types and the maximum was reached at 16 h after irradiation regardless of X-rays or high-LET carbon beams. OCM-1 cells overrode the G 2 arrest and reentered the cell cycle right after reaching the maximum, whereas 92-1 could not. Upon 10 Gy of radiation, the cell cycle of 92-1 was suspended and remained unchanged for up to 5 d. The cell cycle suspension is a unique process lurking in G 2 arrest and related to cellular radiosensitivity. Its induction is dose-dependent and there is a dose threshold for it. The degradation of Cyclin B1 has been found related to the cell cycle suspension though, the mechanism of cell cycle suspension is still under investigation. Basing on our knowledge, this is the first report on cell cycle suspension and we present here a de novo mechanism to cellular radiosensitivity. Further clarification of the mechanism underlying cell cycle suspension is believed to be of significance in tumor radiosensitization or even direct tumor control.
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Control and optimization of Clostridium tyrobutyricum ATCC 25755 adhesion into fibrous matrix in a fibrous bed bioreactor.
Appl. Biochem. Biotechnol.
PUBLISHED: 03-28-2011
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The great performance of a fibrous bed bioreactor (FBB) is mainly dependent on the cell adhesion and immobilization into the fibrous matrix. Therefore, understanding the mechanism and factors controling cell adhesion in the fibrous matrix is necessary to optimize the FBB setup and further improve the fermentability. The adhesion behavior of a strain of Clostridium tyrobutyricum isolated from an FBB was studied, which was proven to be affected by the different environmental conditions, such as growth phase of cells, pH, ionic strength, ionic species, and composition of media. Our results also suggested that electrostatic interactions played an important role on bacteria adhesion into the fibrous matrix. This study demonstrated that the compositions of fermentation broth would have a significant effect on cell adhesion. Consequently, a two-stage glucose supply control strategy was developed to improve the performance of FBB with higher viable cell density in the operation of the FBB setup.
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Mechanisms of increased risk of tumorigenesis in Atm and Brca1 double heterozygosity.
Radiat Oncol
PUBLISHED: 03-17-2011
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Both epidemiological and experimental studies suggest that heterozygosity for a single gene is linked with tumorigenesis and heterozygosity for two genes increases the risk of tumor incidence. Our previous work has demonstrated that Atm/Brca1 double heterozygosity leads to higher cell transformation rate than single heterozygosity. However, the underlying mechanisms have not been fully understood yet. In the present study, a series of pathways were investigated to clarify the possible mechanisms of increased risk of tumorigenesis in Atm and Brca1 heterozygosity.
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Overexpression of ornithine decarboxylase decreases ventricular systolic function during induction of cardiac hypertrophy.
Amino Acids
PUBLISHED: 03-16-2011
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Ornithine decarboxylase (ODC), the first enzyme of polyamine metabolism, is rapidly upregulated in response to agents that induce a pathological cardiac hypertrophy. Transgenic mice overexpressing ODC in the heart (MHC-ODC mice) experience a much more dramatic left ventricular hypertrophy in response to ?-adrenergic stimulation with isoproterenol (ISO) compared to wild-type (WT) controls. ISO also induced arginase activity in transgenic hearts but not in controls. The current work studies the cooperation between the cardiac polyamines and L-arginine (L-Arg) availability in MHC-ODC mice. Although ISO-induced hypertrophy is well-compensated, MHC-ODC mice administered L-Arg along with ISO showed a rapid onset of systolic dysfunction and died within 48 h. Myocytes isolated from MHC-ODC mice administered L-Arg/ISO exhibited reduced contractility and altered calcium transients, suggesting an alteration in [Ca(2+)] homeostasis, and abbreviated action potential duration, which may contribute to arrhythmogenesis. The already elevated levels of spermidine and spermine were not further altered in MHC-ODC hearts by L-Arg/ISO treatment, suggesting alternative L-Arg utilization pathways lead to dysregulation of intracellular calcium. MHC-ODC mice administered an arginase inhibitor (Nor-NOHA) along with ISO died almost as rapidly as L-Arg/ISO-treated mice, while the iNOS inhibitor S-methyl-isothiourea (SMT) was strongly protective against L-Arg/ISO. These results point to the induction of arginase as a protective response to ?-adrenergic stimulation in the setting of high polyamines. Further, NO generated by exogenously supplied L-Arg may contribute to the lethal consequences of L-Arg/ISO treatment. Since considerable variations in human cardiac polyamine and L-Arg content are likely, it is possible that alterations in these factors may influence myocyte contractility.
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Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques.
Nat. Biotechnol.
PUBLISHED: 03-08-2011
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The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.
