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Biomedical subjects

L Samson

Publications and source records attributed to L Samson.

At least 19 recordsLinked to original sources

[Systemic learning planification for residents during the 6-month pediatric radiology training].

Diagnostic Radiology and Medical Imaging residents of French University Hospitals benefit from a wide array of learning tools. However, the professors' clinical workload, lack of sufficient proper formal training, lack of practical applicability of existing standards, and absence of rigorous assessment during the specialized training, reduce their very good quality formation to the level of accidental learning. This study originates from the need to apply a systemic planification of learning activities to the French residents' formal training curriculum, such as the one originally designed and applied within the Quebec educational system. This presentation also benefits from the recent introduction of the CanMEDS 2000 objectives by the Royal College of Physicians and Surgeons of Canada, in order to design an adapted version for the professional and academic activities of French radiologists. Needless to say this work will respect the outlined systematic planning of any learning activity: learning requirements, definition of learning objectives, selection of learning methods, assessment of the participants' achievement of these objectives, and evaluation of the learning activity itself.

Canada↗

Role of nucleotide- and base-excision repair in genotoxin-induced neuronal cell death.

Base-excision (BER) and nucleotide-excision (NER) repair play pivotal roles in protecting the genomes of dividing cells from damage by endogenous and exogenous agents (i.e. environmental genotoxins). However, their role in protecting the genome of post-mitotic neuronal cells from genotoxin-induced damage is less clear. The present study examines the role of the BER enzyme 3-alkyladenine DNA glycosylase (AAG) and the NER protein xeroderma pigmentosum group A (XPA) in protecting cerebellar neurons and astrocytes from chloroacetaldehyde (CAA) or the alkylating agent 3-methyllexitropsin (Me-Lex), which produce ethenobases or 3-methyladenine (3-MeA), respectively. Neuronal and astrocyte cell cultures prepared from the cerebellum of wild type (C57BL/6) mice or Aag(-/-) or Xpa(-/-) mice were treated with 0.1-50 microM CAA for 24h to 7 days and examined for cell viability, DNA fragmentation (TUNEL labeling), nuclear changes, and glutathione levels. Aag(-/-) neurons were more sensitive to the acute (>20 microM) and long-term (>5 microM) effects of CAA than comparably treated wild type neurons and this sensitivity correlated with the extent of DNA fragmentation and nuclear changes. Aag(-/-) neurons were also sensitive to Me-Lex at comparable concentrations of CAA. In contrast, Xpa(-/-) neurons were more sensitive than either wild type or Aag(-/-) neurons to CAA (>10 microM), but less sensitive than Aag(-/-) neurons to Me-Lex. Astrocytes from the cerebellum of wild type, Aag(-/-) or Xpa(-/-) mice were essentially insensitive to CAA at the concentrations tested. These studies demonstrate that BER and NER are required to protect neurons from genotoxin-induced cell death.

Acetaldehyde↗

Base excision repair in yeast and mammals.

Base excision repair (BER), as initiated by at least seven different DNA glycosylases or by enzymes that cleave DNA at abasic sites, executes the repair of a wide variety of DNA damages. Many of these damages arise spontaneously because DNA interacts with the cellular milieu, and so BER profoundly influences spontaneous mutation rates. In addition, BER provides significant protection against the toxic and mutagenic effects of DNA damaging agents present in the external environment, and as such is likely to prevent the adverse health effects of such agents. BER pathways have been studied in a wide variety of organisms (including yeasts) and here we review how these varied studies have shaped our current view of human BER.

Animals↗

A multi-centre rapid assessment of injecting drug use in India.

In 1998, a series of five rapid situation assessments (RSA) of injecting drug use were undertaken by The Society for Service to Urban Poverty (SHARAN) covering the major Metropolitan cities of Mumbai, Chennai, Calcutta, Delhi and Imphal. The RSA determined the extent and patterns of injecting drug use (IDU), the available responses, current and planned interventions, and drug users' perceptions of injecting and sexual-related risk behaviour. The RSA was necessary as there are a lack of data on IDU in India. This has resulted in the denial of injecting drug use except for the north-eastern states by official sources, thereby affecting the inputs for IDU-related interventions. The draft assessment reports were disseminated though city workshops, held between April 1998 and January 1999. Local NGOs involved in drug treatment and HIV related interventions, government officials, and the relevant State AIDS Cells were invited to the workshops in order to contribute to final city assessment reports, so as to promote ownership and to enhance coverage. While the data obtained from the RSA were largely as anticipated, the outcome of the dissemination workshops was phenomenal.

Journal Article↗

Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe.

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.

Adenine↗

Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision.

