Electrosynthesis of binuclear ruthenium complexes from [RuCl3(dppb)(L)] precursors [L = pyridine, 4-methylpyridine or dimethyl sulfoxide; dppb=1,4-bis(diphenylphosphino)butane]

Electrolysis has been examined as a method of synthesis for [(L)(dppb)Ru(mu-Cl)(3)RuCl(dppb)] complexes, where dppb = 1,4-bis(diphenylphosphino)butane and L = pyridine (py), 4-methylpyridine (4-pic) or dimethyl sulfoxide (DMSO), by using [RuCl3(dppb)(L)] as precursors. The products of the electrolysis were characterized by P-31-{H-1} NMR, cyclic voltammetry and near infrared spectroscopy. The presence of the [Ru2Cl5(dppb)(2)] complex in the electrochemical cell suggests a mechanism by which the starting original species from the bulk solution reacts with the reduced form [RuCl2(dppb)(L)] generated at the surface of the electrode. The crystal structure of the precursor mer-[RuCl3(dppb)(4-pic)] was determined by X-ray diffraction.

The effect of dimethyl sulfoxide (DMSO) on dentin bonding and nanoleakage of etch-and-rinse adhesives

Objective The objective was to examine the effect of a solvent dimethyl sulfoxide (DMSO) on resin-dentin bond durability, as well as potential functional mechanisms behind the effect. Methods Microtensile bond strength (μTBS) was evaluated in extracted human teeth in two separate experiments. Dentin specimens were acid-etched and assigned to pre-treatment with 0.5 mM (0.004%) DMSO as additional primer for 30 s and to controls with water pre-treatment. Two-step etch-and-rinse adhesive (Scotchbond 1XT, 3M ESPE) was applied and resin composite build-ups were created. Specimens were immediately tested for μTBS or stored in artificial saliva for 6 and 12 months prior to testing. Additional immediate and 6-month specimens were examined for interfacial nanoleakage analysis under SEM. Matrix metalloproteinase (MMP) inhibition by DMSO was examined with gelatin zymography. Demineralized dentin disks were incubated in 100% DMSO to observe the optical clearing effect. Results The use of 0.5 mM DMSO had no effect on immediate bond strength or nanoleakage. In controls, μTBS decreased significantly after storage, but increased significantly in DMSO-treated group. The control group had significantly lower μTBS than DMSO-group after 6 and 12 months. DMSO also eliminated the increase in nanoleakage seen in controls. 5% and higher DMSO concentrations significantly inhibited the gelatinases. DMSO induced optical clearing effect demonstrating collagen dissociation. Significance DMSO as a solvent may be useful in improving the preservation of long-term dentin-adhesive bond strength. The effect may relate to dentinal enzyme inhibition or improved wetting of collagen by adhesives. The collagen dissociation required much higher DMSO concentrations than the 0.5 mM DMSO used for bonding. © 2013 Academy of Dental Materials.

Cytotoxicity of dimethyl sulfoxide (DMSO) in direct contact with odontoblast-like cells

To evaluate the cytotoxicity of dimethyl sulfoxide (DMSO) on the repair-related activity of cultured odontoblast-like MDPC-23 cells. Methods Solutions with different concentrations of DMSO (0.05, 0.1, 0.3, 0.5 and 1.0 mM), diluted in culture medium (DMEM), were placed in contact with MDPC-23 cells (5 × 104 cells/cm2) for 24 h. Eight replicates (n = 8) were prepared for each solutions for the following methods of analysis: violet crystal dye for cell adhesion (CA), quantification of total protein (TP), alizarin red for mineralization nodules formation (MN) and cell death by necrosis (flow cytometry); while twelve replicates (n = 12) were prepared for viable cell number (Trypan Blue) and cell viability (MTT assay). Data were analyzed by ANOVA and Tukey or Kruskal–Wallis and Mann–Whitney's tests (p < 0.05). Results Cell viability, adhesion and percentage of cell death by necrosis were not affected by DMSO at any concentration, with no statistical significant difference among the groups. A significant reduction in total protein production was observed for 0.5 and 1.0 mM of DMSO compared to the control while increased mineralized nodules formation was seen only for 1.0 mM DMSO. Significance: DMSO caused no or minor cytotoxic effects on the pulp tissue repair-related activity of odontoblast-like cells.

