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Sample records for cysteine desulfhydrase du

  1. Co-expression of Arabidopsis thaliana phytochelatin synthase and Treponema denticola cysteine desulfhydrase for enhanced arsenic accumulation.

    PubMed

    Tsai, Shen-Long; Singh, Shailendra; Dasilva, Nancy A; Chen, Wilfred

    2012-02-01

    Arsenic is one of the most hazardous pollutants found in aqueous environments and has been shown to be a carcinogen. Phytochelatins (PCs), which are cysteine-rich and thio-reactive peptides, have high binding affinities for various metals including arsenic. Previously, we demonstrated that genetically engineered Saccharomyces cerevisiae strains expressing phytochelatin synthase (AtPCS) produced PCs and accumulated arsenic. In an effort to further improve the overall accumulation of arsenic, cysteine desulfhydrase, an aminotransferase that converts cysteine into hydrogen sulfide under aerobic condition, was co-expressed in order to promote the formation of larger AsS complexes. Yeast cells producing both AtPCS and cysteine desulfhydrase showed a higher level of arsenic accumulation than a simple cumulative effect of expressing both enzymes, confirming the coordinated action of hydrogen sulfide and PCs in the overall bioaccumulation of arsenic. Copyright © 2011 Wiley Periodicals, Inc.

  2. Assessing the transcriptional regulation of L-cysteine desulfhydrase 1 in Arabidopsis thaliana.

    PubMed

    Laureano-Marín, Ana M; García, Irene; Romero, Luis C; Gotor, Cecilia

    2014-01-01

    Hydrogen sulfide is an important signaling molecule that functions as a physiological gasotransmitter of comparable importance to NO and CO in mammalian systems. In plants, numerous studies have shown that sulfide increases tolerance/resistance to stress conditions and regulates essential processes. The endogenous production of hydrogen sulfide in the cytosol of Arabidopsis thaliana occurs by the enzymatic desulfuration of L-cysteine, which is catalyzed by the L-cysteine desulfhydrase enzyme DES1. To define the functional role of DES1 and the role that the sulfide molecule may play in the regulation of physiological processes in plants, we studied the localization of the expression of this gene at the tissue level. Transcriptional data reveal that DES1 is expressed at all developmental stages and is more abundant at the seedling stage and in mature plants. At the tissue level, we analyzed the expression of a GFP reporter gene fused to promoter of DES1. The GFP fluorescent signal was detected in the cytosol of both epidermal and mesophyll cells, including the guard cells. GFP fluorescence was highly abundant around the hydathode pores and inside the trichomes. In mature plants, fluorescence was detected in floral tissues; a strong GFP signal was detected in sepals, petals, and pistils. When siliques were examined, the highest GFP fluorescence was observed at the bases of the siliques and the seeds. The location of GFP expression, together with the identification of regulatory elements within the DES1 promoter, suggests that DES1 is hormonally regulated. An increase in DES1 expression in response to ABA was recently demonstrated; in the present work, we observe that in vitro auxin treatment significantly repressed the expression of DES1.

  3. ENZYME DISTRIBUTION AS A FACTOR IN THE INDEPENDENCE OF BACILLUS CEREUS SPORE GERMINATION FROM L- AND D-CYSTEINE DESULFHYDRASE ACTIVITY

    DTIC Science & Technology

    The release of H2S, pyruvate, and NH3 from L- and D- cysteine by extracts from B. cereus strain T spores is ascribed to the mediation of specific L...and D- cysteine desulfhydrases. D-isomer activity is differentiated by pyridoxal phosphate independence, five-fold greater activity, and relative...resistance to inhibition by semicarbazide. Inhibition by NH2OH does not permit differentiation. Low recoveries of pyruvate (26 to 47%) are associated with a nonenzymic reaction between pyruvate and L- or D- cysteine .

  4. Hydrogen sulfide is a novel potential virulence factor of Mycoplasma pneumoniae: characterization of the unusual cysteine desulfurase/desulfhydrase HapE.

    PubMed

    Großhennig, Stephanie; Ischebeck, Till; Gibhardt, Johannes; Busse, Julia; Feussner, Ivo; Stülke, Jörg

    2016-04-01

    Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP-ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide-producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae.

  5. Hydrogen sulfide generated by L-cysteine desulfhydrase acts upstream of nitric oxide to modulate abscisic acid-dependent stomatal closure.

    PubMed

    Scuffi, Denise; Álvarez, Consolación; Laspina, Natalia; Gotor, Cecilia; Lamattina, Lorenzo; García-Mata, Carlos

    2014-12-01

    Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

  6. Hydrogen Sulfide Generated by l-Cysteine Desulfhydrase Acts Upstream of Nitric Oxide to Modulate Abscisic Acid-Dependent Stomatal Closure1[C][W

    PubMed Central

    Scuffi, Denise; Álvarez, Consolación; Laspina, Natalia; Gotor, Cecilia; Lamattina, Lorenzo; García-Mata, Carlos

    2014-01-01

    Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells. PMID:25266633

  7. Fermentative Production of Cysteine by Pantoea ananatis

    PubMed Central

    Takumi, Kazuhiro; Ziyatdinov, Mikhail Kharisovich; Samsonov, Viktor

    2016-01-01

    ABSTRACT Cysteine is a commercially important amino acid; however, it lacks an efficient fermentative production method. Due to its cytotoxicity, intracellular cysteine levels are stringently controlled via several regulatory modes. Managing its toxic effects as well as understanding and deregulating the complexities of regulation are crucial for establishing the fermentative production of cysteine. The regulatory modes include feedback inhibition of key metabolic enzymes, degradation, efflux pumps, and the transcriptional regulation of biosynthetic genes by a master cysteine regulator, CysB. These processes have been extensively studied using Escherichia coli for overproducing cysteine by fermentation. In this study, we genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to identify key factors required for cysteine production. According to this and our previous studies, we identified a major cysteine desulfhydrase gene, ccdA (formerly PAJ_0331), involved in cysteine degradation, and the cysteine efflux pump genes cefA and cefB (formerly PAJ_3026 and PAJ_p0018, respectively), which may be responsible for downregulating the intracellular cysteine level. Our findings revealed that ccdA deletion and cefA and cefB overexpression are crucial factors for establishing fermentative cysteine production in P. ananatis and for obtaining a higher cysteine yield when combined with genes in the cysteine biosynthetic pathway. To our knowledge, this is the first demonstration of cysteine production in P. ananatis, which has fundamental implications for establishing overproduction in this microbe. IMPORTANCE The efficient production of cysteine is a major challenge in the amino acid fermentation industry. In this study, we identified cysteine efflux pumps and degradation pathways as essential elements and genetically engineered Pantoea ananatis, an emerging host for the fermentative production of bio-based materials, to

  8. Cysteine degradation gene yhaM, encoding cysteine desulfidase, serves as a genetic engineering target to improve cysteine production in Escherichia coli.

    PubMed

    Nonaka, Gen; Takumi, Kazuhiro

    2017-12-01

    Cysteine is an important amino acid for various industries; however, there is no efficient microbial fermentation-based production method available. Owing to its cytotoxicity, bacterial intracellular levels of cysteine are stringently controlled via several modes of regulation, including cysteine degradation by cysteine desulfhydrases and cysteine desulfidases. In Escherichia coli, several metabolic enzymes are known to exhibit cysteine degradative activities, however, their specificity and physiological significance for cysteine detoxification via degradation are unclear. Relaxing the strict regulation of cysteine is crucial for its overproduction; therefore, identifying and modulating the major degradative activity could facilitate the genetic engineering of a cysteine-producing strain. In the present study, we used genetic screening to identify genes that confer cysteine resistance in E. coli and we identified yhaM, which encodes cysteine desulfidase and decomposes cysteine into hydrogen sulfide, pyruvate, and ammonium. Phenotypic characterization of a yhaM mutant via growth under toxic concentrations of cysteine followed by transcriptional analysis of its response to cysteine showed that yhaM is cysteine-inducible, and its physiological role is associated with resisting the deleterious effects of cysteine in E. coli. In addition, we confirmed the effects of this gene on the fermentative production of cysteine using E. coli-based cysteine-producing strains. We propose that yhaM encodes the major cysteine-degrading enzyme and it has the most significant role in cysteine detoxification among the numerous enzymes reported in E. coli, thereby providing a core target for genetic engineering to improve cysteine production in this bacterium.

  9. L-Cysteine Metabolism and Fermentation in Microorganisms.

    PubMed

    Takagi, Hiroshi; Ohtsu, Iwao

    L-Cysteine is an important amino acid both biologically and commercially. Although most amino acids are industrially produced by microbial fermentation, L-cysteine has been mainly produced by protein hydrolysis. Due to environmental and safety problems, synthetic or biotechnological products have been preferred in the market. Here, we reviewed L-cysteine metabolism, including biosynthesis, degradation, and transport, and biotechnological production (including both enzymatic and fermentation processes) of L-cysteine. The metabolic regulation of L-cysteine including novel sulfur metabolic pathways found in microorganisms is also discussed. Recent advancement in biochemical studies, genome sequencing, structural biology, and metabolome analysis has enabled us to use various approaches to achieve direct fermentation of L-cysteine from glucose. For example, worldwide companies began to supply L-cysteine and its derivatives produced by bacterial fermentation. These companies successfully optimized the original metabolism of their private strains. Basically, a combination of three factors should be required for improving L-cysteine fermentation: that is, (1) enhancing biosynthesis: overexpression of the altered cysE gene encoding feedback inhibition-insensitive L-serine O-acetyltransferase (SAT), (2) weakening degradation: knockout of the genes encoding L-cysteine desulfhydrases, and (3) exploiting export system: overexpression of the gene involved in L-cysteine transport. Moreover, we found that "thiosulfate" is much more effective sulfur source than commonly used "sulfate" for L-cysteine production in Escherichia coli, because thiosulfate is advantageous for saving consumption of NADPH and relating energy molecules.

  10. Control of Clostridium difficile Physiopathology in Response to Cysteine Availability

    PubMed Central

    Dubois, Thomas; Dancer-Thibonnier, Marie; Monot, Marc; Hamiot, Audrey; Bouillaut, Laurent; Soutourina, Olga; Martin-Verstraete, Isabelle

    2016-01-01

    The pathogenicity of Clostridium difficile is linked to its ability to produce two toxins: TcdA and TcdB. The level of toxin synthesis is influenced by environmental signals, such as phosphotransferase system (PTS) sugars, biotin, and amino acids, especially cysteine. To understand the molecular mechanisms of cysteine-dependent repression of toxin production, we reconstructed the sulfur metabolism pathways of C. difficile strain 630 in silico and validated some of them by testing C. difficile growth in the presence of various sulfur sources. High levels of sulfide and pyruvate were produced in the presence of 10 mM cysteine, indicating that cysteine is actively catabolized by cysteine desulfhydrases. Using a transcriptomic approach, we analyzed cysteine-dependent control of gene expression and showed that cysteine modulates the expression of genes involved in cysteine metabolism, amino acid biosynthesis, fermentation, energy metabolism, iron acquisition, and the stress response. Additionally, a sigma factor (SigL) and global regulators (CcpA, CodY, and Fur) were tested to elucidate their roles in the cysteine-dependent regulation of toxin production. Among these regulators, only sigL inactivation resulted in the derepression of toxin gene expression in the presence of cysteine. Interestingly, the sigL mutant produced less pyruvate and H2S than the wild-type strain. Unlike cysteine, the addition of 10 mM pyruvate to the medium for a short time during the growth of the wild-type and sigL mutant strains reduced expression of the toxin genes, indicating that cysteine-dependent repression of toxin production is mainly due to the accumulation of cysteine by-products during growth. Finally, we showed that the effect of pyruvate on toxin gene expression is mediated at least in part by the two-component system CD2602-CD2601. PMID:27297391

  11. Cysteine homeostasis plays an essential role in plant immunity.

    PubMed

    Álvarez, Consolación; Bermúdez, M Ángeles; Romero, Luis C; Gotor, Cecilia; García, Irene

    2012-01-01

    Cysteine is the metabolic precursor of essential biomolecules such as vitamins, cofactors, antioxidants and many defense compounds. The last step of cysteine metabolism is catalysed by O-acetylserine(thiol)lyase (OASTL), which incorporates reduced sulfur into O-acetylserine to produce cysteine. In Arabidopsis thaliana, the main OASTL isoform OAS-A1 and the cytosolic desulfhydrase DES1, which degrades cysteine, contribute to the cytosolic cysteine homeostasis. • Meta-analysis of the transcriptomes of knockout plants for OAS-A1 and for DES1 show a high correlation with the biotic stress series in both cases. • The study of the response of knockout mutants to plant pathogens shows that des1 mutants behave as constitutive systemic acquired resistance mutants, with high resistance to biotrophic and necrotrophic pathogens, salicylic acid accumulation and WRKY54 and PR1 induction, while oas-a1 knockout mutants are more sensitive to biotrophic and necrotrophic pathogens. However, oas-a1 knockout mutants lack the hypersensitive response associated with the effector-triggered immunity elicited by Pseudomonas syringae pv. tomato DC3000 avrRpm1. • Our results highlight the role of cysteine as a crucial metabolite in the plant immune response. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  12. Cysteine-Generated Sulfide in the Cytosol Negatively Regulates Autophagy and Modulates the Transcriptional Profile in Arabidopsis[W

    PubMed Central

    Álvarez, Consolación; García, Irene; Moreno, Inmaculada; Pérez-Pérez, María Esther; Crespo, José L.; Romero, Luis C.; Gotor, Cecilia

    2012-01-01

    In Arabidopsis thaliana, DES1 is the only identified l-Cysteine desulfhydrase located in the cytosol, and it is involved in the degradation of cysteine and the concomitant production of H2S in this cell compartment. Detailed characterization of the T-DNA insertion mutants des1-1 and des1-2 has provided insight into the role of sulfide metabolically generated in the cytosol as a signaling molecule. Mutations of L-CYS DESULFHYDRASE 1 (DES1) impede H2S generation in the Arabidopsis cytosol and strongly affect plant metabolism. Senescence-associated vacuoles are detected in mesophyll protoplasts of des1 mutants. Additionally, DES1 deficiency promotes the accumulation and lipidation of the ATG8 protein, which is associated with the process of autophagy. The transcriptional profile of the des1-1 mutant corresponds to its premature senescence and autophagy-induction phenotypes, and restoring H2S generation has been shown to eliminate the phenotypic defects of des1 mutants. Moreover, sulfide is able to reverse ATG8 accumulation and lipidation, even in wild-type plants when autophagy is induced by carbon starvation, suggesting a general effect of sulfide on autophagy regulation that is unrelated to sulfur or nitrogen limitation stress. Our results suggest that cysteine-generated sulfide in the cytosol negatively regulates autophagy and modulates the transcriptional profile of Arabidopsis. PMID:23144183

  13. Mechanisms of H2S Production from Cysteine and Cystine by Microorganisms Isolated from Soil by Selective Enrichment

    PubMed Central

    Morra, Matthew J.; Dick, Warren A.

    1991-01-01

    Hydrogen sulfide (H2S) is a major component of biogenic gaseous sulfur emissions from terrestrial environments. However, little is known concerning the pathways for H2S production from the likely substrates, cysteine and cystine. A mixed microbial culture obtained from cystine-enriched soils was used in assays (50 min, 37°C) with 0.05 M Tris-HCl (pH 8.5), 25 μmol of l-cysteine, 25 μmol of l-cystine, and 0.04 μmol of pyridoxal 5′-phosphate. Sulfide was trapped in a center well containing zinc acetate, while pyruvate was measured by derivatization with 2,4-dinitrophenylhydrazine. Sulfide and total pyruvate production were 17.6 and 17.2 nmol mg of protein-1 min-1, respectively. Dithiothreitol did not alter reaction stoichiometry or the amount of H2S and total pyruvate, whereas N-ethylmaleimide reduced both H2S and total pyruvate production equally. The amount of H2S produced was reduced by 96% when only l-cystine was included as the substrate in the assay and by 15% with the addition of propargylglycine, a specific suicide inhibitor of cystathionine γ-lyase. These data indicate that the substrate for the reaction was cysteine and the enzyme responsible for H2S and pyruvate production was cysteine desulfhydrase (EC 4.4.1.1). The enzyme had a Km of 1.32 mM and was inactivated by temperatures greater than 60°C. Because cysteine is present in soil and cysteine desulfhydrase is an inducible enzyme, the potential for H2S production by this mechanism exists in terrestrial environments. The relative importance of this mechanism compared with other processes involved in H2S production from soil is unknown. PMID:16348483

  14. Mechanisms of H sub 2 S production from cysteine and cystine by microorganisms isolated from soil by selective enrichment

    SciTech Connect

    Morra, M.J.; Dick, W.A. )

    1991-05-01

    Hydrogen sulfide (H{sub 2}S) is a major component of biogenic gaseous sulfur emissions from terrestrial environments. However, little is known concerning the pathways for H{sub 2}S production from the likely substrates, cysteine and cystine. A mixed microbial culture obtained from Cystine-enriched soils was used in assays (50 min, 37C) with 0.05 M Tris-HCI (pH 8.5), 25 {mu}mol of L-cysteine, 25 {mu}mol of L-cystine, and 0.04 {mu}mol of pyridoxal 5 feet-phosphate. Sulfide and total pyruvate production were 17.6 and 17.2 nmol mg of protein{sup {minus}1} min{sup {minus}1}, respectively. Dithiothreitol did not alter reaction stoichiometry or the amount of H{sub 2}S and total pyruvate, whereas N-ethylmaleimide reduced both H{sub 2}S and total pyruvate production in the assay and by 15% with the addition of propargylglycine, a specific suicide inhibitor of cystathionine {gamma}-lyase. These data indicate that the substrate for the reaction was cysteine and the enzyme responsible for H{sub 2}S and pyruvate production was cysteine desulfhydrase. The enzyme had a K{sub m} of 1.32 mM and was inactivated by temperatures greater that 60C. Because cysteine is present in soil and cysteine desulfhydrase is an inducible enzyme, the potential for H{sub 2}S production by this mechanism exists in terrestrial environments.

  15. Reaction of Cysteine(s) with Phenyldichloroarsine

    DTIC Science & Technology

    1990-01-01

    acetyl -L- cysteine reacted like the two Cys-l 3 residucs are spatially not In close L-Cys teine- a nd Iformed a 1:1 adduct when the ratio proximity...were obtained when L- cysteine methyl ester and N- acetyl -L- cysteine 0.0 were used in our studies. For the N- acetyl -L- cysteine , the sample decomposed...the N- acetyl derivatives of L- cysteine .... ... .. , also formed 1:1 adducts, Another possibility is that solvent plays a role in the adducts fornmd

  16. Signaling in the plant cytosol: cysteine or sulfide?

    PubMed

    Gotor, Cecilia; Laureano-Marín, Ana M; Moreno, Inmaculada; Aroca, Ángeles; García, Irene; Romero, Luis C

    2015-10-01

    Cysteine (Cys) is the first organic compound containing reduced sulfur that is synthesized in the last stage of plant photosynthetic assimilation of sulfate. It is a very important metabolite not only because it is crucial for the structure, function and regulation of proteins but also because it is the precursor molecule of an enormous number of sulfur-containing metabolites essential for plant health and development. The biosynthesis of Cys is accomplished by the sequential reaction of serine acetyltransferase (SAT) and O-acetylserine(thiol)synthase (OASTL). In Arabidopsis thaliana, the analysis of specific mutants of members of the SAT and OASTL families has demonstrated that the cytosol is the compartment where the bulk of Cys synthesis takes place and that the cytosolic OASTL enzyme OAS-A1 is the responsible enzyme. Another member of the OASTL family is DES1, a novel L-cysteine desulfhydrase that catalyzes the desulfuration of Cys to produce sulfide, thus acting in a manner opposite to that of OAS-A1. Detailed studies of the oas-a1 and des1 null mutants have revealed the involvement of the DES1 and OAS-A1 proteins in coordinate regulation of Cys homeostasis and the generation of sulfide in the cytosol for signaling purposes. Thus, the levels of Cys in the cytosol strongly affect plant responses to both abiotic and biotic stress conditions, while sulfide specifically generated from the degradation of Cys negatively regulates autophagy induced in different situations. In conclusion, modulation of the levels of Cys and sulfide is likely critical for plant performance.

  17. The Cysteine Proteome

    PubMed Central

    Go, Young-Mi; Chandler, Joshua D.; Jones, Dean P.

    2015-01-01

    The cysteine (Cys) proteome is a major component of the adaptive interface between the genome and the exposome. The thiol moiety of Cys undergoes a range of biologic modifications enabling biological switching of structure and reactivity. These biological modifications include sulfenylation and disulfide formation, formation of higher oxidation states, S-nitrosylation, persulfidation, metallation, and other modifications. Extensive knowledge about these systems and their compartmentalization now provides a foundation to develop advanced integrative models of Cys proteome regulation. In particular, detailed understanding of redox signaling pathways and sensing networks is becoming available to discriminate network structures. This research focuses attention on the need for atlases of Cys modifications to develop systems biology models. Such atlases will be especially useful for integrative studies linking the Cys proteome to imaging and other omics platforms, providing a basis for improved redox-based therapeutics. Thus, a framework is emerging to place the Cys proteome as a complement to the quantitative proteome in the omics continuum connecting the genome to the exposome. PMID:25843657

  18. Assay of cysteine dioxygenase activity

    SciTech Connect

    Bagley, P.J.; Stipanuk, M.H. )

    1990-02-26

    It has been proposed that rat liver contains two cysteine dioxygenase enzymes which convert cysteine to cysteinesulfinic acid, one which is stimulated by NAD{sup +} and has a pH optimum of 6.8 and one which is not stimulated by NAD{sup +} and has a pH optimum of 9.0. This led the authors to reinvestigate assay conditions for measuring cysteine dioxygenase activity in rat liver homogenate. An HPLC method, using an anion exchange column (Dionex Amino-Pac{trademark} PA1 (4x250 mm)) was used to separate the ({sup 35}S)cysteinesulfinic acid produced from ({sup 35}S)cysteine in the incubation mixture. They demonstrated that inclusion of hydroxylamine prevented further metabolism of cysteinesulfinic acid. which occurred rapidly in the absence of hydroxylamine.

  19. Chemical Protein Modification through Cysteine.

    PubMed

    Gunnoo, Smita B; Madder, Annemieke

    2016-04-01

    The modification of proteins with non-protein entities is important for a wealth of applications, and methods for chemically modifying proteins attract considerable attention. Generally, modification is desired at a single site to maintain homogeneity and to minimise loss of function. Though protein modification can be achieved by targeting some natural amino acid side chains, this often leads to ill-defined and randomly modified proteins. Amongst the natural amino acids, cysteine combines advantageous properties contributing to its suitability for site-selective modification, including a unique nucleophilicity, and a low natural abundance--both allowing chemo- and regioselectivity. Native cysteine residues can be targeted, or Cys can be introduced at a desired site in a protein by means of reliable genetic engineering techniques. This review on chemical protein modification through cysteine should appeal to those interested in modifying proteins for a range of applications.

  20. The antihypertensive effect of cysteine

    PubMed Central

    Vasdev, Sudesh; Singal, Pawan; Gill, Vicki

    2009-01-01

    Hypertension is a leading cause of morbidity and mortality worldwide. Individuals with hypertension are at an increased risk for stroke, heart disease and kidney failure. Essential hypertension results from a combination of genetic and lifestyle factors. One such lifestyle factor is diet, and its role in the control of blood pressure has come under much scrutiny. Just as increased salt and sugar are known to elevate blood pressure, other dietary factors may have antihypertensive effects. Studies including the Optimal Macronutrient Intake to Prevent Heart Disease (OmniHeart) study, Multiple Risk Factor Intervention Trial (MRFIT), International Study of Salt and Blood Pressure (INTERSALT) and Dietary Approaches to Stop Hypertension (DASH) study have demonstrated an inverse relationship between dietary protein and blood pressure. One component of dietary protein that may partially account for its antihypertensive effect is the nonessential amino acid cysteine. Studies in hypertensive humans and animal models of hypertension have shown that N-acetylcysteine, a stable cysteine analogue, lowers blood pressure, which substantiates this idea. Cysteine may exert its antihypertensive effects directly or through its storage form, glutathione, by decreasing oxidative stress, improving insulin resistance and glucose metabolism, lowering advanced glycation end products, and modulating levels of nitric oxide and other vasoactive molecules. Therefore, adopting a balanced diet containing cysteine-rich proteins may be a beneficial lifestyle choice for individuals with hypertension. An example of such a diet is the DASH diet, which is low in salt and saturated fat; includes whole grains, poultry, fish and nuts; and is rich in vegetables, fruits and low-fat dairy products. PMID:22477470

  1. S-carboxymethyl-L-cysteine.

    PubMed

    Mitchell, Steve C; Steventon, Glyn B

    2012-05-01

    S-carboxymethyl-L-cysteine, the side-chain carboxymethyl derivative of the sulfur-containing amino acid, cysteine, has been known and available for almost 80 years. During this time, it has been put to a variety of uses, but it is within the field of respiratory medicine that, presently, it has found a clinical niche. Early studies indicated that this compound underwent a rather simplistic, predictable pattern of metabolism, whereas later investigations alluded to more subtle interactions with the pathways of intermediary metabolism, as may be expected for an amino acid derivative. In addition, suggestions of polymorphic influences and circadian rhythms within metabolic profiles have emerged. These latter factors may underlie the conflicting reports regarding the therapeutic efficacy of this compound: that it appears to work well in some patients, but has no measurable effects in others. The relevant literature pertaining to the fate of this compound within living systems has been reviewed and a comprehensive précis advanced. Hopefully, this article will serve as a vade mecum for those interested in S-carboxymethyl-L-cysteine and as a catalyst for future research.

  2. Renouvellement des eaux du fjord du Saguenay

    NASA Astrophysics Data System (ADS)

    Belzile, Melany

    Le fjord du Saguenay, localise dans la region subarctique de l'est du Canada, ala particularite d' etre connecte a un estuaire tres energetique plutot que directement a 1' ocean. L'embouchure du fjord est situee ala rencontre d'un chenal profond et d'un seuil de 20m de profondeur qui limite les echanges d'eau entre le fjord et l'estuaire du Saint-Laurent. Cependant, les grandes amplitudes de maree a son embouchure ont le potentiel d'entrainer des eaux denses du Saint-Laurent a l'interieur du fjord renouvelant ainsi les eaux des differents bassins. Dans le but d' a voir une meilleure comprehension de la dynamique et de la saisonnalite des renouvellements dans le bassin interne, deux mouillages y ont ete deployes pour recolter les premieres donnees de courant, sur toute la colonne d' eau et sur plusieurs mois, ainsi que des observations de temperature et de salinite a differentes profondeurs. L'un de ces mouillages n' a malheureusement pas pu etre analyse en detail dfi a la complexite de ses resultats et au manque de temps. Des profils ont ete recoltes le long de section transversales (transects), couvrant plusieurs saisons et plusieurs annees, ce qui a contribue a une meilleure comprehension de la distribution spatiale des masses d'eau dans le fjord. Les resultats montrent que ladynamique du fjord est plus complexe que ce qui avait ete precedemment presente dans la litterature. Un changement saisonnier abrupte dans la circulation du fjord a ete observe a la rni-fevrier. De plus, des renouvellements non-anticipes juste en-dessous de la thermohalocline (˜ 10 m de profondeur) ont ete observes entre la fin de 1' hiver et le milieu de 1' ete. En fonction des saisons, trois types de renouvellement peuvent etre observes : renouvellements profonds a l'automne et au debut de l'hiver suivis des renouvellements de sous-surface et enfin les renouvellements a des profondeurs intermediaires pendant l'ete. Le changement saisonnier abrupte observe au milieu de l'hiver ainsi que la

  3. "Cirque du Freak."

    ERIC Educational Resources Information Center

    Rivett, Miriam

    2002-01-01

    Considers the marketing strategies that underpin the success of the "Cirque du Freak" series. Describes how "Cirque du Freak" is an account of events in the life of schoolboy Darren Shan. Notes that it is another reworking of the vampire narrative, a sub-genre of horror writing that has proved highly popular with both adult and…

  4. "Cirque du Freak."

    ERIC Educational Resources Information Center

    Rivett, Miriam

    2002-01-01

    Considers the marketing strategies that underpin the success of the "Cirque du Freak" series. Describes how "Cirque du Freak" is an account of events in the life of schoolboy Darren Shan. Notes that it is another reworking of the vampire narrative, a sub-genre of horror writing that has proved highly popular with both adult and…

  5. Cysteine Cathepsins in Human Carious Dentin

    PubMed Central

    Nascimento, F.D.; Minciotti, C.L.; Geraldeli, S.; Carrilho, M.R.; Pashley, D.H.; Tay, F.R.; Nader, H.B.; Salo, T.; Tjäderhane, L.; Tersariol, I.L.S.

    2011-01-01

    Matrix metalloproteinases (MMPs) are important in dentinal caries, and analysis of recent data demonstrates the presence of other collagen-degrading enzymes, cysteine cathepsins, in human dentin. This study aimed to examine the presence, source, and activity of cysteine cathepsins in human caries. Cathepsin B was detected with immunostaining. Saliva and dentin cysteine cathepsin and MMP activities on caries lesions were analyzed spectrofluorometrically. Immunostaining demonstrated stronger cathepsins B in carious than in healthy dentin. In carious dentin, cysteine cathepsin activity increased with increasing depth and age in chronic lesions, but decreased with age in active lesions. MMP activity decreased with age in both active and chronic lesions. Salivary MMP activities were higher in patients with active than chronic lesions and with increasing lesion depth, while cysteine cathepsin activities showed no differences. The results indicate that, along with MMPs, cysteine cathepsins are important, especially in active and deep caries. PMID:21248362

  6. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors.

    PubMed

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; Roush, William R

    2008-11-15

    The importance of cysteine proteases in parasites, compounded with the lack of redundancy compared to their mammalian hosts makes proteases attractive targets for the development of new therapeutic agents. The binding mode of K11002 to cruzain, the major cysteine protease of Trypanosoma cruzi was used in the design of conformationally constrained inhibitors. Vinyl sulfone-containing macrocycles were synthesized via olefin ring-closing metathesis and evaluated against cruzain and the closely related cysteine protease, rhodesain.

  7. Cysteine sensing by plasmons of silver nanocubes

    SciTech Connect

    Elfassy, Eitan Mastai, Yitzhak Salomon, Adi

    2016-09-15

    Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 µM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM). - Highlights: • Silver nanocubes (50 nm length) are used to detect low concentrations of cysteine. • A redshift in the plasmonic modes was observed following cysteine adsorption onto the nanocubes. • The cysteine growth onto the nanocubes is also characterized by TEM.

  8. Cysteine sensing by plasmons of silver nanocubes

    NASA Astrophysics Data System (ADS)

    Elfassy, Eitan; Mastai, Yitzhak; Salomon, Adi

    2016-09-01

    Noble metal nanoparticles are considered to be valuable nanostructures in the field of sensors due to their spectral response sensitivity to small changes in the surrounding refractive index which enables them to detect a small amount of molecules. In this research, we use silver nanocubes of about 50 nm length to detect low concentrations of cysteine, a semi-essential amino acid. Following cysteine adsorption onto the nanocubes, a redshift in the plasmonic modes was observed, enabling the detection of cysteine down to 10 μM and high sensitivity of about 125 nm/RIU (refractive index units). Furthermore, we found that multilayer adsorption of cysteine leads to the stabilization of the silver nanocubes. The cysteine growth onto the nanocubes was also characterized by high-resolution transmission electron microscopy (HR-TEM).

  9. A novel role for methyl cysteinate, a cysteine derivative, in cesium accumulation in Arabidopsis thaliana

    NASA Astrophysics Data System (ADS)

    Adams, Eri; Miyazaki, Takae; Hayaishi-Satoh, Aya; Han, Minwoo; Kusano, Miyako; Khandelia, Himanshu; Saito, Kazuki; Shin, Ryoung

    2017-02-01

    Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were investigated. Through chemical library screening, 14 chemicals were isolated as ‘cesium accumulators’ in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation.

  10. A novel role for methyl cysteinate, a cysteine derivative, in cesium accumulation in Arabidopsis thaliana

    PubMed Central

    Adams, Eri; Miyazaki, Takae; Hayaishi-Satoh, Aya; Han, Minwoo; Kusano, Miyako; Khandelia, Himanshu; Saito, Kazuki; Shin, Ryoung

    2017-01-01

    Phytoaccumulation is a technique to extract metals from soil utilising ability of plants. Cesium is a valuable metal while radioactive isotopes of cesium can be hazardous. In order to establish a more efficient phytoaccumulation system, small molecules which promote plants to accumulate cesium were investigated. Through chemical library screening, 14 chemicals were isolated as ‘cesium accumulators’ in Arabidopsis thaliana. Of those, methyl cysteinate, a derivative of cysteine, was found to function within the plant to accumulate externally supplemented cesium. Moreover, metabolite profiling demonstrated that cesium treatment increased cysteine levels in Arabidopsis. The cesium accumulation effect was not observed for other cysteine derivatives or amino acids on the cysteine metabolic pathway tested. Our results suggest that methyl cysteinate, potentially metabolised from cysteine, binds with cesium on the surface of the roots or inside plant cells and improve phytoaccumulation. PMID:28230101

  11. La naissance du parsec

    NASA Astrophysics Data System (ADS)

    Arenou, F.

    2010-01-01

    Les définitions du parsec et de la magnitude absolue sont le fruit de compromis pour régler trois problèmes entremêlés au début du XXème siècle: quelle unité de distance stellaire adopter? Quel nom lui donner? Comment comparer les luminosités intrinsèques des différentes étoiles?

  12. Entamoeba invadens: characterization of cysteine proteinases.

    PubMed

    Sharma, M; Hirata, K; Herdman, S; Reed, S

    1996-10-01

    Cysteine proteinases have a number of important functions in the life cycle of protozoan parasites. Based on our previous studies demonstrating the role of cysteine proteinases in invasion by Entamoeba histolytica, we evaluated the cysteine proteinases of E. invadens, a related protozoan which causes invasive disease of reptiles. E. invadens readily encysts in axenic culture and provides a model to investigate the role of cysteine proteinases in encystation. Broad bands of approximately 130-230, 55, and 35 kDa were detected on gelatin substrate gels and were inhibited with specific cysteine proteinase inhibitors. Maximal enzymatic activity was detected with peptide substrates containing arginine in the P2 position. A 567-bp fragment containing the active site of an E. invadens cysteine proteinase gene was amplified by PCR and had 37.7, 79.1, and 67.9% identity to the derived amino acid sequences of the acp 1, 2, and 3 genes, respectively, of E. histolytica. The PCR product hybridized with a single band of 1.1 kb on a Southern blot of EcoRI-restricted E. invadens genomic DNA. Long-term inhibition of cysteine proteinase activity during encystation resulted in significantly fewer cysts (P < 0.02); however, this effect appeared to be secondary to decreased trophozoite cell division. No difference in chitin synthase activity was detected between controls and encysting cells with inhibited cysteine proteinases, suggesting that these proteinases are not critical for activation of a zymogen form of chitin synthase. These studies demonstrate that cysteine proteinases may be critical for the survival of E. invadens, and specific inhibition may ultimately interrupt transmission.

  13. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  14. Blends of cysteine-containing proteins

    NASA Astrophysics Data System (ADS)

    Barone, Justin

    2005-03-01

    Many agricultural wastes are made of proteins such as keratin, lactalbumin, gluten, and albumin. These proteins contain the amino acid cysteine. Cysteine allows for the formation of inter-and intra-molecular sulfur-sulfur bonds. Correlations are made between the properties of films made from the proteins and the amino acid sequence. Blends of cysteine-containing proteins show possible synergies in physical properties at intermediate concentrations. FT-IR spectroscopy shows increased hydrogen bonding at intermediate concentrations suggesting that this contributes to increased physical properties. DSC shows limited miscibility and the formation of new crystalline phases in the blends suggesting that this too contributes.

  15. Decreased cysteine and proline synthesis in parenterally fed, premature infants.

    PubMed

    Miller, R G; Jahoor, F; Jaksic, T

    1995-07-01

    Little is known about the amino acid (AA) biosynthetic capacity and requirements of premature infants. This study assessed the synthesis of seven biochemically nonessential AA from a universal precursor, glucose, in stable, parenterally fed, premature neonates. Seven infants (six boys, one girl) were studied at a mean age of 6.3 +/- 0.6 (SEM) days; mean gestational age was 29.7 +/- 1.3 (SEM) weeks, and mean birth weight was 1,222.8 +/- 176.5 (SEM) grams. All infants were parenterally fed a mixture of 7.5% to 12.5% dextrose and 2.2% Trophamine, with or without lipid. Mean caloric intake was 93 +/- 8.4 (SEM) kcal/kg/d, and total AA intake was standardized at 2.86 g/kg/d AA, plus supplemental cysteine (30 mg/g AA/d). Each infant received a 4-hour continuous, unprimed intravenous infusion of a stable isotope tracer of D(-)[U13C] glucose (200 mg/kg). Blood samples were obtained before and at the end of the infusion. Conversion of the glucose tracer into seven biochemically nonessential AA (cysteine [Cys], proline [Pro], aspartate [Asp], serine [Ser], glutamate [Glu], alanine [Ala], and glycine [Gly]) was assessed by measuring their isotopic enrichment in plasma, using gas chromatography/mass spectrometry (GC/MS), and expressed as mole percent excess (MPE) (mean +/- SEM). The isotopic enrichment of plasma glucose was also measured using GC/MS. Free plasma AA concentrations (mean +/- SD) were measured using an automated amino acid analyzer. Mean MPE for M + 1, M + 2 and M + 3 Cys, and for M + 1 and M + 3 Pro were not significantly different from 0; M + 2 Pro barely achieved statistical significance (P = .048).(ABSTRACT TRUNCATED AT 250 WORDS)

  16. Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acid.

    PubMed

    Salsbury, Freddie R; Knutson, Stacy T; Poole, Leslie B; Fetrow, Jacquelyn S

    2008-02-01

    Cysteine sulfenic acid (Cys-SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. This post-translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys-SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKaS of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys-SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites.

  17. Cysteine S-conjugate β-lyases

    PubMed Central

    Cooper, Arthur J. L.; Krasnikov, Boris F.; Pinto, John T.; Bruschi, Sam A.

    2010-01-01

    Cysteine S-conjugate β-lyases are pyridoxal 5′-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2−] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2−] that contain a leaving group in the β position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze β-elimination reactions with such cysteine S-conjugates. All are enzymes involved in amino acid metabolism that do not normally catalyze a β-lyase reaction, but catalyze a non-physiological β-lyase side reaction that depends on the electron-withdrawing properties of the –SR or –SeR moiety. In the case of the cysteine S-conjugates, if the eliminated RSH is stable the compound may be S-thiomethylated and excreted (thiomethyl shunt) or S-glucuronidated and harmlessly excreted [the possibility that RSeH compounds may be similarly metabolized has not been extensively studied]. If, however, RSH is chemically reactive the cysteine S-conjugate may be toxic as a result of the β-lyase reaction. The cysteine S-conjugate β-lyase pathway is of particular interest to toxicologists because it is involved in the bioactivation (toxification) of halogenated alkenes and certain drugs. PMID:20949433

  18. The effect of cysteine oxidation on isolated hepatocytes.

    PubMed Central

    Viña, J; Saez, G T; Wiggins, D; Roberts, A F; Hems, R; Krebs, H A

    1983-01-01

    Isolated hepatocytes incubated with 4mM-cysteine lose reduced glutathione, adenine nucleotides and intracellular enzymes, thus showing extensive membrane damage. The toxic effects of cysteine are enhanced by NH4Cl. Lactate, ethanol and unsaturated fatty acids afford significant protection against cysteine-induced cytoxicity. Addition of catalase to the incubation medium also protected against cysteine toxicity, indicating that H2O2 formed during the oxidation of cysteine is involved in the toxic effects observed. Under anaerobic conditions cysteine did not cause leakage of lactate dehydrogenase from cells, confirming that rapid autoxidation is an essential condition for development of the toxic effects of cysteine. PMID:6870855

  19. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils.

    PubMed

    Hasan, Md Ashraful; Ahn, Won-Gyun; Song, Dong-Keun

    2016-09-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca(2+) signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca(2+)]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca(2+)]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca(2+)]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca(2+)]i in human neutrophils was observed. In Ca(2+)-free buffer, NAC- and cysteine-induced [Ca(2+)]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca(2+)]i in human neutrophils occur through Ca(2+) influx. NAC- and cysteine-induced [Ca(2+)]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na(+)-free HEPES, both NAC and cysteine induced a marked increase in [Ca(2+)]i in human neutrophils, arguing against the possibility that Na(+)-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca(2+)]i increasing activity. Our results show that NAC and cysteine induce [Ca(2+)]i increase through Ca(2+) influx in human neutrophils via SKF96365- and ruthenium red-dependent way.

  20. N-acetyl-L-cysteine and cysteine increase intracellular calcium concentration in human neutrophils

    PubMed Central

    Hasan, Md. Ashraful; Ahn, Won-Gyun

    2016-01-01

    N-acetyl-L-cysteine (NAC) and cysteine have been implicated in a number of human neutrophils' functional responses. However, though Ca2+ signaling is one of the key signalings contributing to the functional responses of human neutrophils, effects of NAC and cysteine on intracellular calcium concentration ([Ca2+]i) in human neutrophils have not been investigated yet. Thus, this study was carried out with an objective to investigate the effects of NAC and cysteine on [Ca2+]i in human neutrophils. We observed that NAC (1 µM ~ 1 mM) and cysteine (10 µM ~ 1 mM) increased [Ca2+]i in human neutrophils in a concentration-dependent manner. In NAC pre-supplmented buffer, an additive effect on N-formyl-methionine-leucine-phenylalanine (fMLP)-induced increase in [Ca2+]i in human neutrophils was observed. In Ca2+-free buffer, NAC- and cysteine-induced [Ca2+]i increase in human neutrophils completely disappeared, suggesting that NAC- and cysteine-mediated increase in [Ca2+]i in human neutrophils occur through Ca2+ influx. NAC- and cysteine-induced [Ca2+]i increase was effectively inhibited by calcium channel inhibitors SKF96365 (10 µM) and ruthenium red (20 µM). In Na+-free HEPES, both NAC and cysteine induced a marked increase in [Ca2+]i in human neutrophils, arguing against the possibility that Na+-dependent intracellular uptake of NAC and cysteine is necessary for their [Ca2+]i increasing activity. Our results show that NAC and cysteine induce [Ca2+]i increase through Ca2+ influx in human neutrophils via SKF96365- and ruthenium red-dependent way. PMID:27610031

  1. Cysteine Transport into Cultured Tobacco Cells

    PubMed Central

    Harrington, H. Michael; Smith, Ivan K.

    1977-01-01

    Cysteine transport by tobacco cells (Nicotiana tabacum L. var. Xanthi) cultured on liquid B-5 medium was examined. Transport was linear with time or amount of tissue and had a pH optimum of 4.5. Cysteine transport over a wide concentration range was biphasic. The isotherm, for descriptive convenience, was divided into two segments both of which obeyed Michaelis-Menten kinetics. The Km for high affinity transport was in the range 1.7 × 10−5m(±0.17) while the Km for low affinity transport was in the range 3.5 × 10−4m(±0.13). Maximum velocities were 3 to 6 nmoles/g fresh weight/minute and 13 to 16 nmoles/g fresh weight/minute, respectively. Azide and 2,4-dinitrophenol caused more than 90% inhibition of net transport by either system. N,N′-Dicyclohexylcarbodiimide was not inhibitory while the inhibition by carbonylcyanide m-chlorophenylhydrazone was dependent on the cysteine concentration. Only those compounds that were inhibitory to transport caused significant efflux of labeled material from preloaded cells. Tobacco cells that had been preincubated in iodoacetamide or N-ethylmaleimide did not transport cysteine while similar treatments with dithiothreitol were only slightly inhibitory or had no effect on transport. Transport by either system was, to some extent, inhibited by all other tested amino acids and analogs. Alanine, methionine, and S-methyl cysteine were most effective in inhibiting cysteine transport. Both alanine and methionine were competitive inhibitors of cysteine transport by either system with inhibition constants that were similar to the Km for the particular system. PMID:16660190

  2. Crystal Structure of Mammalian Cysteine dioxygenase: A Novel Mononuclear Iron Center for Cysteine Thiol Oxidation

    SciTech Connect

    Simmons,C.; Liu, Q.; Huang, Q.; Hao, Q.; Begley, T.; Karplus, P.; Stipanuk, M.

    2006-01-01

    Cysteine dioxygenase is a mononuclear iron-dependent enzyme responsible for the oxidation of cysteine with molecular oxygen to form cysteinesulfinate. This reaction commits cysteine to either catabolism to sulfate and pyruvate or to the taurine biosynthetic pathway. Cysteine dioxygenase is a member of the cupin superfamily of proteins. The crystal structure of recombinant rat cysteine dioxygenase has been determined to 1.5 Angstroms resolution, and these results confirm the canonical cupin {beta}-sandwich fold and the rare cysteinyl-tyrosine intramolecular crosslink (between Cys93 and Tyr157) seen in the recently reported murine cysteine dioxygenase structure. In contrast to the catalytically inactive mononuclear Ni(II) metallocenter present in the murine structure, crystallization of a catalytically competent preparation of rat cysteine dioxygenase revealed a novel tetrahedrally coordinated mononuclear iron center involving three histidines (His86, His88, and His140) and a water molecule. Attempts to acquire a structure with bound ligand using either co-crystallization or soaks with cysteine revealed the formation of a mixed disulfide involving Cys164 near the active site, which may explain previously observed substrate inhibition. This work provides a framework for understanding the molecular mechanisms involved in thiol dioxygenation and sets the stage for exploring the chemistry of both the novel mononuclear iron center and the catalytic role of the cysteinyl-tyrosine linkage.

  3. Reconstruction of Cysteine Biosynthesis Using Engineered Cysteine-Free and Methionine-Free Enzymes

    NASA Technical Reports Server (NTRS)

    Wang, Kendrick; Fujishima, Kosuke; Abe, Nozomi; Nakahigashi, Kenji; Endy, Drew; Rothschild, Lynn J.

    2016-01-01

    Ten of the proteinogenic amino acids can be generated abiotically while the remaining thirteen require biology for their synthesis. Paradoxically, the biosynthesis pathways observed in nature require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine. Here, we substituted alternate amino acids for cysteine and also methionine, which is biosynthesized from cysteine, in serine acetyl transferase (CysE) and O-acetylserine sulfhydrylase (CysM). CysE function was rescued by cysteine-and-methionine-free enzymes and CysM function was rescued by cysteine-free enzymes. Structural modeling suggests that methionine stabilizes CysM and is present in the active site of CysM. Cysteine is not conserved among CysE and CysM protein orthologs, suggesting that cysteine is not functionally important for its own synthesis. Engineering biosynthetic enzymes that lack the amino acids being synthesized provides insights into the evolution of amino acid biosynthesis and pathways for bioengineering.

  4. Cysteine biosynthesis in Trichomonas vaginalis involves cysteine synthase utilizing O-phosphoserine

    PubMed Central

    Westrop, Gareth D.; Goodall, Gordon; Mottram, Jeremy C.; Coombs, Graham H.

    2009-01-01

    Trichomonas vaginalis is an early divergent eukaryote with many unusual biochemical features. It is an anaerobic protozoan parasite of humans that is thought to rely heavily on cysteine as a major redox buffer, as it lacks glutathione. We report here that for synthesis of cysteine from sulphide, T. vaginalis relies upon cysteine synthase. The enzyme (TvCS1) can use as substrates either O-acetylserine or O-phosphoserine. The Kms of the enzyme for sulphide is very low (0.02 mM), suggesting that the enzyme may be a means of ensuring that sulphide in the parasite is maintained at a low level. T. vaginalis appears to lack serine acetyltransferase, the source of O-acetylserine in many cells, but has a functional 3-phosphoglycerate dehydrogenase and an O-phosphoserine aminotransferase that together result in the production of O-phosphoserine, suggesting that this is the physiological substrate. TvCS1 can also use thiosulphate as substrate. Overall, TvCS1 has substrate specificities similar to those reported for cysteine synthases of Aeropyrum pernix and Escherichia coli and this is reflected by sequence similarities around the active site. We suggest that these enzymes are classified together as type B cysteine synthases and we hypothesise that the use of O-phosphoserine is a common characteristic of these cysteine synthases. The level of cysteine synthase in T. vaginalis is regulated according to need, such that parasites growing in an environment rich in cysteine have low activity, whereas exposure to propargylglycine results in elevated cysteine synthase activity. Humans lack cysteine synthase, thus this parasite enzyme could be an exploitable drug target. PMID:16735516

  5. π-Clamp-mediated cysteine conjugation

    NASA Astrophysics Data System (ADS)

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; van Voorhis, Troy; Pentelute, Bradley L.

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the ‘π-clamp’, that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  6. Π-Clamp-mediated cysteine conjugation.

    PubMed

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J; Santos, Michael S; Van Voorhis, Troy; Pentelute, Bradley L

    2016-02-01

    Site-selective functionalization of complex molecules is one of the most significant challenges in chemistry. Typically, protecting groups or catalysts must be used to enable the selective modification of one site among many that are similarly reactive, and general strategies that selectively tune the local chemical environment around a target site are rare. Here, we show a four-amino-acid sequence (Phe-Cys-Pro-Phe), which we call the 'π-clamp', that tunes the reactivity of its cysteine thiol for site-selective conjugation with perfluoroaromatic reagents. We use the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues. These examples include antibodies and cysteine-based enzymes that would be difficult to modify selectively using standard cysteine-based methods. Antibodies modified using the π-clamp retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach to mediate site-selective chemistry and provides new avenues to modify biomolecules for research and therapeutics.

  7. π-Clamp Mediated Cysteine Conjugation

    PubMed Central

    Zhang, Chi; Welborn, Matthew; Zhu, Tianyu; Yang, Nicole J.; Santos, Michael S.; Van Voorhis, Troy; Pentelute, Bradley L.

    2016-01-01

    Site-selective functionalization of complex molecules is a grand challenge in chemistry. Protecting groups or catalysts must be used to selectively modify one site among many that are similarly reactive. General strategies are rare such the local chemical environment around the target site is tuned for selective transformation. Here we show a four amino acid sequence (Phe-Cys-Pro-Phe), which we call the “π-clamp”, tunes the reactivity of its cysteine thiol for the site-selective conjugation with perfluoroaromatic reagents. We used the π-clamp to selectively modify one cysteine site in proteins containing multiple endogenous cysteine residues (e.g. antibodies and cysteine-based enzymes), which was impossible with prior cysteine modification methods. The modified π-clamp antibodies retained binding affinity to their targets, enabling the synthesis of site-specific antibody-drug conjugates (ADCs) for selective killing of HER2-positive breast cancer cells. The π-clamp is an unexpected approach for site-selective chemistry and provides opportunities to modify biomolecules for research and therapeutics. PMID:26791894

  8. Determining Cysteines Available for Covalent Inhibition Across the Human Kinome.

    PubMed

    Zhao, Zheng; Liu, Qingsong; Bliven, Spencer; Xie, Lei; Bourne, Philip E

    2017-04-13

    Covalently bound protein kinase inhibitors have been frequently designed to target noncatalytic cysteines at the ATP binding site. Thus, it is important to know if a given cysteine can form a covalent bond. Here we combine a function-site interaction fingerprint method and DFT calculations to determine the potential of cysteines to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site cysteine data set was assembled. The orientation of the cysteine thiol group indicates which cysteines can potentially form covalent bonds. These covalent inhibitor easy-available cysteines are located within five regions: P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop, and DFG-3 close to the DFG peptide. In an independent test set these cysteines covered 95% of covalent kinase inhibitors. This study provides new insights into cysteine reactivity and preference which is important for the prospective development of covalent kinase inhibitors.

  9. Review stapling peptides using cysteine crosslinking.

    PubMed

    Fairlie, David P; Dantas de Araujo, Aline

    2016-11-01

    Stapled peptides are an emerging class of cyclic peptide molecules with enhanced biophysical properties such as conformational and proteolytic stability, cellular uptake and elevated binding affinity and specificity for their biological targets. Among the limited number of chemistries available for their synthesis, the cysteine-based stapling strategy has received considerable development in the last few years driven by facile access from cysteine-functionalized peptide precursors. Here we present some recent advances in peptide and protein stapling where the side-chains of cysteine residues are covalently connected with a range of different crosslinkers affording bisthioether macrocyclic peptides of varying topology and biophysical properties. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 843-852, 2016.

  10. The cysteine proteinases of the pineapple plant.

    PubMed Central

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-01-01

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct. Images Fig. 4. Fig. 5. PMID:2327970

  11. The cysteine proteinases of the pineapple plant.

    PubMed

    Rowan, A D; Buttle, D J; Barrett, A J

    1990-03-15

    The pineapple plant (Ananas comosus) was shown to contain at least four distinct cysteine proteinases, which were purified by a procedure involving active-site-directed affinity chromatography. The major proteinase present in extracts of plant stem was stem bromelain, whilst fruit bromelain was the major proteinase in the fruit. Two additional cysteine proteinases were detected only in the stem: these were ananain and a previously undescribed enzyme that we have called comosain. Stem bromelain, fruit bromelain and ananain were shown to be immunologically distinct. Enzymic characterization revealed differences in both substrate-specificities and inhibition profiles. A study of the cysteine proteinase derived from the related bromeliad Bromelia pinguin (pinguinain) indicated that in many respects it was similar to fruit bromelain, although it was found to be immunologically distinct.

  12. Cysteine Prevents Menopausal Syndromes in Ovariectomized Mouse.

    PubMed

    Han, Na-Ra; Kim, Na-Rae; Kim, Hyung-Min; Jeong, Hyun-Ja

    2016-05-01

    Cysteine (Cys) is well known to be involved in oxidation-reduction reactions, serving as a source of sulfides in the body. Amino acids are known to improve menopausal symptoms and significantly reduce morbidity. This study aims to find an unrevealed effect of Cys with estrogenic and osteogenic actions. Ovariectomized (OVX) mice were treated with Cys daily for 8 weeks. Estrogen-related and osteoporosis-related factors were analyzed in the vagina, serum, and tibia. Cys was treated in estrogen receptor (ER)-positive human osteoblast-like MG-63 cells and ER-positive human breast cancer Michigan Cancer Foundation-7 (MCF-7) cells. Cysteine administration ameliorated overweightness of the body and vaginal atrophy in the OVX mice. Cysteine increased the levels of alkaline phosphatase (ALP) and 17β-estradiol in the serum of the OVX mice and improved the bone mineral density in the OVX mice. In MG-63 cells, Cys increased the proliferation, ERβ messenger RNA (mRNA) expression, and estrogen response element (ERE) activity. Cysteine increased the ALP activity and the phosphorylation of extracellular signal-regulated kinase. In MCF-7 cells, Cys also increased the proliferation, ERβ mRNA expression, and ERE activity. Taken together, these results demonstrated that Cys has estrogenic and osteogenic activities in OVX mice, MG-63 cells, and MCF-7 cells. The novel insights gained here strongly imply the potential use of Cys as a new agent for postmenopausal women.

  13. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements...

  14. Cysteine Modifications in the Pathogenesis of ALS

    PubMed Central

    Valle, Cristiana; Carrì, Maria Teresa

    2017-01-01

    Several proteins are found misfolded and aggregated in sporadic and genetic forms of amyotrophic lateral sclerosis (ALS). These include superoxide dismutase (SOD1), transactive response DNA-binding protein (TDP-43), fused in sarcoma/translocated in liposarcoma protein (FUS/TLS), p62, vasolin-containing protein (VCP), Ubiquilin-2 and dipeptide repeats produced by unconventional RAN-translation of the GGGGCC expansion in C9ORF72. Up to date, functional studies have not yet revealed a common mechanism for the formation of such diverse protein inclusions. Consolidated studies have demonstrated a fundamental role of cysteine residues in the aggregation process of SOD1 and TDP43, but disturbance of protein thiols homeostatic factors such as protein disulfide isomerases (PDI), glutathione, cysteine oxidation or palmitoylation might contribute to a general aberration of cysteine residues proteostasis in ALS. In this article we review the evidence that cysteine modifications may have a central role in many, if not all, forms of this disease. PMID:28167899

  15. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  16. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 6 2011-04-01 2011-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  17. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  18. 21 CFR 582.5271 - Cysteine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 6 2014-04-01 2014-04-01 false Cysteine. 582.5271 Section 582.5271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS, FEEDS, AND RELATED PRODUCTS SUBSTANCES GENERALLY RECOGNIZED AS SAFE Nutrients and/or Dietary Supplements 1...

  19. Characterization of the Cysteine Content in Proteins Utilizing Cysteine Selenylation with 266 nm Ultraviolet Photodissociation (UVPD)

    NASA Astrophysics Data System (ADS)

    Parker, W. Ryan; Brodbelt, Jennifer S.

    2016-08-01

    Characterization of the cysteine content of proteins is a key aspect of proteomics. By defining both the total number of cysteines and their bound/unbound state, the number of candidate proteins considered in database searches is significantly constrained. Herein we present a methodology that utilizes 266 nm UVPD to count the number of free and bound cysteines in intact proteins. In order to attain this goal, proteins were derivatized with N-(phenylseleno)phthalimide (NPSP) to install a selectively cleavable Se-S bond upon 266 UVPD. The number of Se-S bonds cleaved upon UVPD, a process that releases SePh moieties, corresponds to the number of cysteine residues per protein.

  20. Chasing Cysteine Oxidative Modifications: Proteomic Tools for Characterizing Cysteine Redox-Status

    PubMed Central

    Murray, Christopher I.; Van Eyk, Jennifer E.

    2012-01-01

    Redox-proteomics involves the large scale analysis of oxidative protein post-translational modifications. In particular, cysteine residues have become the subject of intensifying research interest because of their redox-reactive thiol side chain. Certain reactive cysteine residues can function as redox-switches, which sense changes in the local redox-environment by flipping between the reduced and oxidized state. Depending on the reactive oxygen or nitrogen species, cysteine residues can receive one of several oxidative modifications, each with the potential to confer a functional effect. Modification of these redox-switches has been found to play an important role in oxidative-signaling in the cardiovascular system and elsewhere. Due to the labile and dynamic nature of these modifications, several targeted approaches have been developed to enrich, identify and characterize the status of these critical residues. Here, we review the various proteomic strategies and limitations for the large scale analysis of the different oxidative cysteine modifications. PMID:23074338

  1. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in §...

  2. Cysteine transport through excitatory amino acid transporter 3 (EAAT3).

    PubMed

    Watts, Spencer D; Torres-Salazar, Delany; Divito, Christopher B; Amara, Susan G

    2014-01-01

    Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1-5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1-3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest

  3. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in § 170.3(o...

  4. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3... of total L-cysteine per 100 parts of flour in dough as a dough strengthener as defined in § 170.3(o...

  5. Determining cysteine oxidation status using differential alkylation

    NASA Astrophysics Data System (ADS)

    Schilling, Birgit; Yoo, Chris B.; Collins, Christopher J.; Gibson, Bradford W.

    2004-08-01

    Oxidative damage to proteins plays a major role in aging and in the pathology of many degenerative diseases. Under conditions of oxidative stress, reactive oxygen and nitrogen species can modify key redox sensitive amino acid side chains leading to altered biological activities or structures of the targeted proteins. This in turn can affect signaling or regulatory control pathways as well as protein turnover and degradation efficiency in the proteasome. Cysteine residues are particularly susceptible to oxidation, primarily through reversible modifications (e.g., thiolation and nitrosylation), although irreversible oxidation can lead to products that cannot be repaired in vivo such as sulfonic acid. This report describes a strategy to determine the overall level of reversible cysteine oxidation using a stable isotope differential alkylation approach in combination with mass spectrometric analysis. This method employs 13C-labeled alkylating reagents, such as N-ethyl-[1,4-13C2]-maleimide, bromo-[1,2-13C2]-acetic acid and their non-labeled counterparts to quantitatively assess the level of cysteine oxidation at specific sites in oxidized proteins. The differential alkylation protocol was evaluated using standard peptides and proteins, and then applied to monitor and determine the level of oxidative damage induced by diamide, a mild oxidant. The formation and mass spectrometric analysis of irreversible cysteine acid modification will also be discussed as several such modifications have been identified in subunits of the mitochondrial electron transport chain complexes. This strategy will hopefully contribute to our understanding of the role that cysteine oxidation plays in such chronic diseases such as Parkinson's disease, where studies in animal and cell models have shown oxidative damage to mitochondrial Complex I to be a specific and early target.

  6. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl...

  7. Cysteine cathepsin activity regulation by glycosaminoglycans.

    PubMed

    Novinec, Marko; Lenarčič, Brigita; Turk, Boris

    2014-01-01

    Cysteine cathepsins are a group of enzymes normally found in the endolysosomes where they are primarily involved in intracellular protein turnover but also have a critical role in MHC II-mediated antigen processing and presentation. However, in a number of pathologies cysteine cathepsins were found to be heavily upregulated and secreted into extracellular milieu, where they were found to degrade a number of extracellular proteins. A major role in modulating cathepsin activities play glycosaminoglycans, which were found not only to facilitate their autocatalytic activation including at neutral pH, but also to critically modulate their activities such as in the case of the collagenolytic activity of cathepsin K. The interaction between cathepsins and glycosaminoglycans will be discussed in more detail.

  8. Quantitative reactivity profiling predicts functional cysteines in proteomes

    PubMed Central

    Weerapana, Eranthie; Wang, Chu; Simon, Gabriel M.; Richter, Florian; Khare, Sagar; Dillon, Myles B.D.; Bachovchin, Daniel A.; Mowen, Kerri; Baker, David; Cravatt, Benjamin F.

    2010-01-01

    Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here, we describe a proteomics method to quantitatively profile the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyperreactivity was a rare feature among cysteines and found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyperreactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and involved in iron-sulfur protein biogenesis. Finally, we demonstrate that quantitative reactivity profiling can also form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs. PMID:21085121

  9. Identification of non-peptidic cysteine reactive fragments as inhibitors of cysteine protease rhodesain.

    PubMed

    McShan, Danielle; Kathman, Stefan; Lowe, Brittiney; Xu, Ziyang; Zhan, Jennifer; Statsyuk, Alexander; Ogungbe, Ifedayo Victor

    2015-10-15

    Rhodesain, the major cathepsin L-like cysteine protease in the protozoan Trypanosoma brucei rhodesiense, the causative agent of African sleeping sickness, is a well-validated drug target. In this work, we used a fragment-based approach to identify inhibitors of this cysteine protease, and identified inhibitors of T. brucei. To discover inhibitors active against rhodesain and T. brucei, we screened a library of covalent fragments against rhodesain and conducted preliminary SAR studies. We envision that in vitro enzymatic assays will further expand the use of the covalent tethering method, a simple fragment-based drug discovery technique to discover covalent drug leads.

  10. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K.

    2009-10-13

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  11. Cysteine-containing peptides having antioxidant properties

    DOEpatents

    Bielicki, John K.

