msrA Gene Research Articles

P84 Identification of erm and msrA genes in inducible clindamycin resistance of clinical isolates of Staphylococcus aureus by PCR and and D-test

P84 Identification of erm and msrA genes in inducible clindamycin resistance of clinical isolates of Staphylococcus aureus by PCR and and D-test

Molecular cloning and characterisation of the methionine sulphoxide reductase A (msrA) gene locus in Campylobacter lari organisms

The methionine sulphoxide reductase A (msrA) gene and its adjacent genetic loci from urease-negative (UN) Campylobacter lari RM2100 and urease-positive thermophilic Campylobacter (UPTC) CF89–12 strains appear to be composed of a msrA structure gene (507 base pairs [bp]) and another five-gene cluster (approximately 6300 bp) in the same strand and direction. A primer pair (F1/R4-msrA) for polymerase chain reaction (PCR) amplification was designed to generate a product of approximately 900 bp of the msrA gene, including its adjacent genetic loci for the thermophilic Campylobacter organisms and generate an amplicon with 16 C. lari isolates (n=4 for UN C. lari; n=12 for UPTC). Following direct nucleotide sequencing, sequence analysis and nucleotide sequence alignment analysis, the putative full-length msrA gene from the 16 C. lari isolates showed high nucleotide sequence similarities (91.8–100%) to each other and relatively low similarity (69.3–71.8%) to three reference C. jejuni and C. coli strains. In addition, the msrA gene was transcribed in both the UPTC CF89–12 and NCTC12893 cells using reverse transcription PCR. An immunoreactively positive signal was identified in the UPTC CF89–12 and NCTC12893 cells with anti-UPTC MsrA synthetic peptide antibodies.

A Genome-Wide Association Study Provides Evidence for Association of Chromosome 8p23 (MYP10) and 10q21.1 (MYP15) with High Myopia in the French Population

Myopia is a complex trait affected by both genetic and environmental factors. High myopia is associated with increased risk of sight threatening eye disorders such as retinal detachment. The purpose of this genome-wide association study was to identify susceptibility genes contributing to high myopia in the French population. High myopic cases were genotyped using Affymetrix SNP 6.0 chips and population controls were selected from the GABRIEL French dataset, in which samples were genotyped by Illumina Human610 quad array. The association study was conducted using 152,234 single nucleotide polymorphisms that were present on both manufacturers' chips in 192 high myopic cases and 1064 controls to identify associated regions. Imputation was performed on peak regions. Associations were found at known myopia locus MYP10 on chromosome 8p23 and MYP15 on chromosome 10q21.1. Rs189798 (8p23), and rs10825992 (10q21.1) showed the strongest associations in these regions (P = 6.32 × 10(-7) and P = 2.17 × 10(-5), respectively). The imputed results at 8p23 showed two peaks of interest. The first spanned 30 kb including rs189798 between MIR4660 and PPP1R3B with the most significant association at rs17155227 (P = 1.07 × 10(-10)). The second novel peak was 4 kb in length, encompassing MIR124-1 and the MSRA gene, with the strongest association at rs55864141 (P = 1.30 × 10(-7)). The peak of imputed data at 10q21.1 was 70 kb in length between ZWINT and MIR3924, with rs3107503 having the lowest P value (P = 1.54 × 10(-7)). We provide evidence for the association of MYP10 at 8p23 and MYP15 at 10p21.1 with high myopia in the French population and refine these regions of association.

Caracterização fenotípica, produção de biofilme e resistência aos antimicrobianos em isolados de Staphylococcus spp. obtidos de casos de mastite em bovinos e bubalinos