Should the standard dimethyl sulfoxide concentration be reduced? Results of a European Group for Blood and Marrow Transplantation prospective noninterventional study on usage and side effects of dimethyl sulfoxide.

BACKGROUND Dimethyl sulfoxide (DMSO) is essential for the preservation of liquid nitrogen-frozen stem cells, but is associated with toxicity in the transplant recipient. STUDY DESIGN AND METHODS In this prospective noninterventional study, we describe the use of DMSO in 64 European Blood and Marrow Transplant Group centers undertaking autologous transplantation on patients with myeloma and lymphoma and analyze side effects after return of DMSO-preserved stem cells. RESULTS While the majority of centers continue to use 10% DMSO, a significant proportion either use lower concentrations, mostly 5 or 7.5%, or wash cells before infusion (some for selected patients only). In contrast, the median dose of DMSO given (20 mL) was much less than the upper limit set by the same institutions (70 mL). In an accompanying statistical analysis of side effects noted after return of DMSO-preserved stem cells, we show that patients in the highest quartile receiving DMSO (mL and mL/kg body weight) had significantly more side effects attributed to DMSO, although this effect was not observed if DMSO was calculated as mL/min. Dividing the myeloma and lymphoma patients each into two equal groups by age we were able to confirm this result in all but young myeloma patients in whom an inversion of the odds ratio was seen, possibly related to the higher dose of melphalan received by young myeloma patients. CONCLUSION We suggest better standardization of preservation method with reduced DMSO concentration and attention to the dose of DMSO received by patients could help reduce the toxicity and morbidity of the transplant procedure.

The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo

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, ΔmsrA::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.

Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress

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.

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

Chromosomal localization of the mammalian peptide-methionine sulfoxide reductase gene and its differential expression in various tissues.

Peptide methionine sulfoxide reductase (MsrA; EC 1.8.4.6) is a ubiquitous protein that can reduce methionine sulfoxide residues in proteins as well as in a large number of methyl sulfoxide compounds. The expression of MsrA in various rat tissues was determined by using immunocytochemical staining. Although the protein was found in all tissues examined, it was specifically localized to renal medulla and retinal pigmented epithelial cells, and it was prominent in neurons and throughout the nervous system. In addition, blood and alveolar macrophages showed high expression of the enzyme. The msrA gene was mapped to the central region of mouse chromosome 14, in a region of homology with human chromosomes 13 and 8p21.

The critical role of tryptophan-116 in the catalytic cycle of dimethylsulfoxide reductase from Rhodobacter capsulatus

In dimethylsulfoxide reductase of Rhodobacter capsulatus tryptophan-116 forms a hydrogen bond with a single oxo ligand bound to the molybdenum ion. Mutation of this residue to phenylalanine affected the UV/visible spectrum of the purified Mo-VI form of dimethylsulfoxide reductase resulting in the loss of the characteristic transition at 720 nm. Results of steady-state kinetic analysis and electrochemical studies suggest that tryptophan 116 plays a critical role in stabilizing the hexacoordinate monooxo Mo-VI form of the enzyme and prevents the formation of a dioxo pentacoordinate Mo-VI species, generated as a consequence of the dissociation of one of the dithiolene ligands of the molybdopterin cofactor from the Mo ion. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Chemoenzymatic Synthesis of Chiral Organosulfur Compounds