    2008-10-21

    Cysteine containing amphipathic alpha helices of the exchangeable apolipoproteins, as exemplified by apolipoprotein (apo) A-I.sub.Milano (R173C) and apoA-I.sub.Paris, (R151C) were found to exhibit potent antioxidant activity on phospholipid surfaces. The addition of a free thiol, at the hydrophobic/hydrophilic interface of an amphipathic alpha helix of synthetic peptides that mimic HDL-related proteins, imparts a unique antioxidant activity to these peptides which inhibits lipid peroxidation and protects phospholipids from water-soluble free radical initiators. These peptides can be used as therapeutic agents to combat cardiovascular disease, ischemia, bone disease and other inflammatory related diseases.

  12. Characterization of Two Cysteine Transfer RNA Genes from Xenopus Laevis

    DTIC Science & Technology

    1984-07-12

    author hereby certifies that the use of any copyrighted material in the dissertation manuscript entitled: "Characterization of two cysteine tRNA genes...Uniformed Services University of the Health Sciences 11 ABSTRACT Title of Thesis: Characterization of Two Cysteine Transfer RNA Genes from Xenopus...method after constructing a set of deletions and reclonlng into the plasmid pUC 8. The DNA fragment is 1737 bp long and contains two cysteine tRNA genes

  13. Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans

    PubMed Central

    Hennicke, Florian; Grumbt, Maria; Lermann, Ulrich; Ueberschaar, Nico; Palige, Katja; Böttcher, Bettina; Jacobsen, Ilse D.; Staib, Claudia; Morschhäuser, Joachim; Monod, Michel; Hube, Bernhard; Hertweck, Christian

    2013-01-01

    The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity. PMID:23417561

  14. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 3 2012-04-01 2012-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl H2O...

  15. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 3 2013-04-01 2013-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl H2O...

  16. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 3 2011-04-01 2011-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184... Listing of Specific Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical L-2-amino-3-mercaptopropanoic acid monohydrochloride monohydrate (C3H7O2NS HCl H2O...

  17. Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis.

    PubMed

    Wawrzyńska, Anna; Kurzyk, Agata; Mierzwińska, Monika; Płochocka, Danuta; Wieczorek, Grzegorz; Sirko, Agnieszka

    2013-06-01

    Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation.

  18. Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

    PubMed Central

    Watts, Spencer D.; Torres-Salazar, Delany; Divito, Christopher B.; Amara, Susan G.

    2014-01-01

    Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest

  19. Peptide-formation on cysteine-containing peptide scaffolds

    NASA Technical Reports Server (NTRS)

    Chu, B. C.; Orgel, L. E.

    1999-01-01

    Monomeric cysteine residues attached to cysteine-containing peptides by disulfide bonds can be activated by carbonyldiimidazole. If two monomeric cysteine residues, attached to a 'scaffold' peptide Gly-Cys-Glyn-Cys-Glu10, (n = 0, 1, 2, 3) are activated, they react to form the dipeptide Cys-Cys. in 25-65% yield. Similarly, the activation of a cysteine residue attached to the 'scaffold' peptide Gly-Cys-Gly-Glu10 in the presence of Arg5 leads to the formation of Cys-Arg5 in 50% yield. The significance of these results for prebiotic chemistry is discussed.

  20. Structure of Leishmania major cysteine synthase

    PubMed Central

    Fyfe, Paul K.; Westrop, Gareth D.; Ramos, Tania; Müller, Sylke; Coombs, Graham H.; Hunter, William N.

    2012-01-01

    Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the asymmetric unit. Diffraction data extending to 1.8 Å resolution were measured and the structure was solved by molecular replacement. A fragment of γ-poly-d-glutamic acid, a constituent of the crystallization mixture, was bound in the enzyme active site. Although a d-­glutamate tetrapeptide had insignificant inhibitory activity, the enzyme was competitively inhibited (K i = 4 µM) by DYVI, a peptide based on the C-­terminus of the partner serine acetyltransferase with which the enzyme forms a complex. The structure surprisingly revealed that the cofactor pyridoxal phosphate had been lost during crystallization. PMID:22750854

  1. DBU-catalyzed transprotection of N-Fmoc-cysteine di- and tripeptides into S-Fm-cysteine di- and tripeptides.

    PubMed

    Katritzky, Alan R; Abo-Dya, Nader E; Abdelmajeid, Abdelmotaal; Tala, Srinivasa R; Amine, M S; El-Feky, Said A

    2011-01-21

    The transprotection of N-Fmoc-cysteine containing di- and tripeptides possessing a free SH group to produce the corresponding S-Fm-cysteine di- and tripeptides bearing a free amino group is accomplished efficiently with DBU in dry THF. The N-Fmoc to S-Fm transformation mechanism is discussed. S-Fm-Cysteine di- and tripeptides readily form amide bonds on coupling with N-(Pg-α-aminoacyl)benzotriazoles and N-(Pg-α-dipeptidoyl)benzotriazoles to give larger peptides.

  2. Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification.

    PubMed

    Wible, Ryan S; Sutter, Thomas R

    2017-03-20

    The unique biophysical and electronic properties of cysteine make this molecule one of the most biologically critical amino acids in the proteome. The defining sulfur atom in cysteine is much larger than the oxygen and nitrogen atoms more commonly found in the other amino acids. As a result of its size, the valence electrons of sulfur are highly polarizable. Unique protein microenvironments favor the polarization of sulfur, thus increasing the overt reactivity of cysteine. Here, we provide a brief overview of the endogenous generation of reactive oxygen and electrophilic species and specific examples of enzymes and transcription factors in which the oxidation or covalent modification of cysteine in those proteins modulates their function. The perspective concludes with a discussion of cysteine chemistry and biophysics, the hard and soft acids and bases model, and the proposal of the Soft Cysteine Signaling Network: a hypothesis proposing the existence of a complex signaling network governed by layered chemical reactivity and cross-talk in which the chemical modification of reactive cysteine in biological networks triggers the reorganization of intracellular biochemistry to mitigate spikes in endogenous or exogenous oxidative or electrophilic stress.

  3. Cri du Chat syndrome

    PubMed Central

    Cerruti Mainardi, Paola

    2006-01-01

    The Cri du Chat syndrome (CdCS) is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-). The incidence ranges from 1:15,000 to 1:50,000 live-born infants. The main clinical features are a high-pitched monochromatic cry, microcephaly, broad nasal bridge, epicanthal folds, micrognathia, abnormal dermatoglyphics, and severe psychomotor and mental retardation. Malformations, although not very frequent, may be present: cardiac, neurological and renal abnormalities, preauricular tags, syndactyly, hypospadias, and cryptorchidism. Molecular cytogenetic analysis has allowed a cytogenetic and phenotypic map of 5p to be defined, even if results from the studies reported up to now are not completely in agreement. Genotype-phenotype correlation studies showed a clinical and cytogenetic variability. The identification of phenotypic subsets associated with a specific size and type of deletion is of diagnostic and prognostic relevance. Specific growth and psychomotor development charts have been established. Two genes, Semaphorin F (SEMAF) and δ-catenin (CTNND2), which have been mapped to the "critical regions", are potentially involved in cerebral development and their deletion may be associated with mental retardation in CdCS patients. Deletion of the telomerase reverse transcriptase (hTERT) gene, localised to 5p15.33, could contribute to the phenotypic changes in CdCS. The critical regions were recently refined by using array comparative genomic hybridisation. The cat-like cry critical region was further narrowed using quantitative polymerase chain reaction (PCR) and three candidate genes were characterised in this region. The diagnosis is based on typical clinical manifestations. Karyotype analysis and, in doubtful cases, FISH analysis will confirm the diagnosis. There is no specific therapy for CdCS but early rehabilitative and educational interventions improve the prognosis and considerable progress has been made in

  4. Cri du Chat syndrome.

    PubMed

    Cerruti Mainardi, Paola

    2006-09-05

    The Cri du Chat syndrome (CdCS) is a genetic disease resulting from a deletion of variable size occurring on the short arm of chromosome 5 (5p-). The incidence ranges from 1:15,000 to 1:50,000 live-born infants. The main clinical features are a high-pitched monochromatic cry, microcephaly, broad nasal bridge, epicanthal folds, micrognathia, abnormal dermatoglyphics, and severe psychomotor and mental retardation. Malformations, although not very frequent, may be present: cardiac, neurological and renal abnormalities, preauricular tags, syndactyly, hypospadias, and cryptorchidism. Molecular cytogenetic analysis has allowed a cytogenetic and phenotypic map of 5p to be defined, even if results from the studies reported up to now are not completely in agreement. Genotype-phenotype correlation studies showed a clinical and cytogenetic variability. The identification of phenotypic subsets associated with a specific size and type of deletion is of diagnostic and prognostic relevance. Specific growth and psychomotor development charts have been established. Two genes, Semaphorin F (SEMAF) and delta-catenin (CTNND2), which have been mapped to the "critical regions", are potentially involved in cerebral development and their deletion may be associated with mental retardation in CdCS patients. Deletion of the telomerase reverse transcriptase (hTERT) gene, localised to 5p15.33, could contribute to the phenotypic changes in CdCS. The critical regions were recently refined by using array comparative genomic hybridisation. The cat-like cry critical region was further narrowed using quantitative polymerase chain reaction (PCR) and three candidate genes were characterised in this region. The diagnosis is based on typical clinical manifestations. Karyotype analysis and, in doubtful cases, FISH analysis will confirm the diagnosis. There is no specific therapy for CdCS but early rehabilitative and educational interventions improve the prognosis and considerable progress has been made

  5. Electrons initiate efficient formation of hydroperoxides from cysteine.

    PubMed

    Gebicki, Janusz M

    2016-09-01

    Amino acid and protein hydroperoxides can constitute a significant hazard if formed in vivo. It has been suggested that cysteine can form hydroperoxides after intramolecular hydrogen transfer to the commonly produced cysteine sulfur-centered radical. The resultant cysteine-derived carbon-centered radicals can react with oxygen at almost diffusion-controlled rate, forming peroxyl radicals which can oxidize other molecules and be reduced to hydroperoxides in the process. No cysteine hydroperoxides have been found so far. In this study, dilute air-saturated cysteine solutions were exposed to radicals generated by ionizing radiation and the hydroperoxides measured by an iodide assay. Of the three primary radicals present, the hydroxyl, hydrogen atoms and hydrated electrons, the first two were ineffective. However, electrons did initiate the generation of hydroperoxides by removing the -SH group and forming cysteine-derived carbon radicals. Under optimal conditions, 100% of the electrons reacting with cysteine produced the hydroperoxides with a 1:1 stoichiometry. Maximum hydroperoxide yields were at pH 5.5, with fairly rapid decline under more acid or alkaline conditions. The hydroperoxides were stable between pH 3 and 7.5, and decomposed in alkaline solutions. The results suggest that formation of cysteine hydroperoxides initiated by electrons is an unlikely event under physiological conditions.

  6. Fluorescent nitrile-based inhibitors of cysteine cathepsins.

    PubMed

    Frizler, Maxim; Mertens, Matthias D; Gütschow, Michael

    2012-12-15

    Cysteine cathepsins play an important role in many (patho)physiological conditions. Among them, cathepsins L, S, K and B are subjects of several drug discovery programs. Besides their role as drug targets, cysteine cathepsins are additionally considered to be possible biomarkers for inflammation and cancer. Herein, we describe the design, synthesis, biological evaluation and spectral properties of fluorescently labeled dipeptide- and azadipeptide nitriles.

  7. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... Substances Affirmed as GRAS § 184.1271 L-Cysteine. (a) L-Cysteine is the chemical L-2-amino-3-mercaptopropanoic acid (C3H7O2NS). (b) The ingredient meets the appropriate part of the specification set forth in...

  8. Role of cysteines in mammalian VDAC isoforms' function.

    PubMed

    De Pinto, Vito; Reina, Simona; Gupta, Ankit; Messina, Angela; Mahalakshmi, Radhakrishnan

    2016-08-01

    In this mini-review, we analyze the influence of cysteines in the structure and activity of mitochondrial outer membrane mammalian VDAC isoforms. The three VDAC isoforms show conserved sequences, similar structures and the same gene organization. The meaning of three proteins encoded in different chromosomes must thus be searched for subtle differences at the amino acid level. Among others, cysteine content is noticeable. In humans, VDAC1 has 2, VDAC2 has 9 and VDAC3 has 6 cysteines. Recent works have shown that, at variance from VDAC1, VDAC2 and VDAC3 exhibit cysteines predicted to protrude towards the intermembrane space, making them a preferred target for oxidation by ROS. Mass spectrometry in VDAC3 revealed that a disulfide bridge can be formed and other cysteine oxidations are also detectable. Both VDAC2 and VDAC3 cysteines were mutagenized to highlight their role in vitro and in complementation assays in Δporin1 yeast. Chemico-physical techniques revealed an important function of cysteines in the structural stabilization of the pore. In conclusion, the works available on VDAC cysteines support the notion that the three proteins are paralogs with a similar pore-function and slightly different, but important, ancillary biological functions. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

  9. A novel cysteine desulfurase influencing organosulfur compounds in Lentinula edodes

    PubMed Central

    Liu, Ying; Lei, Xiao-Yu; Chen, Lian-Fu; Bian, Yin-Bing; Yang, Hong; Ibrahim, Salam A.; Huang, Wen

    2015-01-01

    Organosulfur compounds are the basis for the unique aroma of Lentinula edodes, and cysteine sulfoxide lyase (C-S lyase) is the key enzyme in this trait. The enzyme from Alliium sativum has been crystallized and well-characterized; however, there have been no reports of the characterization of fungi C-S lyase at the molecular level. We identified a L. edodes C-S lyase (Lecsl), cloned a gene of Csl encoded Lecsl and then combined modeling, simulations, and experiments to understand the molecular basis of the function of Lecsl. Our analysis revealed Lecsl to be a novel cysteine desulfurase and not a type of cysteine sulfoxide lyase. The pyridoxal-5-phosphate (PLP) molecule bonded tightly to Lecsl to form a Lecsl-PLP complex. Moreover, the Lecsl had one active center that served to bind two kinds of substrates, S-methyl-L-cysteine sulfoxide and L-cysteine, and had both cysteine sulfoxide lyase and cysteine desulfurase activity. We found that the amino acid residue Asn393 was essential for the catalytic activity of Lecsl and that the gene Csl encoded a novel cysteine desulfurase to influence organosulfur compounds in L. edodes. Our results provide a new insight into understanding the formation of the unique aroma of L. edodes. PMID:26054293

  10. Applications attract DuPont

    SciTech Connect

    Rotman, D.

    1996-08-07

    Scientists at DuPont say they have demonstrated the first chemical processing application for high-temperature superconducting (HTS) magnets. DuPont says the work, which uses a HTS magnet to separate mineral contaminants from kaolin, points to the feasibility of a range of HTS applications in industrial processing, including those involving polymerization. DuPont`s success comes after 10 years of work to commercialize high-temperature superconductors. And while superconductors have lost much of their luster since the late 1980s, the company says it is still bullish on their prospects. {open_quotes}At the moment, there`s no real market for superconductors,{close_quotes} says Alan Lauder, general manager/superconductivity. But, he says, several potentially lucrative applications could be commercialized within the next several years.

  11. A cysteine protease of Dieffenbachia maculata.

    PubMed

    Chitre, A; Padmanabhan, S; Shastri, N V

    1998-12-01

    Plants of the genus Dieffenbachia, very popular as indoor ornamental plants, are known for their toxic as well as therapeutic properties. Their toxic manifestations have been partly attributed to their proteolytic activity. The work described in the present paper shows that stem leaves and petiole of Dieffenbachia maculata Schott, a commonly grown species, contain significant proteolytic activity, different parts showing different types of protease activities. Stem showed the highest enzyme activity and this protease was purified about 55 fold by solvent precipitation, gel filtration and ion exchange chromatography. The enzyme has a relative molecular mass of 61 kDa as determined by SDS-PAGE and has an optimum pH of 8.0 and optimum temperature of 50 degrees C. Effects of various substrates, inhibitors and activators indicate that the enzyme is a cysteine protease with leucylpeptidase activity.

  12. Cysteine sulfoxide derivatives in Petiveria alliacea.

    PubMed

    Kubec, R; Musah, R A

    2001-11-01

    Two diastereomers of S-benzyl-L-cysteine sulfoxide have been isolated from fresh roots of Petiveria alliacea. Their structures and absolute configurations have been determined by NMR, MALDI-HRMS, IR and CD spectroscopy and confirmed by comparison with authentic compounds. Both the R(S) and S(S) diastereomers of the sulfoxide are present in all parts of the plant (root, stem, and leaves) with the latter diastereomer being predominant. Their total content greatly varied in different parts of the plant between 0.07 and 2.97 mg g(-1) fr. wt, being by far the highest in the root. S-Benzylcysteine has also been detected in trace amounts (<10 microg g(-1) fr. wt) in all parts of the plant. This represents the first report of the presence of S-benzylcysteine derivatives in nature.

  13. L'Aventure du LHC

    SciTech Connect

    2010-06-11

    Cette présentation s’adressera principalement aux personnes qui ont construit le LHC. La construction du LHC fut longue et difficile. De nombreux problèmes sont apparus en cours de route. Tous ont été résolus grâce au dévouement et à l’engagement du personnel et des collaborateurs. Je reviendrai sur les coups durs et les réussites qui ont marqués ces 15 dernières années et je vous montrerai combien cette machine, le fruit de vos efforts, est extraordinaire.

  14. DuPont hikes butanediol

    SciTech Connect

    Morris, G.D.L.

    1997-05-14

    Butanediol (BDO) and its derivatives continue to be strong, a positive sign for the many companies planning expansions. DuPont - one of only two global producers not planning capacity additions - has announced that it will discontinue all off-schedule pricing for BDO and two important derivatives, tetrahydrofuran (THF) and polytetramethylene ether glycol (PTMEG). DuPont`s list prices are $1.00/lb fob for BDO, about $1.40/lb for THF, and $2.00/lb for PTMEG. The price adjustment is effective this month or as contracts allow.

  15. Cysteine analogues potentiate glucose-induced insulin release in vitro

    SciTech Connect

    Ammon, H.P.; Hehl, K.H.; Enz, G.; Setiadi-Ranti, A.; Verspohl, E.J.

    1986-12-01

    In rat pancreatic islets, cysteine analogues, including glutathione, acetylcysteine, cysteamine, D-penicillamine, L-cysteine ethyl ester, and cysteine-potentiated glucose (11.1 mM) induced insulin secretion in a concentration-dependent manner. Their maximal effects were similar and occurred at approximately 0.05, 0.05, 0.1, 0.5, 1.0, 1.0 mM, respectively. At substimulatory glucose levels (2.8 mM), insulin release was not affected by these compounds. In contrast, thiol compounds, structurally different from cysteine and its analogues, such as mesna, tiopronin, meso-2,3-dimercaptosuccinic acid (DMSA), dimercaprol (BAL), beta-thio-D-glucose, as well as those cysteine analogues that lack a free-thiol group, including L-cystine, cystamine, D-penicillamine disulfide, S-carbocysteine, and S-carbamoyl-L-cysteine, did not enhance insulin release at stimulatory glucose levels (11.1 mM); cystine (5 mM) was inhibitory. These in vitro data indicate that among the thiols tested here, only cysteine and its analogues potentiate glucose-induced insulin secretion, whereas thiols that are structurally not related to cysteine do not. This suggests that a cysteine moiety in the molecule is necessary for the insulinotropic effect. For their synergistic action to glucose, the availability of a sulfhydryl group is also a prerequisite. The maximal synergistic action is similar for all cysteine analogues tested, whereas the potency of action is different, suggesting similarity in the mechanism of action but differences in the affinity to the secretory system.

  16. CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

  17. CHARACTERIZATION OF DANSYLATED CYSTEINE, GLUTATHIONE DISULFIDE, CYSTEINE AND CYSTINE BY NARROW BORE LIQUID CHROMATOGRAPHY/ELECTROSPRAY IONIZATION MASS SPECTROMETRY

    EPA Science Inventory

    A method using reversed phase high performance liquid chromatography/electrospray ionization-mass spectrometric (RP-LC/ESI-MS) method has been developed to confirm the identity of dansylated derivatives of cysteine and glutathione, and their respective dimers. Cysteine, GSH, CSSC...

  18. Poumon du puisatier

    PubMed Central

    Elidrissi, Amal Moustarhfir; Zaghba, Nahid; Benjelloun, Hanane; Yassine, Najiba

    2016-01-01

    Le puisatier a pour profession le creusement et l'entretien des puits pour fournir de l'eau. Il est au contact de divers minerais, particulièrement la silice, particule qui présente un risque certain de développement des maladies pulmonaires connues sous le nom de silicose. Le but de notre travail est de préciser le profil épidémiologique, clinique, radiologique et évolutif des patients puisatiers silicotiques. C'est une étude rétrospective concernant 54 cas de puisatiers ayant une silicose, colligés au service des maladies respiratoires du CHU Ibn Rochd de Casablanca, de Mars 1997 à Janvier 2016. Tous les malades étaient des puisatiers, de sexe masculin, avec une moyenne d'âge de 50 ans. Le tabagisme était retrouvé dans 36 cas et un antécédent de tuberculose était noté dans huit cas. La radiographie thoracique retrouvait des grandes opacités dans 39 cas, des petites opacités dans 15 cas, et un épaississement des septats dans 11 cas. Ce tableau de silicose s'était compliqué d'une surinfection bactérienne dans 37% des cas, d' un pneumothorax dans 4% des cas et d'une tuberculose dans 20% des cas. La prise en charge thérapeutique était celle des complications. La déclaration de la maladie professionnelle et de l'indemnisation était faite. L'évolution était bonne dans 12 cas, stationnaire dans 17 cas et mauvaise dans 16 cas. La silicose est une pneumoconiose fréquente chez les puisatiers. Elle retentit sur la fonction respiratoire. Nous soulignons l'association fréquente de tuberculose et nous insistons sur la prévention qui reste le meilleur traitement. PMID:28292119

  19. Du Pont Information Flow System

    ERIC Educational Resources Information Center

    Hoffman, Warren S.

    1972-01-01

    The Information Flow System is a large-scale information retrieval system developed for processing of Du Pont information files. As currently implemented, the system stores and retrieves information on company technical reports. Extensions of the system for handling chemical structure information and on-line processing are also discussed. (3…

  20. Probes of the Catalytic Site of Cysteine Dioxygenase

    SciTech Connect

    Chai,S.; Bruyere, J.; Maroney, M.

    2006-01-01

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the a-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ a-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by {alpha}-ketoglutarate.

  1. L-Cysteine metabolism and its nutritional implications.

    PubMed

    Yin, Jie; Ren, Wenkai; Yang, Guan; Duan, Jielin; Huang, Xingguo; Fang, Rejun; Li, Chongyong; Li, Tiejun; Yin, Yulong; Hou, Yongqing; Kim, Sung Woo; Wu, Guoyao

    2016-01-01

    L-Cysteine is a nutritionally semiessential amino acid and is present mainly in the form of L-cystine in the extracellular space. With the help of a transport system, extracellular L-cystine crosses the plasma membrane and is reduced to L-cysteine within cells by thioredoxin and reduced glutathione (GSH). Intracellular L-cysteine plays an important role in cellular homeostasis as a precursor for protein synthesis, and for production of GSH, hydrogen sulfide (H(2)S), and taurine. L-Cysteine-dependent synthesis of GSH has been investigated in many pathological conditions, while the pathway for L-cysteine metabolism to form H(2)S has received little attention with regard to prevention and treatment of disease in humans. The main objective of this review is to highlight the metabolic pathways of L-cysteine catabolism to GSH, H(2)S, and taurine, with special emphasis on therapeutic and nutritional use of L-cysteine to improve the health and well-being of animals and humans.

  2. Chlorhexidine inhibits the activity of dental cysteine cathepsins.

    PubMed

    Scaffa, P M C; Vidal, C M P; Barros, N; Gesteira, T F; Carmona, A K; Breschi, L; Pashley, D H; Tjäderhane, L; Tersariol, I L S; Nascimento, F D; Carrilho, M R

    2012-04-01

    The co-expression of MMPs and cysteine cathepsins in the human dentin-pulp complex indicates that both classes of enzymes can contribute to the endogenous proteolytic activity of dentin. Chlorhexidine (CHX) is an efficient inhibitor of MMP activity. This study investigated whether CHX could also inhibit cysteine cathepsins present in dentin. The inhibitory profile of CHX on the activity of dentin-extracted and recombinant cysteine cathepsins (B, K, and L) was monitored in fluorogenic substrates. The rate of substrate hydrolysis was spectrofluorimetrically measured, and inhibitory constants were calculated. Molecular docking was performed to predict the binding affinity between CHX and cysteine cathepsins. The results showed that CHX inhibited the proteolytic activity of dentin-extracted cysteine cathepsins in a dose-dependent manner. The proteolytic activity of human recombinant cathepsins was also inhibited by CHX. Molecular docking analysis suggested that CHX strongly interacts with the subsites S2 to S2' of cysteine cathepsins B, K, and L in a very similar manner. Taken together, these results clearly showed that CHX is a potent inhibitor of the cysteine cathepsins-proteolytic enzymes present in the dentin-pulp complex.

  3. Modification of Keap1 Cysteine Residues by Sulforaphane

    PubMed Central

    Hu, Chenqi; Eggler, Aimee L.; Mesecar, Andrew D.; van Breemen, Richard B.

    2011-01-01

    Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conflicting in vitro and in vivo data regarding which of these cysteine residues react. Although most biological data indicate that modification of C151 is essential for sulforaphane action, some recent studies using mass spectrometry have failed to identify C151 as a site of Keap1 sulforaphane reaction. We have reconciled these conflicting data using mass spectrometry with a revised sample preparation protocol and confirmed that C151 is indeed among the most readily modified cysteines of Keap1 by sulforaphane. Previous mass spectrometry-based studies used iodoacetamide during sample preparation to derivatize free cysteine sulfhydryl groups causing loss of sulforaphane from highly reactive and reversible cysteine residues on Keap1 including C151. By omitting iodoacetamide from the protocol and reducing sample preparation time, our mass spectrometry-based studies now confirm previous cell-based studies which showed that sulforaphane reacts with at least four cysteine residues of Keap1 including C151. PMID:21391649

  4. Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

    PubMed

    Yao, Chunxiang; Behring, Jessica B; Shao, Di; Sverdlov, Aaron L; Whelan, Stephen A; Elezaby, Aly; Yin, Xiaoyan; Siwik, Deborah A; Seta, Francesca; Costello, Catherine E; Cohen, Richard A; Matsui, Reiko; Colucci, Wilson S; McComb, Mark E; Bachschmid, Markus M

    2015-01-01

    Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

  5. A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

    PubMed

    Gu, Liqing; Robinson, Renã A S

    2016-04-01

    Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall

  6. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  7. Rupture sous-cutanée du tendon long extenseur du pouce: à propos de 5 cas

    PubMed Central

    Abdelillah, Rachid; Abbassi, Najib; Erraji, Moncef; Abdeljawad, Najib; Yacoubi, Hicham; Daoudi, Abdelkrim

    2014-01-01

    La rupture spontanée du muscle long extenseur du pouce (EPL) du tendon au niveau du poignet est rare et principalement rapportés après fracture du radius distal à tubercule de Lister, dans la synovite, ténosynovite ou la polyarthrite rhumatoïde. Nous rapportons 5 cas de rupture spontanée du tendon long extenseur du pouce, traités par une greffe ou un transfert tendineux. PMID:25317233

  8. Cysteinoyl- and cysteine-containing dipeptidoylbenzotriazoles with free sulfhydryl groups: easy access to N-terminal and internal cysteine peptides.

    PubMed

    Ibrahim, Tarek S; Tala, Srinivasa R; El-Feky, Said A; Abdel-Samii, Zakaria K; Katritzky, Alan R

    2012-08-01

    N-Protected cysteines 4a-c each with a free sulfhydryl group were prepared in 70-75% yields by treatment of L-cysteine with 1-(benzyloxycarbonyl) benzotriazole (Cbz-Bt) 1a, N-(tert-butyloxy-carbonyl)benzotriazole (Boc-Bt) 1b, and 1-(9-fluorenylmethoxy-carbonyl)benzotriazole (Fmoc-Bt) 1c, respectively. N-Protected, free sulfhydryl cysteines 4a-c were then converted into the corresponding N-protected, free sulfhydryl cysteinoylbenzotriazoles 7a-c (70-85%), which on treatment with diverse amino acids and dipeptides afforded the corresponding N-protected, free sulfhydryl N-terminal cysteine dipeptides 8a-e and tripeptides 8f-h in 73-80% yields. N-Protected, free sulfhydryl cysteine-containing dipeptides 9a,b were converted into the corresponding N-protected, free sulfhydryl dipeptidoylbenzotriazoles 10a,b (69-81%), which on treatment with amino acids, dipeptides, and a tripeptide afforded internal cysteine tripeptides 11a-c, tetrapeptides 11d,e and pentapeptide 11f, each containing a N-protected, free sulfhydryl groups in 70-90% yields under mild conditions. Treatment of N-protected, free sulfhydryl cysteinoylbenzotriazole 7a with diamines 12a,b afforded directly the cysteine-containing disulfide-bridged cyclic peptides 14a,b in 50% yields.

  9. Developing novel anthelmintics from plant cysteine proteinases

    PubMed Central

    Behnke, Jerzy M; Buttle, David J; Stepek, Gillian; Lowe, Ann; Duce, Ian R

    2008-01-01

    Intestinal helminth infections of livestock and humans are predominantly controlled by treatment with three classes of synthetic drugs, but some livestock nematodes have now developed resistance to all three classes and there are signs that human hookworms are becoming less responsive to the two classes (benzimidazoles and the nicotinic acetylcholine agonists) that are licensed for treatment of humans. New anthelmintics are urgently needed, and whilst development of new synthetic drugs is ongoing, it is slow and there are no signs yet that novel compounds operating through different modes of action, will be available on the market in the current decade. The development of naturally-occurring compounds as medicines for human use and for treatment of animals is fraught with problems. In this paper we review the current status of cysteine proteinases from fruits and protective plant latices as novel anthelmintics, we consider some of the problems inherent in taking laboratory findings and those derived from folk-medicine to the market and we suggest that there is a wealth of new compounds still to be discovered that could be harvested to benefit humans and livestock. PMID:18761736

  10. Organometallic Palladium Reagents for Cysteine Bioconjugation

    PubMed Central

    Vinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.

    2015-01-01

    Transition-metal based reactions have found wide use in organic synthesis and are used frequently to functionalize small molecules.1,2 However, there are very few reports of using transition-metal based reactions to modify complex biomolecules3,4, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature, and mild pH) and the existence of multiple, reactive functional groups found in biopolymers. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation reactions. The bioconjugation reaction is rapid and robust under a range of biocompatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants, and external thiol nucleophiles. The broad utility of the new bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as a new set of benchtop reagents for diverse bioconjugation applications. PMID:26511579

  11. Organometallic palladium reagents for cysteine bioconjugation

    NASA Astrophysics Data System (ADS)

    Vinogradova, Ekaterina V.; Zhang, Chi; Spokoyny, Alexander M.; Pentelute, Bradley L.; Buchwald, Stephen L.

    2015-10-01

    Reactions based on transition metals have found wide use in organic synthesis, in particular for the functionalization of small molecules. However, there are very few reports of using transition-metal-based reactions to modify complex biomolecules, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature and mild pH) and the existence of multiple reactive functional groups found in biomolecules. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications.

  12. Organometallic palladium reagents for cysteine bioconjugation.

    PubMed

    Vinogradova, Ekaterina V; Zhang, Chi; Spokoyny, Alexander M; Pentelute, Bradley L; Buchwald, Stephen L

    2015-10-29

    Reactions based on transition metals have found wide use in organic synthesis, in particular for the functionalization of small molecules. However, there are very few reports of using transition-metal-based reactions to modify complex biomolecules, which is due to the need for stringent reaction conditions (for example, aqueous media, low temperature and mild pH) and the existence of multiple reactive functional groups found in biomolecules. Here we report that palladium(II) complexes can be used for efficient and highly selective cysteine conjugation (bioconjugation) reactions that are rapid and robust under a range of bio-compatible reaction conditions. The straightforward synthesis of the palladium reagents from diverse and easily accessible aryl halide and trifluoromethanesulfonate precursors makes the method highly practical, providing access to a large structural space for protein modification. The resulting aryl bioconjugates are stable towards acids, bases, oxidants and external thiol nucleophiles. The broad utility of the bioconjugation platform was further corroborated by the synthesis of new classes of stapled peptides and antibody-drug conjugates. These palladium complexes show potential as benchtop reagents for diverse bioconjugation applications.

  13. Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles

    PubMed Central

    Serafimova, Iana M.; Pufall, Miles A.; Krishnan, Shyam; Duda, Katarzyna; Cohen, Michael S.; Maglathlin, Rebecca L.; McFarland, Jesse M.; Miller, Rand M.; Frödin, Morten; Taunton, Jack

    2013-01-01

    Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins’ intrinsic reactivity, yet paradoxically eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase, RSK. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides. PMID:22466421

  14. Cysteine-functional polymers via thiol-ene conjugation.

    PubMed

    Kuhlmann, Matthias; Reimann, Oliver; Hackenberger, Christian P R; Groll, Jürgen

    2015-03-01

    A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Application of L-cystein derivative to DNA microarray.

    PubMed

    Nakauchi, Gen; Inaki, Yoshiaki; Kitaoka, Shiho; Yokoyama, Chieko; Tanabe, Tadashi

    2002-01-01

    S-carboxymethyl-L-cystein derivatives of nucleic acid bases were prepared as DNA chip probe. These compounds in vitro have been found to form stable complex with oligo-DNA and RNA. This paper deals with preparing new DNA chip using L-cystein derivative synthetic nucleotides as probe and immobilized it to quartz plate by photosensitive PVA. Then the chip exposed with FITC labeled target DNA was observed by confocal fluorescence microscope.

  16. Methionine-to-Cysteine Recycling in Klebsiella aerogenes

    PubMed Central

    Seiflein, Thomas A.; Lawrence, Jeffrey G.

    2001-01-01

    In the enteric bacteria Escherichia coli and Salmonella enterica, sulfate is reduced to sulfide and assimilated into the amino acid cysteine; in turn, cysteine provides the sulfur atom for other sulfur-bearing molecules in the cell, including methionine. These organisms cannot use methionine as a sole source of sulfur. Here we report that this constraint is not shared by many other enteric bacteria, which can use either cysteine or methionine as the sole source of sulfur. The enteric bacterium Klebsiella aerogenes appears to use at least two pathways to allow the reduced sulfur of methionine to be recycled into cysteine. In addition, the ability to recycle methionine on solid media, where cys mutants cannot use methionine as a sulfur source, appears to be different from that in liquid media, where they can. One pathway likely uses a cystathionine intermediate to convert homocysteine to cysteine and is induced under conditions of sulfur starvation, which is likely sensed by low levels of the sulfate reduction intermediate adenosine-5′-phosphosulfate. The CysB regulatory proteins appear to control activation of this pathway. A second pathway may use a methanesulfonate intermediate to convert methionine-derived methanethiol to sulfite. While the transsulfurylation pathway may be directed to recovery of methionine, the methanethiol pathway likely represents a general salvage mechanism for recovery of alkane sulfide and alkane sulfonates. Therefore, the relatively distinct biosyntheses of cysteine and methionine in E. coli and Salmonella appear to be more intertwined in Klebsiella. PMID:11114934

  17. Cysteine aggravates palmitate-induced cell death in hepatocytes

    PubMed Central

    Dou, Xiaobing; Wang, Zhigang; Yao, Tong; Song, Zhenyuan

    2011-01-01

    Aims Lipotoxicity, defined as cell death induced by excessive fatty acids, especially saturated fatty acids, is critically involved in the development of non-alcoholic steatohepatitis (NASH). Recent studies report that plasma cysteine concentrations is elevated in the subjects with either alcoholic steatohepatitis (ASH) or NASH than normal subjects. The present study was conducted to determine if elevation of cysteine could be a deleterious factor in palmitate-induced hepatocyte cell death. Main methods HepG2 and Hep3B cells were treated with palmitate with/without the inclusion of cysteine in the media for 24 hours. The effects of cysteine inclusion on palmitate induced cell death were determined by lactate dehydrogenase (LDH) release and MTT assay. Oxidative stress was evaluated by intracellular glutathione (GSH) level, malondialdehyde (MDA) formation, and DCFH-DA assay. Western blotting was performed to detect the changes of endoplasmic reticulum(ER) stress markers: C/EBP homologous transcription factor (CHOP), GRP-78, and phosphorylated c-jun N-terminal kinase (p-JNK). Key findings Elevated intracellular cysteine aggravates hepatocytes to palmitate-induced cell death. Enhancement of ER stress, specifically increased activation of JNK pathway, contributed to this cell death process. Significance Increase of plasma cysteine levels, as observed in both ASH and NASH patients, may play a pathological role in the development of the liver diseases. Manipulation of dietary amino acids supplementation could be a therapeutic choice. PMID:22008477

  18. Cysteine transporter SLC3A1 promotes breast cancer tumorigenesis

    PubMed Central

    Jiang, Yang; Cao, Yuan; Wang, Yongbin; Li, Wei; Liu, Xinyi; Lv, Yixuan; Li, Xiaoling; Mi, Jun

    2017-01-01

    Cysteine is an essential amino acid for infants, aged people as well as patients with metabolic disorders. Although the thiol group of cysteine side chain is active in oxidative reactions, the role of cysteine in cancer remains largely unknown. Here, we report that the expression level of the solute carrier family 3, member 1 (SLC3A1), the cysteine carrier, tightly correlated with clinical stages and patients' survival. Elevated SLC3A1 expression accelerated the cysteine uptake and the accumulation of reductive glutathione (GSH), leading to reduced reactive oxygen species (ROS). ROS increased the stability and activity of PP2Ac, resulting in decreased AKT activity. Hence, SLC3A1 activated the AKT signaling through inhibiting PP2A phosphatase activity. Consistently, overexpression of SLC3A1 enhanced tumorigenesis of breast cancer cells, whereas blocking SLC3A1 either with specific siRNA or SLC3A1 specific inhibitor sulfasalazine suppressed tumor growth and also abolished dietary NAC-promoted tumor growth. Collectively, our data demonstrate that SLC3A1 promotes cysteine uptake and determines cellular response to antioxidant N-acetylcysteine, suggesting SLC3A1 is a potential therapeutic target for breast cancer. PMID:28382174

  19. Inhibition of a Plasmodium vinckei cysteine proteinase cures murine malaria.

    PubMed Central

    Rosenthal, P J; Lee, G K; Smith, R E

    1993-01-01

    Intraerythrocytic malaria parasites degrade hemoglobin as a principal source of amino acids for parasite protein synthesis. We have previously identified a Plasmodium falciparum trophozoite cysteine proteinase as a putative hemoglobinase and shown that specific inhibitors of this proteinase block the hydrolysis of globin and the development of cultured parasites. We now show that the murine malaria parasite Plasmodium vinckei has an analogous cysteine proteinase with similar biochemical properties to the P. falciparum proteinase, including an acid pH optimum, a preference for the peptide proteolytic substrate benzyloxycarbonyl (Z)-Phe-Arg-7-amino-4-methylcoumarin, and nonomolar inhibition by seven peptide fluoromethyl ketone proteinase inhibitors. Thus, P. vinckei offers a model system for the in vivo testing of the antimalarial properties of cysteine proteinase inhibitors. One of the proteinase inhibitors studied, morpholine urea (Mu)-Phe-Homophenylalanine (HPhe)-CH2F strongly inhibited the P. vinckei cysteine proteinase in vitro and rapidly blocked parasite cysteine proteinase activity in vivo. When administered four times a day for 4 d to P. vinckei-infected mice, Mu-Phe-HPhe-CH2F elicited long-term cures in 80% of the treated animals. These results show that peptide proteinase inhibitors can be effective antimalarial compounds in vivo and suggest that the P. falciparum cysteine proteinase is a promising target for chemotherapy. Images PMID:8450035

  20. Getting a Knack for NAC: N-Acetyl-Cysteine.

    PubMed

    Sansone, Randy A; Sansone, Lori A

    2011-01-01

    N-acetyl-cysteine, N-acetylcysteine, N-acetyl cysteine, and N-acetyl-L-cysteine are all designations for the same compound, which is abbreviated as NAC. NAC is a precursor to the amino acid cysteine, which ultimately plays two key metabolic roles. Through its metabolic contribution to glutathione production, cysteine participates in the general antioxidant activities of the body. Through its role as a modulator of the glutamatergic system, cysteine influences the reward-reinforcement pathway. Because of these functions, NAC may exert a therapeutic effect on psychiatric disorders allegedly related to oxidative stress (e.g., schizophrenia, bipolar disorder) as well as psychiatric syndromes characterized by impulsive/compulsive symptoms (e.g., trichotillomania, pathological nail biting, gambling, substance misuse). While the dosages, pharmacological strategies (monotherapy versus augmentation), and long-term risks are not fully evident, NAC appears to be a promising, relatively low-risk intervention. If so, NAC might be an ideal treatment strategy for a variety of psychiatric conditions in both psychiatric and primary care settings.

  1. Synthesis and antimicrobial activity of cysteine-free coprisin nonapeptides.

    PubMed

    Lee, Jaeho; Lee, Daeun; Choi, Hyemin; Kim, Ha Hyung; Kim, Ho; Hwang, Jae Sam; Lee, Dong Gun; Kim, Jae Il

    2014-01-10

    Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. CopA3 (LLCIALRKK-NH₂), a 9-mer peptide containing a single free cysteine residue at position 3 of its sequence, was derived from the α-helical region of coprisin and exhibits potent antibacterial and anti-inflammatory activities. The single cysteine implies a tendency for dimerization; however, it remains unknown whether this cysteine residue is indispensible for CopA3's antimicrobial activity. To address this issue, in the present study we synthesized eight cysteine-substituted monomeric CopA3 analogs and two dimeric analogs, CopA3 (Dimer) and CopIK (Dimer), and evaluated their antimicrobial effects against bacteria and fungi, as well as their hemolytic activity toward human erythrocytes. Under physiological conditions, CopA3 (Mono) exhibits a 6/4 (monomer/dimer) molar ratio in HPLC area percent, indicating that its effects on bacterial strains likely reflect a CopA3 (Mono)/CopA3 (Dimer) mixture. We also report the identification of CopW, a new cysteine-free nonapeptide derived from CopA3 that has potent antimicrobial activity with virtually no hemolytic activity. Apparently, the cysteine residue in CopA3 is not essential for its antimicrobial function. Notably, CopW also exhibited significant synergistic activity with ampicillin and showed more potent antifungal activity than either wild-type coprisin or melittin.

  2. L'Aventure du LHC

    ScienceCinema

    None

    2016-07-12

    Cette présentation s’adressera principalement aux personnes qui ont construit le LHC. La construction du LHC fut longue et difficile. De nombreux problèmes sont apparus en cours de route. Tous ont été résolus grâce au dévouement et à l’engagement du personnel et des collaborateurs. Je reviendrai sur les coups durs et les réussites qui ont marqués ces 15 dernières années et je vous montrerai combien cette machine, le fruit de vos efforts, est extraordinaire.

  3. Construction, purification, and immunogenicity of recombinant cystein-cystein type chemokine receptor 5 vaccine.

    PubMed

    Wu, Kongtian; Xue, Xiaochang; Wang, Zenglu; Yan, Zhen; Shi, Jihong; Han, Wei; Zhang, Yingqi

    2006-09-01

    Cystein-Cystein type chemokine receptor 5 (CCR5) is a seven-transmembrane, G-protein coupled receptor. It is a major coreceptor with CD4 glycoprotein mediating cellular entry of CCR5 strains of HIV-1. A lack of cell-surface expression of CCR5 found in the homozygous Delta32 CCR5 mutation, upregulation of CC chemokines and antibodies to CCR5 are associated with resistance to HIV infection. In addition, CCR5 can be blocked by three CC chemokines and antibodies to three extracellular domains of CCR5. Consequently, CCR5 is considered an attractive therapeutic target against HIV infection. In the current study, we constructed a recombinant vaccine by coupling a T helper epitope AKFVAAWTLKAA (PADRE) to the N terminus of CCR5 extracellular domains (PADRE-CCR5) and expressed this protein in Escherichia coli. We have developed an inexpensive and scalable purification process for the fusion protein from inclusion bodies and the final yields of 6mg purified fusion protein per gram of cell paste was obtained. The immunogenicity of the recombinant vaccine generated was examined in BALB/c mice. Sera from the vaccinated mice demonstrated high-titer specific antibodies to the recombinant vaccine, suggesting that PADRE-rCCR5 may be used as a candidate of active CCR5 vaccine.

  4. Cysteine effects on the pharmacokinetics of etoposide in protein-calorie malnutrition rats: increased gastrointestinal absorption by cysteine.

    PubMed

    Suh, J H; Kang, H E; Yoon, I S; Yang, S H; Kim, S H; Lee, H J; Shim, C-K; Lee, M G

    2011-10-01

    Protein-calorie malnutrition (PCM) occurs frequently in advanced cancer patients and has a profound impact on the toxicity of many drugs. Thus, the pharmacokinetics of etoposide were evaluated in control, control with cysteine (CC), PCM, and PCM with cysteine (PCMC) rats. Etoposide was administered intravenously (2 mg/kg) or orally (10 mg/kg). Changes in hepatic and intestinal cytochrome P450s (CYPs) and effects of cysteine on intestinal P-glycoprotein (P-gp)-mediated efflux were also measured. In PCM rats, the CL(NR) (AUC(0-∞)) of intravenous etoposide was significantly slower (greater) than that in controls, because of the significant decrease in the hepatic CYP3A subfamily and P-gp. In PCMC rats, the slowed CL(NR) of etoposide in PCM rats was restored to the control level by cysteine treatment. PCMC rats showed a significantly greater AUC(0-6 h) of oral etoposide than PCM rats, primarily because of the increased gastrointestinal absorption of etoposide as a result of the inhibition of intestinal P-gp by cysteine. The gastrointestinal absorption of an oral anticancer drug, which is a substrate of P-gp, may be improved by co-administration of cysteine in advanced cancer patients if the present rat data can be extrapolated to patients.

  5. A Quantitative Mass-Spectrometry Platform to Monitor Changes in Cysteine Reactivity

    PubMed Central

    Qian, Yu; Weerapana, Eranthie

    2017-01-01

    Summary Cysteine residues on proteins serve diverse functional roles in catalysis and regulation and are susceptible to numerous posttranslational modifications. Methods to monitor the reactivity of cysteines within the context of a complex proteome have facilitated the identification and functional characterization of cysteine residues on disparate proteins. Here, we describe the use of a cysteine-reactive iodoacetamide probe coupled to isotopically labeled, cleavable linkers to identify and quantify cysteine-reactivity changes from two biological samples. PMID:27778278

  6. Recombinant disintegrin domain of human ADAM9 inhibits migration and invasion of DU145 prostate tumor cells

    PubMed Central

    Martin, Ana Carolina Baptista Moreno; Cardoso, Ana Carolina Ferreira; Selistre-de-Araujo, Heloisa Sobreiro; Cominetti, Márcia Regina

    2015-01-01

    One of the most important features of malignant cells is their capacity to invade adjacent tissues and metastasize to distant organs. This process involves the creation, by tumor and stroma cells, of a specific microenvironment, suitable for proliferation, migration and invasion of tumor cells. The ADAM family of proteins has been involved in these processes. This work aimed to investigate the role of the recombinant disintegrin domain of the human ADAM9 (rADAM9D) on the adhesive and mobility properties of DU145 prostate tumor cells. rADAM9D was able to support DU145 cell adhesion, inhibit the migration of DU145 cells, as well as the invasion of this cell line through matrigel in vitro. Overall this work demonstrates that rADAM9D induces specific cellular migratory properties when compared with different constructs having additional domains, specially those of metalloproteinase and cysteine-rich domains. Furthermore, we showed that rADAM9D was able to inhibit cell adhesion, migration and invasion mainly through interacting with α6β1 in DU145 tumor cell line. These results may contribute to the development of new therapeutic strategies for prostate cancer. PMID:26211476

  7. Métastases gastro-intestinales du cancer du sein: à propos de 2 cas

    PubMed Central

    Loubna, Mezouar; Mohamed, El Hfid; Tijani, El Harroudi; Fouzia, Ghadouani; Hanane, Haj Kacem; Zouhour, Bourhaleb; Asmae, Ouabdelmoumen

    2013-01-01

    Le cancer du sein est le cancer le plus fréquent chez la femme, notamment au Maroc, avec un taux de mortalité élevé. Les métastases gastro-intestinales d'un carcinome canalaire du sein sont rares. Leur diagnostic est difficile du fait de la nature non spécifique des symptômes. Nous rapportons deux observations originales de métastases gastroduodénales d'un cancer canalaire infiltrant du sein. Les métastases gastro-intestinales du cancer du sein sont très rares; la présence de symptômes gastro-intestinaux chez une malade ayant un antécédent de cancer du sein doit faire suspecter une atteinte métastatique gastro-intestinale. PMID:24198876

  8. Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis.

    PubMed

    Allan, Euan Ramsay Orr; Yates, Robin Michael

    2015-01-01

    The cysteine cathepsins B, S, and L are functionally linked to antigen processing, and hence to autoimmune disorders such as multiple sclerosis. Stemming from several studies that demonstrate that mice can be protected from experimental autoimmune encephalomyelitis (EAE) through the pharmacologic inhibition of cysteine cathepsins, it has been suggested that targeting these enzymes in multiple sclerosis may be of therapeutic benefit. Utilizing mice deficient in cysteine cathepsins both individually and in combination, we found that the myelin-associated antigen myelin oligodendrocyte glycoprotein (MOG) was efficiently processed and presented by macrophages to CD4+ T cells in the individual absence of cathepsin B, S or L. Similarly, mice deficient in cathepsin B or S were susceptible to MOG-induced EAE and displayed clinical progression and immune infiltration into the CNS, similar to their wild-type counterparts. Owing to a previously described CD4+ T cell deficiency in mice deficient in cathepsin L, such mice were protected from EAE. When multiple cysteine cathepsins were simultaneously inhibited via genetic deletion of both cathepsins B and S, or by a cathepsin inhibitor (LHVS), MHC-II surface expression, MOG antigen presentation and EAE were attenuated or prevented. This study demonstrates the functional redundancy between cathepsin B, S and L in EAE, and suggests that the inhibition of multiple cysteine cathepsins may be needed to modulate autoimmune disorders such as multiple sclerosis.

  9. Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization

    PubMed Central

    Clark, Hillary M.; Hagedorn, Tara D.; Landino, Lisa M.

    2013-01-01

    Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9 mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions. PMID:24215946

  10. Cysteines under ROS attack in plants: a proteomics view.

    PubMed

    Akter, Salma; Huang, Jingjing; Waszczak, Cezary; Jacques, Silke; Gevaert, Kris; Van Breusegem, Frank; Messens, Joris

    2015-05-01

    Plants generate reactive oxygen species (ROS) as part of their metabolism and in response to various external stress factors, potentially causing significant damage to biomolecules and cell structures. During the course of evolution, plants have adapted to ROS toxicity, and use ROS as signalling messengers that activate defence responses. Cysteine (Cys) residues in proteins are one of the most sensitive targets for ROS-mediated post-translational modifications, and they have become key residues for ROS signalling studies. The reactivity of Cys residues towards ROS, and their ability to react to different oxidation states, allow them to appear at the crossroads of highly dynamic oxidative events. As such, a redox-active cysteine can be present as S-glutathionylated (-SSG), disulfide bonded (S-S), sulfenylated (-SOH), sulfinylated (-SO2H), and sulfonylated (-SO3H). The sulfenic acid (-SOH) form has been considered as part of ROS-sensing pathways, as it leads to further modifications which affect protein structure and function. Redox proteomic studies are required to understand how and why cysteines undergo oxidative post-translational modifications and to identify the ROS-sensor proteins. Here, we update current knowledge of cysteine reactivity with ROS. Further, we give an overview of proteomic techniques that have been applied to identify different redox-modified cysteines in plants. There is a particular focus on the identification of sulfenylated proteins, which have the potential to be involved in plant signal transduction.

  11. Conversion of cysteine to 3-mercaptopyruvic acid by bacterial aminotransferases.

    PubMed

    Andreeßen, Christina; Gerlt, Vanessa; Steinbüchel, Alexander

    2017-04-01

    3-Mercaptopyruvate (3MPy), a structural analog of 3-mercaptopropionic acid, is a precursor compound for biosynthesis of polythioesters in bacteria. The cost-effectiveness and sustainability of the whole process could be greatly improved by using the cysteine degradation pathway for an intracellular supply of 3MPy. Transamination of cysteine to its corresponding α-keto acid 3MPy is catalyzed by cysteine aminotransferases (CAT). However, CAT activity has so far not been described for bacterial aminotransferases (AT), and it was unknown whether they can be applied for the conversion of cysteine to 3MPy. In this study, we selected eight bacterial aminotransferases based on sequence homology to CAT of Rattus norvegicus (Got1). The aminotransferases included four aspartate aminotransferases (AATs) and four aromatic amino acid aminotransferases (ArATs) from Advenella mimigardefordensis DPN7, Escherichia coli MG1655, Shimwellia blattae ATCC 33430, Ralstonia eutropha H16 and Paracoccus denitrificans PD1222. For a more detailed characterization, all selected AAT or ArAT encoding genes were heterologously expressed in E. coli and purified. CAT activity was detected for all aminotransferases when a novel continuous coupled enzyme assay was applied. Kinetic studies revealed the highest catalytic efficiency of 5.1mM/s for AAT from A. mimigardefordensis. Formation of 3MPy from cysteine could additionally be verified by an optimized approach using derivatization of 3MPy with the Girard T reagent and liquid chromatography-mass spectrometry analyses.

  12. Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

    PubMed

    Li, R; Kast, J

    2017-01-01

    Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics. © 2017 Elsevier Inc. All rights reserved.

  13. Conservation of cysteine residues in fungal histidine acid phytases.

    PubMed

    Mullaney, Edward J; Ullah, Abul H J

    2005-03-11

    Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

  14. The spectrum character of photoreaction of Hypocrellin A and cysteine

    NASA Astrophysics Data System (ADS)

    Zhang, Jucheng; Liu, Wei; Li, Ying; Zhang, Pei; Yi, Zhongzhou; Min, Yong; Huang, Zhaolong; Yao, Lihua; Lu, Haiju

    2008-12-01

    In the current work, Hypocrellin A (HA) is one of the nature photosensitizer was recognized by researchers, and it used as a probe to research the molecular recognition and interaction with protein, the work suggested the HA can as the medicine to treat some disease. This paper study the spectrum character of photoreaction of Hypocrellin A and cysteine in different pH value, the spectrum show an isosbestic point at 495nm, and the absorption peak at 478nm was red-shifted to about 500nm. The result suggested the HA can react with cysteine in this condition, and farther illuminated the cysteine residue may is one of the target of the interaction of HA or HB with protein.

  15. Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins.

    PubMed

    Zhao, Kai-Hong; Su, Ping; Tu, Jun-Ming; Wang, Xing; Liu, Hui; Plöscher, Matthias; Eichacker, Lutz; Yang, Bei; Zhou, Ming; Scheer, Hugo

    2007-09-04

    Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.

  16. Browning inhibition mechanisms by cysteine, ascorbic acid and citric acid, and identifying PPO-catechol-cysteine reaction products.

    PubMed

    Ali, Hussein M; El-Gizawy, Ahmed M; El-Bassiouny, Rawia E I; Saleh, Mahmoud A

    2015-06-01

    The titled compounds were examined as PPO inhibitors and antibrowning agents; their various mechanisms were investigated and discussed. All compounds reduced significantly both the browning process and PPO activity. Browning index gave strong correlation with PPO activity (r(2) = 0.96, n = 19) indicating that the browning process is mainly enzymatic. Ascorbic acid could reduce the formed quinone instantly to the original substrate (catechol) at high concentration (>1.5 %) while at lower concentrations acted as competitive inhibitor (KI = 0.256 ± 0.067 mM). Cysteine, at higher concentrations (≥1.0 %), reacted with the resulted quinone to give a colorless products while at the low concentrations, cysteine worked as competitive inhibitor (KI = 1.113 ± 0.176 mM). Citric acid acted only as PPO non-competitive inhibitor with KI = 2.074 ± 0.363 mM. The products of PPO-catechole-cysteine reaction could be separation and identification by LC-ESI-MS. Results indicated that the product of the enzymatic oxidation of catechol, quinone, undergoes two successive nucleophilic attacks by cysteine thiol group. Cysteine was condensed with the resulted mono and dithiocatechols to form peptide side chains.

  17. Diverse two-cysteine photocycles in phytochromes and cyanobacteriochromes.

    PubMed

    Rockwell, Nathan C; Martin, Shelley S; Feoktistova, Kateryna; Lagarias, J Clark

    2011-07-19

    Phytochromes are well-known as photoactive red- and near IR-absorbing chromoproteins with cysteine-linked linear tetrapyrrole (bilin) prosthetic groups. Phytochrome photoswitching regulates adaptive responses to light in both photosynthetic and nonphotosynthetic organisms. Exclusively found in cyanobacteria, the related cyanobacteriochrome (CBCR) sensors extend the photosensory range of the phytochrome superfamily to shorter wavelengths of visible light. Blue/green light sensing by a well-studied subfamily of CBCRs proceeds via a photolabile thioether linkage to a second cysteine fully conserved in this subfamily. In the present study, we show that dual-cysteine photosensors have repeatedly evolved in cyanobacteria via insertion of a second cysteine at different positions within the bilin-binding GAF domain (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and formate hydrogen lyase transcription activator FhlA) shared by CBCRs and phytochromes. Such sensors exhibit a diverse range of photocycles, yet all share ground-state absorbance of near-UV to blue light and a common mechanism of light perception: reversible photoisomerization of the bilin 15,16 double bond. Using site-directed mutagenesis, chemical modification and spectroscopy to characterize novel dual-cysteine photosensors from the cyanobacterium Nostoc punctiforme ATCC 29133, we establish that this spectral diversity can be tuned by varying the light-dependent stability of the second thioether linkage. We also show that such behavior can be engineered into the conventional phytochrome Cph1 from Synechocystis sp. PCC6803. Dual-cysteine photosensors thus allow the phytochrome superfamily in cyanobacteria to sense the full solar spectrum at the earth surface from near infrared to near ultraviolet.

  18. Degradation to sulphate of S-methyl-L-cysteine sulphoxide and S-carboxymethyl-L-cysteine sulphoxide in man.

    PubMed

    Waring, R H; Harris, R M; Steventon, G B; Mitchell, S C

    2003-01-01

    A nearly complete recovery of radioactivity was achieved over 14 days following the oral administration of [35S]-S-methyl-L-cysteine sulphoxide and [35S]-S-carboxymethyl-L-cysteine sulphoxide to four healthy male volunteers. The urine was the major pathway of excretion of radioactivity (c. 96% in 0-14 days; c. 59% in 0-24 hours), with the faecal route being relatively unimportant (c. 1.7% in 0-3 days). Inorganic sulphate was an important degradation product, incorporating a substantial proportion of radioactive sulphur derived from these molecules (c. 40% in 0-14 days; c. 20% in 0-24 hours). Subtle differences were noted in the pattern of radioactive sulphate excretion following administration of the two cysteine-sulphoxide compounds, suggesting that their sulphur-containing moieties may enter different catabolic routes.

  19. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residues in ficin

    PubMed Central

    Husain, S. S.; Lowe, G.

    1970-01-01

    Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-14C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and α-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined. PMID:5420043

  20. The amino acid sequence around the active-site cysteine and histidine residues, and the buried cysteine residue in ficin.

    PubMed

    Husain, S S; Lowe, G

    1970-04-01

    Ficin that had been prepared from the latex of Ficus glabrata by salt fractionation and chromatography on carboxymethylcellulose was completely and irreversibly inhibited with 1,3-dibromo[2-(14)C]acetone and then treated with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide in 6m-guanidinium chloride. After reduction and carboxymethylation of the labelled protein, it was digested with trypsin and alpha-chymotrypsin. Two radioactive peptides and two coloured peptides were isolated chromatographically and their sequences determined. The radioactive peptides revealed the amino acid sequences around the active-site cysteine and histidine residues and showed a high degree of homology with the omino acid sequence around the active-site cysteine and histidine residues in papain. The coloured peptides allowed the amino acid sequence around the buried cysteine residue in ficin to be determined.

  1. Approche de prise en charge du trouble du spectre de l’autisme

    PubMed Central

    Lee, Patrick F.; Thomas, Roger E.; Lee, Patricia A.

    2015-01-01

    Résumé Objectif Se pencher sur les critères diagnostiques du trouble du spectre de l’autisme (TSA) comme les définit le Manuel diagnostique et statistique des troubles mentaux, cinquième édition (DSM-V), et concevoir une approche de prise en charge du TSA à l’aide du cadre CanMEDS–Médecine familiale (CanMEDS-MF). Sources d’information Le DSM-V, publié par l’American Psychiatric Association en mai 2013, énonce de nouveaux critères diagnostiques du TSA. Le cadre CanMEDS-MF du Collège des médecins de famille du Canada fournit un plan d’orientation pour la prise en charge complexe du TSA. Nous avons utilisé des données recueillies par le Centers for Disease Control and Prevention afin de déterminer la prévalence du TSA, ainsi que la revue systématique et méta-analyse détaillée effectuée par le National Institute for Health and Care Excellence du R.-U. pour ses lignes directrices sur le TSA dans le but d’évaluer les données probantes issues de plus de 100 interventions. Message principal Selon les données du Centers for Disease Control and Prevention, la prévalence du TSA se chiffrait à 1 sur 88 en 2008 aux États-Unis. La classification du TSA dans la quatrième édition du DSM incluait l’autisme, le syndrome d’Asperger, le trouble envahissant du développement et le trouble désintégratif de l’enfance. La dernière révision du DSM-V réunit tous ces troubles sous la mention TSA, avec différents niveaux de sévérité. La prise en charge du TSA est complexe; elle exige les efforts d’une équipe multidisciplinaire ainsi que des soins continus. Les rôles CanMEDS-MF fournissent un cadre de prise en charge. Conclusion Les médecins de famille sont au cœur de l’équipe de soins multidisciplinaire pour le TSA, et le cadre CanMEDS-MF tient lieu de plan détaillé pour guider la prise en charge d’un enfant atteint de TSA et aider la famille de cet enfant.

  2. A cysteine-selective fluorescent probe for the cellular detection of cysteine.

    PubMed

    Jung, Hyo Sung; Han, Ji Hye; Pradhan, Tuhin; Kim, Sooyeon; Lee, Seok Won; Sessler, Jonathan L; Kim, Tae Woo; Kang, Chulhun; Kim, Jong Seung

    2012-01-01

    A series of coumarin fluorophores (1-3), each bearing a double bond conjugated quinoline unit that can undergo a Michael-type reaction with thiol-containing compounds, is reported. These systems, designed to provide so-called turn-on changes in fluorescence response when exposed to thiols, act as fluorescent chemical sensors for cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). In the case of 1, selectivity for Cys over Hcy and GSH is observed, both in terms of analyte-induced signal enhancement and response time. On the basis of fluorescence spectroscopic analyses, DFT calculations, and pH dependent studies this substrate selectivity is ascribed to steric interactions between the substituents on the quinolone units present in 1 and the targeted thiols, as well as to the comparatively lower pK(a) value of Cys relative to Hcy and GSH. In aqueous solution, probe 1 was found capable of detecting Cys with a detection limit of 10(-7) m. This system was successfully applied to the fluorescence imaging of intracellular Cys in HepG2 cells.

