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Sample records for plant cysteine proteinases

  1. Cysteine proteinases and cystatins

    Directory of Open Access Journals (Sweden)

    Adeliana S. Oliveira

    2003-01-01

    Full Text Available This review describeds the definition, localization, functions and examples of cysteine proteinases and their protein inhibitors in vertebrate, non-vertebrate animals and plants. These inhibitors are related with defense mechanisms of plant against pests. It also describes the factors involved in the specific cysteine proteinase-cystatin interaction and high degree of affinity and large specificity in this interaction which are not only represented by the compatibility between amino acid residues of the active site involved in catalysis, but also of all amino acid residues that participante in the enzyme-inhibitor interaction.Nesta revisão foram descritas definições, localizações, funções e exemplos de proteinases cisteínicas e suas proteinas inibidoras em animais vertebrados e invertebrados e plantas. Tratamos principalmente com aqueles inibidores que são relatados com o mecanismo de defesa da planta contra pestes. Em adição, comentamos sobre recentes trabalhos que contribuíram para uma melhor compreenção dos fatores envolvidos na interação específica proteinase cisteínica-cistatina. Por outro lado, chamamos atenção para o alto grau de afinidade e grande especificidade na interação que não são apenas representadas pela compatibilidade entre os residuos de aminoácidos do sítio ativo envolvidos na catalise, mas também de todos os resíduos de aminoácidos que participam da interação enzima-inibidor.

  2. The relative anthelmintic efficacy of plant-derived cysteine proteinases on intestinal nematodes.

    Science.gov (United States)

    Luoga, W; Mansur, F; Buttle, D J; Duce, I R; Garnett, M C; Lowe, A; Behnke, J M

    2015-03-01

    We examined the in vitro and in vivo efficacy of plant cysteine proteinases (CPs) derived from pineapple (Ananas comosus) and kiwi fruit (Actinidia deliciosa), and compared their efficacy as anthelmintics to the known effects of CPs from the latex of papaya (Carica papaya) against the rodent intestinal nematode, Heligmosomoides bakeri. Both fruit bromelain and stem bromelain had significant in vitro detrimental effects on H. bakeri but in comparison, actinidain from kiwi fruit had very little effect. However, in vivo trials indicated far less efficacy of stem bromelain and fruit bromelain than that expected from the in vitro experiments (24.5% and 22.4% reduction in worm burdens, respectively) against H. bakeri. Scanning electron microscopy revealed signs of cuticular damage on worms incubated in fruit bromelain, stem bromelain and actinidain, but this was far less extensive than on those incubated in papaya latex supernatant. We conclude that, on the basis of presently available data, CPs derived from pineapples and kiwi fruits are not suitable for development as novel anthelmintics for intestinal nematode infections. PMID:24176056

  3. Cysteine proteinase inhibitors in psoriatic epidermis.

    Science.gov (United States)

    Hopsu-Havu, V K; Joronen, I A; Järvinen, M; Rinne, A

    1983-01-01

    Human psoriatic epidermis and scales were demonstrated to contain two antigenically separate cysteine proteinase inhibitors, one acidic with an isoelectric point of 4.7-5.0 (ACPI) and one neutral with an isoelectric point of 6.0-6.5 (NCPI), while normal epidermis contains only ACPI. The total papain (cysteine proteinase) inhibiting activity of the psoriatic epidermis as calculated per mg protein was higher than that in normal epidermis. Both ACPI and NCPI were localized immunocytochemically, mainly in the highest spinous cell layers with less activity in the parakeratotic cells and lower layers of spinous cells. Basal cells were essentially negative. PMID:6607037

  4. The proregion of papaya proteinase IV inhibits Colorado potato beetle digestive cysteine proteinases.

    Science.gov (United States)

