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91.
Chapdelaine P Moisset PA Campeau P Asselin I Vilquin JT Tremblay JP 《Protein engineering》2000,13(9):611-615
Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP-mini- (240 kDa) and EGFP-full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP-mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients. 相似文献
92.
Silverman GL Qin K Moore RC Yang Y Mastrangelo P Tremblay P Prusiner SB Cohen FE Westaway D 《The Journal of biological chemistry》2000,275(35):26834-26841
The Prnd gene encodes a homolog of the cellular prion protein (PrP(C)) called doppel (Dpl). Up-regulation of Prnd mRNA in two distinct lines of PrP gene ablated (Prnp(0/0)) mice, designated Rcm0 and Ngsk, is associated with death of Purkinje cells. Using recombinant Dpl expressed in Escherichia coli and mouse neuroblastoma cells we demonstrate that wild type (wt) Dpl, like PrP(C), adopts a predominantly alpha-helical conformation, forms intramolecular disulfide bonds, has two N-linked oligosaccharides, and is presented on the cell surface via a glycosylphosphatidylinositol anchor. Dpl protein was detected in testis of wt mice. Using Triton X-114 phase partitioning to enrich for glycosylphosphatidylinositol-anchored proteins, Dpl was detected in brain samples from Rcm0 Prnp(0/0) mice but was absent in equivalent samples from wt mice and ZrchI Prnp(0/0) mice, indicating that ectopic expression of this protein may cause cerebellar pathology in Rcm0 mice. Biochemical and structural similarities between PrP(C) and Dpl documented here parallel the observation that ataxic Ngsk Prnp(0/0) mice can be rescued by overexpression of wild-type PrP transgenes, and suggest that cell surface PrP(C) can antagonize the toxic effect of Dpl expressed in the central nervous system. 相似文献
93.
Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo 总被引:23,自引:0,他引:23
Doherty AS Mann MR Tremblay KD Bartolomei MS Schultz RM 《Biology of reproduction》2000,62(6):1526-1535
The H19 gene is imprinted with preferential expression from the maternal allele. The putative imprinting control region for this locus is hypermethylated on the repressed paternal allele. Although maternal-specific expression of H19 is observed in mouse blastocysts that develop in vivo, biallelic expression has been documented in embryos and embryonic stem cells experimentally manipulated by in vitro culture conditions. In this study the effect of culture on imprinted H19 expression and methylation was determined. After culture of 2-cell embryos to the blastocyst stage in Whitten's medium, the normally silent paternal H19 allele was aberrantly expressed, whereas little paternal expression was observed following culture in KSOM containing amino acids (KSOM+AA). Analysis of the methylation status of a CpG dinucleotide located in the upstream imprinting control region revealed a loss in methylation in embryos cultured in Whitten's medium but not in embryos cultured in KSOM+AA. Thus, H19 expression and methylation were adversely affected by culture in Whitten's medium, while the response of H19 to culture in KSOM+AA approximated more closely the in vivo situation. It is unlikely that biallelic expression of H19 following culture in Whitten's medium is a generalized effect of lower methylation levels, since the amount of DNA methyltransferase activity and the spatial distribution of Dnmt1 protein were similar in in vivo-derived and cultured embryos. Moreover, imprinted expression of Snrpn was maintained following culture in either medium, indicating that not all imprinted genes are under the same stringent imprinting controls. The finding that culture conditions can dramatically, but selectively, affect the expression of imprinted genes provides a model system for further study of the linkage between DNA methylation and gene expression. 相似文献
94.
Beauséjour CM Tremblay G Momparler RL 《Biochemical and biophysical research communications》2000,278(3):569-575
Hematopoietic toxicity is the dose-limiting side effect produced in cancer chemotherapy with deoxycytidine nucleoside analogs. Deletion of the deoxycytidine kinase (dCK), results in a drug resistance phenotype to these analogs. An interesting gene therapy strategy to confer drug resistance to cytosine nucleoside analogs would be to specifically inactivate the dCK in normal hematopoietic stem cell. In this study, we designed hammerhead ribozymes that can specifically cut and downregulate the murine dCK mRNA. Three different ribozymes were identified and shown to cleave in vitro the dCK RNA. After introduction of ribozyme cDNA into murine L1210 leukemic cells by retroviral transfer, two of the ribozymes showed some capacity in reducing dCK activity. However, analysis of transduced L1210 clones showed that the significant reduction in the dCK mRNA was not sufficient to confer drug resistance to cytosine arabinoside. Nevertheless, these results provide a new avenue of modulating the dCK enzyme activity and with improved modifications may have the potential for use in gene therapy to confer drug resistance to deoxycytidine analogs. 相似文献
95.
Granzyme activity in the inflamed lung is not controlled by endogenous serine proteinase inhibitors 总被引:1,自引:0,他引:1
Tremblay GM Wolbink AM Cormier Y Hack CE 《Journal of immunology (Baltimore, Md. : 1950)》2000,165(7):3966-3969
Numerous lung diseases, such as hypersensitivity pneumonitis (HP), are characterized by the presence of activated alveolar CTL and NK cells. Since these cells produce granzymes, granzyme A and B levels in bronchoalveolar lavage (BAL) fluids from 14 normal subjects and 12 patients with HP were measured by ELISA. Median (range) BAL granzyme A and B levels were 4 (0-37) and 0 (0-6) pg/ml in normal subjects. BAL granzyme levels were significantly higher in HP patients, being at 74 (0-1,889) and 10 (0-78) pg/ml for granzymes A and B, respectively. In vitro, neither of the three main serine protease inhibitors of the lung, namely alpha1-antitrypsin, secretory leukocyte protease inhibitor, and elafin, showed any effect on granzyme A or B activity. In addition, granzyme A was shown to be fully active in BAL fluids. Hence, these data show that granzyme activity may be poorly controlled by protease inhibitors in inflamed tissues. Thus, granzymes could contribute to tissue remodeling and inflammation characterizing HP. 相似文献
96.
