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Transfection Studies (transfection + studies)
Selected AbstractsIdentification of Novel Regulators Associated With Early-Phase Osteoblast Differentiation,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Diana S de Jong Abstract Key regulatory components of the BMP-induced osteoblast differentiation cascade remain to be established. Microarray and subsequent expression analyses in mice identified two transcription factors, Hey1 and Tcf7, with in vitro and in vivo expression characteristics very similar to Cbfa1. Transfection studies suggest that Tcf7 modulates BMP2-induced osteoblast differentiation. This study contributes to a better definition of the onset of BMP-induced osteoblast differentiation. Introduction: Elucidation of the genetic cascade guiding mesenchymal stem cells to become osteoblasts is of extreme importance for improving the treatment of bone-related diseases such as osteoporosis. The aim of this study was to identify regulators of the early phases of bone morphogenetic protein (BMP)2-induced osteoblast differentiation. Materials and Methods: Osteoblast differentiation of mouse C2C12 cells was induced by treatment with BMP2, and regulation of gene expression was studied during the subsequent 24 h using high-density microarrays. The regulated genes were grouped by means of model-based clustering, and protein functions were assigned. Real-time quantitative RT-PCR analysis was used to validate BMP2-induced gene expression patterns in C2C12 cells. Osteoblast specificity was studied by comparing these expression patterns with those in C3H10T1/2 and NIH3T3 cells under similar conditions. In situ hybridization of mRNA in embryos at embryonic day (E)14.5 and E16.5 of gestation and on newborn mouse tails were used to study in vivo expression patterns. Cells constitutively expressing the regulated gene Tcf7 were used to investigate its influence on BMP-induced osteoblast differentiation. Results and Conclusions: A total of 184 genes and expressed sequence tags (ESTs) were differentially expressed in the first 24 h after BMP2 treatment and grouped in subsets of immediate early, intermediate early, and late early response genes. Signal transduction regulatory factors mainly represented the subset of immediate early genes. Regulation of expression of these genes was direct, independent of de novo protein synthesis and independent of the cell type studied. The intermediate early and late early genes consisted primarily of genes related to processes that modulate morphology, basement membrane formation, and synthesis of extracellular calcified matrix. The late early genes require de novo protein synthesis and show osteoblast specificity. In vivo and in vitro experiments showed that the transcription factors Hey1 and Tcf7 exhibited expression characteristics and cell type specificity very similar to those of the osteoblast specific transcription factor Cbfa1, and constitutive expression of Tcf7 in C2C12 cells differentially regulated osteoblast differentiation marker genes. [source] Molecular characterization of a variant virus that caused de novo hepatitis B without elevation of hepatitis B surface antigen after chemotherapy with rituximabJOURNAL OF MEDICAL VIROLOGY, Issue 12 2008Masami Miyagawa Abstract Hepatitis B virus (HBV) reactivation in hepatitis B surface antigen (HBsAg)-negative patients following treatment with rituximab has been reported increasingly. The aim of this study was to investigate the molecular mechanisms underlying HBV reactivation in an HBsAg-negative patient. HBV was reactivated in a 75-year-old man following chemotherapy with rituximab, without elevation of HBsAg. The patient's full-length HBV genome was cloned and the entire sequence was determined. Transfection studies were performed in vitro using recombinant wild-type HBV (wild-type), the patient's HBV (patient), and two chimeric HBV constructs, in which the preS/S region of the patient and wild-type virus had been exchanged with one another. Secreted HBsAg and intra- and extra-cellular HBV DNA were measured. The number of amino acid substitutions in HBV from this patient was much higher than in previous reports of HBV mutants, such as occult HBV and vaccine escape HBV mutants. Levels of HBsAg and HBV DNA production in vitro were significantly lower in the patient compared to wild-type transfections. From analyses of the chimeric constructs, the altered preS/S region was responsible mainly for this impairment. These results show that highly mutated HBV can reactivate after chemotherapy with rituximab, despite an unusually large number of mutations, resulting in impaired viral replication in vitro. Severe immune suppression, probably caused by rituximab, may permit reactivation of highly mutated HBV. These findings have important clinical implications for the prevention and management of HBV reactivation and may explain partially the mechanism of recent, unusual cases of HBV reactivation. J. Med. Virol. 