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Plasmid Constructs (plasmid + construct)
Selected AbstractsInhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: Experiments with a model systemJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Olga N. Umanskaya Abstract We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context. J. Cell. Biochem. 99: 598,608, 2006. © 2006 Wiley-Liss, Inc. [source] Human liver-derived cells stably modified for regulated proinsulin secretion function as bioimplants in vivoTHE JOURNAL OF GENE MEDICINE, Issue 4 2002Xiang Chen Abstract Background Insulin deficiency is currently treated with pharmacological insulin secretagogues, insulin injections or islet transplants. Secondary failure of pharmacological agents is common; insulin injections often fail to achieve euglycemic control; and islet transplants are rare. Non-, cells capable of regulated insulin secretion in vivo could be a functional cure for diabetes. Hepatocytes are good candidates, being naturally glucose-responsive, protein-secreting cells, while the liver is positioned to receive direct nutrient signals that regulate insulin production. Methods Human liver-derived Chang cells were modified with a plasmid construct in which a bifunctional promoter comprising carbohydrate response elements and the human metallothionein IIA promoter controlled human proinsulin cDNA expression. Secretory responses of stable cell clones were characterized in vitro and in vivo by proinsulin radioimmunoassay. Results Transfected Chang cells secreted 5,8,pmol proinsulin/106 cells per 24,h in continuous passage for at least a year in response to 5,25,mM glucose and 10,90,µM zinc in vitro. Glucose and zinc synergistically increased proinsulin production by up to 30-fold. Non-glucose secretagogues were also active. Glucose transporter 2 (GLUT2) and glucokinase cDNA co-transfection enhanced glucose responsiveness. Intraperitoneally implanted Chang cells secreted proinsulin in scid and Balb/c mice. Serum proinsulin levels were further increased 1.3-fold (p<0.05) after glucose and 1.4- to 1.6-fold (p<0.005) after zinc administration in vivo. Conclusions These results are the first to demonstrate stable proinsulin production in a human liver-derived cell line with activity in vitro and in vivo and provide a basis for engineering hepatocytes as in vivo bioimplants for future diabetes treatment. Copyright © 2002 John Wiley & Sons, Ltd. [source] The Arabidopsis class VIII myosin ATM2 is involved in endocytosisCYTOSKELETON, Issue 6 2008Amirali Sattarzadeh Abstract Members of the class XI of the myosin superfamily comprising higher plant, actin-based molecular motors have been shown to be involved in peroxisome and Golgi vesicle trafficking comparable to yeast and animal class V myosins. The tasks of the second class of myosins of higher plants, class VIII, are unclear. In this study the class VIII myosin ATM2 from the model plant Arabidopsis thaliana was selected for the examination of cargo specificity in vivo. Fluorescent protein-fusion plasmid constructs with fragments of the ATM2 cDNA were generated and used for Agrobacterium tumefaciens -based transient transformation of Nicotiana benthamiana leaves. The resulting subcellular localization patterns were recorded by live imaging with confocal laser scanning microscopy (CLSM) in epidermal leaf cells. Expression of a nearly full-length construct displayed labeling of filaments and vesicles, a head + neck fragment led to decoration of filaments only. However, expression of fluorescent protein-tagged C-terminal tail domain constructs labeled vesicular structures of different appearance. Most importantly, coexpression of different RFP/YFP-ATM2 tail fusion proteins showed colocalization and, hence, binding to the same type of vesicular target. Further coexpression experiments of RFP/YFP-ATM2 tail fusion proteins with the endosomal marker FYVE and the endosomal tracer FM4-64 demonstrated colocalization with endosomes. Colocalization was also detected by expression of the CFP-tagged membrane receptor BRI1 as marker, which is constantly recycled via endosomes. Occasionally the ATM2 tail targeted to sites at the plasma membrane closely resembling the pattern obtained upon expression of the YFP-ATM1 C-terminal tail. ATM1 is known for its localization at the plasma membrane at sites of plasmodesmata. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entryEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2000J. Russell Burley Abstract The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+ -dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+ -channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2B,3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 ,m FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+ -dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 m m BAPTA or 1 ,m FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels. [source] Transcriptionally mediated inhibition of telomerase of fungal immunomodulatory protein from Ganoderma tsugae in A549 human lung adenocarcinoma cell lineMOLECULAR CARCINOGENESIS, Issue 4 2006Chien-Huang Liao Abstract Telomerase expression is the hallmark of tumor cells, and activation of this ribonucleoprotein complex may be a rate-limiting or critical step in cellular immortalization and oncogenesis. Fungal immunomodulatory protein, FIP-gts, has been isolated from Ganoderma tsugae. In the present study, we expressed and purified the recombinant fungal immunomodulatory protein reFIP-gts in E. coli. We found that reFIP-gts significantly and selectively inhibits the growth of A549 cancer cells while not affecting the growth of normal MRC-5 fibroblasts. The reFIP-gts suppression of telomerase activity is concentration-dependent, due to the downregulation of the telomerase catalytic subunit (hTERT). It also happens at the mRNA level. These results were confirmed by transient transfections of A549 cells with pGL3-Basic plasmid constructs containing the functional hTERT promoter and its E-box-deleted sequences cloned upstream of a luciferase reporter gene. With electrophoretic mobility shift assays and Western blotting, we demonstrated that in response to reFIP-gts, binding of c- myc transcriptional factor to the E-box sequence on the hTERT promoter is inhibited. These results show that reFIP-gts suppresses telomerase activity and inhibits transcriptional regulation of hTERT via a c- myc -responsive element-dependent mechanism. Our findings provide new insight into both the anticancer function of reFIP-gts and the regulation of hTERT/telomerase expression, which may be valuable in the development of a promising chemopreventive agent. © 2006 Wiley-Liss, Inc. [source] Neonatal severe hyperparathyroidism associated with a novel de novo heterozygous R551K inactivating mutation and a heterozygous A986S polymorphism of the calcium-sensing receptor geneCLINICAL ENDOCRINOLOGY, Issue 3 2007Judit Tőke Summary Introduction, Neonatal severe hyperparathyroidism (NSHPT) is induced by inactivating mutations of human calcium-sensing receptor (CaSR). Only three heterozygous de novo inactivating mutations of CaSR causing NSHPT have been described. We report the case of a now 11-year-old boy with NSHPT and we characterize a novel inactivating mutation along with the results of some functional analyses. Patient and methods, As a neonate the patient presented the clinical syndrome of NSHPT. At 6 years of age persisting hypercalcaemia without clinical symptoms was documented, and the patient remained completely symptom free without parathyroid surgery until his present age of 11 years. The entire coding region of the CaSR gene of the patient and his family members was sequenced. Functional investigation was performed in HEK-293 cells, transiently transfected with wild type and mutant CaSR plasmid constructs. Results, Sequence analysis revealed a novel de novo heterozygous mutation at codon 551 (AGG,AAG), predicting a change of arginine to lysine (R551K) and a known heterozygous polymorphism (A986S) on the same allele, which was inherited from the father. We demonstrated that the novel R551K mutation significantly reduced the calcium sensitivity of CaSR (EC50: from 3·38 ± 0·62,6·10 ± 0·83 mmol/l), which was not alleviated by the simultaneous presence of A986S polymorphism. Conclusions, We present the fourth NSHPT case induced by a novel de novo heterozygous inactivating mutation (R551K) of the CaSR gene. The disease gradually reverted to a symptomless, benign condition resembling familial hypocalciuric hypercalcaemia without any surgical intervention. [source] | |||||||||||||