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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 19 recordsLinked to original sources
PubMed1995

Increased granzyme B mRNA after alloincompatible myoblast transplantation.

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.

Animals↗
PubMed1995

The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase.

Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Genetic linkage for Darier disease (keratosis follicularis).

Darier disease is an autosomal dominant skin disorder characterized by abnormal keratinocyte adhesion. Recent data have provided evidence for linkage of the Darier disease locus to 12q23-24.1 in British families. We have carried out linkage analysis using the 12q markers D12S58, D12S84, D12S79, D12S86, PLA2, and D12S63 in 6 Canadian families. Pairwise linkage analysis generated positive lod scores at all 6 markers at various recombination fractions, and each family showed positive lod scores with more than one marker. The peak lod score in the multipoint analysis (Zmax) was 5.5 in the interval between markers D12S58 and D12S84. These positive lod scores in North American families of varied European ancestry confirm the location of the Darier disease gene, and suggest genetic homogeneity. The future identification and sequencing of the gene responsible for Darier disease should lead to improved understanding of the disease and of keratinocyte adhesion in general.

Adolescent↗
PubMed1995

Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice.

A mutagenesis RT-PCR method was used to detect normal dystrophin mRNA following the injection of normal myoblasts in mdx mice using two immunosuppressors. A specific sequence of the dystrophin mRNA (257 bp) was amplified by RT-PCR from the muscle total RNA. MaeIII digestion of the amplified products allowed us to distinguish the normal messenger of dystrophin from the dystrophic one and to establish the percentage of normal and of dystrophic (mdx) dystrophin mRNA. Normal dystrophin mRNA was detected using this technique in mdx muscles transplanted with histocompatible normal myoblasts. For this type of transplantation, no significant difference in the percentage of normal dystrophin mRNA was observed between immunosuppressed mice and those not immunosuppressed. No normal dystrophin mRNA was, however, observed in mdx mice following histoincompatible normal myoblast transplantation without immunosuppression. When such transplantations were done in mice immunosuppressed with cyclosporine or FK-506, normal dystrophin mRNA accounted for 31% and 36% of the total dystrophin mRNA, respectively. In fact, one animal immunosuppressed with FK-506 expressed as much as 57% of normal dystrophin mRNA. These results thus show that FK-506 makes it possible to restore dystrophin expression to a level comparable to that observed in DMD carriers that are usually asymptomatic.

Animals↗
PubMed1995

An approach towards the development of progesterone antagonists: synthesis of 7 alpha/7 beta-aryl androstene derivatives.

Synthesis of 3 beta,17 beta-dihydroxy-7 alpha/7 beta-(4-hydroxyphenyl)-androst-5-ene 3,17-diacetate (4 and 5, R = H) and 3 beta,17 beta-dihydroxy-7 alpha/7 beta-(4-methoxyphenyl)-androst-5-ene 3,17-diacetate (4 and 5, R = Me) have been carried out by Friedel-Crafts reaction on 3 beta,7,17 beta-trihydroxy-androst-5-ene 3,17-diacetate (3, R = H) with phenol and anisole, respectively. Compounds 4 (R = H) and 5 (R = H) have been separated and their stereochemistry assigned on the basis of COSY and NOE experiments.

Androstenols↗
PubMed1995

Enhanced host cell reactivation capacity and expression of DNA repair genes in human breast cancer cells resistant to bi-functional alkylating agents.

Human breast carcinoma (MCF7-MLNr) cells resistant to the bifunctional drugs L-phenylalanine mustard (L-PAM, 5-fold resistance), mechlorethamine (9-fold), cisplatin (3-fold), and BCNU (3-fold) were used to investigate the role of DNA repair in the development of resistance to alkylating agents. We have previously shown that neither L-PAM transport and metabolism nor glutathione-associated enzymes were altered in MCF7-MLNr cells, compared to the sensitive cells MCF7-WT. This study shows that treatment of pRSV-CAT plasmid with L-PAM at concentrations up to 1 microM proportionally inhibit the expression of chloramphenicol acetyl transferase (CAT) activity, while higher concentrations abolished CAT activity. pRSV-CAT reactivation was significantly increased when plasmid was transfected into MCF7-MLNr cells, compared to MCF7-WT cells. This indicates that resistant cells have more efficient capacity to recognize and repair L-PAM induced DNA damage. The mRNA expression of DNA nucleotide excision repair genes ERCC1, XPD (ERCC2), XPB (ERCC3), and polymerase beta was found to be similar in both the MCF7-WT and MCF7-MLNr cells. Western blot analysis also reveals no difference in the expression of ERCC1, AP endonuclease, poly (ADP-ribose) polymerase, and alkyl-N-purine-DNA glycosylase proteins. The lack of correlation between enhanced host cell reactivation capacity in resistant cells, and the expression of these specific DNA repair genes suggests that proteins encoded by these genes are not rate limiting steps for resistance to bi-functional alkylating drugs in human breast cancer cells.