No presente estudo, objetivou-se avaliar a sussuscetibilidade aos principais antimicrobianos e realizar uma caracterização fenotípica e genotípica de isolados de Staphylococcus spp. obtidos de casos de mastite em vacas (n=30) e búfalas (n=30). A suscetibilidade foi avaliada pela técnica de disco-difusão e a presença de bomba de efluxo foi avaliada utilizando-se Ágar Mueller Hinton (MH) adicionado de brometo de etídeo e pesquisa do gene msrA. Pela técnica da Reação em Cadeia da Polimerase (PCR) ainda foram identificados os genes mecA, blaZ e ermA, B e C, que posteriormente foram associados com os métodos fenotípicos para a identificação de resistência a antimicrobianos. A caracterização da formação de biofilme foi realizada utilizandose os métodos Ágar Vermelho Congo (CRA), Aderência em Placa e a identificação do gene icaD. Pelo método de discodifusão, os Staphylococcus spp. apresentaram alta sensibilidade aos antimicrobianos. O índice de resistência múltipla aos antimicrobianos (IRMA) apresentou variação de 0 a 0,5. Na pesquisa de bomba de efluxo, 26,7% das amostras foram positivas ao método fenotípico e 6,7% ao método genotípico (gene msrA). Os genes erm, mecA e blaZ foram detectados, respectivamente, em 1,7%, 6,7% e 11,7% das amostras de Staphylococcus spp. Na produção de biofilme, 23,3% dos isolados foram considerados positivos no CRA, 50,0% na Aderência em Placas e 8,3% na PCR pela detecção do gene icaD. Observou-se que os isolados obtidos de amostras bovinas apresentaram uma menor sensibilidade aos antimicrobianos no teste de disco-difusão quando comparados com as amostras bubalinas. A caracterização destes isolados é importante para orientar uma antibioticoterapia bem planejada. A presença de biofilme nos isolados pode estar associada a outros fatores que não a resistência às drogas antimicrobianas.

Treatment factors rather than genetic variation determine metabolic syndrome in childhood cancer survivors

BackgroundGenetic variation that regulates insulin resistance, blood pressure and adiposity in the normal population might determine differential vulnerability for metabolic syndrome after treatment for childhood cancer. ObjectiveTo evaluate the contribution of candidate single nucleotide polymorphisms (SNPs) relevant for metabolic syndrome in our single centre cohort of adult long-term childhood cancer survivors. MethodsIn this retrospective study 532 survivors were analysed. Median age at diagnosis was 5.7years (range 0.0–17.8years), median follow-up time was 17.9years (range 5.0–48.8) and median age at follow-up was 25.6years (range 18.0–50.8). JAZF1 gene rs864745, THADA gene rs7578597, IRS1 gene rs2943641, TFAP2B gene rs987237, MSRA gene rs7826222, ATP2B1 gene rs2681472 and rs2681492 were genotyped. The association of genotypes with total cholesterol levels, blood pressure, body mass index, waist circumference and frequency of diabetes were assessed. ResultsMetabolic syndrome was more frequent in cranially (23.3%, P=0.002) and abdominally (23.4%, P=0.009) irradiated survivors as compared with non-irradiated survivors (10.0%). Association of allelic variants in rs2681472 and rs2681492 with hypertension, rs987237 and rs7826222 with waist circumference and rs864745, rs7578597 and rs2943641 with diabetes were not significant. None of the SNPs was associated with the metabolic syndrome. Adjusting for age, sex, follow-up time, cranial irradiation and abdominal irradiation did not change these results. ConclusionsTreatment factors and not genetic variation determine hypertension, waist circumference, diabetes and metabolic syndrome in adult long-term survivors of childhood cancer.

Protective effects of taurine against oxidative stress in the heart of MsrA knockout mice

Taurine has been shown to have potent anti-oxidant properties under various pathophysiological conditions. We reported previously a cellular dysfunction and mitochondrial damage in cardiac myocytes of methionine sulfoxide reductase A (MsrA) gene knockout mice (MsrA(-/-)). In the present study, we have explored the protective effects of taurine against oxidative stress in the heart of MsrA(-/-) mice with or without taurine treatment. Cardiac cell contractility and Ca(2+) dynamics were measured using cell-based assays and in vivo cardiac function was monitored using high-resolution echocardiography in the tested animals. Our data have shown that MsrA(-/-) mice exhibited a progressive cardiac dysfunction with a significant decrease of ejection fraction (EF) and fraction shortening (FS) at age of 8 months compared to the wild type controls at the same age. However, the dysfunction was corrected in MsrA(-/-) mice treated with taurine supplement in the diet for 5 months. We further investigated the cellular mechanism underlying the protective effect of taurine in the heart. Our data indicated that cardiac myocytes from MsrA(-/-) mice treated with taurine exhibited an improved cell contraction and could tolerate oxidative stress better. Furthermore, taurine treatment reduced significantly the protein oxidation levels in mitochondria of MsrA(-/-) hearts, suggesting an anti-oxidant effect of taurine in cardiac mitochondria. Our study demonstrates that long-term treatment of taurine as a diet supplement is beneficial to a heart that is vulnerable to environmental oxidative stresses.

