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871.
Lin-Li Lv Yuhan Cao Dan Liu Min Xu Hong Liu Ri-Ning Tang Kun-Ling Ma Bi-Cheng Liu 《International journal of biological sciences》2013,9(10):1021-1031
Recent studies indicate that microRNA (miRNA) is contained within exosome. Here we sought to optimize the methodologies for the isolation and quantification of urinary exosomal microRNA as a prelude to biomarker discovery studies. Exosomes were isolated through ultracentrifugation and characterized by immunoelectron microscopy. To determine the RNA was confined inside exosomes, the pellet was treated with RNase before RNA isolation. The minimum urine volume, storage conditions for exosomes and exosomal miRNA was evaluated. The presence of miRNAs in patients with various kidney diseases was validated with real-time PCR. The result shows that miRNAs extracted from the exosomal fraction were resistant to RNase digestion and with high quality confirmed by agarose electrophoresis. 16ml of urine was sufficient for miRNA isolation by absolute quantification with 4.15×105 copies/ul for miR-200c. Exosomes was stable at 4℃ 24h for shipping before stored at -80℃ and was stable in urine when stored at -80°C for 12months. Exosomal miRNA was detectable despite 5 repeat freeze-thaw cycles. The detection of miRNA by quantitative PCR showed high reproducibility (>94% for intra-assay and >76% for inter-assay), high sensitivity (positive call 100% for CKD patients), broad dynamic range (8-log wide) and good linearity for quantification (R2>0.99). miR-29c and miR-200c showed different expression in different types of kidney disease. In summary, the presence of urinary exosomal miRNA was confirmed for patients with a diversity of chronic kidney disease. The conditions of urine collection, storage and miRNA detection determined in this study may be useful for future biomarker discovery efforts. 相似文献
872.
Hualin Ma Yaojiong Wu Wanfan Zhang Yong Dai Furong Li Ying Xu Yunshuai Wang Huiying Tu Wuxian Li Xinzhou Zhang 《Cytotherapy》2013,15(6):703-711
Background aimsThe potential protective effects of mesenchymal stromal cells (MSCs) on some kidney diseases has been reported. However, the effect of MSCs on doxorubicin-induced nephropathy is still poorly understood.MethodsRats with doxorubicin-induced kidney injuries were treated with human cord-derived MSCs. Human MSCs were first labeled with 5-bromo-2′-deoxyuridine to track their homing in kidneys after infusion.ResultsAlleviation of proteinuria, decreased serum albumin, alleviation of lipid disorders and histologic alterations were found in rats 4 weeks after treatment with MSCs, particularly in rats that were given repeat doses. Decreases in serum levels of interleukin-6, tumor necrosis factor-α and prostaglandin E2 and decreases in messenger RNA levels of kidney tissue cylooxygenase-2 and EP4 were found in MSC-treated rats. MSC-treated rats also displayed an increase in serum interleukin-10 levels.ConclusionsThese results indicate that MSCs ameliorate doxorubicin-induced kidney injuries and inflammation, suggesting a potential clinical treatment for inflammatory kidney diseases. 相似文献
873.
874.
Nonunion of fractured bones is a common clinical problem for orthopedic surgeons. This study aimed to investigate the effects of simvastatin locally applied from calcium sulfate (CS) combined with a mesenchymal stem cell (MSC) sheet on fracture healing. In vitro, the proliferation and differentiation of rat bone marrow–derived MSCs stimulated by simvastatin were investigated. In vivo, an osteotomy model was made in rat tibia, and fractured tibias were treated with CS, CS/simvastatin, CS/MSC sheet or simvastatin-loaded CS with MSC or untreated (control). Tibias were harvested at 2 or 8 weeks and underwent real-time quantitative polymerase chain reaction, x-ray, micro-CT and histological analysis. The expression levels of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor of simvastatin-induced MSCs increased with the concentrations of the simvastatin, significantly higher than those in the MSCs group. At 2 weeks, the CS/simvastatin/MSC sheet group showed significantly higher expressions of bone morphogenetic protein 2, alkaline phosphatase, osteocalcin, osteoprotegerin and vascular endothelial growth factor, with more callus formation around the fracture site compared with the other four groups. At 8 weeks, complete bone union was obtained in the CS/simvastatin/MSC sheet group. By contrast, newly regenerated bone tissue partially bridged the gap in the CS/simvastatin group and the CS/MSC sheet group; the control and CS group showed nonunion of the tibia. These results show that both simvastatin and the MSC sheet contributed to the formation of new bone and that the tibia fracture was completely healed by transplantation of the MSC sheet with locally applied simvastatin. Such MSC sheet with locally applied simvastatin might contribute to the treatment of fractures, bone delayed unions or nonunions in clinical practice. 相似文献
875.
