New insights about the structural rearrangements required for substrate translocation in the bovine mitochondrial oxoglutarate carrier.

The oxoglutarate carrier (OGC) belongs to the mitochondrial carrier family and plays a key role in important metabolic pathways. Here, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine, in order to investigate new structural rearrangements required for substrate translocation. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2-oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation. Our results are interpreted in the light of the homology model of BtOGC, built by using as a template the X-ray structure of the bovine ADP/ATP carrier isoform 1 (AAC1).

Arabidopsis EDM2 promotes IBM1 distal polyadenylation and regulates genome DNA methylation patterns.

DNA methylation is important for the silencing of transposons and other repetitive elements in many higher eukaryotes. However, plant and mammalian genomes have evolved to contain repetitive elements near or inside their genes. How these genes are kept from being silenced by DNA methylation is not well understood. A forward genetics screen led to the identification of the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) as a cellular antisilencing factor and regulator of genome DNA methylation patterns. EDM2 contains a composite Plant Homeo Domain that recognizes both active and repressive histone methylation marks at the intronic repeat elements in genes such as the Histone 3 lysine 9 demethylase gene Increase in BONSAI Methylation 1 (IBM1) and is necessary for maintaining the expression of these genes by promoting mRNA distal polyadenylation. Because of its role in maintaining IBM1 expression, EDM2 is required for preventing CHG methylation in the bodies of thousands of genes. Our results thus increase the understanding of antisilencing, genome methylation patterns, and regulation of alternative RNA processing by intronic heterochromatin.

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene.

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh 'Charentais' type melon line that accumulates ß-carotene. One mutagenized M2 family segregated for a novel recessive trait, a yellow-orange fruit flesh ('yofI'). HPLC analysis revealed that 'yofI' accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of 'yofI' is associated with a significant change of the fruit aroma since cleavage of ß-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to ß-carotene in melon fruit. Cloning and sequencing of 'yofI' CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A-T transversion in 'yofI' which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.

Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines.

The nervous system senses peripheral damage through nociceptive neurons that transmit a pain signal. TRPA1 is a member of the Transient Receptor Potential (TRP) family of ion channels and is expressed in nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent natural compounds, and environmental irritants. How such diverse stimuli activate TRPA1 is not known. We observed that most compounds known to activate TRPA1 are able to covalently bind cysteine residues. Here we use click chemistry to show that derivatives of two such compounds, mustard oil and cinnamaldehyde, covalently bind mouse TRPA1. Structurally unrelated cysteine-modifying agents such as iodoacetamide (IA) and (2-aminoethyl)methanethiosulphonate (MTSEA) also bind and activate TRPA1. We identified by mass spectrometry fourteen cytosolic TRPA1 cysteines labelled by IA, three of which are required for normal channel function. In excised patches, reactive compounds activated TRPA1 currents that were maintained at least 10 min after washout of the compound in calcium-free solutions. Finally, activation of TRPA1 by disulphide-bond-forming MTSEA is blocked by the reducing agent dithiothreitol (DTT). Collectively, our data indicate that covalent modification of reactive cysteines within TRPA1 can cause channel activation, rapidly signalling potential tissue damage through the pain pathway.

The basic property of Lys385 is important for potentiation of the human α1 glycine receptor by ethanol.