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Regulation of in vivo cardiac contractility by phospholemman: role of Na+/Ca2+ exchange.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 12-30-2010
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Phospholemman (PLM), when phosphorylated at serine 68, relieves its inhibition on Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger 1 (NCX1) in cardiac myocytes. Under stress when catecholamine levels are high, enhanced Na(+)-K(+)-ATPase activity by phosphorylated PLM attenuates intracellular Na(+) concentration ([Na(+)](i)) overload. To evaluate the effects of PLM on NCX1 on in vivo cardiac contractility, we injected recombinant adeno-associated virus (serotype 9) expressing either the phosphomimetic PLM S68E mutant or green fluorescent protein (GFP) directly into left ventricles (LVs) of PLM-knockout (KO) mice. Five weeks after virus injection, ?40% of isolated LV myocytes exhibited GFP fluorescence. Expression of S68E mutant was confirmed with PLM antibody. There were no differences in protein levels of ?(1)- and ?(2)-subunits of Na(+)-K(+)-ATPase, NCX1, and sarco(endo)plasmic reticulum Ca(2+)-ATPase between KO-GFP and KO-S68E LV homogenates. Compared with KO-GFP myocytes, Na(+)/Ca(2+) exchange current was suppressed, but resting [Na(+)](i), Na(+)-K(+)-ATPase current, and action potential amplitudes were similar in KO-S68E myocytes. Resting membrane potential was slightly lower and action potential duration at 90% repolarization (APD(90)) was shortened in KO-S68E myocytes. Isoproterenol (Iso; 1 ?M) increased APD(90) in both groups of myocytes. After Iso, [Na(+)](i) increased monotonically in paced (2 Hz) KO-GFP but reached a plateau in KO-S68E myocytes. Both systolic and diastolic [Ca(2+)](i) were higher in Iso-stimulated KO-S68E myocytes paced at 2 Hz. Echocardiography demonstrated similar resting heart rate, ejection fraction, and LV mass between KO-GFP and KO-S68E mice. In vivo closed-chest catheterization demonstrated enhanced contractility in KO-S68E compared with KO-GFP hearts stimulated with Iso. We conclude that under catecholamine stress when [Na(+)](i) is high, PLM minimizes [Na(+)](i) overload by relieving its inhibition of Na(+)-K(+)-ATPase and preserves inotropy by simultaneously inhibiting Na(+)/Ca(2+) exchanger.
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Cloning and characterization of the 5-flanking region of the pig cocaine- and amphetamine-regulated transcript gene.
DNA Cell Biol.
PUBLISHED: 11-22-2010
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The cocaine- and amphetamine-regulated transcript (CART) gene encodes an anorexigenic peptide. It has a key role in the hypothalamic regulation of energy balance through reducing food intake and enhancing lipid substrate utilization. To detect the CART expression pattern in pigs, reverse transcription (RT)-polymerase chain reaction (PCR) and real-time PCR were performed in various tissues. Our RT-PCR results revealed that the pig CART gene was ubiquitously expressed in all examined tissues including hypothalamus, m. longissimus, backfat, heart, liver, spleen, lung, kidney, stomach, bladder, belly fat, brain, large intestine, lymph, and skin. Real-time quantitative PCR experiments revealed that the cDNA level of CART in both the hypothalamus and backfat of adult Landrace pig (lean-type) was significantly higher than that of Chinese indigenous Lantang pig (fat-type), and it was in the hypothalamus where the highest expression of CART was observed for both adult Lantang and Landrace pigs, compared with backfat and m. longissimus muscle. To understand the regulation of the pig CART gene, the 5-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient CART expression was identified at nucleotides -73 to -53, using 5-serial deletion of the promoter. Electrophoretic mobility shift assays with competing oligonucleotides revealed that the critical region contained a cis-acting element for the zinc-binding protein factor, a zinc-finger transcription factor of the Kruppel family. This element has not been reported in human or mouse CART genes. Our results indicated that zinc-binding protein factor might be an essential regulatory factor for transcription of pig CART, providing important insight into mechanisms involved in energy homeostasis regulation in the porcine and human brain.
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Three-dimensional culture of human mesenchymal stem cells in a polyethylene terephthalate matrix.
Biomed Mater
PUBLISHED: 11-15-2010
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Polyethylene terephthalate (PET) was used as the scaffold material to support the proliferation of human mesenchymal stem cells (hMSCs). The cells were cultured either statically in multi-wells or in a spinner flask agitated at 80 rpm for up to 20 days. To optimize the cell expansion condition, effects of the initial cell density and basic fibroblast growth factor (bFGF) were examined. During culture, cell growth and metabolism were tested. After 20 days, cells were harvested and surface markers were identified and quantified with flow cytometry. The results showed that hMSCs seeded at the lowest density gave the highest expansion fold. hMSCs grown in porous three-dimensional (3D) matrices displayed significantly different characteristics in terms of their proliferation and metabolism. PET matrices with 3D space could sustain cell proliferation for a long time. In addition, a low concentration (5 ng mL(-1)) of bFGF significantly enhanced the expansion of hMSCs in PET. Cell attachment and distribution in PET matrices were studied with confocal laser microscopy and scanning electron microscopy, which also confirmed cell proliferation. Furthermore, most of the cells in PET matrices were CD29, CD44 and CD105 positive, and CD34, CD45 and CD14 negative, confirming that hMSCs cultured in 3D PET matrices can be expanded and maintained in their undifferentiated state for at least 20 days without subculturing.
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Phospholemman: a novel cardiac stress protein.