Antimicrobial resistance of Staphylococcus spp. from small ruminant mastitis in Brazil

The study aimed to determine the antimicrobial resistance patterns and to identify molecular resistance markers in Staphylococcus spp. (n=210) isolated from small ruminant mastitis in Brazil. The antimicrobial resistance patterns were evaluated by the disk diffusion test and by detection of the presence of mecA, blaZ, ermA, ermB, ermC and msrA genes by PCR. The efflux pump test was performed using ethidium bromide and biofilm production was determined by Congo red agar test along with PCR for detection of the icaD gene. The isolates were most resistant to amoxicillin (50.0%), streptomycin (42.8%), tetracycline (40.4%), lincomycin (39.0%) and erythromycin (33.8%). Pan-susceptibility to all tested drugs was observed in 71 (33.8%) isolates and 41 Staphylococcus isolates were positive for the efflux pump. Although phenotypic resistance to oxacillin was observed in 12.8% of the isolates, none harbored the mecA gene. However, 45.7% of the isolates harbored blaZ indicating that beta-lactamase production was the main mechanism associated with staphylococci resistance to beta-lactams in the present study. The other determinants of resistance to antimicrobial agents ermA, ermB, ermC, and msrA were observed in 1.4%, 10.4%, 16.2%, and 0.9% of the isolates, respectively. In addition, the icaD gen was detected in 32.9% of the isolates. Seventy three isolates (54 from goats and 19 from sheep) were negative for all resistance genes tested and 69 isolates presented two or more resistance genes. Association among blaZ, ermA, ermB, ermC and efflux pump were observed in 17 isolates, 14 of which originated from goats and three from sheep. The data obtained in this study show the resistance of the isolates to beta-lactamics, which may be associated with the use of antimicrobial drugs without veterinary control.

Isolation and identification of methicillin-resistant Staphylococcus aureus from students’ coins

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen of increasing risk on community. This study aims at determining the risk of MRSA transfer from coins as silent and underestimated reservoir in community. One hundred swabs from coins were collected from college students in Malaysia. A series of identification and differentiating tests were conducted for precise identification of MRSA bacteria. Moreover, this study compared the efficacy of the different identification tests with gold standard, polymerase chain reaction (PCR) assay. The tests used were tube coagulase, DNase agar test, antibiogram, several routine biochemical identification tests and PCR assays. PCR assay used specific primers for resistance or ID -related genes: mecA, ermA, ermB, ermC, msrA, linA, femA, and nuc genes. A total of 37 bacterial isolates were isolated from college students’ coins; non-PCR assays of identification and resistance detection revealed the presence and spread of MRSA in coins of 2 college students. PCR-amplification of the nuc gene was used as a baseline test to detect S. aureus. PCR showed only one isolate as true MRSA. False positive MRSA by disc diffusion assay might be attributed to low pH and high thymidine content of Muller Hinton agar medium. Collectively, coins proved to be possible source for the transfer of MRSA in community of college students in South East Asia. Moreover, PCR assay for identification of S. aureus resistance proved to be superior on other methods. Key words : Staphylococcus aureus , MRSA, nail, PCR, resistance.

