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Biomedical subjects

G Duménil

Publications and source records attributed to G Duménil.

At least 19 recordsLinked to original sources
PubMed2005

Extracellular bacterial pathogens and small GTPases of the Rho family: an unexpected combination.

Even in the case of extracellular bacterial pathogens, it is becoming increasingly clear that successful colonization does not limit itself to passive attachment on the surface of human cells; a dialogue takes place between bacteria and infected cells. These pathogens modulate cellular functions to their advantage, leading to survival and proliferation at the cell surface. Furthermore, there is increasing evidence that a variety of extracellular pathogens activate small GTPases of the Rho family during adhesion, placing these regulators at the center of the interaction between these bacteria and their infected host.

Animals↗
PubMed2003

Implication of nitro group reduction in the mutagenic and chromosome damaging activities of 22 new 5-nitroisoquinolines by the Salmonella mutagenicity test and the cytokinesis-blocked micronucleus assay.

The mutagenic (MUT) and chromosome-damaging (CHR) activities of 22 potential antimalarial drugs (5-nitroisoquinoline derivatives) were evaluated by the Salmonella test and the cytokinesis-blocked micronucleus assay (CBMN). The Salmonella mutagenicity test was performed with and without metabolic activation (S9 mix) in S. typhimurium strains TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain). The CBMN was carried out on human lymphocytes without metabolic activation. Four concentrations were tested: 1, 10, 100 and 1000 ng/ml. MUT was expressed as minimal mutagenic concentrations (MMC, microM) and CHR was expressed as minimal chromosome-damaging concentrations (MCDC, nM) to compare both activities. All the 5-nitroisoquinoline compounds were mutagenic in TA100. MMC ranged from 0.1 to 52.9 microM in TA100. A statistically significant decrease in MMC was observed in YG1042 (8 x 10(-3) to 3.5 microM), implicating reduction of the nitro group. Modulation of MUT by S9 mix was not significant in TA100 and YG1042. CHR was detected in 13 products for at least one concentration. Among the chromosome-damaging compounds, the MCDC ranged from 2.9 x 10(-3) to 3.6 nM. No relationship was found between MUT and CHR, suggesting two distinct pathways of DNA damage.

Adolescent↗
PubMed2001

The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high-molecular-weight complexes.

A key event in legionellosis is the ability of Legionella pneumophila to survive and proliferate inside alveolar macrophages. The dot/icm genes, which are necessary for intracellular growth, show sequence similarity to genes encoding conjugative transfer systems, and it is believed that they are responsible for the formation of a secretion apparatus. Evidence is provided here that the IcmR and IcmQ proteins participate in a chaperone-substrate relationship similar to that observed for translocated proteins in type III and type IV secretion apparatuses. Immobilized IcmQ was found to bind IcmR from crude bacterial extracts efficiently. Furthermore, purified IcmR and IcmQ bind with high affinity. This interaction was also observed in vivo by co-immunoprecipitation. The presence of IcmR was found to affect the physical state of IcmQ directly. In the absence of IcmR, IcmQ formed high-molecular-weight complexes both in vivo and in vitro, whereas IcmR prevented and reversed the formation of these complexes.

Amino Acid Sequence↗
PubMed2001

Evaluation of sunscreen protection in human melanocytes exposed to UVA or UVB irradiation using the alkaline comet assay.

The in vivo assessment of sunscreen protection does not include the photogenotoxicity of UVA or UVB solar radiation. Using the comet assay we have developed a simple and rapid technique to quantify sunscreen efficacy against DNA damage induced by UV light. Cutaneous human melanocytes from primary cultures were embedded in low-melting point (LPM) agarose and exposed to UVA (0.8 J/cm2) or to UVB (0.06 J/cm2) through a quartz slide covered with 10 microL volumes of sunscreens. DNA single-strand breaks induced directly by UVA at 4 degrees C and indirectly through nucleotide excision repair by UVB following a 35 min incubation period at 37 degrees C were quantified using the comet assay. Tail moments (TM) (tail length x %tail DNA) of 100 cells/sample were determined by image analysis. DNA damage was evaluated with a nonlinear regression analysis on the normalized distribution frequencies of TM using a chi 2 function. The coefficients of genomic protection (CGP) were defined as the percentage of inhibition of DNA lesions caused by the sunscreens. Twenty-one sunscreens were evaluated, and the calculated CGP were compared with the in vivo sun protective factor (SPF) and with the protection factor UVA (PFA). Nonlinear relationships were found between SPF and CGPUVB and between PFA and CGPUVA.