Ethanol alters the function of several members of the Cys-loop ligand-gated ion channel superfamily. Recent studies have shown that the sensitivity of the α1 glycine receptor (GlyR) to ethanol can be affected by the state of G protein activation mediated by the interaction of Gßγ with intracellular amino acids in the GlyR. Here, we evaluated the physicochemical property of Lys385 that contributes to ethanol modulation by using mutagenesis, patch-clamp, and biochemical techniques. A conserved substitution (K385R) did not affect either the apparent glycine EC50 (40 ± 1 versus 41 ± 0.5 µM) or the ethanol-induced potentiation (53 ± 5 versus 46 ± 5%) of the human α1 GlyR. On the other hand, replacement of this residue with glutamic acid (K385E), an acidic amino acid, reduced the potentiation of the GlyR to 10 ± 1%. Furthermore, mutations with a hydrophobic leucine (K385L), a hydrogen bond donor glutamine (K385Q), or a neutral residue (K385A) also reduced ethanol modulation. Finally, substitution by a large and hydrophobic residue (K385F) and deletion of 385 (Lys385_) reduced ethanol modulation to 10 ± 4 and 17 ± 0.4%, respectively. Experiments using dynamic cysteine substitution with a methanethiosulfonate reagent and homology modeling indicate that the basic property and the position of Lys385, probably because of its interaction with Gßγ, is critical for ethanol potentiation of the receptor.

In vivo antigenotoxic potential and possible mechanism of action of selected 4-hydroxy-2H-chromen-2-one derivatives.

The in vivo sex-linked recessive lethal test was carried out in Drosophila melanogaster to investigate whether or not five substituted 4-hydroxy-2H-chromen-2-ones can modulate the genotoxicity of the well-established mutagenic agent ethyl methanesulfonate (EMS). For this purpose, 3 days old Canton S males were treated with the potent mutagen EMS alone in concentration of 0.75 ppm, as well as in combination with one of the five 4-hydroxycoumarins, namely diethyl 2-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)malonate (2b), 3-(1-(4-hydroxy-2-oxo-2H-chromen-3-yl)ethylidene)pentane-2,4-dione (6b), 4-(4-(4-hydroxy-2-oxo-2H-chromen-3-yl)thiazol-2-ylamino) benzenesulfonic acid (4c), 4-hydroxy-3-(2-(2-nitropheny lamino)thiazol-4-yl)-2H-chromen-2-one (9c), and (E)-4-hydroxy-3-(1-(m-tolylimino)ethyl)-2H-chromen-2-one (5d), in concentration of 70 ppm. The frequency of germinative mutations increased significantly after the treatment with EMS and decreased after treatments with coumarins. The maximum reduction was observed after treatments with 2b, 6b, 4c, and 5d. By the formation of hydrogen bonds or electrostatic interactions with O(6) of DNA guanine, tested coumarins prevent EMS-induced alkylation. The results indicate a protective role of five 4-hydroxycoumarins under the action of a strong mutagen.

'Site of contact genotoxicity' assessment for implants - Potential use of single cell gel electrophoresis in biocompatibility testing of medical devices.

Toxicological risk assessment of medical devices requires genotoxicity assessment as per ISO 10993, Part 3, which is designed to address gene mutations, clastogenicity and/or aneugenicity endpoints. 'Site of contact genotoxicity' is a potential genotoxic risk especially for medical implants, that is currently not addressed in biocompatibility standards. We therefore performed initial validation study on the use of alkaline single cell gel electrophoresis (comet assay) for detecting 'site of contact genotoxicity' of medical devices, using test items made of acrylic implants impregnated with ethyl methanesulphonate (EMS). Comet assay detected increased DNA migration at the site of implantation, but not in the liver. The same implants also failed to show any genotoxicity potentials, when tested on the standard test battery using Salmonella/microsome and chromosome aberration assays. The study suggested that some medical implants can cause 'site of contact genotoxicity', without producing systemic genotoxicity. In conclusion, comet assay will add new dimension to safety assessment of medical devices, and this assay can be added to the battery of genetic toxicology tests for evaluating biocompatibility of medical implants.

Identification of Novel Molecular Regulators Modulating Ethylene Biosynthesis Using EMS-Based Genetic Screening.

