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Biomedical subjects

C Hamelin

Publications and source records attributed to C Hamelin.

At least 19 recordsLinked to original sources
PubMed2004

Apoptotic volume decrease, pH acidification and chloride channel activation during apoptosis requires CD45 expression in HPB-ALL T cells.

Mitochondrial-perturbating agents such as toxic coumponds induce apoptosis. We note that the loss of CD45 expression in the lymphoblastic leukemia cell line HPB-ALL (HPB45.0) leads to an inhibition of nuclear apoptosis. Our hypothesis is that the absence of CD45 disturbs protein function regulated by a proto-oncogene of the Src family playing a significant role in nuclear apoptosis. In this work we explore the importance of a chloride efflux on DNA fragmentation. The role of tyrosine kinase in the function and regulation of the chloride channels was determined. Our results showed a disturbance of ionic homeostasis in CD45 deficient lymphocytes (CD45-) in contrast to normal lymphocytes (CD45+). The phosphorylation levels of the chloride channels are considerably inhibited in CD45-, while the expression levels of these channels are similar in the two types of cells. A hypertonic medium inhibits DNA fragmentation in CD45+ while a hypotonic medium increases DNA fragmentation in CD45-. Thus CD45 plays a significant role in nuclear apoptosis by the regulation of the chloride channels responsible for ionic homeostasis of the cell essential for the DFF40 activation.

Antigens, CD↗
PubMed2003

No detrimental effect from chronic exposure to buprenorphine on corticosteroid-binding globulin and corticosensitive immune parameters.

Opioid drugs reportedly regulate the immune system via their effects on the hypothalamic- pituitary-adrenal (HPA) axis. The present study was carried out to assess the effects of chronic exposure to buprenorphine on HPA axis activation, corticosteroid-binding globulin (CBG), the main glucocorticoid (GC) carrier, and the immune system. Results show that buprenorphine, delivered by osmotic pump subcutaneously in C57BL/6 male mice during a 10-day period, caused a marked decrease in total corticosterone (CORT) levels at day 1 of exposure. CORT levels then increased with maximal values observed at day 5 of exposure. After day 5, total CORT levels gradually decreased and returned to control values. No significant changes were observed in CBG protein levels and mRNA expression in the liver. Since CBG levels remained unchanged, the percentage of free CORT values in buprenorphine mice did not differ from control values. Thus, the variations observed in the amount of free CORT were related only to changes measured in total CORT. These endocrine changes did not have a significant impact on the immune parameters measured. Total CD(4)+ and CD(8)+ splenic and thymic populations were not modulated by buprenorphine. However, splenocytes from mice exposed to buprenorphine after 5 days exhibited greater proliferation upon anti-TCR monoclonal antibody stimulation than saline-exposed mice. These results indicate that buprenorphine can be safely used because it did not have significant effects on GC availability for immune corticosensitive cells.

Analgesics, Opioid↗
PubMed2000

H19 sense and antisense transgenes modify insulin-like growth factor-II mRNA levels.

The oppositely-imprinted genes insulin-like growth factor-II (IGF2) and H19, a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and gamma-actin mRNA were quantified by competitive RT-PCR analysis. Although H19 sense transgene overexpression (n = 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to control cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P < 0.0001, n = 40 clones). Furthermore, the increase in IGF2 mRNA level was accompanied by an elevation of IGF-II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcription, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA sequences under control of a cytomegalovirus promoter. We show a lower reporter gene activity from reporter gene constructs in the presence of sense H19 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effects on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.

Actins↗
PubMed1994

Genetic relationship between mouse adenovirus-2 (strain K87) and human adenovirus-2.

The DNA of mouse adenovirus strain K87 (MAd-2) was cloned and mapped with restriction endonucleases BglII, ClaI, EcoRI, HindIII and SphI. Large differences were found between the MAd-2 and MAd-1 (strain FL) DNA molecules in terms of number and location of restriction sites. The MAd-2 genome also appeared as larger in size than the MAd-1 genome (34.72 kb vs. 30.14 kb). Our results confirm the existence of two distinct adenovirus species in the mouse. Hybridization experiments, on the other hand, indicate that both MAd-1 and MAd-2 are genetically related to human adenovirus type 2 (HAd-2). Overlapping regions of DNA homology are located in genes coding for HAd-2 structural components which could explain serological relationships observed between the human and the murine adenoviruses.

Adenoviruses, Human↗
PubMed1994

Efficient recovery of cloned human cytomegalovirus DNA fragments from agarose gels.

