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Fang Gu Xiuli Li Jian Kong Bing Pan Min Sun Lemin Zheng Yuanqing Yao 《Biochemical and biophysical research communications》2013
Vascular endothelial growth factor (VEGF-A) stimulating angiogenesis is required for tumor growth and progression. The conventional VEGF-A isoforms have been considered as pro-angiogenic factors. Another family of VEGF-A isoforms generated by alternative splicing, termed VEGFxxxb isoforms, has anti-angiogenic property, exemplified by VEGF165b. Here, we identify a new number of VEGFxxx family-VEGF111b induced by mitomycin C, although not detected in mitomycin C-unexposed ovarian cancer cells. SKOV3 cells were transfected with pcDNA3.1 empty vector, pcDNA3.1-VEGF111b or pcDNA3.1-VEGF165b to collect conditioned mediums respectively. VEGF111b overexpression inhibits proliferation, migration and tube formation of endothelial cell by inhibiting VEGF-R2 phosphorylation and its downstream signaling, similar to VEGF165b but slightly lower than VEGF165b. The anti-angiogenic property depends on the six amino acids of exon 8b of the VEGFxxxb isoforms. Our results show that VEGF111b is a novel potent anti-angiogenic agent that can target the VEGF-R2 and its signaling pathway to inhibit ovarian tumor growth. 相似文献
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The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg334, Arg396 and Arg638 were directly assigned to the ETF and ii) Arg198 was assigned as the only tryptic site to the LTF. Arg671, Lys712/Lys713 and Lys728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl2, EGTA or AlF4− strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg1002 as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes. 相似文献
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Stacy A. Amico-Ruvio 《Biophysical journal》2010,98(7):1160-1169
NMDA receptors are heteromeric glutamate-gated channels composed of GluN1 and GluN2 subunits. Receptor isoforms that differ in their GluN2-subunit type (A-D) are expressed differentially throughout the central nervous system and have distinct kinetic properties in recombinant systems. How specific receptor isoforms contribute to the functions generally attributed to NMDA receptors remains unknown, due in part to the incomplete functional characterization of individual receptor types and unclear molecular composition of native receptors. We examined the stationary gating kinetics of individual rat recombinant GluN1/GluN2B receptors in cell-attached patches of transiently transfected HEK293 cells and used kinetic analyses and modeling to describe the full range of this receptor's gating behaviors. We found that, like GluN1/GluN2A receptors, GluN1/GluN2B receptors have three gating modes that are distinguishable by their mean open durations. However, for GluN1/GluN2B receptors, the modes also differed markedly in their mean closed durations and thus generated a broader range of open probabilities. We also found that regardless of gating mode, glutamate dissociation occurred ∼4-fold more slowly (k− = 15 s−1) compared to that observed in GluN1/GluN2A receptors. On the basis of these results, we suggest that slow glutamate dissociation and modal gating underlie the long heterogeneous activations of GluN1/GluN2B receptors. 相似文献
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Huiyu Li Yi-Mei Du Linlin Guo Shenghua Jie Wen Du Wei Liu Jiang Zhu Shiang Huang 《Experimental cell research》2009,315(13):2256-914
Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K+ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K+ channels. Our results showed that E-4031, a specific hERG1 K+ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K+ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K+ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K+ channels functionally linked to cell migration induced by SDF-1. 相似文献
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Human embryonic kidney 293 (HEK293) cells with glycosylation machinery have emerged as an alternative host cell line for stable expression of therapeutic glycoproteins. To characterize dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification in HEK293 cells, an expression vector containing dhfr and monoclonal antibody (mAb) gene was transfected into dhfr-deficient HEK293 cells generated by knocking out dhfr and dhfrl1 in HEK293E cells. Due to the improved selection stringency, mAb-producing parental cell pools could be generated in the absence of MTX. When subjected to stepwise selection for increasing MTX concentrations such as 1, 10, and 100 nM, there was an increase in the specific mAb productivity (qmAb) of the parental cell pool upon DHFR/MTX-mediated gene amplification. High producing (HP) clones with a qmAb of more than 2-fold of the corresponding cell pool could be obtained using the limiting dilution method. The qmAb of most HP clones obtained from cell pools at elevated MTX concentrations significantly decreased during long-term culture (3 months) in the absence of selection pressure. However, some HP clones could maintain high qmAb during long-term culture. Taken together, a stable HP recombinant HEK293 cell line can be established using DHFR/MTX-mediated gene amplification together with dhfr− HEK293 host cells. 相似文献
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A. Huanosta-Gutiérrez A.E. Espino-SaldañaJ.P. Reyes A. PétrizR. Miledi A. Martínez-Torres 《Biochemical and biophysical research communications》2014
Oocytes of Xenopus tropicalis elicit a Ca2+-dependent outwardly rectifying, low-activating current (ICl,Ca) that is inhibited by Cl− channel blockers. When inactivated, ICl,Ca shows an exponentially decaying tail current that is related to currents generated by TMEM16A ion channels. Accordingly, RT-PCR revealed the expression of five alternatively spliced isoforms of TMEM16A in oocytes, which, after expression in HEK-293 cells, gave rise to fully functional Cl− channels. Upon hyperpolarization to −80 mV a transient current was observed only in isoforms that carry the exon 1d, coding for two potentially phosphorylatable Threonine residues. The identified isoforms are differentially expressed in several tissues of the frog. Thus, it appears that X. tropicalis oocytes express TMEM16A that gives rise to a Ca2+-dependent Cl− current, which is different from the previously reported voltage-dependent outwardly rectifying Cl− current. 相似文献