The gaseous hormone ethylene regulates a diverse range of plant development and stress responses. Ethylene biosynthesis is tightly regulated by the transcriptional and posttranscriptional regulation of ethylene biosynthetic enzymes. ACC synthase (ACS) is the rate-limiting enzyme that controls the speed of ethylene biosynthesis in plant tissues, thus serving as a primary target for biotic and abiotic stresses to modulate ethylene production. Despite the critical role of ACS in ethylene biosynthesis, only a few regulatory components regulating ACS stability or ACS transcript levels have been identified and characterized. Here we show a genetic approach for identifying novel regulatory components in ethylene biosynthesis by screening EMS-mutagenized Arabidopsis seeds.

Accessibility of the CLC-0 pore to charged methanethiosulfonate reagents.

Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.

The ionization state of D37 in E. coli porin OmpF and the nature of conductance fluctuations in D37 mutants.

Understanding the flow of ions through E. coli porin outer membrane protein F (OmpF) requires knowledge of the charge state of all titratable residues located along the permeation pathway. Earlier theoretical studies proved successful in the calculation of the pK values of most residues. The (apparent) pK of Asp37 (D37), on the other hand, appeared rather sensitive to the (unknown) protein dielectric used. We addressed the protonation state of D37 experimentally by replacing D37 with a (neutral) valine. This D37V mutant expressed reduced cation selectivity, in agreement with the view that D37 in wild-type (WT) OmpF is fully ionized, i.e., deprotonated. The introduction of a (positively charged) arginine at position 37 evoked current fluctuations. Similar behavior was observed in the D37K mutant and the cysteine mutants D37C-MTSEA and D37C-MTSET. Nontitratable [2-(trimethylammonium)ethyl]-methanethiosulfonate (MTSET) carries a permanent and pH-independent charge of 1e, implying that the fluctuations of the D37C-MTSET mutant do not represent (de)protonation reactions of MTSET. We therefore conclude that these fluctuations reflect transitions between conformational substates evoked by structural instabilities due to the positive charge at that particular position in the pore lumen. Based on the similarities between D37C-MTSET fluctuations and those seen in the other mutants, notably D37K, the underlying mechanism of these fluctuations may be (essentially) the same in all four mutants studied.

Substituted cysteine scanning in D1-S6 of the sodium channel hNav1.4 alters kinetics and structural interactions of slow inactivation.

Slow inactivation in voltage-gated Na+ channels (Navs) plays an important physiological role in excitable tissues (muscle, heart, nerves) and mutations that disrupt Nav slow inactivation can result in pathophysiologies (myotonia, arrhythmias, epilepsy). While the molecular mechanisms responsible for slow inactivation remain elusive, previous studies have suggested a role for the pore-lining D1-S6 helix. The goals of this research were to determine if (1) cysteine substitutions in D1-S6 affect gating kinetics and (2) methanethiosulfonate ethylammonium (MTSEA) accessibility changes in different kinetic states. Site-directed mutagenesis in the human skeletal muscle isoform hNav1.4 was used to substitute cysteine for eleven amino acids in D1-S6 from L433 to L443. Mutants were expressed in HEK cells and recorded from with whole-cell patch clamp. All mutations affected one or more baseline kinetics of the sodium channel, including activation, fast inactivation, and slow inactivation. Substitution of cysteine (for nonpolar residues) adjacent to polar residues destabilized slow inactivation in G434C, F436C, I439C, and L441C. Cysteine substitution without adjacent polar residues enhanced slow inactivation in L438C and N440C, and disrupted possible H-bonds involving Y437:D4 S4-S5 and N440:D4-S6. MTSEA exposure in closed, fast-inactivated, or slow-inactivated states in most mutants had little-to-no effect. In I439C, MTSEA application in closed, fast-inactivated, and slow-inactivated states produced irreversible reduction in current, suggesting I439C accessibility to MTSEA in all three kinetic states. D1-S6 is important for Nav gating kinetics, stability of slow-inactivated state, structural contacts, and state-dependent positioning. However, prominent reconfiguration of D1-S6 may not occur in slow inactivation.

