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Cites findings of a study which reveals that plant stress hormones are capable of suppressing apoptosis in various transformed human cells including leukemia, melanoma, prostate and breast cell cancer. Revelation that methyl jasmonate (MJ) was determined to be the most potent agent in suppressing apoptosis; Information that MJ killed Molt-4 human lymphoblastic leukemia cells in efficient manner; Observation that MJ did not affect normal human peripheral blood lymphocytes; Details of methodology adopted for the study to assess the effectiveness of MJ against lymphoblastic leukemia cells.

Presents a corrected reprint of an article published in the periodical "Leukemia".

Background: Obesity remains a high-level risk factor for various cancers. Yet, some research has shown that higher BMIs may improve survival outcomes, particularly for head and neck squamous cell carcinoma (HNSCC). While this is a clear deviation from the norm, it raises the question of what other effects obesity may have on head and neck cancer patients, including surgical outcomes. Choosing the most appropriate flap for patients can be difficult for even experienced surgeons. Head and neck reconstructive surgeries are no exception to this rule and can be especially challenging. To produce the most favorable outcomes, a physician must be able to combine all flap attributes including donor and recipient site location, flap thickness, as well as each patient's individual risk factors such as prior radiation. Purpose: The purpose of this study was to compare the outcomes of vertical rectus femoris myocutaneous (VRAM) and anterolateral thigh (ALT) flaps in overweight and obese individuals with varying head and neck cancers leaving large-sized defects to the outcomes in normal and underweight patients. Methods: A retrospective chart review was conducted of VRAM and ALT flaps performed over a period of 12 months at our university hospital for large head and neck reconstruction. Results: Of the 17 ALT patients, 80% (4/5) of the underweight patients, 57% (4/7) of the normal weight patients, 50% (1/2) of the overweight patients, and 33% (1/3) of the obese patients experienced complications. Of the 15 VRAM patients, 40% (2/5) of the underweight patients, 83% (5/6) of the overweight patients, and 50% (1/2) of the obese patients experienced complications. Conclusions: In our small sample size, a smaller percentage of obese patients with head and neck cancer who underwent flap reconstruction surgery had complications when undergoing ALT flaps than VRAM flaps, which contrasts with the normal and underweight patients, who had less complications with VRAM flaps than ALT flaps., Competing Interests: The authors have no conflicts of interest to declare.

Mice with constitutive disruption of the Selenop gene have been key to delineate the importance of selenoproteins in neurobiology. However, the phenotype of this mouse model is exquisitely dependent on selenium supply and timing of selenium supplementation. Combining biochemical, histological, and behavioral methods, we tested the hypothesis that parvalbumin-expressing interneurons in the primary somatosensory cortex and hippocampus depend on dietary selenium availability in Selenop -/- mice. Selenop-deficient mice kept on adequate selenium diet (0.15 mg/kg, i.e. the recommended dietary allowance, RDA) developed ataxia, tremor, and hyperexcitability between the age of 4-5 weeks. Video-electroencephalography demonstrated epileptic seizures in Selenop -/- mice fed the RDA diet, while Selenop ± heterozygous mice behaved normally. Both neurological phenotypes, hyperexcitability/seizures and ataxia/dystonia were successfully prevented by selenium supplementation from birth or transgenic expression of human SELENOP under a hepatocyte-specific promoter. Selenium supplementation with 10 μM selenite in the drinking water on top of the RDA diet increased the activity of glutathione peroxidase in the brains of Selenop -/- mice to control levels. The effects of selenium supplementation on the neurological phenotypes were dose- and time-dependent. Selenium supplementation after weaning was apparently too late to prevent ataxia/dystonia, while selenium withdrawal from rescued Selenop -/- mice eventually resulted in ataxia. We conclude that SELENOP expression is essential for preserving interneuron survival under limiting Se supply, while SELENOP appears dispensable under sufficiently high Se status., Competing Interests: Declaration of competing interest L.S. holds shares of selenOmed GmbH, a company involved in Se status assessment. All other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The brain during Alzheimer's disease (AD) is under severe oxidative attack by reactive oxygen species that may lead to methionine oxidation. Oxidation of the sole methionine (Met35) of beta-amyloid (A β ), and possibly methionine residues of other extracellular proteins, may be one of the earliest events contributing to the toxicity of A β and other proteins in vivo. In the current study, we immunized transgenic AD (APP/PS1) mice at 4 months of age with a recombinant methionine sulfoxide (MetO)-rich protein from Zea mays (antigen). This treatment induced the production of anti-MetO antibody in blood-plasma that exhibits a significant titer up to at least 10 months of age. Compared to the control mice, the antigen-injected mice exhibited the following significant phenotypes at 10 months of age: better short and long memory capabilities; reduced A β levels in both blood-plasma and brain; reduced A β burden and MetO accumulations in astrocytes in hippocampal and cortical regions; reduced levels of activated microglia; and elevated antioxidant capabilities (through enhanced nuclear localization of the transcription factor Nrf2) in the same brain regions. These data collected in a preclinical AD model are likely translational, showing that active immunization could give a possibility of delaying or preventing AD onset. This study represents a first step toward the complex way of starting clinical trials in humans and conducting the further confirmations that are needed to go in this direction.