Cells, Cultured↗
PubMed2000

Bacterial signals and cell responses during Shigella entry into epithelial cells.

Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial-host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy-terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC-induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella-induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.

Bacterial Proteins↗
PubMed2000

Src tyrosine kinase activity down-regulates Rho-dependent responses during Shigella entry into epithelial cells and stress fibre formation.

Invasion of epithelial cells by Shigella, the causative agent of bacillary dysentery, is dependent upon the formation of characteristic membrane ruffles that engulf the bacteria in a macropinocytic-like process. We show here that Cdc42 and Rac GTPases, but not Rho;, are critical for actin polymerisation, whereas Rho; is necessary for the recruitment of ezrin and Src at the site of entry. Remarkably, cells expressing constitutively active Src did not show ezrin recruitment at Shigella entry foci. In these cells, formation of stress fibres induced by LPA stimulation, or microinjection of activated Rho; (V14Rho), was inhibited. Src-mediated tyrosyl-phosphorylation of p190RhoGAP correlated with changes in the ability of p190RhoGAP to interact with Rho;, suggesting that Src regulates Rho; function via p190RhoGAP. We propose that Rho; activation is required for proper organisation of Shigella entry foci and for Src recruitment, and that Src tyrosine kinase activity, in turn, down-regulates the function of Rho; at the site of Shigella entry. The significance of this negative regulatory loop on Rho;-dependent responses is discussed.

ADP Ribose Transferases↗
PubMed1999

The protective activity of alpha-hederine against H2O2 genotoxicity in HepG2 cells by alkaline comet assay.

This study was designed to evaluate the protective effect of alpha-hederine (alpha-hed) against H2O2-mediated DNA damage on HepG2 cell line by the alkaline comet assay. For the protective effect of alpha-hed study, cells were treated according to three protocols: pre-treatment, simultaneous treatment and post-treatment. The effect of alpha-hed on catalase activity was evaluated after treating the cells with 3.36 mg/ml of 3-amino-1,2,4-triazole (AMT) singly or in combination with alpha-hed (1.5 or 3 microg/ml) and H2O2 (8.8 microM) during 1 h. The catalase activity was also biochemically measured after treating cells with alpha-hed at 1.5, 3, or 15 microg/ml during 1 h. Additionally, the influence of alpha-hed on membrane RedOx potential, pool of reduced glutathione and total protein content was evaluated by flow cytometry. In the pre-treatment, the two concentrations of alpha-hed (1.5 and 3 microg/ml) decreased the lesions induced by H2O2 (8.8 microM) significantly. This decrease was about 57.2% and 66.1%, respectively. Similar results were observed when cells were treated with alpha-hed and H2O2 simultaneously. The decrease of H2O2-induced lesions was about 78.2% and 83.2% (alpha-hed 1.5 and 3 microg/ml, respectively). In the post-treatment protocol, this decrease was not significant. The combination of AMT and H2O2 induced more DNA damage than H2O2 alone (tail moment (TM) means was 31.4% and 21.8%, respectively). When alpha-hed was added to this mixture, TM means were reduced significantly (17.4% for alpha-hed 1. 5 microg/ml and 15.5% for alpha-hed 3 microg/ml). Up to 6.9 microg/ml, alpha-hed enhanced catalase activity (60.5%), followed by a decrease of the activity. Total protein content and membrane RedOx potential were slightly increased up to 11 microg/ml (14% and 3.6%, respectively) followed by a drop and a plateau. Pool of reduced glutathione remained unchanged up to 10 microg/ml then dropped and reached a plateau. In conclusion, alpha-hed could exert its protective effect against H2O2-mediated DNA damage by scavenging free radicals or by enhancing the catalase activity.

Catalase↗
PubMed1998

Interferon alpha inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.

Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-alpha inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-alpha. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-alpha treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-alpha inhibits this Src-dependent signaling pathway.