H2O2Accumulation, Host Cell Death and Differential Levels of Proteins Related to Photosynthesis, Redox Homeostasis, and Required for Viral Replication Explain the Resistance of EMS-mutagenized Cowpea to Cowpea Severe Mosaic Virus.

Infection with Cowpea severe mosaic virus (CPSMV) represents one of the main limitations for cowpea (Vigna unguiculata L. Walp.) productivity due to the severity of the disease symptoms, frequency of incidence, and difficulties in dissemination control. This study aimed to identify the proteins and metabolic pathways associated with the susceptibility and resistance of cowpea plants to CPSMV. Therefore, we treated the seeds of a naturally susceptible cowpea genotype (CE-31) with the mutagenic agent ethyl methane sulfonate (EMS) and compared the secondary leaf proteomic profile of the mutagenized resistant plants inoculated with CPSMV (MCPI plant group) to those of the naturally susceptible cowpea genotype CE-31 inoculated (CPI) and noninoculated (CPU) with CPSMV. MCPI responded to CPSMV by accumulating proteins involved in the oxidative burst, increasing H2O2 generation, promoting leaf cell death (LCD), increasing the synthesis of defense proteins, and decreasing host factors important for the establishment of CPSMV infection. In contrast, CPI accumulated several host factors that favor CPSMV infection and did not accumulate H2O2 or present LCD, which allowed CPSMV replication and systemic dissemination. Based on these results, we propose that the differential abundance of defense proteins and proteins involved in the oxidative burst, LCD, and the decrease in cowpea protein factors required for CPSMV replication are associated with the resistance trait acquired by the MCPI plant group.

Retention of induced mutations in a Drosophila reverse-genetic resource.

Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.

Conjugal transformation and transposon and chemical mutagenesis of gram-negative selenate-respiring Citrobacter sp. strain JSA.

Conjugal mating between selenate-reducing Citrobacter sp. strain JSA and Escherichia coli S17-1 bearing pSUP2021 allowed transposon mutagenesis and chromosomal transformation. Kanamycin-resistant transconjugants were obtained successfully by this method from a freshwater selenate-respiring Citrobacter sp. strain JSA. The maximum frequency of kanamycin-resistant Tn5 transconjugants was 3.6 x 10(-6) per recipient of this strain. Of these transconjugants, eight strains of selenate reduction-deficient transconjugants living by nitrate reduction were obtained in the strain JSA. Moreover, the same phenotype of deficient mutant was created by chemical mutagenesis with ethylmethanesulfonate. The results strongly indicate that selenate reducing anaerobic respiration was independent of nitrate reduction in the Citrobacter sp. isolate strain JSA.

Drug substances presented as sulfonic acid salts: overview of utility, safety and regulation.

OBJECTIVES: Controlling genotoxic impurities represents a significant challenge to both industry and regulators. The potential for formation of genotoxic short-chain alkyl esters of sulfonic acids during synthesis of sulfonic acid salts is a long-standing regulatory concern. This review provides a general overview of the utility of sulfonic acids as salt-forming moieties and discusses strategies for effectively minimizing the potential for alkyl sulfonate formation during the synthesis and processing of sulfonate salt active pharmaceutical ingredients. The potential implications of the recent establishment of a substantial human threshold dose for ethyl methanesulfonate for the safety assessment of alkyl sulfonates in general are also discussed. KEY FINDINGS: The formation of alkyl sulfonates requires highly acidic conditions, possibly combined with long reaction times and/or elevated temperatures, to generate significant amounts, and these conditions are most unlikely to be present in the synthesis of active pharmaceutical ingredient sulfonate salts. It is possible to design salt formation conditions, using a short-chain alcohol as solvent, to manufacture sulfonate salts that are essentially free of alkyl sulfonate impurities. Processes using non-acidic conditions such as ethanol recrystallization or wet granulation should not raise any concerns of alkyl sulfonate formation. SUMMARY: An understanding of the mechanism of formation of alkyl sulfonates is critical in order to avoid restricting or over-controlling sulfonic acid salts, which have many technical advantages as pharmaceutical counterions. Recent regulatory acceptance of a human threshold limit dose of 2 mg/kg per day for ethyl methanesulfonate, indicating that its toxicological risks have previously been considerably overestimated, could signal the beginning of the end over safety concerns on alkyl sulfonate residues, thus removing a major constraint from the exploitation of sulfonic acid counterions.