Purpose: Concurrent radiotherapy with cetuximab, an anti-EGFR mAb, is a standard treatment for locally advanced head and neck squamous carcinoma (HNSCC). Cytotoxic T lymphocyte antigen-4-positive (CTLA-4+) regulatory T cells (Treg) dampen cellular immunity and correlate negatively with clinical outcomes. This phase I study added ipilimumab, an anti-CTLA-4 mAb, to cetuximab-radiotherapy., Patients and Methods: A (3 + 3) design was used to establish the recommended phase II dose (RP2D) of ipilimumab, added at week 5 for four, every-3-week doses to fixed, standard cetuximab-radiotherapy. Eligible subjects had stage III to IVb, high-risk [human papillomavirus-negative (HPV-)] or intermediate-risk HPV-positive (HPV+)] HNSCC. Dose-limiting toxicity (DLT) was defined as any grade 4 adverse event (AE) except in-field radiation dermatitis or immune-related (ir) AE requiring ≥2 weeks of systemic steroids. Baseline tumor and serial blood specimens were collected for immune correlatives., Results: From July 2013 to May 2016, 18 patients enrolled. Two of 6 in cohort 1 (ipilimumab 3 mg/kg) experienced grade 3 dermatologic DLTs, triggering deescalation of ipilimumab to 1 mg/kg. Dose level -1 was expanded to N  =  12 without DLT. irAE included: grade 1, 2, and 3 dermatitis (2, 1, and 3 cases), grade 4 colitis (1), and grade 1 hyperthyroidism (1). Three-year disease-free survival (DFS) and overall survival were 72% [90% confidence interval (CI), 57-92] and 72% (90% CI, 56-92). High expression of coinhibitory receptors PD1/LAG3/CD39 on baseline tumor-infiltrating Treg was associated with worse DFS (HR = 5.6; 95% CI, 0.83-37.8; P = 0.08)., Conclusions: The RP2D for ipilimumab plus standard cetuximab-radiotherapy is 1 mg/kg in weeks 5, 8, 11, and 14. The regimen is tolerable and yields acceptable survival without cytotoxic chemotherapy., (©2022 American Association for Cancer Research.)

Background: Clinician champions are front-line clinicians who advocate for and influence practice change in their local context. The strategies they use when leading efforts to reduce the use of low-value care have not been well described. The purpose of this study is to identify and describe strategies used by six clinician champions who led a low-value care initiative in their clinical setting., Methods: Qualitative data collected during an overuse reduction initiative led by clinician champions were used to identify strategies, guided by the Expert Recommendations for Implementing Change compilation of strategies. Clinician champions were asked to rank the importance of these activities and indicate which one of the six most important activities they would be willing to discuss in an interview. A 30-min semi-structured interview was conducted with each clinician about the activity they selected and thematically analyzed., Results: Twelve Expert Recommendations for Implementing Change strategies were identified. The top six strategies discussed during interviews were: build a coalition, conduct a local needs assessment, develop a formal implementation blueprint, conduct educational meetings, use facilitation, and develop clinical reminders. Common themes that emerged across all interviews were the use of data to engage clinicians in conversations, including the patient's perspective in designing the interventions, and investing the time upfront to plan and launch the initiative because of the inherent challenges of relinquishing a service., Conclusions: Clinician champions identified multiple strategies as important when de-implementing a low-value service. Many were used to engage in conversations with stakeholders, including leadership, providers, and patients, to increase buy-in and support, challenge beliefs, promote behavior change, and gather insights about next steps in their effort. Future work is needed to better understand how prepare clinicians for this role and to understand the mechanisms through which these strategies might be effective., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Methionine sulfoxide (MetO) is an oxidative posttranslational modification that primarily occurs under oxidative stress conditions, leading to alteration of protein structure and function. This modification is regulated by MetO reduction through the evolutionarily conserved methionine sulfoxide reductase (Msr) system. The Msr type A enzyme (MsrA) plays an important role as a cellular antioxidant and promotes cell survival. The ubiquitin- (Ub) like neddylation pathway, which is controlled by the c-Jun activation domain-binding protein-1 (Jab1), also affects cell survival. Jab1 negatively regulates expression of the cell cycle inhibitor cyclin-dependent kinase inhibitor 1B (P27) through binding and targeting P27 for ubiquitination and degradation. Here we report the finding that MsrA interacts with Jab1 and enhances Jab1's deneddylase activity (removal of Nedd8). In turn, an increase is observed in the level of deneddylated Cullin-1 (Cul-1, a component of E3 Ub ligase complexes). Furthermore, the action of MsrA increases the binding affinity of Jab1 to P27, while MsrA ablation causes a dramatic increase in P27 expression. Thus, an interaction between MsrA and Jab1 is proposed to have a positive effect on the function of Jab1 and to serve as a means to regulate cellular resistance to oxidative stress and to enhance cell survival.