  3. Les plaies du tendon patellaire

    PubMed Central

    Mechchat, Atif; Elidrissi, Mohammed; Mardy, Abdelhak; Elayoubi, Abdelghni; Shimi, Mohammed; Elibrahimi, Abdelhalim; Elmrini, Abdelmajid

    2014-01-01

    Les plaies du tendon patellaire sont peu fréquentes et sont peu rapportés dans la littérature, contrairement aux ruptures sous cutanées. Les sections du tendon patellaire nécessitent une réparation immédiate afin de rétablir l'appareil extenseur et de permettre une récupération fonctionnelle précoce. A travers ce travail rétrospectif sur 13 cas, nous analysons les aspects épidémiologiques, thérapeutiques et pronostiques de ce type de pathologie en comparant différents scores. L’âge moyen est de 25 ans avec une prédominance masculine. Les étiologies sont dominées par les accidents de la voie publique (68%) et les agressions par agent tranchant (26%) et contendant (6 %). Tous nos patients ont bénéficié d'un parage chirurgical avec suture tendineuse direct protégée par un laçage au fils d'aciers en légère flexion. La rééducation est débutée après sédation des phénomènes inflammatoires. Au dernier recul les résultats sont excellents et bon à 92%. Nous n'avons pas noté de différence de force musculaire et d'amplitude articulaire entre le genou sain et le genou lésé. Les lésions ouvertes du tendon patellaire est relativement rare. La prise en charge chirurgicale rapide donne des résultats assez satisfaisants. La réparation est généralement renforcée par un semi-tendineux, synthétique ou métallique en forme de cadre de renfort pour faciliter la réadaptation et réduire le risque de récidive après la fin de l'immobilisation. PMID:25170379

  4. Cysteine functionalized copper organosol: synthesis, characterization and catalytic application

    NASA Astrophysics Data System (ADS)

    Panigrahi, Sudipa; Kundu, Subrata; Basu, Soumen; Praharaj, Snigdhamayee; Jana, Subhra; Pande, Surojit; Ghosh, Sujit Kumar; Pal, Anjali; Pal, Tarasankar

    2006-11-01

    We herein report a facile one-pot synthesis, stabilization, redispersion and Cu-S interaction of L-cysteine and dodecanethiol (DDT) protected copper organosol in toluene from precursor copper stearate using sodium borohydride in toluene under a nitrogen atmosphere. Surface modification of the synthesized copper organosol with an amino acid L-cysteine and an alkanethiol (dodecanethiol, DDT) is accomplished by a thiolate bond between the used ligands and nanoparticle surface. The cysteine molecule binds the copper surface via a thiolate and amine linkage but not through electrostatic interaction with the carboxylate group due to the solvent polarity and dielectric medium. Fourier transform infrared (FTIR) analysis was performed to confirm the surface functionalization of the amino acid and DDT to the copper surface. Copper organosol has been characterized by optical spectroscopy (UV/vis), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS) and x-ray diffraction (XRD). The as-synthesized particles are spherical in shape and exhibit a Mie scattering profile with an absorption maxima in the visible range. Copper nanoparticles capped by cysteine and/or DDT in non-aqueous media are found to represent an interesting catalytic approach for the synthesis of octylphenyl ether.

  5. Temporal dependence of cysteine protease activation following excitotoxic hippocampal injury.

    PubMed

    Berry, J N; Sharrett-Field, L J; Butler, T R; Prendergast, M A

    2012-10-11

    Excitotoxic insults can lead to intracellular signaling cascades that contribute to cell death, in part by activation of proteases, phospholipases, and endonucleases. Cysteine proteases, such as calpains, are calcium (Ca(2+))-activated enzymes which degrade cytoskeletal proteins, including microtubule-associated proteins, tubulin, and spectrin, among others. The current study used the organotypic hippocampal slice culture model to examine whether pharmacologic inhibition of cysteine protease activity inhibits N-methyl-D-aspartate- (NMDA-) induced excitotoxic (20 μM NMDA) cell death and changes in synaptophysin immunoreactivity. Significant NMDA-induced cytotoxicity (as measured by propidium iodide [PI] uptake) was found in the CA1 region of the hippocampus at all timepoints examined (24, 72, 120 h), an effect significantly attenuated by co-exposure to the selective NMDA receptor antagonist DL-2-Amino-5-phosphonopentanoic acid (APV), but not MDL-28170, a potent cysteine protease inhibitor. Results indicated sparing of NMDA-induced loss of the synaptic vesicular protein synaptophysin in all regions of the hippocampus by MDL-28170, though only at early timepoints after injury. These results suggest Ca(2+)-dependent recruitment of cysteine proteases within 24h of excitotoxic insult, but activation of alternative cellular degrading mechanisms after 24h. Further, these data suggest that synaptophysin may be a substrate for calpains and related proteases.

  6. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine

    PubMed Central

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C.

    2015-01-01

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases. PMID:26400108

  7. Methylene Blue Inhibits Caspases by Oxidation of the Catalytic Cysteine.

    PubMed

    Pakavathkumar, Prateep; Sharma, Gyanesh; Kaushal, Vikas; Foveau, Bénédicte; LeBlanc, Andrea C

    2015-09-24

    Methylene blue, currently in phase 3 clinical trials against Alzheimer Disease, disaggregates the Tau protein of neurofibrillary tangles by oxidizing specific cysteine residues. Here, we investigated if methylene blue can inhibit caspases via the oxidation of their active site cysteine. Methylene blue, and derivatives, azure A and azure B competitively inhibited recombinant Caspase-6 (Casp6), and inhibited Casp6 activity in transfected human colon carcinoma cells and in serum-deprived primary human neuron cultures. Methylene blue also inhibited recombinant Casp1 and Casp3. Furthermore, methylene blue inhibited Casp3 activity in an acute mouse model of liver toxicity. Mass spectrometry confirmed methylene blue and azure B oxidation of the catalytic Cys163 cysteine of Casp6. Together, these results show a novel inhibitory mechanism of caspases via sulfenation of the active site cysteine. These results indicate that methylene blue or its derivatives could (1) have an additional effect against Alzheimer Disease by inhibiting brain caspase activity, (2) be used as a drug to prevent caspase activation in other conditions, and (3) predispose chronically treated individuals to cancer via the inhibition of caspases.

  8. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    NASA Astrophysics Data System (ADS)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  9. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  10. Plaque Enhancement of Enteroviruses by Magnesium Chloride, Cysteine, and Pancreatin

    PubMed Central

    Wallis, Craig; Morales, Fred; Powell, Joycelyn; Melnick, Joseph L.

    1966-01-01

    Wallis, Craig (Baylor University College of Medicine, Houston, Tex.), Fred Morales, Joycelyn Powell, and Joseph L. Melnick. Plaque enhancement of enteroviruses by magnesium chloride, cysteine, and pancreatin. J. Bacteriol. 91:1932–1935. 1966.—Plaque formation of 21 echoviruses (types 1–6, 9, 13, 15–19, 23–26, 29–32) and 8 coxsackieviruses (B1–6, A7, and A9) was enhanced by increased concentrations of MgCl2, l-cysteine, and pancreatin in agar overlay medium. In most cases, cationic and anionic polymers (diethylaminoethyl dextran, dextran sulfate, or protamine sulfate) were ineffective. All strains of poliovirus and group B coxsackieviruses were enhanced under agar by MgCl2. Five of the eight coxsackieviruses tested were also enhanced by cysteine or pancreatin. Certain enteroviruses, which have been difficult to assay by plaque method, can now be quantified effectively by incorporation of additives such as MgCl2, cysteine, or pancreatin into the overlay medium. PMID:4287074

  11. 21 CFR 184.1271 - L-Cysteine.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false L-Cysteine. 184.1271 Section 184.1271 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD... ingredient meets the appropriate part of the specification set forth in the “Food Chemicals Codex,” 3d Ed...

  12. 21 CFR 184.1272 - L-Cysteine monohydrochloride.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false L-Cysteine monohydrochloride. 184.1272 Section 184.1272 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) DIRECT FOOD SUBSTANCES AFFIRMED AS GENERALLY RECOGNIZED AS SAFE Listing of Specific Substances Affirmed...

  13. IDENTIFYING CRITICAL CYSTEINE RESIDUES IN ARSENIC (+3 OXIDATION STATE) METHYLTRANSFERASE

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes methylation of inorganic arsenic to mono, di, and trimethylated arsenicals. Orthologous AS3MT genes in genomes ranging from simple echinoderm to human predict a protein with five conserved cysteine (C) residues. In ...

  14. Electricity generation from cysteine in a microbial fuel cell.

    PubMed

    Logan, Bruce E; Murano, Cassandro; Scott, Keith; Gray, Neil D; Head, Ian M

    2005-03-01

    In a microbial fuel cell (MFC), power can be generated from the oxidation of organic matter by bacteria at the anode, with reduction of oxygen at the cathode. Proton exchange membranes used in MFCs are permeable to oxygen, resulting in the diffusion of oxygen into the anode chamber. This could either lower power generation by obligate anaerobes or result in the loss in electron donor from aerobic respiration by facultative or other aerobic bacteria. In order to maintain anaerobic conditions in conventional anaerobic laboratory cultures, chemical oxygen scavengers such as cysteine are commonly used. It is shown here that cysteine can serve as a substrate for electricity generation by bacteria in a MFC. A two-chamber MFC containing a proton exchange membrane was inoculated with an anaerobic marine sediment. Over a period of a few weeks, electricity generation gradually increased to a maximum power density of 19 mW/m(2) (700 or 1000 Omega resistor; 385 mg/L of cysteine). Power output increased to 39 mW/m(2) when cysteine concentrations were increased up to 770 mg/L (493 Omega resistor). The use of a more active cathode with Pt- or Pt-Ru, increased the maximum power from 19 to 33 mW/m(2) demonstrating that cathode efficiency limited power generation. Power was always immediately generated upon addition of fresh medium, but initial power levels consistently increased by ca. 30% during the first 24 h. Electron recovery as electricity was 14% based on complete cysteine oxidation, with an additional 14% (28% total) potentially lost to oxygen diffusion through the proton exchange membrane. 16S rRNA-based analysis of the biofilm on the anode of the MFC indicated that the predominant organisms were Shewanella spp. closely related to Shewanella affinis (37% of 16S rRNA gene sequences recovered in clone libraries).

  15. Cysteine cathepsins: from structure, function and regulation to new frontiers.

    PubMed

    Turk, Vito; Stoka, Veronika; Vasiljeva, Olga; Renko, Miha; Sun, Tao; Turk, Boris; Turk, Dušan

    2012-01-01

    It is more than 50 years since the lysosome was discovered. Since then its hydrolytic machinery, including proteases and other hydrolases, has been fairly well identified and characterized. Among these are the cysteine cathepsins, members of the family of papain-like cysteine proteases. They have unique reactive-site properties and an uneven tissue-specific expression pattern. In living organisms their activity is a delicate balance of expression, targeting, zymogen activation, inhibition by protein inhibitors and degradation. The specificity of their substrate binding sites, small-molecule inhibitor repertoire and crystal structures are providing new tools for research and development. Their unique reactive-site properties have made it possible to confine the targets simply by the use of appropriate reactive groups. The epoxysuccinyls still dominate the field, but now nitriles seem to be the most appropriate "warhead". The view of cysteine cathepsins as lysosomal proteases is changing as there is now clear evidence of their localization in other cellular compartments. Besides being involved in protein turnover, they build an important part of the endosomal antigen presentation. Together with the growing number of non-endosomal roles of cysteine cathepsins is growing also the knowledge of their involvement in diseases such as cancer and rheumatoid arthritis, among others. Finally, cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes. The current challenge is to identify their endogenous substrates, in order to gain an insight into the mechanisms of substrate degradation and processing. In this review, some of the remarkable advances that have taken place in the past decade are presented. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Molybdenum but not copper counteracts cysteine-induced tibial dyschondroplasia in broiler chicks.

    PubMed

    Bai, Y; Sunde, M L; Cook, M E

    1994-04-01

    Studies were conducted to evaluate the ability of copper and molybdenum to prevent cysteine-induced tibial dyschondroplasia in broiler chicks. Experiment 1 was a 3 x 3 factorial arrangement of treatments used to investigate the interaction between Cu (0, 150 or 300 mg/kg diet) and Mo (0, 10, or 100 mg/kg diet) on cysteine-induced tibial dyschondroplasia. Molybdenum at both supplemental levels, but not Cu, prevented cysteine-induced tibial dyschondroplasia. In Experiment 2 (a 3 x 3 factorial arrangement of treatments with 0, 5 or 10 g/kg diet of cysteine and 0, 10 or 100 mg/kg diet of Mo), Mo prevented cysteine-induced but not spontaneous tibial dyschondroplasia. Cysteine and Mo did not affect the mechanical properties of the tibiotarsus. In Experiment 3, cysteine (0 or 10 g/kg diet) and Mo (0 or 100 mg/kg diet) were used to study the tissue concentrations of mineral and hepatic sulfite oxidase activity. Supplemental Mo increased Mo concentrations in the plasma and liver. Cysteine prevented these increases; however, cysteine, in the absence of supplemental Mo, did not affect concentrations of Mo in these tissues. Dietary cysteine and/or Mo did not affect tissue levels of Cu. We conclude that Mo prevents cysteine-induced tibial dyschondroplasia and that the induction of tibial dyschondroplasia by cysteine is not related to the Mo and Cu deficiency.

  17. Lack of congruence between cysteine dioxygenase activity and S-carboxymethyl-L-cysteine S-oxidation activity in rat cytosol.

    PubMed

    Khan, Samera; Mitchell, Stephen C; Steventon, Glyn B

    2004-08-01

    The identity of the enzyme(s) responsible for the S-oxidation of the mucoactive drug S-carboxymethyl-L-cysteine (SCMC) is unknown but the protein(s) are a susceptibility factor for a number of chronic degenerative diseases. The structural similarities between the amino acid L-cysteine and SCMC have raised the possibility that cysteine dioxygenase (CDO) may be responsible for this biotransformation reaction. Both CDO and SCMC S-oxygenase were found to require Fe2+ for enzymatic activity, and both enzyme activities were inhibited by Fe2+ and Fe3+ chelators. However, sulphydryl group modification of the enzymes resulted in the activation of the S-oxidation of SCMC but inhibition of the S-oxidation of L-cysteine. When the two enzyme activities were quantified in 20 female hepatic cytosolic fractions no linear correlation in the production of their respective metabolites was seen. The results of this investigation indicate that CDO is not responsible for the S-oxidation of SCMC in the rat.

  18. Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

    PubMed

    Sallmann, Madleen; Kumar, Suresh; Chernev, Petko; Nehrkorn, Joscha; Schnegg, Alexander; Kumar, Devesh; Dau, Holger; Limberg, Christian; de Visser, Sam P

    2015-05-11

    Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.

  19. DU-127090 Solvay/H Lundbeck.

    PubMed

    Wolf, William

    2003-01-01

    DU-127090 is a mixed dopamine antagonist/serotonin agonist in development by Solvay and H Lundbeck as a potential treatment for psychosis and schizophrenia, for which it is in phase II clinical trials. In August 2002, phase II trials were ongoing and Lundbeck expected to commence phase III trials in 2003 and file an NDA after 2004. DU-127090 is also under development for Parkinson's disease, for which it is in phase I clinical trials.

  20. Synthesis, screening, and sequencing of cysteine-rich one-bead one-compound peptide libraries.

    PubMed

    Juskowiak, Gary L; McGee, Christopher J; Greaves, John; Van Vranken, David L

    2008-01-01

    Cysteine-rich peptides are valued as tags for biarsenical fluorophores and as environmentally important reagents for binding toxic heavy metals. Due to the inherent difficulties created by cysteine, the power of one-bead one-compound (OBOC) libraries has never been applied to the discovery of short cysteine-rich peptides. We have developed the first method for the synthesis, screening, and sequencing of cysteine-rich OBOC peptide libraries. First, we synthesized a heavily biased cysteine-rich OBOC library, incorporating 50% cysteine at each position (Ac-X8-KM-TentaGel). Then, we developed conditions for cysteine alkylation, cyanogen bromide cleavage, and direct MS/MS sequencing of that library at the single bead level. The sequencing efficiency of this library was comparable to a traditional cysteine-free library. To validate screening of cysteine-rich OBOC libraries, we reacted a library with the biarsenical FlAsH and identified beads bearing the known biarsenical-binding motif (CCXXCC). These results enable OBOC libraries to be used in high-throughput discovery of cysteine-rich peptides for protein tagging, environmental remediation of metal contaminants, or cysteine-rich pharmaceuticals.

  1. Electrostatics of cysteine residues in proteins: Parameterization and validation of a simple model

    PubMed Central

    Salsbury, Freddie R.; Poole, Leslie B.; Fetrow, Jacquelyn S.

    2013-01-01

    One of the most popular and simple models for the calculation of pKas from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pKas. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pKas; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pKas. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pKa values (where the calculation should reproduce the pKa within experimental error). Both the general behavior of cysteines in proteins and the perturbed pKa in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pKa should be shifted, and validation of force field parameters for cysteine residues. PMID:22777874

  2. Biokinetics and dosimetry of depleted uranium (DU) in rats implanted with DU fragments.

    SciTech Connect

    Guilmette, Ray A.; Hahn, Fletcher F.; Durbin, P. W.

    2004-01-01

    A number of U. S. veterans of the Persian Gulf War were wounded with depleted uranium (DU) metal fragments as a result of 'friendly fire' incidents, in which Abrams tanks and Bradley fighting vehicles were struck by DU anti-armor munitions. Some of the crew members who survived were left with multiple small fragments of DU in their muscles and soft tissues. The number, size and location of the fragments made them inoperable in general, and therefore subject to long-term retention. Because there was inadequate data to predict the potential carcinogenicity of DU fragments in soft tissues, Hahn et al. (2003) conducted a lifespan cancer study in rats. As part of that study, a number of rats were maintained to study the biokinetics and dosimetry of DU implanted intramuscularly in male Wistar rats. Typically, four metal fragments, either as cylindrical pellets or square wafers were implanted into the biceps femoris muscles of the rats. Urine samples were collected periodically during their lifespans, and DU was analyzed in kidneys and eviscerated carcass (minus the implant sites) at death. The daily DU urinary excretion rate increased steeply during the first 30 d after implantation peaking at about 90 d at 3-10 x 10{sup -3}%/d. During the first 150 d, the average excretion rate was 2.4 x 10{sup -3}%/d, decreasing thereafter to about 1 x 10{sup -3}%/d. Serial radiographs were made of the wound sites to monitor gross morphologic changes in the DU implant and the surrounding tissue. As early as 1 w after implantation, radiographs showed the presence of surface corrosion and small, dense bodies near the original implant, presumably DU. This corrosion from the surface of the implant continued with time, but did not result in an increasing amount of DU reaching the blood and urine after the first 3 mo. During this 3-mo period, connective tissue capsules formed around the implants, and are hypothesized to have reduced the access of DU to tissue fluids by limiting the diffusion

  3. A Review on Various Uses of N-Acetyl Cysteine

    PubMed Central

    Mokhtari, Vida; Afsharian, Parvaneh; Shahhoseini, Maryam; Kalantar, Seyed Mehdi; Moini, Ashraf

    2017-01-01

    N-acetyl cysteine (NAC), as a nutritional supplement, is a greatly applied antioxidant in vivo and in vitro. NAC is a precursor of L-cysteine that results in glutathione elevation biosynthesis. It acts directly as a scavenger of free radicals, especially oxygen radicals. NAC is a powerful antioxidant. It is also recommended as a potential treatment option for different disorders resulted from generation of free oxygen radicals. Additionally, it is a protected and endured mucolytic drug that mellows tenacious mucous discharges. It has been used for treatment of various diseases in a direct action or in a combination with some other medications. This paper presents a review on various applications of NAC in treatment of several diseases. PMID:28367412

  4. Expression and purification of cysteine introduced recombinant saporin.

    PubMed

    Günhan, Emine; Swe, Mimi; Palazoglu, Mine; Voss, John C; Chalupa, Leo M

    2008-04-01

    Saporin, a ribosome inactivating protein is widely used for immunotoxin construction. Here we describe a mutation of saporin (sap)-3 DNA by introducing a cysteine residue, followed by protein expression and purification by ion exchange chromatography. The purified Cys255sap-3, sap-3 isomer and commercially purchased saporin, were tested for toxicity using assays measuring inhibition for protein synthesis. The IC(50) values showed that the toxicity of the Cys255sap-3 is equivalent to the sap-3 isomer and commercial saporin. Reactivity of Cys255sap-3 was confirmed by labeling with a thio-specific fluorescent probe as well as conjugation with a nonspecific mouse IgG. We have found that a single cysteine within saporin provides a method for antibody conjugation that ensures a uniform and reproducible modification of a saporin variant retaining high activity.

  5. Prediction of the disulfide-bonding state of cysteines in proteins based on dipeptide composition.

    PubMed

    Song, Jiang-Ning; Wang, Ming-Lei; Li, Wei-Jiang; Xu, Wen-Bo

    2004-05-21

    In this paper, a novel approach has been introduced to predict the disulfide-bonding state of cysteines in proteins by means of a linear discriminator based on their dipeptide composition. The prediction is performed with a newly enlarged dataset with 8114 cysteine-containing segments extracted from 1856 non-homologous proteins of well-resolved three-dimensional structures. The oxidation of cysteines exhibits obvious cooperativity: almost all cysteines in disulfide-bond-containing proteins are in the oxidized form. This cooperativity can be well described by protein's dipeptide composition, based on which the prediction accuracy of the oxidation form of cysteines scores as high as 89.1% and 85.2%, when measured on cysteine and protein basis using the rigorous jack-knife procedure, respectively. The result demonstrates the applicability of this new relatively simple method and provides superior prediction performance compared with existing methods for the prediction of the oxidation states of cysteines in proteins.

  6. Fluoresence quenching of riboflavin in aqueous solution by methionin and cystein

    NASA Astrophysics Data System (ADS)

    Drössler, P.; Holzer, W.; Penzkofer, A.; Hegemann, P.

    2003-01-01

    The fluorescence quantum distributions, fluorescence quantum yields, and fluorescence lifetimes of riboflavin in methanol, DMSO, water, and aqueous solutions of the sulphur atom containing amino acids methionin and cystein have been determined. In methanol, DMSO, and water (pH=4-8) only dynamic fluorescence reduction due to intersystem crossing and internal conversion is observed. In aqueous methionin solutions of pH=5.25-9 a pH independent static and dynamic fluorescence quenching occurs probably due to riboflavin anion-methionin cation pair formation. In aqueous cystein solutions (pH range from 4.15 to 9) the fluorescence quenching increases with rising pH due to cystein thiolate formation. The cystein thiol form present at low pH does not react with neutral riboflavin. Cystein thiolate present at high pH seems to react with neutral riboflavin causing riboflavin deprotonation (anion formation) by cystein thiolate reduction to the cystein thiol form.

  7. Thiazolidine prodrugs of cysteamine and cysteine as radioprotective agents

    SciTech Connect

    Roberts, J.C.; Koch, K.E.; Detrick, S.R.

    1995-08-01

    The need for protection against the toxic effects of ionizing radiation comes from many different directions: occupational exposure, nuclear accidents, environmental sources and protection of normal tissue during the therapeutic irradiation of cancer. Sulfhydryl-containing compounds, including cysteamine and L-cysteine, have long been known to possess radioprotective properties, but their therapeutic utility is limited by their side effects at radioprotective doses. To avoid this drawback, thiazolidine prodrugs of cysteamine and L-cysteine were prepared by the condensation of each thiolamine with the aldose monosaccharides, D-ribose and D-glucose, producing RibCyst, GlcCyst, Rib-Cys and GlcCys. The prodrugs were designed to liberate the parent thiolamine nonenzymatically, after ring opening and hydrolysis, which is then available e to function as a radioprotective agent. Cysteamine`s inherent toxicity, measured using Chinese hamster V79 cells growing in culture, was completely eliminated, even at concentrations as high as 25 mM, by providing the thiolamine in the form of a prodrug. Good protection against radiation-induced lethality was demonstrated by the cysteamine prodrugs using a clonogenic assay. Protection against radiation-induced DNA single-strand breaks, as measured by alkaline elution, was also shown by both RibCyst and GlcCyst; this activity was higher than that exhibited by either cysteamine or WR-1065. The L-cysteine prodrugs, RibCys and GlcCys, also possessed radioprotective abilities under most of the conditions studied. Protection against DNA damage was comparable between L-cystein, WR-1065 and RibCys. 42 refs., 7 figs., 2 tabs.

  8. Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

    PubMed Central

    El Ridi, Rashika; Tallima, Hatem; Selim, Sahar; Donnelly, Sheila; Cotton, Sophie; Gonzales Santana, Bibiana; Dalton, John P.

    2014-01-01

    Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (P<0.02, 50%) against an experimental challenge infection with Schistosoma mansoni. Since schistosomes express and secrete papain-like cysteine peptidases we reasoned that these could be employed as vaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (P<0.0001) protection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines PMID:24465551

  9. Mechanical and chemical properties of cysteine-modified kinesin molecules.

    PubMed

    Iwatani, S; Iwane, A H; Higuchi, H; Ishii, Y; Yanagida, T

    1999-08-10

    To probe the structural changes within kinesin molecules, we made the mutants of motor domains of two-headed kinesin (4-411 aa) in which either all the five cysteines or all except Cys45 were mutated. A residual cysteine (Cys45) of the kinesin mutant was labeled with an environment-sensitive fluorescent probe, acrylodan. ATPase activity, mechanical properties, and fluorescence intensity of the mutants were measured. Upon acrylodan-labeled kinesin binding to microtubules in the presence of 1 mM AMPPNP, the peak intensity was enhanced by 3.4-fold, indicating the structural change of the kinesin head by the binding. Substitution of cysteines decreased both the maximum microtubule-activated ATPase and the sliding velocity to the same extent. However, the maximum force and the step size were not affected; the force produced by a single molecule was 6-6.5 pN, and a step size due to the hydrolysis of one ATP molecule by kinesin molecules was about 10 nm for all kinesins. This step size was close to a unitary step size of 8 nm. Thus, the mechanical events of kinesin are tightly coupled with the chemical events.

  10. Aminothienopyridazines and methylene blue affect Tau fibrillization via cysteine oxidation.

    PubMed

    Crowe, Alex; James, Michael J; Lee, Virginia M-Y; Smith, Amos B; Trojanowski, John Q; Ballatore, Carlo; Brunden, Kurt R

    2013-04-19

    Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents.

  11. Aminothienopyridazines and Methylene Blue Affect Tau Fibrillization via Cysteine Oxidation*

    PubMed Central

    Crowe, Alex; James, Michael J.; Lee, Virginia M.-Y.; Smith, Amos B.; Trojanowski, John Q.; Ballatore, Carlo; Brunden, Kurt R.

    2013-01-01

    Alzheimer disease and several other neurodegenerative disorders are characterized by the accumulation of intraneuronal fibrils comprised of the protein Tau. Tau is normally a soluble protein that stabilizes microtubules, with splice isoforms that contain either three (3-R) or four (4-R) microtubule binding repeats. The formation of Tau fibrils is thought to result in neuronal damage, and inhibitors of Tau fibrillization may hold promise as therapeutic agents. The process of Tau fibrillization can be replicated in vitro, and a number of small molecules have been identified that inhibit Tau fibril formation. However, little is known about how these molecules affect Tau fibrillization. Here, we examined the mechanism by which the previously described aminothieno pyridazine (ATPZ) series of compounds inhibit Tau fibrillization. Active ATPZs were found to promote the oxidation of the two cysteine residues within 4-R Tau by a redox cycling mechanism, resulting in the formation of a disulfide-containing compact monomer that was refractory to fibrillization. Moreover, the ATPZs facilitated intermolecular disulfide formation between 3-R Tau monomers, leading to dimers that were capable of fibrillization. The ATPZs also caused cysteine oxidation in molecules unrelated to Tau. Interestingly, methylene blue, an inhibitor of Tau fibrillization under evaluation in Alzheimer disease clinical trials, caused a similar oxidation of cysteines in Tau and other molecules. These findings reveal that the ATPZs and methylene blue act by a mechanism that may affect their viability as potential therapeutic agents. PMID:23443659

  12. Cysteine cathepsins in human dentin-pulp complex.

    PubMed

    Tersariol, Ivarne L; Geraldeli, Saulo; Minciotti, Christiane L; Nascimento, Fábio D; Pääkkönen, Virve; Martins, Marília T; Carrilho, Marcela R; Pashley, David H; Tay, Franklin R; Salo, Tuula; Tjäderhane, Leo

    2010-03-01

    Collagen-degrading matrix metalloproteinases (MMPs) are expressed by odontoblasts and present in dentin. We hypothesized that odontoblasts express other collagen-degrading enzymes such as cysteine cathepsins, and their activity would be present in dentin, because odontoblasts are known to express at least cathepsin D. Effect of transforming growth factor beta (TGF-beta) on cathepsin expression was also analyzed. Human odontoblasts and pulp tissue were cultured with and without TGF-beta, and cathepsin gene expression was analyzed with DNA microarrays. Dentin cathepsin and MMP activities were analyzed by degradation of respective specific fluorogenic substrates. Both odontoblasts and pulp tissue demonstrated a wide range of cysteine cathepsin expression that gave minor responses to TGF-beta. Cathepsin and MMP activities were observed in all dentin samples, with significant negative correlations in their activities with tooth age. These results demonstrate for the first time the presence of cysteine cathepsins in dentin and suggest their role, along with MMPs, in dentin modification with aging. Copyright (c) 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  13. Human Lamin B Contains a Farnesylated Cysteine Residue*

    PubMed Central

    Farnsworth, Christopher C.; Wolda, Sharon L.; Gelb, Michael H.; Glomset, John A.

    2012-01-01

    We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of link-age to the mevalonic acid derivative, and established the derivative’s structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H] mevalonic acid or [35S] cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B. PMID:2684976

  14. Cysteine as a Biological Probe for Comparing Plasma Sources

    NASA Astrophysics Data System (ADS)

    Lackmann, Jan-Wilm; Golda, Judith; Kogelheide, Friederike; Held, Julian; Schulz-von-der-Gathen, Volker; Stapelmann, Katharina

    2016-09-01

    A large variety of plasma sources are available in the plasma medicine community. While enabling to choose the most promising source for a certain biomedical application, comparison of the different sources with a focus on their effect on biological targets is rather challenging. To allow for better comparison of various sources, the recent European COST action MP1101 was used to design the COST reference microplasma jet. Cysteine is a promising candidate investigate the impact of plasma from various sources on a standardized biological molecule, which is especially relevant for the investigations on a molecular level after plasma treatment. The simple structure of cysteine allows for a more in-depth analysis of each chemical group after plasma treatment and enables a comparison between different plasma sources and treatment parameters on each chemical group. The model itself has already been successfully established using a dielectric barrier discharge. Here, additional plasma sources are compared by the means of their impact on cysteine samples, showing e.g. the influence of feed-gas variations by adding oxygen or nitrogen admixture This work was supported by the German Research Foundation (DFG) with the packet grant PAK816 (PlaCID).

  15. Analysis of S-nitrosothiols via Copper Cysteine (2C) and Copper Cysteine - Carbon Monoxide (3C) Methods

    PubMed Central

    Rogers, Stephen C.; Gibbons, Lindsey B.; Griffin, Sherraine; Doctor, Allan

    2012-01-01

    This chapter summarizes the principles of RSNO measurement in the gas phase, utilizing ozone-based chemiluminescence and the copper cysteine (2C) ± carbon monoxide (3C) reagent. Although an indirect method for quantifying RSNOs, this assay represents one of the most robust methodologies available. It exploits the NO• detection sensitivity of ozone based chemiluminscence, which is within the range required to detect physiological concentrations of RSNO metabolites. Additionally, the specificity of the copper cysteine (2C and 3C) reagent for RSNOs negates the need for sample pretreatment, thereby minimizing the likelihood of sample contamination (false positive results), NO species inter-conversion, or the loss of certain highly labile RSNO species. Herein, we outline the principles of this methodology, summarizing key issues, potential pitfalls and corresponding solutions. PMID:23116707

  16. Cirque du Monde as a health intervention

    PubMed Central

    Fournier, Cynthia; Drouin, Mélodie-Anne; Marcoux, Jérémie; Garel, Patricia; Bochud, Emmanuel; Théberge, Julie; Aubertin, Patrice; Favreau, Gil; Fleet, Richard

    2014-01-01

    Abstract Objective To present Cirque du Soleil’s social circus program, Cirque du Monde, to explore its potential as a primary health care tool for family physicians. Data sources A review of the literature in PubMed, the Cochrane Library, PsycINFO, LaPresse, Eureka, Google Scholar, and Érudit using the key words circus, social circus, Cirque du Monde, and Cirque du Soleil; a Montreal-based initiative, Espace Transition, modeled on Cirque du Monde; and personal communication with Cirque du Soleil’s Social Circus Training Advisor. Study selection The first 50 articles or websites identified for each key word in each of the databases were examined on the basis of their titles and abstracts in the case of articles, and on the basis of their titles and page content in the case of websites. Articles and websites that explored an aspect of social circuses or that described an intervention that involved circuses were then retained for analysis. Because all literature on social circuses was searched, no criterion for year of publication was used. Synthesis No articles on the social circus as a health intervention were found. One study on the use of the circus as an intervention in schools was identified. It demonstrated an increase in self-esteem in the children who took part. One study on the use of the circus in a First Nations community was found; it contained nonspecific, qualitative findings. The other articles identified were merely descriptions of social circuses. One website was identified on the use of the social circus to help youth who had been treated in a hospital setting for major psychiatric disorders to re-enter the community. The team in the pediatric psychiatry department at Centre Hospitalier Universitaire Sainte-Justine, the children’s hospital in Montreal, Que, was contacted; they were leading this project, called Espace Transition. The unpublished preliminary findings of its pilot project demonstrate substantial improvements in overall patient

  17. The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

    PubMed

    Santiago, Margarita; Gardner, Richard C

    2015-07-01

    Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l-cystine and some other cysteine conjugates, but not l-cystathionine or l-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l-cysteine as a nitrogen source, and that overexpression of the gene results in increased H2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-l-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis.

  18. Mapping protein cysteine sulfonic acid modifications with specific enrichment and mass spectrometry: an integrated approach to explore the cysteine oxidation.

    PubMed

    Chang, Yuan-Chang; Huang, Chien-Ning; Lin, Chia-Hung; Chang, Huan-Cheng; Wu, Chih-Che

    2010-08-01

    Oxidation of thiol proteins, which results in conversion of cysteine residues to cysteine sulfenic, sulfinic or sulfonic acids, is an important posttranslational control of protein function in cells. To facilitate the analysis of this process with MALDI-MS, we have developed a method for selective enrichment and identification of peptides containing cysteine sulfonic acid (sulfopeptides) in tryptic digests of proteins based on ionic affinity capture using polyarginine-coated nanodiamonds as high-affinity probes. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a complex peptide mixture in which the abundance of the sulfonated analyte is as low as 0.02%. The polyarginine-coated probes exhibit a higher affinity for peptides containing multiple sulfonic acids than peptides containing single sulfonic acid. The limit of the detection is in the femtomole range, with the MALDI-TOF mass spectrometer operating in the negative ion mode. The results show that the new approach has good specificity even in the presence of phosphopeptides. An application of this method for selective enrichment and structural identification of sulfopeptides is demonstrated with the tryptic digests of performic-acid-oxidized BSA.

  19. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    PubMed

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  20. Analysis of transmembrane segment 8 of the GLUT1 glucose transporter by cysteine-scanning mutagenesis and substituted cysteine accessibility.

    PubMed

    Mueckler, Mike; Makepeace, Carol

    2004-03-12

    The GLUT1 glucose transporter has been proposed to form an aqueous substrate translocation pathway via the clustering of several amphipathic transmembrane helices (Mueckler, M., Caruso, C., Baldwin, S. A., Panico, M., Blench, I., Morris, H. R., Allard, W. J., Lienhard, G. E., and Lodish, H. F. (1985) Science 229, 941-945). The possible role of transmembrane helix 8 in the formation of this permeation pathway was investigated using cysteine-scanning mutagenesis and the membrane-impermeant sulfhydryl-specific reagent, p-chloromercuribenzenesulfonate (pCMBS). Twenty-one GLUT1 mutants were created from a fully functional cysteine-less parental GLUT1 molecule by successively changing each residue along transmembrane segment 8 to a cysteine. The mutant proteins were then expressed in Xenopus oocytes, and their membrane concentrations, 2-deoxyglucose uptake activities, and sensitivities to pCMBS were determined. Four positions within helix 8, alanine 309, threonine 310, serine 313, and glycine 314, were accessible to pCMBS as judged by the inhibition of transport activity. All four of these residues are clustered along one face of a putative alpha-helix. These results suggest that transmembrane segment 8 of GLUT1 forms part of the sugar permeation pathway. Updated two-dimensional models for the orientation of the 12 transmembrane helices and the conformation of the exofacial glucose binding site of GLUT1 are proposed that are consistent with existing experimental data and homology modeling based on the crystal structures of two bacterial membrane transporters.

  1. The glutamate transporters EAAT2 and EAAT3 mediate cysteine uptake in cortical neuron cultures.

    PubMed

    Chen, Yongmei; Swanson, Raymond A

    2003-03-01

    Cysteine availability is normally the rate-limiting factor in glutathione synthesis. How neurons obtain cysteine from extracellular space is not well established. Here we used mouse cortical neuron cultures to examine the role of the excitatory amino acid transporters (EAATs) in neuronal cysteine uptake. The cultured neurons expressed both EAAT2 and EAAT3. Cysteine uptake was predominantly (> 85%) Na+-dependent, with an apparent Km of 37 microm. Cysteine uptake was reduced by the EAAT substrates l-glutamate and l-aspartate and by synthetic EAAT inhibitors. The non-selective EAAT inhibitor threo-beta-hydroxyaspartate had a significantly greater maximal inhibitory effect than did the EAAT2-selective inhibitor, dihydrokainate, indicating uptake by both EAAT2 and EAAT3. Serine, a substrate of ASC uptake system, had negligible effects on cysteine uptake at 10-fold excess concentrations. To assess the functional importance of EAAT-mediated cysteine uptake in neuronal glutathione synthesis, cultures were treated with diethylmaleate to deplete glutathione, then incubated with cysteine in the presence or absence of EAAT inhibitors. Threo-beta-benzyloxyaspartate and the non-transportable inhibitor threo-beta-hydroxyaspartate both inhibited the cysteine-dependent glutathione synthesis. The findings suggest that neuronal EAAT activity can be a rate-limiting step for neuronal glutathione synthesis and that the primary function of EAATs expressed by neurons in vivo may be to transport cysteine.

  2. Effects of dietary cysteine on blood sulfur amino acid, glutathione, and malondialdehyde concentrations in cats.

    PubMed

    Fettman, M J; Valerius, K D; Ogilvie, G K; Bedwell, C L; Richardson, K L; Walton, J A; Hamar, D W

    1999-03-01

    To determine effects of dietary cysteine on blood sulfur amino acids (SAA), reduced glutathione (GSH), oxidized glutathione (GSSG), and malondialdehyde (MDA) concentrations in cats. 12 healthy adult cats. Cats were fed diets with a nominal (0.50 g/100 g dry matter [DM]), moderate (1.00 g/100 g DM), or high (1.50 g/100 g DM) cysteine content in a 3 X 3 Latin square design with blocks of 8 weeks' duration. Venous blood samples were collected after each diet had been fed for 4 and 8 weeks, and a CBC and serum biochemical analyses were performed; poikilocyte, reticulocyte, and Heinz body counts were determined; and MDA, GSH, GSSG, and SAA concentrations were measured. Blood cysteine and MDA concentrations were not significantly affected by dietary cysteine content. Blood methionine, homocysteine, and GSSG concentrations were significantly increased when cats consumed the high cysteine content diet but not when they consumed the moderate cysteine content diet, compared with concentrations obtained when cats consumed the nominal cysteine content diet. Blood GSH concentrations were significantly increased when cats consumed the moderate or high cysteine content diet. Increased dietary cysteine content promotes higher blood methionine, homocysteine, GSH, and GSSG concentrations in healthy cats. Supplemental dietary cysteine may be indicated to promote glutathione synthesis and ameliorate adverse effects of oxidative damage induced by disease or drugs.

  3. Cysteine-mediated reductive dissolution of poorly crystalline iron(III) oxides by Geobacter sulfurreducens.

    PubMed

    Doong, Ruey-An; Schink, Bernhard

    2002-07-01

    The reductive dissolution of poorly crystalline ferric oxides in the presence of cysteine was investigated to evaluate the potential of cysteine as a possible electron carrier to stimulate the reduction of iron(III) oxides by Geobacter sulfurreducens. The extent and rate of biotic and abiotic reduction of iron(III) oxides in the presence of cysteine at various concentrations were compared. Iron(III) oxides were reduced abiotically by cysteine. The initial rate and extent of iron(III) oxide reduction were correlated linearly with the cysteine concentration ranging from 0 to 6 mM. Also, addition of 0.5-2 mM cysteine significantly stimulated the rate and the extent of iron(III) oxide reduction in cultures of G. sulfurreducens. The cysteine concentration decreased in accordance with the increase of Fe(II) concentration and reached a nearly constant residual concentration. Cysteine depletion followed first-order kinetics and increased linearly with the cysteine concentration. An 8- to 11-fold increase in the extent of iron(III) oxide reduction relative to the abiotic system was observed. Comparison of sorbed and dissolved Fe(II) concentrations between cultures amended with cysteine and with other organic chelators showed that solubilization is not the main factor in cysteine-stimulated Fe(III) reduction. Addition of cystine could enhanced the extent of iron(III) oxide reduction, concomitant with the increase of the regenerated cysteine concentration and support the hypothesis that cysteine could serve as an electron carrier to transfer electrons from G. sulfurreducens to poorly crystalline iron(III) oxides.

  4. tRNA synthase suppression activates de novo cysteine synthesis to compensate for cystine and glutathione deprivation during ferroptosis.

    PubMed

    Shimada, Kenichi; Stockwell, Brent R

    2016-03-01

    Glutathione is a major endogenous reducing agent in cells, and cysteine is a limiting factor in glutathione synthesis. Cysteine is obtained by uptake or biosynthesis, and mammalian cells often rely on either one or the other pathway. Because of the scarcity of glutathione, blockade of cysteine uptake causes oxidative cell death known as ferroptosis. A new study suggests that tRNA synthetase suppression activates the endogenous biosynthesis of cysteine, compensates such cysteine loss, and thus makes cells resistant to ferroptosis.

  5. Mass Spectrometric Analysis of l-Cysteine Metabolism: Physiological Role and Fate of l-Cysteine in the Enteric Protozoan Parasite Entamoeba histolytica

    PubMed Central

    Jeelani, Ghulam; Sato, Dan; Soga, Tomoyoshi; Watanabe, Haruo

    2014-01-01

    ABSTRACT l-Cysteine is essential for virtually all living organisms, from bacteria to higher eukaryotes. Besides having a role in the synthesis of virtually all proteins and of taurine, cysteamine, glutathione, and other redox-regulating proteins, l-cysteine has important functions under anaerobic/microaerophilic conditions. In anaerobic or microaerophilic protozoan parasites, such as Entamoeba histolytica, l-cysteine has been implicated in growth, attachment, survival, and protection from oxidative stress. However, a specific role of this amino acid or related metabolic intermediates is not well understood. In this study, using stable-isotope-labeled l-cysteine and capillary electrophoresis-time of flight mass spectrometry, we investigated the metabolism of l-cysteine in E. histolytica. [U-13C3, 15N]l-cysteine was rapidly metabolized into three unknown metabolites, besides l-cystine and l-alanine. These metabolites were identified as thiazolidine-4-carboxylic acid (T4C), 2-methyl thiazolidine-4-carboxylic acid (MT4C), and 2-ethyl-thiazolidine-4-carboxylic acid (ET4C), the condensation products of l-cysteine with aldehydes. We demonstrated that these 2-(R)-thiazolidine-4-carboxylic acids serve for storage of l-cysteine. Liberation of l-cysteine occurred when T4C was incubated with amebic lysates, suggesting enzymatic degradation of these l-cysteine derivatives. Furthermore, T4C and MT4C significantly enhanced trophozoite growth and reduced intracellular reactive oxygen species (ROS) levels when it was added to cultures, suggesting that 2-(R)-thiazolidine-4-carboxylic acids are involved in the defense against oxidative stress. PMID:25370494

  6. Cri du Chat: report of a case.

    PubMed

    Sparks, S; Hutchinson, B

    1980-01-01

    Since Lejeune et al. (1963) first described the syndrome of Cri du Chat (Cry of the Cat), cases have been described in the literature in terms of genetic abnormalities. All cases were severely retarded and the mental impairment has been believed to be progressive, although no longitudinal studies have been reported. Descriptions of speech and language behavior have been scarce. This paper presents a case of a 7-yr, 6 mo-old girl with Cri du Chat who has received speech and language therapy for five years. Her speech, language, and mental development are noted and are not consistent with cases reported previously.

  7. Atomes du big bang : premiere detection

    NASA Astrophysics Data System (ADS)

    Bonnet-Bidaud, J. M.

    1994-09-01

    Ce fut au Keck d'ouvrir le bal, en debusquant du deuterium a plus de 10 milliards d'annees-lumiere. Puis, en juillet, vint l'annonce qu'Hubble avait apercu de l'helium flottant dans le milieu intergalactque lontain. Que ces deux prouesses realisees a la lumiere de quasars se confirment et elles ouvriraient enfin la chasse aux atomes primordiaux nes dans les trois premieres minutes de l'Univers. Avec toutefois un probleme : l'une apporterait de l'eau au moulin du Big Bang, mais l'autre ferait plutot figure de pave dans la mare ...

  8. Anaerobic Cysteine Degradation and Potential Metabolic Coordination in Salmonella enterica and Escherichia coli.

    PubMed

    Loddeke, Melissa; Schneider, Barbara; Oguri, Tamiko; Mehta, Iti; Xuan, Zhenyu; Reitzer, Larry

    2017-08-15

    Salmonella enterica has two CyuR-activated enzymes that degrade cysteine, i.e., the aerobic CdsH and an unidentified anaerobic enzyme; Escherichia coli has only the latter. To identify the anaerobic enzyme, transcript profiling was performed for E. coli without cyuR and with overexpressed cyuR Thirty-seven genes showed at least 5-fold changes in expression, and the cyuPA (formerly yhaOM) operon showed the greatest difference. Homology suggested that CyuP and CyuA represent a cysteine transporter and an iron-sulfur-containing cysteine desulfidase, respectively. E. coli and S. enterica ΔcyuA mutants grown with cysteine generated substantially less sulfide and had lower growth yields. Oxygen affected the CyuR-dependent genes reciprocally; cyuP-lacZ expression was greater anaerobically, whereas cdsH-lacZ expression was greater aerobically. In E. coli and S. enterica, anaerobic cyuP expression required cyuR and cysteine and was induced by l-cysteine, d-cysteine, and a few sulfur-containing compounds. Loss of either CyuA or RidA, both of which contribute to cysteine degradation to pyruvate, increased cyuP-lacZ expression, which suggests that CyuA modulates intracellular cysteine concentrations. Phylogenetic analysis showed that CyuA homologs are present in obligate and facultative anaerobes, confirming an anaerobic function, and in archaeal methanogens and bacterial acetogens, suggesting an ancient origin. Our results show that CyuA is the major anaerobic cysteine-catabolizing enzyme in both E. coli and S. enterica, and it is proposed that anaerobic cysteine catabolism can contribute to coordination of sulfur assimilation and amino acid synthesis.IMPORTANCE Sulfur-containing compounds such as cysteine and sulfide are essential and reactive metabolites. Exogenous sulfur-containing compounds can alter the thiol landscape and intracellular redox reactions and are known to affect several cellular processes, including swarming motility, antibiotic sensitivity, and biofilm

  9. Silver(I) complex formation with cysteine, penicillamine, and glutathione.

    PubMed

    Leung, Bonnie O; Jalilehvand, Farideh; Mah, Vicky; Parvez, Masood; Wu, Qiao

    2013-04-15

    The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen), and glutathione (H3Glu) in alkaline aqueous solution was examined using extended X-ray absorption fine structure (EXAFS) and (109)Ag NMR spectroscopic techniques. The complexes formed in 0.1 mol dm(-3) Ag(I) solutions with cysteine and penicillamine were investigated for ligand/Ag(I) (L/Ag) mole ratios increasing from 2.0 to 10.0. For the series of cysteine solutions (pH 10-11) a mean Ag-S bond distance of 2.45 ± 0.02 Å consistently emerged, while for penicillamine (pH 9) the average Ag-S bond distance gradually increased from 2.40 to 2.44 ± 0.02 Å. EXAFS and (109)Ag NMR spectra of a concentrated Ag(I)-cysteine solution (C(Ag(I)) = 0.8 mol dm(-3), L/Ag = 2.2) showed a mean Ag-S bond distance of 2.47 ± 0.02 Å and δ((109)Ag) 1103 ppm, consistent with prevailing, partially oligomeric AgS3 coordinated species, while for penicillamine (C(Ag(I)) = 0.5 mol dm(-3), L/Ag = 2.0) the mean Ag-S bond distance of 2.40 ± 0.02 Å and δ((109)Ag) 922 ppm indicate that mononuclear AgS2 coordinated complexes dominate. For Ag(I)-glutathione solutions (C(Ag(I)) = 0.01 mol dm(-3), pH ∼11), mononuclear AgS2 coordinated species with a mean Ag-S bond distance of 2.36 ± 0.02 Å dominate for L/Ag mole ratios from 2.0 to 10.0. The crystal structure of the silver(I)-cysteine compound (NH4)Ag2(HCys)(Cys)·H2O (1) precipitating at pH ∼10 was solved and showed a layer structure with both AgS3 and AgS3N coordination to the cysteinate ligands. A redetermination of the crystal structure of Ag(HPen)·H2O (2) confirmed the proposed digonal AgS2 coordination environment to bridging thiolate sulfur atoms in polymeric intertwining chains forming a double helix. A survey of Ag-S bond distances for crystalline Ag(I) complexes with S-donor ligands in different AgS2, AgS2(O/N), and AgS3 coordination environments was used, together with a survey of (109)Ag NMR chemical shifts, to assist assignments of the Ag

  10. Cadmium(II) complex formation with cysteine and penicillamine.

    PubMed

    Jalilehvand, Farideh; Leung, Bonnie O; Mah, Vicky

    2009-07-06

    The complex formation between cadmium(II) and the ligands cysteine (H(2)Cys) and penicillamine (H(2)Pen = 3,3'-dimethylcysteine) in aqueous solutions, having C(Cd(II)) approximately 0.1 mol dm(-3) and C(H(2)L) = 0.2-2 mol dm(-3), was studied at pH = 7.5 and 11.0 by means of (113)Cd NMR and Cd K- and L(3)-edge X-ray absorption spectroscopy. For all cadmium(II)-cysteine molar ratios, the mean Cd-S and Cd-(N/O) bond distances were found in the ranges 2.52-2.54 and 2.27-2.35 A, respectively. The corresponding cadmium(II)-penicillamine complexes showed slightly shorter Cd-S bonds, 2.50-2.53 A, but with the Cd-(N/O) bond distances in a similar wide range, 2.28-2.33 A. For the molar ratio C(H(2)L)/C(Cd(II)) = 2, the (113)Cd chemical shifts, in the range 509-527 ppm at both pH values, indicated complexes with distorted tetrahedral CdS(2)N(N/O) coordination geometry. With a large excess of cysteine (molar ratios C(H(2)Cys)/C(Cd(II)) >or= 10), complexes with CdS(4) coordination geometry dominate, consistent with the (113)Cd NMR chemical shifts, delta approximately 680 ppm at pH 7.5 and 636-658 ppm at pH 11.0, and their mean Cd-S distances were 2.53 +/- 0.02 A. At pH 7.5, the complexes are almost exclusively sulfur-coordinated as [Cd(S-cysteinate)(4)](n-), while at higher pH, the deprotonation of the amine groups promotes chelate formation. At pH 11.0, a minor amount of the [Cd(Cys)(3)](4-) complex with CdS(3)N coordination is formed. For the corresponding penicillamine solutions with molar ratios C(H(2)Pen)/C(Cd(II)) >or= 10, the (113)Cd NMR chemical shifts, delta approximately 600 ppm at pH 7.5 and 578 ppm at pH 11.0, together with the average bond distances, Cd-S 2.53 +/- 0.02 A and Cd-(N/O) 2.30-2.33 A, indicate that [Cd(penicillaminate)(3)](n-) complexes with chelating CdS(3)(N/O) coordination dominate already at pH 7.5 and become mixed with CdS(2)N(N/O) complexes at pH 11.0. The present study reveals differences between cysteine and penicillamine as ligands to the

  11. Cysteine 904 Is Required for Maximal Insulin Degrading Enzyme Activity and Polyanion Activation

    PubMed Central

    Song, Eun Suk; Melikishvili, Manana; Fried, Michael G.; Juliano, Maria A.; Juliano, Luiz; Rodgers, David W.; Hersh, Louis B.

    2012-01-01

    Cysteine residues in insulin degrading enzyme have been reported as non-critical for its activity. We found that converting the twelve cysteine residues in rat insulin degrading enzyme (IDE) to serines resulted in a cysteine-free form of the enzyme with reduced activity and decreased activation by polyanions. Mutation of each cysteine residue individually revealed cysteine 904 as the key residue required for maximal activity and polyanion activation, although other cysteines affect polyanion binding to a lesser extent. Based on the structure of IDE, Asn 575 was identified as a potential hydrogen bond partner for Cys904 and mutation of this residue also reduced activity and decreased polyanion activation. The oligomerization state of IDE did not correlate with its activity, with the dimer being the predominant form in all the samples examined. These data suggest that there are several conformational states of the dimer that affect activity and polyanion activation. PMID:23077523

  12. Cysteine and hydrogen sulphide in the regulation of metabolism: insights from genetics and pharmacology.

    PubMed

    Carter, Roderick N; Morton, Nicholas M

    2016-01-01

    Obesity and diabetes represent a significant and escalating worldwide health burden. These conditions are characterized by abnormal nutrient homeostasis. One such perturbation is altered metabolism of the sulphur-containing amino acid cysteine. Obesity is associated with elevated plasma cysteine, whereas diabetes is associated with reduced cysteine levels. One mechanism by which cysteine may act is through its enzymatic breakdown to produce hydrogen sulphide (H2S), a gasotransmitter that regulates glucose and lipid homeostasis. Here we review evidence from both pharmacological studies and transgenic models suggesting that cysteine and hydrogen sulphide play a role in the metabolic dysregulation underpinning obesity and diabetes. We then outline the growing evidence that regulation of hydrogen sulphide levels through its catabolism can impact metabolic health. By integrating hydrogen sulphide production and breakdown pathways, we re-assess current hypothetical models of cysteine and hydrogen sulphide metabolism, offering new insight into their roles in the pathogenesis of obesity and diabetes.

  13. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    SciTech Connect

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M. )

    1989-12-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.

  14. Tissue glutathione and cysteine levels in methionine-restricted rats.

    PubMed

    Richie, John P; Komninou, Despina; Leutzinger, Yvonne; Kleinman, Wayne; Orentreich, Norman; Malloy, Virginia; Zimmerman, Jay A

    2004-09-01

    Previously, we demonstrated that lifelong methionine (Met) restriction (MR) increases lifespan, decreases the incidence of aging-related diseases, increases blood glutathione (GSH) levels, and prevents loss of GSH during aging in rats. Our present objective was to elucidate the effects of MR on GSH metabolism and transport by determining the time course and nature of GSH and cysteine changes in blood and other tissues in young and mature rats. Male F-344 rats were placed on control (0.86% Met) or MR (0.17% Met) defined amino acid diets at age 7 wk and killed at different times thereafter. MR was also initiated in adult (12-mo-old) rats. Throughout the first 2 mo of MR, blood GSH levels increased 84% and liver GSH decreased 66% in relation to controls. After this period, liver GSH levels remained constant through at least 6 mo. GSH levels also decreased in the pancreas (80%) and kidney (22%) but remained unchanged in other tissues examined after 11 wk of MR. The increase in blood GSH was evident as soon as 1 wk after initiating MR and reached a plateau by 6 wk. A similar increase in erythrocyte GSH levels was observed when MR was administered to mature adult rats. Fasting decreased liver GSH in controls but had no further effect in MR animals. By 1 mo, cysteine levels had decreased in all tissues except brain. These results suggest that adaptive changes occur in the metabolism of Met, cysteine, and/or GSH as a result of MR in young and adult rats. These early metabolic changes lead to conservation of GSH levels in most extrahepatic tissues and increased GSH in erythrocytes by depleting liver GSH to a critical level.

  15. Gas-phase interactions of organotin compounds with cysteine.

    PubMed

    Latrous, Latifa; Salpin, Jean-Yves; Haldys, Violette; Léon, Emmanuelle; Correia, Catarina; Lamsabhi, Al Mokhtar

    2016-11-01

    The gas-phase interactions of cysteine with di-organotin and tri-organotin compounds have been studied by mass spectrometry experiments and quantum calculations. Positive-ion electrospray spectra show that the interaction of di- and tri-organotins with cysteine results in the formation of [(R)2 Sn(Cys-H)](+) and [(R)3 Sn(Cys)](+) ions, respectively. MS/MS spectra of [(R)2 Sn(Cys-H)](+) complexes are characterized by numerous fragmentation processes, notably associated with elimination of NH3 and (C,H2 ,O2 ). Several dissociation routes are characteristic of each given organic species. Upon collision, both the [(R)3 Sn(Gly)](+) and [(R)3 Sn(Cys)](+) complexes are associated with elimination of the intact amino acid, leading to the formation of [(R)3 Sn](+) cation. But for the latter complex, two additional fragmentation processes are observed, associated with the elimination of NH3 and C3 H4 O2 S. Calculations indicate that the interaction between organotins and cysteine is predominantly electrostatic but also exhibits a considerable covalent character, which is slightly more pronounced in tri-organotin complexes. A preferred bidentate interaction of the type -η(2) -S-NH2 , with sulfur and the amino group, is observed. As for the [(R)3 Sn(Cys)](+) complexes, their stability is due to the combination of the hydrogen bond taking place between the amino group and the sulfur lone pair and the interaction between the carboxylic oxygen atom and the metal. Copyright © 2016 John Wiley & Sons, Ltd.

  16. Decavanadate interactions with actin: cysteine oxidation and vanadyl formation.

    PubMed

    Ramos, Susana; Duarte, Rui O; Moura, José J G; Aureliano, Manuel

    2009-10-14

    Incubation of actin with decavanadate induces cysteine oxidation and oxidovanadium(IV) formation. The studies were performed combining kinetic with spectroscopic (NMR and EPR) methodologies. Although decavanadate is converted to labile oxovanadates, the rate of deoligomerization can be very slow (half-life time of 5.4 h, at 25 degrees C, with a first order kinetics), which effectively allows decavanadate to exist for some time under experimental conditions. It was observed that decavanadate inhibits F-actin-stimulated myosin ATPase activity with an IC(50) of 0.8 microM V(10) species, whereas 50 microM of vanadate or oxidovanadium(IV) only inhibits enzyme activity up to 25%. Moreover, from these three vanadium forms, only decavanadate induces the oxidation of the so called "fast" cysteines (or exposed cysteine, Cys-374) when the enzyme is in the polymerized and active form, F-actin, with an IC(50) of 1 microM V(10) species. Decavanadate exposition to F- and G-actin (monomeric form) promotes vanadate reduction since a typical EPR oxidovanadium(IV) spectrum was observed. Upon observation that V(10) reduces to oxidovanadium(IV), it is proposed that this cation interacts with G-actin (K(d) of 7.48 +/- 1.11 microM), and with F-actin (K(d) = 43.05 +/- 5.34 microM) with 1:1 and 4:1 stoichiometries, respectively, as observed by EPR upon protein titration with oxidovanadium(IV). The interaction of oxidovanadium(IV) with the protein may occur close to the ATP binding site of actin, eventually with lysine-336 and 3 water molecules.

  17. Biosynthesis and Reactivity of Cysteine Persulfides in Signaling.

    PubMed

    Yadav, Pramod K; Martinov, Michael; Vitvitsky, Victor; Seravalli, Javier; Wedmann, Rudolf; Filipovic, Milos R; Banerjee, Ruma

    2016-01-13

    Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and γ-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.

  18. Development of a biosensor specific for cysteine sulfoxides.

    PubMed

    Keusgen, Michael; Jünger, Martina; Krest, Ingo; Schöning, Michael J

    2003-05-01

    S-Alk(en)yl cysteine sulfoxides have been observed in several plants, mainly belonging to the onion family (Alliaceae), which are of high commercial interest (e.g. garlic, Allium sativum). The quality of most garlic containing herbal remedies produced from garlic powder is determined by their content of the cysteine sulfoxide alliin. Therefore, a comprehensive method for the documentation of alliin amounts present in the fresh plant material through to the final remedy is desirable. The newly developed biosensoric method described in this paper was designed in order to fulfil these demands. In contrast to conventional HPLC-methods, neither a pre-column derivatization nor a chromatographic separation are required allowing a high throughput of samples. This technique is based on immobilized alliinase (EC 4.4.1.4), which was combined with an ammonia-gas electrode. The enzyme was either placed in a small cartridge or was immobilized in direct contact of the electrode surface giving detection limits of 3.7 x 10(-7) and 5.9 x 10(-6) M. Founded on these experiments, a pH-sensitive electrolyte/insulator/semiconductor (EIS) layer structure made of Al/p-Si/SiO(2)/Si(3)N(4) was also combined with immobilized alliinase. Measurements could be performed in a range between 1 x 10(-5) and 1 x 10(-3) M alliin. All sensors were operated in the flow-through modus. A high specificity for alliin could be demonstrated for the electrode and a number of garlic samples were analyzed. Results gained with the new method showed a good correlation with those obtained with conventional HPLC-methods. In addition, onion and a variety of wild Allium species were analyzed in order to determine the amount of isoalliin or total cysteine sulfoxides present, respectively.

