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Acta Crystallographica. Section F,... Jun 2019Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of...
Chagas disease, which is caused by Trypanosoma cruzi, affects more than six million people worldwide. Cruzain is the major cysteine protease involved in the survival of this parasite. Here, the expression, purification and crystallization of this enzyme are reported. The cruzain crystals diffracted to 1.2 Å resolution, yielding two novel cruzain structures: apocruzain and cruzain bound to the reversible covalent inhibitor S-methyl thiomethanesulfonate. Mass-spectrometric experiments confirmed the presence of a methylthiol group attached to the catalytic cysteine. Comparison of these structures with previously published structures indicates the rigidity of the cruzain structure. These results provide further structural information about the enzyme and may help in new in silico studies to identify or optimize novel prototypes of cruzain inhibitors.
Topics: Apoproteins; Crystallography, X-Ray; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Drug Design; Methyl Methanesulfonate; Models, Molecular; Protein Conformation; Protozoan Proteins
PubMed: 31204688
DOI: 10.1107/S2053230X19006320 -
Mutation Research Oct 1981Human lymphocytes were treated in G1 with 4-nitroquinoline-N-oxide (4NQO) and methyl methanesulfonate (MMS) and then incubated in the presence or absence of cytosine...
Human lymphocytes were treated in G1 with 4-nitroquinoline-N-oxide (4NQO) and methyl methanesulfonate (MMS) and then incubated in the presence or absence of cytosine arabinoside (ara-C). There was an increase in aberration frequency in those cells incubated with ara-C compared with those treated with 4NQO or MMS alone. This increase was restricted to chromosome-type aberrations. When cells were treated in G2 with 4NQO and then incubated with ara-C until fixation, there was an increase in deletions compared with cells treated with 4NQO alone. No exchange aberrations were observed following any treatment even when deletion frequencies were high, as in the case with 4NQO plus ara-C treatment. These results suggest that ara-C can inhibit the repair of DNA damage induced by 4NQO and MMS that is converted into aberrations. They also show that the terms "S-dependent' and "S-independent' used to describe the modes of action of chemical clastogens are not valid.
Topics: 4-Nitroquinoline-1-oxide; Cells, Cultured; Chromosome Aberrations; Chromosomes; Cytarabine; Drug Therapy, Combination; Humans; Interphase; Lymphocytes; Methyl Methanesulfonate; Mutagens
PubMed: 6799812
DOI: 10.1016/0027-5107(81)90021-x -
Mutation Research. Genetic Toxicology... Mar 2014The search for substances able to inhibit and/or diminish the effects of genotoxic and mutagenic substances has been the target of several investigations performed in...
The search for substances able to inhibit and/or diminish the effects of genotoxic and mutagenic substances has been the target of several investigations performed in recent times. Hymenoptera venoms constitute a considerable source of substances with pharmacological potential. The present study aimed to evaluate the cytotoxic, genotoxic and anti-genotoxic, mutagenic and anti-mutagenic potentials of Apis mellifera venom in HepG2 cells. In this evaluation, the MTT test was applied to determine the most appropriate concentrations for the genotoxicity and mutagenicity tests. It was verified that the concentrations of 0.1, 0.05 and 0.01μg/mL were not cytotoxic, hence these concentrations were used in the experiments. For the evaluation of the genotoxic and mutagenic potential of the bee venom the comet assay and the micronucleus test were applied, respectively. The concentrations mentioned above presented both genotoxic and mutagenic potential for HepG2 cells and it was necessary to test lower concentrations of the venom (10pg/mL, 1pg/mL and 0.1pg/mL) for the anti-genotoxicity and anti-mutagenicity tests, which were performed subjecting the cells to the action of MMS (methyl methanesulfonate) in order to verify the ability of the venom to inhibit or diminish the action of this compound, which has a recognized action on the genetic material. Pre-, post-treatment and simultaneous treatment with and without incubation with the venom were performed. It was observed that the lowest three concentrations tested did not present any anti-genotoxic and anti-mutagenic activity on the cells. The use of bee venom for pharmacological purposes in treatments such as cancer must be done with extreme caution, since it was observed that even at very low concentrations the venom can induce genotoxicity and mutagenicity in human cells, as was verified for the HepG2 cells.
Topics: Animals; Antimutagenic Agents; Bee Venoms; Bees; Cell Survival; DNA Damage; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Methyl Methanesulfonate; Micronuclei, Chromosome-Defective; Mutagenicity Tests; Mutagens
PubMed: 24525379
DOI: 10.1016/j.mrgentox.2013.11.005 -
Biochimica Et Biophysica Acta. Proteins... Nov 2019S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of...