The transportation of mental health patients between facilities by emergency medical services personnel poses a unique risk to both patients and their providers. Increasingly, common injuries are occurring and difficulties are arising during this transition in care. Proximal causes exist that could be addressed to help mitigate many of the complexities that occur during this shift in care. Patient safety, quality of care, and provider safety are all at risk if improvements are not made and problems not identified or rectified., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. JACEP Open published by Wiley Periodicals, Inc. on behalf of the American College of Emergency Physicians.)

Objective : Sporadic Alzheimer's disease (AD) is an oxidative, stress-dependent neurodegenerative disease. We investigated whether the levels of protein-methionine sulfoxide (MetO) in plasma could be a possible marker for AD in individuals with mild cognitive impariment (MCI). Design : We evaluated blood samples from patients with AD or MCI, as well as from normal controls, testing their MetO levels and superoxide dismutase (SOD) specific activity. Results : An increase of MetO levels of a particular protein of human plasma and a decrease of SOD activity were observed only in AD plasma. Conclusion : Monitoring the patterns of these plasma markers in patients with MCI could provide a warning sign for disease progression into AD., Competing Interests: FUNDING:Support for the studies were provided by the KU ADC P30 AG035982 and the Landon Center on Aging and the Hedwig Miller Fund for Aging Research. DISCLOSURES:The authors have no conflicts of interest relevant to the content of this article., (Copyright © 2019. Matrix Medical Communications. All rights reserved.)

Methionine sulfoxide reductase enzymes are a protective system against biological oxidative stress in aerobic organisms. Modifications to this antioxidant system have been shown to impact the lifespan of several model system organisms. In humans, methionine oxidation of critical proteins and deficiencies in the methionine sulfoxide reductase system have been linked to age-related diseases, including cancer and neurodegenerative disease. Substrates for methionine sulfoxide reductases have been reviewed multiple times, and are still an active area of discovery. In contrast, less is known about the genetic regulation of methionine sulfoxide reductases. In this review, we discuss studies on the genetic regulation of the methionine sulfoxide reductase system with relevance to longevity and age-related diseases. A better understanding of genetic regulation for methionine sulfoxide reductases may lead to new therapeutic approaches for age-related diseases in the future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This review article describes and discusses the current knowledge on the general role of the methionine sulfoxide reductase (MSR) system and the particular role of MSR type A (MSRA) in mammals. A powerful tool to investigate the contribution of MSRA to molecular processes within a mammalian system/organism is the MSRA knockout. The deficiency of MSRA in this mouse model provides hints and evidence for this enzyme function in health and disease. Accordingly, the potential involvement of MSRA in the processes leading to neurodegenerative diseases, neurological disorders, cystic fibrosis, cancer, and hearing loss will be deliberated and evaluated.

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1 + TIL has been reported in human papillomavirus (HPV) + HNC patients, despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact, because PD-1 high T cells may be more exhausted than PD-1 low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study, PD-1 expression was indeed upregulated on HNC patient TIL, and the frequency of these PD-1 + TIL was higher in HPV + patients ( P = 0.006), who nonetheless experienced significantly better clinical outcome. However, PD-1 high CD8 + TILs were more frequent in HPV - patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover, HNC patients with higher frequencies of PD-1 high CD8 + TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence ( P < 0.001), while higher fractions of PD-1 low T cells associated with HPV positivity and better outcome. In a murine HPV + HNC model, anti-PD-1 mAb therapy differentially modulated PD-1 high/low populations, and tumor rejection associated with loss of dysfunctional PD-1 high CD8 + T cells and a significant increase in PD-1 low TIL. Thus, the extent of PD-1 expression on CD8 + TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Methionine sulfoxide reductase A (MsrA) is an antioxidant enzyme found in all domains of life that catalyzes the reduction of methionine- S -sulfoxide (MSO) to methionine in proteins and free amino acids. We demonstrate that archaeal MsrA has a ubiquitin-like (Ubl) protein modification activity that is distinct from its stereospecific reduction of MSO residues. MsrA catalyzes this Ubl modification activity, with the Ubl-activating E1 UbaA, in the presence of the mild oxidant dimethyl sulfoxide (DMSO) and in the absence of reductant. In contrast, the MSO reductase activity of MsrA is inhibited by DMSO and requires reductant. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis reveals that MsrA-dependent Ubl conjugates are associated with DNA replication, protein remodeling, and oxidative stress and include the Ubl-modified MsrA, Orc3 (Orc1/Cdc6), and Cdc48d (Cdc48/p97 AAA+ ATPase). Overall, we found archaeal MsrA to have opposing MSO reductase and Ubl modifying activities that are associated with oxidative stress responses and controlled by exposure to mild oxidant. IMPORTANCE Proteins that are damaged by oxidative stress are often targeted for proteolysis by the ubiquitin-proteasome system (UPS). The mechanisms that control this response are poorly understood, especially under conditions of mild oxidative stress when protein damage is modest. Here, we discovered a novel function of archaeal MsrA in guiding the Ubl modification of target proteins in the presence of mild oxidant. This newly reported activity of MsrA is distinct from its stereospecific reduction of methionine- S -sulfoxide to methionine residues. Our results are significant steps forward, first, in elucidating a protein factor that guides Ubl modification in archaea, and second, in providing an insight into oxidative stress responses that can trigger Ubl modification in a cell., (Copyright © 2017 Fu et al.)

