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Biomedical subjects

L Samson

Publications and source records attributed to L Samson.

At least 37 records · Page 2Linked to original sources
PubMed1996

DNA repair functions in heterologous cells.

Our genetic information is constantly challenged by exposure to endogenous and exogenous DNA-damaging agents, by DNA polymerase errors, and thereby inherent instability of the DNA molecule itself. The integrity of our genetic information is maintained by numerous DNA repair pathways, and the importance of these pathways is underscored by their remarkable structural and functional conservation across the evolutionary spectrum. Because of the highly conserved nature of DNA repair, the enzymes involved in this crucial function are often able to function in heterologous cells; as an example, the E. coli Ada DNA repair methyltransferase functions efficiently in yeast, in cultured rodent and human cells, in transgenic mice, and in ex vivo-modified mouse bone marrow cells. The heterologous expression of DNA repair functions has not only been used as a powerful cloning strategy, but also for the exploration of the biological and biochemical features of numerous enzymes involved in DNA repair pathways. In this review we highlight examples where the expression of DNA repair enzymes in heterologous cells was used to address fundamental questions about DNA repair processes in many different organisms.

Animals↗
PubMed1996

[Urinary excretion of free and total deoxypyridinoline during secondary hyperparathyroidism in the elderly. Comparison of chromatographic (HPLC) and immunoenzymatic (Pyrilinks-D) methods].

The measurement of urinary deoxypyridinoline (DPD) constitutes a specific and sensitive marker of bone resorption. Total and free forms of DPD are determined by chromatographic method (HPLC) after or without hydrolysis of urine, respectively. Pyrilinks-D, a new immunoassay, allows to assess directly the free forms and needs an appropriate hydrolysis step for measuring the total form. We have compared the values of free (F), total (T) and conjugated (NF) forms of DPD determined by HPLC and Pyrilinks-D, in elderly women (n = 21, mean age: 83.5 +/- 1.5 years) with vitamin D insufficiency (25 OH D < 6 ng/mL) and Ca insufficiency responsible for a secondary hyperparathyroidism (iPTH = 45.3 +/- 22.7 pg/mL) and in healthy elderly women (n = 25, mean age: 76.6 +/- 3.1 years) with a normal vit D status (25 OH D > 10 ng/mL) as control group. We have also measured DPD during the course of vit D and Ca supplementation. At baseline, the HPLC and Pyrilinks-D values of DPD/Cr are highly correlated (DPD-T: r = 0.92, p < 0.001 and DPD-F: r = 0.76, p < 0.001), DPD-F and -NF values are correlated with those of DPD-T, while DPD-F and -NF are not correlated between themselves. In elderly with vit D insufficiency, the values obtained with Pyrilinks-D as compared to control subjects, show a significant increase of urinary excretion of DPD-F (8.5 +/- 3.1 vs 5.7 +/- 1.9 nmol/mmol, Cr, p < 0.0001), DPD-T (16.8 +/- 10.2 vs 9.9 +/- 3.5 nmol/mmol, Cr; p < 0.001) and DPD-NF (8.3 +/- 9.0 vs 4.5 +/- 3.3 nmol/mmol, Cr, p < 0.05). The administration of 800 IU of vit D and 1 g of elemental Ca during a course of 6 months normalize the iPTH values (24.4 +/- 11.8 and 30.9 +/- 14.6 pg/mL at 3 and 6 months). Simultaneously, the urinary excretion at 3 and 6 months of DPD-T (12.9 +/- 6.0 and 13.6 +/- 6.5 nmol/mmol, Cr) and of DPD-NF (4.5 +/- 3.3 and 5.5 +/- 4.8 nmol/mmol Cr) assessed by Pyrilinks-D as well as by HPLC decreased significantly, while no change was seen with DPD-F assessed by both methods. The decreases expressed as percent of baseline values were about 20% for DPD-T and more than 30% for DPD-NF, while DPD-F levels remain unchanged. We conclude that the Pyrilinks-D immunoassay presents reliable characteristics and allows to assess either free or total forms of DPD, like the HPLC technique. It constitutes an excellent reflection of bone resorption in elderly with vit D insufficiency. However its application to monitor therapy like vit D and Ca supplementation, needs a hydrolysis step to determine DPD-T which appears in this study more sensitive to the treatment than DPD-F.