97.
Laberge L Carrier J Lespérance P Lambert C Vitaro F Tremblay RE Montplaisi J 《Chronobiology international》2000,17(4):489-501
Our aim was to compare the circadian phase characteristics of healthy adolescent and young adult males in a naturalistic summertime condition. A total of 19 adolescents (mean age 15.7 years) and 18 young adults (mean age 24.5 years) with no sleep problems took part in this study. Two-night polysomnographic (PSG) sleep recordings and 24h secretion patterns of urinary 6-sulfatoxymelatonin were monitored in all 37 subjects. Sleep-wake patterns were initially assessed at home using a standard sleep diary. Circadian assessment included the measure of dim light melatonin offset (DLMOff) and the morningness-eveningness (M/E) questionnaire. As expected, compared to young adults, adolescents habitually spent more nocturnal time in bed and spent more time (and percentage) in delta sleep. No difference was found between adolescents and young adults on multiple sleep latency test (MSLT) sleep onset latencies, M/E, melatonin secretion measures (24h total, nighttime, daytime, and night ratio), and DLMOff. For the subjects as a whole, correlational analyses revealed a significant association between the DLMOff and M/E and between both these phase markers and habitual bedtimes, habitual rising times, and melatonin secretion measures (daytime levels and the night ratio). No association was found between phase markers and daytime sleepiness or sleep consolidation parameters such as sleep efficiency or number of microarousals. These results together indicate that adolescents and young adults investigated during summertime showed similar circadian phase characteristics, and that, in these age groups, an evening phase preference is associated with a delayed melatonin secretion pattern and delayed habitual sleep patterns without a decrease in sleep consolidation or vigilance. (Chronobiology International, 17(4), 489-501, 2000) 相似文献
98.
A common molecular signature unifies the chitosanases belonging to families 46 and 80 of glycoside hydrolases 总被引:1,自引:0,他引:1
The 3D structure-oriented alignment of the primary sequences of fourteen chitosanases, mainly of bacterial origin and belonging to families 46 and 80 of glycoside hydrolases, resulted in the identification of the following pattern common to all these enzymes: E-[DNQ]-x(8,17)-Y-x(7)-D-x-[RD]-[GP]-x-[TS]-x(3)-[AIVFLY]-G- x(5,11)-D. This pattern is proposed as the molecular signature of the chitosanases from families 46 and 80. It includes several amino acids essential for enzyme activity and (or) stability as shown by site-directed mutagenesis studies on the chitosanase from Streptomyces sp. N174. In particular, it includes two carboxylic residues directly involved in catalysis. We suggest that there is a continuum of sequence similarity between all the analyzed chitosanases, and that all these enzymes should probably be classified in one family. 相似文献
99.
Nader Rahimi Eric Tremblay Laura McAdam Anita Roberts Bruce Elliott 《In vitro cellular & developmental biology. Animal》1998,34(5):412-420
Summary We have developed an in vitro system to examine the influence of adipocytes, a major mammary stromal cell type, on the growth of a murine mammary carcinoma,
SP1. Previously, we have shown that 3T3-L1 adipocytes release a mitogenic factor, hepatocyte growth factor, which strongly
stimulates proliferation of SP1 cells. We now show that 3T3-L1 pre-adipocytes secrete active inhibitory molecules which inhibit
DNA synthesis in SP1 cells. In addition, latent inhibitory activity is present in conditioned media (CM) from both pre-adipocytes
and adipocytes, and is activated following acid treatment. CM also inhibited DNA synthesis in Mv1Lu wild type epithelial cells,
but not DR27 mutant epithelial cells which lack TGF-β type II receptor. Inhibitory activity of CMs was partially abrogated
by neutralizing anti-TGF-β1 and anti-TGF-β2 antibodies, and was removed following ultrafiltration through membranes of 10
000 Mr but not 30 000 Mr pore size. These results show that the inhibitory effect on DNA synthesis is mediated by TGF-β1-like and TGF-β2-like molecules.
In addition, acid-treated CM as well as purified TGF-β inhibited differentiation of pre-adipocytes. Untreated pre-adipocyte
CM, but not mature adipocyte CM, spontaneously inhibited adipocyte differentiation. Together, these findings indicate that
pre-adipocytes spontaneously activate their own secreted TGF-β, whereas mature adipocytes do not, and suggest that activation
of TGF-β has a potent negative regulatory effect on adipocyte differentiation and tumor growth. Thus, TGF-β may be an important
modulator of tumor growth and adipocyte differentiation via both paracrine and autocrine mechanisms. These findings emphasize
the importance of adipocyte-tumor interactions in the regulation of tumor microenvironment. 相似文献
100.
David J. Herre J. Blake Norman Ruchi Anderson Michel L. Tremblay Anne-Cecile Huby Eric J. Belin de Chantemèle 《PloS one》2015,10(5)
Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI2 levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI2, in T1DM mice. 相似文献