Actins↗
PubMed1998

Specific contribution of Tyk2 JH regions to the binding and the expression of the interferon alpha/beta receptor component IFNAR1.

Cytokine signaling involves the activation of the Janus kinase (JAK) family of tyrosine kinases. These enzymes are physically associated with cytokine receptor components. Here, we sought to define the molecular basis of the interaction between Tyk2 and IFNAR1, a component of the interferon alpha/beta receptor, by delimiting a minimal IFNAR1 binding region in the Tyk2 protein. Using an in vitro assay system, we narrowed down the interaction domain to a region comprising the JH7 and part of the JH6 homology boxes (amino acids 22-221). When expressed in Tyk2-negative cells, the JH7-6 region was unable to stabilize IFNAR1 protein levels, a critical function that we previously attributed to the N region (amino acids 1-591) of Tyk2. Moreover, substitution of the JH7-JH6 domain in JAK1 with that of Tyk2 did not restore IFNAR1 level nor interferon alpha signaling in Tyk2-negative cells. Thus, the major interaction surface lies within JH7-6, but additional JH regions (JH5-4-3) contribute in a specific manner to the in vivo assembly of Tyk2 and IFNAR1. Evidence is also provided of the lack of specificity of the Tyk2 kinase-like and tyrosine kinase domains in interferon alpha/beta receptor signaling.

Amino Acid Sequence↗
PubMed1998

Evaluation of the mutagenicity and antimutagenicity of forty-two 3-substituted flavones in the Ames test.

The mutagenic and antimutagenic activities of forty-two synthetic flavones were assessed by the Ames test. The tested flavones included twenty-three 3-nitroflavones, eighteen 3-aminoflavones and the 3-chloroflavone. The mutagenicity was evaluated with Salmonella typhimurium TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain) with and without metabolic activation (S9 mix). The antimutagenicity of the non mutagenic derivatives was evaluated against 11 known reference mutagens. A total of 39 synthetic flavones were mutagenic. The mutagenic activities ranged from 0.1 rev/nmole (4'-chloro-6-methoxy-3-nitroflavone) to 6240 rev/nmole (4'-methoxy-3, 3'-diaminoflavone). Two differences were found between the 3-amino and the 3-nitroflavones: (i) the mutagenicity of the 3-aminoflavones required the presence of the metabolic activation; (ii) the 3-amino derivatives were more mutagenic than their 3-nitro counterparts. Increased mutagenicity, as assessed with strain YG1042, was limited to 17/39 derivatives. The mutagenic activity was induced by the presence of the double bond at the 2,3-position for conjugation of the lone-pair electron with the carbonyl group on the 'C' ring. This mutagenicity was modulated by substituents at the 2'-position. Additional mutagenicity was brought by the aminoaromatic and nitroaromatic group reduction by bacterial nitroreductases and by the S9 mix; it was modulated by different substituents on the aromatic rings of the flavones. Three flavones: 3-chloroflavone (1C), 4'-hydroxy-3-nitroflavone (23N) and 2',3-diaminoflavone (2A) showed antimutagenic properties. Compound 1C was efficient against benzo(a)pyrene (BaP), 2-aminofluorene (2AF), 2-aminoanthracene (2AA), 4-nitroquinoline-1-oxide (4NQO) and 1-methyl-3'-nitro-1-nitrosoguanidine (MNNG). Compound 23N inhibited the mutagenicity of BaP and MNNG. The antimutagenic activity of 2A was limited to MNNG.

Antimutagenic Agents↗
PubMed1997

Identification of signalling components in tyrosine kinase cascades using phosphopeptide affinity chromatography.

Various methods are now available to identify the molecular partners of the component of a signal transduction pathway. Some interactions, however, can be technically difficult to detect because they depend upon transient tyrosine phosphorylation. Here, we present a simple affinity chromatography approach based on synthetic phosphopeptides to purify potential partners of phosphotyrosine-containing proteins. With this approach, we confirm the previously characterized interaction between Grb2 and the EGF receptor, and we identify novel partners of the IGF-1 receptor and of the JAK proteins. Methenyltetrahydrofolate synthetase (MTHFS) was identified as a potential mediator of IGF-1R dependent transformation. P85alpha, the regulatory subunit of PI3 kinase, was identified as one of four proteins recruited by a phosphopeptide mimicking a motif conserved in all JAK family members.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Evaluation of the genotoxic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay.