Adenocarcinoma↗
PubMed1995

p53 modulation of TFIIH-associated nucleotide excision repair activity.

p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH-associated factors, including transcription-repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand-specific DNA repair, via its C-terminal domain. We also found that wild-type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV-induced dimers is slower in Li-Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

Adenosine Triphosphatases↗
PubMed1995

Successful histocompatible myoblast transplantation in dystrophin-deficient mdx mouse despite the production of antibodies against dystrophin.

Myoblast transplantation has been considered a potential treatment for some muscular disorders. It has proven very successful, however, only in immunodeficient or immunosuppressed mice. In this study, myoblasts from C57BL10J +/+ mice were transplanted, with no immunosuppressive treatment, in the tibialis anterior of fully histocompatible but dystrophin-deficient C57BL10J mdx/mdx mice. One to 9 months after transplantation, the success of the graft was evaluated by immunohistochemistry. All the transplanted mice (n = 24) developed dystrophin-positive fibers following transplantation. Depending on myoblast cultures, transplantations, and time of analysis, the mice presented 15 to 80% of dystrophin-positive fibers in transplanted muscles. These fibers were correctly oriented and they were either from donor or hybrid origin. The dystrophin-positive fibers remained stable up to 9 months. Possible humoral and cellular immune responses were investigated after grafting. Antibodies directed against dystrophin and/or muscle membrane were developed by 58% of the mice as demonstrated by immunohistochemistry and Western blotting. Despite the presence of these antibodies, dystrophin-positive fibers were still present in grafted muscles 9 months after transplantation. Moreover, the muscles did not show massive infiltration by CD4 cells, CD8 cells, or macrophages, as already described in myoblast allotransplantations. This lack of rejection was attributed to the sequestrated nature of dystrophin after fiber formation. These results indicate that myoblast transplantation leads to fiber formation when immunocompetent but fully histocompatible donors and recipients are used and that dystrophin incompatibility alone is not sufficient to induce an immunological rejection reaction.

Animals↗
PubMed1995

A comparative study of the biochemical properties of human and mouse recombinant O6-methylguanine-DNA methyltransferases.

The O6-methylguanine-DNA methyltransferase (MGMT) repairs mutagenic and carcinogenic O6-alkylguanine in DNA by accepting stoichiometrically the alkyl group from the base. Although the mouse MGMT is larger than the human protein because of an additional tetrapeptide sequence, these proteins are 70% homologous. Recombinant MGMTs of the human, the mouse and a mouse mutant with the tetrapeptide deleted were purified to homogeneity from Escherichia coli. The N-terminal amino acid sequences of these proteins are identical to those predicted from the nucleotide sequences, and their molecular masses determined by SDS-PAGE agreed with the predicted values. However, the observed isoelectric points of 9.3, 9.2 and 9.3, for the human, mouse and mutant mouse proteins respectively were significantly different from the values, 8.09, 7.47 and 7.49 calculated from the amino acid composition. The extinction coefficients E280 nm1% of human, mouse and mutant mouse protein were calculated from amino acid composition to be 18.2, 11.1 and 11.3 respectively. These values agree fairly well with calculated values. Human and wild-type mouse MGMTs react with the alkylated base in a synthetic DNA substrate poly(dC, dG, m6dG) with comparable second-order rate constants of 2.2 x 10(8) and 3.7 x 10(8) l/M/min at 37 degrees C respectively and were inactivated by O6-benzylguanine at similar rates. The initial reaction rate (Kin) and rate of inactivation (kinact) constants for reaction with the base were calculated to be 1.8 x 10(-4) M and 1.4 x 10(-3)/s for the human protein, 2.3 x 10(-4) M and 1.1 x 10(-3)/s for the wild-type mouse protein, and 2.1 x 10(-4) M and 1.4 x 10(-3)/s for the mutant mouse protein respectively. The MGMTs were inactivated to the extent of 55-65% after heating at 50 degrees C in 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM DTT and 10% glycerol. However, in the presence of DNA (200 micrograms/ml), only 25-35% of the protein was inactivated. Both DNA and RNA inhibited all three enzymes in a concentration-dependent fashion, although DNA was a better inhibitor than RNA. High salt (0.2 M NaCl) inhibited human MGMT by 80%, while the wild-type and the mutant mouse MGMTs were inhibited by 55%. The human protein had higher affinity for binding to duplex DNAs than the mouse proteins. Immunoprecipitation (69%) and affinity constant (19.4 nM) of human MGMT with a human-specific monoclonal antibody 4.A1 significantly discriminated the human protein from either of the mouse proteins.

Amino Acid Sequence↗
PubMed1995

Immune response to sulfamethoxazole in patients with AIDS.

Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Characterization of intracranial mass lesions with in vivo proton MR spectroscopy.