  19. Cysteine Modification of a Putative Pore Residue in Clc-0

    PubMed Central

    Lin, Chia-Wei; Chen, Tsung-Yu

    2000-01-01

    The ClC channel family consists of chloride channels important for various physiological functions. Two members in this family, ClC-0 and ClC-1, share ∼50–60% amino acid identity and show similar gating behaviors. Although they both contain two subunits, the number of pores present in the homodimeric channel is controversial. The double-barrel model proposed for ClC-0 was recently challenged by a one-pore model partly based on experiments with ClC-1 exploiting cysteine mutagenesis followed by modification with methanethiosulfonate (MTS) reagents. To investigate the pore stoichiometry of ClC-0 more rigorously, we applied a similar strategy of MTS modification in an inactivation-suppressed mutant (C212S) of ClC-0. Mutation of lysine 165 to cysteine (K165C) rendered the channel nonfunctional, but modification of the introduced cysteine by 2-aminoethyl MTS (MTSEA) recovered functional channels with altered properties of gating-permeation coupling. The fast gate of the MTSEA-modified K165C homodimer responded to external Cl− less effectively, so the Po-V curve was shifted to a more depolarized potential by ∼45 mV. The K165C-K165 heterodimer showed double-barrel–like channel activity after MTSEA modification, with the fast-gating behaviors mimicking a combination of those of the mutant and the wild-type pore, as expected for the two-pore model. Without MTSEA modification, the heterodimer showed only one pore, and was easier to inactivate than the two-pore channel. These results showed that K165 is important for both the fast and slow gating of ClC-0. Therefore, the effects of MTS reagents on channel gating need to be carefully considered when interpreting the apparent modification rate. PMID:11004203

  20. Lysosomal cysteine proteases: structure, function and inhibition of cathepsins.

    PubMed

    Roberts, Rebecca

    2005-12-01

    Lysosomal cysteine proteases, a subgroup of the cathepsin family, are critical for normal cellular functions such as general protein turnover, antigen processing and bone remodeling. In the past decade, the number of identified human cathepsins has more than doubled and their known role in several pathologies has expanded rapidly. Increased understanding of the structure and mechanism of this class of enzymes has brought on a new fervor in the design of small molecule inhibitors with the hope of producing specific, therapeutic drugs for diseases such as arthritis, allergy, multiple sclerosis, atherosclerosis, Alzheimer's disease and cancer.

  1. Teratogenicity of patulin and patulin adducts formed with cysteine.

    PubMed Central

    Ciegler, A; Beckwith, A C; Jackson, L K

    1976-01-01

    The mean lethal dose of patulin for the chicken embryo injected in the air cell before incubation was determined to be 68.7 mug and that for the 4-day-old embryo was 2.35 mug. Both patulin (1 to 2 mug/egg) and the reaction mixture between patulin and cysteine (15 to 150 mug of patulin equivalents) were teratogenic to the chicken embryo. At least two ninhydrin-negative and four ninhydrin-positive products were formed during the latter reaction. Our explanation of the reaction mechanism remains to be elaborated. PMID:1275488

  2. Efficient Preparation of Site-Specific Antibody-Drug Conjugates Using Cysteine Insertion.

    PubMed

    Dimasi, Nazzareno; Fleming, Ryan; Zhong, Haihong; Bezabeh, Binyam; Kinneer, Krista; Christie, Ronald J; Fazenbaker, Christine; Wu, Herren; Gao, Changshou

    2017-05-01

    Antibody-drug conjugates (ADCs) are a class of biopharmaceuticals that combine the specificity of antibodies with the high-potency of cytotoxic drugs. Engineering cysteine residues in the antibodies using mutagenesis is a common method to prepare site-specific ADCs. With this approach, solvent accessible amino acids in the antibody have been selected for substitution with cysteine for conjugating maleimide-bearing cytotoxic drugs, resulting in homogeneous and stable site-specific ADCs. Here we describe a cysteine engineering approach based on the insertion of cysteines before and after selected sites in the antibody, which can be used for site-specific preparation of ADCs. Cysteine-inserted antibodies have expression level and monomeric content similar to the native antibodies. Conjugation to a pyrrolobenzodiazepine dimer (SG3249) resulted in comparable efficiency of site-specific conjugation between cysteine-inserted and cysteine-substituted antibodies. Cysteine-inserted ADCs were shown to have biophysical properties, FcRn, and antigen binding affinity similar to the cysteine-substituted ADCs. These ADCs were comparable for serum stability to the ADCs prepared using cysteine-mutagenesis and had selective and potent cytotoxicity against human prostate cancer cells. Two of the cysteine-inserted variants abolish binding of the resulting ADCs to FcγRs in vitro, thereby potentially preventing non-target mediated uptake of the ADCs by cells of the innate immune system that express FcγRs, which may result in mitigating off-target toxicities. A selected cysteine-inserted ADC demonstrated potent dose-dependent anti-tumor activity in a xenograph tumor mouse model of human breast adenocarcinoma expressing the oncofetal antigen 5T4.

  3. A New Class of Serine and Cysteine Protease Inhibitor with Chemotherapeutic Potential

    DTIC Science & Technology

    1999-06-01

    also be used to produce a serine protease inhibitor. Similar to the cysteine inhibitors, a dipeptide side chain is attached to the ring which is...which relieves the 7 strain (Figure 3). Serine and cysteine proteases use a mechanism to cleave peptide bonds which involves addition of a catalytic...serine and cysteine proteases share a similar mechanism for hydrolyzing amide bonds , we expect that 4-heterocyclohexanones should be good inhibitors

  4. Variete Technique du Lambeau Sural dans les Brulures Profondes du Pied

    PubMed Central

    Ezzoubi, M.; Ettalbi, S.; Elmounjid, S.; Fassi, J.; Benchamckha, F.J.; Sakhi, M.; Boukind, E.

    2005-01-01

    Summary Les couvertures des pertes de substance de la jambe, du talon et du pied font souvent appel au lambeau sural, qui reste, de part ses dimensions, une indication limitée. Les Auteurs présentent, à travers deux cas cliniques, une variété technique pour la levée du lambeau sural, permettant d'obtenir des palettes cutanées de grande surface avec une bonne sécurité vasculaire. C'est un lambeau fasciocutané remontant jusqu'à un centimètre du creux poplité et incluant, lors de la levée, l'aponévrose, le nerf sural, la petite veine saphène et le nerf sural latéral. PMID:21990988

  5. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice

    PubMed Central

    Niewiadomski, Julie; Zhou, James Q.; Roman, Heather B.; Liu, Xiaojing; Hirschberger, Lawrence L.; Locasale, Jason W.; Stipanuk, Martha H.

    2016-01-01

    To gain further insights into the effect of elevated cysteine levels on energy metabolism and the possible mechanisms by which cysteine may have these effects, we conducted studies in cysteine dioxygenase (Cdo1)–null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression holds in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggested impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated cysteine levels with excess H2S production and impairments in energy metabolism suggest that H2S signaling could be involved. PMID:26995761

  6. Diurnal variation in the metabolism of S-carboxymethyl-L-cysteine in humans.

    PubMed

    Steventon, G B

    1999-09-01

    The routes of metabolism of S-carboxymethyl-L-cysteine in humans are dependent on the time of dosing. Administration of 750 mg of S-carboxymethyl-L-cysteine (Day 1) during the day at 8:00 AM followed by a 8:00 AM to 4:00 PM urine collection revealed that S-carboxymethyl-L-cysteine S-oxide was the major urinary metabolite produced. The 4:00 PM to midnight urine collection resulted in S-(carboxymethylthio)-L-cysteine being identified as the major urinary metabolite. However, the administration of 750 mg of S-carboxymethyl-L-cysteine (day 15) during the night at midnight and analysis of the midnight to 8:00 AM urine collection found that thiodiglycolic acid was the major urinary metabolite, whereas thiodiglycolic S-oxide was identified as the major urinary metabolite in the 8:00 AM to 4:00 PM urine collection. A diurnal variation in the metabolism of S-carboxymethyl-L-cysteine was seen and, in particular, the timing of S-carboxymethyl-L-cysteine administration had a profound effect on the identity of urinary S-oxide metabolites produced. After administration at 8:00 AM the urinary S-oxides produced were S-carboxymethyl-L-cysteine S-oxide and S-methyl-L-cysteine S-oxide but at midnight the major urinary S-oxide metabolite produced was thiodiglycolic acid S-oxide.

  7. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase.

    PubMed

    Elshorbagy, Amany K; Valdivia-Garcia, Maria; Mattocks, Dwight A L; Plummer, Jason D; Smith, A David; Drevon, Christian A; Refsum, Helga; Perrone, Carmen E

    2011-01-01

    Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation.

  8. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation

    PubMed Central

    Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process. PMID:26764487

  9. Cysteine supplementation reverses methionine restriction effects on rat adiposity: significance of stearoyl-coenzyme A desaturase

    PubMed Central

    Elshorbagy, Amany K.; Valdivia-Garcia, Maria; Mattocks, Dwight A. L.; Plummer, Jason D.; Smith, A. David; Drevon, Christian A.; Refsum, Helga; Perrone, Carmen E.

    2011-01-01

    Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in fatty acid and energy metabolism, but little is known about its nutritional regulation. Dietary methionine restriction in rats decreases hepatic Scd1 mRNA and protein, increases energy expenditure, and decreases fat-pad mass/body-weight% (FM/BW%). In humans, plasma concentrations of the methionine product, cysteine, are associated with obesity. To determine which consequences of methionine-restriction are mediated by decreased cysteine availability, we monitored obesity-related variables in 4 dietary groups for 12 weeks: control-fed (CF), methionine-restricted (MR), MR supplemented with 0.5% l-cysteine (MR+Cys) and CF+Cys rats. MR lowered weight gain and FM/BW% despite higher food intake/weight than CF, and lowered serum cysteine. Hepatic Scd1 expression was decreased, with decreased serum SCD1 activity indices (calculated from serum fatty acid profile), decreased serum insulin, leptin and triglycerides, and higher adiponectin. Cysteine supplementation (MR+Cys) essentially reversed all these phenotypes and raised serum cysteine but not methionine to CF levels. Adding extra cysteine to control diet (CF+Cys) increased serum taurine but did not affect serum cysteine, lipids, proteins, or total weight gain. FM/BW% and serum leptin were modestly decreased. Our results indicate that anti-obesity effects of MR are caused by low cysteine and that dietary sulfur amino acid composition contributes to SCD1 regulation. PMID:20871132

  10. Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer.

    PubMed

    Edgington-Mitchell, Laura E; Rautela, Jai; Duivenvoorden, Hendrika M; Jayatilleke, Krishnath M; van der Linden, Wouter A; Verdoes, Martijn; Bogyo, Matthew; Parker, Belinda S

    2015-09-29

    Cysteine cathepsin proteases contribute to many normal cellular functions, and their aberrant activity within various cell types can contribute to many diseases, including breast cancer. It is now well accepted that cathepsin proteases have numerous cell-specific functions within the tumor microenvironment that function to promote tumor growth and invasion, such that they may be valid targets for anti-metastatic therapeutic approaches. Using activity-based probes, we have examined the activity and expression of cysteine cathepsins in a mouse model of breast cancer metastasis to bone. In mice bearing highly metastatic tumors, we detected abundant cysteine cathepsin expression and activity in myeloid-derived suppressor cells (MDSCs). These immature immune cells have known metastasis-promoting roles, including immunosuppression and osteoclastogenesis, and we assessed the contribution of cysteine cathepsins to these functions. Blocking cysteine cathepsin activity with multiple small-molecule inhibitors resulted in enhanced differentiation of multinucleated osteoclasts. This highlights a potential role for cysteine cathepsin activity in suppressing the fusion of osteoclast precursor cells. In support of this hypothesis, we found that expression and activity of key cysteine cathepsins were downregulated during MDSC-osteoclast differentiation. Another cysteine protease, legumain, also inhibits osteoclastogenesis, in part through modulation of cathepsin L activity. Together, these data suggest that cysteine protease inhibition is associated with enhanced osteoclastogenesis, a process that has been implicated in bone metastasis.

  11. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    PubMed

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  12. Chicken scFvs with an Artificial Cysteine for Site-Directed Conjugation.

    PubMed

    Yoon, Aerin; Shin, Jung Won; Kim, Soohyun; Kim, Hyori; Chung, Junho

    2016-01-01

    For the site-directed conjugation of chemicals and radioisotopes to the chicken-derived single-chain variable fragment (scFv), we investigated amino acid residues replaceable with cysteine. By replacing each amino acid of the 157 chicken variable region framework residues (FR, 82 residues on VH and 75 on VL) with cysteine, 157 artificial cysteine mutants were generated and characterized. At least 27 residues on VL and 37 on VH could be replaced with cysteine while retaining the binding activity of the original scFv. We prepared three VL (L5, L6 and L7) and two VH (H13 and H16) mutants as scFv-Ckappa fusion proteins and showed that PEG-conjugation to the sulfhydryl group of the artificial cysteine was achievable in all five mutants. Because the charge around the cysteine residue affects the in vivo stability of thiol-maleimide conjugation, we prepared 16 charge-variant artificial cysteine mutants by replacing the flanking residues of H13 with charged amino acids and determined that the binding activity was not affected in any of the mutants except one. We prepared four charge-variant H13 artificial cysteine mutants (RCK, DCE, ECD and ECE) as scFv-Ckappa fusion proteins and confirmed that the reactivity of the sulfhydryl group on cysteine is active and their binding activity is retained after the conjugation process.

  13. Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat.

    PubMed Central

    Stipanuk, M H; Beck, P W

    1982-01-01

    The contribution of cystathionine gamma-lyase, cystathionine beta-synthase and cysteine aminotransferase coupled to 3-mercaptopyruvate sulphurtransferase to cysteine desulphhydration in rat liver and kidney was assessed with four different assay systems. Cystathionine gamma-lyase and cystathionine beta-synthase were active when homogenates were incubated with 280 mM-L-cysteine and 3 mM-pyridoxal 5'-phosphate at pH 7.8. Cysteine aminotransferase in combination with 3-mercaptopyruvate sulphurtransferase catalysed essentially all of the H2S production from cysteine at pH 9.7 with 160 mM-L-cysteine, 2 mM-pyridoxal 5'-phosphate, 3 mM-2-oxoglutarate and 3 mM-dithiothreitol. At more-physiological concentrations of cysteine (2 mM) cystathionine gamma-lyase and cystathionine beta-synthase both appeared to be active in cysteine desulphhydration, whereas the aminotransferase pathway did not. The effect of inhibition of cystathionine gamma-lyase by a suicide inactivator, propargylglycine, in the intact rat was also investigated; there was no significant effect of propargylglycine administration on the urinary excretion of total 35S, 35SO4(2-) or [35S]taurine formed from labelled dietary cysteine. PMID:7150244

  14. Influence of cysteine doping on photoluminescence intensity from semiconducting single-walled carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Kurnosov, N. V.; Leontiev, V. S.; Linnik, A. S.; Karachevtsev, V. A.

    2015-03-01

    Photoluminescence (PL) from semiconducting single-walled carbon nanotubes can be applied for detection of cysteine. It is shown that cysteine doping (from 10-8 to 10-3 M) into aqueous suspension of nanotubes with adsorbed DNA leads to increase of PL intensity. The PL intensity was enhanced by 27% at 10-3 M cysteine concentration in suspension. Most likely, the PL intensity increases due to the passivation of p-defects on the nanotube by the cysteine containing reactive thiol group. The effect of doping with other amino acids without this group (methionine, serine, aspartic acid, lysine, proline) on the PL intensity is essentially weaker.

  15. Direct evidence that two cysteines in the dopamine transporter form a disulfide bond.

    PubMed

    Chen, Rong; Wei, Hua; Hill, Erik R; Chen, Lucy; Jiang, Liying; Han, Dawn D; Gu, Howard H

    2007-04-01

    We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.

  16. Electrochemical cysteine determination in serum samples by Hg thin film sensor.

    PubMed

    Sezginturk, Mustafa Kemal; Dinckaya, Erhan

    2011-01-01

    Cysteine is a nonessential aminoacid, meaning that cysteine can be made in the human body. It is one of the few amino acids that contain sulfur. This allows cysteine to bond in a special way and maintain the structures of proteins in the body. Cysteine strengthens the protective lining of the stomach and intestines, which may help prevent damage caused by aspirin and similar drugs. In addition, cysteine may play an important role in the communication between immune system cells. In this study, glassy carbon electrodes modified with mercury (Hg) were used as working electrode. Mercury thin film on glassy carbon electrode was deposited by holding the electrode potential at -0.7 V; the measurement period for the coating process was 2 minutes. pH and temperature effects on the electrode response were carried out by working at different pHs and temperatures. The calibration graph for cysteine was drawn in the range of 5-120 μM cysteine. Repeatability and interferences studies were investigated. GSH had an interference effect of about 13% of cysteine response. Finally, the sensor was applied to real samples for cysteine determination and the method was validated by Ellman's reagent.

  17. Effect of L-cysteine on the oxidation of cyclohexane catalyzed by manganeseporphyrin.

    PubMed

    Zhou, Wei-You; Tian, Peng; Chen, Yong; He, Ming-Yang; Chen, Qun; Chen, Zai Xin

    2015-06-01

    Effect of L-cysteine as the cocatalyst on the oxidation of cyclohexane by tert-butylhydroperoxide (TBHP) catalyzed by manganese tetraphenylporphyrin (MnTPP) has been investigated. The results showed that L-cysteine could moderately improve the catalytic activity of MnTPP and significantly increase the selectivity of cyclohexanol. Different from imidazole and pyridine, the L-cysteine may perform dual roles in the catalytic oxidation of cyclohexane. Besides as the axial ligand for MnTPP, the L-cysteine could also react with cyclohexyl peroxide formed as the intermediate to produce alcohol as the main product.

  18. Rick and Becky DuFour

    ERIC Educational Resources Information Center

    McLester, Susan

    2012-01-01

    In 1969, a concern with the deep inequity of students' experiences and opportunities in traditional school systems first drove social studies teacher Rick DuFour to begin advocating for the kind of reforms that would jell into his transformative model, Professional Learning Communities (PLC) at Work, some 16 years later. The core belief of the PLC…

  19. Sign Communication in Cri du Chat Syndrome

    ERIC Educational Resources Information Center

    Erlenkamp, Sonja; Kristoffersen, Kristian Emil

    2010-01-01

    This paper presents findings from a study on the use of sign supported Norwegian (SSN) in two individuals with Cri du chat syndrome (CCS). The study gives a first account of some selected aspects of production and intelligibility of SSN in CCS. Possible deviance in manual parameters, in particular inter- and/or intra-subject variation in the use…

  20. Prejudice: From Allport to DuBois.

    ERIC Educational Resources Information Center

    Gaines, Stanley O., Jr.; Reed, Edward S.

    1995-01-01

    Examines the differences between Gordon Allport's and W. E. B. DuBois's theories on the origins of prejudice and the impact of discrimination on the personality and social development of blacks. The article argues that prejudice is a historically developed process, not a universal feature of human psychology. Implications for U.S. race relations…

  1. The Du Pont OZ Creative Thinking Network.

    ERIC Educational Resources Information Center

    Tanner, David

    1994-01-01

    The OZ Creative Thinking Network is a volunteer group of Du Pont Corporation employees devoted to educating themselves and others concerning creativity and innovation. This network, organized in 1986, has a current membership of over 600 employees and has produced a book that couples essays expressing concepts in creativity and innovation with…

  2. Prejudice: From Allport to DuBois.

    ERIC Educational Resources Information Center

    Gaines, Stanley O., Jr.; Reed, Edward S.

    1995-01-01

    Examines the differences between Gordon Allport's and W. E. B. DuBois's theories on the origins of prejudice and the impact of discrimination on the personality and social development of blacks. The article argues that prejudice is a historically developed process, not a universal feature of human psychology. Implications for U.S. race relations…

  3. Rick and Becky DuFour

    ERIC Educational Resources Information Center

    McLester, Susan

    2012-01-01

    In 1969, a concern with the deep inequity of students' experiences and opportunities in traditional school systems first drove social studies teacher Rick DuFour to begin advocating for the kind of reforms that would jell into his transformative model, Professional Learning Communities (PLC) at Work, some 16 years later. The core belief of the PLC…

  4. Growth study of cri du chat syndrome.

    PubMed

    Collins, M S; Eaton-Evans, J

    2001-10-01

    We compared the growth of children with cri du chat (5p-) syndrome with the 1990 UK growth curves. Most subjects had impaired growth, particularly of head circumference. The more emaciated the child the more pronounced the microcephaly, showing the need for growth and nutrition monitoring.

  5. DuSable High School Program Flourishes.

    ERIC Educational Resources Information Center

    Graff, Pat

    1995-01-01

    Describes the fall and rise of the Panther Press, the scholastic newspaper of the DuSable High School in Chicago. States that despite being located in the midst of public housing projects, the school's newspaper is thriving where others in similar circumstances have failed. Describes how the school's principal and an advisor revitalized and…

  6. Sign Communication in Cri du Chat Syndrome

    ERIC Educational Resources Information Center

    Erlenkamp, Sonja; Kristoffersen, Kristian Emil

    2010-01-01

    This paper presents findings from a study on the use of sign supported Norwegian (SSN) in two individuals with Cri du chat syndrome (CCS). The study gives a first account of some selected aspects of production and intelligibility of SSN in CCS. Possible deviance in manual parameters, in particular inter- and/or intra-subject variation in the use…

  7. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster.

    PubMed

    Menger, Katja E; James, Andrew M; Cochemé, Helena M; Harbour, Michael E; Chouchani, Edward T; Ding, Shujing; Fearnley, Ian M; Partridge, Linda; Murphy, Michael P

    2015-06-30

    Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster.

  8. Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons.

    PubMed

    Himi, T; Ikeda, M; Yasuhara, T; Nishida, M; Morita, I

    2003-12-01

    Cysteine uptake is the rate-limiting process in glutathione synthesis. Previously we have shown that the inhibitors of excitatory amino acid transporters (EAATs) significantly enhance glutamate toxicity via depletion of intracellular glutathione. In this study we show evidence that the neuronal glutamate transporter EAAT3 is directly enrolled in cysteine uptake in cultured neurons. Neuronal cysteine uptake was dependent on the extracellular sodium, and was suppressed by EAAT inhibitors. Cysteine uptake was suppressed by extracellular glutamate and aspartate, substrates of EAATs, and not by substrates of cysteine transporters. Intracellular glutathione levels were reduced by EAAT inhibitors, and not by inhibitors of cysteine transporters. Knock down of EAAT3 expression using antisense oligonucleotide significantly reduced cysteine uptake, intracellular glutathione level, and neuronal viability against oxidative stress. These facts indicate that EAAT3 functions as a cysteine transporter, and this function seems to be unique and distinct from cysteine transporters that have been reported.

  9. S-sulfhydration: a cysteine posttranslational modification in plant systems.

    PubMed

    Aroca, Ángeles; Serna, Antonio; Gotor, Cecilia; Romero, Luis C

    2015-05-01

    Hydrogen sulfide is a highly reactive molecule that is currently accepted as a signaling compound. This molecule is as important as carbon monoxide in mammals and hydrogen peroxide in plants, as well as nitric oxide in both eukaryotic systems. Although many studies have been conducted on the physiological effects of hydrogen sulfide, the underlying mechanisms are poorly understood. One of the proposed mechanisms involves the posttranslational modification of protein cysteine residues, a process called S-sulfhydration. In this work, a modified biotin switch method was used for the detection of Arabidopsis (Arabidopsis thaliana) proteins modified by S-sulfhydration under physiological conditions. The presence of an S-sulfhydration-modified cysteine residue on cytosolic ascorbate peroxidase was demonstrated using liquid chromatography-tandem mass spectrometry analysis, and a total of 106 S-sulfhydrated proteins were identified. Immunoblot and enzyme activity analyses of some of these proteins showed that the sulfide added through S-sulfhydration reversibly regulates the functions of plant proteins in a manner similar to that described in mammalian systems.

  10. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    NASA Astrophysics Data System (ADS)

    Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

    2014-09-01

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10-4 cm s-1. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

  11. Thiazolidine derivatives from fluorescent dithienyl-BODIPY-carboxaldehydes and cysteine.

    PubMed

    Poirel, Arnaud; De Nicola, Antoinette; Ziessel, Raymond

    2014-12-05

    Fluorescent dithienyl-borondipyrromethene (BODIPY) dyes formylated in the β'-position (2b, 2c) have been treated with L-cysteine to provide thiazolidine derivatives. N-Protection of the thiazolidine unit by ethoxycarbonylation facilitated isolation of the two major diasteroisomers 6 and 7. These stereoisomers have been fully characterized by (1)H NMR spectroscopy, allowing assignment of their stereochemistry as 2R,4R,aS and 2S,4R,aR, respectively. The optical properties of the thiazolidine dyes differ markedly in both absorption (λ(abs) = 612 nm for 6 and 615 nm for 7) and emission (λ(em) = 669 nm, Φ(F) = 62% for 6 and λ(em) = 672 nm, Φ(F) = 19% for 7) from those of the BODIPY-carboxaldehydes 2b (λ(abs) = 643 nm and λ(em) = 719 nm, Φ(F) = 26%) and 2c (λ(abs) = 636 nm and λ(em) = 710 nm, Φ(F) = 36%). In a mixed solvent [phosphate buffer saline (PBS), pH = 7.4/ethanol 1:9], the fluorescence response of the dyes in the presence of L-cysteine is slow, but a ratiometric detection process in the therapeutic window (650 to 800 nm) is evident.

  12. Enzyme structure captures four cysteines aligned for disulfide relay

    PubMed Central

    Gat, Yair; Vardi-Kilshtain, Alexandra; Grossman, Iris; Major, Dan Thomas; Fass, Deborah

    2014-01-01

    Thioredoxin superfamily proteins introduce disulfide bonds into substrates, catalyze the removal of disulfides, and operate in electron relays. These functions rely on one or more dithiol/disulfide exchange reactions. The flavoenzyme quiescin sulfhydryl oxidase (QSOX), a catalyst of disulfide bond formation with an interdomain electron transfer step in its catalytic cycle, provides a unique opportunity for exploring the structural environment of enzymatic dithiol/disulfide exchange. Wild-type Rattus norvegicus QSOX1 (RnQSOX1) was crystallized in a conformation that juxtaposes the two redox-active di-cysteine motifs in the enzyme, presenting the entire electron-transfer pathway and proton-transfer participants in their native configurations. As such a state cannot generally be enriched and stabilized for analysis, RnQSOX1 gives unprecedented insight into the functional group environments of the four cysteines involved in dithiol/disulfide exchange and provides the framework for analysis of the energetics of electron transfer in the presence of the bound flavin adenine dinucleotide cofactor. Hybrid quantum mechanics/molecular mechanics (QM/MM) free energy simulations based on the X-ray crystal structure suggest that formation of the interdomain disulfide intermediate is highly favorable and secures the flexible enzyme in a state from which further electron transfer via the flavin can occur. PMID:24888638

  13. Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates

    SciTech Connect

    Nagelkerke, J.F.; Boogaard, P.J. )

    1991-01-01

    In 1916 a relationship was postulated between the occurrence of aplastic anemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-D-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloakenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity is outlined.

  14. Harnessing redox cross-reactivity to profile distinct cysteine modifications

    PubMed Central

    Majmudar, Jaimeen D.; Konopko, Aaron M.; Labby, Kristin J.; Tom, Christopher T.M.B.; Crellin, John E.; Prakash, Ashesh; Martin, Brent R.

    2016-01-01

    Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids. PMID:26780921

  15. Nephrotoxicity of halogenated alkenyl cysteine-S-conjugates.

    PubMed

    Nagelkerke, J F; Boogaard, P J

    1991-01-01

    In 1916 a relationship was postulated between the occurrence of aplastic anaemia in cattle and the soy bean meal that they had been fed, which had been extracted with trichloroethylene. The toxic compound was later identified as S-(1,2-dichlorovinyl)-L-cysteine (DCV-Cys). In addition to effects on the hemopoietic system it also produced nephrotoxicity in calves. In rats only renal tubular necrosis was found. Further research demonstrated that other halogenated hydrocarbons produced similar nephrotoxicity. The haloalkenyl cysteine-S-conjugates (Cys-S-conjugates) have extensively been studied; this has provided new insight into the biochemical processes that lead to nephrotoxicity. It has been shown that a combination of transport processes and specific metabolic pathways, resulting in reactive intermediates that bind to cellular macromolecules, makes the kidney vulnerable to the noxious effects of the haloalkenyl Cys-S-conjugates. The first part of this review gives a brief overview of the bioactivation of the haloalkenes; in the second part the present knowledge of the underlying mechanisms of cytotoxicity will be outlined.

  16. [Isolation and properties of cysteine protease from Serratia proteamaculans 94].

    PubMed

    Mozhina, N V; Burmistrova, O A; Pupov, D V; Rudenskaia, G N; Dunaevskiĭ, Ia E; Demidiuk, I V; Kostrov, S V

    2008-01-01

    A new cysteine protease (SpCP) with a molecular mass of about 50 kDa and optimal functioning at pH 8.0 was isolated from the culture medium of a Serratia proteamaculans 94 psychrotolerant strain using affinity and gel permeation chromatography. The enzyme N terminal amino acid sequence (SPVEEAEGDGIVLDV-) exhibits a reliable similarity to N terminal sequences of gingipains R, cysteine proteases from Polphyromonas gingivalis. Unlike gingipains R, SpCP displays a double substrate specificity and cleaves bonds formed by carboxylic groups of Arg, hydrophobic amino acid residues (Val, Leu, Ala, Tyr, and Phe), Pro, and Gly. SpCP can also hydrolyze native collagen. The enzyme catalysis is effective in a wide range of temperatures. Kinetic studies of Z-Ala-Phe-Arg-pNA hydrolysis catalyzed by the protease at 4 and 37 degrees C showed that a decrease in temperature by more than 30 degrees C causes a 1.3-fold increase in the kcat/Km ratio. Thus, SpCP is an enzyme adapted to low positive temperatures. A protease displaying such properties was found in microorganisms of the Serratia genus for the first time and may serve as a virulent factor for these bacteria.

  17. Salt Effect Accelerates Site-Selective Cysteine Bioconjugation

    PubMed Central

    2016-01-01

    Highly efficient and selective chemical reactions are desired. For small molecule chemistry, the reaction rate can be varied by changing the concentration, temperature, and solvent used. In contrast for large biomolecules, the reaction rate is difficult to modify by adjusting these variables because stringent biocompatible reaction conditions are required. Here we show that adding salts can change the rate constant over 4 orders of magnitude for an arylation bioconjugation reaction between a cysteine residue within a four-residue sequence (π-clamp) and a perfluoroaryl electrophile. Biocompatible ammonium sulfate significantly enhances the reaction rate without influencing the site-specificity of π-clamp mediated arylation, enabling the fast synthesis of two site-specific antibody–drug conjugates that selectively kill HER2-positive breast cancer cells. Computational and structure–reactivity studies indicate that salts may tune the reaction rate through modulating the interactions between the π-clamp hydrophobic side chains and the electrophile. On the basis of this understanding, the salt effect is extended to other bioconjugation chemistry, and a new regioselective alkylation reaction at π-clamp cysteine is developed. PMID:27725962

  18. Gold nanoparticles surface modification using BSA and cysteine

    NASA Astrophysics Data System (ADS)

    Cardoso-Avila, P. E.; Pichardo-Molina, J. L.; Upendra Kumar, K.; Barbosa-Sabanero, G.; Barbosa-Garcia, O.

    2011-08-01

    Metal nanometer-size particles show intriguing optical properties which depend on their shape, size and local environment. For these reasons, these materials have received a lot of attention in different scientific areas, and several applications can be found, for example: fabrication of bio-sensor, electronic devices, catalysis and new drugs. However, in the case of biomedical applications, metallic nanoparticles need to satisfy several requirements: bio-compatibility, stability and functionality. To satisfy these requirements, metallic nanoparticles need to be modified in their surfaces. In this work we report the synthesis and the modification of gold nanoparticles (GNPs) surface. GNPs were fabricated following the Turkevich's method, and the bio-conjugation (surface modification) was done using cysteine and bovine serum albumin (BSA). Our results of Uv-vis spectroscopy show that BSA and cysteine permit to increase the stability of GNPs in presence of NaCl, the stability is function of BSA concentration. Also to verify the bio-conjugation we used Raman spectroscopy and gel electrophoresis.

  19. Mechanical Strength of 17 134 Model Proteins and Cysteine Slipknots

    PubMed Central

    2009-01-01

    A new theoretical survey of proteins' resistance to constant speed stretching is performed for a set of 17 134 proteins as described by a structure-based model. The proteins selected have no gaps in their structure determination and consist of no more than 250 amino acids. Our previous studies have dealt with 7510 proteins of no more than 150 amino acids. The proteins are ranked according to the strength of the resistance. Most of the predicted top-strength proteins have not yet been studied experimentally. Architectures and folds which are likely to yield large forces are identified. New types of potent force clamps are discovered. They involve disulphide bridges and, in particular, cysteine slipknots. An effective energy parameter of the model is estimated by comparing the theoretical data on characteristic forces to the corresponding experimental values combined with an extrapolation of the theoretical data to the experimental pulling speeds. These studies provide guidance for future experiments on single molecule manipulation and should lead to selection of proteins for applications. A new class of proteins, involving cystein slipknots, is identified as one that is expected to lead to the strongest force clamps known. This class is characterized through molecular dynamics simulations. PMID:19876372

  20. Cysteine rich secretory proteins in reproduction and venom.

    PubMed

    Gibbs, Gerard M; O'Bryan, Moira K

    2007-01-01

    The cysteine rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract and in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionarily diverse N-terminal domain and a characteristic cysteine rich C-terminal domain which we refer to as the Crisp domain. Since their identification 30 years ago Crisp research in mammals has focused on the characterisation of their expression localization to infer function. While no doubt important observations, these have not substantially led to an understanding of the biochemical activity of the Crisps and their role in sperm function or fertilisation. Recently, we demonstrated that the Crisp-2 Crisp domain has a structure similar to ion channel toxins ShK and BgK and was itself able to regulate Ca2+ flux through ryanodine receptors. These data build upon the previous characterizations of reptile venom Crisps as regulators of several types of ion channels and permits for the first time a dissection of the biochemical activity of mammalian Crisps.

  1. Methylation of cysteine in hemoglobin following exposure to methylating agents

    SciTech Connect

    Bailey, E.; Connors, T.A.; Farmer, P.B.; Gorf, S.M.; Rickard, J.

    1981-06-01

    In addition to reacting with biologically important nucleophilic sites in DNA, alkylating agents also interact with amino acids in proteins. Measurements of the extent of formation of these alkyl amino acids may be used as a means of determining exposure to these compounds. The degree of S-methylation of cysteine in hemoglobin was studied following in vivo exposure of rats to methyl methanesulfonate, dimethylnitrosamine, and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide. A linear dose-response curve was observed for methyl methanesulfonate over a 100-fold dose range. For dimethylnitrosamine, there was a threshold of doses where no methylation could be detected, and a curved dose-response curve was obtained. At high doses, the degree of methylation of hemoglobin cysteine was 7-fold lower than that with methyl methanesulfonate. In vivo, no alkylation could be observed with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide; however, the existence of naturally occurring S-methylcysteine in the rat hemoglobin may have overshadowed small increases in alkylation arising from exposure to this compound. The natural occurrence of S-methylcysteine was studied in 13 species, and amounts ranging from 5.6 nmol/g globin (hamster) to 481 nmol/g globin (partridge) were observed. The reason for its occurrence is unknown but is under investigation.

  2. Effect of supplementation with a cysteine donor on muscular performance.

    PubMed

    Lands, L C; Grey, V L; Smountas, A A

    1999-10-01

    Oxidative stress contributes to muscular fatigue. GSH is the major intracellular antioxidant, the biosynthesis of which is dependent on cysteine availability. We hypothesized that supplementation with a whey-based cysteine donor [Immunocal (HMS90)] designed to augment intracellular GSH would enhance performance. Twenty healthy young adults (10 men, 10 women) were studied presupplementation and 3 mo postsupplementation with either Immunocal (20 g/day) or casein placebo. Muscular performance was assessed by whole leg isokinetic cycle testing, measuring peak power and 30-s work capacity. Lymphocyte GSH was used as a marker of tissue GSH. There were no baseline differences (age, ht, wt, %ideal wt, peak power, 30-s work capacity). Follow-up data on 18 subjects (9 Immunocal, 9 placebo) were analyzed. Both peak power [13 +/- 3.5 (SE) %, P < 0.02] and 30-s work capacity (13 +/- 3.7%, P < 0.03) increased significantly in the Immunocal group, with no change (2 +/- 9.0 and 1 +/- 9.3%) in the placebo group. Lymphocyte GSH also increased significantly in the Immunocal group (35.5 +/- 11.04%, P < 0.02), with no change in the placebo group (-0.9 +/- 9.6%). This is the first study to demonstrate that prolonged supplementation with a product designed to augment antioxidant defenses resulted in improved volitional performance.

  3. Photochemical and Nonphotochemical Transformations of Cysteine with Dissolved Organic Matter.

    PubMed

    Chu, Chiheng; Erickson, Paul R; Lundeen, Rachel A; Stamatelatos, Dimitrios; Alaimo, Peter J; Latch, Douglas E; McNeill, Kristopher

    2016-06-21

    Cysteine (Cys) plays numerous key roles in the biogeochemistry of natural waters. Despite its importance, a full assessment of Cys abiotic transformation kinetics, products and pathways under environmental conditions has not been conducted. This study is a mechanistic evaluation of the photochemical and nonphotochemical (dark) transformations of Cys in solutions containing chromophoric dissolved organic matter (CDOM). The results show that Cys underwent abiotic transformations under both dark and irradiated conditions. Under dark conditions, the transformation rates of Cys were moderate and were highly pH- and temperature-dependent. Under UVA or natural sunlight irradiations, Cys transformation rates were enhanced by up to two orders of magnitude compared to rates under dark conditions. Product analysis indicated cystine and cysteine sulfinic acid were the major photooxidation products. In addition, this study provides an assessment of the contributions of singlet oxygen, hydroxyl radical, hydrogen peroxide, and triplet dissolved organic matter to the CDOM-sensitized photochemical oxidation of Cys. The results suggest that another unknown pathway was dominant in the CDOM-sensitized photodegradation of Cys, which will require further study to identify.

  4. Metabolic Fate of Cysteine and Methionine in Rumen Digesta

    PubMed Central

    Nader, C. J.; Walker, D. J.

    1970-01-01

    Estimates were obtained of the extent to which cysteine and methionine were incorporated into the protein of the microbes of rumen digesta without prior degradation and resynthesis. By using the amino acids labeled with both 35S and 14C, it was observed that a large proportion of the 35S appeared in the sulfide pool and of the 14C appeared in volatile fatty acids. By isolating the appropriate amino acid, obtaining the 14C to 35S ratio, and comparing this with the ratio in the added amino acid, the degree of direct incorporation was calculated. For cysteine it was estimated that at most 1% and for methionine, at most 11% of the amino acid in the free pool was incorporated unchanged into microbial protein. As a consequence of these findings, it is considered that the method for measuring microbial protein synthesis in rumen digesta based upon incorporation of 35S from the free sulfide pool is not seriously affected by direct utilization of sulfur amino acids arising from dietary sources. PMID:5485079

  5. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    SciTech Connect

    Pandey, Chandra Mouli; Sumana, Gajjala; Tiwari, Ida

    2014-09-08

    The present work describes the promising electrochemical immunosensing strategy based on copper (II) assisted hierarchical cysteine structures (CuCys) varying from star to flower like morphology. The CuCys having average size of 10 μm have been synthesised using L-Cysteine as initial precursor in presence of copper oxide under environmentally friendly conditions in aqueous medium. To delineate the synthesis mechanism, detailed structural investigations have been carried out using characterization techniques such as X-ray diffraction, transmission electron microscopy, and Fourier transform infrared spectroscopy. The electrochemical behaviour of self-assembled CuCys on gold electrode shows surface controlled electrode reaction with an apparent electron transfer rate constant of 3.38 × 10{sup −4 }cm s{sup −1}. This innovative platform has been utilized to fabricate an immunosensor by covalently immobilizing monoclonal antibodies specific for Escherichia coli O157:H7 (E. coli). Under the optimal conditions, the fabricated immunosensor is found to be sensitive and specific for the detection of E. coli with a detection limit of 10 cfu/ml.

  6. Redox interactions between Fe and cysteine: Spectroscopic studies and multiplet calculations

    NASA Astrophysics Data System (ADS)

    Bhattacharyya, Amrita; Stavitski, Eli; Dvorak, Joseph; Martínez, Carmen Enid

    2013-12-01

    The biogeochemical cycle of Fe is intricately linked with that of organic matter. Cysteine represents an organic molecule with functionalities (O, S, N functional groups) and a C backbone that may mimic the functional groups present in organic matter from terrestrial and aquatic environments. In the present study we explore the redox speciation and coordination environment of Fe and the roles of the various ligand atoms of cysteine (C, N, S) in iron-organic redox coupling and transformations. The changes in oxidation state of Fe, C, N, and S in laboratory-synthesized Fe(II)-cysteine (synthesized from ferrous sulfate) and Fe(III)-cysteine (synthesized from ferric nitrate) complexes are monitored as a function of time using synchrotron X-ray absorption spectroscopy (Fe L2,3-edge XANES; C, N and S K-edge XANES; Fe K-edge EXAFS) and theoretical multiplet calculations using the program CTM4XAS (Charge Transfer Multiplet for X-ray Absorption Spectroscopy). CTM4XAS calculations show that 80% of the total Fe in both the Fe(II)-cysteine and the Fe(III)-cysteine complexes is present as Fe2+ initially (t = 0), thus indicating preservation of Fe(II) in Fe(II)-cysteine and reduction of Fe(III) in Fe(III)-cysteine at initial conditions, the latter caused by an internal electron transfer reaction from S of -SH on the cysteine molecule. After 12 months, however, ∼60% of the total Fe is present as Fe3+ in the Fe(II)-cysteine complex whereas ∼67% of the total Fe is present as Fe2+ in the Fe(III)-cysteine complex. The fact that a larger proportion of the Fe in the Fe(III)-cysteine complex remained reduced after 12 months than that in the Fe(II)-cysteine complex suggests that the reduced Fe in Fe(III)-cysteine after 12 months is further stabilized via preferential binding with the donor atoms of cysteine. Stabilization via preferential binding is supported by a coordination environment that changed from tetrahedral Fe2+ binding to S at a distance of 2.3 Å at t = 0 for both Fe(II,III)-cysteine

  7. Gluten gel and film properties in the presence of cysteine and sodium alginate.

    PubMed

    Yuno-Ohta, Naoko; Yamada, Mariko; Inomata, Masako; Konagai, Hiromi; Kataoka, Tomomi

    2009-08-01

    Wheat flour has an ability of forming dough by mixing with water, which exhibits a rheological property required for making bread. The major protein is gluten, which is a valuable protein material for food industry. In this study, gluten protein gels and films were formed with cysteine and sodium alginate. Adding cysteine improved gel and film properties (stress relaxation behavior, bending strength). The gel containing 0.01 M cysteine had a longer relaxation time and was more rigid than the gel without cysteine. Although adding sodium alginate to the gluten suspension containing cysteine improved the water-holding ability and homogeneity of the gel network, the film from this gel was more brittle than the gluten film with cysteine alone. Microstructural observations of the gels and films with scanning electron microscopy suggested that water evaporation was more heterogeneous from the gel containing sodium alginate than from the gel with cysteine alone. Fourier transform-infrared (FT-IR) analysis during film formation suggested that the presence of cysteine encourages interaction between gluten molecules and results in intermolecular beta-sheet formation in earlier stages than in the no additive condition. FT-IR results also suggested that the combined effect of sodium alginate and cysteine on the protein secondary structure was remarkably different from that of cysteine alone. Our results suggest that addition of a suitable amount of cysteine (0.01 M) and heat treatment to 80 degrees C during gluten gel and film formation induces a homogenous network in the gel and film by regulating disulfide-sulfide interactions.

  8. Emission of hydrogen sulfide by leaf tissue in response to L-cysteine

    SciTech Connect

    Sekiya, J.; Schmidt, A.; Wilson, L.G.; Filner, P.

    1982-08-01

    Leaf discs and detached leaves exposed to L-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H/sub 2/S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to L-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H/sub 2/S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar L-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar L-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar L-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H/sub 2/S emission was a specific consequence of exposure to L-cysteine; neither D-cysteine nor L-cysteine elicited H/sub 2/S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H/sub 2/S formation and its release occurred in response to L-cysteine. Feeding experiments with (/sup 35/S)t-cysteine showed that most of the sulfur in H/sub 2/S was derived from sulfur in the L-cysteine supplied.

  9. Functional characterization of enzymes involved in cysteine biosynthesis and H(2)S production in Trypanosoma cruzi.

    PubMed

    Marciano, Daniela; Santana, Marianela; Nowicki, Cristina

    2012-10-01

    Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the two-step pathway involving serine acetyltransferase and cysteine synthase (CS) is comprised. Also, cystathionine β synthase (CBS) might contribute to the de novo generation of cysteine in addition to catalyze the first step of the reverse transsulfuration route producing cystathionine. However, neither the functionality of CS nor that of cystathionine γ lyase (CGL) has been assessed. Our results show that T. cruzi CS could participate notably more actively than CBS in the de novo synthesis of cysteine. Interestingly, at the protein level T. cruzi CS is more abundant in amastigotes than in epimastigotes. Unlike the mammalian homologues, T. cruzi CGL specifically cleaves cystathionine into cysteine and is unable to produce H(2)S. The expression pattern of T. cruzi CGL parallels that of CBS, which unexpectedly suggests that in addition to the de novo synthesis of cysteine, the reverse transsulfuration pathway could be operative in the mammalian and insect stages. Besides, T. cruzi CBS produces H(2)S by decomposing cysteine or via condensation of cysteine with homocysteine. The latter reaction leads to cystathionine production, and is catalyzed remarkably more efficiently than the breakdown of cysteine. In T. cruzi like in other organisms, H(2)S could exert regulatory effects on varied metabolic processes. Notably, T. cruzi seems to count on stage-specific routes involved in cysteine production, the multiple cysteine-processing alternatives could presumably reflect this parasite's high needs of reducing power for detoxification of reactive oxygen species.

  10. Electrostatics of cysteine residues in proteins: parameterization and validation of a simple model.

    PubMed

    Salsbury, Freddie R; Poole, Leslie B; Fetrow, Jacquelyn S

    2012-11-01

    One of the most popular and simple models for the calculation of pK(a) s from a protein structure is the semi-macroscopic electrostatic model MEAD. This model requires empirical parameters for each residue to calculate pK(a) s. Analysis of current, widely used empirical parameters for cysteine residues showed that they did not reproduce expected cysteine pK(a) s; thus, we set out to identify parameters consistent with the CHARMM27 force field that capture both the behavior of typical cysteines in proteins and the behavior of cysteines which have perturbed pK(a) s. The new parameters were validated in three ways: (1) calculation across a large set of typical cysteines in proteins (where the calculations are expected to reproduce expected ensemble behavior); (2) calculation across a set of perturbed cysteines in proteins (where the calculations are expected to reproduce the shifted ensemble behavior); and (3) comparison to experimentally determined pK(a) values (where the calculation should reproduce the pK(a) within experimental error). Both the general behavior of cysteines in proteins and the perturbed pK(a) in some proteins can be predicted reasonably well using the newly determined empirical parameters within the MEAD model for protein electrostatics. This study provides the first general analysis of the electrostatics of cysteines in proteins, with specific attention paid to capturing both the behavior of typical cysteines in a protein and the behavior of cysteines whose pK(a) should be shifted, and validation of force field parameters for cysteine residues. Copyright © 2012 Wiley Periodicals, Inc.

  11. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    USDA-ARS?s Scientific Manuscript database

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  12. Enhanced incorporation yield of cysteine for glutathione overproduction by fed-batch fermentation of Saccharomyces cerevisiae.

    PubMed

    Lorenz, Eric; Schmacht, Maximilian; Stahl, Ulf; Senz, Martin

    2015-12-20

    In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).

  13. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    USDA-ARS?s Scientific Manuscript database

    Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-Y, wh...

  14. Effects of a block in cysteine catabolism on energy balance and fat metabolism in mice.

    PubMed

    Niewiadomski, Julie; Zhou, James Q; Roman, Heather B; Liu, Xiaojing; Hirschberger, Lawrence L; Locasale, Jason W; Stipanuk, Martha H

    2016-01-01

    To gain further insights into the effects of elevated cysteine levels on energy metabolism and the possible mechanisms underlying these effects, we conducted studies in cysteine dioxygenase (Cdo1)-null mice. Cysteine dioxygenase (CDO) catalyzes the first step of the major pathway for cysteine catabolism. When CDO is absent, tissue and plasma cysteine levels are elevated, resulting in enhanced flux of cysteine through desulfhydration reactions. When Cdo1-null mice were fed a high-fat diet, they gained more weight than their wild-type controls, regardless of whether the diet was supplemented with taurine. Cdo1-null mice had markedly lower leptin levels, higher feed intakes, and markedly higher abundance of hepatic stearoyl-CoA desaturase 1 (SCD1) compared to wild-type control mice, and these differences were not affected by the fat or taurine content of the diet. Thus, reported associations of elevated cysteine levels with greater weight gain and with elevated hepatic Scd1 expression are also seen in the Cdo1-null mouse model. Hepatic accumulation of acylcarnitines suggests impaired mitochondrial β-oxidation of fatty acids in Cdo1-null mice. The strong associations of elevated cysteine levels with excess H2 S production and impairments in energy metabolism suggest that H2 S signaling could be involved.

  15. A label-free fluorescence turn-on sensor for rapid detection of cysteine.

    PubMed

    Chen, Xia; Liu, Hongli; Wang, Chen; Hu, Hui; Wang, Yuhui; Zhou, Xiaodong; Hu, Jiming

    2015-06-01

    A Hg(2+)-mediated fluorescence turn-on sensor for cysteine (Cys) detection was developed using the nucleic acid minor groove binding dye DAPI. In this work, two fully complementary DNA sequences, a T-rich single-stranded molecule (ssDNA) and an A-rich single-stranded molecule, were employed to constitute consecutive "AT/TA" base pairs, which could strongly enhance the fluorescence of DAPI. In the absence of cysteine, Hg(2+) reacted with T-rich single-stranded DNA and "T-Hg(2+)-T" base pairs formed, this seriously disrupted consecutive AT base pairs. As a result, the fluorescence of DAPI was not increased efficiently. However, considering that cysteine binds strongly to Hg(2+), the structure of the "T-Hg(2+)-T" complexes was destroyed in the presence of cysteine, resulting in the re-formation of consecutive AT base pairs and increased DAPI fluorescence. Obviously, the amount of cysteine could be easily measured based on the enhancement of DAPI fluorescence, and it took only 20 min to complete the whole cysteine-sensing process. Therefore, a label-free fluorescent "turn-on" sensor for the rapid detection of cysteine was designed, and the detection limit of this sensor was as low as 2.4 nM, which was much lower than those of the most of the previously reported cysteine sensors.

  16. Bio-functionalized silver nanoparticles: a novel colorimetric probe for cysteine detection.

    PubMed

    Borase, Hemant P; Patil, Chandrashekhar D; Salunkhe, Rahul B; Suryawanshi, Rahul K; Kim, Beom S; Bapat, Vishwas A; Patil, Satish V

    2015-04-01

    Chemical interactions between nanoparticles and biomolecules are vital for applying nanoparticles in medicine and life science. Development of sensitive, rapid, low-cost, and eco-friendly sensors for the detection of molecules acting as disease indicator is need of an hour. In the present investigation, a green trend for silver nanoparticle synthesis was followed using leaf extract of Calotropis procera. Silver nanoparticles exhibited surface plasmon absorption peak at 421 nm, spherical shape with average size of 10 nm, and zeta potential of -22.4 mV. The as-synthesized silver nanoparticles were used for selective and sensitive detection of cysteine. Cysteine induces aggregation in stable silver nanoparticles owing to selective and strong interaction of -SH group of cysteine with silver nanoparticle surface. Cysteine-induced silver nanoparticle aggregation can be observed visually by change in color of silver nanoparticles from yellow to pink. Cysteine concentration was estimated colorimetrically by measuring absorption at surface plasmon wavelength. Limit of detection for cysteine using silver nanoparticles is ultralow, i.e., 100 nM. The mechanistic insight into cysteine detection by silver nanoparticles was investigated using FT-IR, TEM, DLS, and TLC analysis. Proposed method can be applied for the detection of cysteine in blood plasma and may give rise to a new insight into development of eco-friendly fabricated nanodiagnostic device in future.

  17. Gamma-Radiolysis of Cysteine-Cysteamine Disulfide in Aqueous Solution,

    DTIC Science & Technology

    Gamma-radiolysis of a mixed disulfide, cysteine- cysteamine disulfide, in unbuffered aqueous solution (0.3 mM) was investigated in the presence and...absence of oxygen. The principal products were the thiols (cysteine and cysteamine ), the corresponding sulfinic and sulfonic acids, the symmetrical

  18. Purification and characterization of the cysteine proteinases in the latex of Vasconcellea spp.

    PubMed

    Kyndt, Tina; Van Damme, Els J M; Van Beeumen, Jozef; Gheysen, Godelieve

    2007-01-01

    Latex of all Vasconcellea species analyzed to date exhibits higher proteolytic amidase activities, generally attributed to cysteine proteinases, than the latex of Carica papaya. In the present study, we show that this higher activity is correlated with a higher concentration of enzymes in the latex of Vasconcellea fruits, but in addition also results from the presence of other cysteine proteinases or isoforms. In contrast to the cysteine proteinases present in papaya latex, which have been extensively studied, very little is known about the cysteine proteinases of Vasconcellea spp. In this investigation, several cDNA sequences coding for cysteine proteinases in Vasconcellea x heilbornii and Vasconcellea stipulata were determined using primers based on conserved sequences. In silico translation showed that they hold the characteristic features of all known papain-class cysteine proteinases, and a phylogenetic analysis revealed the existence of several papain and chymopapain homologues in these species. Ion-exchange chromatography and gel filtration procedures were applied on latex of V. x heilbornii in order to characterize its cysteine proteinases at the protein level. Five major protein fractions (VXH-I-VXH-V) revealing very high amidase activities (between 7.5 and 23.3 nkat x mg protein(-1)) were isolated. After further purification, three of them were N-terminally sequenced. The observed microheterogeneity in the N-terminal and cDNA sequences reveals the presence of several distinct cysteine proteinase isoforms in the latex of Vasconcellea spp.

  19. Polycarbophil-cysteine conjugates as platforms for oral polypeptide delivery systems.

    PubMed

    Bernkop-Schnürch, A; Thaler, S C

    2000-07-01

    The purpose of the present study was to evaluate the potential of polycarbophil-cysteine conjugates as carrier systems for orally administered peptide and protein drugs. Mediated by a carbodiimide, cysteine was covalently attached to polycarbophil. The properties of resulting conjugates, displaying 35-50 microM thiol groups per gram of polymer, to bind polypeptides and to inhibit pancreatic proteases was evaluated in vitro. Results demonstrated that only some polypeptides are immobilized to the polycarbophil-cysteine conjugate. Due to the covalent attachment of cysteine to polycarbophil, the inhibitory effect of the polymer toward carboxypeptidase A (EC 3.4. 17.1) and carboxypeptidase B (EC 3.4.17.2) could be significantly (p < 0.05) improved. As the zinc binding affinity of polycarbophil could be improved by the covalent attachment of cysteine, the raised inhibitory effect seems to be based on the complexation of this divalent cation from the enzyme structure. Whereas the covalent attachment of cysteine on polycarbophil had no influence on the enzymatic activity of trypsin (EC 3.4.21.4) and elastase (EC 3.4.21. 36), the inhibitory effect of the polymer-cysteine conjugate toward chymotrypsin (EC 3.4.21.1) was significantly (p < 0.05) higher than that of the unmodified polymer. Because of these inhibitory features, polycarbophil-cysteine conjugates seem to be a promising tool in protecting orally administered therapeutic polypeptides, which are not bound to the polymer, from presystemic metabolism in the intestine.

  20. First-pass splanchnic metabolism of dietary cysteine in weanling pigs

    USDA-ARS?s Scientific Manuscript database

    Cysteine is a semi-indispensable amino acid in neonates and is synthesized from the essential amino acid methionine by transsulfuration. We previously showed that the gastrointestinal tract (GIT) is a metabolically significant site of methionine transsulfuration to cysteine, yet the metabolic fate o...

  1. An iron-oxygen intermediate formed during the catalytic cycle of cysteine dioxygenase.

    PubMed

    Tchesnokov, E P; Faponle, A S; Davies, C G; Quesne, M G; Turner, R; Fellner, M; Souness, R J; Wilbanks, S M; de Visser, S P; Jameson, G N L

    2016-07-07

    Cysteine dioxygenase is a key enzyme in the breakdown of cysteine, but its mechanism remains controversial. A combination of spectroscopic and computational studies provides the first evidence of a short-lived intermediate in the catalytic cycle. The intermediate decays within 20 ms and has absorption maxima at 500 and 640 nm.

  2. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery

    PubMed Central

    Hallenbeck, Kenneth K.; Turner, David M.; Renslo, Adam R.; Arkin, Michelle R.

    2017-01-01

    The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an ‘allele specific’ manner. For the medicinal chemist, the structure-guided design of cysteine-reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteine-modifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues. PMID:27449257

  3. Identification of Covalent Binding Sites Targeting Cysteines Based on Computational Approaches.

    PubMed

    Zhang, Yanmin; Zhang, Danfeng; Tian, Haozhong; Jiao, Yu; Shi, Zhihao; Ran, Ting; Liu, Haichun; Lu, Shuai; Xu, Anyang; Qiao, Xin; Pan, Jing; Yin, Lingfeng; Zhou, Weineng; Lu, Tao; Chen, Yadong

    2016-09-06

    Covalent drugs have attracted increasing attention in recent years due to good inhibitory activity and selectivity. Targeting noncatalytic cysteines with irreversible inhibitors is a powerful approach for enhancing pharmacological potency and selectivity because cysteines can form covalent bonds with inhibitors through their nucleophilic thiol groups. However, most human kinases have multiple noncatalytic cysteines within the active site; to accurately predict which cysteine is most likely to form covalent bonds is of great importance but remains a challenge when designing irreversible inhibitors. In this work, FTMap was first applied to check its ability in predicting covalent binding site defined as the region where covalent bonds are formed between cysteines and irreversible inhibitors. Results show that it has excellent performance in detecting the hot spots within the binding pocket, and its hydrogen bond interaction frequency analysis could give us some interesting instructions for identification of covalent binding cysteines. Furthermore, we proposed a simple but useful covalent fragment probing approach and showed that it successfully predicted the covalent binding site of seven targets. By adopting a distance-based method, we observed that the closer the nucleophiles of covalent warheads are to the thiol group of a cysteine, the higher the possibility that a cysteine is prone to form a covalent bond. We believe that the combination of FTMap and our distance-based covalent fragment probing method can become a useful tool in detecting the covalent binding site of these targets.

  4. Cysteine sulfinate desulfinase, a NIFS-like protein of Escherichia coli with selenocysteine lyase and cysteine desulfurase activities. Gene cloning, purification, and characterization of a novel pyridoxal enzyme.

    PubMed

    Mihara, H; Kurihara, T; Yoshimura, T; Soda, K; Esaki, N

    1997-09-05

    Selenocysteine lyase (EC 4.4.1.16) exclusively decomposes selenocysteine to alanine and elemental selenium, whereas cysteine desulfurase (NIFS protein) of Azotobacter vinelandii acts indiscriminately on both cysteine and selenocysteine to produce elemental sulfur and selenium respectively, and alanine. These proteins exhibit some sequence homology. The Escherichia coli genome contains three genes with sequence homology to nifS. We have cloned the gene mapped at 63.4 min in the chromosome and have expressed, purified to homogeneity, and characterized the gene product. The enzyme comprises two identical subunits with 401 amino acid residues (Mr 43,238) and contains pyridoxal 5'-phosphate as a coenzyme. The enzyme catalyzes the removal of elemental sulfur and selenium atoms from L-cysteine, L-cystine, L-selenocysteine, and L-selenocystine to produce L-alanine. Because L-cysteine sulfinic acid was desulfinated to form L-alanine as the preferred substrate, we have named this new enzyme cysteine sulfinate desulfinase. Mutant enzymes having alanine substituted for each of the four cysteinyl residues (Cys-100, Cys-176, Cys-323, and Cys-358) were all active. Cys-358 corresponds to Cys-325 of A. vinelandii NIFS, which is conserved among all NIFS-like proteins and catalytically essential (Zheng, L., White, R. H., Cash, V. L., and Dean, D. R. (1994) Biochemistry 33, 4714-4720), is not required for cysteine sulfinate desulfinase. Thus, the enzyme is distinct from A. vinelandii NIFS in this respect.

  5. Cysteine proteases as therapeutic targets: does selectivity matter? A systematic review of calpain and cathepsin inhibitors

    PubMed Central

    Siklos, Marton; BenAissa, Manel; Thatcher, Gregory R.J.

    2015-01-01

    Cysteine proteases continue to provide validated targets for treatment of human diseases. In neurodegenerative disorders, multiple cysteine proteases provide targets for enzyme inhibitors, notably caspases, calpains, and cathepsins. The reactive, active-site cysteine provides specificity for many inhibitor designs over other families of proteases, such as aspartate and serine; however, a) inhibitor strategies often use covalent enzyme modification, and b) obtaining selectivity within families of cysteine proteases and their isozymes is problematic. This review provides a general update on strategies for cysteine protease inhibitor design and a focus on cathepsin B and calpain 1 as drug targets for neurodegenerative disorders; the latter focus providing an interesting query for the contemporary assumptions that irreversible, covalent protein modification and low selectivity are anathema to therapeutic safety and efficacy. PMID:26713267

  6. Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

    SciTech Connect

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

    2012-06-05

    A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

  7. A fluorescence enhancement probe based on BODIPY for the discrimination of cysteine from homocysteine and glutathione.

    PubMed

    Gong, Deyan; Tian, Yuejun; Yang, Chengduan; Iqbal, Anam; Wang, Zhiping; Liu, Weisheng; Qin, Wenwu; Zhu, Xiangtao; Guo, Huichen

    2016-11-15

    Herein, a fluorescent probe BODIPY-based glyoxal hydrazone (BODIPY-GH) (1) for cysteine based on inhibiting of intramolecular charge transfer (ICT) quenching process upon reaction with the unsaturated aldehyde has been synthesized, which exhibits longer excitation wavelength, selective and sensitive colorimetric and fluorimetric response toward cysteine in natural media. The probe shows highly selectivity towards cysteine over homocysteine and glutathione as well as other amino acids with a significant fluorescence enhancement response within 15min In the presence of 50 equiv. of homocysteine, the emission increased slightly within 15min and completed in 2.5h to reach its maximum intensity. Therefore, the discrimination of cysteine from homocysteine and glutathione can be achieved through detection of probe 1. It shows low cytotoxicity and excellent membrane permeability toward living cells, which was successfully applied to detect and image intracellular cysteine effectively by confocal fluorescence imaging.

  8. Granulosain I, a cysteine protease isolated from ripe fruits of Solanum granuloso-leprosum (Solanaceae).

    PubMed

    Vallés, Diego; Bruno, Mariela; López, Laura M I; Caffini, Néstor O; Cantera, Ana María B

    2008-08-01

    A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family.