Susceptibility Variants for Waist Size in Relation to Abdominal, Visceral, and Hepatic Adiposity in Postmenopausal Women

Genome-wide association studies have identified common genetic variants that can contribute specifically to the risk of abdominal adiposity, as measured by waist circumference or waist-to-hip ratio. However, it is unknown whether these genetic risk factors affect relative body fat distribution in the abdominal visceral and subcutaneous compartments. The association between imaging-based abdominal fat mass and waist-size risk variants in the FTO, LEPR, LYPLAL1, MSRA, NRXN3, and TFAP2B genes was investigated. A cross-sectional sample of 60 women was selected among study participants of The Multiethnic Cohort, who were aged 60 to 65 years, of European or Japanese descent, and with a body mass index (calculated as kg/m2) between 18.5 and 40. Dual-energy x-ray absorptiometry and abdominal magnetic resonance imaging scans were used to measure adiposity. After adjustments for age, ethnicity, and total fat mass, the FTO variants showed an association with less abdominal subcutaneous fat and a higher visceral-to-subcutaneous abdominal fat ratio, with the variant rs9941349 showing significant associations most consistently (P=0.003 and 0.03, respectively). Similarly, the LEPR rs1137101 variant was associated with less subcutaneous fat (P=0.01) and a greater visceral-to-subcutaneous fat ratio (P=0.03) and percent liver fat (P=0.007). MSRA rs545854 variant carriers had a lower percent of leg fat. Our findings provide initial evidence that some of the genetic risk factors identified for larger waist size might also contribute to disproportionately greater intra-abdominal and liver fat distribution in postmenopausal women. If replicated, these genetic variants can be incorporated with other biomarkers to predict high-risk body fat distribution.

Presence of erythromycin and tetracycline resistance genes in lactic acid bacteria from fermented foods of Indian origin

Lactic acid bacteria (LAB) resistant to erythromycin were isolated from different food samples on selective media. The isolates were identified as Enterococcus durans, Enterococcus faecium, Enterococcus lactis, Enterococcus casseliflavus, Lactobacillus salivarius, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus and Leuconostoc mesenteroides. Of the total 60 isolates, 88% harbored the ermB gene. The efflux gene msrA was identified in E. faecium, E. durans, E. lactis, E. casseliflavus, P. pentosaceus and L. fermentum. Further analysis of the msrA gene by sequencing suggested its homology to msrC. Resistance to tetracycline due to the genes tetM, tetW, tetO, tetK and tetL, alone or in combination, were identified in Lactobacillus species. The tetracycline efflux genes tetK and tetL occurred in P. pentosaceus and Enterococcus species. Since it appeared that LAB had acquired these genes, fermented foods may be a source of antibiotic resistance.

ErmA, ermC, tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia

The objective of this study was to determine the expression and transferability of tetracycline and erythromycin resistance among 188 MRSA strains from a Malaysian tertiary hospital. The minimum inhibitory concentrations (MICs) for oxacillin, erythromycin, tetracycline and ciprofloxacin ranged from 4 to 512 μg/ml, 0.25 to 256 μg/ml, 0.5 to 256 μg/ml and 0.5 to 512 μg/ml, respectively. Tetracycline-resistant strains showed co-resistance towards ciprofloxacin and erythromycin. There was a significant increase (P<0.05) of high-level tetracycline (≥MIC 256 μg/ml) and erythromycin (≥MIC 128 μg/ml) resistant strains in between the years 2003 and 2008. All erythromycin-resistant strains harboured ermA or ermC gene and all tetracycline-resistant strains harboured tetM or tetK gene. The blaZ was detected in all MRSA strains, whereas ermA, tetM, ermC, tetK and msrA genes were detected in 157 (84%), 92 (49%), 40 (21%), 39 (21%) and 4 (2%) MRSA strains, respectively. The blaZ, tetM, ermC and tetK genes were plasmid-encoded, with ermC gene being easily transmissible. Tn5801-like transposon was present in 78 tetM-positive strains. ermA and tetM genes were the most prevalent erythromycin and tetracycline resistance determinants, respectively, in MRSA strains. The association of resistance genes with mobile genetic elements possibly enhances the spread of resistant traits in MRSA.