PURPOSE: To assess the use of in vivo proton MR spectroscopy for characterization of intracranial mass lesions and to ascertain its reliability in grading of gliomas. METHODS: One hundred twenty patients with intracranial masses were subjected to volume selective spectroscopy using stimulated echo acquisition mode (echo time, 20 and 270 milliseconds) and spin echo (echo time, 135 milliseconds) sequences. The intracranial lesions were grouped into intraaxial and extraaxial, as judged with MR imaging. Assignment of resonances was confirmed in two samples each of brain abscess, epidermoid cyst, and tuberculoma using ex vivo high-resolution MR spectroscopy. RESULTS: The in vivo spectra appeared distinct compared with normal brain in all the cases. All high-grade gliomas (n = 37) showed high choline and low or absent N-acetyl-L-aspartate and creatine along with lipid and/or lactate, whereas low-grade gliomas (n = 23) were characterized by low N-acetyl-aspartate and creatine and high choline and presence of only lactate. N-acetyl-aspartate/choline ratio was significantly lower and choline/creatine ratio was significantly higher in high-grade gliomas than in low-grade gliomas. Presence of lipids suggested a higher grade of malignancy. All metastases (n = 7) showed lipid and lactate, whereas choline was visible in only four cases. Epidermoids showed resonances from lactate and an unassigned resonance at 1.8 ppm. Meningiomas could be differentiated from schwannomas by the presence of alanine in the former. Among the infective masses, pyogenic abscesses (n = 6) showed resonances only from cytosolic amino acids, lactate, alanine, and acetate; and tuberculomas (n = 11) showed only lipid resonances. CONCLUSIONS: In vivo proton MR spectroscopy, helps in tissue characterization of intracranial mass lesions. Spectroscopy is a reliable technique for grading of gliomas when N-acetyl-aspartate/choline and choline/creatine ratios and presence of lipids are used in combination.

Adolescent↗
PubMed1995

AmpliType PM and HLA DQ alpha typing from pap smear, semen smear, and postcoital slides.

Deoxyribonucleic acid (DNA) samples extracted from stained pap smear, semen smear and postcoital slides were amplified by the polymerase chain reaction (PCR) and typed for the AmpliType PM and HLA DQ alpha alleles. HLA DQ alpha and PM types consistent with blood controls from the donors were obtained. Stained cells fixed to slides can provide a valuable source of material for determining a genetic profile of the sample donor, particularly in sexual assault cases and possibly in missing person cases involving women.

Coitus↗
PubMed1995

[Infanticide committed by the mother].

Using data gathered at the Institut Philippe Pinel in Montréal, we shall describe the sociodemographic and psychiatric profile of a sample of 17 women who have killed (n = 14) or attempted to kill (n = 3) one of their children. Our data indicate that women who have committed this type of offence generally come from a disadvantaged socioeconomic environment. Most have a psychiatric history (evaluation and/or hospitalization). Review of the offence demonstrates that most women do not use a weapon to kill their child; the preferred methods are strangulation or drowning. Most of these offences may be classified as extended suicide or altruistic acts. Several of the women present with a severe personality disorder and an additional depressive episode in the context of the offence. We hope our study will help clarify understanding of filicide and assist in the development of certain prevention axes. These results indicate that the population at large and various intervenors in our society (family physicians, psychiatrists, criminologists, social workers, pediatricians, psychologists, gynecologists) must become increasingly vigilant and avoid trivialization of signals such as verbalization of homicidal thoughts about the child or recourse to certain disorganized behaviours.

Adult↗
PubMed1994

The DNA-dependent ATPase activity associated with the class II basic transcription factor BTF2/TFIIH.

BTF2/TFIIH from human, delta from rat, and factor b from yeast are multisubunit basal transcription factors that have been shown to be closely associated with a protein kinase capable of phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (Lu, H., Zawel, L., Fischer, L., Egly, J. M., and Reinberg, D. (1992) Nature 358, 641-645; Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480; Feaver, W. J., Gileadi, O., and Kornberg, R. D. (1991) Cell 67, 1223-1230). We report here that a DNA-dependent ATPase and the previously characterized helicase (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J., Chambon, P., and Egly, J. M. (1993) Science 260, 58-63) are both associated with BTF2 and reside with the p89 polypeptide subunit. The DNA requirement, the effect of Sarkosyl and staurosporine inhibitors, as well as nucleotide competition experiments, clearly distinguished ATPase/helicase from the carboxyl-terminal domain kinase. Using recombinant wild type or mutated p89/ERCC3 polypeptides and different forms of DNA template, we show the connection between ATPase and the helicase.

Adenosine Triphosphatases↗
PubMed1994

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression.

Transgenic CD1 mice expressing beta-galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty-eight days after myoblast transplantation, the percentage of muscle fibers beta-glactosidase-positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing beta-galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted.

Animals↗
PubMed1994

A chalcone glycoside from Clerodendron phlomidis.

A new chalcone glycoside, together with pectolinarigenin, 7-hydroxyflavone and 7-hydroxyflavanone 7-O-glucoside have been isolated from the flowers and leaves of Clerodendron phlomidis. The structure of the chalcone glycoside has been established as 4,2',4'-trihydroxy-6'-methoxychalcone 4,4'-D-diglucoside by spectroscopic and degradative methods.

Carbohydrate Sequence↗