Aged↗
PubMed1995

Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo.

Severe and delayed myelosuppression is a major side effect encountered with the clinical use of nitrosourea-type chemotherapeutic drugs. The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) has been shown to repair nitrosourea-induced DNA damage. We therefore investigated the effect of expressing MGMT in hematopoietic cells (via retrovirus-mediated gene transfer) on nitrosourea-induced toxicity. A retroviral vector (N2/ZipPGK-MGMT) expressing the human MGMT cDNA from the phosphoglycerate kinase promoter was constructed. Infection of murine bone marrow with the N2/ZipPGK-MGMT retrovirus significantly increased the survival of murine bone marrow-committed progenitor cells following in vitro exposure to N-N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine). MGMT gene transfer also protected murine hematopoietic cells in vivo in a murine model of BCNU-induced myelosuppression. The infusion of 4-6 x 10(6) N2/ZipPGK-MGMT-transduced bone marrow cells into mice every 2 weeks significantly increased peripheral leukocyte counts, platelet counts, and hematocrits compared to infusions of mock-infected bone marrow cells. In addition, bone marrow-committed progenitor cells from some recipient animals demonstrated increased resistance to BCNU in vitro when analyzed 2.5 months after initial treatment. The integration of the N2/ZipPGK-MGMT provirus in the spleen DNA from these animals correlated with committed progenitor cell resistance to BCNU. These data suggest that MGMT expression in hematopoietic progenitor and precursor cells protects against nitrosourea-induced toxicity and that gene transfer may prove useful in attempts to reduce nitrosourea-induced myelosuppression in the clinical setting.

3T3 Cells↗
PubMed1995

Replication protein A binds to regulatory elements in yeast DNA repair and DNA metabolism genes.

Saccharomyces cerevisiae responds to DNA damage by arresting cell cycle progression (thereby preventing the replication and segregation of damaged chromosomes) and by inducing the expression of numerous genes, some of which are involved in DNA repair, DNA replication, and DNA metabolism. Induction of the S. cerevisiae 3-methyladenine DNA glycosylase repair gene (MAG) by DNA-damaging agents requires one upstream activating sequence (UAS) and two upstream repressing sequences (URS1 and URS2) in the MAG promoter. Sequences similar to the MAG URS elements are present in at least 11 other S. cerevisiae DNA repair and metabolism genes. Replication protein A (Rpa) is known as a single-stranded-DNA-binding protein that is involved in the initiation and elongation steps of DNA replication, nucleotide excision repair, and homologous recombination. We now show that the MAG URS1 and URS2 elements form similar double-stranded, sequence-specific, DNA-protein complexes and that both complexes contain Rpa. Moreover, Rpa appears to bind the MAG URS1-like elements found upstream of 11 other DNA repair and DNA metabolism genes. These results lead us to hypothesize that Rpa may be involved in the regulation of a number of DNA repair and DNA metabolism genes.

Base Sequence↗
PubMed1995

Multiple molecular forms of pyridinolines cross-links excreted in human urine evaluated by chromatographic and immunoassay methods.