    Visal, S; Taylor, M A; Michaud, D

    1998-09-01

    Three distinct digestive protease systems were induced in larvae of the herbivorous pest, Colorado potato beetle (CPB; Leptinotarsa decemlineata Say), and used as a model to assess the ability of the proregion of papaya proteinase IV (PPIV; glycyl endopeptidase, EC 3.4.22.25) to act as an inhibitor of insect digestive cysteine proteinases. As shown by gelatin/PAGE and complementary inhibition assays, a recombinant form of the proregion produced in Escherichia coli inhibited a fraction of the insect proteases also inhibited by the well-characterized inhibitor of cysteine proteinases, oryzacystatin I (OCI). In contrast with OCI, the inhibitory potency of the proregion was affected by an increase of the temperature, suggesting a certain alteration of its structural integrity by the insect non-target proteases. This apparent susceptibility to proteolysis was confirmed by SDS-PAGE, after challenging the proregion with the different insect extracts. As seen on gel, selective inhibition of the insect aspartate proteinase, cathepsin D, with the inhibitor pepstatin A preserved the activity of the proregion against cysteine proteinases by preventing its hydrolysis. Taken together, these observations suggest the potential of plant protease proregions as regulators of cysteine proteinases in biotechnological systems, and show the ability of protease inhibitors to preserve the integrity of 'companion' defense-related proteins from the action of insensitive proteases in target pests. PMID:9742962

  5. Cysteine proteinase inhibitors produced by mononuclear phagocytes.

    Science.gov (United States)

    Hopsu-Havu, V K; Joronen, I A; Järvinen, M; Rinne, A; Aalto, M

    1984-01-01

    Monocytes were separated from human peripheral blood and allowed to attach to culture flasks, after which the content and production of a number of cysteine proteinase inhibitors was assayed. These were: a low molecular weight (MW 12000) acid cysteine proteinase inhibitor (ACPI); a low-molecular weight inhibitor of the same size with neutral pH (NCPI), and alpha-cysteine proteinase inhibitor with a molecular weight around 90 000 (alpha-CPI). Only NCPI was detectable in the cultures at the beginning of the incubation, and it was synthesized and released into the incubation mixture during the incubation, especially if the cells were stimulated with silica. The amount of NCPI contained in and released from the cells was drastically decreased by puromycin. Immunoblots after cell electrophoresis in polyacrylamide gel revealed only one molecular form of NCPI with a molecular weight of 12 000 both in the cells and in the culture medium. No ACPI or alpha-CPI could be detected. PMID:6424942

  6. Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

    OpenAIRE

    Gustavo Coelho Correa; Márcia Margis-Pinheiro; Rogério Margis

    2001-01-01

    Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as memb...

  7. Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

    Scientific Electronic Library Online (English)

    Gustavo Coelho, Correa; Márcia, Margis-Pinheiro; Rogério, Margis.

    2001-12-01

    Full Text Available Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um gr [...] ande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB) como pertencentes à sub-sub-classe E.C.3.4.22, foram usadas na busca de clusters no banco de dados do SUCEST (SUgarCane EST project), utilizando-s o programa T-BLAST-n. Homologia de seqüências foram encontradas com 76 clusters que correspondem a prováveis proteinases cisteínicas. O alinhamento destas seqüências com a de outras proteases cisteínicas, de diversas origens, forneceu informação quanto à classificação e possível função das proteinases de cana-de-açúcar. Além disso, o padrão de expressão de cada gene foi postulado a partir da correlação direta com as bibliotecas de cDNA do SUCEST dos quais os clusters foram derivados. Uma vez que nenhum gene de protease cisteínica foi anteriormente evidenciado em cana-de-açúcar, este estudo representa uma etapa inicial para o estudo de novos aspectos bioquímicos, fisiológicos e biotecnológicos destas enzimas. Abstract in english Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that t [...] hey are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST) data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.

  8. Identification, classification and expression pattern analysis of sugarcane cysteine proteinases

    Directory of Open Access Journals (Sweden)