Accumulation of oxidized proteins, and especially β-amyloid (Aβ), is thought to be one of the common causes of Alzheimer's disease (AD). The current studies determine the effect of an in vivo methionine sulfoxidation of Aβ through ablation of the methionine sulfoxide reductase A (MsrA) in a mouse model of AD, a mouse that overexpresses amyloid precursor protein (APP) and Aβ in neurons. Lack of MsrA fosters the formation of methionine sulfoxide in proteins, and thus its ablation in the AD-mouse model will increase the formation of methionine sulfoxide in Aβ. Indeed, the novel MsrA-deficient APP mice (APP(+)/MsrAKO) exhibited higher levels of soluble Aβ in brain compared with APP(+) mice. Furthermore, mitochondrial respiration and the activity of cytochrome c oxidase were compromised in the APP(+)/MsrAKO compared with control mice. These results suggest that lower MsrA activity modifies Aβ solubility properties and causes mitochondrial dysfunction, and augmenting its activity may be beneficial in delaying AD progression., (Copyright © 2016 the American Physiological Society.)

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca2+-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.

Staphylococcus aureus is a major human pathogen and emergence of antibiotic resistance in clinical staphylococcal isolates raises concerns about our ability to control these infections. Cell wall-active antibiotics cause elevated synthesis of methionine sulfoxide reductases (Msrs: MsrA1 and MsrB) in S. aureus. MsrA and MsrB enzymes reduce S-epimers and R-epimers of methionine sulfoxide, respectively, that are generated under oxidative stress. In the S. aureus chromosome, there are three msrA genes (msrA1, msrA2 and msrA3) and one msrB gene. To understand the precise physiological roles of Msr proteins in S. aureus, mutations in msrA1, msrA2 and msrA3 and msrB genes were created by site-directed mutagenesis. These mutants were combined to create a triple msrA (msrA1, msrA2 and msrA3) and a quadruple msrAB (msrA1, msrA2, msrA3, msrB) mutant. These mutants were used to determine the roles of Msr proteins in staphylococcal growth, antibiotic resistance, adherence to human lung epithelial cells, pigment production, and survival in mice relative to the wild-type strains. MsrA1-deficient strains were sensitive to oxidative stress conditions, less pigmented and less adherent to human lung epithelial cells, and showed reduced survival in mouse tissues. In contrast, MsrB-deficient strains were resistant to oxidants and were highly pigmented. Lack of MsrA2 and MsrA3 caused no apparent growth defect in S. aureus. In complementation experiments with the triple and quadruple mutants, it was MsrA1 and not MsrB that was determined to be critical for adherence and phagocytic resistance of S. aureus. Overall, the data suggests that MsrA1 may be an important virulence factor and MsrB probably plays a balancing act to counter the effect of MsrA1 in S. aureus.

Catechol-O-methyl transferase (COMT) plays a key role in the degradation of brain dopamine (DA). Specifically, low COMT activity results in higher DA levels in the prefrontal cortex (PFC), thereby reducing the vulnerability for attentional and cognitive deficits in both psychotic and healthy individuals. COMT activity is markedly reduced by a non-synonymous single-nucleotide polymorphism (SNP) that generates a valine-to-methionine substitution on the residue 108/158, by means of as-yet incompletely understood post-translational mechanisms. One post-translational modification is methionine sulfoxide, which can be reduced by the methionine sulfoxide reductase (Msr) A and B enzymes. We used recombinant COMT proteins (Val/Met108) and mice (wild-type (WT) and MsrA knockout) to determine the effect of methionine oxidation on COMT activity and COMT interaction with Msr, through a combination of enzymatic activity and Western blot assays. Recombinant COMT activity is positively regulated by MsrA, especially under oxidative conditions, whereas brains of MsrA knockout mice exhibited lower COMT activity (as compared with their WT counterparts). These results suggest that COMT activity may be reduced by methionine oxidation, and point to Msr as a key molecular determinant for the modulation of COMT activity in the brain. The role of Msr in modulating cognitive functions in healthy individuals and schizophrenia patients is yet to be determined.