The measurement of the collagen cross-links, hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), excreted in urine either in free or peptide-bound forms represents the most extensively investigated biochemical marker of bone collagen degradation. We studied the urinary molecular forms of pyridinolines after separation in free and peptide-linked fractions by chromatography and serial dialysis. The pyridinoline amounts of molecular species (free, < 1000 D, 1000-3500 D, 3500-10,000 D, and > 10,000 D) were evaluated by high performance liquid chromatography (HPLC) as well as with the two newly introduced enzyme-linked immunosorbent assay (ELISA) methods for determination of free pyridinolines (collagen Pyrilinks and collagen Pyrilinks-D). The variability of urinary pyridinoline forms were studied in healthy adult control subjects (n = 10, 38.4 +/- 7.5) years), in adolescents (n = 10, 16 +/- 3.3 years), and in elderly subjects with vitamin D insufficiency (n = 10, 87.3 +/- 4.3 years). Free and peptide-conjugated pyridinolines with MW < 1000 D constitute the major part of urinary cross-links in all groups, with a significantly lesser excretion in elderly patients than in adolescent groups. Expressed as a percent of total cross-links, urinary free pyridinolines assessed by direct HPLC are less in elderly subjects (HP = 34.2 +/- 6.2%, LP = 32.7 +/- 7.6%) than in adolescents (HP = 45.8 +/- 10.8%, p = 0.0065 and LP = 47.8 +/- 12.1%, p = 0.012) and in healthy adults (HP = 39.3 +/- 11.5%, NS and LP = 38.1 +/- 9.3%, NS).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Suppression of Escherichia coli alkB mutants by Saccharomyces cerevisiae genes.

The alkB gene is one of a group of alkylation-inducible genes in Escherichia coli, and its product protects cells from SN2-type alkylating agents such as methyl methanesulfonate (MMS). However, the precise biochemical function of the AlkB protein remains unknown. Here, we describe the cloning, sequencing, and characterization of three Saccharomyces cerevisiae genes (YFW1, YFW12, and YFW16) that functionally complement E. coli alkB mutant cells. DNA sequence analysis showed that none of the three gene products have any amino acid sequence homology with the AlkB protein. The YFW1 and YFW12 proteins are highly serine and threonine rich, and YFW1 contains a stretch of 28 hydrophobic residues, indicating that it may be a membrane protein. The YFW16 gene turned out to be allelic with the S. cerevisiae STE11 gene. STE11 is a protein kinase known to be involved in pheromone signal transduction in S. cerevisiae; however, the kinase activity is not required for MMS resistance because mutant STE11 proteins lacking kinase activity could still complement E. coli alkB mutants. Despite the fact that YFW1, YFW12, and YFW16/STE11 each confer substantial MMS resistance upon E. coli alkB cells, S. cerevisiae null mutants for each gene were not MMS sensitive. Whether these three genes provide alkylation resistance in E. coli via an alkB-like mechanism remains to be determined, but protection appears to be specific for AlkB-deficient E. coli because none of the genes protect other alkylation-sensitive E. coli strains from killing by MMS.

Alkylation↗
PubMed1995

The radiologic report: a realistic approach.

The radiologic report records all of the important steps taken in the practice of radiology and should be evaluated with special interest. The literature reflects the subjectivity inherent to a field left to personal interpretation, and few prospective studies of radiologic reporting are available. To determine the characteristics of the ideal radiologic report, the authors held workshops with two groups of radiologists and residents in radiology. The following items (in order of priority) were considered of prime importance by the participants and are discussed here: expression of an opinion and presentation of a workable differential diagnosis; an answer to the referring physician's question; a list of the limitations of the examination, if any; open-ended recommendations to the referring physician; integration of the findings with the clinical context; a synthesis of the findings obtained with various imaging modalities, when pertinent; and a complete description of the findings. The radiologic report has been compared to a scientific paper, and this comparison suggests the format for bringing these items together. The resulting realistic blueprint for the ideal radiologic report can be tailored to fit every imaging situation. Moreover, it can be a useful tool for evaluating the whole imaging process.

Humans↗
PubMed1994

All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase.

We have previously reported that human cells and tissues contain a 1,N6-ethenoadenine (epsilon A) binding protein, which, through glycosylase activity, releases both 3-methyladenine (m3A) and epsilon A from DNA treated with methylating agents or the vinyl chloride metabolite chloroacetaldehyde, respectively. We now find that both the partially purified human epsilon A-binding protein and cell-free extracts containing the cloned human m3A-DNA glycosylase release all four cyclic etheno adducts--namely epsilon A, 3,N4-ethenocytosine (epsilon C), N2,3-ethenoguanine (N2,3-epsilon G), and 1,N2-ethenoguanine (1,N2-epsilon G). Base release was both time and protein concentration dependent. Both epsilon A and epsilon C were excised at similar rates, while 1,N2-epsilon G and N2,3-epsilon G were released much more slowly under identical conditions. The cleavage of glycosyl bonds of several heterocyclic adducts as well as those of simple methylated adducts by the same human glycosylase appears unusual in enzymology. This raises the question of how such a multiple, divergent activity evolved in humans and what may be its primary substrate.