  9. L-cystein protects the pigment epithelium from acute sodium iodate toxicity.

    PubMed

    Heike, M; Marmor, M F

    1990-08-01

    Intravenous sodium iodate damages the retinal pigment epithelium, causing immediate loss of the electroretinogram c-wave and eventual pigmentary retinopathy. L-cystein, an agent that enhances the c-wave, has been reported to prevent the late development of pigmentary degeneration. We found in rabbits that L-cystein given 30 min before, or simultaneously with (but not 30 min after) sodium iodate also blocks the loss of the c-wave. This result occurred at doses of L-cystein lower than those needed to produce enhancement of the c-wave, suggesting that these two actions of L-cystein may be independent. The iodate-blocking action of L-cystein may depend on chemical interaction.

  10. A colorimetric sensor for determination of cysteine by carboxymethyl cellulose-functionalized gold nanoparticles.

    PubMed

    Wei, Xiaoyi; Qi, Li; Tan, Junjun; Liu, Ruigang; Wang, Fuyi

    2010-06-25

    A simple and sensitive colorimetric method for cysteine detection was established based on the carboxymethyl cellulose-functionalized gold nanoparticles (CMC-AuNPs). The nanoparticles were directly synthesized with sodium carboxymethyl cellulose by a simple approach, which would protect particles against salt-induced aggregation. Then the CMC-AuNPs solution exhibited a high colorimetric selectivity to cysteine. The assay results indicated that the introduction of cysteine could induce the aggregation of the colloidal solutions at the presence of sodium chloride, displaying changes in color and in UV-vis absorption spectra. Thus an exceptionally simple, rapid method for detecting cysteine was obtained at the linear range of 10.0-100.0 microM with the relative coefficient of 0.997. The proposed method possessed the advantages of simplicity and sensitivity, and was applied to real urine sample detection. The results were satisfying and the proposed method was especially appropriate for detection of cysteine in biological samples.

  11. Dextran coated silver nanoparticles - Chemical sensor for selective cysteine detection.

    PubMed

    Davidović, Slađana; Lazić, Vesna; Vukoje, Ivana; Papan, Jelena; Anhrenkiel, S Phillip; Dimitrijević, Suzana; Nedeljković, Jovan M

    2017-09-12

    A simple, fast and non-costly method for selective cysteine (Cys) detection, based on optical changes of silver colloids, is developed. For that purpose, stable colloids consisting of silver nanoparticles (Ag NPs) coated with polysaccharide dextran (Dex), isolated from bacterium species Leuconostoc mesenteroides T3, were prepared. The synthesized samples were thoroughly characterized including absorption and FTIR spectroscopy, as well as transmission electron microscopy and X-ray diffraction analysis. The silver colloids display high sensitivity and selectivity towards Cys detection in aqueous solutions. The Ag NPs coated with Dex provide possibility to detect Cys among a dozen amino acids and its detection limit was found to be 12.0μM. The sensing mechanism - red shift of optical absorption - is discussed in terms of the agglomeration of Ag NPs due to formation of hydrogen bonds between Cys molecules attached to different Ag NPs. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Supported oligomethionine sulfoxide and Ellman's reagent for cysteine bridges formation.

    PubMed

    Ronga, Luisa; Verdié, Pascal; Sanchez, Pierre; Enjabal, Christine; Maurras, Amélie; Jullian, Magalie; Puget, Karine; Martinez, Jean; Subra, Gilles

    2013-02-01

    A large number of bioactive peptides are cyclized through a disulfide bridge. This structural feature is very important for both bioactivity and stability. The oxidation of cysteine side chains is challenging not only to avoid intermolecular reaction leading to oligomers and oxidation of other residues but also to remove solvents and oxidant such as dimethyl sulfoxide. Supported reagents advantageously simplify the work-up of such disulfide bond formation, but may lead to a significant decrease in yield of the oxidized product. In this study, two resins working through different mechanisms were evaluated: Clear-Ox, a supported version of Ellman's reagent and Oxyfold, consisting in a series of oxidized methionine residues. The choice of the supported reagent is discussed on the light of reaction speed, side-products formation and yield considerations.

  13. Efficient expression systems for cysteine proteases of malaria parasites

    PubMed Central

    Sarduy, Emir Salas; de los A. Chávez Planes, María

    2013-01-01

    Papain-like cysteine proteases of malaria parasites are considered important chemotherapeutic targets or valuable models for the evaluation of drug candidates. Consequently, many of these enzymes have been cloned and expressed in Escherichia coli for their biochemical characterization. However, their expression has been problematic, showing low yield and leading to the formation of insoluble aggregates. Given that highly-productive expression systems are required for the high-throughput evaluation of inhibitors, we analyzed the existing expression systems to identify the causes of such apparent issues. We found that significant divergences in codon and nucleotide composition from host genes are the most probable cause of expression failure, and propose several strategies to overcome these limitations. Finally we predict that yeast hosts Saccharomyces cerevisiae and Pichia pastoris may be better suited than E. coli for the efficient expression of plasmodial genes, presumably leading to soluble and active products reproducing structural and functional characteristics of the natural enzymes. PMID:23018863

  14. Immunodiagnosis of fasciolosis using recombinant procathepsin L cystein proteinase.

    PubMed

    Carnevale, S; Rodríguez, M I; Guarnera, E A; Carmona, C; Tanos, T; Angel, S O

    2001-01-01

    Cathepsin L1, a cysteine protease secreted by the gastrodermis of juvenile and adult Fasciola hepatica, was expressed in Escherichia coli as a fusion protein containing the proregion, supplied with six histidyl residues at the N-terminal end (rproCL1). In this study we tested its potential as antigen for the serologic diagnosis of F. hepatica infections by enzyme-linked immunosorbent assay (ELISA). The analyzed human sera included 16 positive samples, 99 negative controls and 111 from individuals affected by other parasitic and non parasitic diseases. The sensitivity and specificity of the rproCL1-ELISA were 100%. We also assessed the ability to detect antibodies in sera from 10 experimentally infected sheep, obtaining preliminary results that shown a response since the third week post infection in all the studied animals. Therefore, the recombinant rproCL1-based ELISA could be a standardized test for the accurate diagnosis of fasciolosis.

  15. N-acetyl-L-Cysteine as prophylaxis against sulfur mustard.

    PubMed

    Bobb, Andrew J; Arfsten, Darryl P; Jederberg, Warren W

    2005-01-01

    Sulfur mustard (HD) is a blister agent targeting the eyes, respiratory system, skin, and possibly other organs. Extensive exposure can destroy the immune system by destruction of bone marrow cells. There is no antidote for HD or effective treatment other than rapid decontamination. Clinical trials have demonstrated activity for N-acetyl-L-cysteine (NAC) against a number of significant human pathologies involving free radicals, and animal and tissue studies have suggested efficacy for NAC as a chemoprotectant against many toxic chemicals. Among these are studies demonstrating that NAC significantly reduces the effects of HD and HD simulants, both in cultured cells and animals. Given the historical effectiveness of HD, the lack of any effective treatment, the demonstrated chemoprotective properties of NAC, its low toxicity, the lack of regulatory controls, and the data supporting efficacy against HD effects, we suggest daily oral administration of the maximum safe dose of NAC to personnel entering combat zones.

  16. Secreted Protein Acidic and Rich in Cysteine in Ocular Tissue

    PubMed Central

    Scavelli, Kurt; Chatterjee, Ayan

    2015-01-01

    Abstract Secreted protein acidic and rich in cysteine (SPARC), also known as osteonectin or BM-40, is the prototypical matricellular protein. Matricellular proteins are nonstructural secreted proteins that provide an integration between cells and their surrounding extracellular matrix (ECM). Regulation of the ECM is important in maintaining the physiologic function of tissues. Elevated levels of SPARC have been identified in a variety of diseases involving pathologic tissue remodeling, such as hepatic fibrosis, systemic sclerosis, and certain carcinomas. Within the eye, SPARC has been identified in the trabecular meshwork, lens, and retina. Studies have begun to show the role of SPARC in these tissues and its possible role, specifically in primary open-angle glaucoma, cataracts, and proliferative vitreoretinopathy. SPARC may, therefore, be a therapeutic target in the treatment of certain ocular diseases. Further investigation into the mechanism of action of SPARC will be necessary in the development of SPARC-targeted therapy. PMID:26167673

  17. The Future of Cysteine Cathepsins in Disease Management.

    PubMed

    Kramer, Lovro; Turk, Dušan; Turk, Boris

    2017-10-01

    Since the discovery of the key role of cathepsin K in bone resorption, cysteine cathepsins have been investigated by pharmaceutical companies as drug targets. The first clinical results from targeting cathepsins by activity-based probes and substrates are paving the way for the next generation of molecular diagnostic imaging, whereas the majority of antibody-drug conjugates currently in clinical trials depend on activation by cathepsins. Finally, cathepsins have emerged as suitable vehicles for targeted drug delivery. It is therefore timely to review the future of cathepsins in drug discovery. We focus here on inflammation-associated diseases because dysregulation of the immune system accompanied by elevated cathepsin activity is a common feature of these conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Characterization of cysteine proteases in Malian medicinal plants.

    PubMed

    Bah, Sékou; Paulsen, Berit S; Diallo, Drissa; Johansen, Harald T

    2006-09-19

    Extracts form 10 different Malian medicinal plants with a traditional use against schistosomiasis were investigated for their possible content of proteolytic activity. The proteolytic activity was studied by measuring the hydrolysis of two synthetic peptide substrates Z-Ala-Ala-Asn-NHMec and Z-Phe-Arg-NHMec. Legumain- and papain-like activities were found in all tested crude extracts except those from Entada africana, with the papain-like activity being the strongest. Cissus quadrangularis, Securidaca longepedunculata and Stylosanthes erecta extracts showed high proteolytic activities towards both substrates. After gel filtration the proteolytic activity towards the substrate Z-Ala-Ala-Asn-NHMec in root extract of Securidaca longepedunculata appeared to have Mr of 30 and 97kDa, while the activity in extracts from Cissus quadrangularis was at 39kDa. Enzymatic activity cleaving the substrate Z-Phe-Arg-NHMec showed apparent Mr of 97 and 26kDa in extracts from roots and leaves of Securidaca longepedunculata, while in Cissus quadrangularis extracts the activity eluted at 39 and 20kDa, with the highest activity in the latter. All Z-Phe-Arg-NHMec activities were inhibited by E-64 but unaffected by PMSF. The legumain activity was unaffected by E-64 and PMSF. The SDS-PAGE analysis exhibited five distinct gelatinolytic bands for Cissus quadrangularis extracts (115, 59, 31, 22 and 20kDa), while two bands (59 and 30kDa) were detected in Securidaca longepedunculata extracts. The inhibition profile of the gelatinolytic bands and that of the hydrolysis of the synthetic substrates indicate the cysteine protease class of the proteolytic activities. Several cysteine protease activities with different molecular weights along with a strong variability of these activities between species as well as between plant parts from the same species were observed.

  19. Unusual hydrogen bonding in L-cysteine hydrogen fluoride.

    PubMed

    Minkov, V S; Ghazaryan, V V; Boldyreva, E V; Petrosyan, A M

    2015-08-01

    L-Cysteine hydrogen fluoride, or bis(L-cysteinium) difluoride-L-cysteine-hydrogen fluoride (1/1/1), 2C3H8NO2S(+)·2F(-)·C3H7NO2S·HF or L-Cys(+)(L-Cys···L-Cys(+))F(-)(F(-)...H-F), provides the first example of a structure with cations of the 'triglycine sulfate' type, i.e. A(+)(A···A(+)) (where A and A(+) are the zwitterionic and cationic states of an amino acid, respectively), without a doubly charged counter-ion. The salt crystallizes in the monoclinic system with the space group P2(1). The dimeric (L-Cys···L-Cys(+)) cation and the dimeric (F(-)···H-F) anion are formed via strong O-H···O or F-H···F hydrogen bonds, respectively, with very short O···O [2.4438 (19) Å] and F···F distances [2.2676 (17) Å]. The F···F distance is significantly shorter than in solid hydrogen fluoride. Additionally, there is another very short hydrogen bond, of O-H···F type, formed by a L-cysteinium cation and a fluoride ion. The corresponding O···F distance of 2.3412 (19) Å seems to be the shortest among O-H···F and F-H···O hydrogen bonds known to date. The single-crystal X-ray diffraction study was complemented by IR spectroscopy. Of special interest was the spectral region of vibrations related to the above-mentioned hydrogen bonds.

  20. Cysteine modified polyaniline films improve biocompatibility for two cell lines.

    PubMed

    Yslas, Edith I; Cavallo, Pablo; Acevedo, Diego F; Barbero, César A; Rivarola, Viviana A

    2015-06-01

    This work focuses on one of the most exciting application areas of conjugated conducting polymers, which is cell culture and tissue engineering. To improve the biocompatibility of conducting polymers we present an easy method that involves the modification of the polymer backbone using l-cysteine. In this publication, we show the synthesis of polyaniline (PANI) films supported onto Polyethylene terephthalate (PET) films, and modified using cysteine (PANI-Cys) in order to generate a biocompatible substrate for cell culture. The PANI-Cys films are characterized by Fourier Transform infrared and UV-visible spectroscopy. The changes in the hydrophilicity of the polymer films after and before the modification were tested using contact angle measurements. After modification the contact angle changes from 86°±1 to 90°±1, suggesting a more hydrophylic surface. The adhesion properties of LM2 and HaCaT cell lines on the surface of PANI-Cys films in comparison with tissue culture plastic (TCP) are studied. The PANI-Cys film shows better biocompatibility than PANI film for both cell lines. The cell morphologies on the TCP and PANI-Cys film were examined by florescence and Atomic Force Microscopy (AFM). Microscopic observations show normal cellular behavior when PANI-Cys is used as a substrate of both cell lines (HaCaT and LM2) as when they are cultured on TCP. The ability of these PANI-Cys films to support cell attachment and growth indicates their potential use as biocompatible surfaces and in tissue engineering.

  1. The inhibitory effects of silver diamine fluorides on cysteine cathepsins.

    PubMed

    Mei, May L; Ito, L; Cao, Y; Li, Q L; Chu, C H; Lo, Edward C M

    2014-03-01

    The expression of cysteine cathepsins in human carious dentine suggests that this enzyme contributes to the collagen degradation in caries progress. This study investigated whether silver diamine fluoride (SDF) inhibited the activity of cysteine cathepsins. Three commercial SDF solutions with concentrations at 38%, 30% and 12% were studied. Two fluoride solutions with the same fluoride ion (F(-)) concentrations as the 38% and 12% SDF solutions, and 2 silver solutions with the same silver ion (Ag(+)) concentrations as the 38% and 12% SDF solutions were prepared. Five samples of each experimental solution were used to study their inhibitory effect on two cathepsins (B and K) using cathepsin assay kits. Positive control contained assay buffer and cathepsins dilution was used to calculate the percentage inhibition (difference between the mean readings of the test solution and control solution divided by that of the control group). The percentage inhibition of 38%, 30% and 12% SDF on cathepsin B were 92.0%, 91.5% and 90.3%, respectively (p<0.001); on cathepsin K were 80.6%, 78.5% and 77.9%, respectively (p<0.001). Ag(+) exhibited the inhibitory effect against both cathepsin B and K with or without the presence of F(-) (p<0.01). The solutions containing Ag(+) have significantly higher inhibitory effect than the solutions containing F(-) only (p<0.01). According to this study, SDF solution at all 3 tested concentrations significantly inhibited the activity of cathepsin B and K. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. A viable synthesis of N-methyl cysteine.

    PubMed

    Ruggles, Erik L; Flemer, Stevenson; Hondal, Robert J

    2008-01-01

    While a number of methods exist for the production of N-methyl amino acid derivatives, the methods for the production of N-methyl cysteine (MeCys) derivatives are suboptimal as they either have low yields or lead to significant sulfhydryl deprotection during the synthetic protocol. This article focuses on the generation of MeCys and its subsequent use in Fmoc solid-phase peptide synthesis for the generation of N-methyl cystine containing peptides. Various methods for amino methylation of cysteine, in the presence of acid labile or acid stable sulfhydryl protecting groups, are compared and contrasted. Production of MeCys is best attained through formation of an oxazolidinone precursor obtained via cyclization of Fmoc--Cys(StBu)--OH. Following oxazolidinone ring opening, iminium ion reduction generates Fmoc--MeCys(StBu)--OH with an overall yield of 91%. The key to this procedure is using an electronically neutral Cys-derivative, as other polar Cys-derivatives gave poor results using the oxazolidinone procedure. Subsequently, the Fmoc--MeCys(StBu)--OH building block was used to replace a Cys residue with a MeCys residue in two peptide fragments that correspond to the active sites of glutaredoxin and thioredoxin reductase. The examples used here highlight the use of a MeCys(StBu) derivative, which allows for facile on-resin conversion to a MeCys(5-Npys) residue that can be subsequently used for intramolecular disulfide bond formation with concomitant cleavage of the peptide from the solid support. (c) 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 61-68, 2008. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.

  3. CP30, a Cysteine Proteinase Involved in Trichomonas vaginalis Cytoadherence

    PubMed Central

    Mendoza-López, M. Remedios; Becerril-Garcia, Cecilia; Fattel-Facenda, Loriz V.; Avila-Gonzalez, Leticia; Ruíz-Tachiquín, Martha E.; Ortega-Lopez, Jaime; Arroyo, Rossana

    2000-01-01

    We describe here the participation of a Trichomonas vaginalis 30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells. PMID:10948104

  4. DU-AGG pilot plant design study

    SciTech Connect

    Lessing, P.A.; Gillman, H.

    1996-07-01

    The Idaho National Engineering Laboratory (INEL) is developing new methods to produce high-density aggregate (artificial rock) primarily consisting of depleted uranium oxide. The objective is to develop a low-cost method whereby uranium oxide powder (UO[sub 2], U[sub 3]O[sub ]8, or UO[sub 3]) can be processed to produce high-density aggregate pieces (DU-AGG) having physical properties suitable for disposal in low-level radioactive disposal facilities or for use as a component of high-density concrete used as shielding for radioactive materials. A commercial company, G-M Systems, conducted a design study for a manufacturing pilot plant to process DU-AGG. The results of that study are included and summarized in this report. Also explained are design considerations, equipment capacities, the equipment list, system operation, layout of equipment in the plant, cost estimates, and the proposed plan and schedule.

  5. Formation of three N-acetyl-L-cysteine monoadducts and one diadduct by the reaction of S-(1,2-dichlorovinyl)-L-cysteine sulfoxide with N-acetyl-L-cysteine at physiological conditions: chemical mechanisms and toxicological implications.

    PubMed

    Barshteyn, Nella; Elfarra, Adnan A

    2007-10-01

    Previously, our laboratory has shown that S-(1,2-dichlorovinyl)-L-cysteine sulfoxide (DCVCS), a Michael acceptor produced by a flavin-containing monooxygenase 3 (FMO3)-mediated oxidation of S-(1,2-dichlorovinyl)-L-cysteine (DCVC), is a more potent nephrotoxicant than DCVC. In the present study, we characterized reactions of DCVCS with nucleophilic amino acids. DCVCS incubations with N-acetyl-L-cysteine (NAC) at pH 7.4 and 37 degrees C for 1 h resulted in the formation of three monoadducts and one diadduct characterized by LC/MS, 1H NMR, and 1H-detected heteronuclear single quantum correlation. The formation of all adducts (with relative ratios of 29, 31, 24, and 12%, respectively) was rapid and time-dependent; the half-lives of the two DCVCS diastereomers in the presence of NAC were 13.8 (diastereomer I) and 9.4 min (diastereomer II). Adducts 1 and 2 were determined to be diastereomers of S-[1-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed by Michael addition of NAC to the terminal vinylic carbon of DCVCS followed by loss of HCl. Adduct 4 was determined to be S-[2-chloro-2-(N-acetyl-L-cystein- S-yl)vinyl]-L-cysteine sulfoxide formed from the initial Michael addition product followed by a less favorable loss of HCl and/or by a rearrangement of adduct 2 through the formation of a cyclic chloronium ion. The addition of another molecule of NAC to monoadducts 1, 2, or 4 resulted in the formation of the novel diadduct, S-[2,2-( N-acetyl-L-cystein-S-yl)vinyl]-L-cysteine sulfoxide (adduct 3), whose detection in relatively large amount suggests that DCVCS could act as a cross-linking agent. DCVCS was not reactive with N-acetyl-L-lysine or L-valinamide at similar incubation conditions. Collectively, the results suggest selective reactivity of DCVCS toward protein sulfhydryl groups. Furthermore, the cross-linking properties of DCVCS may in part explain its high nephrotoxic potency.

  6. Sustainable growth, the DuPont way.

    PubMed

    Holliday, C

    2001-09-01

    Like many manufacturers, DuPont traditionally has grown by making more and more "stuff." And its business growth has been proportional to the amount of raw materials and energy used--as well as the resulting waste and emissions from operations. Over the years, though, DuPont became aware that cheap supplies of nonrenewable resources wouldn't be endlessly available and that the earth's ecosystems couldn't indefinitely absorb the waste and emissions of production and consumption. Chad Holliday, chairman and CEO of DuPont, believes strongly in the challenge of sustainable growth and makes the business case for it: By using creativity and scientific knowledge effectively, he says, companies can provide strong returns for shareholders and grow their businesses--while also meeting the human needs of societies around the world and reducing the environmental footprint of their operations and products. In fact, a focus on sustainability can help identify new products, markets, partnerships, and intellectual property and lead to substantial business growth. Holliday describes how DuPont developed a three-pronged strategy to translate the concept of sustainability into nuts-and-bolts business practices. Focusing on integrated science, knowledge intensity, and productivity improvement, the strategy was accompanied by a new way to measure progress quantitatively. Sustainable growth should be viewed not as a program for stepped-up environmental performance but as a comprehensive way of doing business, one that delivers tremendous economic value and opens up new opportunities. Ultimately, companies will find that they can generate substantial business value through sustainability while both enhancing the quality of life around the world and protecting the environment.

  7. Pourfour Du Petit syndrome after interscalene block

    PubMed Central

    Santhosh, Mysore Chandramouli Basappji; Pai, Rohini B.; Rao, Raghavendra P.

    2013-01-01

    Interscalene block is commonly associated with reversible ipsilateral phrenic nerve block, recurrent laryngeal nerve block, and cervical sympathetic plexus block, presenting as Horner's syndrome. We report a very rare Pourfour Du Petit syndrome which has a clinical presentation opposite to that of Horner's syndrome in a 24-year-old male who was given interscalene block for open reduction and internal fixation of fracture upper third shaft of left humerus. PMID:23956726

  8. Cri du Chat syndrome: a case report.

    PubMed

    Torres, Carolina Paes; Borsatto, Maria Cristina; de Queiroz, Alexandra Mussolino; Lessa, Fernanda Campos Rosetti; Orsi, Iara Agusta

    2005-01-01

    Cri du Chat Syndrome occurs as a result of a partial deletion in the short arm of chromosome 5. Among the consequent abnormalities are low birth weight, a striking catlike cry in infancy, mental retardation, epicanthal folds, hypertelorism and dental malocclusions. This paper presents a case report on the dental treatment of a 23-year-old patient who received multidisciplinary treatment, including special education and precocious stimulation for carriers of this syndrome.

  9. DuPont Chemical Vapor Technical Report

    SciTech Connect

    MOORE, T.L.

    2003-10-03

    DuPont Safety Resources was tasked with reviewing the current chemical vapor control practices and providing preventive recommendations on best commercial techniques to control worker exposures. The increased focus of the tank closure project to meet the 2024 Tri-Party Agreement (TPA) milestones has surfaced concerns among some CH2MHill employees and other interested parties. CH2MHill is committed to providing a safe working environment for employees and desires to safely manage the tank farm operations using appropriate control measures. To address worker concerns, CH2MHill has chartered a ''Chemical Vapors Project'' to integrate the activities of multiple CH2MHill project teams, and solicit the expertise of external resources, including an independent Industrial Hygiene expert panel, a communications consultant, and DuPont Safety Resources. Over a three-month time period, DuPont worked with CH2MHill ESH&Q, Industrial Hygiene, Engineering, and the independent expert panel to perform the assessment. The process included overview presentations, formal interviews, informal discussions, documentation review, and literature review. DuPont Safety Resources concluded that it is highly unlikely that workers in the tank farms are exposed to chemicals above established standards. Additionally, the conventional and radiological chemistry is understood, the inherent chemical hazards are known, and the risk associated with chemical vapor exposure is properly managed. The assessment highlighted management's commitment to addressing chemical vapor hazards and controlling the associated risks. Additionally, we found the Industrial Hygiene staff to be technically competent and well motivated. The tank characterization data resides in a comprehensive database containing the tank chemical compositions and relevant airborne concentrations.

  10. Pourfour Du Petit syndrome after interscalene block.

    PubMed

    Santhosh, Mysore Chandramouli Basappji; Pai, Rohini B; Rao, Raghavendra P

    2013-04-01

    Interscalene block is commonly associated with reversible ipsilateral phrenic nerve block, recurrent laryngeal nerve block, and cervical sympathetic plexus block, presenting as Horner's syndrome. We report a very rare Pourfour Du Petit syndrome which has a clinical presentation opposite to that of Horner's syndrome in a 24-year-old male who was given interscalene block for open reduction and internal fixation of fracture upper third shaft of left humerus.

  11. Proprietes de Transport Electronique du Rutile Stoechiometrique

    NASA Astrophysics Data System (ADS)

    Keroack, Danielle

    Le rutile est un oxyde metallique qui presente beaucoup de similitudes avec certains perovskites notamment le titanate de barium ou de strontium. Il est comme ces derniers, compose d'un agencement d'octaedres d'oxygene centres sur l'atome de titane. Ces octaedres sont responsables de la forte polarisabilite de ces cristaux et de leur grande constante dielectrique. Leurs proprietes optiques, seuil d'absorption et spectre de phonons, sont par exemple forts semblables. La presente etude vise a determiner la nature du transport electronique dans le rutile stoechiometrique pur et d'en comparer les resultats avec les proprietes de certains perovskites. Nous determinerons par differentes mesures optoelectroniques les parametres caracteristiques des pieges et leur influence sur le transport des electrons et des trous. Les resultats de conductivite et de capacitance de meme que les spectres de photoconductivite dans nos echantillons ont mis en evidence la presence d'au moins cinq niveaux energetiques dans la bande interdite du rutile agissant comme pieges pour les electrons ou pour les trous et qui jouent un role de premiere importance dans le comportement electrique du rutile. Par la technique de charge transitoire, nous determinerons pour la premiere fois dans le rutile stoechiometrique la grandeur de la mobilite de derive des trous a la temperature ambiante soit 3,4 cm^2/V cdots et nous etablierons une borne superieure a la mobilite des electrons soit 0,1 cm^2 /Vcdots.

  12. Adsorption Study of Cysteine, N-Acetylcysteamine, Cysteinesulfinic Acid and Cysteic Acid on a Polycrystalline Gold Electrode

    DTIC Science & Technology

    1993-04-15

    effectively increases packing density of cysteine at the electrode surface. Films of N- acetyl - cysteamine are more hydrophobic than those of cysteine ... Cysteine , N-acetylcysteamine, CysteinesulImic Acid and Cysteic Acid on a Polycrystalline Gold Electrode by W. Ronald Fawcett, Milan Fedurco, Zuzana...200 words) Capacity against potential data for the adsorption of cysteine and Other organosulfur Compounds on polyc~rysraline gold are reported

  13. Emission of Hydrogen Sulfide by Leaf Tissue in Response to l-Cysteine 1

    PubMed Central

    Sekiya, Jiro; Schmidt, Ahlert; Wilson, Lloyd G.; Filner, Philip

    1982-01-01

    Leaf discs and detached leaves exposed to l-cysteine emitted a volatile sulfur compound which was proven by gas chromatography to be H2S. This phenomenon was demonstrated in all nine species tested (Cucumis sativus, Cucurbita pepo, Nicotiana tabacum, Coleus blumei, Beta vulgaris, Phaseolus vulgaris, Medicago sativa, Hordeum vulgare, and Gossypium hirsutum). The emission of volatile sulfur by cucumber leaves occurred in the dark at a similar rate to that in the light. The emission of leaf discs reached the maximal rate, more than 40 picomoles per minute per square centimeter, 2 to 4 hours after starting exposure to l-cysteine; then it decreased. In the case of detached leaves, the maximum occurred 5 to 10 h after starting exposure. The average emission rate of H2S during the first 4 hours from leaf discs of cucurbits in response to 10 millimolar l-cysteine, was usually more than 40 picomoles per minute per square centimeter, i.e. 0.24 micromoles per hour per square decimeter. Leaf discs exposed to 1 millimolar l-cysteine emitted only 2% as much as did the discs exposed to 10 millimolar l-cysteine. The emission from leaf discs and from detached leaves lasted for at least 5 and 15 hours, respectively. However, several hours after the maximal emission, injury of the leaves, manifested as chlorosis, was evident. H2S emission was a specific consequence of exposure to l-cysteine; neither d-cysteine nor l-cystine elicited H2S emission. Aminooxyacetic acid, an inhibitor of pyridoxal phosphate dependent enzymes, inhibited the emission. In a cell free system from cucumber leaves, H2S formation and its release occurred in response to l-cysteine. Feeding experiments with [35S]l-cysteine showed that most of the sulfur in H2S was derived from sulfur in the l-cysteine supplied and that the H2S emitted for 9 hours accounted for 7 to 10% of l-cysteine taken up. 35S-labeled SO32− and SO42− were found in the tissue extract in addition to internal soluble S2−. These findings

  14. s-Ethyl Cysteine and s-Methyl Cysteine Protect Human Bronchial Epithelial Cells Against Hydrogen Peroxide Induced Injury.

    PubMed

    Hsia, Te-chun; Yin, Mei-chin

    2015-09-01

    Protective effects and actions from s-ethyl cysteine (SEC) and s-methyl cysteine (SMC) for BEAS-2B cells were examined. BEAS-2B cells were pretreated with SEC or SMC at 4, 8, or 16 μmol/L, and followed by hydrogen peroxide (H2 O2 ) treatment. Data showed that H2 O2 enhanced Bax, caspase-3 and caspase-8 expression, and declined Bcl-2 expression. However, SEC or SMC dose-dependently decreased caspase-3 expression and reserved Bcl-2 expression. H2 O2 increased reactive oxygen species (ROS) production, and lowered glutathione level, glutathione peroxide, and glutathione reductase activities in BEAS-2B cells. SEC or SMC pretreatments reduced ROS generation, and maintained glutathione redox cycle in those cells. H2 O2 upregulated the expression of both p47(phox) and gp91(phox) . SEC and SMC downregulated p47(phox) expression. SEC or SMC at 8 and 16 μmol/L decreased H2 O2 -induced release of inflammatory cytokines. H2 O2 stimulated the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase. SEC and SMC pretreatments dose-dependently downregulated NF-κB p65 and p-p38 expression. Pyrrolidine dithiocarbamate or SB203580 inhibited NF-κB activation and p38 phosphorylation; thus, SEC or SMC pretreatments failed to affect protein expression of these factors. These novel findings suggest that SEC or SMC could protect bronchial cells and benefit respiratory epithelia stability and functions.

  15. Metabolism of cysteine, cysteinesulfinate and cysteinesulfonate in rats fed adequate and excess levels of sulfur-containing amino acids

    SciTech Connect

    Stipanuk, M.H.; Rotter, M.A.

    1984-08-01

    The oxidation of cysteine, cysteinesulfinate and cysteinesulfonate labeled with 14C in the 1- and 3-positions was studied in rats that had been fed diets with adequate or excess cysteine. Consumption of excess cysteine for 5 or 10 days resulted in an increase in hepatic cysteine dioxygenase activity and a decrease in hepatic cysteinesulfinate decarboxylase activity but had no effect on the oxidation of the C-1 or C-3 of cysteine, cysteinesulfinate or cysteinesulfonate. When the labeled compounds were administered by intraperitoneal injection, 41% of cysteine, 100% of cysteinesulfinate and 37% of cysteinesulfonate were oxidized over an 8-hour period. The percentage of the oxidized cysteine, cysteinesulfinate and cysteinesulfonate that was converted to taurine was calculated to be 83, 70 and 100%, respectively. When these same compounds were administered intragastrically, the relative flux to taurine was lower for all compounds; 41% of the oxidized cysteine, none of the cysteinesulfinate and 11% of the oxidized cysteinesulfonate appeared to be converted to taurine. Metabolism of intragastrically administered cysteine may be more indicative of what happens to dietary cysteine, whereas metabolism of intraperitoneally administered cysteine and cysteinesulfinate may be more indicative of liver metabolism and of the metabolism of endogenous cysteine and cysteinesulfinate.

  16. Specific detection of cysteine and homocysteine: recognizing one-methylene difference using fluorosurfactant-capped gold nanoparticles.

    PubMed

    Lu, Chao; Zu, Yanbing

    2007-10-07

    Aggregation of fluorosurfactant-capped gold nanoparticles could be induced selectively by cysteine and homocysteine and, when solution ionic strength was low, the kinetics of homocysteine-induced aggregation of large size nanoparticles (approximately 40 nm) was much faster than that induced by cysteine, leading to specific detection of homocysteine in the presence of excess cysteine.

  17. The cysteine dioxgenase knockout mouse: altered cysteine metabolism in nonhepatic tissues leads to excess H2S/HS(-) production and evidence of pancreatic and lung toxicity.

    PubMed

    Roman, Heather B; Hirschberger, Lawrence L; Krijt, Jakub; Valli, Alessandro; Kožich, Viktor; Stipanuk, Martha H

    2013-10-20

    To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS(-) (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO(-/-) mice that were fed either a taurine-free or taurine-supplemented diet. CDO(-/-) mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO(-/-) mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS(-). Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthionine in pancreas of CDO(-/-) mice were observed, suggesting a unique cysteine metabolism in the pancreas. The CDO(-/-) mouse model provides new insights into tissue-specific cysteine metabolism, particularly the role of pancreas in metabolism of excess cysteine by CBS-catalyzed reactions, and will be a useful model for studying the effects of excess endogenous production of H2S/HS(-). The CDO(-/-) mouse clearly demonstrates that H2S/HS(-) production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS(-)production than are liver and kidney.

  18. Knockout of the murine cysteine dioxygenase gene results in severe impairment in ability to synthesize taurine and an increased catabolism of cysteine to hydrogen sulfide

    PubMed Central

    Ueki, Iori; Roman, Heather B.; Valli, Alessandro; Fieselmann, Krista; Lam, Jimmy; Peters, Rachel; Hirschberger, Lawrence L.

    2011-01-01

    Cysteine homeostasis is dependent on the regulation of cysteine dioxygenase (CDO) in response to changes in sulfur amino acid intake. CDO oxidizes cysteine to cysteinesulfinate, which is further metabolized to either taurine or to pyruvate plus sulfate. To gain insight into the physiological function of CDO and the consequence of a loss of CDO activity, mice carrying a null CDO allele (CDO+/− mice) were crossed to generate CDO−/−, CDO+/−, and CDO+/+ mice. CDO−/− mice exhibited postnatal mortality, growth deficit, and connective tissue pathology. CDO−/− mice had extremely low taurine levels and somewhat elevated cysteine levels, consistent with the lack of flux through CDO-dependent catabolic pathways. However, plasma sulfate levels were slightly higher in CDO−/− mice than in CDO+/− or CDO+/+ mice, and tissue levels of acid-labile sulfide were elevated, indicating an increase in cysteine catabolism by cysteine desulfhydration pathways. Null mice had lower hepatic cytochrome c oxidase levels, suggesting impaired electron transport capacity. Supplementation of mice with taurine improved survival of male pups but otherwise had little effect on the phenotype of the CDO−/− mice. H2S has been identified as an important gaseous signaling molecule as well as a toxicant, and pathology may be due to dysregulation of H2S production. Control of cysteine levels by regulation of CDO may be necessary to maintain low H2S/sulfane sulfur levels and facilitate the use of H2S as a signaling molecule. PMID:21693692

  19. The Cysteine Dioxgenase Knockout Mouse: Altered Cysteine Metabolism in Nonhepatic Tissues Leads to Excess H2S/HS− Production and Evidence of Pancreatic and Lung Toxicity

    PubMed Central

    Roman, Heather B.; Hirschberger, Lawrence L.; Krijt, Jakub; Valli, Alessandro; Kožich, Viktor

    2013-01-01

    Abstract Aims: To define the consequences of loss of cysteine dioxygenase (CDO) on cysteine metabolism at the tissue level, we determined levels of relevant metabolites and enzymes and evidence of H2S/HS− (gaseous hydrogen sulfide and its conjugate base) toxicity in liver, pancreas, kidney, and lung of CDO−/− mice that were fed either a taurine-free or taurine-supplemented diet. Results: CDO−/− mice had low tissue and serum taurine and hypotaurine levels and high tissue levels of cysteine, consistent with the loss of CDO. CDO−/− mice had elevated urinary excretion of thiosulfate, high tissue and serum cystathionine and lanthionine levels, and evidence of inhibition and destabilization of cytochrome c oxidase, which is consistent with excess production of H2S/HS−. Accumulation of cystathionine and lanthionine appeared to result from cystathionine β-synthase (CBS)-mediated cysteine desulfhydration. Very high levels of hypotaurine in pancreas of wild-type mice and very high levels of cystathionine and lanthionine in pancreas of CDO−/− mice were observed, suggesting a unique cysteine metabolism in the pancreas. Innovation: The CDO−/− mouse model provides new insights into tissue-specific cysteine metabolism, particularly the role of pancreas in metabolism of excess cysteine by CBS-catalyzed reactions, and will be a useful model for studying the effects of excess endogenous production of H2S/HS−. Conclusion: The CDO−/− mouse clearly demonstrates that H2S/HS− production in tissues can exceed the capacity of the animal to oxidize sulfide to sulfate and demonstrates that pancreas and lung are more susceptible to toxicity from endogenous H2S/HS−production than are liver and kidney. Antioxid. Redox Signal. 19, 1321–1336. PMID:23350603

  20. Influence du traitement thermique sur l'usinabilite du laiton monophase

    NASA Astrophysics Data System (ADS)

    Cholley, Airy

    La connaissance des proprietes et du comportement d'un materiau en usinage est primordiale pour optimiser son utilisation et obtenir une usinabilite maximale. Dans ce but, l'etude de la modification de la ductilite par traitement thermiques sur plusieurs criteres d'usinabilite tels que les efforts de coupe, la rugosite, les bavures et la formation du copeau a ete menee sur le laiton monophase. A cette fin, l'influence du traitement thermique sur la microstructure du laiton a d'abord ete etudiee. La taille des grains et la durete ont permis de determiner les proprietes mecaniques des etats metallurgiques. Des essais de percage ont ensuite ete effectues sur les etats metallurgiques H01 (99HV), OS100 (88HV) et OS250 (47HV) pour regarder l'influence du traitement thermique sur l'usinabilite. Cette etude experimentale a permis de comprendre l'influence du taux de laminage et de la temperature de recuit sur les proprietes mecaniques. Les essais d'usinabilite ont ensuite permis de prouver que les efforts de coupe sont dependants des conditions de coupe mais pas de la ductilite dans les etats metallurgiques testes. La taille des bavures augmente avec la ductilite et la vitesse de coupe, et diminue avec l'avance. La rugosite de la surface des trous apres l'usinage a egalement ete etudiee. Il a aussi ete prouve par une etude sur les copeaux que la temperature croit avec la vitesse de coupe. L'analyse de la segmentation des copeaux n'a en revanche pas permis de trouver une correlation significative avec les traitements thermiques testes. Enfin, le revetement de l'outil a montre une grande importance sur l'usinabilite du materiau. Il a ete conclu que l'usinabilite du laiton C26000 (CuZn30) est meilleure lorsqu'on travaille dans un etat metallurgique dur. Ces conclusions sont valables pour le laiton monophase etudie, il serait interessant d'examiner aussi le cas des laitons biphases.

  1. Role of Saccharomyces cerevisiae serine O-acetyltransferase in cysteine biosynthesis.

    PubMed

    Takagi, Hiroshi; Yoshioka, Kenji; Awano, Naoki; Nakamori, Shigeru; Ono, Bun ichiro

    2003-01-28

    Some strains of Saccharomyces cerevisiae have detectable activities of L-serine O-acetyltransferase (SATase) and O-acetyl-L-serine/O-acetyl-L-homoserine sulfhydrylase (OAS/OAH-SHLase), but synthesize L-cysteine exclusively via cystathionine by cystathionine beta-synthase and cystathionine gamma-lyase. To untangle this peculiar feature in sulfur metabolism, we introduced Escherichia coli genes encoding SATase and OAS-SHLase into S. cerevisiae L-cysteine auxotrophs. While the cells expressing SATase grew on medium lacking L-cysteine, those expressing OAS-SHLase did not grow at all. The cells expressing both enzymes grew very well without L-cysteine. These results indicate that S. cerevisiae SATase cannot support L-cysteine biosynthesis and that S. cerevisiae OAS/OAH-SHLase produces L-cysteine if enough OAS is provided by E. coli SATase. It appears as if S. cerevisiae SATase does not possess a metabolic role in vivo either because of very low activity or localization. For example, S. cerevisiae SATase may be localized in the nucleus, thus controlling the level of OAS required for regulation of sulfate assimilation, but playing no role in the direct synthesis of L-cysteine.

  2. Development of Cysteine-Free Fluorescent Proteins for the Oxidative Environment

    PubMed Central

    Suzuki, Takahisa; Arai, Seisuke; Takeuchi, Mayumi; Sakurai, Chiye; Ebana, Hideaki; Higashi, Tsunehito; Hashimoto, Hitoshi; Hatsuzawa, Kiyotaka; Wada, Ikuo

    2012-01-01

    Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology. PMID:22649538

  3. Development of cysteine-free fluorescent proteins for the oxidative environment.

    PubMed

    Suzuki, Takahisa; Arai, Seisuke; Takeuchi, Mayumi; Sakurai, Chiye; Ebana, Hideaki; Higashi, Tsunehito; Hashimoto, Hitoshi; Hatsuzawa, Kiyotaka; Wada, Ikuo

    2012-01-01

    Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.

  4. Hierarchical effect behind the supramolecular chirality of silver(I)-cysteine coordination polymers.

    PubMed

    Randazzo, Rosalba; Di Mauro, Alessandro; D'Urso, Alessandro; Messina, Gabriele C; Compagnini, Giuseppe; Villari, Valentina; Micali, Norberto; Purrello, Roberto; Fragalà, Maria Elena

    2015-04-09

    Cysteine is a sulfur-containing amino acid that easily coordinates to soft metal ions and grafts to noble metal surfaces. Recently, chiroptical activity of Ag(+)/cysteine coordination polymers has been widely studied, while, on the other hand, the appearance of a plasmon-enhanced circular dichroic signal (PECD) at the plasmonic spectral region (λ > 400 nm) has been observed for AgNPs capped with chiral sulfur-containing amino acids. These two events are both potentially exploited for sensing applications. However, the presence of Ag(+) ions in AgNP colloidal solution deals with the competition of cysteine grafting at the metal NP surface and/or metal ion coordination. Herein we demonstrate that the chiroptical activity observed by adding cysteine to AgNP colloids prepared by pulsed laser ablation in liquids (PLAL) is mainly related to the formation of CD-active Ag(+)/cysteine supramolecular polymers. The strict correlation between supramolecular chirality and hierarchical effects, driven by different chemical environments experienced by cysteine when different titration modalities are used, is pivotal to validate cysteine as a fast and reliable probe to characterize the surface oxidation of AgNPs prepared by pulsed laser ablation in liquids by varying the laser wavelengths.

  5. Targeting Non-Catalytic Cysteine Residues Through Structure-Guided Drug Discovery.

    PubMed

    Hallenbeck, Kenneth K; Turner, David M; Renslo, Adam R; Arkin, Michelle R

    2017-01-01

    The targeting of non-catalytic cysteine residues with small molecules is drawing increased attention from drug discovery scientists and chemical biologists. From a biological perspective, genomic and proteomic studies have revealed the presence of cysteine mutations in several oncogenic proteins, suggesting both a functional role for these residues and also a strategy for targeting them in an 'allele specific' manner. For the medicinal chemist, the structure-guided design of cysteine- reactive molecules is an appealing strategy to realize improved selectivity and pharmacodynamic properties in drug leads. Finally, for chemical biologists, the modification of cysteine residues provides a unique means to probe protein structure and allosteric regulation. Here, we review three applications of cysteinemodifying small molecules: 1) the optimization of existing drug leads, 2) the discovery of new lead compounds, and 3) the use of cysteine-reactive molecules as probes of protein dynamics. In each case, structure-guided design plays a key role in determining which cysteine residue(s) to target and in designing compounds with the proper geometry to enable both covalent interaction with the targeted cysteine and productive non-covalent interactions with nearby protein residues.

  6. Cysteine protects rabbit spermatozoa against reactive oxygen species-induced damages

    PubMed Central

    Fan, Xiaoteng; Pan, Yang; Lv, Shan; Pan, Chuanying; Lei, Anmin

    2017-01-01

    The process of cryopreservation results in over-production of reactive oxygen species, which is extremely detrimental to spermatozoa. The aim of this study was to investigate whether addition of cysteine to freezing extender would facilitate the cryosurvival of rabbit spermatozoa, and if so, how cysteine protects spermatozoa from cryodamages. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of cysteine. The motility, intact acrosomes, membrane integrity, mitochondrial potentials, 8-hydroxyguanosine level and sperm-zona pellucida binding capacity were examined. Furthermore, glutathione peroxidase (GPx) activity, glutathione content (GSH), and level of reactive oxygen species (ROS) and hydrogen peroxide of spermatozoa were analyzed. The values of motility, intact acrosomes, membrane integrity, mitochondrial potentials and sperm-zona pellucida binding capacity of the frozen-thawed spermatozoa in the treatment of cysteine were significantly higher than those of the control. Addition of cysteine to extenders improved the GPx activity and GSH content of spermatozoa, while lowered the ROS, DNA oxidative alterations and lipid peroxidation level, which makes spermatozoa avoid ROS to attack DNA, the plasma membrane and mitochondria. In conclusion, cysteine protects spermatozoa against ROS-induced damages during cryopreservation and post-thaw incubation. Addition of cysteine is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry. PMID:28700739

  7. Positional isotope exchange analysis of the Mycobacterium smegmatis cysteine ligase (MshC).

    PubMed

    Williams, LaKenya; Fan, Fan; Blanchard, John S; Raushel, Frank M

    2008-04-22

    MshC catalyzes the ATP-dependent condensation of GlcN-Ins and cysteine to form Cys-GlcN-Ins, which is an intermediate in the biosynthetic pathway of mycothiol, i.e., 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH or AcCys-GlcN-Ins). MSH is produced by Mycobacterium tuberculosis, members of the Actinomycetes family, to maintain an intracellular reducing environment and protect against oxidative and antibiotic induced stress. The biosynthesis of MSH is essential for cell growth, and therefore, the MSH biosynthetic enzymes present potential targets for inhibitor design. The formation of kinetically competent adenylated intermediates was suggested by the observation of positional isotope exchange (PIX) reaction using [betagamma-(18)O6]-ATP in the presence of cysteine. The PIX rate depends on the presence of cysteine and increases with concentrations of cysteine. The loss of PIX activity upon the addition of small concentrations of pyrophosphatase suggests that the PP(i) is free to dissociate from the active site of cysteine ligase into the bulk solution. The PIX activity is also eliminated at high concentrations of GlcN-Ins, consistent with the mechanism in which GlcN-Ins binds after cysteine-adenylate formation. This PIX analysis confirms that MshC catalyzes the formation of a kinetically competent cysteinyl-adenylate intermediate after the addition of ATP and cysteine.

  8. Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump.

    PubMed

    Grumbt, Maria; Monod, Michel; Yamada, Tsuyoshi; Hertweck, Christian; Kunert, Jiri; Staib, Peter

    2013-06-01

    Millions of people suffer from superficial infections caused by dermatophytes. Intriguingly, these filamentous fungi exclusively infect keratin-rich host structures such as hair, nails, and skin. Keratin is a hard, compact protein, and its utilization by dermatophytes for growth has long been discussed as a major virulence attribute. Here, we provide strong support for the hypothesis that keratin degradation is facilitated by the secretion of the reducing agent sulfite, which can cleave keratin-stabilizing cystine bonds. We discovered that sulfite is produced by dermatophytes from environmental cysteine, which at elevated concentrations is toxic for microbes and humans. We found that sulfite formation from cysteine relies on the key enzyme cysteine dioxygenase Cdo1. Sulfite secretion is supported by the sulfite efflux pump Ssu1. Targeted mutagenesis proved that dermatophyte mutants in either Cdo1 or Ssu1 were highly growth-sensitive to cysteine, and mutants in Ssu1 were specifically sensitive to sulfite. Most notably, dermatophyte mutants in Cdo1 and Ssu1 were specifically growth-defective on hair and nails. As keratin is rich in cysteine, our identified mechanism of cysteine conversion and sulfite efflux supports both cysteine and sulfite tolerance per se and progression of keratin degradation. These in vitro findings have implications for dermatophyte infection pathogenesis.

  9. Tying everything together: the multiple roles of cysteine string protein (CSP) in regulated exocytosis.

    PubMed

    Evans, Gareth J O; Morgan, Alan; Burgoyne, Robert D

    2003-10-01

    In addition to the core vesicle fusion machinery, the SNARE proteins, a large number of regulatory proteins have been implicated in the process of Ca2+-dependent exocytosis. How these exocytotic proteins are properly targeted and how their myriad interactions are temporally and spatially coordinated is poorly understood. Cysteine string protein (CSP), a secretory vesicle membrane protein and a member of the dnaJ family of co-chaperones, may assist in performing this function. Through its interaction with the ubiquitous chaperone, Hsc70, it is thought that cysteine string protein targets chaperone complexes to the exocytotic machinery to facilitate the correct folding of polypeptides or to regulate the assembly of protein complexes. Since its discovery, there have been conflicting reports from different systems concerned with whether cysteine string protein exerts its effects on exocytosis either up- or down-stream of Ca2+-influx. In this review, we summarize recent experiments that associate cysteine string protein with the regulation of vesicle filling, vesicle docking, Ca2+-channels and the SNARE proteins themselves, hence supporting a role for cysteine string protein as a multifunctional secretory co-chaperone. In addition, we provide an update on the mammalian isoforms of cysteine string protein following the recent discovery of two novel cysteine string proteins.

  10. Acetaldehyde removal from indoor air through chemical absorption using L-cysteine.

    PubMed

    Yamashita, Kyoko; Noguchi, Miyuki; Mizukoshi, Atsushi; Yanagisawa, Yukio

    2010-09-01

    The irreversible removal of acetaldehyde from indoor air via a chemical reaction with amino acids was investigated. To compare effectiveness, five types of amino acid (glycine, l-lysine, l-methionine, l-cysteine, and l-cystine) were used as the reactants. First, acetaldehyde-laden air was introduced into aqueous solutions of each amino acid and the removal abilities were compared. Among the five amino acids, l-cysteine solution showed much higher removal efficiency, while the other amino acids solutions didn't show any significant differences from the removal efficiency of water used as a control. Next, as a test of the removal abilities of acetaldehyde by semi-solid l-cysteine, a gel containing l-cysteine solution was put in a fluororesin bag filled with acetaldehyde gas, and the change of acetaldehyde concentration was measured. The l-cysteine-containing gel removed 80% of the acetaldehyde in the air within 24 hours. The removal ability likely depended on the unique reaction whereby acetaldehyde and l-cysteine rapidly produce 2-methylthiazolidine-4-carboxylic acid. These results suggested that the reaction between acetaldehyde and l-cysteine has possibilities for irreversibly removing toxic acetaldehyde from indoor air.

  11. Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein.

    PubMed

    Camporeale, Gabriela; Lorenzo, Juan R; Thomas, Maria G; Salvatierra, Edgardo; Borkosky, Silvia S; Risso, Marikena G; Sánchez, Ignacio E; de Prat Gay, Gonzalo; Alonso, Leonardo G

    2017-04-01

    Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn(+2) binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism.

  12. Acetaminophen-induced liver injury is attenuated in male glutamate-cysteine ligase transgenic mice.

    PubMed

    Botta, Dianne; Shi, Shengli; White, Collin C; Dabrowski, Michael J; Keener, Cassie L; Srinouanprachanh, Sengkeo L; Farin, Federico M; Ware, Carol B; Ladiges, Warren C; Pierce, Robert H; Fausto, Nelson; Kavanagh, Terrance J

    2006-09-29

    Acetaminophen overdose is a leading cause of drug-related acute liver failure in the United States. Glutathione, a tripeptide antioxidant protects cells against oxidative damage from reactive oxygen species and plays a crucial role in the detoxification of xenobiotics, including acetaminophen. Glutathione is synthesized in a two-step enzymatic reaction. Glutamate-cysteine ligase carries out the rate-limiting and first step in glutathione synthesis. We have generated C57Bl/6 mice that conditionally overexpress glutamate-cysteine ligase, and report here their resistance to acetaminophen-induced liver injury. Indices of liver injury included histopathology and serum alanine aminotransferase activity. Male transgenic mice induced to overexpress glutamate-cysteine ligase exhibited resistance to acetaminophen-induced liver injury when compared with acetaminophen-treated male mice carrying, but not expressing glutamate-cysteine ligase transgenes, or to female glutamate-cysteine ligase transgenic mice. We conclude that glutamate-cysteine ligase activity is an important factor in determining acetaminophen-induced liver injury in C57Bl/6 male mice. Because people are known to vary in their glutamate-cysteine ligase activity, this enzyme may also be an important determinant of sensitivity to acetaminophen-induced liver injury in humans.

  13. Molecular Biology, Biochemistry and Cellular Physiology of Cysteine Metabolism in Arabidopsis thaliana

    PubMed Central

    Hell, Rüdiger; Wirtz, Markus

    2011-01-01

    Cysteine is one of the most versatile molecules in biology, taking over such different functions as catalysis, structure, regulation and electron transport during evolution. Research on Arabidopsis has contributed decisively to the understanding of cysteine synthesis and its role in the assimilatory pathways of S, N and C in plants. The multimeric cysteine synthase complex is present in the cytosol, plastids and mitochondria and forms the centre of a unique metabolic sensing and signaling system. Its association is reversible, rendering the first enzyme of cysteine synthesis active and the second one inactive, and vice-versa. Complex formation is triggered by the reaction intermediates of cysteine synthesis in response to supply and demand and gives rise to regulation of genes of sulfur metabolism to adjust cellular sulfur homeostasis. Combinations of biochemistry, forward and reverse genetics, structural- and cell-biology approaches using Arabidopsis have revealed new enzyme functions and the unique pattern of spatial distribution of cysteine metabolism in plant cells. These findings place the synthesis of cysteine in the centre of the network of primary metabolism. PMID:22303278

  14. The mechanism of cysteine detection in biological media by means of vanadium oxide nanoparticles

    NASA Astrophysics Data System (ADS)

    Bezerra, A. G.; Barison, A.; Oliveira, V. S.; Foti, L.; Krieger, M. A.; Dhalia, R.; Viana, I. F. T.; Schreiner, W. H.

    2012-09-01

    We report on the interaction of vanadate nanoparticles, produced using the laser ablation in liquids synthesis, with cysteine in biological molecules. Cysteine is a very important amino acid present in most proteins, but also because cysteine and the tripeptide glutathione are the main antioxidant molecules in our body system. Detailed UV-Vis absorption spectra and dynamic light scattering measurements were done to investigate the detection of cysteine in large biological molecules. The intervalence band of the optical absorption spectra shows capability for quantitative cysteine sensing in the μM range in biological macromolecules. Tests included cytoplasmic repetitive antigen and flagellar repetitive antigen proteins of the Trypanosoma cruzi protozoa, as well as the capsid p24 proteins from Human Immunodeficiency Virus type 1 and type 2. Detailed NMR measurements for hydrogen, carbon, and vanadium nuclei show that cysteine in contact with the vanadate looses hydrogen of the sulphydryl side chain, while the vanadate is reduced. The subsequent detachment of two deprotonated molecules to form cystine and the slow return to the vanadate complete the oxidation-reduction cycle. Therefore, the vanadate acts as a charge exchanging catalyst on cysteine to form cystine. The NMR results also indicate that the nanoparticles are not formed by the common orthorhombic V2O5 form.

  15. Isd11p Protein Activates the Mitochondrial Cysteine Desulfurase Nfs1p Protein*

    PubMed Central

    Pandey, Alok; Yoon, Heeyong; Lyver, Elise R.; Dancis, Andrew; Pain, Debkumar

    2011-01-01

    Cysteine desulfurases perform pyridoxal phosphate (PLP)-dependent desulfuration of cysteine. The key steps of the enzymatic cycle include substrate binding to PLP, formation of a covalent persulfide intermediate at the active site cysteine, and transfer of sulfur to recipients for use in various metabolic pathways. In Saccharomyces cerevisiae, the cysteine desulfurase Nfs1p and an accessory protein, Isd11p, are found primarily in mitochondria, and both are essential for cell viability. Although cysteine desulfurases are conserved from bacteria to humans, Isd11p is found only in eukaryotes and not in prokaryotes. Here we show that Isd11p activates Nfs1p. The enzyme without Isd11p was inactive and did not form the [35S]persulfide intermediate from the substrate [35S]cysteine. Addition of Isd11p to inactive Nfs1p induced formation of the persulfide. Remarkably, in a two-step assay, [35S]cysteine could be bound to the inactive Nfs1p in a PLP-dependent manner, and the enzyme could be subsequently induced to form the persulfide by addition of Isd11p. A mutant form of Isd11p with the 15LYK17 motif changed to 15AAA17 was able to bind but failed to activate Nfs1p, thus separating these two functions of Isd11p. Finally, compared with Nfs1p with or without the bound Isd11p mutant, the Nfs1p·Isd11p complex was more resistant to inactivation by an alkylating agent. On the basis of these novel findings, we propose that interaction of Isd11p with Nfs1p activates the enzyme by inducing a conformational change, thereby promoting formation of the persulfide intermediate at the active site cysteine. Such a conformational change may protect the active site cysteine from alkylating agents. PMID:21908622

  16. l-cysteine suppresses ghrelin and reduces appetite in rodents and humans

    PubMed Central

    McGavigan, A K; O'Hara, H C; Amin, A; Kinsey-Jones, J; Spreckley, E; Alamshah, A; Agahi, A; Banks, K; France, R; Hyberg, G; Wong, C; Bewick, G A; Gardiner, J V; Lehmann, A; Martin, N M; Ghatei, M A; Bloom, S R; Murphy, K G

    2015-01-01

    Background: High-protein diets promote weight loss and subsequent weight maintenance, but are difficult to adhere to. The mechanisms by which protein exerts these effects remain unclear. However, the amino acids produced by protein digestion may have a role in driving protein-induced satiety. Methods: We tested the effects of a range of amino acids on food intake in rodents and identified l-cysteine as the most anorexigenic. Using rodents we further studied the effect of l-cysteine on food intake, behaviour and energy expenditure. We proceeded to investigate its effect on neuronal activation in the hypothalamus and brainstem before investigating its effect on gastric emptying and gut hormone release. The effect of l-cysteine on appetite scores and gut hormone release was then investigated in humans. Results: l-Cysteine dose-dependently decreased food intake in both rats and mice following oral gavage and intraperitoneal administration. This effect did not appear to be secondary to behavioural or aversive side effects. l-Cysteine increased neuronal activation in the area postrema and delayed gastric emptying. It suppressed plasma acyl ghrelin levels and did not reduce food intake in transgenic ghrelin-overexpressing mice. Repeated l-cysteine administration decreased food intake in rats and obese mice. l-Cysteine reduced hunger and plasma acyl ghrelin levels in humans. Conclusions: Further work is required to determine the chronic effect of l-cysteine in rodents and humans on appetite and body weight, and whether l-cysteine contributes towards protein-induced satiety. PMID:25219528

  17. Diversity, abundance and characterization of ruminal cysteine phytases suggest their important role in phytate degradation.

    PubMed

    Huang, Huoqing; Zhang, Rui; Fu, Dawei; Luo, Jianjie; Li, Zhongyuan; Luo, Huiying; Shi, Pengjun; Yang, Peilong; Diao, Qiyu; Yao, Bin

    2011-03-01

    A novel class of cysteine phytase showing ability to degrade phytate has recently been isolated from rumen bacteria. To expand our knowledge of this enzyme class, a total of 101 distinct cysteine phytase gene fragments were identified from the ruminal genomic DNA of Bore goats and Holstein cows, and most of them shared low identities (< 50%) with known sequences. By phylogenetic analysis, these sequences were separated into three clusters that showed substantial diversity. The two most abundant cysteine phytase genes of goat rumens were cloned and their protein products were characterized. Four findings were revealed based on our results. (i) Compared with soil and water environment, where β-propeller phytase is the most important phytate-degrading enzyme, cysteine phytase is the major phytate-degrading enzyme in the anaerobic ruminal environment. (ii) Cysteine phytase fragments in the rumen contents of goat and cow have the same diversity profile, although most of the sequences and their abundance differ in the two species. (iii) Each species has their respective high-abundance genes, which may play major roles for phytate degradation. (iv) Compared with previously reported cysteine phytases that have pH optimum at 4.5, the pH optima of the two most abundant secreted goat cysteine phytases are 6.5 and 6.0, which are within the pH range found in the rumens. This study provides valuable information about the diversity, abundance and enzymatic properties of the ruminal cysteine phytases and emphasizes the important role(s) of these cysteine phytases probably in the terrestrial cycle of phosphorus.

  18. Deficiency of filaggrin regulates endogenous cysteine protease activity, leading to impaired skin barrier function.

    PubMed

    Wang, X W; Wang, J J; Gutowska-Owsiak, D; Salimi, M; Selvakumar, T A; Gwela, A; Chen, L Y; Wang, Y J; Giannoulatou, E; Ogg, G

    2017-08-01

    Atopic dermatitis (AD) is a common inflammatory skin disorder, characterized by skin barrier defects and enhanced allergen priming. Null mutations in the filaggrin gene (FLG) are strongly associated with moderate to severe AD, but the pathways linking barrier dysfunction and cutaneous inflammation are still largely unknown. To assess alteration of endogenous cysteine protease activity in FLG-deficient keratinocytes, and to determine whether the alteration in cysteine protease activity affects epidermal barrier function and associated gene and protein expression. We established a stable FLG knockdown cell line, and reconstructed epidermal equivalents in vitro. Barrier function of the reconstructed epidermis, the barrier-associated genes and proteins, and the activity of endogenous cysteine proteases were tested. Inhibitors of cysteine proteases were used to further evaluate the role of endogenous cysteine proteases in epidermal barrier function. FLG knockdown induced impaired epidermal barrier function. Microarray, western blotting and fluorescence staining showed reduced expression of K10, ZO-1, E-cadherin, claudin-1 and occludin in FLG knockdown keratinocytes. Compared with cysteine protease activity in control cells, protease activity was dramatically enhanced in FLG knockdown keratinocytes. Furthermore, administration of cysteine protease inhibitors significantly recovered expression of K10 and tight junction proteins, and the barrier defect induced by FLG deficiency. This is the first observation of elevated endogenous cysteine protease activity in FLG-deficient keratinocytes, which may play an important role in impaired barrier function in AD skin. Modulation of cysteine protease activity might be a novel therapeutic approach for AD treatment. © 2017 British Association of Dermatologists.