80:2069,2078, 2008. © 2008 Wiley-Liss, Inc. [source] Identification of hypoxia-inducible factor-1, (HIF-1,) polymorphism as a mutation in prostate cancer that prevents normoxia-induced degradationTHE PROSTATE, Issue 3 2005Xinyu S. Fu Abstract BACKGROUND Hypoxia-inducible factor-1, (HIF-1,) regulates cellular responses to hypoxia and is rapidly degraded under normoxia through von Hippel-Lindau (VHL) mediated ubiquitination. Although HIF-1, stabilization appears to be the molecular basis for VHL-associated cancers, stabilizing mutations in HIF-1, have not been reported. METHODS A series of 15 metastatic androgen independent prostate cancers were examined for mutations in the oxygen-dependent domain (ODD) of HIF-1, by PCR amplification and DNA sequencing. RESULTS A somatic proline to serine mutation in codon 582 (P582S) was identified in one sample. Transfection studies with a HIF-1, regulated reporter gene showed increased transcriptional activity that correlated with higher mutant HIF-1, protein expression. Increased expression of the P582S mutant induced by iron chelation, which blocks proline hydroxylation of wild-type HIF-1,, was markedly attenuated. The mutant also showed increased stability under normoxic versus hypoxic conditions. CONCLUSION The P582S HIF-1, is a stable variant and HIF-1, mutation is a mechanism for enhancing HIF-1, activity in human cancer. The recent identification of the identical P582S HIF-1, as a polymorphism suggests that this variant may increase tumor susceptibility or cause more aggressive biological behavior. © 2004 Wiley-Liss, Inc. [source] Myosin16b: The COOH-tail region directs localization to the nucleus and overexpression delays S-phase progressionCYTOSKELETON, Issue 1 2007Richard S. Cameron Abstract Rat Myo16a and Myo16b comprise the founding members of class XVI myosin and are characterized by an N-terminal ankyrin repeat domain thought to mediate an association with protein phosphatase 1 catalytic subunits 1, and 1,. Myo16b is the principal isoform and reveals predominant expression in developing neural tissue. Here, we use COS-7 cells as a model system to develop an understanding of Myo16b function. We find that Myo16b displays predominant localization in the nucleus of cells transitioning through interphase, but is not associated with processes of mitosis. Using a panel of EGFP-Myo16b-expression plasmids in transient transfection studies, we identified the COOH-terminal residues 1616,1912 as necessary and solely sufficient to target Myo16b to the nucleus. We show that the Myo16b-tail region directs localization to a nuclear compartment containing profilin and polymerized actin, which appears to form a three-dimensional meshwork through the depth of the nucleus. Further, we demonstrate that this compartment localizes within euchromatic regions of the genome and contains proliferating cell nuclear antigen (PCNA) and cyclin A, both markers of S-phase of the cell cycle. Cells transiently expressing Myo16b or Myo16b-tail region show limited incorporation of BrdU, delayed progression through S-phase of the cell cycle, and curtailed cellular proliferation. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] DAP kinase activity is critical for C2 -ceramide-induced apoptosis in PC12 cellsFEBS JOURNAL, Issue 1 2002Mutsuya Yamamoto Exposure of PC12 cells to C2 -ceramide results in dose-dependent apoptosis. Here, we investigate the involvement of death-associated protein (DAP) kinase, initially identified as a positive mediator of the interferon-,-induced apoptosis of HeLa cells, in the C2 -ceramide-induced apoptosis of PC12 cells. DAP kinase is endogenously expressed in these cells. On exposure of PC12 cells to 30 µm C2 -ceramide, both the total (assayed in the presence of Ca2+/calmodulin) and Ca2+/calmodulin-independent (assayed in the presence of EGTA) DAP kinase activities were transiently increased 5.0- and 12.2-fold, respectively, at 10 min, and then decreased to 1.7- and 3.4-fold at 90 min. After 10 min exposure to 30 µm C2 -ceramide, the Ca2+/calmodulin independent activity/ total activity ratio increased