Dandan Chen Fengxia Ma Shuxia Xu Shaoguang Yang Fang Chen Lijuan Rong Ying Chi Qinjun Zhao Shihong Lu Zhibo Han Aiming Pang Zhongchao Han 《Cytotherapy》2013,15(4):423-433
Background aimsToll-like receptors (TLRs) play an important role in innate and adaptive immunity by recognizing pathogen-associated molecular patterns (PAMPs).MethodsIn the present study, we investigated the expression and role of TLRs on human umbilical cord mesenchymal stromal cells (UC-MSCs). The proliferation, differentiation and immunoregulatory activity of UC-MSCs primed with or without TLR ligands were determined.ResultsAt the RNA level, the expression of TLR2, 4, 6 and 9 was relatively higher than that of other TLRs. However, TLR3 and TLR4 expression were relatively higher at the protein level. UC-MSCs expressed functional TLRs by nuclear factor-κB activation and cytokine expression assay. Poly-inosinic acid:cytidylic acid [Poly(I:C)] stimulation inhibited the proliferation of UC-MSCs, but the ligand of other TLRs had no significant effect. Poly(I:C) stimulation enhanced the adipogenic differentiation capability of UC-MSCs, but lipopolysaccharide inhibited the adipogenic differentiation. Poly(I:C) and CpG-oligonucleotide promoted the immunosuppressive potentiality of UC-MSCs, accompanied with the phosphorylation of interferon regulatory factor 3 (IRF3) and increased expression of indoleamine 2,3-dioxygenase and interferon β, whereas activation of other TLR ligands (synthetic analog fibroblast-stimulating lipopeptide-1 and lipopolysaccharide) failed to affect the immunoregulatory activity of UC-MSCs.ConclusionsTaken together, our data demonstrated that TLR activation influenced the function of UC-MSCs, which might have important implications in future efforts to explore the clinical potentials of UC-MSCs. 相似文献
876.
Altered expression of the PTR/NRT1 homologue OsPTR9 affects nitrogen utilization efficiency,growth and grain yield in rice 总被引:2,自引:0,他引:2
Zhongming Fang Kuaifei Xia Xin Yang Marianne Suter Grotemeyer Stefan Meier Doris Rentsch Xinlan Xu Mingyong Zhang 《Plant biotechnology journal》2013,11(4):446-458
The plant PTR/NRT1 (peptide transporter/nitrate transporter 1) gene family comprises di/tripeptide and low‐affinity nitrate transporters; some members also recognize other substrates such as carboxylates, phytohormones (auxin and abscisic acid), or defence compounds (glucosinolates). Little is known about the members of this gene family in rice (Oryza sativa L.). Here, we report the influence of altered OsPTR9 expression on nitrogen utilization efficiency, growth, and grain yield. OsPTR9 expression is regulated by exogenous nitrogen and by the day‐night cycle. Elevated expression of OsPTR9 in transgenic rice plants resulted in enhanced ammonium uptake, promotion of lateral root formation and increased grain yield. On the other hand, down‐regulation of OsPTR9 in a T‐DNA insertion line (osptr9) and in OsPTR9‐RNAi rice plants had the opposite effect. These results suggest that OsPTR9 might hold potential for improving nitrogen utilization efficiency and grain yield in rice breeding. 相似文献
877.
Lei Wang Jie Zheng Yanzhong Luo Tao Xu Qiuxue Zhang Lan Zhang Miaoyun Xu Jianmin Wan Ming‐Bo Wang Chunyi Zhang Yunliu Fan 《Plant biotechnology journal》2013,11(8):997-1005
Long hairpin RNA (hpRNA) transgenes are a powerful tool for gene function studies in plants, but a genomewide RNAi mutant library using hpRNA transgenes has not been reported for plants. Here, we report the construction of a hpRNA library for the genomewide identification of gene function in rice using an improved rolling circle amplification‐mediated hpRNA (RMHR) method. Transformation of rice with the library resulted in thousands of transgenic lines containing hpRNAs targeting genes of various function. The target mRNA was down‐regulated in the hpRNA lines, and this was correlated with the accumulation of siRNAs corresponding to the double‐stranded arms of the hpRNA. Multiple members of a gene family were simultaneously silenced by hpRNAs derived from a single member, but the degree of such cross‐silencing depended on the level of sequence homology between the members as well as the abundance of matching siRNAs. The silencing of key genes tended to cause a severe phenotype, but these transgenic lines usually survived in the field long enough for phenotypic and molecular analyses to be conducted. Deep sequencing analysis of small RNAs showed that the hpRNA‐derived siRNAs were characteristic of Argonaute‐binding small RNAs. Our results indicate that RNAi mutant library is a high‐efficient approach for genomewide gene identification in plants. 相似文献
878.