Variation in MSRA Modifies Risk of Neonatal Intestinal Obstruction in Cystic Fibrosis

Meconium ileus (MI), a life-threatening intestinal obstruction due to meconium with abnormal protein content, occurs in approximately 15 percent of neonates with cystic fibrosis (CF). Analysis of twins with CF demonstrates that MI is a highly heritable trait, indicating that genetic modifiers are largely responsible for this complication. Here, we performed regional family-based association analysis of a locus that had previously been linked to MI and found that SNP haplotypes 5′ to and within the MSRA gene were associated with MI (P = 1.99×10−5 to 1.08×10−6; Bonferroni P = 0.057 to 3.1×10−3). The haplotype with the lowest P value showed association with MI in an independent sample of 1,335 unrelated CF patients (OR = 0.72, 95% CI [0.53–0.98], P = 0.04). Intestinal obstruction at the time of weaning was decreased in CF mice with Msra null alleles compared to those with wild-type Msra resulting in significant improvement in survival (P = 1.2×10−4). Similar levels of goblet cell hyperplasia were observed in the ilea of the Cftr −/− and Cftr −/− Msra −/− mice. Modulation of MSRA, an antioxidant shown to preserve the activity of enzymes, may influence proteolysis in the developing intestine of the CF fetus, thereby altering the incidence of obstruction in the newborn period. Identification of MSRA as a modifier of MI provides new insight into the biologic mechanism of neonatal intestinal obstruction caused by loss of CFTR function.

Isolation and Identification of Methicillin-Resistant Staphylococcus aureus from Keys of College Students Using Different Detection Methods

Aims: In this study, the methicillin-resistant Staphylococcus aureus (MRSA) were isolated and identified by using biochemical tests, antibiogram and polymerase chain reaction (PCR) to explore the circulation of MRSA among college students. Study design: Cross-sectional study. Place and Duration of Study: Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Research Article British Biotechnology Journal, 2(1): 13-25, 2012 14 between June 2010 and December 2010. Methodology: A total of 100 samples were collected from keys of college students. There were 39 isolates (39 %) Gram-positive cocci and Catalase positive. 29 (74.36%) were glucose oxidation and fermentation positive. From the 39 isolates, 16 (43.24%) were shown Mannitol Salt Agar (MSA) tests positive. The deoxyribonuclease (DNase) tests and tube coagulase tests with human and rabbit plasma were carried out to improve the efficiency of the MSA test. Results: 7 (43.75%) DNase positive and 2 (12.5%) tube coagulase positive. Both human and rabbit plasma showed similar sensitivity for the tube coagulase tests in this study. However, both isolates with tube coagulase positive were confirmed as S. aureus but not resistant to oxacillin, methicillin, erythromycin and cefoxitin. 2 (66.67%) of 3 (18.75%) isolates which is tube coagulase negative were resistant to erythromycin and 1 (33.33%) of them was resistant to methicillin. Rare strains of S. aureus can be coagulase negative. PCR assay was used. 1 (33.33%) of the coagulase negative isolate resistant to erythromycin was found to have nuc gene, mecA gene, ermC gene, msrA gene, linA gene, and femA gene. The isolate was confirmed as MRSA. Conclusion: In conclusion, PCR technique is more sensitive and reliable than tube coagulase test or antibiogram for the detection of MRSA. And keys were shown to be an important source of MRSA and other bacteria circulation in the community.

Detection of the antibiotic resistance genes in Staphylococcus aureus isolated from human infections and bovine mastitis

The present study was carried out in an attempt to detect the distribution of antibiotic-resistant genes of Staphylococcus aureus isolates from human infections and bovine mastitis. mecA, msrA, msrB, aacA-D, tetK and tetM genes were selected in order to detect the distribution of antibiotic-resistant genes by multiplex PCR technique. According to the biochemical analysis and detection of 16S-rDNA by PCR method, 108 isolates of 300 human infections samples and 18 strains from 150 bovine mastitis milks were recognized as S. aureus. Distributions of antibiotic-resistant genes in human isolates were as follows: (85.18%) mecA, (46.29%) msrA, (49.07%) msrB, (33.33%) aacA-D, (80.50%) tetK and (66.66%) tetM and in bovine mastitis, isolates were seen to be ranging from: (22.22%) mecA, (66.66%) msrA, (77.77%) msrB, (33.33%) aacA-D, (55.55%) tetKand (50.00%) tetM, respectively. Results indicated that all S. aureus strains have one or more of the antibiotic-resistant genes. Also, multiplex PCR technique is a fast, practical and appropriate technique for determining antibiotic-resistant genes. Hence, it was possible that the treatment and the right antibiotics were used. Key words: Antibiotic-resistant genes, bovine mastitis, human infections, multiplex PCR,Staphylococcus aureus.