S-Methyl methanethiosulfonate (MMTS) is used in experimental biochemistry for alkylating thiol groups of protein cysteines. Its applications include mainly trapping of natural thiol-disulfide states of redox-sensitive proteins and proteins which have undergone S-nitrosylation. The reagent can also be employed as an inhibitor of enzymatic activity, since nucleophilic cysteine thiolates are commonly present at active sites of various enzymes. The advantage of using MMTS for this purpose is the reversibility of the formation of methylthio mixed disulfides, compared to irreversible alkylation using conventional agents. Additional benefits include good accessibility of MMTS to buried protein cysteines due to its small size and the simplicity of the protection and deprotection procedures. In this study we report examples of MMTS application in experiments involving oxidoreductase (glyceraldehyde-3-phosphate dehydrogenase, GAPDH), redox-regulated protein (recoverin) and cysteine protease (triticain-α). We demonstrate that on the one hand MMTS can modify functional cysteines in the thiol enzyme GAPDH, thereby preventing thiol oxidation and reversibly inhibiting the enzyme, while on the other hand it can protect the redox-sensitive thiol group of recoverin from oxidation and such modification produces no impact on the activity of the protein. Furthermore, using the example of the papain-like enzyme triticain-α, we report a novel application of MMTS as a protector of the primary structure of active cysteine protease during long-term purification and refolding procedures. Based on the data, we propose new lines of MMTS employment in research, pharmaceuticals and biotechnology for reversible switching off of undesirable activity and antioxidant protection of proteins with functional thiol groups.
Topics: Animals; Cysteine Proteases; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Methyl Methanesulfonate; Oxidation-Reduction; Plant Proteins; Rabbits; Recoverin; Sulfhydryl Compounds; Triticum
PubMed: 31376523
DOI: 10.1016/j.bbapap.2019.07.012 -
Mutation Research Jul 1979CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In... (Comparative Study)
Comparative Study
CHO cells were synchronized in G1 phase and treated with MMS or HN2. The subsequent rate of DNA replication was found to be reduced in a dose-dependent manner. In addition, 2 X 10(-3 M and 3 X 10(-3) M MMS resulted in a 3--4 h delay prior to the initiation of S phase. If the cells were held for 8 h in hydroxyurea after MMS treatment, no subsequent lag in DNA synthesis was seen after removal of the hydroxyurea. The entry of confluent cells into S phase was found to be delayed 7 h upon trypsinizing and replating. Treatment of these cells with MMS resulted in a reduced rate of DNA replication, but no further delay in its initiation. Repair replication was found to continue at a constant rate for at least 12 h following MMS treatment of cells under all of these conditions. At the concentrations used in these experiments MMS severely inhibited the rate of protein synthesis, but HN2 had little effect. By comparing both the kinetics of repair replication and recovery of protein synthesis with the rate of DNA replication, it was concluded that the initial, severe reduction in rate following MMS treatment was probably due to an inhibition of protein synthesis.
Topics: Animals; Cell Line; Cricetinae; Cricetulus; DNA; DNA Repair; DNA Replication; Female; Methyl Methanesulfonate; Nitrogen Mustard Compounds; Ovary
PubMed: 481440
DOI: 10.1016/0027-5107(79)90138-6 -
Mutation Research Jul 1999We have developed an in vivo micronucleus (MN) test that uses rat skin as the target organ. Sample preparation involves cold-treating the epidermis with trypsin, peeling...
We have developed an in vivo micronucleus (MN) test that uses rat skin as the target organ. Sample preparation involves cold-treating the epidermis with trypsin, peeling it off with a fine forceps, treating it in hypotonic solution, and staining it with acridine orange (A.O.). We evaluated the assay using mitomycin C (MMC) and methyl methanesulfonate (MMS) as model clastogens, applying them as single and repeat treatments. Both chemicals induced a significant, dose-dependent increase in MN frequency in basal cells. One treatment per day for 3 days was optimal for MN induction.
Topics: Acridine Orange; Animals; Dose-Response Relationship, Drug; Fluorescent Dyes; Male; Methyl Methanesulfonate; Micronucleus Tests; Mitomycin; Models, Biological; Mutagens; Rats; Rats, Sprague-Dawley; Skin
PubMed: 10477350
DOI: 10.1016/s1383-5718(99)00084-4 -
Mutation Research Jul 1981Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2...
Unscheduled DNA synthesis and excision of pyrimidine dimers in human cells exposed to ultraviolet let were inhibited by exposure to methyl methanesulfonate (MMS, 1-2 mM), but repair of MMS damage was not inhibited by UV light. Because the pathways for excision of pyrimidine dimers and alkylation damage have previously been shown to be different, this observation implies a direct effect of alkylation on repair enzymes. We estimate that if inhibition is due to protein alkylation, the UV repair system must present an extremely large target to alkylation and may involve a complex of protein subunits in the order of 1 million daltons such that 1 or more alkylations occur per complex at the concentrations used. These results also indicate that the method of exposing cells to 2 DNA-damaging agents to determine whether they are repaired by common or different pathways can be quite unreliable because of other effects on the repair systems themselves.
Topics: Animals; Cell Line; Cricetinae; Cricetulus; DNA; DNA Repair; DNA Replication; Depression, Chemical; Female; Fibroblasts; Humans; Methyl Methanesulfonate; Ovary; Ultraviolet Rays; Xeroderma Pigmentosum
PubMed: 7196494
DOI: 10.1016/0027-5107(81)90165-2 -
Journal of Biomolecular Structure &... Oct 2022Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple...