Mian Cao Zhuo Mao Chuen Kam Nan Xiao Xiaoxing Cao Chong Shen Kenneth K. Y. Cheng Aimin Xu Kwong-Man Lee Liwen Jiang Jun Xia 《PLoS biology》2013,11(4)
Diabetes is a metabolic disorder characterized by hyperglycemia. Insulin, which is secreted by pancreatic beta cells, is recognized as the critical regulator of blood glucose, but the molecular machinery responsible for insulin trafficking remains poorly defined. In particular, the roles of cytosolic factors that govern the formation and maturation of insulin granules are unclear. Here we report that PICK1 and ICA69, two cytosolic lipid-binding proteins, formed heteromeric BAR-domain complexes that associated with insulin granules at different stages of their maturation. PICK1-ICA69 heteromeric complexes associated with immature secretory granules near the trans-Golgi network (TGN). A brief treatment of Brefeldin A, which blocks vesicle budding from the Golgi, increased the amount of PICK1 and ICA69 at TGN. On the other hand, mature secretory granules were associated with PICK1 only, not ICA69. PICK1 deficiency in mice caused the complete loss of ICA69 and led to increased food and water intake but lower body weight. Glucose tolerance tests demonstrated that these mutant mice had high blood glucose, a consequence of insufficient insulin. Importantly, while the total insulin level was reduced in PICK1-deficient beta cells, proinsulin was increased. Lastly, ICA69 knockout mice also displayed similar phenotype as the mice deficient in PICK1. Together, our results indicate that PICK1 and ICA69 are key regulators of the formation and maturation of insulin granules.
Author Summary
Insulin is a key regulator of blood glucose and insufficient insulin leads to diabetes. Insulin is synthesized as proinsulin, processed in endoplasmic reticulum and Golgi, and eventually packaged into insulin granules, a type of dense core vesicles. Despite its importance, the molecular mechanisms governing the biogenesis and maturation of insulin granules are not fully understood. In this study, we identified two cytosolic proteins, PICK1 and ICA69, as important regulators of insulin granule biogenesis and maturation. Both PICK1 and ICA69 have the banana-shaped BAR domain that can bend the lipid membrane and help the formation of dense core vesicles. We show that without PICK1 or ICA69, insulin granules cannot be properly formed and, as a result, proinsulin cannot be effectively processed into mature insulin. Mice lacking functional PICK1 or ICA69 genes have reduced insulin but increased proinsulin. Consequently, these mice have high levels of glucose, a prominent feature found in diabetes patients. These results add to previous findings that PICK1 is important for the generation of proacrosomal granules found in cells of the testis, and thereby support a wider role for PICK1 and ICA69 in regulating dense core vesicle biogenesis and maturation. 相似文献879.
Background
Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis.Methodology/Principal Findings
A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0–10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg−1 and 228 U mg−1) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel.Conclusion
RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei. 相似文献880.
Cardiomyocyte apoptosis is a common pathological alteration in heart disease which results in systolic dysfunction or sudden death. Klotho is a novel anti-aging hormone. We tested the effects of klotho on cell apoptosis in isoproterenol-treated cardiomyocytes. In BALB/c mice, cardiac injury was induced by subcutaneous injection of isoproterenol (5mg/kg, for 9days, sc). Klotho (0.01 mg/kg, every other day for 4days, ip) was administered to determine the changes in isoproterenol-induced apoptosis. Mouse heart was harvested at day 2, day 5, and day 9 after isoproterenol injection. Isoproterenol induced cardiac apoptosis and endoplasmic reticulum (ER) stress in a time-dependent manner. However, klotho partly reversed isoproterenol-induced cardiac apoptosis and ER stress. These same effects were observed in cultured cardiomyocytes. Furthermore, the results also showed that SB203580, a p38 inhibitor, and SP600125, a c-Jun NH2-terminal kinase (JNK) inhibitor, reduced cardiomyocyte apoptosis and ER stress, however, klotho suppressed isoproterenol-induced activation of p38 and JNK. Taken together, these results indicated that cardioprotection by klotho was related to the attenuation of ER stress and ER stress-induced apoptosis, at least partly, through suppressing activation of the p38 and JNK pathway. 相似文献