A high prevalence of mupirocin and macrolide resistance determinant among Staphylococcus aureus strains isolated from burnt patients

Infections due to Staphylococcus aureus have become increasingly common among burn patients. The antibiotic resistance profile of S. aureus isolates and inducible resistance against clindamycin were investigated in this study. The presence of mecA gene, mupA gene and macrolide resistance genes were detected using PCR and multiplex-PCR. The resistance rate to methicillin, erythromycin and mupirocin were 58.5%, 58% and 40%, respectively. The prevalence of constitutive and inducible resistance among macrolide resistant isolates was 75% and 25%, respectively. Ninety five percent of the isolates were positive for one or more erm genes. The most common genes were ermA (75%), ermC (72%) and ermB (69%), respectively. The ermA gene predominated in the strains with the inducible phenotype, while ermC was more common in the isolates with the constitutive phenotype. The msrA gene was only found in one MRSA isolate with the constitutive phenotype. A total of 27 isolates (25%) carried the mupA gene. All the mupirocin resistant isolates and almost all the erythromycin resistant isolates were also resistant against methicillin which may indicate an outbreak of MRSA isolates with high-level mupirocin and erythromycin resistance in the burn unit assessed.

Mechanism of adhesion maintenance by methionine sulphoxide reductase in Streptococcus gordonii

Methionine sulphoxide reductase maintains adhesin function during oxidative stress. Using Streptococcus gordonii as a model, we now show the mechanistic basis of adhesin maintenance provided by MsrA. In biofilms, S. gordonii selectively expresses the msrA gene. When the wild-type strain was grown with exogenous hydrogen peroxide (H(2)O(2)), msrA-specific mRNA expression significantly increased, while acid production was unaffected. In the presence of H(2)O(2), a msrA-deletion mutant (ΔMsrA) showed a 6 h delay in lag phase growth, a 30% lower yield of H(2)O(2), significantly greater inhibition by H(2)O(2) on agar plates (reversed by complementation), 30% less adhesion to saliva-coated hydroxyapatite, 87% less biofilm formation and an altered electrophoretic pattern of SspAB protein adhesins. Using mass spectrometry, methionine residues in the Met-rich central region of SspB were shown to be oxidized by H(2)O(2) and reduced by MsrA. In intact wild-type cells, MsrA colocalized with a cell wall-staining dye, and MsrA was detected in both cell wall and cytosolic fractions. To maintain normal adhesion and biofilm function of S. gordonii in response to exogenous oxidants therefore msrA is upregulated, methionine oxidation of adhesins and perhaps other proteins is reversed, and adhesion and biofilm formation is maintained.

Identification of the oxidative stress–related gene MSRA as a rheumatoid arthritis susceptibility locus by genome‐wide pathway analysis

Genome-wide association studies carried out in rheumatoid arthritis (RA) have led to the discovery of several genetic associations with this disease. Still, the current associated genetic variations can explain only part of the genetic risk involved in RA, and it is well recognized that these genome-wide association studies are likely underpowered to detect all common disease variants. This study was undertaken to explore the genomic regions showing low-significance associations in previous genome-wide association studies of RA. To reduce the false-positive signal fraction, we exploited pathway analysis to prioritize regions containing genes most likely to be implicated in RA. We hypothesized that true disease genes would be in a similar pathway. Therefore, genes from similar pathways but located in different regions were prioritized for replication using Prioritizer software. A total of 384 genetic variants selected from previous RA genome-wide association studies were tested in a Spanish case-control discovery cohort comprising 376 RA patients and 478 healthy controls for replication. Statistically significant associations were further validated in replication cohorts from Spain and The Netherlands. The study consisted of a total of 1,818 RA patients and 2,498 controls. We detected a novel genetic association between RA and the MSRA gene (rs10903323) in the Spanish combined population (P = 2.91 × 10(-5) , odds ratio [OR] 1.51). This association was further tested in our independent Dutch replication cohort. Combined analysis showed an overall association of MSRA with RA (P = 3.19 × 10(-4) , OR 1.28). Our findings indicate that a novel association in the MSRA gene is related to oxidative stress and support the notion of a major role for this process in RA.

