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31.
Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair. 相似文献
32.
Bell JR Kennington E Fuller W Dighe K Donoghue P Clark JE Jia LG Tucker AL Moorman JR Marber MS Eaton P Dunn MJ Shattock MJ 《American journal of physiology. Heart and circulatory physiology》2008,294(2):H613-H621
Phospholemman (PLM, FXYD1), abundantly expressed in the heart, is the primary cardiac sarcolemmal substrate for PKA and PKC. Evidence supports the hypothesis that PLM is part of the cardiac Na-K pump complex and provides the link between kinase activity and pump modulation. PLM has also been proposed to modulate Na/Ca exchanger activity and may be involved in cell volume regulation. This study characterized the phenotype of the PLM knockout (KO) mouse heart to further our understanding of PLM function in the heart. PLM KO mice were bred on a congenic C57/BL6 background. In vivo conductance catheter measurements exhibited a mildly depressed cardiac contractile function in PLM KO mice, which was exacerbated when hearts were isolated and Langendorff perfused. There were no significant differences in action potential morphology in paced Langendorff-perfused hearts. Depressed contractile function was associated with a mild cardiac hypertrophy in PLM KO mice. Biochemical analysis of crude ventricular homogenates showed a significant increase in Na-K-ATPase activity in PLM KO hearts compared with wild-type controls. SDS-PAGE and Western blot analysis of ventricular homogenates revealed small, nonsignificant changes in Na- K-ATPase subunit expression, with two-dimensional gel (isoelectric focusing, SDS-PAGE) analysis revealing minimal changes in ventricular protein expression, indicating that deletion of PLM was the primary reason for the observed PLM KO phenotype. These studies demonstrate that PLM plays an important role in the contractile function of the normoxic mouse heart. Data are consistent with the hypothesis that PLM modulates Na-K-ATPase activity, indirectly affecting intracellular Ca and hence contractile function. 相似文献
33.
FSHbeta mRNA has a unique 3' untranslated region (3'UTR) that is highly conserved across the species. Sequence analyses of the mouse, rat, human, bovine, and ovine 3'UTRs revealed the presence of elements implicated in mRNA instability and translational control such as AU-Rich Element (ARE) and lipoxygenase differentiation control elements. Bovine FSHbeta 3'UTR down-regulated reporter expression in alphaT3-1 and NIH3T3 cells, but not in HEK 293 cells, suggesting the involvement of a cell-specific factor or mechanism. The presence of a 3'UTR did not influence reporter mRNA stability, but it did decrease its association with polysomes, indicating that the downregulatory effect may be exerted at the translational level. The segment spanning 601-800 bases (U4) of the bovine FSHbeta 3'UTR was found to be the most effective downregulating segment, its effect being equal to that of the full-length 3'UTR. RNA electrophoretic mobility shift assay with U4 showed the presence of specific transfactors in the cytosolic preparations of bovine pituitary and the cell lines. U4 contained an ARE that appeared to be functional, because the mutated U4 ARE was ineffective in downregulating the reporter expression and inhibiting [(32)P]-labeled U4-transfactor complex formation. Downregulation of reporter activity by the full-length 3'UTR and U4 could be overcome by overexpression of HuR, a protein known to stabilize ARE-containing mRNAs in NIH3T3 cells, but not in the alphaT3-1 cells, once again indicating that factors other than HuR may also be involved in the regulation of FSHbeta in the pituitary. 相似文献
34.
Dighe A Rodriguez M Sabastian P Xie X McVoy M Brown MG 《Journal of immunology (Baltimore, Md. : 1950)》2005,175(10):6820-6828
Human CMV infections are a major health risk in patients with dysfunctional or compromised immunity, especially in patients with NK cell deficiencies, as these are frequently associated with high morbidity and mortality. In experimental murine CMV (MCMV) infections, Ly49H activation receptors on C57BL/6 (B6) NK cells engage m157 viral ligands on MCMV-infected cells and initiate dominant virus control. In this study, we report that MCMV resistance in MA/My relies on Ly49H-independent NK cell-mediated control of MCMV infection as NK cells in these mice do not bind anti-Ly49H mAb or soluble m157 viral ligands. We genetically compared MA/My resistance with MCMV susceptibility in genealogically and NK gene complex-Ly49 haplotype-related C57L mice. We found that MCMV resistance strongly associated with polymorphic H2k-linked genes, including MHC and non-MHC locations by analysis of backcross and intercross progeny. The H2b haplotype most frequently, but not absolutely, correlated with MCMV susceptibility, thus confirming a role for non-MHC genes in MCMV control. We also demonstrate a definite role for NK cells in H2k-type MCMV resistance because their removal from C57L.M-H2k mice before MCMV infection diminished immunity. NK gene complex-linked polymorphisms, however, did not significantly influence MCMV control. Taken together, effective NK cell-mediated MCMV control in this genetic system required polymorphic H2k genes without need of Ly49H-m157 interactions. 相似文献
35.