    Gustavo Coelho Correa

    2001-12-01

    Full Text Available Cysteine proteases are peptidyl hydrolyses dependent on a cysteine residue at the active center. The physical and chemical properties of cysteine proteases have been extensively characterized, but their precise biological functions have not yet been completely understood, although it is known that they are involved in a number of events such as protein turnover, cancer, germination, programmed cell death and senescence. Protein sequences from different cysteine proteinases, classified as members of the E.C.3.4.22 sub-sub-class, were used to perform a T-BLAST-n search on the Brazilian Sugarcane Expressed Sequence Tags project (SUCEST data bank. Sequence homology was found with 76 cluster sequences that corresponded to possible cysteine proteinases. The alignments of these SUCEST clusters with the sequence of cysteine proteinases of known origins provided important information about the classification and possible function of these sugarcane enzymes. Inferences about the expression pattern of each gene were made by direct correlation with the SUCEST cDNA libraries from which each cluster was derived. Since no previous reports of sugarcane cysteine proteinases genes exists, this study represents a first step in the study of new biochemical, physiological and biotechnological aspects of sugarcane cysteine proteases.Proteinases cisteínicas são peptidil-hidrolases dependentes de um resíduo de cisteína em seu sítio ativo. As propriedades físico-químicas destas proteinases têm sido amplamente caracterizadas, entretanto suas funções biológicas ainda não foram completamente elucidadas. Elas estão envolvidas em um grande número de eventos, tais como: processamento e degradação protéica, câncer, germinação, morte celular programada e processos de senescência. Diferentes proteinases cisteínicas, classificadas pelo Comitê de Nomenclatura da União Internacional de Bioquímica e Biologia Molecular (IUBMB como pertencentes à sub-sub-classe E.C.3.4.22, foram usadas na busca de clusters no banco de dados do SUCEST (SUgarCane EST project, utilizando-s o programa T-BLAST-n. Homologia de seqüências foram encontradas com 76 clusters que correspondem a prováveis proteinases cisteínicas. O alinhamento destas seqüências com a de outras proteases cisteínicas, de diversas origens, forneceu informação quanto à classificação e possível função das proteinases de cana-de-açúcar. Além disso, o padrão de expressão de cada gene foi postulado a partir da correlação direta com as bibliotecas de cDNA do SUCEST dos quais os clusters foram derivados. Uma vez que nenhum gene de protease cisteínica foi anteriormente evidenciado em cana-de-açúcar, este estudo representa uma etapa inicial para o estudo de novos aspectos bioquímicos, fisiológicos e biotecnológicos destas enzimas.

  9. Natural cysteine proteinase inhibitors reduce lectin induced lymphocyte stimulation.

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    Hopsu-Havu, V K; Joronen, I A; Rinne, A; Järvinen, M

    1986-01-01

    Lymphocyte stimulation by lectins can be inhibited by several synthetic inhibitors of proteolytic enzymes, notably those of cysteine proteinases. The effects of naturally occurring enzyme inhibitors are less well known. The effect of the neutral low-molecular weight cysteine proteinase inhibitor (NCPI) recently purified from lymph nodes and spleen was therefore investigated. Cultured peripheral blood lymphocytes were stimulated by PHA or ConA in the presence or absence of NCPI and the incorporation of 3H-thymidine was measured. NCPI was found to inhibit these lymphocyte responses in these circumstances. PMID:3090834

  10. Cysteine proteinase inhibitors in human melanoma transplanted into nude mice.

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    Tsushima, H; Sumi, H; Mihara, H; Joronen, I; Hopsu-Havu, V K

    1988-05-01

    Cysteine proteinase inhibitors in the human melanoma tissue transplanted into nude mice were found to increase in concentration during tumor growth. The activity (unit/g) at 8 weeks was about 3 times higher than the activity at 4 weeks after transplantation. The inhibitors were separated into two main forms (Mr about 76,000 and 10,000) with Sephacryl S-200 and/or Sephadex G-75 gel chromatography. The activities of the inhibitors of both molecular masses increased parallely during tumor growth. The high molecular mass inhibitor fractions reacted with antisera made against alpha-cysteine proteinase inhibitor (alpha-CPI, human kininogen) and against neutral low-molecular mass proteinase inhibitor (cystatin B). Free cystatin B appeared to be liberated in SDS-polyacrylamide gel electrophoresis following electroimmunoblotting with an antiserum to cystatin B. Similarly, free cystatin B was detected in gel chromatography on Sephadex G-75 after alkali treatment at pH 11.5. It may thus represent a cystatin B--cysteine proteinase complex mixed with alpha-CPI. The low molecular mass inhibitor fractions reacted with antisera made against cystatin A and cystatin B. When the low-molecular mass inhibitor fraction was subjected to isoelectric focusing, it was separated into three peaks with pIs 8.0, 7.4, and 6.0. The inhibitors with pI 8.0 and 7.4 reacted with antisera made against cystatin B, while the inhibitor with pI 6.0 reacted with antisera made against cystatin B and cystatin A. PMID:3144293

  11. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    Energy Technology Data Exchange (ETDEWEB)

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  12. Protective Role of Purified Cysteine Proteinases against Fasciola gigantica Infection in Experimental Animals

    OpenAIRE

    El-Ahwany, Eman; Rabia, Ibrahim; Nagy, Faten; Zoheiry, Mona; Diab, Tarek; Zada, Suher

    2012-01-01

    Fascioliasis is one of the public health problems in the world. Cysteine proteinases (CP) released by Fasciola gigantica play a key role in parasite feeding, migration through host tissues, and in immune evasion. There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infections. Cysteine proteinases were purified and tested in vaccine trials of sheep infected with the liver fluke. Multiple doses (2 mg of CP in ...