  19. A process for the preparation of cysteine from cystine

    DOEpatents

    Chang, Shih-Ger; Liu, David K.; Griffiths, Elizabeth A.; Littlejohn, David

    1989-01-01

    The present invention in one aspect relates to a process for the simultaneous removal of NO.sub.x and SO.sub.2 from a fluid stream comprising mixtures thereof and in another aspect relates to the separation, use and/or regeneration of various chemicals contaminated or spent in the process and which includes the steps of: (A) contacting the fluid stream at a temperature of between about 105.degree. and 180.degree. C. with a liquid aqueous slurry or solution comprising an effective amount of an iron chelate of an amino acid moiety having at least one --SH group; (B) separating the fluid stream from the particulates formed in step (A) comprising the chelate of the amino acid moiety and fly ash; (C) washing and separating the particulates of step (B) with an aqeous solution having a pH value of between about 5 to 8; (D) subsequently washing and separating the particulates of step (C) with a strongly acidic aqueous solution having a pH value of between about 1 to 3; (E) washing and separating the particulates of step (D) with an basic aqueous solution having a pH value of between about 9 to 12; (F) optionally adding additional amino acid moiety, iron (II) and alkali to the aqueous liquid from step (D) to produce an aqueous solution or slurry similar to that in step (A) having a pH value of between about 4 to 12; and (G) recycling the aqueous slurry of step (F) to the contacting zone of step (A). Steps (D) and (E) can be carried out in the reverse sequence, however the preferred order is (D) and then (E). In a preferred embodiment the present invention provides an improved process for the preparation (regeneration) of cysteine from cystine, which includes reacting an aqueous solution of cystine at a pH of between about 9 to 13 with a reducing agent selected from hydrogen sulfide or alkali metal sulfides, sulfur dioxide, an alkali metal sulfite or mixtures thereof for a time and at a temperature effective to cleave and reduce the cystine to cysteine with subsequent

  20. Evidence that d-cysteine protects mice from gastric damage via hydrogen sulfide produced by d-amino acid oxidase.

    PubMed

    Souza, Luan Kelves M; Araújo, Thiago S L; Sousa, Nayara A; Sousa, Francisca Beatriz M; Nogueira, Kerolayne M; Nicolau, Lucas A D; Medeiros, Jand Venes R

    2017-04-01

    Hydrogen sulfide (H2S) is a signaling molecule in the gastrointestinal tract. H2S production can derive from d-cysteine via various pathways, thus pointing to a new therapeutic approach: delivery of H2S to specific tissues. This study was designed to evaluate the concentration and effects of H2S (generated by d-amino acid oxidase [DAO] from d-cysteine) in the gastric mucosa and the protective effects against ethanol-induced lesions in mice. Mice were treated with l-cysteine or d-cysteine (100 mg/kg per os). Other groups received oral l-propargylglycine (cystathionine γ-lyase inhibitor, 100 mg/kg) or indole-2-carboxylate (DAO inhibitor), and 30 min later, received d- or l-cysteine. After 30 min, 50% ethanol (2.5 mL/kg, per os) was administered. After 1 h, the mice were euthanized and their stomachs excised and analyzed. Pretreatment with either l-cysteine or d-cysteine significantly reduced ethanol-induced lesions. Pretreatment of d-cysteine- or l-cysteine-treated groups with indole-2-carboxylate reversed the gastroprotective effects of d-cysteine but not l-cysteine. Histological analysis revealed that pretreatment with d-cysteine decreased hemorrhagic damage, edema, and the loss of the epithelium, whereas the administration of indole-2-carboxylate reversed these effects. d-Cysteine also reduced malondialdehyde levels but maintained the levels of reduced glutathione. Furthermore, pretreatment with d-cysteine increased the synthesis of H2S. Thus, an H2S-generating pathway (involving d-cysteine and DAO) is present in the gastric mucosa and protects this tissue from ethanol-induced damage by decreasing direct oxidative damage.

  1. Chikungunya nsP2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine.

    PubMed

    Saisawang, Chonticha; Saitornuang, Sawanan; Sillapee, Pornpan; Ubol, Sukathida; Smith, Duncan R; Ketterman, Albert J

    2015-11-24

    Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target.

  2. Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide.

    PubMed

    Haag, Andreas F; Kerscher, Bernhard; Dall'Angelo, Sergio; Sani, Monica; Longhi, Renato; Baloban, Mikhail; Wilson, Heather M; Mergaert, Peter; Zanda, Matteo; Ferguson, Gail P

    2012-03-30

    The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide (S-S) bridges. However, the natural configuration of NCR AMP S-S bridges and the role of these in the activity of the peptide are unknown. In this study, we found that either cysteine replacements or S-S bond modifications influenced the activity of NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S-S bridges from cysteines 1-2, 3-4 to 1-3, 2-4 or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.

  3. Preparation, crystallization and X-ray diffraction analysis to 1.5 Å resolution of rat cysteine dioxygenase, a mononuclear iron enzyme responsible for cysteine thiol oxidation

    SciTech Connect

    Simmons, Chad R.; Hao, Quan; Stipanuk, Martha H.

    2005-11-01

    Recombinant rat cysteine dioxygenase (CDO) has been expressed, purified and crystallized and X-ray diffraction data have been collected to 1.5 Å resolution. Cysteine dioxygenase (CDO; EC 1.13.11.20) is an ∼23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O{sub 2}, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Å resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Å, α = β = γ = 90°. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  4. Chikungunya nsP2 protease is not a papain-like cysteine protease and the catalytic dyad cysteine is interchangeable with a proximal serine

    PubMed Central

    Saisawang, Chonticha; Saitornuang, Sawanan; Sillapee, Pornpan; Ubol, Sukathida; Smith, Duncan R.; Ketterman, Albert J.

    2015-01-01

    Chikungunya virus is the pathogenic alphavirus that causes chikungunya fever in humans. In the last decade millions of cases have been reported around the world from Africa to Asia to the Americas. The alphavirus nsP2 protein is multifunctional and is considered to be pivotal to viral replication, as the nsP2 protease activity is critical for proteolytic processing of the viral polyprotein during replication. Classically the alphavirus nsP2 protease is thought to be papain-like with the enzyme reaction proceeding through a cysteine/histidine catalytic dyad. We performed structure-function studies on the chikungunya nsP2 protease and show that the enzyme is not papain-like. Characterization of the catalytic dyad cysteine residue enabled us to identify a nearby serine that is catalytically interchangeable with the dyad cysteine residue. The enzyme retains activity upon alanine replacement of either residue but a replacement of both cysteine and serine residues results in no detectable activity. Protein dynamics appears to allow the use of either the cysteine or the serine residue in catalysis. This switchable dyad residue has not been previously reported for alphavirus nsP2 proteases and would have a major impact on the nsP2 protease as an anti-viral target. PMID:26597768

  5. Papillomavirus Assembly Requires Trimerization of the Major Capsid Protein by Disulfides between Two Highly Conserved Cysteines

    PubMed Central

    Sapp, Martin; Fligge, Claudia; Petzak, Ingrid; Harris, J. Robin; Streeck, Rolf E.

    1998-01-01

    We have used viruslike particles (VLPs) of human papillomaviruses to study the structure and assembly of the viral capsid. We demonstrate that mutation of either of two highly conserved cysteines of the major capsid protein L1 to serine completely prevents the assembly of VLPs but not of capsomers, whereas mutation of all other cysteines leaves VLP assembly unaffected. These two cysteines form intercapsomeric disulfides yielding an L1 trimer. Trimerization comprises about half of the L1 molecules in VLPs but all L1 molecules in complete virions. We suggest that trimerization of L1 is indispensable for the stabilization of intercapsomeric contacts in papillomavirus capsids. PMID:9621087

  6. Prediction of the disulfide-bonding state of cysteines in proteins at 88% accuracy

    PubMed Central

    Martelli, Pier Luigi; Fariselli, Piero; Malaguti, Luca; Casadio, Rita

    2002-01-01

    The task of predicting the cysteine-bonding state in proteins starting from the residue chain is addressed by implementing a new hybrid system that combines a neural network and a hidden Markov model (hidden neural network). Training is performed using 4136 cysteine-containing segments extracted from 969 nonhomologous proteins of well-resolved three-dimensional structure. After a 20-fold cross-validation procedure, the efficiency of the prediction scores as high as 88% and 84%, when measured on cysteine and protein basis, respectively. These results outperform previously described methods for the same task. PMID:12381855

  7. La reconstruction du sourcil par greffon composite du cuir chevelu: une astuce pour faciliter la technique

    PubMed Central

    El Omari, Mounia; El Mazouz, Samir; Gharib, Noureddine; EL Abbassi, Abdallah

    2015-01-01

    Les sourcils jouent un rôle important dans l’équilibre esthétique du visage. Leur reconstruction ou ophriopoïése, après séquelle de brûlure fait partie intégrante du programme de réhabilitation de la face brûlée. Plusieurs techniques ont été décrites. Nous insistons ici sur l'intérêt d'une technique simple, à la portée de tous les chirurgiens, et dont la méthode et les résultats peuvent être améliorés par un dessin bien planifié des zones donneuse et receveuse: la greffe composite prélevée au niveau du cuir chevelu dessinée à l'aide d'un calque du sourcil controlatéral. PMID:26401195

  8. Suivi après le traitement du cancer du sein

    PubMed Central

    Sisler, Jeffrey; Chaput, Geneviève; Sussman, Jonathan; Ozokwelu, Emmanuel

    2016-01-01

    Résumé Objectif Offrir aux médecins de famille un résumé des recommandations fondées sur les données probantes pour guider les soins aux survivantes traitées pour le cancer du sein. Qualité des données Une recherche documentaire a été effectuée dans MEDLINE entre 2000 et 2016 à l’aide des mots-clés anglais suivants : breast cancer, survivorship, follow-up care, aftercare, guidelines et survivorship care plans, en se concentrant sur la revue des lignes directrices publiées récemment par les organismes nationaux de cancérologie. Les données étaient de niveaux I à III. Message principal Les soins aux survivantes comportent 4 facettes : surveillance et dépistage, prise en charge des effets à long terme, promotion de la santé et coordination des soins. La surveillance des récidives ne se traduit que par une mammographie annuelle, et le dépistage d’autres cancers doit suivre les lignes directrices basées sur la population. La prise en charge des effets à long terme du cancer et de son traitement aborde des problèmes courants tels la douleur, la fatigue, le lymphœdème, la détresse et les effets indésirables des médicaments, de même que les préoccupations à long terme comme la santé du cœur et des os. La promotion de la santé met en relief les bienfaits de l’activité chez les survivantes du cancer, avec l’accent mis sur l’activité physique. Les soins aux survivantes sont de meilleure qualité lorsque divers services et professionnels de la santé participent aux soins, et le médecin de famille joue un rôle important dans la coordination des soins. Conclusion Les médecins de famille sont de plus en plus souvent les principaux fournisseurs de soins de suivi après le traitement du cancer du sein. Le cancer du sein doit être considéré comme une affection médicale chronique, même chez les femmes en rémission, et les patientes profitent de la même approche que celle utilisée pour les autres affections chroniques en

  9. Differential Expression of Cysteine Dioxygenase 1 in Complex Karyotype Liposarcomas

    PubMed Central

    Shaker, Mohammed; Pascarelli, Kara M; Plantinga, Matthew J; Love, Miles A; Lazar, Alexander J; Ingram, Davis R; von Mehren, Margaret; Lev, Dina; Kipling, David; Broccoli, Dominique

    2014-01-01

    Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P < 0.001). Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation. PMID:24741338

  10. Plasma total cysteine and cardiovascular risk burden: action and interaction.

    PubMed

    De Chiara, Benedetta; Sedda, Valentina; Parolini, Marina; Campolo, Jonica; De Maria, Renata; Caruso, Raffaele; Pizzi, Gianluigi; Disoteo, Olga; Dellanoce, Cinzia; Corno, Anna Rosa; Cighetti, Giuliana; Parodi, Oberdan

    2012-01-01

    We hypothesized that redox analysis could provide sensitive markers of the oxidative pathway associated to the presence of an increasing number of cardiovascular risk factors (RFs), independently of type. We classified 304 subjects without cardiovascular disease into 4 groups according to the total number of RFs (smoking, hypertension, hypercholesterolaemia, hyperhomocysteinaemia, diabetes, obesity, and their combination). Oxidative stress was evaluated by measuring plasma total and reduced homocysteine, cysteine (Cys), glutathione, cysteinylglycine, blood reduced glutathione, and malondialdehyde. Twenty-seven percent of subjects were in group 0 RF, 26% in 1 RF, 31% in 2 RF, and 16% in ≥ 3 RF. By multivariable ordinal regression analysis, plasma total Cys was associated to a higher number of RF (OR = 1.068; 95% CI = 1.027-1.110, P = 0.002). Total RF burden is associated with increased total Cys levels. These findings support a prooxidant effect of Cys in conjunction with RF burden, and shed light on the pathophysiologic role of redox state unbalance in preclinical atherosclerosis.

  11. Effect of N-acetyl cysteine on Helicobacter pylori.

    PubMed

    Gurbuz, Ahmet Kemal; Ozel, A Melih; Ozturk, Ramazan; Yildirim, Sukru; Yazgan, Yusuf; Demirturk, Levent

    2005-11-01

    Use of mucolytic agents that result in reduced mucous viscosity of the gastric mucous has been suggested to have an additive effect in curing Helicobacter pylori infection. Seventy Hpylori-positive patients were given either eradication treatment consisting of 500 mg clarithromycin bid and 30 mg lansoprazole bid for 10 days plus 10 mL (400 mg) N-acetyl cysteine (NAC) liquid tid (AC group) or eradication treatment only (control group). The results were compared 1 month after the completion of the treatment. Fifty-eight patients were available for statistical analysis. Of the 28 patients in the AC group, 14 (50.0%) eradicated the infection after treatment, whereas only 7 of 30 (23.3%) patients in the control group had negative results. The difference between the AC group and the control group was statistically significant (P = 0.034). In both groups, there was no difference in the number of smokers and in the eradication rates between smokers and nonsmokers. Eradication treatment with or without NAC caused no significant side effects in either group. Our findings suggest that NAC has an additive effect on the eradication rates of H pylori obtained with dual therapy with lansoprazole and clarithromycin. NAC does not have any known activity against H pylori, but it may improve the delivery of antibiotics at the site of infection due to its ability to reduce the thickness of the mucus.

  12. Extracellular Cysteine in Connexins: Role as Redox Sensors

    PubMed Central

    Retamal, Mauricio A.; García, Isaac E.; Pinto, Bernardo I.; Pupo, Amaury; Báez, David; Stehberg, Jimmy; Del Rio, Rodrigo; González, Carlos

    2016-01-01

    Connexin-based channels comprise hemichannels and gap junction channels. The opening of hemichannels allow for the flux of ions and molecules from the extracellular space into the cell and vice versa. Similarly, the opening of gap junction channels permits the diffusional exchange of ions and molecules between the cytoplasm and contacting cells. The controlled opening of hemichannels has been associated with several physiological cellular processes; thereby unregulated hemichannel activity may induce loss of cellular homeostasis and cell death. Hemichannel activity can be regulated through several mechanisms, such as phosphorylation, divalent cations and changes in membrane potential. Additionally, it was recently postulated that redox molecules could modify hemichannels properties in vitro. However, the molecular mechanism by which redox molecules interact with hemichannels is poorly understood. In this work, we discuss the current knowledge on connexin redox regulation and we propose the hypothesis that extracellular cysteines could be important for sensing changes in redox potential. Future studies on this topic will offer new insight into hemichannel function, thereby expanding the understanding of the contribution of hemichannels to disease progression. PMID:26858649

  13. Simulation of Gold Functionalization with Cysteine by Reactive Molecular Dynamics.

    PubMed

    Monti, Susanna; Carravetta, Vincenzo; Ågren, Hans

    2016-01-21

    The anchoring mechanism of cysteine to gold in water solution is characterized in detail by means of a combination of quantum chemistry (QC) and reactive classical molecular dynamics (RC-MD) calculations. A possible adsorption-reaction route is proposed, through RC-MD simulations based on a modified version of the protein reactive force field (ReaxFF), in which gold-protein interactions have been included after accurate parametrization at the QC level. The computational results confirm recent experimental findings regarding the mechanism as a two-step binding, namely, a slow physisorption followed by a fast chemisorption. The reaction barriers are estimated through the nudged elastic band approach and checked by QC calculations. Surface reconstructions, induced by the strong adsorption of the molecule, are identified, and their role, as further adsorbate stabilizers, is properly disclosed. The satisfactory agreement with QC data and experiments confirm the reliability of the simulations and the unique opportunity they provide to follow locally molecule adsorption on selected materials.

  14. N-acetyl cysteine therapy in acute viral hepatitis

    PubMed Central

    Gunduz, Huseyin; Karabay, Oguz; Tamer, Ali; Özaras, Resat; Mert, Ali; Tabak, Ömer Fehmi

    2003-01-01

    AIM: To investigate the effect of N-acetyl cysteine (NAC) on acute viral hepatitis (AVH). METHODS: We administered 200 mg oral NAC three times daily (600 mg/day) to the study group and placebo capsules to the control group. All patients were hospitalized and diagnosed as AVH. Blood total and direct bilirubin, ALT, AST, alkaline phosphatese, albumin and globulin levels of each patient were measured twice weekly until total bilirubin level dropped under 2 mg/dl, ALT level under 100 U/L, follow up was continued and then the patients were discharged. RESULTS: A total of 41(13 female and 28 male) AVH patients were included in our study. The period for normalization of ALT and total bilirubin in the study group was 19.7 ± 6.9 days and 13.7 ± 8.5 days respectively. In the control group it was 20.4 ± 6.5 days and 16.9 ± 7.8 days respectively (P > 0.05). CONCLUSION: NAC administration effected neither the time necessary for normalization of ALT and total bilirubin values nor duration of hospitalization, so we could not suggest NAC for the treatment of icteric AVH cases. However, our results have shown that this drug is not harmful to patients with AVH. PMID:14669316

  15. Cysteine-rich protein 2 accelerates actin filament cluster formation

    PubMed Central

    Shinohara, Satoko; Takaoka, Shunpei; Miyake, Jun

    2017-01-01

    Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation. PMID:28813482

  16. Rapid sensitive analysis of cysteine rich peptide venom components.

    PubMed

    Ueberheide, Beatrix M; Fenyö, David; Alewood, Paul F; Chait, Brian T

    2009-04-28

    Disulfide-rich peptide venoms from animals such as snakes, spiders, scorpions, and certain marine snails represent one of nature's great diversity libraries of bioactive molecules. The various species of marine cone shells have alone been estimated to produce >50,000 distinct peptide venoms. These peptides have stimulated considerable interest because of their ability to potently alter the function of specific ion channels. To date, only a small fraction of this immense resource has been characterized because of the difficulty in elucidating their primary structures, which range in size between 10 and 80 aa, include up to 5 disulfide bonds, and can contain extensive posttranslational modifications. The extraordinary complexity of crude venoms and the lack of DNA databases for many of the organisms of interest present major analytical challenges. Here, we describe a strategy that uses mass spectrometry for the elucidation of the mature peptide toxin components of crude venom samples. Key to this strategy is our use of electron transfer dissociation (ETD), a mass spectrometric fragmentation technique that can produce sequence information across the entire peptide backbone. However, because ETD only yields comprehensive sequence coverage when the charge state of the precursor peptide ion is sufficiently high and the m/z ratio is low, we combined ETD with a targeted chemical derivatization strategy to increase the charge state of cysteine-containing peptide toxins. Using this strategy, we obtained full sequences for 31 peptide toxins, using just 7% of the crude venom from the venom gland of a single cone snail (Conus textile).

  17. Ectopie thyroïdienne: apport du scanner

    PubMed Central

    Abdoulaye, Traore Ababacar; Zakaria, Traore; Ousmane, Camara; Meryem, Boubbou; Moustapha, Maaroufi; Siham, Tizniti; Imane, Kamaoui

    2017-01-01

    L’ectopie thyroïdienne est une malformation pathologique rare. Nous rapportons un cas supplémentaire d’ectopie du lobe thyroïdien droit, découvert lors du bilan tomodensitométrique d’une masse latéro cervicale gauche. PMID:28450999

  18. Prenatal diagnosis of cri du chat syndrome with encephalocele.

    PubMed

    Bakkum, Jamie N; Watson, William J; Johansen, Keith L; Brost, Brian C

    2005-10-01

    A 19-year-old primigravida was found to have an encephalocele on screening ultrasound study. Amniocentesis indicated cri du chat syndrome, 5p-. Although cri du chat syndrome has been noted in association with central nervous system malformations, encephalocele is a rare finding in this syndrome.

  19. 33 CFR 117.443 - Du Large Bayou.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 33 Navigation and Navigable Waters 1 2013-07-01 2013-07-01 false Du Large Bayou. 117.443 Section 117.443 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Louisiana § 117.443 Du Large Bayou. The draw of...

  20. 33 CFR 117.443 - Du Large Bayou.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 33 Navigation and Navigable Waters 1 2011-07-01 2011-07-01 false Du Large Bayou. 117.443 Section 117.443 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Louisiana § 117.443 Du Large Bayou. The draw of...

  1. 33 CFR 117.443 - Du Large Bayou.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 33 Navigation and Navigable Waters 1 2010-07-01 2010-07-01 false Du Large Bayou. 117.443 Section 117.443 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Louisiana § 117.443 Du Large Bayou. The draw of...

  2. 33 CFR 117.443 - Du Large Bayou.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 33 Navigation and Navigable Waters 1 2014-07-01 2014-07-01 false Du Large Bayou. 117.443 Section 117.443 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Louisiana § 117.443 Du Large Bayou. The draw of...

  3. 33 CFR 117.443 - Du Large Bayou.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 33 Navigation and Navigable Waters 1 2012-07-01 2012-07-01 false Du Large Bayou. 117.443 Section 117.443 Navigation and Navigable Waters COAST GUARD, DEPARTMENT OF HOMELAND SECURITY BRIDGES DRAWBRIDGE OPERATION REGULATIONS Specific Requirements Louisiana § 117.443 Du Large Bayou. The draw of...

  4. Cysteine dietary supplementation reverses the decrease in mitochondrial ROS production at complex I induced by methionine restriction.

    PubMed

    Gomez, A; Gomez, J; Lopez Torres, M; Naudi, A; Mota-Martorell, N; Pamplona, R; Barja, G

    2015-06-01

    It has been described that dietary cysteine reverses many of the beneficial changes induced by methionine restriction in aging rodents. In this investigation male Wistar rats were subjected to diets low in methionine, supplemented with cysteine, or simultaneously low in methionine and supplemented with cysteine. The results obtained in liver showed that cysteine supplementation reverses the decrease in mitochondrial ROS generation induced by methionine restriction at complex I. Methionine restriction also decreased various markers of oxidative and non-oxidative stress on mitochondrial proteins which were not reversed by cysteine. Instead, cysteine supplementation also lowered protein damage in association with decreases in mTOR activation. The results of the present study add the decrease in mitochondrial ROS production to the various beneficial changes induced by methionine restriction that are reversed by cysteine dietary supplementation.

  5. Modelisation par elements finis du muscle strie

    NASA Astrophysics Data System (ADS)

    Leonard, Mathieu

    Ce present projet de recherche a permis. de creer un modele par elements finis du muscle strie humain dans le but d'etudier les mecanismes engendrant les lesions musculaires traumatiques. Ce modele constitue une plate-forme numerique capable de discerner l'influence des proprietes mecaniques des fascias et de la cellule musculaire sur le comportement dynamique du muscle lors d'une contraction excentrique, notamment le module de Young et le module de cisaillement de la couche de tissu conjonctif, l'orientation des fibres de collagene de cette membrane et le coefficient de poisson du muscle. La caracterisation experimentale in vitro de ces parametres pour des vitesses de deformation elevees a partir de muscles stries humains actifs est essentielle pour l'etude de lesions musculaires traumatiques. Le modele numerique developpe est capable de modeliser la contraction musculaire comme une transition de phase de la cellule musculaire par un changement de raideur et de volume a l'aide des lois de comportement de materiau predefinies dans le logiciel LS-DYNA (v971, Livermore Software Technology Corporation, Livermore, CA, USA). Le present projet de recherche introduit donc un phenomene physiologique qui pourrait expliquer des blessures musculaires courantes (crampes, courbatures, claquages, etc.), mais aussi des maladies ou desordres touchant le tissu conjonctif comme les collagenoses et la dystrophie musculaire. La predominance de blessures musculaires lors de contractions excentriques est egalement exposee. Le modele developpe dans ce projet de recherche met ainsi a l'avant-scene le concept de transition de phase ouvrant la porte au developpement de nouvelles technologies pour l'activation musculaire chez les personnes atteintes de paraplegie ou de muscles artificiels compacts pour l'elaboration de protheses ou d'exosquelettes. Mots-cles Muscle strie, lesion musculaire, fascia, contraction excentrique, modele par elements finis, transition de phase

  6. Oncoplastie avec conservation mammaire dans le traitement du cancer du sein: à propos de 16 cas

    PubMed Central

    Bouzoubaa, Wail; Laadioui, Meryam; Jayi, Sofia; Alaoui, Fatime Zahra Fdili; Bouguern, Hakima; Chaara, Hikmat; Melhouf, Moulay Abdelilah

    2015-01-01

    Le cancer du sein est actuellement le cancer le plus fréquent chez la femme, et pose un véritable problème diagnostique et thérapeutique. Le dépistage des lésions à un stade de plus en plus précoce, a permis une extension des indications du traitement conservateur radiochirurgical, qui était initialement limitées aux tumeurs de moins de 3 cm, unifocales, non inflammatoires. Par ailleurs, l'utilisation de traitements préopératoires permet d’étendre les indications du traitement conservateur à des tumeurs plus volumineuses. Parallèlement à cette extension des indications de conservation mammaire, on a observé le développement de nouvelles approches thérapeutiques notamment la chirurgie oncoplastique, technique du ganglion sentinelle et chirurgie stéréotaxique, dont les résultats initiaux sont très encouragent. A travers cette étude réalisée dans le service de gynécologie et obstétrique II du CHU HASSAN II de FES au MAROC, après l'analyse rétrospective de 16 patientes traitées par traitement conservateur et oncoplastie, nous avons voulus montrer notre aptitude a réalisé ses techniques chirurgicales et a bien prendre en charge ces patientes, mais aussi évaluer ces techniques en termes de résultat carcinologique et de résultat esthétique, aussi en terme de survie globale, survie sans métastase et en termes de récidive locale entre les plasties mammaires et les traitements usuels: mastectomie et traitement conservateur classique. PMID:26430477

  7. La fin du jeûne?

    PubMed Central

    Naugler, Christopher; Sidhu, Davinder

    2014-01-01

    Résumé Objectif Présenter une mise à jour sur l’utilité clinique de ne pas être à jeun par rapport à l’être pour l’analyse des lipides dans le but d’améliorer l’observance par les patients, leur sécurité et l’évaluation clinique dans les tests du cholestérol. Qualité des données Les recommandations sont classées comme étant fondées sur des données probantes fortes, acceptables ou faibles (conflictuelles ou insuffisantes), selon les classifications adoptées par le Groupe d’étude canadien sur les soins de santé préventifs. Message principal Le dépistage de la dyslipidémie comme facteur de risque de coronaropathie et la prescription de médicaments hypolipidémiants sont des activités importantes en soins primaires. De récentes données probantes remettent en question la nécessité d’être à jeun pour la mesure des lipides. Dans des études sur la population, le cholestérol total, le cholestérol à lipoprotéines de haute densité et le cholestérol à lipoprotéines autres qu’à haute densité variaient tous d’en moyenne 2 % à jeun. Pour un dépistage de routine, la mesure du cholestérol sans être à jeun est maintenant une option de rechange raisonnable à l’analyse à jeun. Pour les patients diabétiques, l’exigence d’être à jeun peut représenter un important problème de sécurité en raison des possibilités d’hypoglycémie. Pour la surveillance des triglycérides et du cholestérol à lipoprotéines de basse densité chez les patients qui prennent des médicaments hypolipidémiants, le jeûne devient important. Conclusion Être à jeun pour la détermination routinière des niveaux lipidiques est largement inutile et il est improbable que le jeûne influence la stratification du risque clinique chez le patient, tandis que la mesure sans être à jeun pourrait améliorer l’observance par le patient et sa sécurité.

  8. Continuous improvement journey at Du Pont photomasks

    NASA Astrophysics Data System (ADS)

    Henderson, Robert K.

    1994-02-01

    This paper describes the history and experiences of Du Pont Photomasks in their efforts to integrate the continuous improvement philosophy and practices embodied in the Malcolm Baldrige National Quality Award criteria into their way of doing business. A case study of key learnings in this almost four year long process is presented. Specific topics discussed include the process applied to achieve ISO 9000 certification, the quality systems deployed in this effort, and the use of a balanced set of business and quality metrics to assess and improve upon performance.

  9. Facile and green synthesis of highly stable L-cysteine functionalized copper nanoparticles

    NASA Astrophysics Data System (ADS)

    Kumar, Nikhil; Upadhyay, Lata Sheo Bachan

    2016-11-01

    A simple eco-friendly method for L-cysteine capped copper nanoparticles (CCNPs) synthesis in aqueous solution has been developed. Glucose and L-cysteine were used as reducing agent and capping/functionalizing agent, respectively. Different parameters such as capping agent concentration, pH, reaction temperature, and reducing agent concentration were optimized during the synthesis. The L-cysteine capped copper nanoparticle were characterized by ultraviolet-visible spectroscopy, Fourier-transform infrared spectroscopy, X-ray diffraction, Particle size and zeta potential analyser, and high resolution transmission electron microscopy. Spherical shaped cysteine functionalized/capped copper nanoparticles with an average size of 40 nm were found to be highly stable at room temperature (RT) for a period of 1 month

  10. 7-cysteine-pyrrole conjugate: A new potential DNA reactive metabolite of pyrrolizidine alkaloids.

    PubMed

    He, Xiaobo; Xia, Qingsu; Ma, Liang; Fu, Peter P

    2016-01-01

    Pyrrolizidine alkaloids (PAs) require metabolic activation to exert cytotoxicity, genotoxicity, and tumorigenicity. We previously reported that (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts are responsible for PA-induced liver tumor formation in rats. In this study, we determined that metabolism of riddelliine and monocrotaline by human or rat liver microsomes produced 7-cysteine-DHP and DHP. The metabolism of 7-glutathionyl-DHP by human and rat liver microsomes also generated 7-cysteine-DHP. Further, reaction of 7-cysteine-DHP with calf thymus DNA in aqueous solution yielded the described DHP-derived DNA adducts. This study represents the first report that 7-cysteine-DHP is a new PA metabolite that can lead to DNA adduct formation.

  11. A simple fluorescent probe for sensing cysteine over homocysteine and glutathione based on PET

    NASA Astrophysics Data System (ADS)

    Fan, Wenlong; Huang, Ximing; Shi, Xiaomin; Wang, Zhuo; Lu, Zhengliang; Fan, Chunhua; Bo, Qibing

    2017-02-01

    A big challenge is the discrimination of sulfhydryl-containing amino acids due to their structural similarity. We designed and synthesized a simple fluorescent probe 3 for specific detection of cysteine based on photo-induced electron transfer (PET). The acrylate and BODIPY moieties in probe 3 act as a reaction site and reporter group, respectively. So the synergistic effect of the substituent groups endows probe 3 very strong green fluorescence at 525 nm (λex = 500 nm). The cleavage reaction induced by cysteine leads to acrylate hydrolysis, and thereby triggers PET on, which effectively quench the fluorescence of 3. Probe 3 exhibited a rapid response towards cysteine over homocysteine and glutathione. Probe 3 is successfully applied for sensing and imaging cysteine in vitro or in vivo cells with low cytotoxicity.

  12. Evaluation of the inhibitory effect of N-acetyl-L-cysteine on Babesia and Theileria parasites.

    PubMed

    Rizk, Mohamed Abdo; El-Sayed, Shimaa Abd El-Salam; AbouLaila, Mahmoud; Yokoyama, Naoaki; Igarashi, Ikuo

    2017-08-01

    N-acetyl-L-cysteine is known to have antibacterial, antiviral, antimalarial, and antioxidant activities. Therefore, the in vitro inhibitory effect of this hit was evaluated in the present study on the growth of Babesia and Theileria parasites. The in vitro growth of Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi, and Babesia caballi that were tested was significantly inhibited (P < 0.05) by micromolar concentrations of N-acetyl-L-cysteine. The inhibitory effect of N-acetyl-L-cysteine was synergistically potentiated when used in combination with diminazene aceturate on B. bovis and B. caballi cultures. These results indicate that N-acetyl-L-cysteine might be used as a drug for the treatment of babesiosis, especially when used in combination with diminazene aceturate. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Excretory bladder: the source of cysteine proteases in Paragonimus westermani metacercariae

    PubMed Central

    Yang, Hyun-Jong; Kang, Shin-Yong; Kong, Yoon; Cho, Seung-Yull

    2002-01-01

    The cysteine proteases of Paragonimus westermani metacercariae are involved in metacercarial excystment, host immune modulation, and possibly in tissue penetration. In order to clarify the origin of the enzymes, 28 and 27 kDa cysteine proteases in metacercarial excretory-secretory products were purified through the FPLC system using Mono Q column chromatography. The polyclonal antibodies to the enzymes were produced in BALB/c mice. Immunolocalization studies revealed that both cysteine proteases were distributed at the linings of excretory bladder and excretory concretions of the metacercariae. It was suggested that the excretory epithelium of P. westermani undertake the secretory function of metacercarial cysteine proteases, in addition to its role as a route for eliminating waste products. PMID:12073734

  14. Participation of cysteine and cystine in inactivation of tyrosine aminotransferase in rat liver homogenates.

    PubMed Central

    Buckley, W T; Milligan, L P

    1978-01-01

    1. Inactivation of tyrosine aminotransferase was studied in rat liver homogenates. Under an O2 atmosphere with cysteine added, inactivation was rapid after a lag period of approx. 1h, whereas a N2 atmosphere extended the lag period to approx. 3h. 2. Replacement of cysteine with cystine resulted in rapid inactivation both aerobically and anaerobically. 3. Removal of the particulate fraction by centrifuging rat liver homogenates at 13,000g for 9min resulted in an aerobic lag period of 0.5h in the presence of cystine and approx. 3h in the presence of cysteine. 4. It is proposed that the stimulatory effect of cysteine on tyrosine aminotransferase inactivation occurs largely as a result of oxidation to cystine, which appears to be a more directly effective agent. PMID:33669

  15. A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione.

    PubMed

    Niu, Li-Ya; Guan, Ying-Shi; Chen, Yu-Zhe; Wu, Li-Zhu; Tung, Chen-Ho; Yang, Qing-Zheng

    2013-02-14

    We report a turn-on fluorescent sensor based on nitrothiophenolate boron dipyrromethene (BODIPY) derivatives for the discrimination of cystein (Cys) from homocystein (Hcy) and glutathione (GSH). The sensor was applied for detection of Cys in living cells.

  16. Identification of an active site cysteine residue in Escherichia coli pyruvate oxidase.

    PubMed

    Koland, J G; Gennis, R B

    1982-06-10

    The cysteine-directed reagent N-ethylmaleimide rapidly and completely inactivates pyruvate oxidase. This inactivation is correlated with the reaction of one cysteine residue per enzyme monomer. In the presence of the cofactor, thiamin pyrophosphate, the enzyme is not inhibited by N-ethylmaleimide. Furthermore, the N-ethylmaleimide-inactivated enzyme exhibits a very low affinity for the cofactor as determined by a fluorescence quenching technique. The presence of a reactive cysteine residue at the thiamin pyrophosphate binding site is therefore indicated. Although N-ethylmaleimide completely inactivates the enzyme, a second sulfhydryl reagent methylmethanethiosulfonate is only partially inhibitory. It is shown that methylmethanethiosulfonate and N-ethylmaleimide react with the same cysteine residue. Thus, the N-ethylmaleimide-sensitive residue is probably not directly involved in catalysis.

  17. Anaerobic Degradation of Cyanuric Acid, Cysteine, and Atrazine by a Facultative Anaerobic Bacterium

    PubMed Central

    Jessee, J. A.; Benoit, R. E.; Hendricks, A. C.; Allen, G. C.; Neal, J. L.

    1983-01-01

    A facultative anaerobic bacterium that rapidly degrades cyanuric acid (CA) was isolated from the sediment of a stream that received industrial wastewater effluent. CA decomposition was measured throughout the growth cycle by using a high-performance liquid chromatography assay, and the concomitant production of ammonia was also measured. The bacterium used CA or cysteine as a major, if not the sole, carbon and energy source under anaerobic, but not aerobic, conditions in a defined medium. The cell yield was greatly enhanced by the simultaneous presence of cysteine and CA in the medium. Cysteine was preferentially used rather than CA early in the growth cycle, but all of the CA was used without an apparent lag after the cysteine was metabolized. Atrazine was also degraded by this bacterium under anaerobic conditions in a defined medium. PMID:16346187

  18. How cysteine reacts with citral: an unexpected reaction of beta,beta-disubstituted acroleins with cysteine leading to hexahydro-1,4-thiazepines.

    PubMed

    Starkenmann, Christian; Brauchli, Robert; Maurer, Bruno

    2005-11-16

    The reaction of beta,beta-disubstituted acroleins [3-methyl-2-butenal (1), 3-methyl-2-hexenal (2), and citral (3)] with cysteine gave 1:2 adducts of a novel structural type, namely hexahydro-1,4-thiazepines. To the best of our knowledge, the spontaneous formation of a seven-membered heterocycle from the addition of cysteine to alpha,beta-unsaturated aldehydes is unprecedented. The adduct 6 obtained from citral, under acidic conditions, reacted further to give the new bicyclic compound 8.

  19. Role of Conserved Cysteine Residues in Hepatitis C Virus Glycoprotein E2 Folding and Function

    PubMed Central

    McCaffrey, Kathleen; Boo, Irene; Tewierek, Kevin; Edmunds, Mark L.; Poumbourios, Pantelis

    2012-01-01

    Hepatitis C virus glycoprotein E2 contains 18 conserved cysteines predicted to form nine disulfide pairs. In this study, a comprehensive cysteine-alanine mutagenesis scan of all 18 cysteine residues was performed in E1E2-pseudotyped retroviruses (HCVpp) and recombinant E2 receptor-binding domain (E2 residues 384 to 661 [E2661]). All 18 cysteine residues were absolutely required for HCVpp entry competence. The phenotypes of individual cysteines and pairwise mutation of disulfides were largely the same for retrovirion-incorporated E2 and E2661, suggesting their disulfide arrangements are similar. However, the contributions of each cysteine residue and the nine disulfides to E2 structure and function varied. Individual Cys-to-Ala mutations revealed discordant effects, where removal of one Cys within a pair had minimal effect on H53 recognition and CD81 binding (C486 and C569) while mutation of its partner abolished these functions (C494 and C564). Removal of disulfides at C581-C585 and C452-C459 significantly reduced the amount of E1 coprecipitated with E2, while all other disulfides were absolutely required for E1E2 heterodimerization. Remarkably, E2661 tolerates the presence of four free cysteines, as simultaneous mutation of C452A, C486A, C569A, C581A, C585A, C597A, and C652A (M+C597A) retained wild-type CD81 binding. Thus, only one disulfide from each of the three predicted domains, C429-C552 (DI), C503-C508 (DII), and C607-C644 (DIII), is essential for the assembly of the E2661 CD81-binding site. Furthermore, the yield of total monomeric E2 increased to 70% in M+C597A. These studies reveal the contribution of each cysteine residue and the nine disulfide pairs to E2 structure and function. PMID:22278231

  20. Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

    PubMed Central

    Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

    2015-01-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

  1. Spectroscopic studies on the interaction of cysteine capped CuS nanoparticles with tyrosine

    SciTech Connect

    Prasanth, S.; Raj, D. Rithesh; Kumar, T. V. Vineesh; Sudarsanakumar, C.

    2015-06-24

    Biocompatible cysteine coated CuS nanoparticles were synthesized by a simple aqueous solution method. Hexagonal phase of the samples were confirmed from X-ray diffraction and particle size found to be 9 nm. The possible interaction between the bioactive cysteine capped CuS nanoparticles and tyrosine were investigated using spectroscopic techniques such as UV-Visible absorption and fluorescence spectroscopy. It is observed that the luminescence intensity of tyrosine molecule enhanced by the addition CuS nanoparticles.

  2. Cathodic stripping voltammetry of cysteine using silver and copper solid amalgam electrodes.

    PubMed

    Yosypchuk, B; Novotný, L

    2002-04-01

    Silver and copper solid amalgam electrodes (modified with mercury meniscus and based on amalgamation of fine metallic powder) have been successfully tested for cathodic stripping voltammetry of cysteine. In the case of the silver solid amalgam electrode AgSAE the relative standard deviation (RSD) and the detection limit (3 SD) reached +/-2.3% and 3x10(-9) mol l(-1) cysteine, respectively.

  3. Protein topology determines cysteine oxidation fate: the case of sulfenyl amide formation among protein families.

    PubMed

    Defelipe, Lucas A; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A; Turjanski, Adrián G

    2015-03-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function.

  4. The amino acid sequence around the active-site cysteine and histidine residues of stem bromelain

    PubMed Central

    Husain, S. S.; Lowe, G.

    1970-01-01

    Stem bromelain that had been irreversibly inhibited with 1,3-dibromo[2-14C]-acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin three radioactive peptides were isolated chromatographically. The amino acid sequences around the cross-linked cysteine and histidine residues were determined and showed a high degree of homology with those around the active-site cysteine and histidine residues of papain and ficin. PMID:5420046

  5. Visual determination of trace cysteine based on promoted corrosion of triangular silver nanoplates by sodium thiosulfate.

    PubMed

    Hou, Xin Yan; Chen, Shu; Tang, Jian; Long, Yun Fei

    2014-05-05

    In this study, triangular silver nanoplates (TAg-NPs) were used to detect trace Cysteine concentration in the presence of sodium thiosulfate (Na2S2O3). Study showed that the TAg-NPs could be gently etched by Cysteine with the concentration of 1.0×10(-7) mol L(-1) through forming Ag-S covalent bond at the three corners. However, in the presence of Na2S2O3 (only 3.0×10(-6) mol L(-1)), the corrosion of Cysteine on TAg-NPs can be promoted significantly. It was also found that the color, morphology, and the maximum absorption wavelength of TAg-NPs change clearly with the concentrations of Cysteine as low as 2.5×10(-8) mol L(-1). Furthermore, the wavelength shift values (Δλ) of TAg-NPs solution were proportional to the concentrations of Cysteine in the range of 1.0×10(-9)-1.0×10(-7) mol L(-1), and the linear regression equation is Δλ=-0.89+319.94 c (c, μM, n=5) with the correlation coefficient of 0.990. At the same time, the color change of the TAg-NPs solution could be observed clearly by the naked eyes with increasing Cysteine concentrations in the range of 2.5×10(-8)-1.0×10(-7) mol L(-1). Thus, a novel method for the detection of Cysteine by either UV-vis spectrophotometry detection or naked eyes observation is established. It allows determination of Cysteine content in compound amino acid injection sample of 18AA-V. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Mitochondrial Cysteine Synthase Complex Regulates O-Acetylserine Biosynthesis in Plants*

    PubMed Central

    Wirtz, Markus; Beard, Katherine F. M.; Lee, Chun Pong; Boltz, Achim; Schwarzländer, Markus; Fuchs, Christopher; Meyer, Andreas J.; Heeg, Corinna; Sweetlove, Lee J.; Ratcliffe, R. George; Hell, Rüdiger

    2012-01-01

    Cysteine synthesis is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) in the cytosol, plastids, and mitochondria of plants. Biochemical analyses of recombinant plant SAT and OAS-TL indicate that the reversible association of the proteins in the cysteine synthase complex (CSC) controls cellular sulfur homeostasis. However, the relevance of CSC formation in each compartment for flux control of cysteine synthesis remains controversial. Here, we demonstrate the interaction between mitochondrial SAT3 and OAS-TL C in planta by FRET and establish the role of the mitochondrial CSC in the regulation of cysteine synthesis. NMR spectroscopy of isolated mitochondria from WT, serat2;2, and oastl-C plants showed the SAT-dependent export of OAS. The presence of cysteine resulted in reduced OAS export in mitochondria of oastl-C mutants but not in WT mitochondria. This is in agreement with the stronger in vitro feedback inhibition of free SAT by cysteine compared with CSC-bound SAT and explains the high OAS export rate of WT mitochondria in the presence of cysteine. The predominant role of mitochondrial OAS synthesis was validated in planta by feeding [3H]serine to the WT and loss-of-function mutants for OAS-TLs in the cytosol, plastids, and mitochondria. On the basis of these results, we propose a new model in which the mitochondrial CSC acts as a sensor that regulates the level of SAT activity in response to sulfur supply and cysteine demand. PMID:22730323

  7. Effects of N-acetyl-cysteine and N-acetyl-cysteine-amide supplementation on in vitro matured porcine oocytes.

    PubMed

    Whitaker, B D; Knight, J W

    2010-10-01

    This study was conducted to evaluate the effects of different concentrations of the antioxidant N-acetyl-cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC-amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post-fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development. © 2009 Blackwell Verlag GmbH.

  8. L-Cysteine halogenides: A new family of salts with an L-cysteine⋯L-cysteinium dimeric cation

    NASA Astrophysics Data System (ADS)

    Ghazaryan, V. V.; Minkov, V. S.; Boldyreva, E. V.; Petrosyan, A. M.

    2016-10-01

    Two L-cysteinium-halogenides with (L-cysteine···L-cysteinium) dimeric cations have been obtained, (L-Cys⋯L-Cys+)·Cl-, and (L-Cys⋯L-Cys+)·Br-. Both salts crystallize in monoclinic space group P21. Although these salts have the same dimeric cations and isotypical halogen anions, crystal packing is different. The main difference between the two salts rests in the conformation of (L-Cys⋯L-Cys+) dimeric cation, which also differs from that of the dimeric cation in the previously reported compound L-Cys+(L-Cys⋯L-Cys+)·F-·(F-⋯HF). The dimeric cation is formed by a very short O-H⋯O hydrogen bond with d(O···O) of 2.449(2) Å and 2.435(11) Å in the chloride and bromide, respectively. In addition to crystal structure analysis, Infrared and Raman spectra have been registered and discussed with a particular focus on intermolecular interactions. The L-Cys+·Br-·H2O salt with a simple L-cysteinium cation was also obtained and the crystal structure solved. It resembles its chloride analogue, L-Cys+·Cl-·H2O.

  9. Mesure du taux de la capture radiative du muon par l'hydrogene liquide

    NASA Astrophysics Data System (ADS)

    Jonkmans, Guy

    À basse énergie, l'interaction faible entre leptons et quarks est décrite par une interaction de la forme courant × courant de type V - A. La présence de l'interaction forte induit des couplages additionnels qui doivent être déterminés expérimentalement. De ceux-ci, le couplage pseudoscalaire induit, gp , est mesuré avec la plus grande incertitude et fait l'objet de la présente recherche. L'hypothèse du Courant Axial Partiellement Conservé (CAPC) et l'usage de la relation de Goldberger-Treiman relie gp au couplage axial ga . Cette relation a été vérifiée traditionnellement par la Capture Ordinaire du Muon (COM) à une valeur fixe du moment de transfert q. La Capture Radiative du Muon (CRM), m- p-->nnmg , est un meilleur outil pour l'étude de gp à cause de sa dépendance variable en q2 qui offre une plus grande sensibilité dans la partie à haute énergie du spectre des photons. Toutefois, le petit rapport d'embranchement (~10-8) de la CRM par rapport à la désintégration du muon a retardé cette mesure jusqu'à ce jour. La théorie et les difficultés expérimentales associées à la détection des photons de CRM sont présentées au deuxième chapitre. On décrit ensuite, au troisième chapitre, les composantes du système de détection. Ce détecteur est un spectromètre à paires de grand angle solide (~3p) et qui permet l'observation des photons par l'analyse des électrons et des positrons de photo-conversion. Ainsi, le bruit de fond important des neutrons de la COM ne constitue pas un problème pour cette mesure. Nous décrivons, au quatrième chapitre, toutes les étapes de l'analyse, nécessaires pour la réduction des multiples bruits de fond. Le cinquième chapitre présente le calcul des efficacités ainsi que l'estimation des erreurs systématiques. Le sixième chapitre démontre comment l'on extrait le rapport d'embranchement pour la CRM ainsi que la valeur ae gp . On insiste sur la dépendance de gp en fonction de la valeur de

  10. The cysteine releasing pattern of some antioxidant thiazolidine-4-carboxylic acids.

    PubMed

    Önen Bayram, F Esra; Sipahi, Hande; Acar, Ebru Türköz; Kahveci Ulugöl, Reyhan; Buran, Kerem; Akgün, Hülya

    2016-05-23

    Oxidative stress that corresponds to a significant increase in free radical concentration in cells can cause considerable damage to crucial biological macromolecules if not prevented by cellular defense mechanisms. The low-molecular-weight thiol glutathione (GSH) constitutes one of the main intracellular antioxidants. It is synthesized via cysteine, an amino acid found only in limited amounts in cells because of its neurotoxicity. Thus, to ensure an efficient GSH synthesis in case of an oxidative stress, cysteine should be provided extracellularly. Yet, given its nucleophilic properties and its rapid conversion into cystine, its corresponding disulfide, cysteine presents some toxicity and therefore is usually supplemented in a prodrug approach. Here, some thiazolidine-4-carboxylic acids were synthesized and evaluated for their antioxidant properties via the DDPH and CUPRAC assays. Then, the cysteine releasing capacity of the obtained compounds was investigated in aqueous and organic medium in order to correlate the relevant antioxidant properties of the molecules with their cysteine releasing pattern. As a result, the structures' antioxidative properties were not only attributed to cysteine release but also to the thiazolidine cycle itself.

  11. Cysteine protease inhibitor (AcStefin) is required for complete cyst formation of Acanthamoeba.

    PubMed

    Lee, Jung-Yub; Song, Su-Min; Moon, Eun-Kyung; Lee, Yu-Ran; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Cha, Hee-Jae; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong-Il; Hong, Yeonchul

    2013-04-01

    The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba.

  12. Stereoselective HDAC inhibition from cysteine-derived zinc-binding groups.

    PubMed

    Butler, Kyle V; He, Rong; McLaughlin, Kathryn; Vistoli, Giulio; Langley, Brett; Kozikowski, Alan P

    2009-08-01

    A series of small-molecule histone deacetylase (HDAC) inhibitors, which feature zinc binding groups derived from cysteine, were synthesized. These inhibitors were tested against multiple HDAC isoforms, and the most potent, compound 10, was determined to have IC(50) values below 1 microM. The compounds were also tested in a cellular assay of oxidative stress-induced neurodegeneration. Many of the inhibitors gave near-complete protection against cell death at 10 microM without the neurotoxicity seen with hydroxamic acid-based inhibitors, and were far more neuroprotective than HDAC inhibitors currently in clinical trials. Both enantiomers of cysteine were used in the synthesis of a variety of novel zinc-binding groups (ZBGs). Derivatives of L-cysteine were active in the HDAC inhibition assays, while the derivatives of D-cysteine were inactive. Notably, the finding that both the D- and L-cysteine derivatives were active in the neuroprotection assays suggests that multiple mechanisms are working to protect the neurons from cell death. Molecular modeling was employed to investigate the differences in inhibitory activity between the HDAC inhibitors generated from the two enantiomeric forms of cysteine.

  13. Cd–cysteine precursor nanowire templated microwave-assisted transformation route to CdS nanotubes

    SciTech Connect

    Liu, Xiao-Lin; Zhu, Ying-Jie; Zhang, Qian; Li, Zhi-Feng; Yang, Bin

    2012-12-15

    Graphical abstract: Cadmium sulfide polycrystalline nanotubes have been successfully synthesized by microwave-assisted transformation method using Cd–cysteine precursor nanowires as the source material and template in ethylene glycol at 160 °C or ethanol at 60 °C. Display Omitted Highlights: ► Cd–cysteine precursor nanowires were successfully synthesized in alkaline solution. ► CdS nanotubes were prepared by templated microwave-assisted transformation method. ► CdS nanotubes can well duplicate the size and morphology of precursor nanowires. ► This method has the advantages of the simplicity and low cost. -- Abstract: We report the Cd–cysteine precursor nanowire templated microwave-assisted transformation route to CdS nanotubes. In this method, the Cd–cysteine precursor nanowires are synthesized using CdCl{sub 2}·2.5H{sub 2}O, L-cysteine and ethanolamine in water at room temperature. The Cd–cysteine precursor nanowires are used as the source material and template for the subsequent preparation of CdS nanotubes by a microwave-assisted transformation method using ethylene glycol or ethanol as the solvent. This method has the advantages of the simplicity and low cost, and may be extended to the synthesis of nanotubes of other compounds. The products are characterized by X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

  14. Cysteine Protease Inhibitor (AcStefin) Is Required for Complete Cyst Formation of Acanthamoeba

    PubMed Central

    Lee, Jung-Yub; Song, Su-Min; Moon, Eun-Kyung; Lee, Yu-Ran; Jha, Bijay Kumar; Danne, Dinzouna-Boutamba Sylvatrie; Cha, Hee-Jae; Yu, Hak Sun; Kong, Hyun-Hee; Chung, Dong-Il

    2013-01-01

    The encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions, such as those associated with starvation, low temperatures, and biocides. Furthermore, cysteine proteases have been implicated in the massive turnover of intracellular components required for encystation. Thus, strict modulation of the activities of cysteine proteases is required to protect Acanthamoeba from intracellular damage. However, mechanisms underlying the control of protease activity during encystation have not been established in Acanthamoeba. In the present study, we identified and characterized Acanthamoeba cysteine protease inhibitor (AcStefin), which was found to be highly expressed during encystation and to be associated with lysosomes by fluorescence microscopy. Recombinant AcStefin inhibited various cysteine proteases, including human cathepsin B, human cathepsin L, and papain. Transfection with small interfering RNA against AcStefin increased cysteine protease activity during encystation and resulted in incomplete cyst formation, reduced excystation efficiency, and a significant reduction in cytoplasmic area. Taken together, these results indicate that AcStefin is involved in the modulation of cysteine proteases and that it plays an essential role during the encystation of Acanthamoeba. PMID:23397569

  15. Metacaspase activity of Arabidopsis thaliana is regulated by S-nitrosylation of a critical cysteine residue.

    PubMed

    Belenghi, Beatrice; Romero-Puertas, Maria C; Vercammen, Dominique; Brackenier, Anouk; Inzé, Dirk; Delledonne, Massimo; Van Breusegem, Frank

    2007-01-12

    Nitric oxide (NO) regulates a number of signaling functions in both animals and plants under several physiological and pathophysiological conditions. S-Nitrosylation linking a nitrosothiol on cysteine residues mediates NO signaling functions of a broad spectrum of mammalian proteins, including caspases, the main effectors of apoptosis. Metacaspases are suggested to be the ancestors of metazoan caspases, and plant metacaspases have previously been shown to be genuine cysteine proteases that autoprocess in a manner similar to that of caspases. We show that S-nitrosylation plays a central role in the regulation of the proteolytic activity of Arabidopsis thaliana metacaspase 9 (AtMC9) and hypothesize that this S-nitrosylation affects the cellular processes in which metacaspases are involved. We found that AtMC9 zymogens are S-nitrosylated at their active site cysteines in vivo and that this posttranslational modification suppresses both AtMC9 autoprocessing and proteolytic activity. However, the mature processed form is not prone to NO inhibition due to the presence of a second S-nitrosylation-insensitive cysteine that can replace the S-nitrosylated cysteine residue within the catalytic center of the processed AtMC9. This cysteine is absent in caspases and paracaspases but is conserved in all reported metacaspases.

  16. Effects of cysteine on the pharmacokinetics of docetaxel in rats with protein-calorie malnutrition.

    PubMed

    Choi, Young Hee; Yoon, Insoo; Kim, Yoon Gyoon; Lee, Myung Gull

    2012-05-01

    The objective of this study is to report the effects of cysteine on the pharmacokinetics of intravenous and oral docetaxel in rats with protein-calorie malnutrition (PCM). The in vivo pharmacokinetics and in vitro hepatic/intestinal metabolism of docetaxel were assessed using control, CC (control with cysteine), PCM and PCMC (PCM with cysteine) rats. The effects of cysteine on the intestinal absorption of docetaxel were further investigated through in vitro transport studies using rat intestine and Caco-2 cell monolayers. The AUCs (the areas under the plasma concentration-time curve from time zero to time infinity) of intravenous docetaxel in PCM rats were significantly greater than in the control rats because of the significant decrease in the hepatic CYP3A. In PCMC rats, the elevated AUCs in PCM rats returned to control levels. The AUC(0-6 h)s of oral docetaxel in PCM rats were significantly smaller than that in the control rats, mainly due to the decrease in gastrointestinal absorption. In CC and PCMC rats, oral cysteine supplement enhanced the gastrointestinal absorption of docetaxel probably via intestinal P-gp inhibition. If the present rat data could be expressed to humans, the alterations in docetaxel absorption and metabolism should be considered in designing a dosage regimen for cancer patients with PCM state after cysteine supplement.

  17. Cysteine Inhibits Mercury Methylation by Geobacter sulfurreducens PCA Mutant Δ omcBESTZ

    DOE PAGES

    Lin, Hui; Lu, Xia; Liang, Liyuan; ...

    2015-04-21

    For cysteine enhances Hg uptake and methylation by Geobacter sulfurreducens PCA wild type (WT) strain in short-term assays. The prevalence of this enhancement in other strains remains poorly understood. We examined the influence of cysteine concentration on time-dependent Hg(II) reduction, sorption and methylation by PCA-WT and its c-type cytochrome-deficient mutant ( omcBESTZ) in phosphate buffered saline. Without cysteine, the mutant methylated twice as much Hg(II) as the PCA-WT, whereas addition of cysteine inhibited Hg methylation, regardless of the reaction time. PCA-WT, but, exhibited both time-dependent and cysteine concentration-dependent methylation. In 144 hour assay, nearly complete sorption of the Hg(II) bymore » PCA-WT occurred in the presence of 1 mM cysteine, resulting in our highest observed methylmercury production. Moreover, the chemical speciation modeling and experimental data suggest that uncharged Hg(II) species are more readily taken up, and that this uptake is kinetic limiting, thereby affecting Hg methylation by both mutant and WT.« less

  18. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs.

    PubMed

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  19. Rapid synthesis of DNA-cysteine conjugates for expressed protein ligation

    SciTech Connect

    Lovrinovic, Marina; Niemeyer, Christof M. . E-mail: christof.niemeyer@uni-dortmund.de

    2005-09-30

    We report a rapid method for the covalent modification of commercially available amino-modified DNA oligonucleotides with a cysteine moiety. The resulting DNA-cysteine conjugates are versatile reagents for the efficient preparation of covalent DNA-protein conjugates by means of expressed protein ligation (EPL). The EPL method allows for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins, the latter of which contain a C-terminal thioester enabling the mild and highly specific reaction with N-terminal cysteine compounds. We prepared a cysteine-modifier reagent in a single-step reaction which allows for the rapid and near quantitative synthesis of cysteine-DNA conjugates. The latter were ligated with the green fluorescent protein mutant EYFP, recombinantly expressed as an intein-fusion protein, allowing for the mild and selective formation of EYFP-DNA conjugates in high yields of about 60%. We anticipate many applications of our approach, ranging from protein microarrays to the arising field of nanobiotechnology.

  20. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs

    PubMed Central

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases. PMID:25307852

  1. The Pic du Midi solar survey

    NASA Astrophysics Data System (ADS)

    Koechlin, L.

    2015-12-01

    We carry a long term survey of the solar activity with our coronagraphic system at Pic du Midi de Bigorre in the French Pyrenees (CLIMSO). It is a set of two solar telescopes and two coronagraphs, taking one frame per minute for each of the four channels : Solar disk in H-α (656.28 nm), prominences in H-α, disk in Ca II (393.3 nm), prominences in He I (1083 nm), all year long, weather permitting. Since 2015 we also take images of the FeXIII corona (1074.7 nm) at the rate of one every 10 minutes. These images cover a large field: 1.25 solar diameter, 2k*2K pixels, and are freely downloadable form a database. The improvements made since 2015 concern an autoguiding system for better centering of the solar disk behind the coronagraphic masks, and a new Fe XIII channel at λ=1074.7 nm. In the near future we plan to provide radial velocity maps of the disc and polarimetry maps of the disk and corona. This survey took its present form in 2007 and we plan to maintain image acquisition in the same or better experimental conditions for a long period: one or several solar cycles if possible. During the partial solar eclipse of March 20, 2015, the CLIMSO instruments and the staff at Pic du Midi operating it have provided several millions internet users with real time images of the Sun and Moon during all the phenomenon.

  2. ROSICS: CHEMISTRY AND PROTEOMICS OF CYSTEINE MODIFICATIONS IN REDOX BIOLOGY

    PubMed Central

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1–2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, “ROSics,” for the science which describes the principles of mode of action of ROS at molecular levels. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Rapid Commun. Mass Spec Rev 34:184–208, 2015. PMID:24916017

  3. An Entamoeba cysteine peptidase specifically expressed during encystation.

    PubMed

    Ebert, Frank; Bachmann, Anna; Nakada-Tsukui, Kumiko; Hennings, Ina; Drescher, Babette; Nozaki, Tomoyoshi; Tannich, Egbert; Bruchhaus, Iris

    2008-12-01

    Protozoan parasites of the genus Entamoeba possess a considerable number of cysteine peptidases (CPs), the function of most of these molecules for amoeba biology needs to be established. In order to determine whether CPs may play a role during Entamoeba stage conversion from trophozoites into cysts and vice versa, expression of cp genes was analysed in the reptilian parasite Entamoeba invadens, a model organism for studying Entamoeba cyst development. By homology search, 28 papain-like cp genes were identified in public E. invadens genome databases. For eight of these genes the expression profiles during stage conversion was determined. By Northern blot analysis, transcripts for eicp-a9, -b7, -b8 and -c2, respectively, were detected neither in trophozoites or cysts nor at any of the point of times analysed during stage conversion. On the other hand, eicp-a5 is constitutively expressed during all developmental stages, whereas eicp-a3 and eicp-a11, respectively, are trophozoite-specific. Only eicp-b9 was found to be cyst-specific as it is expressed exclusively 18 to 28 h after cyst induction. Cyst-specific expression was confirmed by immunofluorescence microscopy of the corresponding protein EiCP-B9. In immature cysts, the molecule is located in structures that accumulate near the cyst wall, but which are uniformly distributed in mature cysts. The precise function of EiCP-B9 during Entamoeba encystation remains to be determined. However, colocalisation studies with an Entamoeba marker for autophagosomes suggest that EiCP-B9 is not associated with Entamoeba autophagy.