Acetaldehyde↗
PubMed1994

Urinary excretion of pyridinolines crosslinks measured by immunoassay and HPLC techniques in normal subjects and in elderly patients with vitamin D deficiency.

Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are specific constituents of mature skeletal collagens excreted in urine. Their measurement represents a sensitive index of bone resorption. In this study, we have measured urinary excretion of pyridinolines crosslinks by immunoassay (ELISA) and HPLC methods in 80 patients with different bone resorption rates. We chose a sample of 44 healthy adults (30 men and 14 women) and a sample of 36 elderly patients (7 men and 29 women) presenting a secondary hyperparathyroidism due to a vitamin D deficiency. The correlation between HPLC (x) and ELISA (y) was judged satisfactory (y = 0.794x + 6.947, r = 0.92). The sensitivity of pyridinolines estimation was 50 nmol/l for immunoassay and 20 nmol/l for HPLC. The intra-assay and inter-assay coefficients of variation for the two analytical methods was < 10%. The mean excretion of crosslinks (nmol/mmol of creatinine) measured by both methods in the sample of healthy adults was higher in women than in men. The amount of pyridinolines crosslinks excreted by elderly patients with vitamin D deficiency are three time higher than those of normal adults when measured by ELISA and HPLC methods. The distribution of different molecular forms of urinary pyridinoline crosslinks was investigated. Values of pyridinolines measured by HPLC in our samples of elderly patients have shown that free and peptide-bound pyridinolines with molecular weight (mol. wt.) smaller than 1000 Da represent approximately 80% of the total pyridinolines contained in urinary samples. A study on the evaluation of the antiserum used in the immunoassay for reacting with the different molecular forms isolated from urine showed a high affinity for free and peptide-bound pyridinolines with molecular weight smaller than 10,000 Da and that do not react strongly with peptide-bound with molecular weight greater than 10,000 Da. We conclude that, although this immunoassay does not measure total pyridinolines and does not distinguish between HP and LP, it seems convenient for diagnostic of metabolic bone diseases.

Adult↗
PubMed1994

The Escherichia coli AlkB protein protects human cells against alkylation-induced toxicity.

Escherichia coli can ameliorate the toxic effects of alkylating agents either by preventing DNA alkylation or by repairing DNA alkylation damage. The alkylation-sensitive phenotype of E. coli alkB mutants marks the alkB pathway as an extremely effective defense mechanism against the cytotoxic effects of the SN2, but not the SN1, alkylating agents. Although it is clear that AlkB helps cells to better handle alkylated DNA, no DNA alkylation repair function could be assigned to the purified AlkB protein, suggesting that AlkB either acts as part of a complex or acts to regulate the expression of other genes whose products are directly responsible for alkylation resistance. However, here we present evidence that the provision of alkylation resistance is an intrinsic function of the AlkB protein per se. We expressed the E. coli AlkB protein in two human cell lines and found that it confers the same characteristic alkylation-resistant phenotype in this foreign environment as it does in E. coli. AlkB expression rendered human cells extremely resistant to cell killing by the SN2 but not the SN1 alkylating agents but did not affect the ability of dimethyl sulfate (an SN2 agent) to alkylate the genome. We infer that SN2 agents produce a class of DNA damage that is not efficiently produced by SN1 agents and that AlkB somehow prevents this damage from killing the cell.

AlkB Homolog 1, Histone H2a Dioxygenase↗
PubMed1994

[Radiological changes of talc pleurodesis in cases of effusion].