  13. Serum cysteine proteinase inhibitors with special reference to kidney failure.

    Science.gov (United States)

    Hopsu-Havu, V K; Joronen, I; Havu, S; Rinne, A; Järvinen, M; Forsström, J

    1985-02-01

    Serum levels of proteins reactive in radioimmunoassay with an antiserum prepared in rabbits against purified human spleen neutral cysteine proteinase inhibitor (NCPI) was determined in 70 healthy controls and from 80 patients suffering from suspected or proven kidney failure. The values varied from less than 0.2 mg/l in normal sera to levels over 2 mg/l in some patient sera. Serum level of NCPI was found to roughly correlate with serum creatinine values. However, there were sera with high NCPI levels which did not have increased serum creatinine values. In sera with high NCPI levels subjected to double radial immunodiffusion two precipitin lines, one completely and the other partially identical to NCPI were registered. After fractionating of serum proteins with gel chromatography on Sephadex G 100, two protein peaks of immunological similarity to purified NCPI were found: one low molecular weight (MW around 12,000) and one high molecular weight (MW around 100,000). The low molecular weight NCPI-like material appeared to inhibit human cathepsin B and papain and is thus free serum NCPI. alpha-Cysteine proteinase inhibitor did not increase with serum creatinine as did NCPI. PMID:3919439

  14. The reactive site loop of the serpin SCCA1 is essential for cysteine proteinase inhibition

    OpenAIRE

    Schick, Charles; Brömme, Dieter; Bartuski, Allison J.; Uemura, Yoshiki; Schechter, Norman M.; Silverman, Gary A.

    1998-01-01

    The high-molecular-weight serine proteinase inhibitors (serpins) are restricted, generally, to inhibiting proteinases of the serine mechanistic class. However, the viral serpin, cytokine response modifier A, and the human serpins, antichymotrypsin and squamous cell carcinoma antigen 1 (SCCA1), inhibit different members of the cysteine proteinase class. Although serpins employ a mobile reactive site loop (RSL) to bait and trap their target serine proteinases, the mechanism by which they inacti...

  15. Characterization of the cathepsin-like cysteine proteinases of Schistosoma mansoni.

    OpenAIRE

    Dalton, J. P.; Clough, K A; Jones, M.K.; Brindley, P J

    1996-01-01

    Adult Schistosoma mansoni parasites synthesize and secrete both cathepsin L and cathepsin B cysteine proteinases. These cysteine proteinase activities, believed to be involved in hemoglobin digestion by adult schistosomes, were characterized by using specific fluorogenic peptide substrates and zymography. Both cathepsin L- and B-like activities with pH optima of 5.2 and 6.2, respectively, predominated in soluble extracts of worms, and both these activities were secreted by adult worms into th...

  16. Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase.

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    Goetting-Minesky, M P; Mullin, B C

    1994-10-11

    Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation. Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway. Differential screening of an Alnus glutinosa nodule cDNA library revealed altered levels of gene expression in nodules as compared with roots and allowed isolation of host plant nodule-specific cDNA sequences. The deduced amino acid sequence of one full-length cDNA, AgNOD-CP1, represents a nodule-specific cysteine proteinase similar to cysteine proteinases of the papain superfamily. Residues critical to catalysis, active site, and disulfide bridges are conserved. Suggested roles for this enzyme are as a defense response to Frankia invasion, as a component of tissue remodeling in root and nodule tissues, as a cell cycle component, or as an element of protein turnover. Complexity of hybridization patterns revealed by Southern blot analysis suggests that the gene for AgNOD-CP1 is a member of a multigene family. Northern hybridization results indicate that this gene may have been recruited for a role specific to this symbiosis, a phenomenon observed in the Rhizobium-legume symbioses, perhaps common to many microbe-plant interactions. PMID:7937912

  17. Detection of low molecular weight cysteine proteinase inhibitors by time-resolved fluoroimmunoassay.

    Science.gov (United States)