from 0.22 to 0.60. These effects were dependent on the C2 -ceramide concentration. C8 -ceramide, another active ceramide analog, also induced apoptosis and activated DAP kinase, while C2 -dihydroceramide, an inactive ceramide analog, failed to induce apoptosis and increase DAP kinase activity. Furthermore, transfection studies revealed that overexpression of wild-type DAP kinase enhanced the sensitivity to C2 - and C8 -ceramide, while a catalytically inactive DAP kinase mutant and a construct containing the death domain and C-terminal tail of DAP kinase, which act in a dominant-negative manner, rescued cells from C2 -, and C8 -ceramide-induced apoptosis. These findings demonstrate that DAP kinase is an important component of the apoptotic machinery involved in ceramide-induced apoptosis, and that the intrinsic DAP kinase activity is critical for ceramide-induced apoptosis. [source] Genistein induces cell growth inhibition in prostate cancer through the suppression of telomerase activityINTERNATIONAL JOURNAL OF UROLOGY, Issue 1 2005HIDEKI OUCHI Abstract Aim:, To clarify the mechanism of the anticancer effect of genistein, we examined the effect of genistein on telomerase activity in prostate cancer cells. We hypothesized that genistein may exert its anticancer effect by modifying telomerase activity in prostate cancer cells. Methods:, Prostate cancer (LNCaP) cells were cultured with genistein and the number of viable cells was counted. Growth medium was also collected to measure prostate-specific antigen (PSA) concentration. Polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and reverse transcriptase (RT)-PCR analysis were performed to investigate telomerase activity and the expression of human telomerase reverse transcriptase (hTERT), c-myc and p21 mRNA. To examine the possibility that hTERT transcriptional activity is modulated by genistein, transient cell transfection studies were performed by using luciferase reporter assay. Telomere repeat amplification protocol (TRAP) assay and PCR analysis of hTERT were performed in androgen independent cells, DU-145. Results:, Cell growth of LNCaP was inhibited by genistein and PSA secretion was similarly reduced. In TRAP assay, the telomerase activity of LNCaP cells was reduced by genistein. Reverse transcriptase-PCR analysis revealed that the expression of hTERT and c-myc mRNA was down-regulated by genistein, whereas p21 mRNA increased in response to genistein. Luciferase reporter assay revealed that genistein reduced the transcriptional activity of hTERT. In DU-145 cells, telomerase activity and the expression of hTERT mRNA were also reduced by genistein. Conclusion:, The current study elucidated the molecular mechanism of cell growth inhibition by genistein. The antiproliferative effects of genistein seem to be exerted on the hTERT transcriptional activity via different molecular pathways. [source] Critical amino acid residues of the ,4 subunit for ,4,7 integrin functionJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2001Yvonka Zeller Abstract A characteristic feature of integrin,ligand interactions is the requirement for divalent cations. Putative cation binding sites have been identified in the , and , subunit of the ,4 integrins, ,4,1 and ,4,7, and within their ligands which display the tripeptide LDV in fibronectin and homologous motifs in VCAM-1 and MAdCAM-1. The extracellular domain of the murine and human ,4-subunit contains three conserved LDV motifs, designated LDV-1 to -3. Using site directed mutagenesis and transfection studies, we now examined the functional relevance of the LDV motifs for ,4,7 integrins. We present evidence that LDV-1 mutants (D489N) behave like ,4 wt cells, but LDV-3 mutants (D811N) are impaired in ,4,7 integrin-triggered homotypic cell aggregation and in adhesion and spreading on ,4 specific ligands. Further characterization of LDV-3 mutants revealed a defect in mAb-induced ,4,7-cell surface cluster formation. Mutation of the LDV-2 motif (D698N) caused loss of ,4,7 integrin cell surface expression. Our results indicate: (i) that LDV-3, located proximal to the cell membrane, is important for ,4,7 integrin-triggered functions and for lateral clustering and (ii) that LDV-2 affects ,4,7 heterodimer stability. J. Cell. Biochem. 