  4. Oxidation of hypotaurine and cysteine sulphinic acid by peroxynitrite

    PubMed Central

    2005-01-01

    Peroxynitrite mediates the oxidation of the sulphinic group of both HTAU (hypotaurine) and CSA (cysteine sulphinic acid), producing the respective sulphonates, TAU (taurine) and CA (cysteic acid). The reaction is associated with extensive oxygen uptake, suggesting that HTAU and CSA are oxidized by the one-electron transfer mechanism to sulphonyl radicals, which may initiate an oxygen-dependent radical chain reaction with the sulphonates as final products. Besides the one-electron mechanism, HTAU and CSA can be oxidized by the two-electron pathway, leading directly to sulphonate formation without oxygen consumption. The apparent second-order rate constants for the direct reaction of peroxynitrite with HTAU and CSA at pH 7.4 and 25 °C are 77.4±5 and 76.4±9 M−1·s−1 respectively. For both sulphinates, the apparent second-order rate constants increase sharply with decrease in pH, and the sigmoidal curves obtained are consistent with peroxynitrous acid as the species responsible for sulphinate oxidation. The kinetic data, together with changes in oxygen uptake, sulphinate depletion, sulphonate production, and product distribution of nitrite and nitrate, suggest that oxidation of sulphinates by peroxynitrite may take place by the two reaction pathways whose relative importance depends on reagent concentrations and pH value. In the presence of bicarbonate, the direct reaction of sulphinates with peroxynitrite is inhibited and the oxidative reaction probably involves only the radicals •NO2 and CO3•−, generated by decomposition of the peroxynitrite-CO2 adduct. PMID:15740460

  5. ROSics: chemistry and proteomics of cysteine modifications in redox biology.

    PubMed

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2015-01-01

    Post-translational modifications (PTMs) occurring in proteins determine their functions and regulations. Proteomic tools are available to identify PTMs and have proved invaluable to expanding the inventory of these tools of nature that hold the keys to biological processes. Cysteine (Cys), the least abundant (1-2%) of amino acid residues, are unique in that they play key roles in maintaining stability of protein structure, participating in active sites of enzymes, regulating protein function and binding to metals, among others. Cys residues are major targets of reactive oxygen species (ROS), which are important mediators and modulators of various biological processes. It is therefore necessary to identify the Cys-containing ROS target proteins, as well as the sites and species of their PTMs. Cutting edge proteomic tools which have helped identify the PTMs at reactive Cys residues, have also revealed that Cys residues are modified in numerous ways. These modifications include formation of disulfide, thiosulfinate and thiosulfonate, oxidation to sulfenic, sulfinic, sulfonic acids and thiosulfonic acid, transformation to dehydroalanine (DHA) and serine, palmitoylation and farnesylation, formation of chemical adducts with glutathione, 4-hydroxynonenal and 15-deoxy PGJ2, and various other chemicals. We present here, a review of relevant ROS biology, possible chemical reactions of Cys residues and details of the proteomic strategies employed for rapid, efficient and sensitive identification of diverse and novel PTMs involving reactive Cys residues of redox-sensitive proteins. We propose a new name, "ROSics," for the science which describes the principles of mode of action of ROS at molecular levels.

  6. Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome.

    PubMed

    Song, Jiangning; Wang, Minglei; Burrage, Kevin

    2006-07-21

    High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.

  7. The cysteine-cluster motif of c-Yes, Lyn and FAK as a suppressive module for the kinases.

    PubMed

    Rahman, Mohammad Aminur; Senga, Takeshi; Oo, Myat Lin; Hasegawa, Hitoki; Biswas, Md Helal Uddin; Mon, Naing Naing; Huang, Pengyu; Ito, Satoko; Yamamoto, Tadashi; Hamaguchi, Michinari

    2008-04-01

    The Src family of non-receptor protein tyrosine kinases plays a critical role in the progression of human cancers so that the development of its specific inhibitors is important as a therapeutic tool. We previously reported that cysteine residues in the cysteine-cluster (CC) motif of v-Src were critical for the kinase inactivation by the SH-alkylating agents such as N-(9-acridinyl) maleimide (NAM), whereas other cysteine residues were dispensable. We found similar CC-motifs in other Src-family kinases and a non-Src-family kinase, FAK. In this study, we explored the function of the CC-motif in Yes, Lyn and FAK. While Src has four cysteines in the CC-motif, c-Yes and Lyn have three and two of the four cysteines, respectively. Two conserved cysteines of the Src family kinases, corresponding to Cys487 and Cys498 of Src, were essential for the resistance to the inactivation of the kinase activity by NAM, whereas the first cysteine of c-Yes, which is absent in Lyn, was less important. FAK has similar CC-motifs with two cysteines and both cysteines were again essential for the resistance to the inactivation of the kinase activity by NAM. Taken together, modification of cysteine residues of the CC-motif causes a repressor effect on the catalytic activity of the Src family kinases and FAK.

  8. Preparation, Crystallization and X-ray Diffraction Analysis to 1.5 A Resolution of Rat Cysteine Dioxygenase, a Mononuclear Iron Enzyme Responsible for Cysteine Thiol Oxidation

    SciTech Connect

    Simmons,C.; Hao, Q.; Stipanuk, M.

    2005-01-01

    Cysteine dioxygenase (CDO; EC 1.13.11.20) is an {approx}23 kDa non-heme iron metalloenzyme that is responsible for the oxidation of cysteine by O2, yielding cysteinesulfinate. CDO catalyzes the first step in the conversion of cysteine to taurine, as well as the first step in the catabolism of cysteine to pyruvate plus sulfate. Recombinant rat CDO was heterologously expressed, purified and crystallized. The protein was expressed as a fusion protein bearing a polyhistidine tag to facilitate purification, a thioredoxin tag to improve solubility and a factor Xa cleavage site to permit removal of the entire N-terminus, leaving only the 200 amino acids inherent to the native protein. A multi-step purification scheme was used to achieve >95% purity of CDO. The optimal CDO crystals diffracted to 1.5 Angstroms resolution and belonged to space group P4{sub 3}2{sub 1}2 or P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 57.55, c = 123.06 Angstrom, {alpha} = {beta} = {gamma} = 90. CDO shows little homology to any other proteins; therefore, the structure of the enzyme will be determined by ab initio phasing using a selenomethionyl derivative.

  9. Clitocypin, a fungal cysteine protease inhibitor, exerts its insecticidal effect on Colorado potato beetle larvae by inhibiting their digestive cysteine proteases.

    PubMed

    Šmid, Ida; Rotter, Ana; Gruden, Kristina; Brzin, Jože; Buh Gašparič, Meti; Kos, Janko; Žel, Jana; Sabotič, Jerica

    2015-07-01

    Colorado potato beetle (Leptinotarsa decemlineata Say, CPB) is a major potato pest that adapts readily to insecticides. Several types of protease inhibitors have previously been investigated as potential control agents, but with limited success. Recently, cysteine protease inhibitors from parasol mushroom, the macrocypins, were reported to inhibit growth of CPB larvae. To further investigate the insecticidal potential and mode of action of cysteine protease inhibitors of fungal origin, clitocypin, a cysteine protease inhibitor from clouded agaric (Clitocybe nebularis), was evaluated for its lethal effects on CPB larvae. Clitocypin isolated from fruiting bodies and recombinant clitocypin produced in Escherichia coli slowed growth and reduced survival of CPB larvae in a concentration dependent manner. Clitocypin was also expressed by transgenic potato, but only at low levels. Nevertheless, it reduced larval weight gain and delayed development. We have additionally shown that younger larvae are more susceptible to the action of clitocypin. The inhibition of digestive cysteine proteases, intestains, by clitocypin was shown to be the underlying mode of action. Protease inhibitors from mushrooms are confirmed as promising candidates for biopesticides.

  10. The identification of free cysteine residues within antibodies and a potential role for free cysteine residues in covalent aggregation because of agitation stress.

    PubMed

    Huh, Joon H; White, April J; Brych, Stephen R; Franey, Heather; Matsumura, Masazumi

    2013-06-01

    Human immunoglobulin G1 (IgG1) and immunoglobulin G2 (IgG2) antibodies contain multiple disulfide bonds, which are an integral part of the structure and stability of the protein. Open disulfide bonds have been detected in a number of therapeutic and serum derived antibodies. This report details a method that fluorescently labels free cysteine residues, quantifies, and identifies the proteolytic fragments by liquid chromatography coupled to online mass spectrometry. The majority of the open disulfide bonds in recombinant and serum derived IgG1 and IgG2 antibodies were in the constant domains. This method was applied to the identification of cysteines in an IgG2 antibody that are involved in the formation of covalent intermolecular bonds because of the application of a severe agitation stress. The free cysteine in the CH 1 domain of the IgG2 decreased upon application of the stress and implicates open disulfide bonds in this domain as the likely source of free cysteines involved in the formation of intermolecular disulfide bonds. The presence of comparable levels of open disulfide bonds in recombinant and endogenous antibodies suggests that open disulfide bonds are an inherent feature of antibodies and that the susceptibility of intermolecular disulfide bond formation is similar for recombinant and serum-derived IgG antibodies.

  11. Establishment of the DU.528 human lymphohemopoietic stem cell line

    PubMed Central

    1985-01-01

    We have established the DU.528 cell line from the pretreatment leukemia cells of a patient who underwent a T lymphoblastic-to-promyelocytic phenotype conversion during treatment with the adenosine deaminase inhibitor, deoxycoformycin. The cell line and clones obtained from it by limiting dilution have the same karyotype previously found in the patient's pretreatment T lymphoblasts and post-deoxycoformycin treatment promyelocytes. DU.528 cells in continuous culture for greater than 2 yr display a predominant undifferentiated T lymphoblastoid phenotype. These cells spontaneously generate progeny of at least three lineages, T lymphoid, granulocytic/monocytic, and erythroid. The surface marker most consistently expressed by DU.528 cells in the undifferentiated state is the 3A1 antigen, which has been found on prothymocytes in the embryonic thymus. Some undifferentiated DU.528 cells also expressed the IL-2 receptor, but no other T cell differentiation antigens. Exposure of DU.528 cells to a variety of agents induced myeloid maturation; adenosine and deoxyadenosine, in the presence of deoxycoformycin, induced expression of myeloid differentiation antigens. Our results suggest that DU.528 is a lymphohematopoietic stem cell line and support the hypothesis that differentiation of pluripotent stem cells may be altered by genetic deficiency of adenosine deaminase. DU.528 cells may provide a useful model for examining factors that regulate stem cell proliferation and differentiation. PMID:4056659

  12. The Significance of Hydrogen Sulfide for Arabidopsis Seed Germination

    PubMed Central

    Baudouin, Emmanuel; Poilevey, Aurélie; Hewage, Nishodi Indiketi; Cochet, Françoise; Puyaubert, Juliette; Bailly, Christophe

    2016-01-01

    Hydrogen sulfide (H2S) recently emerged as an important gaseous signaling molecule in plants. In this study, we investigated the possible functions of H2S in regulating Arabidopsis seed germination. NaHS treatments delayed seed germination in a dose-dependent manner and were ineffective in releasing seed dormancy. Interestingly, endogenous H2S content was enhanced in germinating seeds. This increase was correlated with higher activity of three enzymes (L-cysteine desulfhydrase, D-cysteine desulfhydrase, and β-cyanoalanine synthase) known as sources of H2S in plants. The H2S scavenger hypotaurine and the D/L cysteine desulfhydrase inhibitor propargylglycine significantly delayed seed germination. We analyzed the germinative capacity of des1 seeds mutated in Arabidopsis cytosolic L-cysteine desulfhydrase. Although the mutant seeds do not exhibit germination-evoked H2S formation, they retained similar germination capacity as the wild-type seeds. In addition, des1 seeds responded similarly to temperature and were as sensitive to ABA as wild type seeds. Taken together, these data suggest that, although its metabolism is stimulated upon seed imbibition, H2S plays, if any, a marginal role in regulating Arabidopsis seed germination under standard conditions. PMID:27446159

  13. Hydrogen sulfide regulates abiotic stress tolerance and biotic stress resistance in Arabidopsis.

    PubMed

    Shi, Haitao; Ye, Tiantian; Han, Ning; Bian, Hongwu; Liu, Xiaodong; Chan, Zhulong

    2015-07-01

    Hydrogen sulfide (H2S) is an important gaseous molecule in various plant developmental processes and plant stress responses. In this study, the transgenic Arabidopsis thaliana plants with modulated expressions of two cysteine desulfhydrases, and exogenous H2S donor (sodium hydrosulfide, NaHS) and H2S scavenger (hypotaurine, HT) pre-treated plants were used to dissect the involvement of H2S in plant stress responses. The cysteine desulfhydrases overexpressing plants and NaHS pre-treated plants exhibited higher endogenous H2S level and improved abiotic stress tolerance and biotic stress resistance, while cysteine desulfhydrases knockdown plants and HT pre-treated plants displayed lower endogenous H2S level and decreased stress resistance. Moreover, H2S upregulated the transcripts of multiple abiotic and biotic stress-related genes, and inhibited reactive oxygen species (ROS) accumulation. Interestingly, MIR393-mediated auxin signaling including MIR393a/b and their target genes (TIR1, AFB1, AFB2, and AFB3) was transcriptionally regulated by H2S, and was related with H2S-induced antibacterial resistance. Moreover, H2S regulated 50 carbon metabolites including amino acids, organic acids, sugars, sugar alcohols, and aromatic amines. Taken together, these results indicated that cysteine desulfhydrase and H2S conferred abiotic stress tolerance and biotic stress resistance, via affecting the stress-related gene expressions, ROS metabolism, metabolic homeostasis, and MIR393-targeted auxin receptors. © 2014 Institute of Botany, Chinese Academy of Sciences.

  14. The Significance of Hydrogen Sulfide for Arabidopsis Seed Germination.

    PubMed

    Baudouin, Emmanuel; Poilevey, Aurélie; Hewage, Nishodi Indiketi; Cochet, Françoise; Puyaubert, Juliette; Bailly, Christophe

    2016-01-01

    Hydrogen sulfide (H2S) recently emerged as an important gaseous signaling molecule in plants. In this study, we investigated the possible functions of H2S in regulating Arabidopsis seed germination. NaHS treatments delayed seed germination in a dose-dependent manner and were ineffective in releasing seed dormancy. Interestingly, endogenous H2S content was enhanced in germinating seeds. This increase was correlated with higher activity of three enzymes (L-cysteine desulfhydrase, D-cysteine desulfhydrase, and β-cyanoalanine synthase) known as sources of H2S in plants. The H2S scavenger hypotaurine and the D/L cysteine desulfhydrase inhibitor propargylglycine significantly delayed seed germination. We analyzed the germinative capacity of des1 seeds mutated in Arabidopsis cytosolic L-cysteine desulfhydrase. Although the mutant seeds do not exhibit germination-evoked H2S formation, they retained similar germination capacity as the wild-type seeds. In addition, des1 seeds responded similarly to temperature and were as sensitive to ABA as wild type seeds. Taken together, these data suggest that, although its metabolism is stimulated upon seed imbibition, H2S plays, if any, a marginal role in regulating Arabidopsis seed germination under standard conditions.

  15. Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

    PubMed

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

    2016-01-01

    Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC50>1 mM) on the activities of five major isoforms of human CYP in vitro.

  16. Annuaire du Bureau des longitudes - 2006

    NASA Astrophysics Data System (ADS)

    Imcce; Bureau Des Longitudes

    2005-07-01

    This annual publication provides ephemerides and data to the use of professionnal and amateur astronomers. Divided in 11 chapters it covers concordance of various calendars, explanation of fondamental astronomy and various time scales, explanation for the use of ephemerides; tables provide ephemerides (positions, rise/set/passage) of the Sun and the Moon, planets, planetary satellites, asteroids, comets, bright stars; data and explanation for the physical observation of the surface of the Sun, the Moon, and planets; chart of the sky and a list of constellations and galaxies; prediction and ephemerides for astronomical phenomenon: occultation by the moon, stellar occultations by asteroids and appulses, solar eclipses and lunar eclipses; and an additional review about a hot scientific topic, this year: "Legendre et le méridien terrestre, 200 ans après". Cette publication annuelle fournit des éphémérides et des données à l'usage des astronomes professionnels et des astronomes amateurs. Composée de 11 chapitres elle comprend les rubriques sur les différents calendriers et leurs concordance, les fêtes légales en France, les dates et décrets sur les heures légales en France métropolitaine ; une introduction à l'astronomie fondamentale et aux différentes échelles de temps, des explications sur l'utilisation des éphémérides ; des tables fournissent les éphémérides (positions, heures de lever/coucher/passage) du Soleil et de la Lune, de planètes, de satellites naturels, d'astéroïdes, de comètes, d'étoiles brillantes ; des données pour l'observation de la surface du Soleil, de la Lune, et des planètes ; des cartes du ciel ainsi qu'une liste de constellations et de galaxies ; des prédictions des phénomènes astronomiques : occultation par la Lune, occultation stellaires par des astéroïdes et appulses, éclipses de Soleil et de la Lune; la liste et les coordonnées des observatoires astronomiques les plus connus ; et enfin un cahier th

  17. Brain stem hypoplasia associated with Cri-du-Chat syndrome.

    PubMed

    Hong, Jin Ho; Lee, Ha Young; Lim, Myung Kwan; Kim, Mi Young; Kang, Young Hye; Lee, Kyung Hee; Cho, Soon Gu

    2013-01-01

    Cri-du-Chat syndrome, also called the 5p-syndrome, is a rare genetic abnormality, and only few cases have been reported on its brain MRI findings. We describe the magnetic resonance imaging findings of a 1-year-old girl with Cri-du-Chat syndrome who showed brain stem hypoplasia, particularly in the pons, with normal cerebellum and diffuse hypoplasia of the cerebral hemispheres. We suggest that Cri-du-Chat syndrome chould be suspected in children with brain stem hypoplasia, particularly for those with high-pitched cries.

  18. Brain Stem Hypoplasia Associated with Cri-du-Chat Syndrome

    PubMed Central

    Hong, Jin Ho; Lim, Myung Kwan; Kim, Mi Young; Kang, Young Hye; Lee, Kyung Hee; Cho, Soon Gu

    2013-01-01

    Cri-du-Chat syndrome, also called the 5p-syndrome, is a rare genetic abnormality, and only few cases have been reported on its brain MRI findings. We describe the magnetic resonance imaging findings of a 1-year-old girl with Cri-du-Chat syndrome who showed brain stem hypoplasia, particularly in the pons, with normal cerebellum and diffuse hypoplasia of the cerebral hemispheres. We suggest that Cri-du-Chat syndrome chould be suspected in children with brain stem hypoplasia, particularly for those with high-pitched cries. PMID:24265573

  19. Developmental and behavioural characteristics of cri du chat syndrome.

    PubMed Central

    Cornish, K M; Pigram, J

    1996-01-01

    Developmental and behavioural characteristics were assessed in 27 children with cri du chat syndrome using the Society for the Study of Behavioural Phenotypes questionnaire, which gave information on prenatal and perinatal conditions, neurological problems, and developmental and behavioural difficulties. The findings suggest that the behavioural profile of children with cri du chat syndrome incorporates self injurious behaviour, repetitive movements, hypersensitivity to sound, clumsiness, and obsessive attachments to objects. In terms of a developmental profile, children with cri du chat syndrome were able to communicate their needs, socially interact with others, and have some degree of mobility. PMID:8957962

  20. Depleted uranium (DU): a holistic consideration of DU and related matters.

    PubMed

    Hamilton, E I

    2001-12-17

    Following the use of depleted uranium (DU) during the Gulf and Balkan conflicts, unnecessary and costly confusion has existed for some 11 years concerning the hazard it constitutes, despite the fact that sufficient data are available to answer most of the relevant questions. In tracing the significance of uranium in the environment and humans, too much reliance is still placed upon the extrapolation of animal data. The existing radiological nomenclature is far too involved and complex to understand, let alone implement. The excellence of early health physics seems to have been lost, and hence there is a failure to utilise the large body of knowledge, and the manner in which it was obtained, in other disciplines. Health physics has failed to understand the nature of some natural processes that ultimately control radiation dose to the environment and humans. Examination of three types of DU, in particular the highly radioactive and potentially hazardous unprocessed, spent-reactor uranium fuel debris (UDU), alluded to as hot particles, has been poorly studied on the basis of scarcity in the environment. Fundamental geological processes are described which illustrate that, as a consequence of routine operation of nuclear reprocessing plants, especially in the past, and following reactor accidents, natural processes can result in an enrichment of DU particles in most types of sediment. Failure to grasp essential geological processes in relation to the dispersion of radionuclides in the environment is detrimental to public acceptance of an essential form of energy in association with others.

  1. Selective Loss of Cysteine Residues and Disulphide Bonds in a Potato Proteinase Inhibitor II Family

    PubMed Central

    Li, Xiu-Qing; Zhang, Tieling; Donnelly, Danielle

    2011-01-01

    Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution. PMID:21494600

  2. Activation of human acid sphingomyelinase through modification or deletion of C-terminal cysteine.

    PubMed

    Qiu, Huawei; Edmunds, Tim; Baker-Malcolm, Jennifer; Karey, Kenneth P; Estes, Scott; Schwarz, Cordula; Hughes, Heather; Van Patten, Scott M

    2003-08-29

    One form of Niemann-Pick disease is caused by a deficiency in the enzymatic activity of acid sphingomyelinase. During efforts to develop an enzyme replacement therapy based on a recombinant form of human acid sphingomyelinase (rhASM), purified preparations of the recombinant enzyme were found to have substantially increased specific activity if cell harvest media were stored for several weeks at -20 degrees C prior to purification. This increase in activity was found to correlate with the loss of the single free thiol on rhASM, suggesting the involvement of a cysteine residue. It was demonstrated that a variety of chemical modifications of the free cysteine on rhASM all result in substantial activation of the enzyme, and the modified cysteine responsible for this activation was shown to be the C-terminal residue (Cys629). Activation was also achieved by copper-promoted dimerization of rhASM (via cysteine) and by C-terminal truncation using carboxypeptidase Y. The role of the C-terminal cysteine in activation was confirmed by creating mutant forms of rhASM in which this residue was either deleted or replaced by a serine, with both forms having substantially higher specific activity than wild-type rhASM. These results indicate that purified rhASM can be activated in vitro by loss of the free thiol on the C-terminal cysteine via chemical modification, dimerization, or deletion of this amino acid residue. This method of activation is similar to the cysteine switch mechanism described previously for matrix metalloproteinases and could represent a means of posttranslational regulation of ASM activity in vivo.

  3. Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1

    PubMed Central

    Ibarra, Yessenia

    2013-01-01

    The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel. PMID:23478802

  4. Benzoquinone reveals a cysteine-dependent desensitization mechanism of TRPA1.

    PubMed

    Ibarra, Yessenia; Blair, Nathaniel T

    2013-05-01

    The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel.

  5. In silico designing of a new cysteine analogue of hirudin variant 3 for site specific PEGylation

    PubMed Central

    Sajjadi, Seyed Mehdi; Rahimi, Hamzeh; Mohammadi, Saeed; Faranoush, Mohammad; Mirzahoseini, Hasan; Toogeh, Gholamreza

    2017-01-01

    Hirudin is an anticoagulant agent of the salivary glands of the medicinal leech. Recombinant hirudin (r-Hir) displays certain drawbacks including bleeding and immunogenicity. To solve these problems, cysteine-specific PEGylation has been proposed as a successful technique. However, proper selection of the appropriate cysteine residue for substitution is a critical step. This study has, for the first time, used a computational approach aimed at identifying a single potential PEGylation site for replacement by cysteine residue in the hirudin variant 3 (HV3). Homology modeling (HM) was performed using MODELLER. All non-cysteine residues of the HV3 were replaced with the cysteine. The best model was selected based on the results of discrete optimized protein energy score, PROCHECK software, and Verify3D. The receptor binding was investigated using protein-protein docking by ClusPro web tool which was then visualized using LigPlot+ software and PyMOL. Finally, multiple sequence alignment (MSA) using ClustalW software and disulfide bond prediction were performed. According to the results of HM and docking, Q33C, which was located on the surface of the protein, was the best site for PEGylation. Furthermore, MSA showed that Q33 was not a conserved residue and LigPlot+ software showed that it is not involved in the hirudin-thrombin binding pocket. Moreover, prediction softwares established that it is not involved in disulfide bond formation. In this study, for the first time, the utility of the in silico approach for creating a cysteine analogue of HV3 was introduced. Our study demonstrated that the substitution of Q33 by cysteine probably has no effect on the biological activity of the HV3. However, experimental analyses are required to confirm the results. PMID:28255315

  6. Un cas de fracture luxation négligée du coude avec conservation de la fonction du coude

    PubMed Central

    Lahrach, Kamal; Ammoumri, Oussama; Mezzani, Amine; Benabid, Mounir; Marzouki, Amine; Boutayeb, Fawzi

    2015-01-01

    Les fractures luxations du coude sont rares et souvent mal tolérées chez les sujets jeunes actifs. Nous rapportons un cas de fracture-luxation du coude remontant à 20 ans. C'est un jeune de 35 ans, victime il y a 20 ans d'un traumatisme fermé, suite à une chute lors d'un match du football, de son coude gauche occasionnant une fracture-luxation du coude. Le patient a refusé une intervention chirurgicale avec une auto-rééducation. L'examen a mis en évidence une conservation de la fonction du coude. Un bilan radiologique a montré une fracture luxation du coude avec remaniement de la palette humérale. Une abstention thérapeutique a été décidée devant l'ancienneté de la fracture-luxation et la gêne fonctionnelle minime engendrée. Contrairement aux autres séries, la fracture-luxation dans notre cas était bien tolérée malgré le jeune âge du patient. PMID:26113930

  7. Metabolism of cysteine and cysteinesulfinate in rat kidney tubules

    SciTech Connect

    De La Rosa, J.; Stipanuk, M.H.

    1986-05-01

    In studies with rat hepatocytes, hypotaurine plus taurine production accounted for less than 5% of the total amount of cysteine (CYS) catabolized, whereas more than 90% of the metabolized cysteinesulfinate (CSA) was converted to taurine plus hypotaurine. Similar studies have been carried out with kidney tubules isolated from fed rats and incubated with 2 mM (1-/sup 14/C)CYS or 25 mM (1-/sup 14/C)CSA at 37/sup 0/C for up to 40 min. The production of /sup 14/CO/sub 2/ from CSA (3.1 +/- 1.3 nmol/sup ./ min/sup -1//sup ./ mg dry wt/sup -1/) was equivalent to the accumulation of N in NH/sub 4//sup +/ plus glutamate. Substantial oxidation of CYS was observed (16 +/- 11 nmol CO/sub 2/ x min/sup -1/ x mg dry wt/sup -1/), but only 12% of the expected amount of N was recovered as NH/sub 4//sup +/ plus glutamate. Accumulation of hypotaurine plus taurine was equivalent to 20% of the observed rate of /sup 14/CO/sub 2/ production from CSA but accounted for only 2% of the observed rate of /sup 14/CO/sub 2/ production from CYS. Addition of unlabeled CSA to incubations with varying levels of CYS had no effect on production of /sup 14/CO/sub 2/. Addition of 2 mM ..cap alpha..-ketoglutarate to the incubation mixtures resulted in an increased in /sup 14/CO/sub 2/ production from CSA to 290% of the control level but had no effect on CYS oxidation. In agreement with the authors findings for rat hepatocytes, these data suggest that most metabolism of CYS by the rat kidney tubule occurs by a CSA-independent pathway. However, in contrast to the metabolism of CSA almost entirely to taurine in the hepatocyte, kidney tubules appeared to metabolize CSA primarily by the transamination pathway.

  8. Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

    PubMed Central

    2011-01-01

    Background Acetaminophen-cysteine adducts (APAP-CYS) are a specific biomarker of acetaminophen exposure. APAP-CYS concentrations have been described in the setting of acute overdose, and a concentration >1.1 nmol/ml has been suggested as a marker of hepatic injury from acetaminophen overdose in patients with an ALT >1000 IU/L. However, the concentrations of APAP-CYS during therapeutic dosing, in cases of acetaminophen toxicity from repeated dosing and in cases of hepatic injury from non-acetaminophen hepatotoxins have not been well characterized. The objective of this study is to describe APAP-CYS concentrations in these clinical settings as well as to further characterize the concentrations observed following acetaminophen overdose. Methods Samples were collected during three clinical trials in which subjects received 4 g/day of acetaminophen and during an observational study of acetaminophen overdose patients. Trial 1 consisted of non-drinkers who received APAP for 10 days, Trial 2 consisted of moderate drinkers dosed for 10 days and Trial 3 included subjects who chronically abuse alcohol dosed for 5 days. Patients in the observational study were categorized by type of acetaminophen exposure (single or repeated). Serum APAP-CYS was measured using high pressure liquid chromatography with electrochemical detection. Results Trial 1 included 144 samples from 24 subjects; Trial 2 included 182 samples from 91 subjects and Trial 3 included 200 samples from 40 subjects. In addition, we collected samples from 19 subjects with acute acetaminophen ingestion, 7 subjects with repeated acetaminophen exposure and 4 subjects who ingested another hepatotoxin. The mean (SD) peak APAP-CYS concentrations for the Trials were: Trial 1- 0.4 (0.20) nmol/ml, Trial 2- 0.1 (0.09) nmol/ml and Trial 3- 0.3 (0.12) nmol/ml. APAP-CYS concentrations varied substantially among the patients with acetaminophen toxicity (0.10 to 27.3 nmol/ml). No subject had detectable APAP-CYS following exposure to

  9. Sensitive signal-on fluorescent sensing for copper ions based on the polyethyleneimine-capped silver nanoclusters-cysteine system.

    PubMed

    Zhang, Na; Qu, Fei; Luo, Hong Qun; Li, Nian Bing

    2013-08-12

    In this work, we present a label-free sensor for copper ions. This sensor is composed of silver nanoclusters and cysteine. The fluorescence of the silver nanoclusters was quenched by cysteine, which was recovered in the presence of copper ions. This binding of silver nanoclusters to cysteine promoted agglomeration of silver nanoclusters to yield larger non-fluorescent silver nanoparticles. The presence of copper ions resulted in the oxidation of cysteine to form a disulfide compound, leading to recovery of fluorescence of the silver nanoclusters. The fluorescence of the silver nanoclusters in the presence of cysteine increased with increasing concentration of copper ions in the range of 10-200 nM. The detection limit of this sensor for copper ions was 2.3 nM. The silver nanoclusters-cysteine sensor provides a simple, cost-effective, and sensitive platform for the detection of copper ions.

  10. Le mouvement du pôle

    NASA Astrophysics Data System (ADS)

    Bizouard, Christian

    2012-03-01

    Les variations de la rotation terrestre. En conditionnant à la fois notre vie quotidienne, notre perception du ciel, et bon nombre de phénomènes géophysiques comme la formation des cyclones, la rotation de la Terre se trouve au croisement de plusieurs disciplines. Si le phenomena se faisait uniformément, le sujet serait vite discuté, mais c'est parce que la rotation terrestre varie, même imperceptiblement pour nos sens, dans sa vitesse angulaire comme dans la direction de son axe, qu'elle suscite un grand intérêt. D'abord pour des raisons pratiques : non seulement les aléas de la rotation terrestre modi_ent à la longue les pointés astrométriques à un instant donné de la journée mais in_uencent aussi les mesures opérées par les techniques spatiales ; en consequence l'exploitation de ces mesures, par exemple pour déterminer les orbites des satellites impliqués ou pratiquer le positionnement au sol, nécessite une connaissance précise de ces variations. Plus fondamentalement, elles traduisent les propriétés globales de la Terre comme les processus physiques qui s'y déroulent, si bien qu'en analysant les causes des fluctuations observées, on dispose d'un moyen de mieux connaître notre globe. La découverte progressive des fluctuations de la rotation de la Terre a une longue histoire. Sous l'angle des techniques d'observation, trois époques se pro-celle du pointé astrométrique à l'oeil nu, à l'aide d'instruments en bois ou métalliques (quart de cercle muraux par exemple). À partir du XVIIe siècle débute l'astrométrie télescopique dont les pointés sont complétés par des datations de plus en plus précises grâce à l'invention d'horloges régulées par balancier. Cette deuxième époque se termine vers 1960, avec l'avènement des techniques spatiales : les pointés astrométriques sont délaissés au profit de la mesure ultra-précise de durées ou de fréquences de signaux électromagnétiques, grâce à l'invention des horloges

  11. Carte du Ciel, San Fernando zone

    NASA Astrophysics Data System (ADS)

    Abad, C.

    2014-06-01

    An updated summary of a future large astrometric catalogue is presented, based on the two most important astrometric projects carried out by the Real Instituto y Observatorio de la Armada de San Fernando (ROA). The goal is to make a catalogue of positions and proper motions based on ROA's Cart du Ciel (CdC) and the Astrographic Catalogue (AC) San Fernando zone plates, and the HAMC2 meridian circle catalogue. The CdC and AC plates are being reduced together to provide first-epoch positions while HAMC2 will provide second-epoch ones. New techniques have been applied, that range from using a commercial flatbed scanner to the proper reduction schemes to avoid systematics from it. Only thirty plates (out of 540) remain to be processed, due to scanning problems that are being solved.

  12. Diagnostic et prise en charge du psoriasis

    PubMed Central

    Kim, Whan B.; Jerome, Dana; Yeung, Jensen

    2017-01-01

    Résumé Objectif Présenter aux cliniciens en soins primaires un aperçu pratique et à jour du diagnostic et de la prise en charge du psoriasis. Sources des données Une recension a été effectuée dans les bases de données de PubMed, MEDLINE, EMBASE et Cochrane pour trouver des méta-analyses, des études randomisées contrôlées, des revues systématiques et des études observationnelles pertinentes portant sur le diagnostic et la prise en charge du psoriasis. Message principal Le psoriasis est une maladie inflammatoire chronique et multisystémique qui affecte principalement la peau et les articulations. En plus des dimensions physiques de la maladie, le psoriasis a des répercussions émotionnelles et psychosociales considérables sur les patients, et nuit au fonctionnement social et aux relations interpersonnelles. En tant que maladie inflammatoire systémique, le psoriasis est associé à de multiples comorbidités, dont les maladies cardiovasculaires et les cancers. Le diagnostic est principalement d’ordre clinique et une biopsie de la peau est rarement nécessaire. Selon la sévérité de la maladie, un traitement approprié peut être amorcé. Pour les cas de légers à modérés, le traitement de première intention comporte des thérapies topiques, dont les corticostéroïdes, les analogues de la vitamine D3 et des produits combinés. Ces traitements topiques sont efficaces et peuvent être initiés et prescrits en toute sécurité par des médecins de soins primaires. Les patients dont les symptômes sont plus graves et réfractaires pourraient devoir être envoyés en consultation auprès d’un dermatologue pour une évaluation plus approfondie et une thérapie systémique. Conclusion De nombreux patients atteints de psoriasis consultent leur médecin de soins primaires pour une évaluation initiale et pour recevoir un traitement. La reconnaissance du psoriasis, de même que des comorbidités médicales et psychiatriques qui lui sont associ

  13. Classification moléculaire du cancer du sein au Maroc

    PubMed Central

    Fouad, Abbass; Yousra, Akasbi; Kaoutar, Znati; Omar, El Mesbahi; Afaf, Amarti; Sanae, Bennis

    2012-01-01

    Introduction La classification moléculaire des cancers du sein basée sur l'expression génique puis sur le profil protéique a permis de distinguer cinq groupes moléculaires: luminal A, luminal B, Her2/neu, basal-like et non-classées. L'objectif de cette étude réalisée au CHU Hassan II de Fès est de classer 335 cancers du sein infiltrant en groupes moléculaires, puis de les corréler avec les caractéristiques clinicopathologiques. Méthodes Etude rétrospective étalée sur 45 mois, comportant 335 patientes colligées au CHU pour le diagnostic et le suivi. Les tumeurs sont analysées histologiquement et classées après une étude immunohistochimique en groupes: luminal A, luminal B, Her2+, basal-like et non-classées. Résultats 54.3% des tumeurs sont du groupe luminal A, 16% luminal B, 11.3% Her2+, 11.3% basal-like et 7% non-classées. Le groupe luminal A renferme le plus faible taux de grade III, d'emboles vasculaires ainsi que de métastases; alors que le groupe des non-classées et basal-like représentent un taux élevé de grade III, une faible proportion d'emboles vasculaires et d'envahissement ganglionnaire. Ces facteurs sont significativement élevés dans les groupes luminal B et Her2+ avec un taux de survie globale de 78% et 76% respectivement. Dans le groupe luminal A, la survie globale des patientes est élevée (87%) alors qu'elle n'est que de 49% dans le groupe des triples négatifs (basal-like et non-classés). Conclusion Le groupe luminal B est différent du luminal A et il est de pronostic péjoratif vis à vis du groupe Her2+. Les caractéristiques clinicopathologiques concordent avec le profil moléculaire donc devraient être pris en considération comme facteurs pronostiques. PMID:23396646

  14. Impact socio professionnel de la libération chirurgicale du syndrome du canal carpien

    PubMed Central

    Kraiem, Aouatef Mahfoudh; Hnia, Hajer; Bouzgarrou, Lamia; Henchi, Mohamed Adnène; Khalfallah, Taoufik

    2016-01-01

    L’objectif de notre travail était d’étudier les conséquences socioprofessionnelles d’une libération chirurgicale du SCC. Il s’agit d’une étude transversale portant sur les sujets opérés pour un SCC d’origine professionnelle ; recensés dans le Service de Médecine de Travail et de Pathologies Professionnelles au CHU Tahar Sfar de Mahdia en Tunisie sur une période de 8 ans allant du 1 Janvier 2006 au mois Décembre 2013. Le recueil des données s’est basé sur une fiche d’enquête, portant sur la description des caractéristiques socioprofessionnelles, médicales, et sur le devenir professionnel des participants. Pour étudier les contraintes psychosociales au travail, nous avons adopté le questionnaire de Karasek. La durée d’arrêt de travail après libération chirurgicale du SCC était significativement liée à l’existence d’autres troubles musculo-squelettiques autre que le SCC, la déclaration du SCC en maladie professionnelle et à l’ancienneté professionnelle des salariés. Quant au devenir professionnel des salariés opérés, 50,7% ont gardé le même poste, 15,3% ont bénéficié d’un aménagement de poste et 33,8% ont bénéficié d’un changement de poste dans la même entreprise. Le devenir professionnel de ces salariés était corrélé à leurs qualifications professionnelles et au type de l’atteinte sensitive et/ou motrice du nerf médian à l’EMG. Un certain nombre de facteurs non lésionnels déterminaient la durée de l’arrêt de travail, alors que le devenir professionnel des opérés pour SCC dépendait essentiellement de leurs qualifications professionnelles et des données de l’électromyogramme. Il est certain que des travaux beaucoup plus larges permettraient d’affiner encore ces résultats. PMID:27800089

  15. L-Cysteine and L-AP4 microinjections in the rat caudal ventrolateral medulla decrease arterial blood pressure.

    PubMed

    Takemoto, Yumi

    2014-12-01

    The thiol amino acid L-cysteine increases arterial blood pressure (ABP) when injected into the cerebrospinal fluid space in conscious rats, indicating a pressor response to centrally acting L-cysteine. A prior synaptic membrane binding assay suggests that L-cysteine has a strong affinity for the L-2-amino-4-phosphonobutyric acid (L-AP4) binding site. The central action of L-cysteine may be vial-AP4 sensitive receptors. The present study investigated cardiovascular responses to L-cysteine and L-ap4 microinjected into the autonomic area of the caudal ventrolateral medulla (CVLM) where inhibitory neurons regulate ABP via pre-sympathetic vasomotor neurons. Both the injection of L-cysteine and L-AP4 in the CVLM sites identified with L-glutamate produced the same depressor and bradycardic responses in urethane-anesthetized rats. Neither a prior antagonist microinjection of MK801 for the N-methyl-D-aspartate (NMDA) receptor nor CNQX for the non-NMDA receptor attenuated the responses to L-cysteine, but the combination of the two receptor blocking with an additional prior injection abolished the response. In contrast, either receptor blockade alone abolished the response to L-AP4, indicating distinct mechanisms between responses to L-cysteine and L-AP4 in the CVLM. The results indicate that the CVLM is a central active site for L-cysteine's cardiovascular response. Central L-cysteine's action could be independent of the L-AP4 sensitive receptors. Cardiovascular regulation may involve endogenous L-cysteine in the CVLM. Further multidisciplinary examinations are required to elaborate on L-cysteine's functional roles in the CVLM.

  16. DFT study of the adsorption of D-(L-)cysteine on flat and chiral stepped gold surfaces.

    PubMed

    Fajín, José L C; Gomes, José R B; Cordeiro, M Natália D S

    2013-07-16

    The adsorption of cysteine onto the intrinsically chiral gold surface, Au(321)(R,S), was investigated by means of a periodic supercell density functional theory approach. The results are compared to those obtained at the same level of theory with a nonchiral surface having the same terrace orientation, the Au(111) surface. Neutral and zwitterionic cysteine forms of the L and D enantiomers are considered, as are surface coverage effects. It was found that at high coverage the zwitterionic forms of L- and D-cysteine are more stable on the Au(321)(R,S) faces of the stepped surface and also on the flat Au(111) surface, leading to highly organized cysteine monolayers. However, at low coverage the adsorption of cysteine dimers, with the pairs interacting through their carbonyl groups, is more favorable than or at least equally favorable to the adsorption of single cysteine molecules on both surfaces. A comparison between the cysteine adsorption on the two different surface structures shows that the adsorption on the stepped surface is clearly more favorable than on the flat surface, revealing the importance of the low-coordinated gold atoms in the adsorption of these species. Furthermore, non-negligible differences between the adsorption energy of the enantiomers of cysteine were found both at high and low coverage, thus showing the enantiospecificity of this intrinsically chiral surface regarding cysteine adsorption. This adsorption occurs with the cysteine binding the surface through only one contact point (by its sulfur atom), in contrast to previous work where the enantiospecific adsorption of cysteine has been related to two nonequivalent binding sites of the cysteine enantiomers with the surface.

  17. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination

    PubMed Central

    2012-01-01

    Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role

  18. Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination.

    PubMed

    Lepelley, Maud; Amor, Mohamed Ben; Martineau, Nelly; Cheminade, Gerald; Caillet, Victoria; McCarthy, James

    2012-03-01

    Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Two cysteine proteinase (CP) and four cysteine proteinase inhibitor (CPI) gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death

  19. Characterization of biodegradable films obtained from cysteine-mediated polymerized gliadins.

    PubMed

    Hernandez-Munoz, Pilar; Kanavouras, Antonis; Villalobos, Ricardo; Chiralt, Amparo

    2004-12-29

    This study focuses on the effect exerted by interchain disulfide bonds on the functional properties of films made from gliadins when cross-linked with cysteine. Gliadins were extracted from commercial wheat gluten with 70% aqueous ethanol, and cysteine was added to the film-forming solution to promote cross-linking between protein chains. The formation of interchain disulfide bonds was assessed by SDS-PAGE analysis. Gliadin films treated with cysteine maintain their integrity in water and become less extensible while their tensile strength increases as a consequence of the development of a more rigid network. The glass transition temperature of cross-linked films shifts to slightly higher values. The plasticizing effects of glycerol and moisture are also demonstrated. The mechanical behavior of cysteine-cross-linked gliadin films was compared to that of polymeric glutenins. Cross-linked gliadins displayed tensile strength values similar to those of glutenin films but achieved slightly lower elongation values. Cysteine-cross-linked gliadin films present the advantage that they are ethanol soluble, facilitating film fabrication or their application as a coating for food or for any other film or surface.

  20. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A

    PubMed Central

    Mallorquí-Fernández, Noemí; Manandhar, Surya P.; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B.; Enghild, Jan J.; Potempa, Jan; Gomis-Rüth, F.Xavier

    2009-01-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defences and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and its self-processed mature form. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin and a latency flap in the zymogen. Dramatic rearrangement of up to 20Å of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain. PMID:17993455

  1. The effect of cysteine on production of volatile sulphur compounds by cheese-ripening bacteria.

    PubMed

    del Castillo-Lozano, M López; Mansour, S; Tâche, R; Bonnarme, P; Landaud, S

    2008-03-20

    The effect of cysteine on the ability of smear cheese-ripening bacteria (Brevibacterium linens and Arthrobacter spp) to produce volatile sulphur compounds (VSC) from methionine was studied. These bacteria were cultivated in a synthetic medium supplemented with various cysteine concentrations with or without methionine. Cultures with only cysteine showed slightly lower levels of VSC produced and an unpleasant odour like rotten eggs, resulting from hydrogen sulphide production. The levels and profiles of VSC produced with supplemented methionine-cysteine mixtures had strain-dependant behaviours. However, the highest levels of dimethyl disulfide, dimethyl trisulfide and dimethyl tetrasulfide were observed when increasing the cysteine concentration from 0.2 to 1.0 gl(-1) at the same methionine concentration (1.0 gl(-1)). In contrast, production levels of thioesters, especially S-methylthio acetate, were reduced by 50 and 80% under such conditions. An initial sensory approach showed that such an effect could have a strong impact on the global odour of ripened cheeses.

  2. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine.

    PubMed

    Ma, Dong Hee; Kim, Dokyoung; Akisawa, Takuya; Lee, Kyung-Ha; Kim, Kyong-Tai; Ahn, Kyo Han

    2015-04-01

    A novel FRET couple of fluorescein is disclosed, and it was readily constructed by conjugating an amino-BODIPY dye, a new FRET donor, with fluorescein isocyanate. Its potential was demonstrated by a fluorescence sensing system for cysteine, which was prepared by introducing acryloyl groups to the fluorescein moiety. The FRET probe exhibited promising ratiometric response to cysteine with high selectivity and sensitivity in a buffer solution containing acetonitrile at a physiological pH of 7.4, but showed slow reactivity. This slow response was solved by addition of a surfactant, thus allowing ratiometric imaging and determination of the endogenous level of cysteine in cells in HEPES buffer, by confocal fluorescence microscopy. Imaging experiments toward various cells suggested that such aryl acrylate type probes are vulnerable to the ubiquitous esterase activity. For the selected C6 cell line, in which the esterase activity was minimal, the ratiometric quantification of cysteine level was demonstrated. The FRET probe was also applied to determine the level of cysteine in human blood plasma.

  3. Mechanistic study for immobilization of cysteine-labeled oligopeptides on UV-activated surfaces.

    PubMed

    Ong, Lian Hao; Ding, Xiaokang; Yang, Kun-Lin

    2014-10-01

    In this study, we report immobilization of cysteine-labeled oligopeptides on UV activated surfaces decorated with N,N-dimethyl-n-octadecyl-3-aminopropyltrimethoxysilyl chloride (DMOAP). Our result shows that cysteine group, regardless of its position in the oligopeptide, is essential for successful immobilization of oligopeptide on the UV-activated surface. A possible reaction mechanism is nucleophilic addition of thiolates to surface aldehyde groups generated during UV activation. By using this technique, we are able to incorporate anchoring points into oligopeptides through cysteine residues. Furthermore, immobilized oligopeptides on the UV-activated surface is very stable even under harsh washing conditions. Finally, we show that an HPQ-containing oligopeptide can be immobilized on the UV-activated surface, but the final surface density and its ability to bind streptavidin are affected by the position of cysteine and HPQ. An oligopeptide with a cysteine at the N-terminus and a HPQ motif at the C-terminus gives the highest binding signal in the streptavidin-binding assay. This result is potentially useful for the development of functional oligopeptide microarrays for detecting target protein molecules.

  4. Effects of L-cysteine on lead acetate induced neurotoxicity in albino mice.

    PubMed

    Mahmoud, Y I; Sayed, S S

    2016-07-01

    Lead is a toxic heavy metal that adversely affects nervous tissues; it often occurs as an environmental pollutant. We investigated histological changes in the cerebral cortex, hippocampus and cerebellum of adult albino mice following exposure to lead acetate. We also studied the possible ameliorative effect of the chelating agent, L-cysteine, on lead-induced neurotoxicity. We divided albino mice into six groups: 1) vehicle-only control, 2) L-cysteine control, 3 and 4) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, and 5 and 6) treated for 7 days with 20 and 40 mg/kg lead acetate, respectively, followed by 50 mg/kg L-cysteine for 7 days. Lead acetate administration caused disorganization of cell layers, neuronal loss and degeneration, and neuropil vacuolization. Brain sections from lead-intoxicated mice treated with L-cysteine showed fewer pathological changes; the neuropil showed less vacuolization and the neurons appeared less damaged. L-cysteine at the dose we used only marginally alleviated lead-induced toxicity.

  5. Sulfhydryl-specific PEGylation of phosphotriesterase cysteine mutants for organophosphate detoxification.

    PubMed

    Daffu, Gurdip K; Lopez, Patricia; Katz, Francine; Vinogradov, Michael; Zhan, Chang-Guo; Landry, Donald W; Macdonald, Joanne

    2015-11-01

    The catalytic bioscavenger phosphotriesterase (PTE) is experimentally an effective antidote for organophosphate poisoning. We are interested in the molecular engineering of this enzyme to confer additional functionality, such as improved in vivo longevity. To this aim, we developed PTE cysteine mutants with free sulfhydryls to allow macromolecular attachments to the protein. A library of PTE cysteine mutants were assessed for efficiency in hydrolysing the toxic pesticide metabolite paraoxon, and screened for attachment with a sulfhydryl-reactive small molecule, fluorescein 5-maleimide (F5M), to examine cysteine availability. We established that the newly incorporated cysteines were readily available for labelling, with R90C, E116C and S291C displaying the highest affinity for binding with F5M. Next, we screened for efficiency in attaching a large macromolecule, a 30 000 Da polyethylene glycol (PEG) molecule. Using a solid-phase PEGylation strategy, we found the E116C mutant to be the best single-mutant candidate for attachment with PEG30. Kinetic activity of PEGylated E116C, with paraoxon as substrate, displayed activity approaching that of the unPEGylated wild-type. Our findings demonstrate, for the first time, an efficient cysteine mutation and subsequent method for sulfhydryl-specific macromolecule attachment to PTE.

  6. Characterizations of Three Major Cysteine Sensors of Keap1 in Stress Response.

    PubMed

    Saito, Ryota; Suzuki, Takafumi; Hiramoto, Keiichiro; Asami, Soichiro; Naganuma, Eriko; Suda, Hiromi; Iso, Tatsuro; Yamamoto, Hirotaka; Morita, Masanobu; Baird, Liam; Furusawa, Yuki; Negishi, Takaaki; Ichinose, Masakazu; Yamamoto, Masayuki

    2015-11-02

    The Keap1-Nrf2 system plays a central role in cytoprotection against electrophilic/oxidative stresses. Although Cys151, Cys273, and Cys288 of Keap1 are major sensor cysteine residues for detecting these stresses, it has not been technically feasible to evaluate the functionality of Cys273 or Cys288, since Keap1 mutants that harbor substitutions in these residues and maintain the ability to repress Nrf2 accumulation do not exist. To overcome this problem, we systematically introduced amino acid substitutions into Cys273/Cys288 and finally identified Cys273Trp and Cys288Glu mutations that do not affect Keap1's ability to repress Nrf2 accumulation. Utilizing these Keap1 mutants, we generated stable murine embryonic fibroblast (MEF) cell lines and knock-in mouse lines. Our analyses with the MEFs and peritoneal macrophages from the knock-in mice revealed that three major cysteine residues, Cys151, Cys273, and Cys288, individually and/or redundantly act as sensors. Based on the functional necessity of these three cysteine residues, we categorized chemical inducers of Nrf2 into four classes. Class I and II utilizes Cys151 and Cys288, respectively, while class III requires all three residues (Cys151/Cys273/Cys288), while class IV inducers function independently of all three of these cysteine residues. This study thus demonstrates that Keap1 utilizes multiple cysteine residues specifically and/or collaboratively as sensors for the detection of a wide range of environmental stresses.

  7. Cloning and characterization of a cathepsin L-like cysteine protease from Taenia pisiformis.

    PubMed

    Wang, Qiuxia; Zhang, Shaohua; Luo, Xuenong; Hou, Junling; Zhu, Xueliang; Cai, Xuepeng

    2013-05-01

    Rabbit cysticercosis, caused by the larval stage of Taenia pisiformis, is a serious parasitic disease of rabbits. It was reported that some cysteine peptidases have potential roles in the pathogenesis of various parasitic infections. To investigate the biochemical characteristics and roles in the pathogenesis/host-invasion of cysteine peptidases, a cDNA sequence encoding for a cathepsin L-like cysteine protease (TpCP) was cloned and identified from the T. pisiformis metacestodes. This sequence was 1220 bp in its length, which included a 1017 bp open reading frame encoding a 339 amino acid peptide. Multiple sequence alignments revealed a 28.9-88.5% similarity with cathepsin L-like cysteine proteases from other helminth parasites and mammals. The recombinant TpCP expressed in Escherichia coli did not show the proteolytic activity by zymography gel assay. However, the TpCP expressed in Pichia pastoris had typical biochemical activities that could hydrolyze rabbit immunoglobulin G, bovine serum albumin and fibronectin. Substrate studies indicated pronounced cleavage of Z-Phe-Arg-AMC. This activity was sensitive to cysteine protease inhibitor E-64 and immunohistochemistry results also indicated that TpCP was distributed as an intense positive reaction in the bladder wall. Our results gave us insights into future studies of TpCP's roles in the infection.

  8. Cysteine modified rare-earth up-converting nanoparticles for in vitro and in vivo bioimaging.

    PubMed

    Wei, Zuwu; Sun, Lining; Liu, Jinliang; Zhang, Jin Z; Yang, Huiran; Yang, Yang; Shi, Liyi

    2014-01-01

    Cysteine, as a small organic molecule and amino acid, is a basic building block for proteins and has special physiological functions in vivo. Cysteine has strong affinity for cells, which can be taken advantage for various applications. A new and facile surface modification method has been developed for rare-earth doped upconversion nanoparticles (UCNs) using cysteine. Compared with unmodified samples, the water-solubility and biocompatibility of the cysteine modified NaYF4:Yb,Er and NaYF4:Yb,Tm UCNs (termed as UCN-Er-Cys and UCN-Tm-Cys, respectively) have been significantly improved, while their particle size and emission properties did not change substantially. Due to the low cytotoxicity as revealed by methyl thiazolyl tetrazolium assay, the cysteine modified UCNs were successfully applied to imaging of Hela cells in vitro and nude mouse in vivo. Most significant is that the method offers the advantages of ease of synthesis and handling as well as potentially low cost for biomedical emerging applications.

  9. Cysteine scanning of transmembrane domain three of the human dipeptide transporter: implications for substrate transport.

    PubMed

    Links, Jennifer L S; Kulkarni, Ashutosh A; Davies, Daryl L; Lee, Vincent H L; Haworth, Ian S

    2007-04-01

    The human intestinal dipeptide transporter (hPepT1) transports dipeptides and pharmacologically active drugs from the intestine to the blood. The role of transmembrane domain 3 (TMD3) of hPepT1 was studied using cysteine-scanning mutagenesis and methane thiosulfonate (MTS) cysteine modification. Each amino acid in TMD3 was individually mutated to a cysteine and Gly-Sar uptake by each mutated and modified transporter was determined relative to wild-type hPepT1. Uptake data for mutated transporters modified with the lipid-insoluble cysteine-modifying reagent MTSET suggested tilting of TMD3 relative to the substrate translocation pathway; the extracellular region of TMD3 showed little MTSET reactivity, indicative of solvent inaccessibility, whereas the intracellular part of TMD3 was relatively solvent accessible. Modification at 10 positions of TMD3 with MTSEA, a lipid-soluble cysteine-modifying reagent, gave unusual and statistically significant increases in Gly-Sar uptake relative to untreated mutants. We interpret these data in terms of the spatial properties of the hPepT1 substrate translocation channel and possible interactions of TMD3 with other transmembrane domains.

  10. The yeast TUM1 affects production of hydrogen sulfide from cysteine treatment during fermentation.

    PubMed

    Huang, Chien-Wei; Walker, Michelle E; Fedrizzi, Bruno; Roncoroni, Miguel; Gardner, Richard C; Jiranek, Vladimir

    2016-12-01

    The undesirable rotten-egg odour of hydrogen sulfide (H2S) produced by yeast shortly after yeast inoculation of grape musts might be an important source of desirable varietal thiols, which contribute to tropical aromas in varieties such as Sauvign-on Blanc. In this study, we observed that Saccharomyces cerevisiae strains produce an early burst of H2S from cysteine. Both Δmet2 and Δmet17 strains produce a larger burst, likely because they are unable to utilise the H2S in the sulfate assimilation pathway. For the first time, we show that TUM1 is partly responsible for the early production of H2S from cysteine. Overex-pressing TUM1 elevated production of H2S, whilst its deletion yields only half of the H2S. We further confirmed that yeast convert cysteine to H2S by analysing growth of mutants lacking components of the transsulfuration pathway. High concent-rations of cysteine overcame this growth block, but required TUM1 Collectively, the data indicate that S. cerevisiae does not convert cysteine to sulfate or sulfite, but rather to sulfide via a novel pathway that requires the action of Tum1p. The findi-ngs of this study may allow the improvement of commercial yeasts through the manipulation of sulfur metabolism that are better suited towards production of fruit-driven styles. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  11. Novel residues lining the CFTR chloride channel pore identified by functional modification of introduced cysteines.

    PubMed

    Fatehi, Mohammad; Linsdell, Paul

    2009-04-01

    Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current-voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl(-) permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.

  12. Determination of DNA damage in experimental liver intoxication and role of N-acetyl cysteine.

    PubMed

    Aksit, Hasan; Bildik, Aysegül

    2014-11-01

    The present study aimed at detecting DNA damage and fragmentation as well as histone acetylation depending on oxidative stress caused by CCl4 intoxication. Also, the protective role of N-acetyl cysteine, a precursor for GSH, in DNA damage is investigated. Sixty rats were used in this study. In order to induce liver toxicity, CCl4 in was dissolved in olive oil (1/1) and injected intraperitoneally as a single dose (2 ml/kg). N-acetyl cysteine application (intraperitoneal, 50 mg/kg/day) was started 3 days prior to CCl4 injection and continued during the experimental period. Control groups were given olive oil and N-acetyl cysteine. After 6 and 72 h of CCl4 injection, blood and liver tissue were taken under ether anesthesia. Nuclear extracts were prepared from liver. Changes in serum AST and ALT activities as well as MDA, TAS, and TOS levels showed that CCl4 caused lipid peroxidation and liver damage. However, lipid peroxidation and liver damage were reduced in the N-acetyl cysteine group. Increased levels in 8-hydroxy-2-deoxy guanosine and histone acetyltransferase activities, decreased histone deacetylase activities, and DNA breakage detected in nuclear extracts showed that CCl4 intoxication induces oxidative stress and apoptosis in rat liver. The results of the present study indicate that N-acetyl cysteine has a protective effect on CCl4-induced DNA damage.

  13. Cysteine Proteases: Modes of Activation and Future Prospects as Pharmacological Targets

    PubMed Central

    Verma, Sonia; Dixit, Rajnikant; Pandey, Kailash C.

    2016-01-01

    Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria, and parasite) to the higher organisms (mammals). Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases, and metalloproteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress, and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a prodomain (regulatory) and a mature domain (catalytic). The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein–protein interactions (PPIs) and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing) of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases. PMID:27199750

  14. Novel Cysteine-Centered Sulfur Metabolic Pathway in the Thermotolerant Methylotrophic Yeast Hansenula polymorpha

    PubMed Central

    Oh, Doo-Byoung; Kwon, Ohsuk; Lee, Sang Yup; Sibirny, Andriy A.; Kang, Hyun Ah

    2014-01-01

    In yeast and filamentous fungi, sulfide can be condensed either with O-acetylhomoserine to generate homocysteine, the precursor of methionine, or with O-acetylserine to directly generate cysteine. The resulting homocysteine and cysteine can be interconverted through transsulfuration pathway. Here, we systematically analyzed the sulfur metabolic pathway of the thermotolerant methylotrophic yeast Hansenula polymorpha, which has attracted much attention as an industrial yeast strain for various biotechnological applications. Quite interestingly, the detailed sulfur metabolic pathway of H. polymorpha, which was reconstructed based on combined analyses of the genome sequences and validation by systematic gene deletion experiments, revealed the absence of de novo synthesis of homocysteine from inorganic sulfur in this yeast. Thus, the direct biosynthesis of cysteine from sulfide is the only pathway of synthesizing sulfur amino acids from inorganic sulfur in H. polymorpha, despite the presence of both directions of transsulfuration pathway Moreover, only cysteine, but no other sulfur amino acid, was able to repress the expression of a subset of sulfur genes, suggesting its central and exclusive role in the control of H. polymorpha sulfur metabolism. 35S-Cys was more efficiently incorporated into intracellular sulfur compounds such as glutathione than 35S-Met in H. polymorpha, further supporting the cysteine-centered sulfur pathway. This is the first report on the novel features of H. polymorpha sulfur metabolic pathway, which are noticeably distinct from those of other yeast and filamentous fungal species. PMID:24959887

  15. A new autocatalytic activation mechanism for cysteine proteases revealed by Prevotella intermedia interpain A.

    PubMed

    Mallorquí-Fernández, Noemí; Manandhar, Surya P; Mallorquí-Fernández, Goretti; Usón, Isabel; Wawrzonek, Katarzyna; Kantyka, Tomasz; Solà, Maria; Thøgersen, Ida B; Enghild, Jan J; Potempa, Jan; Gomis-Rüth, F Xavier

    2008-02-01

    Prevotella intermedia is a major periodontopathogen contributing to human gingivitis and periodontitis. Such pathogens release proteases as virulence factors that cause deterrence of host defenses and tissue destruction. A new cysteine protease from the cysteine-histidine-dyad class, interpain A, was studied in its zymogenic and self-processed mature forms. The latter consists of a bivalved moiety made up by two subdomains. In the structure of a catalytic cysteine-to-alanine zymogen variant, the right subdomain interacts with an unusual prodomain, thus contributing to latency. Unlike the catalytic cysteine residue, already in its competent conformation in the zymogen, the catalytic histidine is swung out from its active conformation and trapped in a cage shaped by a backing helix, a zymogenic hairpin, and a latency flap in the zymogen. Dramatic rearrangement of up to 20A of these elements triggered by a tryptophan switch occurs during activation and accounts for a new activation mechanism for proteolytic enzymes. These findings can be extrapolated to related potentially pathogenic cysteine proteases such as Streprococcus pyogenes SpeB and Porphyromonas gingivalis periodontain.