Biophysical characterization of structural and conformational changes in methylmethane sulfonate modified DNA leading to the frizzled backbone structure and strand breaks in DNA.
Methyl methanesulfonate (MMS) is a highly toxic DNA-alkylating agent that has a potential to damage the structural integrity of DNA. This work employed multiple biophysical and computational methods to report the MMS mediated structural alterations in the DNA (MMS-DNA). Spectroscopic techniques and gel electrophoresis studies revealed MMS induced exposure of chromophoric groups of DNA; methylation mediated anti→syn conformational change, DNA fragmentation and reduced nucleic acid stability. MMS induced single-stranded regions in the DNA were observed in nuclease S1 assay. FT-IR results indicated MMS mediated loss of the assigned peaks for DNA, partial loss of C-O ribose, loss of deoxyribose region, C-O stretching and bending of the C-OH groups of hexose sugar, a progressive shift in the assigned guanine and adenine peaks, loss of thymine peak, base stacking and presence of C-O-H vibrations of glucose and fructose, indicating direct strand breaks in DNA due to backbone loss. Isothermal titration calorimetry showed MMS-DNA interaction as exothermic with moderate affinity. Dynamic light scattering studies pointed towards methylation followed by the generation of single-stranded regions. Electron microscopy pictured the loss of alignment in parallel base pairs and showed the formation of fibrous aggregates in MMS-DNA. Molecular docking found MMS in close contact with the ribose sugar of DNA backbone having non-bonded interactions. Molecular dynamic simulations confirmed that MMS is capable of interacting with DNA at two levels, one at the level of nitrogenous bases and another at the DNA backbone. The study offers insights into the molecular interaction of MMS and DNA.Communicated by Ramaswamy H. Sarma.
Topics: DNA; DNA Damage; DNA Repair; Methyl Methanesulfonate; Molecular Docking Simulation; Ribose; Spectroscopy, Fourier Transform Infrared
PubMed: 33719845
DOI: 10.1080/07391102.2021.1899051 -
Molecular & General Genetics : MGG Nov 1976Twenty-eight X-linked, recessive mutations of Drosophila melanogaster conferring enhanced sensitivity to the monofunctional alkylating agent, methyl methanesulfonate,...
Twenty-eight X-linked, recessive mutations of Drosophila melanogaster conferring enhanced sensitivity to the monofunctional alkylating agent, methyl methanesulfonate, have been recoered and assigned to five complementation groups. These groups can be distinguished on the basis of map location and variations in the pattern of mutagen sensitivity. Allelism of members of one complementation group with the previously described meiotic mutant, mei-41, (Baker and Carpenter, 1972) as well as the frequent appearance of female infertility with mutagen sensitivity suggests associated defects in meiotic chromosome behavior or early embryogenesis. Examination of the mutagen sensitivity of double mutants has led to the formulation of a working model of DNA repair for this organism. Studies of a similar nature (Boyd et al., 1976) have identified five additional X chromosome complementation groups, suggesting that the genome of Drosophila melanogaster may contain many loci involved with mutagen sensitivity. The continued isolation and characterization of conditional mutants of this type promises future insights into the mechanisms of DNA replication, DNA repair and recombination in this complex higher eucaryote.
Topics: Animals; DNA Repair; Drosophila melanogaster; Drug Resistance; Female; Genes, Recessive; Genetic Complementation Test; Genetic Linkage; Infertility; Male; Mesylates; Methyl Methanesulfonate; Mutation; Sex Chromosomes
PubMed: 189178
DOI: 10.1007/BF00275962 -
Mutation Research Sep 1979Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative...
Unscheduled DNA synthesis (UDS) in the germ cells of male mice after in vivo treatment with X-rays or methyl methanesulfonate (MMS) was assayed by use of a quantitative autoradiographic procedure. MMS induced UDS in meiotic through type III elongating spermatid stages, whereas X-rays induced UDS in meiotic through round spermatid stages. No UDS was detected in the most mature spermatid stages present in the testis with either MMS or X-rays. Taking into account differences in DNA content of the various germ-cell stages studied, we concluded that X-rays induced a maximum UDS response in spermatocytes at diakinesis--metaphase I. The level of UDS induced by MMS was about the same in all the stages capable of repair. Chromosome damage and UDS were measured simultaneously in the same spermatocytes at diakinesis 90 min after X-irradiation or MMS treatment. The level of UDS in most of the X-irradiated cells paralleled the extent of chromosome damage induced. A statistical analysis of these results revealed a positive correlation. As expected, MMS induced no chromosome aberrations above control levels. Therefore no correlation was determined between UDS and chromosome damage in this case. The distribution of UDS over the chromosomes treated at diakinesis with MMS or X-rays was studied. It was found that UDS occurred in clusters in the irradiated cells, whereas it was uniformly distributed in the MMS-treated cells.
Topics: Animals; Autoradiography; Chromosome Aberrations; Chromosomes; DNA Replication; Male; Methyl Methanesulfonate; Mice; Spermatozoa; X-Rays
PubMed: 503097
DOI: 10.1016/0027-5107(79)90086-1