Clin Transl Sci
PUBLISHED: 08-20-2010
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Phospholemman (PLM), a member of the FXYD family of regulators of ion transport, is a major sarcolemmal substrate for protein kinases A and C in cardiac and skeletal muscle. In the heart, PLM co-localizes and co-immunoprecipitates with Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and L-type Ca(2+) channel. Functionally, when phosphorylated at serine(68), PLM stimulates Na(+)-K(+)-ATPase but inhibits Na(+)/Ca(2+) exchanger in cardiac myocytes. In heterologous expression systems, PLM modulates the gating of cardiac L-type Ca(2+) channel. Therefore, PLM occupies a key modulatory role in intracellular Na(+) and Ca(2+) homeostasis and is intimately involved in regulation of excitation-contraction (EC) coupling. Genetic ablation of PLM results in a slight increase in baseline cardiac contractility and prolongation of action potential duration. When hearts are subjected to catecholamine stress, PLM minimizes the risks of arrhythmogenesis by reducing Na(+) overload and simultaneously preserves inotropy by inhibiting Na(+)/Ca(2+) exchanger. In heart failure, both expression and phosphorylation state of PLM are altered and may partly account for abnormalities in EC coupling. The unique role of PLM in regulation of Na(+)-K(+)-ATPase, Na(+)/Ca(2+) exchanger, and potentially L-type Ca(2+) channel in the heart, together with the changes in its expression and phosphorylation in heart failure, make PLM a rational and novel target for development of drugs in our armamentarium against heart failure. Clin Trans Sci 2010; Volume 3: 189-196.
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The correlation coefficient of GC content of the genome-wide genes is positively correlated with animal evolutionary relationships.
FEBS Lett.
PUBLISHED: 06-27-2010
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In this study, we present a new method for evaluating animal evolutionary relationships. We used the GC% levels of genome-wide genes to determine the correlation between the GC% content and evolutionary relationship. The correlation coefficients of the GC% content of the orthologous genes of the paired animal species were calculated for a total of 21 species, and the evolutionary branching dates of these 21 species were derived from fossil records. The correlation coefficient of the GC% content of the orthologous genes of the species pair under study served as an indicator of their evolutionary relationship. Moreover, there was a decreasing linear relationship between the correlation coefficient and evolutionary branching date (R(2)=0.930).
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Disruption of lactate dehydrogenase through homologous recombination to improve bioethanol production in Thermoanaerobacterium aotearoense.
Enzyme Microb. Technol.
PUBLISHED: 05-17-2010
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To enhance ethanol production in Thermoanaerobacterium aotearoense, the lactate dehydrogenase (ldh) gene, which is responsible for lactic acid production in a key branch pathway, was successfully disrupted via homologous recombination. ldh-up and ldh-down were designed and amplified based on JW/SL-YS485-AY 278026, and they were subsequently used as homologous fragments with an inserted erythromycin resistance gene to construct the targeted vector based on pBLUESCRIPT II SK(+). Southern hybridization and PCR-based assay definitely confirmed that the ldh gene in the ?ldh mutant was disrupted by the insertion of the erythromycin resistance gene. Compared with the wild type, the ?ldh mutant exhibited increases of 31.0% and 31.4% in cell yield under glucose and xylose cultivation, respectively, probably because knocking out the ldh gene results in increased acetate and ATP levels. Knockout of lactate dehydrogenase produced 2.37- and 2.1-fold increases in the yield of ethanol (mole/mole substrate) under glucose and xylose cultivation, respectively. Moreover, no lactic acid was detected in ?ldh mutant fermentation mixtures (detection limit of HPLC: 0.5 mM), but lactic acid was readily detected for growth of the wild-type strain on both glucose and xylose, with final concentrations up to 59.24 mM and 56.06 mM, respectively. The success of this process thoroughly demonstrates the methodological possibility of gene knockout through homologous recombination in Thermoanaerobacterium.
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High efficiency hydrogen production from glucose/xylose by the ldh-deleted Thermoanaerobacterium strain.
Bioresour. Technol.
PUBLISHED: 05-06-2010
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A strictly anaerobic, thermoacidophilic, H(2)-producing bacterium was isolated and designated as Thermoanaerobacterium aotearoense. The optimized cultivation conditions for H(2) production are 55 degrees C, pH 6.5 and 10gl(-1) of glucose or xylose. A metabolic pathway analysis showed that lactate occupied most of the liquid metabolites and consumed a large amount of NADH. To increase the efficiency of hydrogen production, the gene encoding the l-lactate dehydrogenase was knocked out to redirect the NADH flow. Genetic manipulation resulted in the 2 and 2.5 folds increase of the H(2) yield and production rate, respectively. The maximum H(2) yields using the Deltaldh mutant were 2.71, 1.45 and 2.28molH(2)mol(-1) sugar under glucose, xylose and glucose/xylose mixture tests, respectively. The recombinant Deltaldh strain could ferment the mixture of glucose and xylose to produce H(2) effectively, indicating that the performance of Thermoanaerobacterium in H(2) production can be significantly improved by metabolic engineering technique.