  16. Cysteine Cathepsins as Regulators of the Cytotoxicity of NK and T Cells

    PubMed Central

    Perišić Nanut, Milica; Sabotič, Jerica; Jewett, Anahid; Kos, Janko

    2014-01-01

    Cysteine cathepsins are lysosomal peptidases involved at different levels in the processes of the innate and adaptive immune responses. Some, such as cathepsins B, L, and H are expressed constitutively in most immune cells. In cells of innate immunity they play a role in cell adhesion and phagocytosis. Other cysteine cathepsins are expressed more specifically. Cathepsin X promotes dendritic cell maturation, adhesion of macrophages, and migration of T cells. Cathepsin S is implicated in major histocompatibility complex class II antigen presentation, whereas cathepsin C, expressed in cytotoxic T lymphocytes and natural killer (NK) cells, is involved in processing pro-granzymes into proteolytically active forms, which trigger cell death in their target cells. The activity of cysteine cathepsins is controlled by endogenous cystatins, cysteine protease inhibitors. Of these, cystatin F is the only cystatin that is localized in endosomal/lysosomal vesicles. After proteolytic removal of its N-terminal peptide, cystatin F becomes a potent inhibitor of cathepsin C with the potential to regulate pro-granzyme processing and cell cytotoxicity. This review is focused on the role of cysteine cathepsins and their inhibitors in the molecular mechanisms leading to the cytotoxic activity of T lymphocytes and NK cells in order to address new possibilities for regulation of their function in pathological processes. PMID:25520721

  17. Paired natural cysteine mutation mapping: aid to constraining models of protein tertiary structure.

    PubMed Central

    Kreisberg, R.; Buchner, V.; Arad, D.

    1995-01-01

    This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur. PMID:8563638

  18. Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax.

    PubMed Central

    Na, Byoung-Kuk; Shenai, Bhaskar R; Sijwali, Puran S; Choe, Youngchool; Pandey, Kailash C; Singh, Ajay; Craik, Charles S; Rosenthal, Philip J

    2004-01-01

    Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity. PMID:14629194

  19. Characterization of cysteine protease-like genes in the striped rice stem borer, Chilo suppressalis.

    PubMed

    Ge, Zhao-Yu; Wan, Pin-Jun; Li, Guo-Qing; Xia, Yong-Gui; Han, Zhao-Jun

    2014-02-01

    The striped rice stem borer, Chilo suppressalis (Walker), is a major pest for rice production in China and the rest of Southeast Asia. Chemical control is the main means to alleviate losses due to this pest, which causes serious environmental pollution. An effective and environmentally friendly approach is needed for the management of the striped rice stem borer. Cysteine proteases in insects could be useful targets for pest management either through engineering plant protease inhibitors, targeting insect digestive cysteine proteases, or through RNA interference-based silencing of cysteine proteases, disrupting developmental regulation of insects. In this study, eight cysteine protease-like genes were identified and partially characterized. The genes CCO2 and CCL4 were exclusively expressed in the larval gut, and their expression was affected by the state of nutrition in the insect. The expression of CCL2, CCL3, and CCO1 was significantly affected by the type of host plant, suggesting a role in host plant - insect interactions. Our initial characterization of the striped rice stem borer cysteine protease-like genes provides a foundation for further research on this important group of genes in this major insect pest of rice.

  20. Identification of semicarbazones, thiosemicarbazones and triazine nitriles as inhibitors of Leishmania mexicana cysteine protease CPB.

    PubMed

    Schröder, Jörg; Noack, Sandra; Marhöfer, Richard J; Mottram, Jeremy C; Coombs, Graham H; Selzer, Paul M

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas' disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases.

  1. Ga2O3 photocatalyzed on-line tagging of cysteine to facilitate peptide mass fingerprinting.

    PubMed

    Qiao, Liang; Su, Fangzheng; Bi, Hongyan; Girault, Hubert H; Liu, Baohong

    2011-09-01

    β-Ga(2)O(3) is a wide-band-gap semiconductor having strong oxidation ability under light irradiation. Herein, the steel target plates modified with β-Ga(2)O(3) nanoparticles have been developed to carry out in-source photo-catalytic oxidative reactions for online peptide tagging during laser desorption/ionization mass spectrometry (LDI-MS) analysis. Under UV laser irradiation, β-Ga(2)O(3) can catalyze the photo-oxidation of 2-methoxyhydroquinone added to a sample mixture to 2-methoxy benzoquinone that can further react with the thiol groups of cysteine residues by Michael addition reaction. The tagging process leads to appearance of pairs of peaks with an m/z shift of 138.1Th. This online labelling strategy is demonstrated to be sensitive and efficient with a detection-limit at femtomole level. Using the strategy, the information on cysteine content in peptides can be obtained together with peptide mass, therefore constraining the database searching for an advanced identification of cysteine-containing proteins from protein mixtures. The current peptide online tagging method can be important for specific analysis of cysteine-containing proteins especially the low-abundant ones that cannot be completely isolated from other high-abundant non-cysteine-proteins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Papain protects papaya trees from herbivorous insects: role of cysteine proteases in latex.

    PubMed

    Konno, Kotaro; Hirayama, Chikara; Nakamura, Masatoshi; Tateishi, Ken; Tamura, Yasumori; Hattori, Makoto; Kohno, Katsuyuki

    2004-02-01

    Many plants contain latex that exudes when leaves are damaged, and a number of proteins and enzymes have been found in it. The roles of those latex proteins and enzymes are as yet poorly understood. We found that papain, a cysteine protease in latex of the Papaya tree (Carica papaya, Caricaceae), is a crucial factor in the defense of the papaya tree against lepidopteran larvae such as oligophagous Samia ricini (Saturniidae) and two notorious polyphagous pests, Mamestra brassicae (Noctuidae) and Spodoptera litura (Noctuidae). Leaves of a number of laticiferous plants, including papaya and a wild fig, Ficus virgata (Moraceae), showed strong toxicity and growth inhibition against lepidopteran larvae, though no apparent toxic factors from these species have been reported. When the latex was washed off, the leaves of these lactiferous plants lost toxicity. Latexes of both papaya and the wild fig were rich in cysteine-protease activity. E-64, a cysteine protease-specific inhibitor, completely deprived the leaves of toxicity when painted on the surface of papaya and fig leaves. Cysteine proteases, such as papain, ficin, and bromelain, all showed toxicity. The results suggest that plant latex and the proteins in it, cysteine proteases in particular, provide plants with a general defense mechanism against herbivorous insects.

  3. X-ray structures of Nfs2, the plastidial cysteine desulfurase from Arabidopsis thaliana

    PubMed Central

    Roret, Thomas; Pégeot, Henri; Couturier, Jérémy; Mulliert, Guillermo; Rouhier, Nicolas; Didierjean, Claude

    2014-01-01

    The chloroplastic Arabidopsis thaliana Nfs2 (AtNfs2) is a group II pyridoxal 5′-phosphate-dependent cysteine desulfurase that is involved in the initial steps of iron–sulfur cluster biogenesis. The group II cysteine desulfurases require the presence of sulfurtransferases such as SufE proteins for optimal activity. Compared with group I cysteine desulfurases, proteins of this group contains a smaller extended lobe harbouring the catalytic cysteine and have a β-hairpin constraining the active site. Here, two crystal structures of AtNfs2 are reported: a wild-type form with the catalytic cysteine in a persulfide-intermediate state and a C384S variant mimicking the resting state of the enzyme. In both structures the well conserved Lys241 covalently binds pyridoxal 5′-phosphate, forming an internal aldimine. Based on available homologous bacterial complexes, a model of a complex between AtNfs2 and the SufE domain of its biological partner AtSufE1 is proposed, revealing the nature of the binding sites. PMID:25195888

  4. Acceleration of Anaerobic Cysteine Transformations to Sulfane Sulfur Consequent to γ-Glutamyl Transpeptidase Inhibition

    PubMed Central

    Kwiecień, Inga; Iciek, Małgorzata; Włodek, Lidia

    2012-01-01

    Toxicity of drugs and radiation in the cells is largely dependent on the level of thiols. In the present studies, an attempt has been made to inhibit γ-glutamyl transpeptidase (γGT) activity in EAT-bearing animals tissue. We have expected that administration of γGT inhibitors: acivicin and 1,2,3,4-tetrahydroisoquinoline (TIQ) may influence GSH/γ–glutamyl transpeptidase (γGT) system in the regulation of cysteine concentration and anaerobic cysteine metabolism in normal and cancer cells. Development of Ehrlich ascites tumor in mice enhances peroxidative processes, diminishes levels of nonprotein thiols (NPSH) and sulfane sulfur, and lowers activities of enzymes involved in its formation and transfer in the liver and kidney. Although γGT inhibitors further decrease NPSH level, they increase cysteine and sulfane sulfur levels. This means that upon γGT inhibition, cysteine can be efficiently acquired by normal liver and kidney cells via another pathway, that is so productive that sulfane sulfur level and intensity of anaerobic cysteine metabolism even rise. PMID:22629124

  5. High Levels of Intracellular Cysteine Promote Oxidative DNA Damage by Driving the Fenton Reaction

    PubMed Central

    Park, Sunny; Imlay, James A.

    2003-01-01

    Escherichia coli is generally resistant to H2O2, with >75% of cells surviving a 3-min challenge with 2.5 mM H2O2. However, when cells were cultured with poor sulfur sources and then exposed to cystine, they transiently exhibited a greatly increased susceptibility to H2O2, with <1% surviving the challenge. Cell death was due to an unusually rapid rate of DNA damage, as indicated by their filamentation, a high rate of mutation among the survivors, and DNA lesions by a direct assay. Cell-permeable iron chelators eliminated sensitivity, indicating that intracellular free iron mediated the conversion of H2O2 into a hydroxyl radical, the direct effector of DNA damage. The cystine treatment caused a temporary loss of cysteine homeostasis, with intracellular pools increasing about eightfold. In vitro analysis demonstrated that cysteine reduces ferric iron with exceptional speed. This action permits free iron to redox cycle rapidly in the presence of H2O2, thereby augmenting the rate at which hydroxyl radicals are formed. During routine growth, cells maintain small cysteine pools, and cysteine is not a major contributor to DNA damage. Thus, the homeostatic control of cysteine levels is important in conferring resistance to oxidants. More generally, this study provides a new example of a situation in which the vulnerability of cells to oxidative DNA damage is strongly affected by their physiological state. PMID:12618458

  6. Mechanism of thiosulfate oxidation in the SoxA family of cysteine-ligated cytochromes.

    PubMed

    Grabarczyk, Daniel B; Chappell, Paul E; Eisel, Bianca; Johnson, Steven; Lea, Susan M; Berks, Ben C

    2015-04-03

    Thiosulfate dehydrogenase (TsdA) catalyzes the oxidation of two thiosulfate molecules to form tetrathionate and is predicted to use an unusual cysteine-ligated heme as the catalytic cofactor. We have determined the structure of Allochromatium vinosum TsdA to a resolution of 1.3 Å. This structure confirms the active site heme ligation, identifies a thiosulfate binding site within the active site cavity, and reveals an electron transfer route from the catalytic heme, through a second heme group to the external electron acceptor. We provide multiple lines of evidence that the catalytic reaction proceeds through the intermediate formation of a S-thiosulfonate derivative of the heme cysteine ligand: the cysteine is reactive and is accessible to electrophilic attack; cysteine S-thiosulfonate is formed by the addition of thiosulfate or following the reverse reaction with tetrathionate; the S-thiosulfonate modification is removed through catalysis; and alkylating the cysteine blocks activity. Active site amino acid residues required for catalysis were identified by mutagenesis and are inferred to also play a role in stabilizing the S-thiosulfonate intermediate. The enzyme SoxAX, which catalyzes the first step in the bacterial Sox thiosulfate oxidation pathway, is homologous to TsdA and can be inferred to use a related catalytic mechanism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

    PubMed Central

    Schröder, Jörg; Noack, Sandra; Marhöfer, Richard J.; Mottram, Jeremy C.; Coombs, Graham H.; Selzer, Paul M.

    2013-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes. They play pivotal roles in the biology of parasites and inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Homology modeling of the mature Leishmania mexicana cysteine protease CPB2.8 suggested that it differs significantly from bovine cathepsin B and thus could be a good drug target. High throughput screening of a compound library against this enzyme and bovine cathepsin B in a counter assay identified four novel inhibitors, containing the warhead-types semicarbazone, thiosemicarbazone and triazine nitrile, that can be used as leads for antiparasite drug design. Covalent docking experiments confirmed the SARs of these lead compounds in an effort to understand the structural elements required for specific inhibition of CPB2.8. This study has provided starting points for the design of selective and highly potent inhibitors of L. mexicana cysteine protease CPB that may also have useful efficacy against other important cysteine proteases. PMID:24146999

  8. Cysteine-Mediated Gene Expression and Characterization of the CmbR Regulon in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Manzoor, Irfan; Kuipers, Oscar P.; Shafeeq, Sulman

    2016-01-01

    In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to cysteine. Transcriptome comparison of the D39 wild-type grown at a restricted concentration of cysteine (0.03 mM) to one grown at a high concentration of cysteine (50 mM) in chemically-defined medium (CDM) revealed elevated expression of various genes/operons, i.e., spd-0150, metQ, spd-0431, metEF, gshT, spd-0618, fhs, tcyB, metB-csd, metA, spd-1898, yvdE, and cysK, likely to be involved in the transport and utilization of cysteine and/or methionine. Microarray-based data were further confirmed by quantitative RT-PCR. Promoter lacZ-fusion studies and quantitative RT-PCR data showed that the transcriptional regulator CmbR acts as a transcriptional repressor of spd-0150, metEF, gshT, spd-0618, tcyB, metA, and yvdE, putatively involved in cysteine uptake and utilization. The operator site of CmbR in the promoter regions of CmbR-regulated genes is predicted and confirmed by mutating or deleting CmbR operator sites from the promoter regions of these genes. PMID:27990139

  9. Growth of different morphologies (quantum dots to nanorod) of Ag-nanoparticles: role of cysteine concentrations.

    PubMed

    Khan, Zaheer; Talib, Abou

    2010-03-01

    This work describes an easy chemical method for the preparation of orange-red color silver sol by the cysteine reduction of silver (I) in the presence of cetyltrimethylammonium bromide (CTAB). The obtained sol was found to have very small particles in the order of quantum dots for the first time. Transmission electron microscope (TEM) images show that the silver sol consists of aggregated as well as cross-linking arrangement of spherical silver quantum dots (size in the range ca. cysteine]. The rate of Ag-nanoparticles formation decreases with the increase in [cysteine] whereas [CTAB] and [Ag(+)] have no effect on the reaction rate. Interestingly, at higher [cysteine] (>or=20.0x10(-4)mol dm(-3)), white precipitate was formed instead of transparent silver sol. Cysteine acts as a reducing, cross-linking, stabilizing and buffering agent during the growth of different shape and size of silver nanoparticles.

  10. Formation of Elemental Sulfur by Chlorella fusca during Growth on l-Cysteine Ethylester 1

    PubMed Central

    Krauss, Friedrich; Schäfer, Wolfram; Schmidt, Ahlert

    1984-01-01

    During growth on l-cysteine ethylester, Chlorella fusca (211-8b) accumulated a substance which contained bound sulfide, which could be liberated by reduction with dithioerythritol (DTE) as inorganic sulfide. This substance was extracted with hot methanol and purified by thin layer chromatography. This substance liberated free sulfide when incubated with mono- and dithiols, and thiocyanate was formed after heating with KCN. The isolated substance cochromatographed with authentic sulfur flower using different solvent systems for thin layer chromatography, high pressure liquid chromatography, and the identical spectrum with a relative λmax at 263 nm was found. The chemical structure was confirmed by mass spectrometry showing a molecular weight of 256 m/e for the S8 configuration. No labeled elemental sulfur was detected when the cells were grown on [35S]sulfate and l-cysteine ethylester indicating the origin of elemental sulfur from l-cysteine ethylester. C. fusca seems to have enzymes for the metabolism of elemental sulfur, since it disappeared after prolonged growth into the stationary phase. Cysteine was formed from O-acetyl-l-serine and elemental sulfur in the presence of thiol groups and purified cysteine synthase from spinach or Chlorella. PMID:16663375

  11. The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase*

    PubMed Central

    Tchesnokov, Egor P.; Fellner, Matthias; Siakkou, Eleni; Kleffmann, Torsten; Martin, Lois W.; Aloi, Sekotilani; Lamont, Iain L.; Wilbanks, Sigurd M.; Jameson, Guy N. L.

    2015-01-01

    Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site. PMID:26272617

  12. Movement of cysteine in intact monkey lenses: the major site of entry is the germinative region.

    PubMed

    Sweeney, Matthew H J; Garland, Donita L; Truscott, Roger J W

    2003-08-01

    Monkey lenses were incubated with 35S-L-cysteine for various times and the movement of label within the lens followed by autoradiography. Cysteine appeared to enter primarily at the germinative region of the lens. No evidence was found for major transport through either the anterior or posterior faces of the lens. The movement of cysteine within different parts of the lens was followed over time. The data suggest that, for cysteine, the major pathway for transport within the lens involves entry at the germinative region followed by movement along the fibre cells. The data were consistent with orthogonal movement across the fibres in the equatorial plane but little or no movement across the fibres at the anterior pole or posterior faces of the lens. Such a scenario is in accord with the distribution of connexons, indicating that this pattern of entry may also be observed for other small molecules. The finding of high permeability at the lens germinative region is in accord with the anatomy of the eye, since this is the lens surface in contact with the posterior chamber. Thus, cysteine secreted by the ciliary body into the aqueous humor would come into contact initially with the region of the lens best able to absorb this amino acid. Although this aspect was not addressed in the current study, the same phenomenon may also be observed with other lens nutrients.

  13. The Astrographic Catalogue and the Carte du Ciel

    NASA Astrophysics Data System (ADS)

    Jaschek, C.

    1985-11-01

    Answers to a circular letter concerning the Astrographic Catalogue and the Carte du Ciel from Alger (A. Ghezloun), Catania (C. Blanco), Cordoba (G. Carranza), Greenwich (P. J. Andrews), Paris (J. Delhaye), Sydney (A. E. Vaughan), Toulouse (R. Nadal).

  14. The Cri-Du-Chat Syndrome: A Case Study.

    ERIC Educational Resources Information Center

    Sykes, Stewart C.; Christie, Margarette A.

    1987-01-01

    The developmental history of a 14-year-old girl with Cri-Du-Chat Syndrome (a genetic disorder characterized by a distinctive cry and severe physical and intellectual disabilities) is reported. (Author/DB)

  15. The Career Development Program at Du Pont's Pioneering Research Laboratory.

    ERIC Educational Resources Information Center

    Nusbaum, H. J.

    1986-01-01

    Describes the Career Development Program, designed to help professional employees accept responsibility for their own careers, located at Du Pont's Pioneering Research Laboratory. Covers the concepts governing the program, program elements, and working with management to address program goals. (CH)

  16. Lymphome primitif du sein: à propos d'un cas

    PubMed Central

    Njoumi, Noureddine; Najih, Mohamed; Haqqi, Laila; Atolou, Gilles; Bougtab, Abdessalm; Hachi, Hafid; Benjelloun, Samir

    2012-01-01

    Le lymphome primitif du sein est une entité histologique très rare du cancer du sein. Les aspects cliniques et radiologiques ne présentent pas de spécificités particulières. Le diagnostic est souvent retardé. Le traitement se base essentiellement sur la chimiothérapie. Le pronostic est globalement péjoratif. Nous rapportons un cas de lymphome malin non Hodgkinien primitif du sein chez une patiente de 38 ans. Parallèlement une revue de la littérature est entreprise évoquant les aspects épidémiologiques, cliniques, histologiques et thérapeutiques de ce néoplasme. PMID:22937198

  17. 1. Historic American Buildings Survey, drawn by Pierre du Simitiere ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    1. Historic American Buildings Survey, drawn by Pierre du Simitiere (papers in Philadelphia Library) DRAWING OF REDWOOD LIBRARY IN 1768. - Redwood Library, 50 Bellevue Avenue, Newport, Newport County, RI

  18. Fasting, but Not Aging, Dramatically Alters the Redox Status of Cysteine Residues on Proteins in Drosophila melanogaster

    PubMed Central

    Menger, Katja E.; James, Andrew M.; Cochemé, Helena M.; Harbour, Michael E.; Chouchani, Edward T.; Ding, Shujing; Fearnley, Ian M.; Partridge, Linda; Murphy, Michael P.

    2015-01-01

    Summary Altering the redox state of cysteine residues on protein surfaces is an important response to environmental challenges. Although aging and fasting alter many redox processes, the role of cysteine residues is uncertain. To address this, we used a redox proteomic technique, oxidative isotope-coded affinity tags (OxICAT), to assess cysteine-residue redox changes in Drosophila melanogaster during aging and fasting. This approach enabled us to simultaneously identify and quantify the redox state of several hundred cysteine residues in vivo. Cysteine residues within young flies had a bimodal distribution with peaks at ∼10% and ∼85% reversibly oxidized. Surprisingly, these cysteine residues did not become more oxidized with age. In contrast, 24 hr of fasting dramatically oxidized cysteine residues that were reduced under fed conditions while also reducing cysteine residues that were initially oxidized. We conclude that fasting, but not aging, dramatically alters cysteine-residue redox status in D. melanogaster. PMID:26095360

  19. Crystal Structure of the Cysteine Desulfurase DndA from Streptomyces lividans Which Is Involved in DNA Phosphorothioation

    PubMed Central

    Qian, Tianle; Zhang, Yan; You, Delin; He, Xinyi; Wang, Zhijun; Liang, Jingdan; Deng, Zixin; Wu, Geng

    2012-01-01

    DNA phosphorothioation is widespread among prokaryotes, and might function to restrict gene transfer among different kinds of bacteria. There has been little investigation into the structural mechanism of the DNA phosphorothioation process. DndA is a cysteine desulfurase which is involved in the first step of DNA phosphorothioation. In this study, we determined the crystal structure of Streptomyces lividans DndA in complex with its covalently bound cofactor PLP, to a resolution of 2.4 Å. Our structure reveals the molecular mechanism that DndA employs to recognize its cofactor PLP, and suggests the potential binding site for the substrate L-cysteine on DndA. In contrast to previously determined structures of cysteine desulfurases, the catalytic cysteine of DndA was found to reside on a β strand. This catalytic cysteine is very far away from the presumable location of the substrate, suggesting that a conformational change of DndA is required during the catalysis process to bring the catalytic cysteine close to the substrate cysteine. Moreover, our in vitro enzymatic assay results suggested that this conformational change is unlikely to be a simple result of random thermal motion, since moving the catalytic cysteine two residues forward or backward in the primary sequence completely disabled the cysteine desulfurase activity of DndA. PMID:22570733

  20. Effects of cysteine on amino acid concentrations and transsulfuration enzyme activities in rat liver with protein-calorie malnutrition.

    PubMed

    Kim, Yoon G; Kim, Sang K; Kwon, Jong W; Park, Ock J; Kim, Sang G; Kim, Young C; Lee, Myung G

    2003-01-24

    The changes in amino acid concentrations and transsulfuration enzyme activities in liver were investigated after 4-week fed on 23% casein diet (control group) and 5% casein diet without (protein-calorie malnutrition, PCM group) or with (PCMC group) oral administration of cysteine, 250 mg/kg (twice daily, starting from the fourth week) using rats as an animal model. By supplementation with cysteine in PCM rats (PCMC group), cysteine level was elevated almost close to the control level, and glutathione (GSH), aspartic acid and serine levels were restored greater than the control levels. The measurement of transsulfuration enzyme activities exhibited that gamma-glutamylcysteine ligase (gamma-GCL) activity was up-regulated in rats with protein restriction (PCM group), and cysteine supplementation (PCMC group) down-regulated to the control level. One-week supplementation of cysteine (PCMC group) significantly down-regulated the cysteine sulfinate decarboxylase activity. These results indicate that the availability of sulfur amino acid(s) especially cysteine appears to play a role in determining the flux of cysteine between cysteine catabolism and GSH synthesis.

  1. Peters anomaly in cri-du-chat syndrome.

    PubMed

    Hope, William C; Cordovez, Jose A; Capasso, Jenina E; Hammersmith, Kristin M; Eagle, Ralph C; Lall-Trail, Joel; Levin, Alex V

    2015-06-01

    The cri-du-chat syndrome is a rare genetic disorder caused by deletions in the short arm of chromosome 5. It presents with a distinctive catlike high-pitched cry, psychomotor delays, microcephaly, craniofacial abnormalities, and, in many cases, ocular findings. We report the first child with cri-du-chat and the findings of unilateral corneal staphyloma due to Peters anomaly and retinal dysplasia. Copyright © 2015 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.

  2. Anaesthetic considerations for the patient with cri du chat syndrome.

    PubMed

    Brislin, R P; Stayer, S A; Schwartz, R E

    1995-01-01

    Cri du chat syndrome is an inherited disease affecting multiple organ systems. Most characteristic is the anatomical abnormality of the larynx resulting in a cat-like cry. Issues important in developing an anaesthetic plan include: anatomical abnormalities of the airway, congenital heart disease, hypotonia, mental retardation, and temperature maintenance. We report the case of a 33-month-old patient with cri du chat syndrome undergoing patent ductus arteriosus (PDA) ligation and discuss the anaesthetic issues.

  3. Oxidation states of uranium in DU particles from Kosovo.

    PubMed

    Salbu, B; Janssens, K; Lind, O C; Proost, K; Danesi, P R

    2003-01-01

    The oxidation states of uranium contained in depleted uranium (DU) particles were determined by synchrotron radiation based micro-XANES, applied to individual particles in soil samples collected at Ceja Mountain, Kosovo. Based on scanning electron microscopy (SEM) with XRMA prior to micro-XANES, DU particles ranging from submicrons to about 30 microm (average size: 2 microm or less) were identified. Compared to well-defined standards, all investigated DU particles were oxidized. About 50% of the DU particles were characterized as UO2, the remaining DU particles present were U3O8 or a mixture of oxidized forms (ca. 2/3 UO2, 1/3 U3O8). Since the particle weathering rate is expected to be higher for U3O8 than for UO2, the presence of respiratory U3O8 and UO2 particles, their corresponding weathering rates and subsequent remobilisation of U from DU particles should be included in the environmental or health impact assessments.

  4. Determination of periodontopathogens in patients with Cri du chat syndrome

    PubMed Central

    Ballesta-Mudarra, Sofía; Torres-Lagares, Daniel; Rodríguez-Caballero, Ángela; Yáñez-Vico, Rosa M.; Solano-Reina, Enrique; Perea-Pérez, Evelio

    2013-01-01

    Objectives: Cri du chat syndrome is a genetic alteration associated with some oral pathologies. However, it has not been described previously any clinical relationship between the periodontal disease and the syndrome. The purpose of this comparative study was to compare periodontopathogenic flora in a group with Cri du chat syndrome and another without the síndrome, to assess a potential microbiological predisposition to suffer a periodontitis. Study Design: The study compared nineteen subjects with Cri du chat Syndrome with a control group of nineteen patients without it. All patients were clinically evaluated by periodontal probing, valuing the pocket depth, the clinical attachmente level and bleeding on probing. There were no significant differences between both groups. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola were detected by multiplex-PCR using 16S rDNA (microIDENT). Results: When A. actinomycetemcomitans, P. gingivalis, P. intermedia and T. denticola were compared, no statistically significant differences were found between the two groups (p>0.05). The value of T. forsythia was significantly higher for Cri du chat syndrome (31.6%) than for the control group (5.3%). The odds ratio for T. forsythia was 8.3. Conclusions: In the present study T. forsythia is associated with Cri du chat syndrome subjects and not with healthy subjects. Key words:Cri du Chat syndrome, periodontal health, microbiology, special care dentistry. PMID:24121919

  5. Tumeur du sac vitellin du testicule au stade IIIc métastatique : à propos d’un cas

    PubMed Central

    Zizi, Mohamed; Ziouziou, Imad; El Yacoubi, Souhail; Khmou, Mouna; Jahid, Ahmed; Mahassini, Najat; Karmouni, Tariq; El Khader, Khalid; Koutani, Abdellatif; Andaloussi, Ahmed Iben Attya

    2014-01-01

    Résumé Les tumeurs du sac vitellin du testicule sont rares chez l’adulte. Ces tumeurs se caractérisent par un mauvais pronostic à un stade métastatique avancé. Cependant, nous rapportons, dans le présent article, le cas clinique d’un adulte de 32 ans qui présentait une tumeur du sac vitellin du testicule au stade IIIc métastatique. Ce patient a subi une orchidectomie haute, accompagnée de quatre cycles de chimiothérapie à base de bléomycine, d’étoposide et de cisplatine. Il a répondu complètement au traitement, moyennant un recul de deux ans. PMID:25295144

  6. Effets du titane et du niobium sur l'oxydation à 950circC d'aciers ferritiques

    NASA Astrophysics Data System (ADS)

    Issartel, C.; Buscail, H.; Caudron, E.; Cueff, R.; Riffard, F.; El Messki, S.; Karimi, N.; Antoni, L.

    2004-11-01

    Nous avons étudié l'effet du titane et du niobium sur l'oxydation à 950circC d'un acier Fe-Cr chrominoformeur. La DRX in situ montre que le titane semble s'oxyder en formant Cr{2}TiO{5} et TiO{2} qui contribuent à une augmentation de la prise de masse des échantillons. Une partie du titane issu de ces oxydes semble doper la couche de chromine. Sa présence augmente la concentration en lacunes cationiques dans la chromine et augmente donc la diffusion du chrome dans la couche. Nous avons aussi montré que le niobium n'a pas d'influence sur l'oxydation de ce type d'acier à 950circC.

  7. The Basics of Thiols and Cysteines in Redox Biology and Chemistry

    PubMed Central

    Poole, Leslie B.

    2014-01-01

    Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts functional sites with their specialized properties (e.g., nucleophilicity, high affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low molecular weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes including signal transduction. PMID:25433365

  8. Regulation of Protein Function and Signaling by Reversible Cysteine S-Nitrosylation*

    PubMed Central

    Gould, Neal; Doulias, Paschalis-Thomas; Tenopoulou, Margarita; Raju, Karthik; Ischiropoulos, Harry

    2013-01-01

    NO is a versatile free radical that mediates numerous biological functions within every major organ system. A molecular pathway by which NO accomplishes functional diversity is the selective modification of protein cysteine residues to form S-nitrosocysteine. This post-translational modification, S-nitrosylation, impacts protein function, stability, and location. Despite considerable advances with individual proteins, the in vivo biological chemistry, the structural elements that govern the selective S-nitrosylation of cysteine residues, and the potential overlap with other redox modifications are unknown. In this minireview, we explore the functional features of S-nitrosylation at the proteome level and the structural diversity of endogenously modified residues, and we discuss the potential overlap and complementation that may exist with other cysteine modifications. PMID:23861393

  9. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

    PubMed Central

    Bernardim, Barbara; Cal, Pedro M.S.D.; Matos, Maria J.; Oliveira, Bruno L.; Martínez-Sáez, Nuria; Albuquerque, Inês S.; Perkins, Elizabeth; Corzana, Francisco; Burtoloso, Antonio C.B.; Jiménez-Osés, Gonzalo; Bernardes, Gonçalo J. L.

    2016-01-01

    Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications. PMID:27782215

  10. Cysteine Proteases Inhibitors with immunoglobulin-like fold in protozoan parasites and their role in pathogenesis.

    PubMed

    Jimenez-Sandoval, Pedro; Lopez-Castillo, Laura Margarita; Trasviña-Arenas, Carlos H; Brieba, Luis G

    2016-08-13

    The number of protein folds in nature is limited, thus is not surprising that proteins with the same fold are able to exert different functions. The cysteine protease inhibitors that adopt an immunoglobulin-like fold (Ig-ICPs) are inhibitors encoded in bacteria and protozoan parasites. Structural studies indicate that these inhibitors resemble the structure of archetypical proteins with an Ig fold, like antibodies, cadherins or cell receptors. The structure of Ig-ICPs from four different protozoan parasites clearly shows the presence of three loops that form part of a protein-ligand interaction surface that resembles the antigen binding sites of antibodies. Thus, Ig-ICPs bind to different cysteine proteases using a tripartite mechanism in which their BC, DE and FG loops are responsible for the main interactions with the target cysteine protease. Ig-ICPs from different protozoan parasites regulate the enzymatic activity of host or parasite&#039;s proteases and thus regulate virulence and pathogenesis.

  11. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

    NASA Astrophysics Data System (ADS)

    Bernardim, Barbara; Cal, Pedro M. S. D.; Matos, Maria J.; Oliveira, Bruno L.; Martínez-Sáez, Nuria; Albuquerque, Inês S.; Perkins, Elizabeth; Corzana, Francisco; Burtoloso, Antonio C. B.; Jiménez-Osés, Gonzalo; Bernardes, Gonçalo J. L.

    2016-10-01

    Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.

  12. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

    PubMed

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R

    2017-02-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  13. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis

    PubMed Central

    Hernández, Hilda M.; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis. PMID:25348828

  14. Four free cysteine residues found in human IgG1 of healthy donors.

    PubMed

    Gevondyan, N M; Volynskaia, A M; Gevondyan, V S

    2006-03-01

    Modifications with different thiol reagents demonstrated that 28 of 32 cysteine residues of human IgG1 are involved in the formation of disulfide bonds, and four cysteines remain free. So IgG1 is a protein possessing both free SH-groups and disulfide bonds. Only one of the four SH-groups is accessible for silver or mercury ions and hydrophobic reagents, whereas the remaining three SH-groups are masked and can be revealed only after deep denaturation of the protein. Detection of the masked cysteine residues was shown to depend on the kinetics of intramolecular changes occurring during denaturation of the protein and on the method of the assay of the SH-groups.

  15. An archaeal ADP-dependent serine kinase involved in cysteine biosynthesis and serine metabolism

    PubMed Central

    Makino, Yuki; Sato, Takaaki; Kawamura, Hiroki; Hachisuka, Shin-ichi; Takeno, Ryo; Imanaka, Tadayuki; Atomi, Haruyuki

    2016-01-01

    Routes for cysteine biosynthesis are still unknown in many archaea. Here we find that the hyperthermophilic archaeon Thermococcus kodakarensis generates cysteine from serine via O-phosphoserine, in addition to the classical route from 3-phosphoglycerate. The protein responsible for serine phosphorylation is encoded by TK0378, annotated as a chromosome partitioning protein ParB. The TK0378 protein utilizes ADP as the phosphate donor, but in contrast to previously reported ADP-dependent kinases, recognizes a non-sugar substrate. Activity is specific towards free serine, and not observed with threonine, homoserine and serine residues within a peptide. Genetic analyses suggest that TK0378 is involved in serine assimilation and clearly responsible for cysteine biosynthesis from serine. TK0378 homologs, present in Thermococcales and Desulfurococcales, are most likely not ParB proteins and constitute a group of kinases involved in serine utilization. PMID:27857065

  16. Decoration of gold nanoparticles with cysteine in solution: reactive molecular dynamics simulations.

    PubMed

    Monti, Susanna; Carravetta, Vincenzo; Ågren, Hans

    2016-07-14

    The dynamics of gold nanoparticle functionalization by means of adsorption of cysteine molecules in water solution is simulated through classical reactive molecular dynamics simulations based on an accurately parametrized force field. The adsorption modes of the molecules are characterized in detail disclosing the nature of the cysteine-gold interactions and the stability of the final material. The simulation results agree satisfactorily with recent experimental and theoretical data and confirm previous findings for a similar system. The covalent attachments of the molecules to the gold support are all slow physisorptions followed by fast chemisorptions. However, a great variety of binding arrangements can be observed. Interactions with the adsorbate caused surface modulations in terms of adatoms and dislocations which contributed to strengthen the cysteine adsorption.

  17. S-Substituted cysteine derivatives and thiosulfinate formation in Petiveria alliacea-part II.

    PubMed

    Kubec, Roman; Kim, Seokwon; Musah, Rabi A

    2002-11-01

    Three cysteine derivatives, (R)-S-(2-hydroxyethyl)cysteine, together with (R(S)R(C))- and (S(S)R(C))-S-(2-hydroxyethyl)cysteine sulfoxides, have been isolated from the roots of Petiveria alliacea. Furthermore, three additional amino acids, S-methyl-, S-ethyl-, and S-propylcysteine derivatives, were detected. They were present only in trace amounts (<3 microg g(-1) fr. wt), precluding determination of their absolute configurations and oxidation states. In addition, four thiosulfinates, S-(2-hydroxyethyl) (2-hydroxyethane)-, S-(2-hydroxyethyl) phenylmethane-, S-benzyl (2-hydroxyethane)- and S-benzyl phenylmethanethiosulfinates, have been found in a homogenate of the roots. The formation pathways of various benzyl/phenyl-containing compounds previously found in the plant were also discussed.

  18. The basics of thiols and cysteines in redox biology and chemistry.

    PubMed

    Poole, Leslie B

    2015-03-01

    Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pKa and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Puzzling over protein cysteine phosphorylation--assessment of proteomic tools for S-phosphorylation profiling.

    PubMed

    Buchowiecka, A K

    2014-09-07

    Cysteine phosphorylation has recently been discovered in both prokaryotic and eukaryotic systems, and is thought to play crucial roles in signaling and regulation of cellular responses. This article explores the topics of chemical stability of this type of structural modification and the resulting issues regarding affinity enrichment of S-phosphopeptides and their mass spectrometry-based detection in the course of general proteomics studies. Together, this work suggests that the current advances in phosphoproteomic methodologies provide adequate tools for investigating protein cysteine phosphorylation and appear to be immediately available for practical implementation. The article provides useful information necessary for designing experiments in the emerging cysteine phosphoproteomics. The examples of methodological proposals for S-linked phosphorylation detection are included herein in order to stimulate development of new approaches by the phosphoproteomic community.

  20. Biological roles of cysteine proteinases in the pathogenesis of Trichomonas vaginalis.

    PubMed

    Hernández, Hilda M; Marcet, Ricardo; Sarracent, Jorge

    2014-01-01

    Human trichomonosis, infection with Trichomonas vaginalis, is the most common non-viral sexually transmitted disease in the world. The host-parasite interaction and pathophysiological processes of trichomonosis remain incompletely understood. This review focuses on the advancements reached in the area of the pathogenesis of T. vaginalis, especially in the role of the cysteine proteinases. It highlights various approaches made in this field and lists a group of trichomonad cysteine proteinases involved in diverse processes such as invasion of the mucous layer, cytoadherence, cytotoxicity, cytoskeleton disruption of red blood cells, hemolysis, and evasion of the host immune response. A better understanding of the biological roles of cysteine proteinases in the pathogenesis of this parasite could be used in the identification of new chemotherapeutic targets. An additional advantage could be the development of a vaccine in order to reduce transmission of T. vaginalis.

  1. Exploring the mechanism of inhibition of human telomerase by cysteine-reactive compounds.

    PubMed

    Kellermann, Guillaume; Dingli, Florent; Masson, Vanessa; Dauzonne, Daniel; Ségal-Bendirdjian, Evelyne; Teulade-Fichou, Marie-Paule; Loew, Damarys; Bombard, Sophie

    2017-03-01

    Telomerase is an almost universal cancer target that consists minimally of a core protein human telomerase reverse transcriptase (hTERT) and a RNA component human telomerase RNA (hTR). Some inhibitors of this enzyme are thought to function by the covalent binding to one or several cysteine residues; however, this inhibition mechanism has never been investigated because of the difficulty in producing telomerase. In this study, we use a recent method to produce recombinant hTERT to analyze the effect of cysteine-reactive inhibitors on telomerase. Using mass spectrometry and mutagenesis analysis, we identify several targeted residues in separated domains of the hTERT protein and show that cysteine-reactive reagents abolish the interaction with the CR4/5 region of hTR. © 2017 Federation of European Biochemical Societies.

  2. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors

    PubMed Central

    Burtoloso, Antonio C. B.; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C.; Gonçalez, Cristiana; Kenny, Peter W.; Leitão, Andrei; Quilles, José Carlos; Ribeiro, Jean F. R.; Rocha, Josmar R.

    2017-01-01

    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases. PMID:28222138

  3. Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae).

    PubMed

    Bruno, Mariela A; Pardo, Marcelo F; Caffini, Néstor O; López, Laura M I

    2003-02-01

    A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5-9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.

  4. Coenzyme A biosynthesis: steric course of 4'-phosphopantothenoyl-L-cysteine decarboxylase.

    PubMed

    Aberhart, D J; Ghoshal, P K; Cotting, J A; Russell, D J

    1985-12-03

    4'-Phosphopantothenoyl-L-cysteine decarboxylase (PPC decarboxylase) was partially purified from rat liver. 4'-Phosphopantothenoyl[2-2H1]-L-cysteine was synthesized and converted by PPC decarboxylase to 4'-phosphol[1-2H1]pantetheine. The product was degraded by reduction with Raney nickel followed by acidic hydrolysis to [1-2H1]ethylamine. The latter was converted to the (-)-camphanamide derivative, NMR studies of which revealed that the deuterium was located in the pro-1S position. Also, unlabeled 4'-phosphopantothenoyl-L-cysteine was incubated with PPC decarboxylase in D2O, giving, after degradation, the (-)-camphanamide of (1R)-[1-2H1]ethylamine. The results show that the decarboxylation takes place with retention of configuration. These results are discussed in terms of possible mechanisms for the decarboxylation.

  5. Contrasting effects of cysteine modification on the transfection efficiency of amphipathic peptides.

    PubMed

    Sharma, Rajpal; Nisakar, Daniel; Shivpuri, Shivangi; Ganguli, Munia

    2014-08-01

    Delivery of DNA to cells remains a key challenge towards development of gene therapy. A better understanding of the properties involved in stability and transfection efficiency of the vector could critically contribute to the improvement of delivery vehicles. In the present work we have chosen two peptides differing only in amphipathicity and explored how presence of cysteine affects DNA uptake and transfection efficiency. We report an unusual observation that addition of cysteine selectively increases transfection efficiency of secondary amphipathic peptide (Mgpe-9) and causes a drop in the primary amphipathic peptide (Mgpe-10). Our results point the effect of cysteine is dictated by the importance of physicochemical properties of the carrier peptide. We also report a DNA delivery agent Mgpe-9 exhibiting high transfection efficiency in multiple cell lines (including hard-to-transfect cell lines) with minimal cytotoxicity which can be further explored for in vivo applications. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Detection of cysteine protease in Taenia solium-induced brain granulomas in naturally infected pigs.

    PubMed

    Mkupasi, Ernatus Martin; Sikasunge, Chummy Sikalizyo; Ngowi, Helena Aminiel; Leifsson, Pall S; Johansen, Maria Vang

    2013-10-18

    In order to further characterize the immune response around the viable or degenerating Taenia solium cysts in the pig brain, the involvement of cysteine protease in the immune evasion was assessed. Brain tissues from 30 adult pigs naturally infected with T. solium cysticercosis were subjected to histopathology using hematoxylin and eosin stain, and immunohistochemistry using caspase-3 antibodies. Histopathological evaluation revealed lesions of stage I which was characterized by presence of viable parasite surrounded with minimal to moderate inflammatory cells and stage III characterized by the presence of a disintegrating parasite surrounded with high inflammatory cells. The results of immunohistochemistry indicated caspase-3 positive cells interspaced between inflammatory infiltrate mainly in stage I lesions, indicating the presence of cysteine protease. This result confirms the earlier hypothesis that cysteine protease may play a role in inducing immune evasion through apoptosis around viable T. solium cysts.

  7. Optical Absorption and Electric Resistivity of an l-Cysteine Film

    NASA Astrophysics Data System (ADS)

    Kamada, Masao; Hideshima, Takuya; Azuma, Junpei; Yamamoto, Isamu; Imamura, Masaki; Takahashi, Kazutoshi

    2016-12-01

    The optical and electric properties of an l-cysteine film have been investigated to understand its applicability to bioelectronics. The fundamental absorption is the allowed transition having the threshold at 5.8 eV and the absorption is due to the charge-transfer type transition from sulfur-3sp to oxygen-2p and/or carbon-2p states, while absorptions more than 9 eV can be explained with intra-atomic transitions in the functional groups. The electric resistivity is 2.0 × 104 Ω m at room temperature and increases as the sample temperature decreases. The results indicate that the l-cysteine film is a p-type semiconductor showing the hole conduction caused by the sulfur-3sp occupied states and unknown impurity or defect states as acceptors. The electron affinity of the l-cysteine film is derived as ≦-0.3 eV.

  8. Prise en charge du cancer du col utérin durant la grossesse: à propos de 05 cas

    PubMed Central

    El Mazghi, Abderrahman; Bouhafa, Touria; Loukili, Kaoutar; El Kacemi, Hanan; Lalya, Issam; Hassouni, Khalid

    2014-01-01

    L'association d'un cancer du col utérin avec une grossesse est une éventualité rare. Son incidence est assez mal évaluée, elle se situe selon les études entre 1 et 2/10 000. Lorsque la découverte en est faite, il faut conjuguer deux impératifs parfois totalement divergents: le traitement de la mère et la prise en charge du foetus. Cette association pose schématiquement quatre grands problèmes, qui sont: Celui du diagnostic, qui est loin d’être évident, compte tenu des remaniements cervicaux observés en début de gestation, le pronostic de l'affection, la date du traitement chirurgical et du délai entre le diagnostic et la prise en charge thérapeutique, enfin et de manière plus accessoire, le devenir de la grossesse et le mode d'accouchement. Nous rapportons une série de 5 cas de cancer du col utérin découverts au cours de la grossesse colligés dans notre service entre 2010 et 2013. La prise en charge thérapeutique est identique à celle des patientes en dehors de la grossesse même si quelques adaptations sont nécessaires du fait de l’état gravide, le pronostic du cancer ne semble pas être modifié par la grossesse. PMID:25852788

  9. Comparative efficacies of 2 cysteine prodrugs and a glutathione delivery agent in a colitis model

    PubMed Central

    OZ, HELIEH S.; CHEN, THERESA S.; NAGASAWA, HERBERT

    2007-01-01

    Oxidant-mediated injury plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Recently, antioxidants were shown to modulate colitis in mice. In this study, the protective effects of L-cysteine and glutathione (GSH) prodrugs are further evaluated against progression of colitis in a murine model. ICR mice were fed compounds incorporated into chow as follows: Group (A) received chow supplemented with vehicle. Group (B) was provided 2-(RS)-n-propylthiazolidine-4(R)-carboxylic-acid (PTCA), a cysteine prodrug. Group (C) received D-ribose-L-cysteine (RibCys), another cysteine prodrug that releases L-cysteine. Group (D) was fed L-cysteine-glutathione mixed sulfide (CySSG), a ubiquitous GSH derivative present in mammalian cells. After 3 days, the animals were further provided with normal drinking water or water supplemented with dextran sodium sulfate (DSS). Mice administered DSS developed severe colitis and suffered weight loss. Colonic lesions significantly improved in animals treated with PTCA and RibCys and, to a lesser extent, with CySSG therapy. Hepatic GSH levels were depleted in colitis animals (control vs DSS, P < 0.001), and normalized with prodrug therapies (control vs treatments, P > 0.05). Protein expressions of serum amyloid A and inflammatory cytokines [interleukin (IL)-6, IL-12, tumor necrosis factor-alpha (TNF-α), osteopontin (OPN)] were significantly increased in colitis animals and improved with therapies. Immunohistochemistry and Western blot analyses showed significant upregulation of the macrophage-specific markers, COX-2 and CD68, which suggests macrophage activation and infiltration in the colonic lamina propria in colitis animals. These abnormalities were attenuated in prodrug-treated mice. In conclusion, these data strongly support the novel action of the PTCA against colitis, which further supports a possible therapeutic application for IBD patients. PMID:17656332

  10. Ethylene-regulated expression of a carnation cysteine proteinase during flower petal senescence.

    PubMed

    Jones, M L; Larsen, P B; Woodson, W R

    1995-06-01

    The senescence of carnation (Dianthus caryophyllus L.) flower petals is regulated by the phytohormone ethylene and is associated with considerable catabolic activity including the loss of protein. In this paper we present the molecular cloning of a cysteine proteinase and show that its expression is regulated by ethylene and associated with petal senescence. A 1600 bp cDNA was amplified by polymerase chain reaction using a 5'-specific primer and 3'-nonspecific primer designed to amplify a 1-aminocyclopropane-1-carboxylate synthase cDNA from reverse-transcribed stylar RNA. The nucleotide sequence of the cloned product (pDCCP1) was found to share significant homology to several cysteine proteinases rather than ACC synthase. A single open reading frame of 428 amino acids was shown to share significant homology with other plant cysteine proteinases including greater than 70% identity with a cysteine proteinase from Arabidopsis thaliana. Amino acids in the active site of cysteine proteinases were conserved in the pDCCP1 peptide. RNA gel blot analysis revealed that the expression of pDCCP1 increased substantially with the onset of ethylene production and senescence of petals. Increased pDCCP1 expression was also associated with ethylene production in other senescing floral organs including ovaries and styles. The pDCCP1 transcript accumulated in petals treated with exogenous ethylene within 3 h and treatment of flowers with 2,5-norbornadiene, an inhibitor of ethylene action, prevented the increase in pDCCP1 expression in petals. The temporal and spatial patterns of pDCCP1 expression suggests a role for cysteine proteinase in the loss of protein during floral senescence.

  11. Sample Multiplexing with Cysteine-Selective Approaches: cysDML and cPILOT

    NASA Astrophysics Data System (ADS)

    Gu, Liqing; Evans, Adam R.; Robinson, Renã A. S.

    2015-04-01

    Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

  12. Plasma homocysteine and cysteine and risk of breast cancer in women

    PubMed Central

    Lin, Jennifer; Lee, I-Min; Song, Yiqing; Cook, Nancy R.; Selhub, Jacob; Manson, JoAnn E.; Buring, Julie E.; Zhang, Shumin M.

    2010-01-01

    Homocysteine and cysteine are associated with oxidative damage and metabolic disorders, which may lead to carcinogenesis. Observational studies assessing the association between circulating homocysteine or cysteine and breast cancer are very limited and findings have been inconsistent. We prospectively evaluated plasma levels of homocysteine and cysteine in relation to breast cancer risk among 812 incident cases of invasive breast cancer and 812 individually matched control subjects from 28,345 women in the Women’s Health Study aged ≥45 years who provided blood samples and had no history of cancer or cardiovascular disease at baseline. Logistic regression controlling for matching factors and risk factors for breast cancer was used to estimate relative risks (RRs) and 95% confidence intervals (CIs). All statistical tests were two sided. Homocysteine levels were not associated with overall risk for breast cancer. However, we observed a positive association between cysteine levels and breast cancer risk; the multivariate RR for the highest quintile group relative to the lowest quintile was 1.65 (95% CI=1.04–2.61, p for trend=0.04). In addition, women with higher levels of homocysteine and cysteine were at a greater risk for developing breast cancer when their folate levels were low (p values for interaction were 0.04 and 0.002, respectively). Although our study offers little support for an association between circulating homocysteine and overall breast cancer risk, higher homocysteine levels may be associated with an increased risk for breast cancer among women with low folate status. The increased risk of breast cancer associated with high cysteine levels warrants further investigation. PMID:20197471

  13. Sample multiplexing with cysteine-selective approaches: cysDML and cPILOT.

    PubMed

    Gu, Liqing; Evans, Adam R; Robinson, Renã A S

    2015-04-01

    Cysteine-selective proteomics approaches simplify complex protein mixtures and improve the chance of detecting low abundant proteins. It is possible that cysteinyl-peptide/protein enrichment methods could be coupled to isotopic labeling and isobaric tagging methods for quantitative proteomics analyses in as few as two or up to 10 samples, respectively. Here we present two novel cysteine-selective proteomics approaches: cysteine-selective dimethyl labeling (cysDML) and cysteine-selective combined precursor isotopic labeling and isobaric tagging (cPILOT). CysDML is a duplex precursor quantification technique that couples cysteinyl-peptide enrichment with on-resin stable-isotope dimethyl labeling. Cysteine-selective cPILOT is a novel 12-plex workflow based on cysteinyl-peptide enrichment, on-resin stable-isotope dimethyl labeling, and iodoTMT tagging on cysteine residues. To demonstrate the broad applicability of the approaches, we applied cysDML and cPILOT methods to liver tissues from an Alzheimer's disease (AD) mouse model and wild-type (WT) controls. From the cysDML experiments, an average of 850 proteins were identified and 594 were quantified, whereas from the cPILOT experiment, 330 and 151 proteins were identified and quantified, respectively. Overall, 2259 unique total proteins were detected from both cysDML and cPILOT experiments. There is tremendous overlap in the proteins identified and quantified between both experiments, and many proteins have AD/WT fold-change values that are within ~20% error. A total of 65 statistically significant proteins are differentially expressed in the liver proteome of AD mice relative to WT. The performance of cysDML and cPILOT are demonstrated and advantages and limitations of using multiple duplex experiments versus a single 12-plex experiment are highlighted.

  14. Preparation and application of L-cysteine-doped Keggin polyoxometalate microtubes

    SciTech Connect

    Shen Yan; Peng Jun; Zhang Huanqiu; Meng Cuili; Zhang Fang

    2012-01-15

    L-cysteine-doped tungstosilicate (Lcys-SiW{sub 12}) microtubes are prepared, and the amount of L-cysteine doped in the microtubes can be tuned to some extent. The as-prepared Lcys-SiW{sub 12} microtubes are sensitive to ammonia gas exhibited through the distinct color change of the microtubes from light purple to dark blue after exposing to ammonia gas. A possible mechanism of the coloration is that the adsorbed ammonia molecules increase the basicity of the Lcys-SiW{sub 12} microtubes and promote the redox reaction between L-cysteine and polyoxometalate. This is a pH-dependent solid-solid redox reaction, which is triggered by proton capture agent. The Lcys-SiW{sub 12} microtubes show application in chemical sensors for alkaline gases. - Graphical abstract: The Lcys-SiW{sub 12} microtubes were formed during transformation of the monolacunary Keggin-type [{alpha}-SiW{sub 11}O{sub 39}]{sup 8-} to the saturated Keggin-type [{alpha}-SiW{sub 12}O{sub 40}]{sup 4-}, meanwhile L-cysteine molecules were doped during the growth of the microtubes. Highlights: Black-Right-Pointing-Pointer L-cysteine-doped polyoxometalate microtubes are prepared. Black-Right-Pointing-Pointer Amount of L-cysteine doped in the microtubes can be tuned to some extent. Black-Right-Pointing-Pointer Lcys-SiW{sub 12} microtubes can be applied as a sensor for detecting alkaline gases. Black-Right-Pointing-Pointer This is a proton capture agent-triggered solid-solid redox reaction.

  15. Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

    PubMed Central

    Jasenec, Ashley; Barasa, Nathaniel; Kulkarni, Samatha; Shaik, Nabeel; Moparthi, Swarnalatha; Konda, Venkataramana; Caguiat, Jonathan

    2009-01-01

    Background Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway. Results Growth curves using M-9 minimal medium containing 40 mM selenite and 1 mM sulfate showed that Enterobacter sp. YSU grew when L-cysteine was present but died when it was absent. Differential protein expression analysis by two dimensional gel electrophoresis showed that CysK was present in cultures containing selenite and lacking L-cysteine but absent in cultures containing both selenite and L-cysteine. Additional RT-PCR studies demonstrated that transcripts for the sulfate permease genes, cysA, cysT and cysW, were down-regulated in the presence of L-cysteine. Conclusion L-cysteine appeared to confer selenite resistance upon Enterobacter sp. YSU by decreasing the level of selenite transport into the cell through the non-specific pathway. PMID:19715574

  16. Phytomonas serpens: cysteine peptidase inhibitors interfere with growth, ultrastructure and host adhesion.

    PubMed

    Santos, André L S; d'Avila-Levy, Claudia M; Dias, Felipe A; Ribeiro, Rachel O; Pereira, Fernanda M; Elias, Camila G R; Souto-Padrón, Thaïs; Lopes, Angela H C S; Alviano, Celuta S; Branquinha, Marta H; Soares, Rosangela M A

    2006-01-01

    In this study, we report the ultrastructural and growth alterations caused by cysteine peptidase inhibitors on the plant trypanosomatid Phytomonas serpens. We showed that the cysteine peptidase inhibitors at 10 microM were able to arrest cellular growth as well as promote alterations in the cell morphology, including the parasites becoming short and round. Additionally, iodoacetamide induced ultrastructural alterations, such as disintegration of cytoplasmic organelles, swelling of the nucleus and kinetoplast-mitochondrion complex, which culminated in parasite death. Leupeptin and antipain induced the appearance of microvillar extensions and blebs on the cytoplasmic membrane, resembling a shedding process. A 40 kDa cysteine peptidase was detected in hydrophobic and hydrophilic phases of P. serpens cells after Triton X-114 extraction. Additionally, we have shown through immunoblotting that anti-cruzipain polyclonal antibodies recognised two major polypeptides in P. serpens, including a 40 kDa component. Flow cytometry analysis confirmed that this cruzipain-like protein has a location on the cell surface. Ultrastructural immunocytochemical analysis demonstrated the presence of the cruzipain-like protein on the surface and in small membrane fragments released from leupeptin-treated parasites. Furthermore, the involvement of cysteine peptidases of P. serpens in the interaction with explanted salivary glands of the phytophagous insect Oncopeltus fasciatus was also investigated. When P. serpens cells were pre-treated with either cysteine peptidase inhibitors or anti-cruzipain antibody, a significant reduction of the interaction process was observed. Collectively, these results suggest that cysteine peptidases participate in several biological processes in P. serpens including cell growth and interaction with the invertebrate vector.

  17. Structure and Function of the Hetero-oligomeric Cysteine Synthase Complex in Plants*

    PubMed Central

    Wirtz, Markus; Birke, Hannah; Heeg, Corinna; Müller, Christopher; Hosp, Fabian; Throm, Christian; König, Stephan; Feldman-Salit, Anna; Rippe, Karsten; Petersen, Gabriele; Wade, Rebecca C.; Rybin, Vladimir; Scheffzek, Klaus; Hell, Rüdiger

    2010-01-01

    Cysteine synthesis in bacteria and plants is catalyzed by serine acetyltransferase (SAT) and O-acetylserine (thiol)-lyase (OAS-TL), which form the hetero-oligomeric cysteine synthase complex (CSC). In plants, but not in bacteria, the CSC is assumed to control cellular sulfur homeostasis by reversible association of the subunits. Application of size exclusion chromatography, analytical ultracentrifugation, and isothermal titration calorimetry revealed a hexameric structure of mitochondrial SAT from Arabidopsis thaliana (AtSATm) and a 2:1 ratio of the OAS-TL dimer to the SAT hexamer in the CSC. Comparable results were obtained for the composition of the cytosolic SAT from A. thaliana (AtSATc) and the cytosolic SAT from Glycine max (Glyma16g03080, GmSATc) and their corresponding CSCs. The hexameric SAT structure is also supported by the calculated binding energies between SAT trimers. The interaction sites of dimers of AtSATm trimers are identified using peptide arrays. A negative Gibbs free energy (ΔG = −33 kcal mol−1) explains the spontaneous formation of the AtCSCs, whereas the measured SAT:OAS-TL affinity (KD = 30 nm) is 10 times weaker than that of bacterial CSCs. Free SAT from bacteria is >100-fold more sensitive to feedback inhibition by cysteine than AtSATm/c. The sensitivity of plant SATs to cysteine is further decreased by CSC formation, whereas the feedback inhibition of bacterial SAT by cysteine is not affected by CSC formation. The data demonstrate highly similar quaternary structures of the CSCs from bacteria and plants but emphasize differences with respect to the affinity of CSC formation (KD) and the regulation of cysteine sensitivity of SAT within the CSC. PMID:20720017

  18. Decoration of gold nanoparticles with cysteine in solution: reactive molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Monti, Susanna; Carravetta, Vincenzo; Ågren, Hans

    2016-06-01

    The dynamics of gold nanoparticle functionalization by means of adsorption of cysteine molecules in water solution is simulated through classical reactive molecular dynamics simulations based on an accurately parametrized force field. The adsorption modes of the molecules are characterized in detail disclosing the nature of the cysteine-gold interactions and the stability of the final material. The simulation results agree satisfactorily with recent experimental and theoretical data and confirm previous findings for a similar system. The covalent attachments of the molecules to the gold support are all slow physisorptions followed by fast chemisorptions. However, a great variety of binding arrangements can be observed. Interactions with the adsorbate caused surface modulations in terms of adatoms and dislocations which contributed to strengthen the cysteine adsorption.The dynamics of gold nanoparticle functionalization by means of adsorption of cysteine molecules in water solution is simulated through classical reactive molecular dynamics simulations based on an accurately parametrized force field. The adsorption modes of the molecules are characterized in detail disclosing the nature of the cysteine-gold interactions and the stability of the final material. The simulation results agree satisfactorily with recent experimental and theoretical data and confirm previous findings for a similar system. The covalent attachments of the molecules to the gold support are all slow physisorptions followed by fast chemisorptions. However, a great variety of binding arrangements can be observed. Interactions with the adsorbate caused surface modulations in terms of adatoms and dislocations which contributed to strengthen the cysteine adsorption. Electronic supplementary information (ESI) available: Different views of the AuNP surface coverage. Distance map describing the position of each molecule in relation to the others on the AuNP (alpha carbon distances). See DOI: 10.1039/C

  19. Stable structure-approximating inverse protein folding in 2D hydrophobic-polar-cysteine (HPC) model.

    PubMed

    Khodabakhshi, Alireza Hadj; Manuch, Ján; Rafiey, Arash; Gupta, Arvind

    2009-01-01

    The inverse protein folding problem is that of designing an amino acid sequence which folds into a prescribed conformation/structure. This problem arises in drug design where a particular structure is necessary to ensure proper protein-protein interactions. Gupta et al. (2005) introduced a design in the two-dimensional (2D) hydrophobic-polar (HP) model of Dill that can be used to approximate any given (2D) shape. They conjectured that the protein sequences of their design are stable but only proved the stability for an infinite class of very basic structures. We introduce a refinement of the HP model, in which the cysteine and non-cysteine hydrophobic monomers are distinguished and SS-bridges, which two cysteines can form, are taken into account in the energy function. We call this model the HPC model. We consider a subclass of linear structures designed in Gupta et al. (2005) which is rich enough to approximate (although more coarsely) any given structure. We refine these structures for the HPC model by setting approximately a half of H amino acids to cysteine ones and call them snake structures. We first prove that the proteins of the snake structures are stable under the strong HPC model in which we make an additional assumption that non-cysteine amino acids act as cysteine ones, i.e., they can form their own bridges to reduce the energy. Then we consider a subclass of snake structures called wave structures that can still approximate any given shape and prove that their proteins are stable under the proper HPC model. This partially confirms the conjecture stated in Gupta et al. (2005). To prove the above results we developed a computational tool, called 2DHPSolver, which we used to perform large case analysis required for the proofs. We conjecture that the proteins of snake structures are stable under the proper HPC model.

  20. The x-ray absorption spectroscopy model of solvation about sulfur in aqueous L-cysteine.

    PubMed

    Sarangi, Ritimukta; Frank, Patrick; Benfatto, Maurizio; Morante, Silvia; Minicozzi, Velia; Hedman, Britt; Hodgson, Keith O

    2012-11-28

    The environment of sulfur in dissolved aqueous L-cysteine has been examined using K-edge x-ray absorption spectroscopy (XAS), extended continuum multiple scattering (ECMS) theory, and density functional theory (DFT). For the first time, bound-state and continuum transitions representing the entire XAS spectrum of L-cysteine sulfur are accurately reproduced by theory. Sulfur K-edge absorption features at