A simple and efficient method for the recovery of DNA fragments from agarose gels is described. After electrophoresis, bands of interest are cut out of the gel and agarose slices pushed through the opening of a syringe needle. The resulting gel slurry is frozen and thawed three times and then centrifuged. DNA in the supernatant is precipitated, resuspended in a small volume of buffer and, finally, desalted. A recombinant pAT153 plasmid carrying the BamHI-P fragment of the human cytomegalovirus (HCMV) genome was detected using purified viral HindIII-E DNA fragment as a probe. Restriction endonuclease analysis was used to confirm the identity of the cloned fragment. Experiments performed with the recombinant pLCR127 plasmid indicate that our freeze/thaw method, with about 80% recovery and very little DNA degradation, is more advantageous than the well known electroelution and NaI/glass methods.

Adsorption↗
PubMed1994

Molecular cloning, physical mapping and cross-hybridization of the murine adenovirus type 1 and type 2 genomes.

Murine adenovirus (MAd) type 1 strain FL and type 2 strain K87 genomes were cloned into plasmid pAT153 as HindIII restriction fragments. The MAd-1 and MAd-2 DNA genomes, 30.10 kb and 34.71 kb in length respectively, were mapped using BglII, ClaI, EcoRI, HindIII and SphI restriction endonuclease cleavage sites. In view of the large differences found between the MAd-1 and MAd-2 genomes in terms of the number and location of restriction sites, cross-hybridization experiments were performed. Homologous DNA sequences were located on the MAd-1 and MAd-2 physical maps. Both viruses are also genetically related to human adenovirus type 2 (HAd-2). Nucleotide sequences shared by HAd-2 and the MAds code for structural proteins, which may explain the antigenic similarities between these viruses from different origins. Our results confirm the existence of two distinct adenovirus species in the mouse.

Animals↗
PubMed1993

Electrophoresis of small DNA molecules in agaroses with different electroendosmotic properties.

The migration rate of DNA standards in agaroses with different electroendosmotic (EEO) properties was compared in order to find an alternative to polyacrylamide slab gels for the separation of small restriction fragments by electrophoresis. Slower migration of DNA molecules only a few hundred of base pairs in length was observed after raising the concentration of unmodified low EEO agarose in gels up to 6%. Resolution of low-molecular-weight DNA fragments was best achieved, however, with hydroxyethylated agaroses showing high or low EEO properties. An immediate application of the above results was to confirm the presence of the human cytomegalovirus BamH I-P subgenomic fragment (7.2 kb) in a recombinant pAT153 plasmid by restriction endonuclease analysis.

DNA↗
PubMed1993

SV40 DNA extracted from persistently infected Vero cells using miniprep columns for plasmids.

A recombinant pAT153 plasmid carrying the whole simian virus 40 (SV40) genome in the form of two consecutive Pst I restriction fragments was rapidly isolated using a commercially available minipreps DNA purification system. This rapid and simple method was used to extract SV40 DNA from infected Vero monkey cells. Viral DNA replication in persistently infected SVP-1 monkey cells was also conveniently followed over a period of 8 d by agarose gel electrophoresis and molecular hybridization using minipreps and the recombinant plasmid (pLCB104) as a probe. Unsatisfactory results were obtained, however, when herpes simplex virus DNA was tentatively extracted from infected Vero cells with the above method. Only covalently closed circular DNA molecules, with two strands unable to separate fully under denaturing conditions, were apparently retained after rapid neutralization by the silicone-based minipreps DNA purification resin.

Animals↗
PubMed1992

Electrophoretic transfer of DNA from gels to filters by using a homemade apparatus.

Electroblotting is a rapid and high-efficiency method of transferring DNA from gels to the variety of membranes available, in very short time frames when compared with capillary-blotting techniques and with much less preparation required. A simple apparatus made of two food savers, one dialysis membrane, one sponge, and two removable electrodes was used to transfer DNA molecules up to 21 kb, from agarose gels to nitrocellulose filters, in less than 30 min. A recombinant pAT153 plasmid carrying the human cytomegalovirus BamH I-P fragment (7.2 kb) was rapidly identified by hybridization, using our simplified transfer procedure.

Blotting, Southern↗
PubMed1992

Characterization of the DNA of rodent herpesviruses by restriction endonuclease analysis and hybridization.

Restriction endonuclease analysis, dot-blot hybridization, and dried gel hybridization were used to differentiate mouse cytomegalovirus, rat cytomegalovirus, and mouse herpesvirus strain 76. Viral DNA was obtained directly from virus-infected mouse or rat cells. Restriction endonuclease digestion was performed by standard methods with BamHI, EcoRI, HindIII, and PstI, and DNA was analyzed by electrophoresis on agarose gels. Cross-hybridization was used to determine the degree of genetic homology among the three viruses. Electrophoretic patterns revealed clear differences between mouse cytomegalovirus and rat cytomegalovirus restriction profiles. Extensive comigration of DNA was observed for rat cytomegalovirus and mouse herpesvirus strain 76. Both viruses also shared common DNA sequences. These results suggest that mouse herpesvirus strain 76 is probably a rat cytomegalovirus strain infecting mice.