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., Competing Interests: The authors have declared that no competing interests exist.

Previously, we have showed that overexpression of methionine-oxidized α-synuclein in methionine sulfoxide reductase A (MsrA) null mutant yeast cells inhibits α-synuclein phosphorylation and increases protein fibrillation. The current studies show that ablation of mouse MsrA gene caused enhanced methionine oxidation of α-synuclein while reducing its own phophorylation levels, especially in the hydrophobic cell-extracted fraction. These data provide supportive evidence that a compromised MsrA function in mammalian brain may cause enhanced pathologies associated with altered α-synuclein oxidation and phosphorylation levels.

While elucidating the peculiar epitope of the alpha-PrP mAb IPC2, we found that PrPSc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the alpha-fold, suggesting oxidation may facilitate the conversion of PrPC into PrPSc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrPC, but could not recognize most PrPSc bands unless they were vigorously reduced. Next, we showed that the alpha-Met pAbs did not recognize newly formed PrPSc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrPC to PrPSc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

Schistosomiasis, also known as Bilharzia, is an infectious disease caused by several species of Schistosoma. Twenty million individuals suffer severe symptoms and 200,000 people die annually from the disease. The host responds to the presence of S. mansoni by producing reactive oxygen species that cause oxidative stress. We hypothesized that schistosomes produce antioxidants in response to oxidative stress. A known antioxidant protein is methionine sulfoxide reductase (Msr). Methionine residues can be oxidized to methionine sulfoxide in the presence of oxidizing agents, and the process is readily reversed by the action of the Msr system. Two S. mansoni MsrB genes (MsrB1 and MsrB2) were cloned and the recombinant proteins were expressed in bacteria and purified. The S. mansoni MsrB proteins contained the common conserved catalytic-and zinc-coordinating cysteines. Analysis of the proteins showed that both proteins promote the reduction of both free methionine sulfoxide (Met[O]) and dabsyl-Met(O) to free methionine (Met) and dabsyl-Met, respectively, while exhibiting differences in their specific activities toward these substrates. Using real-time polymerase-chain reaction (RT-PCR), both proteins were found to be expressed in all stages of the parasite's life cycle, with the highest level of expression of both proteins in the egg stage. This is the first description of MsrB proteins from a parasite.

Selenium is a chemical element participating in the synthesis of selenocysteine residues that play a pivotal role in the enzymatic activity efficiency of selenoproteines. The methionine sulfoxide reductase (Msr) system that reduces methionine sulfoxide (MetO) to methionine comprises the selenoprotein MsrB (MsrB1) and the non-selenoprotein MsrA, which reduce the R- and the S- forms of MetO, respectively. The effects of a selenium deficient (SD) diet, which was administrated to wild type (WT) and MsrA knockout mice (MsrA(-)/(-)), on the expression and function of Msr-related proteins are examined and discussed. Additionally, new data about the levels of selenium in brain, liver, and kidneys of WT and MsrA(-)/(-) mice are presented and discussed.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Symptomatic patients with Gaucher disease (GD) (acid beta-glucosidase [Gcase] deficiency) are treated with injectable human recombinant GCase. Treatment results in significant decreases in lipid storage in liver, spleen, and bone marrow, but the generalized osteopenia and focal bone lesions present in many adult patients are refractory to treatment. A double-blind, 2-arm, placebo-controlled trial of alendronate (40 mg/d) was performed in adults with GD who had been treated with enzyme for at least 24 months. Primary therapeutic endpoints were improvements in (1) bone mineral density (BMD) and content (BMC) at the lumbar spine, and (2) focal lesions in x-rays of long bones assessed by a blinded reviewer. There were 34 patients with GD type 1 (age range, 18-50 years) receiving enzyme therapy who were randomized for this study. After 18 months, DeltaBMD at the lumbar spine was 0.068 +/- 0.21 and 0.015 +/- 0.034 for alendronate and placebo groups, respectively (P =.001). Long-bone x-rays showed no change in focal lesions or bone deformities in any subject in either arm. Alendronate is a useful adjunctive therapy in combination with enzyme replacement therapy (ERT) for the treatment of GD-related osteopenia in adults, but it cannot be expected to improve focal lesions.