Deficiency of methionine sulfoxide reductase A causes cellular dysfunction and mitochondrial damage in cardiac myocytes under physical and oxidative stresses

Methionine sulfoxide reductase A (MsrA) is an enzyme that reverses oxidation of methionine in proteins. Using a MsrA gene knockout (MsrA−/−) mouse model, we have investigated the role of MsrA in the heart. Our data indicate that cellular contractility and cardiac function are not significantly changed in MsrA−/− mice if the hearts are not stressed. However, the cellular contractility, when stressed using a higher stimulation frequency (2Hz), is significantly reduced in MsrA−/− cardiac myocytes. MsrA−/− cardiac myocytes also show a significant decrease in contractility after oxidative stress using H2O2. Corresponding changes in Ca2+ transients are observed in MsrA−/− cardiomyocytes treated with 2Hz stimulation or with H2O2. Electron microscope analyses reveal a dramatic morphological change of mitochondria in MsrA−/− mouse hearts. Further biochemical measurements indicate that protein oxidation levels in MsrA−/− mouse hearts are significantly higher than those in wild type controls. Our study demonstrates that the lack of MsrA in cardiac myocytes reduces myocardial cell’s capability against stress stimulations resulting in a cellular dysfunction in the heart.

Methionine sulfoxide reductase A (MsrA) protects cultured mouse embryonic stem cells from H2O2‐mediated oxidative stress

Methionine sulfoxide reductase A (MsrA), a member of the Msr gene family, can reduce methionine sulfoxide residues in proteins formed by oxidation of methionine by reactive oxygen species (ROS). Msr is an important protein repair system which can also function to scavenge ROS. Our studies have confirmed the expression of MsrA in mouse embryonic stem cells (ESCs) in culture conditions. A cytosol-located and mitochondria-enriched expression pattern has been observed in these cells. To confirm the protective function of MsrA in ESCs against oxidative stress, a siRNA approach has been used to knockdown MsrA expression in ES cells which showed less resistance than control cells to hydrogen peroxide treatment. Overexpression of MsrA gene products in ES cells showed improved survivability of these cells to hydrogen peroxide treatment. Our results indicate that MsrA plays an important role in cellular defenses against oxidative stress in ESCs. Msr genes may provide a new target in stem cells to increase their survivability during the therapeutic applications.

Oxidation of methionine 35 reduces toxicity of the amyloid beta-peptide(1–42) in neuroblastoma cells (IMR-32) via enzyme methionine sulfoxide reductase A expression and function

The beta amyloid peptide (Aβ), the major protein component of brain senile plaques in Alzheimer's disease, is known to be directly responsible for the production of free radicals that may lead to neurodegeneration. Our recent evidence suggest that the redox state of methionine residue in position 35 (Met-35) of Aβ has the ability to deeply modify peptide's neurotoxic actions. Reversible oxidation of methionine in proteins involving the enzyme methionine sulfoxide reductase type A (MsrA) is postulated to serve a general antioxidant role and a decrease in MsrA has been implicated in Alzheimer's disease. In rat neuroblastoma cells (IMR-32), we used Aβ(1–42), in which the Met-35 is present in the reduced state, with a modified peptide with oxidized Met-35 (Aβ(1–42)Met35 OX), as well as an Aβ-derivative in which Met-35 is substituted with norleucine (Aβ(1–42)Nle35) to investigate the relationship between Met-35 redox state, expression and function of MsrA and reactive oxygen species (ROS) generation. The obtained results shown that MsrA activity, as well as mRNA levels, increase in IMR-32 cells treated with Aβ(1–42)Met35 OX, differently to that shown by the reduced derivative. The increase in MsrA function and expression was associated with a decline of ROS levels. None of these effects were observed when cells were exposed to Aβ containing oxidized Met35 (Aβ1–42)Met35 OX. Taken together, the results of the present study indicate that the differential toxicity of Aβ peptides containing reduced or oxidised Met-35 depends on the ability of the latter form to reduce ROS generation by enhancing MsrA gene expression and function and suggests the therapeutic potential of MsrA in Alzheimer's disease.