    Joronen, I; Hopsu-Havu, V K; Manninen, M; Rinne, A; Järvinen, M; Halonen, P

    1986-02-12

    A time-resolved fluoroimmunoassay was developed for the detection of 3 human low molecular weight cysteine proteinase inhibitors, ACPI (cystatin A), NCPI (cystatin B), and gamma-trace (cystatin C). Polystyrene tubes or polystyrene microtitration strips were used as solid phase. The rabbit anti-inhibitor immunoglobulins were used as the capture antibody, and, when labelled with europium, also as the detector antibody. The threshold sensitivity of the tests was 0.1 ng/ml for NCPI and 1 ng/ml for the others. All the 3 cysteine proteinase inhibitors, ACPI, NCPI, and gamma-trace, were detected in pooled serum samples of patients with kidney failure. gamma-Trace seemed to be quantitatively the major and ACPI the minor inhibitor. No other low molecular mass cysteine proteinase inhibitor was detected after isoelectric focusing of the 12 kDa area of gel filtered human serum. PMID:3511154

  18. Cysteine proteinase inhibitor in cultured human medullary thyroid carcinoma cells.

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    Barka, T; van der Noen, H; Patil, S

    1992-06-01

    The TT cell line of human medullary thyroid carcinoma, that retains some of the differentiated functions of thyroid C cells including the synthesis and secretion of calcitonin, was found to contain and release into the culture medium cysteine proteinase inhibitor(s), cystatin(s). The major inhibitor, which is similar to, if not identical with, cystatin C, is constitutively released, or secreted, by TT cells. The rate of secretion of cystatin, quantified by titration of inhibition of papain, was stimulated by dibutyryladenosine 3':5'-cyclic monophosphate, forskolin, the calcium ionophore A 23187, and by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Neither forskolin nor TPA had, however, an effect on the level of the inhibitor in TT cells. Treatment with n-butyrate strongly inhibited the proliferation of TT cells, and led, in 4 to 7 days, to a doubling of the intracellular concentration of cystatins. Northern blot hybridizations to a 32P-labeled riboprobe complementary to human cystatin C cDNA indicated that cAMP, forskolin, and TPA had no effect on the steady-state levels of cystatin C mRNA. These data indicate that release of cystatin(s) from TT cells is regulated by cAMP-calcium-protein kinase C mechanisms that appear to be similar to those that regulate the secretion of calcitonin from these cells. However, in contrast to the calcitonin gene, the expression of the cystatin C gene in these cells is not regulated by cAMP or TPA. By a combination of acetone fractionation, affinity chromatography on Cm-papain-Sepharose, and gel exclusion chromatography a protein of approximately 14 kilodaltons was isolated from TT cells that reacted with antibodies against human cystatin C, and strongly inhibited papain. Cystain secreted by TT cells also had a molecular weight of 14 kilodaltons, and reacted with anti-human cystatin C antibodies. The physiologic and pathologic roles of cystatins in different cell types remain to be established. The TT cells provide a suitable cell type to study the regulation of the expression of the cystatin gene and the mechanism of cystatin release. PMID:1602739

  19. Effects of E-64, a cysteine proteinase inhibitor, on cowpea weevil growth, development, and fecundity

    Energy Technology Data Exchange (ETDEWEB)

    Murdock, L.L.; Shade, R.E.; Pomeroy, M.A.

    1988-06-01

    E-64, a specific inhibitor of cysteine proteinases, was incorporated into artificial seeds at low levels (0.01-0.25% by weight). It prolonged developmental time and increased mortality of the larval cowpea weevil, Callosobruchus maculatus (F.), in direct proportion to its concentration in the artificial seeds. The fecundity of females emerging from the artificial seeds was significantly decreased by E-64 concentrations of 0.06% and higher. These observations are compatible with the hypothesis that the midgut cysteine proteinase in C. maculatus is essential for normal growth and development.