Staphylococcus aureus contains three genes encoding MsrA-specific methionine sulfoxide reductase (Msr) activity (msrA1, msrA2 and msrA3) and an additional gene that encodes MsrB-specific Msr activity. Data presented here suggest that MsrA1 is the major contributor of the MsrA activity in S. aureus. In mutational analysis, while the total Msr activity in msrA2 mutant was comparable to that of the parent, Msr activity was significantly up-regulated in the msrA1 or msrA1 msrA2 double mutant. Assessment of substrate specificity together with increased reactivity of the cell-free protein extracts of the msrA1 mutants to anti-MsrB polyclonal antibodies in Western analysis provided evidence that increased Msr activity was due to elevated synthesis of MsrB in the MsrA1 mutants. Previously, it was reported that oxacillin treatment of S. aureus cells led to induced synthesis of MsrA1 and a mutation in msrA1 increased the susceptibility of the organism to H(2)O(2). A mutation in the msrA2 gene, however, was not significant for the bacterial oxidative stress response. In complementation assays, while the msrA2 gene was unable to complement the msrA1 msrA2 double mutant for H(2)O(2) resistance, the same gene restored H(2)O(2) tolerance in the double mutant when placed under the control of the msrA1 promoter. However, msrA1 which was able to complement the oxidative stress response in msrA1 mutants could not restore the tolerance of the msrA1 msrA2 mutants to H(2)O(2) when placed under the control of the msrA2 promoter. Additionally, although the oxacillin minimum inhibitory concentration of the msrA1 mutant was comparable to that of the wild-type parent, in shaking liquid culture, the msrA1 mutant responded more efficiently to sublethal doses of oxacillin. The data suggest complex regulation of Msr proteins and a more significant physiological role for msrA1/msrB in S. aureus.

Methionine sulfoxide reductase A (MsrA) maintains the function of many proteins by reversing oxidation of methionine residues. Lack of this repair mechanism very likely increases aging-related disease susceptibility. In Saccharomyces cerevisiae, disruption of the msrA gene increases free and protein-bound methionine sulfoxide and decreases cell viability. Although the underlying mechanisms in the induction of the msrA gene are still unknown, a transcriptional regulation may be involved. Hence, a search of nuclear proteins regulating the msrA gene is a major target of the experiments reported in this article. Using protein purification combined with MS, we discovered that calcium phospholipid-binding protein (CPBP), a homologue of elongation factor-1 gamma, is a component of a complex that binds to the msrA promoter. By measuring CPBP cooperative binding to the msrA promoter, we have mapped the CPBP binding site to a 39-bp sequence at the 3' end of the promoter. In a mutant yeast strain lacking the CPBP-encoding gene, the ability to overexpress msrA mRNA and MsrA protein was impaired and MsrA catalytic activity was greatly reduced, suggesting that CPBP may enhance msrA gene expression.

Mammals contain two methionine sulfoxide (MetO) reductases, MsrA and MsrB, that catalyze the thioredoxin-dependent reduction of the S-MetO and R-MetO derivatives, respectively, to methionine. The major mammalian MsrB is a selenoprotein (except in the heart). Here, we show that there is a loss of MsrB activity in the MsrA-/- mouse that correlates with parallel losses in the levels of MsrB mRNA and MsrB protein, suggesting that MsrA might have a role in MsrB transcription. Moreover, mice that were grown on a selenium-deficient (SD) diet showed a substantial decrease in the levels of MsrB-catalytic activity, MsrB protein, and MsrB mRNA in liver and kidney tissues of both WT and MsrA-/- mouse strains. Whereas no significant protein-MetO could be detected in tissue proteins of young mature mice grown on a selenium-adequate diet, growth on the SD diet led to substantial accumulations of MetO in proteins and also of protein carbonyl derivatives in the liver, kidney, cerebrum, and cerebellum, respectively. In addition, accumulation of protein-MetO derivatives increased with age in tissues of mice fed with a selenium-adequate diet. It should be pointed out that even though the total Msr level is at least 2-fold higher in WT than in MsrA-/- mice, SD diet causes an equal elevation of protein-MetO (except in brain cerebellum) and carbonyl levels in both strains, suggesting involvement of other selenoproteins in regulation of the level of cellular protein-MetO accumulation. Furthermore, the development of the "tip-toe" walking behavior previously observed in the MsrA-/- mice occurred earlier when they were fed with the SD diet.

Actinobacillus actinomycetemcomitans is an oral pathogen that is a causative agent for periodontal disease as well as other non-oral infections. The chronic inflammation associated with periodontal diseases suggests that the bacterium must be able to neutralize oxygen intermediates to survive in the host tissues. Methionine sulfoxide reductase (MsrA) is an enzyme that has been demonstrated to have a role in protection against oxidative damage and has also been identified to be required for the proper expression or maintenance of functional adhesins on the surface of several pathogenic bacteria. The A. actinomycetemcomitans homologue of msrA has been isolated and a chromosomal insertion mutant constructed by allele replacement mutagenesis. Inactivation of the gene led to a complete loss of enzymic activity toward a synthetic substrate. However, the isogenic mutant was not more sensitive to oxidative stress or less adherent to epithelial cells as compared with the parent strain. These data suggest that this strain of A. actinomycetemcomitans has redundant systems that compensate for the MsrA activities ascribed for other organisms.