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Effects of cardiac-restricted overexpression of the A(2A) adenosine receptor on adriamycin-induced cardiotoxicity.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 04-02-2010
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Activation of the A(2A) adenosine receptor (A(2A)R) has been shown to be cardioprotective. We hypothesized that A(2A)R overexpression could protect the heart from adriamycin-induced cardiomyopathy. Transgenic (TG) mice overexpressing the A(2A)R and wild-type mice (WT) were injected with adriamycin (5 mg.kg(-1).wk(-1) ip, 4 wk). All WT mice survived adriamycin treatment while A(2A)R TG mice suffered 100% mortality at 4 wk. Telemetry showed progressive prolongation of the QT interval, bradyarrhythmias, heart block, and sudden death in adriamycin-treated A(2A)R TG but not WT mice. Both WT and A(2A)R TG demonstrated similar decreases in heart function at 3 wk after treatment. Adriamycin significantly increased end-diastolic intracellular Ca(2+) concentration in A(2A)R TG but not in WT myocytes (P < 0.05). Compared with WT myocytes, action potential duration increased dramatically in A(2A)R TG myocytes (P < 0.05) after adriamycin treatment. Expression of connexin 43 was decreased in adriamycin treated A(2A)R TG but not WT mice. In sharp contrast, A(2A)R overexpression induced after the completion of adriamycin treatment resulted in no deaths and enhanced cardiac performance compared with WT adriamycin-treated mice. Our results indicate that the timing of A(2A)R activation is critical in terms of exacerbating or protecting adriamycin-induced cardiotoxicity. Our data have direct relevance on the clinical use of adenosine agonists or antagonists in the treatment of patients undergoing adriamycin therapy.
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Cloning and characterization of the 5-flanking region of the pig AgRP gene.
Mol. Biol. Rep.
PUBLISHED: 01-31-2010
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Agouti-related peptide (AgRP), a brain neuropeptide generated by AgRP/neuropeptide Y (NPY) neurons, plays a vital role in the hypothalamic regulation of energy homeostasis. RT-PCR and real-time PCR were carried out in various tissues to detect the AgRP expression pattern in pigs. Our RT-PCR results showed that the pig AgRP gene was ubiquitously expressed in all examined tissues including heart, liver, spleen, lung, kidney, stomach, bladder, m. longissimus, belly fat, brain, large intestine, lymph, back fat, skin, and hypothalamus. Real-time quantitative PCR experiments revealed that it is in the hypothalamus with the highest expression of AgRP both in adult Lantang and Landrace pigs compared to the back fat and m.longissimus muscle and the cDNA level of AgRP in the hypothalamus of adult Chinese indigenous Lantang pig (fat-type) is significantly higher than that of Landrace pig (lean-type). To understand the regulation of the pig AgRP gene, the 5-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient AgRP expression was identified at nucleotides -501 to -479, by 5-serial deletion of the promoter. Electrophoretic mobility-shift assays (EMSA) with competing oligonucleotides revealed that the critical region contained a cis-acting element for Neurogenic Differentiation (NeuroD), which is a member of the NeuroD family of basic-helix-loop-helix transcription factors. This element has not been reported in human or mouse AgRP genes. Our results indicated that NeuroD might be an essential regulatory factor for transcription of pig AgRP, providing an important clue about energy homeostasis regulation in the porcine and human brain.
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Modeling of protein refolding from inclusion bodies.
Acta Biochim. Biophys. Sin. (Shanghai)
PUBLISHED: 12-17-2009
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Overexpression of foreign proteins in Escherichia coli often leads to the formation of inclusion bodies (IBs), which becomes the major bottleneck in the preparation of recombinant proteins and their applications. In the present study, 36 proteins from IBs were refolded using a simple refolding method. Refolding yields of these proteins were defined as the percentage of soluble proteins following dilution refolding in the amount of denatured proteins in the samples before diluting into refolding buffer. Furthermore, a mathematical model was deduced to evaluate the role of biochemical properties in the protein refolding. Our results indicated that under the experimental conditions, isoelectric point of proteins might be mostly contributing to the high efficacy of protein refolding since the increment of one unit resulted in a decrease of 14.83% in the refolding yield. Other important mediators were components of protein secondary structure and the molecular weight (R(2) = 0.98, P = 0.000, F-test). Six proteins with low efficiency in the protein refolding possessed relatively low isoelectric points. Furthermore, refolding yields of six additional proteins from IBs were predicted and further validated by refolding the proteins under the same conditions. Therefore, the model of protein refolding developed here could be used to predict the refolding yields of proteins from IBs through a simple method. Our study will be suggestive to optimize the methods for protein refolding from IBs according to their intrinsic properties.