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Tomohito Matsuo Yukiko Noguchi Mieko Shindo Yoshifumi Morita Yoshie Oda Eiko Yoshida Hiroko Hamada Mine Harada Yuichi Shiokawa Takahiro Nishida Ryuji Tominaga Yoshikane Kikushige Koichi Akashi Jun Kudoh Nobuyoshi Shimizu Yuka Tanaka Tsukuru Umemura Taketoshi Taniguchi Akihiko Yoshimura Takashi Kobayashi Masao Mitsuyama Hironori Kurisaki Hitoshi Katsuta Seiho Nagafuchi 《Gene》2013
Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4+ T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein–Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3′UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3′UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3′UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3′UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation. 相似文献
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Park JY Hwang EM Yarishkin O Seo JH Kim E Yoo J Yi GS Kim DG Park N Ha CM La JH Kang D Han J Oh U Hong SG 《Biochemical and biophysical research communications》2008,368(3):677-683
We identified human TRPC3 protein by yeast two-hybrid screening of a human brain cDNA library with human TRPM4b as a bait. Immunoprecipitation and confocal microscopic analyses confirmed the protein-protein interaction between TRPM4b and TRPC3, and these two TRPs were found to be highly colocalized at the plasma membrane of HEK293T cells. Overexpression of TRPM4b suppressed TRPC3-mediated whole cell currents by more than 90% compared to those in TRPC3-expressed HEK293T cells. Furthermore, HEK293T cells stably overexpressing red fluorescent protein (RFP)-TRPM4b exhibited an almost complete abolition of UTP-induced store-operated Ca2+ entry, which is known to take place via endogenous TRPC channels in HEK293T cells. This study is believed to provide the first clear evidence that TRPM4b interacts physically with TRPC3, a member of a different TRP subfamily, and regulates negatively the channel activity, in turn suppressing store-operated Ca2+ entry through the TRPC3 channel. 相似文献
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Anton Skopin Alexey Shalygin Vladimir Vigont Olga ZiminaLyubov Glushankova Galina N. MozhayevaElena Kaznacheyeva 《Biochimie》2013
TRPC1 is a major component of store-operated calcium entry in many cell types. In our previous studies, three types of endogenous store-operated calcium channels have been described in HEK293 cells, but it remained unknown which of these channels are composed of TRPC1 proteins. Here, this issue has been addressed by performing single-channel analysis in HEK293 cells transfected with anti-TRPC1 siRNA (siTPRC1) or a TPRC1-encoding plasmid. The results show that thapsigargin-or agonist-induced calcium influx is significantly attenuated in siTRPC1-transfected HEK293 cells. TRPC1 knockdown by siRNA results in the disappearance of store-operated Imax channels, while the properties of Imin and INS channels are unaffected. In HEK293 cells with overexpressed TRPC1 protein, the unitary current–voltage relationship of exogenous TRPC1 channels is almost linear, with a slope conductance of about 17 pS. The extrapolated reversal potential of expressed TRPC1 channels is +30 mV. Therefore, the main electrophysiological and regulatory properties of expressed TRPC1 and native Imax channels are identical. Moreover, TRPC1 overexpression in HEK293 cells results in an increased number of store-operated Imax channels. All these data allow us to conclude that TRPC1 protein forms native store-operated Imax channels but is not an essential subunit for other store-operated channel types in HEK293 cells. 相似文献
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Csányi G Cifuentes-Pagano E Al Ghouleh I Ranayhossaini DJ Egaña L Lopes LR Jackson HM Kelley EE Pagano PJ 《Free radical biology & medicine》2011,51(6):1116-1125
In recent years, reactive oxygen species (ROS) derived from the vascular isoforms of NADPH oxidase, Nox1, Nox2, and Nox4, have been implicated in many cardiovascular pathologies. As a result, the selective inhibition of these isoforms is an area of intense current investigation. In this study, we postulated that Nox2ds, a peptidic inhibitor that mimics a sequence in the cytosolic B-loop of Nox2, would inhibit ROS production by the Nox2-, but not the Nox1- and Nox4-oxidase systems. To test our hypothesis, the inhibitory activity of Nox2ds was assessed in cell-free assays using reconstituted systems expressing the Nox2-, canonical or hybrid Nox1-, or Nox4-oxidase. Our findings demonstrate that Nox2ds, but not its scrambled control, potently inhibited superoxide (O2•−) production in the Nox2 cell-free system, as assessed by the cytochrome c assay. Electron paramagnetic resonance confirmed that Nox2ds inhibits O2•− production by Nox2 oxidase. In contrast, Nox2ds did not inhibit ROS production by either Nox1- or Nox4-oxidase. These findings demonstrate that Nox2ds is a selective inhibitor of Nox2-oxidase and support its utility to elucidate the role of Nox2 in organ pathophysiology and its potential as a therapeutic agent. 相似文献
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Kazuya Sakata Masaki Ohmuraya Kimi Araki Chigure Suzuki Satoshi Ida Daisuke Hashimoto Jung Wang Yasuo Uchiyama Hideo Baba Ken-ichi Yamamura 《Experimental Animals》2014,63(1):45-53
Serine protease inhibitor Kazal type 1 (SPINK1; mouse homologue
Spink3) was initially discovered as a trypsin-specific inhibitor in the
pancreas. However, previous studies have suggested that SPINK1/Spink3 is
expressed in a wide range of normal tissues and tumors, although precise characterization
of its gene expression has not been described in adulthood. To further analyze
Spink3 expression, we generated two mouse lines in which either
lacZ or Cre recombinase genes were inserted into the
Spink3 locus by Cre-loxP technology. In
Spink3lacZ mice, β-galactosidase activity was found in
acinar cells of the pancreas and kidney, as well as epithelial cells of the bronchus in
the lung, but not in the gastrointestinal tract or liver.