Purpose: In response to injury, activated neutrophils release tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO). TNFalpha in turn causes human corneal epithelial cells to secrete interleukin (IL)-1alpha, whereas MPO results in formation of HClO/OCl(-). The effect of HClO/OCl(-) on the expression of the IL-1alpha gene and protein is unknown. The current study was undertaken to examine in immortalized human corneal epithelial cells whether NaOCl alters TNFalpha-induced increases in expression of IL-1alpha gene and protein., Methods: Semiquantitative RT-PCR and ELISA characterized IL-1alpha gene and protein expression, respectively. TNFalpha-induced nuclear transfer of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay (EMSA). The alpha isoform of inhibitory protein kappaB (IkappaBalpha) was identified by Western blot analysis., Results: Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNFalpha-induced increases in IL-1alpha mRNA and protein, declines in NFkappaB nuclear transfer, and a modification of IkappaBalpha, based on a bandshift detected by Western blot analysis. Modified IkappaBalpha became resistant to TNFalpha-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 micro M) eliminated the NaOCl-induced IkappaBalpha bandshift. CONCLUSIONS; NaOCl oxidizes IkappaBalpha at methionine residues and thereby suppresses dissociation of IkappaBalpha from NFkappaB. Decreased dissociation could in turn suppress TNFalpha-induced activation of NFkappaB, resulting in declines in expression of IL-1alpha gene and protein. These effects suggest that release of HClO/OCl(-) in vivo by activated neutrophils may counterbalance TNFalpha-induced NFkappaB-dependent secretion if IL-1alpha and suppress an excessive inflammatory reaction.

Previous studies have indicated that human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) are less active at blocking viral replication in HIV-1 infected peripheral blood monocytes/macrophages (M/M) than in HIV-1-infected T cells. We explored the hypothesis that oxidative modification and/or metabolism of the PIs in M/M might account for this reduced potency. We first tested the susceptibility of several PIs (kynostatin-272 [KNI-272], saquinavir, indinavir, ritonavir, or JE-2147) to oxidation after exposure to hydrogen peroxide (H(2)O(2)): only KNI-272 was highly susceptible to oxidation. Treatment of KNI-272 with low millimolar concentrations of H(2)O(2) resulted in mono-oxidation of the sulfur in the S-methyl cysteine (methioalanine) moiety, as determined by reversed-phase high-performance liquid chromatography and mass spectrometry (RP-HPLC/MS). Higher concentrations of H(2)O(2) led to an additional oxidation of the sulfur in the thioproline moiety of KNI-272. None of the PIs were metabolized or oxidized when added to T cells and cultured for up to 12 days. However, when KNI-272 was added to M/M, the concentration of the original KNI-272 steadily decreased with a corresponding increase in the production of three KNI-272 metabolites as identified by RP-HPLC/MS. The structures of these metabolites were different from those produced by H(2)O(2) treatment. The two major products of M/M metabolism of KNI-272 were identified as isomeric forms of KNI-272 oxidized solely on the thioproline ring. Both metabolites had reduced capacities to inhibit HIV-1 protease activity when tested in a standard HIV-1 protease assay. These studies demonstrate that antiviral compounds can be susceptible to oxidative modification in M/M and that this can affect their antiviral potency.

Oxidation of proteins by reactive oxygen species is associated with aging, oxidative stress, and many diseases. Although free and protein-bound methionine residues are particularly sensitive to oxidation to methionine sulfoxide derivatives, these oxidations are readily repaired by the action of methionine sulfoxide reductase (MsrA). To gain a better understanding of the biological roles of MsrA in metabolism, we have created a strain of mouse that lacks the MsrA gene. Compared with the wild type, this mutant: (i) exhibits enhanced sensitivity to oxidative stress (exposure to 100% oxygen); (ii) has a shorter lifespan under both normal and hyperoxic conditions; (iii) develops an atypical (tip-toe) walking pattern after 6 months of age; (iv) accumulates higher tissue levels of oxidized protein (carbonyl derivatives) under oxidative stress; and (v) is less able to up-regulate expression of thioredoxin reductase under oxidative stress. It thus seems that MsrA may play an important role in aging and neurological disorders.