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Phospholemman and beta-adrenergic stimulation in the heart.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 12-11-2009
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Phosphorylation at serine 68 of phospholemman (PLM) in response to beta-adrenergic stimulation results in simultaneous inhibition of cardiac Na(+)/Ca(2+) exchanger NCX1 and relief of inhibition of Na(+)-K(+)-ATPase. The role of PLM in mediating beta-adrenergic effects on in vivo cardiac function was investigated with congenic PLM-knockout (KO) mice. Echocardiography showed similar ejection fraction between wild-type (WT) and PLM-KO hearts. Cardiac catheterization demonstrated higher baseline contractility (+dP/dt) but similar relaxation (-dP/dt) in PLM-KO mice. In response to isoproterenol (Iso), maximal +dP/dt was similar but maximal -dP/dt was reduced in PLM-KO mice. Dose-response curves to Iso (0.5-25 ng) for WT and PLM-KO hearts were superimposable. Maximal +dP/dt was reached 1-2 min after Iso addition and declined with time in WT but not PLM-KO hearts. In isolated myocytes paced at 2 Hz. contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitudes and [Na(+)](i) reached maximum 2-4 min after Iso addition, followed by decline in WT but not PLM-KO myocytes. Reducing pacing frequency to 0.5 Hz resulted in much smaller increases in [Na(+)](i) and no decline in contraction and [Ca(2+)](i) transient amplitudes with time in Iso-stimulated WT and PLM-KO myocytes. Although baseline Na(+)-K(+)-ATPase current was 41% higher in PLM-KO myocytes because of increased alpha(1)- but not alpha(2)-subunit activity, resting [Na(+)](i) was similar between quiescent WT and PLM-KO myocytes. Iso increased alpha(1)-subunit current (I(alpha1)) by 73% in WT but had no effect in PLM-KO myocytes. Iso did not affect alpha(2)-subunit current (I(alpha2)) in WT and PLM-KO myocytes. In both WT and NCX1-KO hearts, PLM coimmunoprecipitated with Na(+)-K(+)-ATPase alpha(1)- and alpha(2)-subunits, indicating that association of PLM with Na(+)-K(+)-ATPase did not require NCX1. We conclude that under stressful conditions in which [Na(+)](i) was high, beta-adrenergic agonist-mediated phosphorylation of PLM resulted in time-dependent reduction in inotropy due to relief of inhibition of Na(+)-K(+)-ATPase.
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Internalization of NK cells into tumor cells requires ezrin and leads to programmed cell-in-cell death.
Cell Res.
PUBLISHED: 09-29-2009
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Cytotoxic lymphocytes are key players in the orchestration of immune response and elimination of defective cells. We have previously reported that natural killer (NK) cells enter target tumor cells, leading to either target cell death or self-destruction within tumor cells. However, it has remained elusive as to the fate of NK cells after internalization and whether the heterotypic cell-in-cell process is different from that of the homotypic cell-in-cell event recently named entosis. Here, we show that NK cells undergo a cell-in-cell process with the ultimate fate of apoptosis within tumor cells and reveal that the internalization process requires the actin cytoskeletal regulator, ezrin. To visualize how NK cells enter into tumor cells, we carried out real-time dual color imaging analyses of NK cell internalization into tumor cells. Surprisingly, most NK cells commit to programmed cell death after their entry into tumor cells, which is distinctively different from entosis observed in the homotypic cell-in-cell process. The apoptotic cell death of the internalized NK cells was evident by activation of caspase 3 and DNA fragmentation. Furthermore, NK cell death after internalization is attenuated by the caspase inhibitor, Z-VAD-FMK, confirming apoptosis as the mode of NK cell death within tumor cells. To determine protein factors essential for the entry of NK cells into tumor cells, we carried out siRNA-based knockdown analysis and discovered a critical role of ezrin in NK cell internalization. Importantly, PKA-mediated phosphorylation of ezrin promotes the NK cell internalization process. Our findings suggest a novel regulatory mechanism by which ezrin governs NK cell internalization into tumor cells.
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[Prokaryotic expression and identification of dual-fluorescence fusion proteins of small ubiquitin-like modifier and sentrin-specific protease].
Sheng Wu Gong Cheng Xue Bao
PUBLISHED: 08-13-2009
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We cloned genes of four sentrin-specific protease (SENP), three small ubiquitin-like modifiers (SUMO), enhanced cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP) by two-step PCR. Then we constructed expression vector B28 for SENP and B13 for ECFP-SUMO-EYFP. The recombinant plasmids were transformed into Escherichia coli BL21 and expression was induced by isopropyl beta-D-thiogalactoside, then recombinant proteins were purified by Ni-NTA agarose column ion-exchange chromatography. The proteins were analyzed with SDS-PAGE and identified with Western blotting. Except that SENP3 catalytic domain (SENP3C) truncated in the C termini and SENP5C expressed in inclusion body, others were expressed as soluble proteins. SDS-PAGE analysis showed that the relative molecular mass of these fusion proteins were consistent with theoretical ones, and the specificity of the fusion proteins were confirmed with Western blotting. The fusion proteins of SENP and ECFP-SUMO-EYFP can be successfully expressed in prokaryotic expression system. It lays the foundation for the fluorescence resonance energy transfer analysis.