Spink3cre knock-in mice were crossed with Rosa26 reporter
(R26R) mice to monitor Spink3 promoter activity. In
Spink3cre;R26R mice, β-galactosidase activity was found in
acinar cells of the pancreas, kidney, lung, and a small proportion of cells in the
gastrointestinal tract and liver. These data suggest that Spink3 is widely expressed in
endoderm-derived tissues, and that Spink3cre knock-in mice are
a useful tool for establishment of a conditional knockout mice to analyze Spink3 function
not only in normal tissues, but also in tumors that express
SPINK1/Spink3. 相似文献
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J.D. Schulzke A.H. Gitter J. Mankertz U. Seidler M. Saitou M. Fromm 《生物化学与生物物理学报:生物膜》2005,1669(1):34-42
Background and Aims
This study aimed at functional characterization of the tight junction protein occludin using the occludin-deficient mouse model.Methods
Epithelial transport and barrier functions were characterized in Ussing chambers. Impedance analysis revealed the ionic permeability of the epithelium (Re, epithelial resistance). Conductance scanning differentiated transcellular (Gc) and tight junctional conductance (Gtj). The pH-stat technique quantified gastric acid secretion.Results
In occludin+/+ mice, Re was 23±5 Ω cm2 in jejunum, 66±5 Ω cm2 in distal colon and 33±6 Ω cm2 in gastric corpus and was not altered in heterozygotic occludin+/− or homozygotic occludin−/− mice. Additionally, [3H]mannitol fluxes were unaltered. In the control colon, Gc and Gtj were 7.6±1.0 and 0.3±0.1 mS/cm2 and not different in occludin deficiency. Epithelial resistance after mechanical perturbation or EGTA exposition (low calcium switch) was not more affected in occludin−/− mice than in control. Barrier function was measured in the urinary bladder, a tight epithelium, and in the stomach. Control Rt was 5.8±0.8 kΩ cm2 in urinary bladder and 33±6 Ω cm2 in stomach and not altered in occludin−/− mice. In gastric corpus mucosa, the glandular structure exhibited a complete loss of parietal cells and mucus cell hyperplasia, as a result of which acid secretion was virtually abolished in occludin−/− mice.Conclusion
Epithelial barrier characterization in occludin-deficiency points against an essential barrier function of occludin within the tight junction strands or to a substitutional redundancy of single tight junction molecules like occludin. A dramatic change in gastric morphology and secretory function indicates that occludin is involved in gastric epithelial differentiation. 相似文献18.
Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents
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Mineur P Colige AC Deroanne CF Dubail J Kesteloot F Habraken Y Noël A Vöö S Waltenberger J Lapière CM Nusgens BV Lambert CA 《The Journal of cell biology》2007,179(6):1261-1273
Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases. 相似文献
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DNA polymerase kappa (Polκ) bypasses planar polycyclic N2-guanine adducts in an error-free manner. Cholesterol derivatives may interact with DNA to form similarly bulky lesions. In accordance, these studies examined whether increased mutagenesis of DNA accompanies hypercholesterolemia in Polk−/− mice. These mice also carried apoE gene knockouts to ensure increased levels of plasma cholesterol following exposure to a high cholesterol diet. The mice carried a reporter transgene (the λ-phage cII gene) for subsequent quantitative analysis of mutagenesis in various tissues. We observed significantly increased mutation frequencies in several organs of apoE−/−Polk−/− mice following a high cholesterol diet, compared to those remaining on a standard diet. Regardless of dietary regime, the mutation frequency in many organs was significantly higher in apoE−/−Polk−/− than in apoE−/−Polk+/+ mice. As expected for polycyclic guanine adducts, the mutations mainly consisted of G:C transversions. The life expectancy of apoE−/−Polk−/− mice maintained on a high cholesterol diet was reduced compared to apoE−/−Polk+/+ mice. Overall, this study demonstrates a role for Polκ in bypass of cholesterol-induced guanine lesions. 相似文献