Mitali Samaddar James F. Catterall Rajan R. Dighe 《Protein expression and purification》1997,10(3):345-355
Follicle-stimulating hormone (FSH), a pituitary gonadotropin, is a heterodimer composed of an α subunit, which is common to all the glycoprotein hormones, noncovalently associated with the hormone-specific β subunit. The objective of the present study is to develop a recombinant DNA expression system for the β subunit of FSH that can be applied to study structure–function relationships while producing large quantities of the hormone subunit for immunocontraceptive, clinical, and veterinary purposes. We report here the expression of biologically active bovine FSHβ (bFSHβ) in the methylotrophic yeastPichia pastoris.ThePichia-expressed FSHβ (pFSHβ) was secreted into the culture medium and was found to be immunologically very similar to pituitary-derived ovine FSHβ. Replacement of the cognate signal peptide with the yeast α mating factor signal peptide increased the level of expression from 230 ng/ml (cognate signal peptide) to 4 μg/ml (α mating factor signal peptide) of the culture supernatant. pFSHβHis.tag(pFSHβ with six histidine residues at the C terminus) was purified to apparent homogeneity using one-step nickel affinity chromatography. The molecular weight of purified pFSHβHis.tagwas approximately 22,000, which was slightly higher than that of the pituitary-derived ovine FSHβ. pFSHβHis.tagcould assemble with the α subunit to yield a heterodimer capable of binding to the FSH receptors and also elicit biological response. These data show that pFSHβHis.tagis properly folded and biologically active. 相似文献
36.
James L Crainey Túllio RR da Silva Fernando Encinas Michel A Marín Ana Carolina P Vicente Sérgio LB Luz 《Memórias do Instituto Oswaldo Cruz》2016,111(1):79-81
We report here the first complete mitochondria genome of Onchocerca
volvulus from a focus outside of Africa. An O. volvulus
mitogenome from the Brazilian Amazonia focus was obtained using a combination of
high-throughput and Sanger sequencing technologies. Comparisons made between this
mitochondrial genome and publicly available mitochondrial sequences identified 46
variant nucleotide positions and suggested that our Brazilian mitogenome is more
closely related to Cameroon-origin mitochondria than West African-origin
mitochondria. As well as providing insights into the origins of Latin American
onchocerciasis, the Brazilian Amazonia focus mitogenome may also have value as an
epidemiological resource. 相似文献
37.
38.
Saha S Majumdar R Dighe RR Chakravarty AR 《Metallomics : integrated biometal science》2010,2(11):754-765
Ternary cobalt(III) complexes [CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at ~690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K(d) value of 81 nM and from the photocleavage of hTSHR-ECD. 相似文献
39.
Basavarajappa DK Gupta VK Dighe R Rajala A Rajala RV 《Molecular and cellular biology》2011,31(19):3975-3987
Growth factor receptor-bound protein 14 (Grb14) is an adapter protein implicated in receptor tyrosine kinase signaling. Grb14(-/-) studies highlight both the positive and negative roles of Grb14 in receptor tyrosine kinase signaling in a tissue-specific manner. In this study, we made a novel finding that Grb14 inhibits the activity of PTP1B, the major negative regulator of insulin receptor (IR) signaling, in a phosphorylation-regulated manner. Phosphorylation of Tyr-347 in the BPS domain of Grb14 is critical for interaction with PTP1B, resulting in the competitive inhibition of PTP1B activity. We also found that rhodopsin-regulated Src kinase activation in retina leads to the phosphorylation of Grb14. Further, ablation of Grb14 resulted in significantly elevated retinal PTP1B activity in vivo. PTP1B is known to be regulated by oxidation, glutathionylation, phosphorylation, and SUMOlyation, and our study for the first time demonstrates the inhibition of PTP1B activity in vivo by protein molecule Grb14 in a tissue-specific manner. 相似文献
40.
Marcia Berrêdo-Pinho Dario E Kalume Paloma R Correa Leonardo HF Gomes Melissa P Pereira Renata F da Silva Luiz RR Castello-Branco Wim M Degrave Leila Mendonça-Lima 《BMC microbiology》2011,11(1):80