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Induced overexpression of Na+/Ca2+ exchanger transgene: altered myocyte contractility, [Ca2+]i transients, SR Ca2+ contents, and action potential duration.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 06-12-2009
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We have produced mice in which expression of the rat cardiac Na(+)/Ca(2+) exchanger (NCX1) transgene was switched on when doxycycline was removed from the feed at 5 wk. At 8 to 10 wk, NCX1 expression in induced (Ind) mouse hearts was 2.5-fold higher but protein levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase, alpha(1)- and alpha(2)-subunits of Na(+)-K(+)-ATPase, phospholamban, ryanodine receptor, calsequestrin, and unphosphorylated and phosphorylated phospholemman were unchanged compared with wild-type (WT) or noninduced (non-Ind) hearts. There was no cellular hypertrophy since WT, non-Ind, and Ind myocytes had similar whole cell membrane capacitance. In Ind myocytes, NCX1 current amplitude was approximately 42% higher, L-type Ca(2+) current amplitude was unchanged, and action potential duration was prolonged compared with WT or non-Ind myocytes. Contraction and intracellular Ca(2+) concentration ([Ca(2+)](i)) transient amplitudes in Ind myocytes were lower at 0.6, not different at 1.8, and higher at 5.0 mM extracellular Ca(2+) concentration ([Ca(2+)](o)) compared with WT or non-Ind myocytes. Despite similar Ca(2+) current amplitude and sarcoplasmic reticulum (SR) Ca(2+) uptake, SR Ca(2+) content at 5.0 mM [Ca(2+)](o) was significantly higher in Ind compared with non-Ind myocytes, indicating that NCX1 directly contributed to SR Ca(2+) loading. Echocardiography demonstrated that heart rate, left ventricular mass, ejection fraction, stroke volume, and cardiac output were similar among the three groups of animals. In vivo close-chest catheterization demonstrated similar contractility and relaxation among the three groups of mice, both at baseline and after stimulation with isoproterenol. We conclude that induced expression of NCX1 transgene resulted in altered [Ca(2+)](i) homeostasis, myocyte contractility, and action potential morphology. In addition, heart failure did not occur 3 to 5 wk after NCX1 transgene was induced to be expressed at levels found in diseased hearts.
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Phosphoenolpyruvate-dependent phosphorylation of sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for the phosphotransferase transport system.
Bioresour. Technol.
PUBLISHED: 05-04-2009
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The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate.
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Antibody-enhanced, Fc gamma receptor-mediated endocytosis of Clostridium difficile toxin A.
Infect. Immun.
PUBLISHED: 03-23-2009
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Toxin A (TcdA) and toxin B (TcdB) are major virulence factors of Clostridium difficile. These two toxins intoxicate cultured cells by similar mechanisms, and TcdB generally is more potent than TcdA in cultured cells. The exact reason for this difference is unclear. Here, we report that the cellular effects of TcdA can be substantially enhanced via an opsonizing antibody through Fc gamma receptor I (FcgammaRI)-mediated endocytosis. A TcdA-specific monoclonal antibody, A1H3, was found to significantly enhance the cytotoxicity of TcdA to macrophages and monocytes. The A1H3-dependent enhancement of glucosyltransferase activity, cytoskeleton disruption, and tumor necrosis factor alpha production induced by TcdA was further demonstrated using RAW 264.7 cells. Subsequent experiments indicated that the interaction of FcgammaRI with A1H3 underlays the antibody-dependent enhancement of the cellular effects of TcdA. While blocking FcgammaRII and FcgammaRIII with anti-CD16/32 antibodies did not affect the TcdA-mediated glucosylation of Rac1 in RAW 264.7 cells, presaturation of FcgammaRI with anti-CD64 antibodies in THP1 cells significantly reduced this activity. Incubation of a TcdA-A1H3 immune complex with recombinant mouse CD64 completely abrogated the A1H3-mediated enhancement of the glucosyltransferase activity of TcdA in RAW 264.7 cells. Moreover, expression of FcgammaRI in CHO cells strikingly enhanced the sensitivity of these cells to TcdA complexed with A1H3. We showed that the presence of A1H3 facilitated cell surface recruitment of TcdA, contributing to the antibody-dependent, FcgammaRI-mediated enhancement of TcdA activity. Finally, studies using chlorpromazine and endosomal acidification inhibitors revealed an important role of the endocytic pathway in the A1H3-dependent enhancement of TcdA activity.
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Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses.
Bioresour. Technol.
PUBLISHED: 02-17-2009
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Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2 g l(-1) with yield of 0.47 g g(-1) and reactor productivity up to 4.13 g l(-1)h(-1) at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6+/-0.8 g l(-1)), yield (0.58+/-0.01 g g(-1)), and sugar utilization (90.8+/-0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2 g l(-1).
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High-level expression of soluble subunit b of F1F0 ATP synthase in Escherichia coli cell-free system.
Appl. Microbiol. Biotechnol.
PUBLISHED: 02-05-2009
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The overexpression of subunit b of F(1)F(0) adenosine triphosphate (ATP) synthase from Escherichia coli is so toxic that it even prevents the transformation of plasmids encoding this protein into E. coli BL21 (DE3). In the present work, E. coli cell-free system was chosen as an alternative to express this highly toxic membrane protein. This protein was either produced as precipitates followed by detergent resolubilization or expressed as a soluble form with detergent addition. Among several types of tested detergents, Brij 58 could effectively solubilize approximately 85% of the target membrane protein within a wide range of concentration (48 to 178 times critical micelle concentration [CMC]) with little effect on the expression level. With the presence of Brij 58 at the final concentration of 96 times CMC in the E. coli cell-free system, 789 microg/mL of soluble subunit b was achieved after 4 h biosynthesis, which is the highest level for the expression of membrane proteins in a batch-mode cell-free expression system. The present work provides a rapid and efficient procedure of expressing one membrane protein with high cytotoxicity in the cell-free system and will be helpful to further exploration of reconstituting F(1)F(0) ATP synthase into liposome or polymer vesicle to design a nanoelectromechanical system device.