83: 304,319, 2001. © 2001 Wiley-Liss, Inc. [source] Ethanol-Induced Up-Regulation of the Urokinase Receptor In Cultured Human Endothelial CellsALCOHOLISM, Issue 2 2001Edlue M. Tabengwa Background: Moderate alcohol consumption has been correlated to reduced coronary artery disease (CAD) risk and mortality. This alcohol effect may be mediated in part by an increased endothelial cell (EC) fibrinolysis. ECs synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase type plasminogen activator (u-PA), and plasminogen activator inhibitor type-1(PAI-1). In addition, they synthesize and regulate receptors for fibrinolytic proteins, namely (t-PA and plasminogen receptor) Annexin II and u-PA receptor (u-PAR). These receptors play an important role in the regulated expression of receptor-bound plasminogen activator conversion of receptor-bound plasminogen to receptor-bound plasmin on the EC surface (surface-localized fibrinolytic activity). Therefore, systemic factors, such as ethanol, that affect the level, or activity or interaction of one or more of these components, resulting in the increased expression of surface-localized EC fibrinolytic activity, will be expected to reduce the risk for thrombosis, CAD, and myocardial infarction (MI). We have previously shown that low ethanol up-regulates t-PA and u-PA gene transcription, while it down-regulates PAI-1, hence resulting in increased (sustained, 24 hr) surface-localized EC fibrinolytic activity. The current studies were carried out to determine whether low ethanol increased u-PAR expression in cultured human umbilical cord vein ECs (HUVECs). Methods: Cultured HUVECs were preincubated (1 hr) in the absence/presence of ethanol (0.025,0.2%, v/v); u-PAR mRNA (RT-PCR), antigen (western blot), and activity (125I-u-PA ligand binding/Scatchard analysis) levels were then measured after 0,24 hr. To determine whether the ethanol-induced changes in the u-PAR expression were transcriptional, transient transfection studies were carried out using a u-PAR/luciferase promoter construct (pu-PAR120/luc [1.2-kb u-PAR promoter fragment ligated to a promoterless luciferase vector]). Results: uPAR mRNA levels increased 2- to 3-fold and antigen levels (western blot) increased 2- to 4-fold while u-PA binding activity increased 36% (1.25 vs. 1.7 , 105 sites/cell, Bmax) without significantly affecting the Kd (1,2 nM). Transient transfection of cultured HUVECs with a pu-PAR120/luc construct resulted in a 2- to 3-fold increase in promoter activity in ethanol-induced cultures, compared with controls. Conclusion: These combined results demonstrate that low ethanol (,0.1%, v/v) induces the up-regulation of u-PAR gene transcription, resulting in increased u-PAR ligand binding activity. These results also further identify/define the contribution and role of another fibrinolytic protein in the overall ethanol-induced increase in surface-localized EC fibrinolysis that may underlie and contribute, in part, to the cardioprotection attributed to moderate alcohol consumption. [source] Analysis of two translocation breakpoints and identification of a negative regulatory element in patients with Rieger's syndromeBIRTH DEFECTS RESEARCH, Issue 2 2004Dimitri G. Trembath Abstract BACKGROUND Rieger's syndrome is an autosomal dominant disorder characterized by eye, tooth, and umbilical anomalies. A gene responsible for Rieger's syndrome, PITX2, has previously been cloned using two patients with balanced translocations, t(4;16) and t(4;11), with breakpoints that lie near the gene, but which do not interrupt it. METHODS We sequenced both breakpoint regions on chromosome 4 and screened this area for novel genes. Fluorescence in situ hybridization (FISH) was used to determine if PITX2 was still present on the 4:16 chromosome. Both the chromosome 16 and chromosome 11 breakpoints were cloned and sequenced using panhandle polymerase chain reaction (PHPCR). Transient transfection studies were performed to compare effects on a reporter gene between native chromosome 4 sequence and chromosome 11 sequence. RESULTS The region surrounding PITX2 on chromosome 4 is rich in repetitive elements, but no novel genes were identified. FISH demonstrated that PITX2 was intact on the 4:16 translocation chromosome. The PHPCR experiments demonstrated that the translocated regions of chromosomes 16 and 11 were repeat-rich, and transfection studies revealed a slight enhancer effect with the chromosome 4 sequence, and a strong silencer effect when the chromosome 11 sequence was present. CONCLUSIONS Given the lack of any novel genes near either breakpoint, changes in potential regulatory elements may be the best model to explain the loss of PITX2 expression in these patients and hence the Rieger's syndrome phenotype. Birth Defects Research (Part A), 2004. © 2004 Wiley-Liss, Inc. [source] | |||||||||||||