The genotoxicity of metronidazole (MZ) and dimetridazole (DZ) has been evaluated in human lymphocytes using the comet assay. The test has been performed using 3 doses (58.4, 175.2 and 292.1 microM for MZ; and 70.9, 212.6 and 354.3 microM for DZ) under 3 experimental protocols: aerobiosis, anaerobiosis (90% N2, 10% CO2) and with the presence of the microsomal fraction S9 mix. The effects of 4 antioxidants (8-hydroxyquinoline (8HQ), vitamin C (VitC), catalase (CAT) and superoxide dismutase (SOD), have been investigated on DNA damage generated by fixed concentrations of MZ (292.1 microM) and DZ (354.4 microM). In aerobic conditions, MZ and DZ produced significant dose-response relationships. The dose-related effects of both drugs decreased or were abolished in anaerobic conditions or in presence of S9 mix. 8HQ, VitC, CAT and SOD induced dose-related protective responses against DNA damage due to MZ and DZ. These findings suggest that MZ and DZ induce DNA damage in human lymphocytes through the futile cycle. The one-electron reduction of the drugs leads to the production of nitro radical anions. In the presence of oxygen, these radicals are reoxidized and generate oxygen-activated species.

Animals↗
PubMed1997

Chemical degradation of wastes of antineoplastic agents: cyclophosphamide, ifosfamide and melphalan.

Handling genotoxic compounds commonly used in cancer chemotherapy generates contaminated wastes that require decontamination before disposal. Chemical methods are an alternative and/or a complement to incineration for the treatment of wastes and spills. As part of a program initiated by the International Agency for Research on Cancer (IARC), three chemical methods readily available in the hospital environment, viz sodium hypochlorite (NaOCl, 5.25%), hydrogen peroxide (H2O2, < or = 30%) and Fenton reagent (FeCl2, 2H2O; 0.3 g in 10 ml H2O2, 30%), were tested for the degradation of three alkylating agents (cyclophosphamide, CP; ifosfamide, IF, and melphalan). Pharmaceutical preparations corresponding to the most highly concentrated administration solutions in either NaCl (0.9%) or dextrose (5%) were inactivated by oxidation volume/volume with each of the methods for at least 1 h. The efficiency of degradation was monitored by high-pressure liquid chromatography. The mutagenicity of the degradation residues was tested by means of the Ames test using tester strains of Salmonella typhimurium TA 97a, TA 98, TA 100 and TA 102 with and without an exogenous metabolic activation system. Complete disappearance of CP was observed after 1 h with all degradation methods. However, direct mutagens were generated by the Fenton oxidation technique in the presence of dextrose (5%). IF was completely degraded by the Fenton reagent and NaOCl methods. No mutagenic residues were detected after 1 h of treatment with the Fenton technique, and after 3 h with the NaOCl method. Direct-acting mutagens remained after the H2O2 treatment in the presence of dextrose (5%). Complete degradation of melphalan was achieved in 1 h by each of the three methods, and no mutagenic residues were produced by any of the treatments. The use of NaOCl (5.25%) proved the most efficient system for degradation of the three alkylating agents.

Animals↗
PubMed1996

The Salmonella sulA-test: a new in vitro system to detect genotoxins.

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.

Bacterial Proteins↗
PubMed1996

Optimization of the Salmonella/mammalian microsome assay for urine mutagenesis by experimental designs.