DNA N-glycosylases are base excision-repair proteins that locate and cleave damaged bases from DNA as the first step in restoring the genetic blueprint. The human enzyme 3-methyladenine DNA glycosylase removes a diverse group of damaged bases from DNA, including cytotoxic and mutagenic alkylation adducts of purines. We report the crystal structure of human 3-methyladenine DNA glycosylase complexed to a mechanism-based pyrrolidine inhibitor. The enzyme has intercalated into the minor groove of DNA, causing the abasic pyrrolidine nucleotide to flip into the enzyme active site, where a bound water is poised for nucleophilic attack. The structure shows an elegant means of exposing a nucleotide for base excision as well as a network of residues that could catalyze the in-line displacement of a damaged base from the phosphodeoxyribose backbone.

Alkylation↗

Evidence-based guidelines for universal counselling and offering of HIV testing in pregnancy in Canada.

OBJECTIVE: To provide Canadian health care workers with evidence-based guidelines for universal counselling about HIV testing and the offering of such testing to all pregnant women. OPTIONS: Universal counselling and offering of HIV testing to all pregnant women versus targeted testing of only pregnant women at high risk for HIV infection. Antiretroviral treatment protocols for HIV-positive mothers and their infants are discussed as the intervention to reduce mother-to-child transmission rates. OUTCOMES: Main outcomes are mother-to-child HIV transmission rates and consequences of HIV testing on the mother and infant. EVIDENCE: Articles published from January 1985 to March 1997 identified through a MEDLINE search; articles published in pertinent medical journals in 1996 and 1997 identified through a manual search; and abstracts presented at international HIV/AIDS conferences. BENEFITS, HARMS AND COSTS: Early diagnosis of HIV infection in a pregnant woman optimizes her medical and psychosocial care, decreases the incidence of mother-to-child transmission and decreases the risk of horizontal transmission to sexual partners. New, third-generation HIV tests have reduced false-positive rates and thus diminished the harm of screening. RECOMMENDATIONS: A screening strategy consisting of universal counselling and offering of HIV testing is recommended for all pregnant women in Canada (grade B recommendation). Targeted testing of only pregnant women at high risk for HIV infection fails to identify a substantial proportion of HIV-positive pregnant women and is therefore not recommended (grade D recommendation). Women who identify themselves as being at high risk and whose initial HIV test result is negative should be counselled about the reduction of high-risk behaviours and retested in 6 months (grade B recommendation). Treatment of seropositive women and infants with zidovudine to prevent mother-to-child transmission is recommended (grade A or B recommendation depending on gestational age and CD4 count). VALIDATION: These guidelines are endorsed by the Canadian Pediatric AIDS Research Group and are in agreement with the recommendations of the Canadian Paediatric Society and the US Public Health Service Task Force.

AIDS Serodiagnosis↗

Hypermutation of immunoglobulin genes in memory B cells of DNA repair-deficient mice.

To investigate the possible involvement of DNA repair in the process of somatic hypermutation of rearranged immunoglobulin variable (V) region genes, we have analyzed the occurrence, frequency, distribution, and pattern of mutations in rearranged Vlambda1 light chain genes from naive and memory B cells in DNA repair-deficient mutant mouse strains. Hypermutation was found unaffected in mice carrying mutations in either of the following DNA repair genes: xeroderma pigmentosum complementation group (XP)A and XPD, Cockayne syndrome complementation group B (CSB), mutS homologue 2 (MSH2), radiation sensitivity 54 (RAD54), poly (ADP-ribose) polymerase (PARP), and 3-alkyladenine DNA-glycosylase (AAG). These results indicate that both subpathways of nucleotide excision repair, global genome repair, and transcription-coupled repair are not required for somatic hypermutation. This appears also to be true for mismatch repair, RAD54-dependent double-strand-break repair, and AAG-mediated base excision repair.

Animals↗

O6-alkylguanine DNA lesions trigger apoptosis.

It is unclear whether alkylating agents induce apoptosis because they damage DNA, or because they damage other cellular targets. Isogenic Chinese hamster ovary (CHO) cell lines varying in the repair of O6-alkylguanine (O6AlkG) were examined for their propensity to undergo alkylation-induced apoptosis. Robust O6AlkG repair virtually eliminated the apoptogenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU, carmustine), as did the expression of BCL-2. O6AlkG repair had no effect on apoptosis induced by tumor necrosis factor alpha or by gamma-irradiation. We conclude that alkylating agents induce apoptosis by virtue of their ability to modify DNA bases and, more specifically, that O6AlkG lesions can trigger such programmed cell death.

Animals↗

Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair.