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Phospholemman regulates cardiac Na+/Ca2+ exchanger by interacting with the exchangers proximal linker domain.
Am. J. Physiol., Cell Physiol.
PUBLISHED: 01-21-2009
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Phospholemman (PLM) belongs to the FXYD family of small ion transport regulators. When phosphorylated at Ser(68), PLM inhibits cardiac Na(+)/Ca(2+) exchanger (NCX1). We previously demonstrated that the cytoplasmic tail of PLM interacts with the proximal intracellular loop (residues 218-358), but not the transmembrane (residues 1-217 and 765-938) or Ca(2+)-binding (residues 371-508) domains, of NCX1. In this study, we used intact Na(+)/Ca(2+) exchanger with various deletions in the intracellular loop to map the interaction sites with PLM. We first demonstrated by Western blotting and confocal immunofluorescence microscopy that wild-type (WT) NCX1 and its deletion mutants were expressed in transfected HEK-293 cells. Cotransfection with PLM and NCX1 (or its deletion mutants) in HEK-293 cells did not decrease expression of NCX1 (or its deletion mutants). Coexpression of PLM with WT NCX1 inhibited NCX1 current (I(NaCa)). Deletion of residues 240-679, 265-373, 250-300, or 300-373 from WT NCX1 resulted in loss of inhibition of I(NaCa) by PLM. Inhibition of I(NaCa) by PLM was preserved when residues 229-237, 270-300, 328-330, or 330-373 were deleted from the intracellular loop of NCX1. These results suggest that PLM mediated inhibition of I(NaCa) by interacting with two distinct regions (residues 238-270 and 300-328) of NCX1. Indeed, I(NaCa) measured in mutants lacking residues 238-270, 300-328, or 238-270 + 300-328 was not affected by PLM. Glutathione S-transferase pull-down assays confirmed that PLM bound to fragments corresponding to residues 218-371, 218-320, 218-270, 238-371, and 300-373, but not to fragments encompassing residues 250-300 and 371-508 of NCX1, indicating that residues 218-270 and 300-373 physically associated with PLM. Finally, acute regulation of I(NaCa) by PLM phosphorylation observed with WT NCX1 was absent in 250-300 deletion mutant but preserved in 229-237 deletion mutant. We conclude that PLM mediates its inhibition of NCX1 by interacting with residues 238-270 and 300-328.
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Development of direct competitive enzyme-linked immunosorbent assay for the determination cadmium residue in farm produce.
Appl. Biochem. Biotechnol.
PUBLISHED: 01-20-2009
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Cadmium, a toxic heavy metal, poses a significant threat to human health. Currently, the methods for detecting cadmium residue in farm produce need expensive equipment, intensive labor, and much time to finish one detection. In this study, a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) based on a cadmium-chelate-specific monoclonal antibody has been developed. The DC-ELISA showed an IC(50) of 2.30 microg/L with a detection limit of 0.20 microg/L for cadmium. The assay has been demonstrated to be highly specific since the monoclonal antibody showed little or no cross-reactivity with all tested metal chelates which include Cd(2+), Pb(2+), Hg(2+), Zn(2+), Na(+), Ca(2+), Fe(3+), Mg(2+), Mn(2+), Cu(2+), Al(3+), Co(2+), Cr(2+), Ni(2+), Sn(2), and K(+). The assay showed that a mean recovery ranged from 100.47% to 103.86%, and the coefficients of variations for intra- and inter-assay were 1.73-7.14% and 3.63-6.81%, respectively. Then, several farm produces including wheat flour, apple juice, rice flour, and tea were analyzed for cadmium residue with DC-ELISA and graphite furnace atomic absorption spectroscopy (GFAAS). The correlation coefficient between the DC-ELISA and GFAAS was 0.99. It was demonstrated that the DC-ELISA can be used as a simple and economic method to detect and quantitate cadmium residue in farm produce.
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The heterogeneous nature of polyethylenimine-DNA complex formation affects transient gene expression.
Cytotechnology
PUBLISHED: 01-13-2009
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Polyethylenimine has been used widely in transient gene expression with mammalian cells. To further understand its mediation of gene transfer, the transfection of HEK 293-F cells with dynamically prepared PEI/DNA complexes was studied with the help of fluorescent labeling. The efficiency of complex endocytosis/phagocytosis was found to correlate with the average sizes of complexes applied and complexes greater than 1 mum in diameter were likely excluded by the cells. Coupled with complex growth in size, the degree of association between PEI and DNA increased with the time of complex formation in the presence of competing ions. The blocking of transcription by complex formation necessitated complex dissociation in the nuclear environment for transcription to happen. Intracellularly, the fates of PEI complexed DNA therefore may be mostly determined by the degree of association. Results also suggested that the uptake of PEI/DNA complexes and subsequent protein expression were independent of the cell cycle stages of HEK 293-F cells.
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An ultrasensitive rapid immunocytotoxicity assay for detecting Clostridium difficile toxins.