Animals↗
PubMed1991

Restriction endonuclease patterns of herpes simplex virus DNA: subtyping of HSV-1 and HSV-2 strains from genital and nongenital lesions.

Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) isolates from genital and nongenital infections were submitted to restriction endonuclease analysis for possible genomic changes in relation with the adaptation of the virus to a new site on the body. HSV-1 and HSV-2 strains were successfully divided into two subgroups using the Hin c II restriction enzyme. No correlation was found, however, between the proposed genomic subtypes H1A, H1B, H2A and H2B, and the genital or nongenital origin of the HSV strains.

Animals↗
PubMed1990

Low tumorigenicity of canine cells transformed by the human cytomegalovirus.

Dog embryo kidney cells transformed by the human cytomegalovirus (HCMV) were obtained after non-permissive infection or transfection with viral DNA digested by restriction endonuclease EcoR I. The transformed cells, growing rapidly and showing an unlimited division potential, could use medium with only 2% serum for growth, contained nuclear virus antigens, and formed small colonies (less than 0.2 mm) in agarose. From 40 mice inoculated with transformed canine cells, only one eventually developed a tumor. Results indicate that dog cells are immortalized but not tumorigenically transformed by the human cytomegalovirus.

Animals↗
PubMed1990

Physical mapping of a temperature-sensitive mutation of human cytomegalovirus by marker rescue.

Physical mapping of a temperature-sensitive (ts) mutation of human cytomegalovirus (HCMV) strain AD-169 was attempted here using cloned HindIII restriction endonuclease fragments and the mutant virus. The DNA-positive mutant tested (HCMV ts 1585) was successfully rescued by viral DNA sequences between 0.277 and 0.303 map units. The product of this gene is apparently a structural protein of molecular weight 40,000. Marker rescue could thus be used to establish the physical location of essential HCMV genes, even if the viral DNA molecule is extremely large and complex.

Chromosome Mapping↗
PubMed1990

Differentiation of vaccine and field strains of bovine herpesvirus type 1 by restriction endonuclease analysis.

Bovine herpesvirus type 1 (BHV-1) DNA molecules obtained from a limited number of infected cells were cleaved with a variety of restriction endonucleases. By use of selected DNA fragments in Bgl 1, Pst 1, Pvu 1 and Pvu 11 restriction patterns, one reference, two vaccine and three wild-type strains of BHV-1 were distinguished from one another. The simplified DNA fingerprinting method described here should be most useful, not only to control the genetic stability of BHV-1 vaccines during production, but also to differentiate the vaccine strains from other isolates in clinical cases.

Animals↗
PubMed1990

Inactivating and mutagenic effects of nitrosoguanide on human cytomegalovirus.

Human cytomegalovirus (HCMV) was exposed for 24 hr to 10-30 micrograms/ml nitrosoguanidine (NG) at different times postinfection. NG treatments performed 24 to 48 hr after infection of human IAFP-1 fibroblasts with HCMV interfered most significantly with the replication of the virus. No correlation was found, however, between the inactivating effect of NG at this particular stage of the replication cycle and the induction of HCMV temperature-sensitive mutants. All the 17 HCMV mutant strains isolated on this occasion showed the same BamHI, Cfo I, Hpa II and Sph I restriction profiles which is indicative of mutations caused by single base changes.

Cytomegalovirus↗
PubMed1990

Gel and buffer effects on the migration of DNA molecules in agarose.

The migration rate of linear and supercoiled DNA molecules in four brands of agarose from two different lots was compared using three buffer systems. Differences in DNA mobility were observed between brands and lots of agarose, in standard as well as in pulsed-field gel electrophoresis. A buffer-related effect on the migration of DNA molecules in agarose gels was also observed. Our results underline the need for an actual lot analysis of agarose, this extraction product varying from one preparation to another in several aspects.

Bacteriophage lambda↗
PubMed1990

[Physical mapping of strain AD169 human cytomegalovirus DNA].

The whole human cytomegalovirus strain AD169 genome was cloned into plasmid pAT153 in the form of 25 HindIII fragments. Double and triple digestions of the recombinant plasmids with restriction endonucleases BamHI, BglII, ClaI, DraI, EcoRI, EcoRV, HindIII, HpaI, KpnI, PaeR7, PstI, SphI and XbaI yielded a detailed restriction map of human cytomegalovirus DNA. Knowing the exact position of numerous restriction sites in the viral DNA molecule, we have been able to examine very closely the heterologous region between the long and the short segments of the human cytomegalovirus genome.

Cloning, Molecular↗