Ethyl methanesulphonate in a parenteral formulation of BMS-214662 mesylate, a selective farnesyltransferase inhibitor: formation and rate of hydrolysis.

The objectives of the present study were to investigate the formation and rate of hydrolysis of ethyl methanesulphonate (EMS) in BMS-214662 mesylate drug substance and parenteral formulation by a gas chromatographic/mass spectrometric (GC/MS) method. EMS levels in the drug substance ranged between 0.3 microg/g and 0.8 microg/g. The parenteral formulation contains ethanol and the reaction between residual free methane sulphonic acid and ethanol may lead to the formation of EMS. Given that EMS is a potent mutagen, it is therefore of vital importance to eliminate or reduce the risk of human exposure. Data indicate no significant increase in the levels of EMS following storage of the drug product for 18 weeks at 25 degrees C or six weeks at 60 degrees C indicating that the potential reaction between ethanol and free methane sulphonic acid may not occur in the BMS-214662 formulation under the storage conditions evaluated and therefore causes no plausible safety concerns of EMS exposure in humans. Kinetic studies were conducted by spiking 200 ppb of EMS into water and the diluted and undiluted parenteral formulation. The rates of hydrolysis of EMS at 25 degrees C followed pseudo-first order kinetics and were determined to be 2.35 x 10(-4)min(-1), 67.4 x 10(-4)min(-1), and 1.32 x 10(-4)min(-1) in water, undiluted, and diluted drug product, respectively.

Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method.

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. We have developed a method to map systematically all the residues forming the surface of this binding-site crevice, and we have applied this method to the third membrane-spanning segment (M3). We mutated, one at a time, 23 residues in and flanking M3 to cysteine and expressed the mutant receptors heterologously. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist. Thus, the side chains of these residues are exposed in the binding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consistent with a short loop followed by six turns of an alpha helix.

Control of inward rectifier K channel activity by lipid tethering of cytoplasmic domains.

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called "slide-helix" is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET(+) or MTSEA(+), but not MTSES(-) or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.

Role of DNA mismatch repair and double-strand break repair in genome stability and antifungal drug resistance in Candida albicans.

Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Delta/mre11Delta, rad50Delta/rad50Delta, and rad52Delta/rad52Delta mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Delta/msh2Delta, pms1Delta/pms1Delta, and yku80Delta/yku80Delta mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Delta/mre11Delta and rad50Delta/rad50Delta mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Delta/mre11Delta, rad50Delta/rad50Delta, or rad52Delta/rad52Delta leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Delta/msh2Delta and pms1Delta/pms1Delta (MMR mutants) and rad50Delta/rad50Delta (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

Development and validation of an automated static headspace gas chromatography-mass spectrometry (SHS-GC-MS) method for monitoring the formation of ethyl methane sulfonate from ethanol and methane sulfonic acid.

An automated sample preparation and analysis procedure was developed to monitor the formation of ethyl methane sulfonate from reaction mixtures containing ethanol and methane sulfonic acid. The system is based on a liquid handling robot combined with a static headspace module. The formed ethyl methane sulfonate is analysed after derivatisation with pentafluorothiophenol using static headspace-gas chromatography-mass spectrometry (SHS-GC-MS). Using the automated reaction-derivatisation-headspace GC-MS system, the formation of ethyl methane sulfonate can be monitored in different reaction mixtures under different reaction conditions, including temperature, water content and pH. Excellent linearity, repeatability and robustness were obtained, allowing the system to be used in kinetic studies.