J. Microbiol. Methods
PUBLISHED: 01-07-2009
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We describe a novel ultrasensitive cell-based immunocytotoxicity assay for detecting Clostridium difficile toxin A and B. The assay is simple to perform with a turnaround time of approximately 3 h . It is particularly sensitive in detecting TcdA at a level less then 1 pg/ml. Using this assay, we were able to detect the presence of C. difficile toxins in the fecal and serum specimens of experimentally infected piglets.
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Cell-free expression of human glucosamine 6-phosphate N-acetyltransferase (HsGNA1) for inhibitor screening.
Protein Expr. Purif.
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Glucosamine 6-phosphate N-acetyltransferase (GNA1; EC 2.3.1.4) is required for the de novo synthesis of N-acetyl-d-glucosamine-6-phosphate (GlcNAc-6P), which is an essential precursor in Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) biosynthesis pathway. Therefore, GNA1 is indispensable for the viability of organisms. Here, a novel cell-free expression strategy was developed to efficiently produce large amounts of human GNA1(HsGNA1) and HsGNA1-sGFP for throughput inhibitor screening. The binding site of inhibitor glucose-6-phosphate (G6P) to hGNA was identified by simulated annealing. Subtle differences to the binding site of Aspergillius GNA1(AfGNA1) can be harnessed for inhibitor design. HsGNA1 may be also useful as an antimicrobial and chemotherapeutic target against cancer. Additionally HsGNA1 inhibitors/modulators can possibly be administered with other drugs in the next generation of personalized medicine.
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Improvement of Vitreoscilla hemoglobin function by Bacillus licheformis glutamate-specific endopeptidase treatment.
Protein Expr. Purif.
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Vitreoscilla hemoglobin (VHb) was widely used in metabolic engineering to improve oxygen utilization in the low oxygen environment. It is sometimes necessary to remove affinity tags because they may impede functions of target proteins. Here we report an efficient method employing Glutamate-specific endopeptidase from Bacillus licheformis (GSE-BL) to perform the cleavage between VHb and His-tag. The optimal length of GSE-BL treatment was 15min. Results of SDS-PAGE and western blot demonstrated that the His-tag of VHb-His(6) was nearly completely removed, the purity of VHb was enhanced from 74% to 99.5%, and the yield of tagless VHb from VHb-His(6) was 92.2%. Results of CO difference spectrum suggested that tagless VHb was more prone to bind to CO compared with VHb-His(6). It was observed that tagless VHb displayed higher catalase activity than VHb-His(6). The enhancement of welan gum yield was more significant by addition of tagless VHb compared with addition of VHb-His(6). This method can be utilized to mass-produce tagless VHb, thus widening the application of VHb in various industries.
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Chemical and cellular antioxidant activity of two novel peptides designed based on glutathione structure.
Food Chem. Toxicol.
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Two novel peptides, ECH (Glu-Cys-His) and YECG (Tyr-Glu-Cys-Gly), were designed based on glutathione (Glu-Cys-Gly, GSH) and their antioxidant activities were studied. Various chemical methods based on single-electron-transfer (SET) and hydrogen-atom-transfer (HAT) were applied to evaluate the antioxidant activities of the peptides. For SET-based assay, tripeptide ECH displayed the highest DPPH radical scavenging activity (80.16%) and the strongest reducing power (A(700)=0.378). Besides, ECH also exhibited the best inhibition activity toward linoleic acid peroxidation with inhibition rate 98.25% at 7th day, which is a HAT-based assay. However, for another two HAT-based assays, it was tetrapeptide YECG that showed extraordinary oxygen radical absorption capacity (ORAC value=2.42 ?M Trolox/?M) and ABTS free radical scavenging ability (8.88 mM Trolox/mM). In vitro cultured PC12 cell model also suggested that YECG gave the best protection for PC12 cells to resist H(2)O(2)-treated necrosis. It was found that the discrepancy of antioxidant capacity between ECH and YECG was caused by the presence of antioxidant amino acids (His/Tyr) and their position in peptide chain. With His located at C-terminal position, ECH demonstrated good electrons donating capacity, while with Tyr at N-terminal position, YECG exhibited strong oxygen radical absorbance capacity.
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miR-3928 activates ATR pathway by targeting Dicer.
RNA Biol
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Alterations in microRNA (miRNA) expression have been observed in cells subjected to exogenous stresses, implying that miRNAs play an important role in cellular stress response; however, the underlying mechanism is still largely unknown. In the present study, we found that miR-3928 was implicated in cellular response to ionizing radiation. After exposed to X-rays, miR-3928 expression increased in 1.5 h and then decreased, meanwhile Dicer, a key component in the miRNA processing machinery, increased gradually. An oscillation was observed in the expression of both mature miR-3928 and Dicer mRNA from 2 h to 3.5 h in irradiated cells. Then, we verified that miR-3928 directly bound to the 3-untranslated region of Dicer mRNA. Consequently, Dicer expression was suppressed and the maturation of other miRNAs including miR-185, miR-300, and miR-663, was inhibited. Overexpression of miR-3928 induced DNA damage, activated ATR, and phosphorylated Chk1 accompanied by G1 arrest. Taken together, these findings replenished ATR/Chk1 pathway by revealing a novel miRNA regulatory network in response to exogenous stress, in which miR-3928 plays an important role in regulating the expression of Dicer.
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