O6-Methylguanine (O6MeG) and O4-methylthymine (O4MeT) are potentially mutagenic DNA lesions that cause G:C-->A:T and A:T-->G:C transition mutations by mispairing during DNA replication, and the repair of O6MeG and O4MeT by DNA repair methyltransferases (MTases) is therefore expected to prevent methylation-induced transitions. The efficiency of O6MeG and O4MeT repair by different MTases can vary by several hundred-fold and the aim of this study was to establish the biological consequences of such differences in the efficiency of repair. The ability of three microbial and two mammalian MTases to prevent methylation-induced G:C-->A:T and A:T-->G:C transitions is taken as a measure of their ability to repair O6MeG and O4MeT in vivo respectively. All five MTases give complete protection against G:C-->A:T transitions. However, while the microbial MTases give complete protection against A:T-->G:C transitions, the mammalian MTases actually sensitize cells to A:T-->G:C transitions. We hypothesize that the mammalian MTases bind O4MeT lesions in vivo but that, because they are extremely slow at subsequent methyl transfer, binding shields O4MeT from repair by the nucleotide excision repair pathway. Results are presented to support this hypothesis.

Animals↗

Cloning and characterization of a cDNA encoding a 3-methyladenine DNA glycosylase from the fission yeast Schizosaccharomyces pombe.

We have begun to develop the fission yeast, Schizosaccharomyces pombe, as a eukaryotic model for cellular defenses against alkylating agents. Here we describe the cloning and characterization of a cDNA, designated mag1, encoding a S. pombe 3-methyladenine (3MeA) DNA glycosylase. 3MeA DNA glycosylases in Escherichia coli are encoded by alkA and tag. S. pombe mag1 was cloned by its ability to reverse the alkylation-sensitive phenotype of an alkA tag E. coli double mutant. The expression of S. pombe mag1 in E. coli confers partial resistance to alkylating agents that produce methyl, ethyl and propyl lesions, and Mag1 production produces 3MeA DNA glycosylase activity. In contrast to the E. coli alkA and Saccharomyces cerevisiae MAG genes, expression of S. pombe mag1 was not appreciably induced by alkylating agents. The mag1 cDNA encodes a protein of 228 amino acids (aa) that shares similarity with 3MeA DNA glycosylases from E. coli (AlkA), Bacillus subtilis (BsAlkA) and S. cerevisiae (MAG). A consensus sequence of 9 aa common to these microbial 3MeA DNA glycosylases is discussed.

Alkylation↗

Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing.

In Escherichia coli, the repair of 3-methyladenine (3MeA) DNA lesions prevents alkylation-induced cell death because unrepaired 3MeA blocks DNA replication. Whether this lesion is cytotoxic to mammalian cells has been difficult to establish in the absence of 3MeA repair-deficient cell lines. We previously isolated and characterized a mouse 3MeA DNA glycosylase cDNA (Aag) that provides resistance to killing by alkylating agents in E. coli. To determine the in vivo role of Aag, we cloned a large fragment of the Aag gene and used it to create Aag-deficient mouse cells by targeted homologous recombination. Aag null cells have no detectable Aag transcripts or 3MeA DNA glycosylase activity. The loss of Aag renders cells significantly more sensitive to methyl methanesulfonate-induced chromosome damage, and to cell killing induced by two methylating agents, one of which produces almost exclusively 3MeAs. Aag null embryonic stem cells become sensitive to two cancer chemotherapeutic alkylating agents, namely 1,3-bis(2-chloroethyl)-1-nitrosourea and mitomycin C, indicating that Aag status is an important determinant of cellular resistance to these agents. We conclude that this mammalian 3MeA DNA glycosylase plays a pivotal role in preventing alkylation-induced chromosome damage and cytotoxicity.

Alkylating Agents↗

Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent.

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.

Animals↗

The Escherichia coli MutS DNA mismatch binding protein specifically binds O(6)-methylguanine DNA lesions.

DNA mismatch repair defects in certain cell types confer resistance to the cytotoxic effects of alkylating agents, suggesting that a normally functioning DNA mismatch repair pathway can actually mediate alkylation-induced cell death. In eukaryotic cells this phenomenon is only observed in cells lacking adequate DNA methyltransferase for the repair of O6-methylguanine (O6MeG) DNA lesions. It has been proposed that O6MeG may act as a substrate for DNA mismatch repair when paired with cytosine and when mispaired with thymine and that repeated futile DNA mismatch repair at O6MeG DNA lesions is cytotoxic. Here we show that the Escherichia coli MutS DNA mismatch repair binding protein does indeed bind specifically to O6MeG DNA lesions. In contrast, MutS does not bind DNA containing another O-alkylated base, namely O4-methylthymine, or another kind of modified guanine, namely 8-oxoguanine. These results provide direct biochemical evidence for the involvement of DNA mismatch repair in specifically processing O6MeG DNA lesions.

Adenosine Triphosphatases↗