Assessing urine mutagenicity with the Salmonella mutagenicity test is often limited by the volumes of the samples. Optimization of the assay was performed with factorial and Doehlert designs. Two fractional factorial designs 2(3-1) (3 factors, 4 experiments) were used to estimate the main effects of the percent S9 in the mix, the time of liquid incubation, the inoculum size and the growth conditions. A Doehlert design (3 factors, 13 experiments) was used to study the main effects and the interactions of the NADP, G6P and S9 in the mix. The positive markers were benzo[a]pyrene (BaP, 0.3 microgram/plate) and a pool of smokers' urine (SU, 1.25 ml equivalent/plate). The response was limited to the induction factor (IF, number of induced revertants/number of spontaneous revertants) with Salmonella typhimurium TA98. The optimal conditions for BaP were: a 60 min period of liquid incubation and a volume of 0.1 ml (approx. 10(8) cells/plate) of an overnight culture grown in 50 ml of Nutrient Broth No. 2 from a 250 ml flask. The S9 mix (0.1 ml, final volume) included 1.5% of S9, 1.0 mM NADP and 4.4 mM G6P. The maximal IF was 15.79. The optimal conditions for SU were: a 60 min period of liquid incubation and a volume of 0.1 ml (approx. 10(8) cells/plate) of an overnight culture grown in 7 ml of Nutrient Broth No. 2 from a 20 x 180 mm tube. The S9 mix (0.1 ml, final volume) included: 4% S9, 4.2 mM NADP and 5.2 mM G6P. The maximal IF was 10.95. These optimal conditions did not modify the spontaneous frequencies of the tester strains: TA97a, TA98, TA100 and TA102. The dose-response curves of mutagenic urine samples were found to be non-linear. This micromethod required 8-fold less urine sample and 12.5-fold less liver homogenate as compared to the standard plate incorporation assay and was from 6.2- to 11.8-fold more sensitive to evaluate urine mutagenicity. The sensitivity of this technique was found to be limited to individuals smoking more than approx. 5 cigarettes/day by the standard extraction-concentration procedure.

Dose-Response Relationship, Drug↗
PubMed1996

Protective effects of alpha-hederin, chlorophyllin and ascorbic acid towards the induction of micronuclei by doxorubicin in cultured human lymphocytes.

The influence of alpha-hederin (a saponin isolated from Hedera helix), chlorophyllin, the sodium-copper salt of chlorophyll, and ascorbic acid (vitamin C) on the direct clastogenicity of doxorubicin (Adriamycin) was investigated in vitro in human lymphocytes for the induction of micronuclei. In order to determine a possible mechanism of action responsible for the antimutagenic activity, treatments were performed for the three substances at different times of the culture (pre-treatment, simultaneous and post-treatment). Alpha-hederin (1.3 x 10(-2), 0.13, 1.3 and 13 nmol/ml) and chlorophyllin (0.14, 1.4 and 14 nmol/ml) were found to exert an antimutagenic effect against the clastogenicity of doxorubicin (1.5 x 10(-2) nmol/ml) in all treatments at all concentrations. Ascorbic acid (10 nmol/ml) was effective in reducing the micronucleus levels only in the simultaneous treatment, when it was previously incubated with doxorubicin for 2 h at 37 degrees C before being introduced into the culture. Our results suggested a desmutagenic effect for alpha-hederin, chlorophyllin and ascorbic acid. Chlorophyllin acted also through a bio-antimutagenic mechanism and alpha-hederin seemed to induce metabolic enzymes, which inactivated doxorubicin. Preliminary studies showed that the effective antimutagenic concentrations of alpha-hederin, chlorophyllin and ascorbic acid had no clastogenic or aneugenic effects in human lymphocytes. No cytotoxicity was observed for the three antimutagenic agents either.

Adult↗
PubMed1995

Monitoring exposure of hospital personnel handling cytostatic drugs and contaminated materials.

A study was undertaken to evaluate the urine mutagenicity of 63 individuals working in four hospital departments. The exposed group included 38 subjects who were exposed to various cytostatic drugs and/or contaminated material from treated patients. The control group included 25 individuals of the hospital personnel. Urine mutagenicity was monitored by the Ames test using tester strains TA 98 + S9 Mix and TA 102-S9 Mix. Urine samples were collected before and after the working periods. A total of 29/116 (25%) urine samples were mutagenic for either strain. Among the mutagenic samples, 24/29 were mutagenic for tester strain TA 98 exclusively. No significant correlation could be found between occupational exposure to cytostatic drugs and urine mutagenicity evaluated by the strain TA 98 + S9 Mix. Smoking was the main environmental factor that modulated urine mutagenicity with TA 98. Three subjects in the exposed group had mutagenic urine samples at the end of the working period with strain TA 102-S9 Mix. This mutagenicity was related to occupational exposure to cisplatin. In the control group, one individual had mutagenic samples before and after the working period. Assessing occupational exposure to cytostatic drugs with strain TA 102 requires additional studies to determine environmental mutagens which can be detected by this strain.

Adult↗