This study is based on the observations of 86 pleurodesis done by talc insufflation during thoracoscopy in 82 patients suffering from benign (8%) and malignant (92%) pleural effusions. Serial chest films were obtained on every patient. Chest computed tomography was obtained in ten patients. The most frequent finding seen in the early phase and one month later was the appearance of loculations (94%) in selective areas of the thorax. Occasionally they take the appearance of airfluid levels (22%). These loculations are characteristically located in the axillary (60%), intrafissural (30%) and paramediastinal (34%) areas of the chest. In the late phase with a mean evolution time of 6 months, these loculations evolve in 77% of patients in areas of pleural thickening. CT of the chest demonstrates the presence of characteristic pleural thickening in the form of coarse (5/12) and/or fine linear densities (7/12) corresponding to talc deposits, on the pleural surface. These modifications are shown by light microscopy examination of the pleural done at the autopsy.

Female↗
PubMed1993

Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.

Animals↗
PubMed1993

Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine.

Human O6-alkylguanine-DNA alkyltransferase was rapidly inactivated by low concentrations of O6-benzylguanine, but the alkyltransferase from the Escherichia coli ogt gene was much less sensitive and alkyltransferases from the E. coli ada gene or from yeast were not affected. O6-Benzyl-2'-deoxyguanosine was less potent than the base, but was still an effective inactivator of the human alkyltransferase and had no effect on the microbial proteins. O6-Allylguanine was somewhat less active, but still gave complete inactivation of both the human and Ogt alkyltransferases at 200 microM in 30 min, slightly affected the Ada protein, and had no effect on the yeast alkyltransferase. O4-Benzylthymidine did not inactivate any of the alkyltransferase proteins tested. Inactivation of the human alkyltransferase by O6-benzylguanine led to the formation of S-benzylcysteine in the protein and to the stoichiometric production of guanine. The rate of guanine formation followed second-order kinetics (k = 600 M-1 s-1). Prior inactivation of the alkyltransferase by reaction with a methylated DNA substrate abolished its ability to convert O6-benzylguanine into guanine. These results indicate that O6-benzylguanine inactivates the protein by acting as a substrate for alkyl transfer and by forming S-benzylcysteine at the acceptor site of the protein. The inability of O6-benzylguanine to inactivate the microbial alkyltransferases may be explained by steric constraints at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

In vivo evidence for endogenous DNA alkylation damage as a source of spontaneous mutation in eukaryotic cells.

Three genes that participate in the repair of DNA alkylation damage were recently cloned from Saccharomyces cerevisiae: the MGT1 O6-methylguanine DNA methyltransferase gene, the MAG 3-methyladenine DNA glycosylase gene, and the APN1 apurinic/apyrimidinic (AP) endonuclease gene. Altering the expression levels of these three genes produced significant changes in the S. cerevisiae spontaneous mutation rate. Spontaneous mutation increased in the absence of the MGT1 DNA methyltransferase, presumably because unrepaired, spontaneously produced, O6-alkylguanine lesions mispair during replication. Moreover, changing the ratios of the MAG 3-methyladenine DNA glycosylase and the APN1 AP endonuclease had profound effects on spontaneous mutation rates. In the absence of APN1, the overexpression of MAG increased spontaneous mutation, and the underexpression of MAG decreased spontaneous mutation. We infer that the MAG glycosylase acts upon spontaneously produced 3-alkyladenine and 7-alkylguanine DNA lesions to produce mutagenic abasic sites, and that if the repair of these abasic sites is not initiated by the APN1 AP endonuclease they cause mutations during replication. Our results indicate that eukaryotic cells harbor endogenous metabolites that alkylate nuclear DNA at both oxygens and nitrogens.

Alkylation↗
PubMed1993

Effects of a physiological dose of cholecystokinin on food intake and postprandial satiation in man.

CCK-33 was infused intravenously to groups of 9 lean and 9 obese volunteers in doses that elicited plasma CCK concentrations in the physiological range. The effect of these infusions on food intake and satiety signals was compared with the effect of saline infusions in the same subjects. Food intake (486 +/- 52 g; mean +/- S.E.M.) was slightly, but not significantly decreased (553 +/- 55 g after saline), and hunger and fullness feelings after eating were unaffected, in both of the two groups. We conclude that the infusion of CCK-33 to plasma levels comparable to those observed after a fatty meal does not have a major effect on food intake and postprandial hunger feelings.

Adult↗