Previous studies employing two-dimensional gel electrophoresis and N-terminal protein sequencing have shown elevated synthesis of the enzyme methionine sulfoxide reductase (MsrA) in Staphylococcus aureus in response to cell-wall-active antibiotics. In the present study, the S. aureus msrA gene was cloned, overexpressed, purified as His-tagged MsrA and shown to have methionine sulfoxide reductase activity. The transcription of msrA was studied by assaying beta-galactosidase activity in an msrA promoter::lacZ fusion strain and by Northern blot analysis. Transcription of msrA was increased by oxacillin; but not by a variety of other stresses including H2O2. Northern blot analysis revealed that the size of the msrA transcript was 2.3 kb, considerably larger than the 531 nt msrA ORF. The msrA transcription start site was mapped 25 nt upstream of the msrA start codon. Computer analysis from database sequences indicated at least three additional ORFs downstream of msrA. The deduced amino acid sequences of two of these three ORFs showed significant sequence homologies to PilB, and enzyme IIA of the phosphotransferase system, respectively. The third ORF could not be identified by homology searches. Northern blot hybridization with probes specific to the msrA downstream region indicated that the S. aureus msrA was transcribed as part of a polycistronic message. Interestingly, purified S. aureus PilB was shown to possess approximately approximately 28-fold higher methionine sulfoxide reductase activity than the MsrA. An insertional knockout mutation in the first gene of this operon resulted in increased susceptibility of the mutant to H2O2 compared to the parent strain, but not to oxacillin.

The yeast peptide-methionine sulfoxide reductase (MsrA) was overexpressed in a Saccharomyces cerevisiae null mutant of msrA by using a high-copy plasmid harboring the msrA gene and its promoter. The resulting strain had about 25-fold higher MsrA activity than its parent strain. When exposed to either hydrogen peroxide, paraquat, or 2,2'-azobis-(2-amidinopropane) dihydrochloride treatment, the MsrA overexpressed strain grew better, had lower free and protein-bound methionine sulfoxide and had a better survival rate under these conditions than did the msrA mutant and its parent strain. Substitution of methionine with methionine sulfoxide in a medium lacking hydrogen peroxide had little effect on the growth pattern, which suggests that the oxidation of free methionine in the growth medium was not the main cause of growth inhibition of the msrA mutant. Ultraviolet A radiation did not result in obvious differences in survival rates among the three strains. An enhanced resistance to hydrogen peroxide treatment was shown in human T lymphocyte cells (Molt-4) that were stably transfected with the bovine msrA and exposed to hydrogen peroxide. The survival rate of the transfected strain was much better than its parent strain when grown in the presence of hydrogen peroxide. These results support the proposition that the msrA gene is involved in the resistance of yeast and mammalian cells to oxidative stress.

A gene homologous to methionine sulfoxide reductase (msrA) was identified as the predicted ORF (cosmid 9379) in chromosome V of Saccharomyces cerevisiae encoding a protein of 184 amino acids. The corresponding protein has been expressed in Escherichia coli and purified to homogeneity. The recombinant yeast MsrA possessed the same substrate specificity as the other known MsrA enzymes from mammalian and bacterial cells. Interruption of the yeast gene resulted in a null mutant, DeltamsrA::URA3 strain, which totally lost its cellular MsrA activity and was shown to be more sensitive to oxidative stress in comparison to its wild-type parent strain. Furthermore, high levels of free and protein-bound methionine sulfoxide were detected in extracts of msrA mutant cells relative to their wild-type parent cells, under various oxidative stresses. These findings show that MsrA is responsible for the reduction of methionine sulfoxide in vivo as well as in vitro in eukaryotic cells. Also, the results support the proposition that MsrA possess an antioxidant function. The ability of MsrA to repair oxidative damage in vivo may be of singular importance if methionine residues serve as antioxidants.

Gaucher disease type 1, a non-neuronopathic lysosomal storage disease, is caused by mutations at the acid beta-glucosidase locus. Periodic infusions of macrophage-targeted acid beta-glucosidase reverse hepatosplenomegaly, hematologic, and bony findings in many patients. Two patients receiving enzyme therapy developed neutralizing antibodies to acid beta-glucosidase that were associated with a lack of improvement or progressive disease. After initial improvement, case 1 had no additional response to 2 years of high-dose (50 U/kg every 2 weeks) enzyme therapy. Similarly, case 2 initially showed a favorable response to enzyme therapy that plateaued after 1 year of treatment. Both patients developed minor allergic reactions and antibodies to acid beta-glucosidase within the first 6 months of treatment. Enzyme therapy was discontinued in case 1, with resultant disease progression and need for splenectomy. An immunosuppression/tolerization protocol was initiated in case 2 because of disease progression and stable neutralizing antibody titers. The IgG neutralizing antibodies rapidly and completely inactivated the wild-type, but not the N370S, acid beta-glucosidase in vitro. Antibodies to human serum albumin and chorionic gonadotropin also developed. The finding of neutralizing antibodies to acid beta-glucosidase during enzyme therapy for Gaucher disease has significant implications for monitoring the therapeutic responses and for potential alternative future therapies for Gaucher disease.