This thesis is primarily concerned with the enzyme- catalysed synthesis of sulfoxides using reductase and dioxygenase enzymes. Chapter 1 provides an introduction to the topic of redox chemistry with particular emphasis on the application of reductase and dioxygenase enzymes in organosulfur chemistry. Earlier literature methods for the production of enantiopure sulfoxides are reviewed. A brief discussion of the methods used for the determination of enantiomeric excess and absolute configuration is provided. Chapter 2 contains results obtained using a range of whole-cell bacteria each using a dimethyl sulfoxide reductase enzyme. The synthesis of a series of racemic sulfoxides and the development of appropriate CSPHPLC analytical methods is discussed. Kinetic resolutions of a series of sulfoxides have been achieved. Chapter 3 contains a presentation of results using dioxygenase enzymes as biocatalysts for the asymmetric sulfoxidation of dialkyl sulfoxides including thioacetal sulfoxides. A new range of monosulfoxides, cis-dihydrodiols and cis- dihydrodiol sulfoxides have been isolated in enantiopure form. Chapter 4 is focussed on the application of chiral sulfoxides in synthesis. A new chemoenzymatic route to diol sulfoxide enantiomers and the derived enantiopure phenols and catechols is discussed. The application of chemically synthesised sulfoxide enantiomers in the production of hydroxy sulfoxides is reported. Chapter 5 provides a full experimental section where the synthesis of sulfides and racemic sulfoxides is included. The methods used in the isolation and characterisation of bioproducts from the biotransformation are discussed and full experimental details given.

Enzyme-catalysed oxygenation and deoxygenation routes to chiral thiosulfinates

Enantioenriched thiosulfinates have been obtained by dioxygenase- and chloroperoxidase-catalysed oxidation of 1,2-disulfides and dimethyl sulfoxide reductase-catalysed deoxygenation.

Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides

The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms. Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp. and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor. The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used. C. braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide. DMSO reductase was purified from the periplasmic fraction of C. braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.

Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins.

An enzyme that reduces methionine sulfoxide [Met(O)] residues in proteins [peptide Met(O) reductase (MsrA), EC 1.8.4.6; originally identified in Escherichia coli] was purified from bovine liver, and the cDNA encoding this enzyme was cloned and sequenced. The mammalian homologue of E. coli msrA (also called pmsR) cDNA encodes a protein of 255 amino acids with a calculated molecular mass of 25,846 Da. This protein has 61% identity with the E. coli MsrA throughout a region encompassing a 199-amino acid overlap. The protein has been overexpressed in E. coli and purified to homogeneity. The mammalian recombinant MsrA can use as substrate, proteins containing Met(O) as well as other organic compounds that contain an alkyl sulfoxide group such as N-acetylMet(O), Met(O), and dimethyl sulfoxide. Northern analysis of rat tissue extracts showed that rat msrA mRNA is present in a variety of organs with the highest level found in kidney. This is consistent with the observation that kidney extracts also contained the highest level of enzyme activity.

Efficient succinic acid production from glycerol by actinobacillus succinogenes using dimethyl sulfoxid as electron acceptor

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Convergencia de vías de señalización en un modelo de apoptosis

La sepsis es un evento inflamatorio generalizado del organismo inducido por un daño causado generalmente por un agente infeccioso. El patógeno más frecuentemente asociado con esta entidad es el Staphylococcus aureus, responsable de la inducción de apoptosis en células endoteliales debida a la producción de ceramida. Se ha descrito el efecto protector de la proteína C activada (PCA) en sepsis y su relación con la disminución de la apoptosis de las células endoteliales. En este trabajo se analizó la activación de las quinasas AKT, ASK1, SAPK/JNK y p38 en un modelo de apoptosis endotelial usando las técnicas de Western Blotting y ELISA. Las células endoteliales (EA.hy926), se trataron con C2-ceramida (130μM) en presencia de inhibidores químicos de cada una de estas quinasas y PCA. La supervivencia de las células en presencia de inhibidores químicos y PCA fue evaluada por medio de ensayos de activación de las caspasas 3, 7 y 9, que verificaban la muerte celular por apoptosis. Los resultados evidencian que la ceramida reduce la activación de AKT y aumenta la activación de las quinasas ASK, SAPK/JNK y p38, en tanto que PCA ejerce el efecto contrario. Adicionalmente se encontró que la tiorredoxina incrementa la activación/fosforilación de AKT, mientras que la quinasa p38 induce la defosforilación de AKT.