  20. Separation and partial characterization of four cysteine proteinases from a human epidermal cell line.

    Science.gov (United States)

    Joronen, I A; Hopsu-Havu, V K

    1987-01-01

    Four different cysteine proteinases from a cultured human epidermal cell line (NCTC 2544) were partially purified and characterized. The biggest hydrolase was an endoaminopeptidase with the molecular weight of several hundred kilodaltons. It was a glycoprotein and had an almost neutral pH optimum. The three other hydrolases resembled lysosomal cathepsins B, H, and L in various respects except for somewhat higher molecular weight for cathepsin B (29 kDa) and the cathepsin H-like (70 kDa) hydrolase than those reported from most other tissues. Low molecular weight cysteine proteinase inhibitors ACPI (cystatin A) and NCPI (cystatin B) inhibited the cathepsins, but not the high molecular weight proteinase. PMID:3501707

  1. Crystal structure of viral serpin crmA provides insights into its mechanism of cysteine proteinase inhibition.

    OpenAIRE

    Simonovic, M; Gettins PGW; Volz, K

    2000-01-01

    CrmA is an unusual viral serpin that inhibits both cysteine and serine proteinases involved in the regulation of host inflammatory and apoptosis processes. It differs from other members of the serpin superfamily by having a reactive center loop that is one residue shorter, and by its apparent inability to form SDS-stable covalent complexes with cysteine proteinases. To obtain insight into the inhibitory mechanism of crmA, we determined the crystal structure of reactive center loop-cleaved crm...

  2. Selective Loss of Cysteine Residues and Disulphide Bonds in a Potato Proteinase Inhibitor II Family

    OpenAIRE

    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) s...

  3. Functional Properties of a Cysteine Proteinase from Pineapple Fruit with Improved Resistance to Fungal Pathogens in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2014-02-01

    Full Text Available In plant cells, many cysteine proteinases (CPs are synthesized as precursors in the endoplasmic reticulum, and then are subject to post-translational modifications to form the active mature proteinases. They participate in various cellular and physiological functions. Here, AcCP2, a CP from pineapple fruit (Ananas comosus L. belonging to the C1A subfamily is analyzed based on the molecular modeling and homology alignment. Transcripts of AcCP2 can be detected in the different parts of fruits (particularly outer sarcocarps, and gradually increased during fruit development until maturity. To analyze the substrate specificity of AcCP2, the recombinant protein was overexpressed and purified from Pichia pastoris. The precursor of purified AcCP2 can be processed to a 25 kDa active form after acid treatment (pH 4.3. Its optimum proteolytic activity to Bz-Phe-Val-Arg-NH-Mec is at neutral pH. In addition, the overexpression of AcCP2 gene in Arabidopsis thaliana can improve the resistance to fungal pathogen of Botrytis cinerea. These data indicate that AcCP2 is a multifunctional proteinase, and its expression could cause fruit developmental characteristics of pineapple and resistance responses in transgenic Arabidopsis plants.

  4. Developing novel anthelmintics from plant cysteine proteinases

    OpenAIRE

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

    2008-01-01

    Abstract 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 dru...

  5. Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase*

    OpenAIRE

    Guo?, Bihong, ??; Lin?, Jinzhong, ??; Ye?, Keqiong, ??

    2011-01-01

    The helper-component proteinase (HC-Pro) of potyvirus is involved in polyprotein processing, aphid transmission, and suppression of antiviral RNA silencing. There is no high resolution structure reported for any part of HC-Pro, hindering mechanistic understanding of its multiple functions. We have determined the crystal structure of the cysteine protease domain of HC-Pro from turnip mosaic virus at 2.0 Å resolution. As a protease, HC-Pro only cleaves a Gly-Gly dipeptide at its own C terminus....

  6. Cathepsin L cysteine proteinase in the diagnosis of bovine Fasciola gigantica infection.

    Science.gov (United States)

    Sriveny, D; Raina, O K; Yadav, S C; Chandra, D; Jayraw, A K; Singh, M; Velusamy, R; Singh, B P

    2006-01-15

    Cathepsin L cysteine proteinase from Fasciola gigantica was evaluated for its potential in the early prepatent detection of this helminth infection in bovine calves. Five cross-bred bovine calves were experimentally infected with 400 metacercariae/calf and evaluated for anti-cathepsin L antibody response. F. gigantica infection in these calves could be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western blotting with 100% sensitivity. The antigen was also used to detect F. gigantica field infection in cattle, by screening 256 sera of these animals by an ELISA, which demonstrated an overall infection rate of 26.95%. Preliminary studies showed that F. gigantica cathepsin L cysteine proteinase does not cross-react with Paramphistomum epiclitum, Gigantocotyle explanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with related helminth parasites of cattle and buffaloes are required, before this antigen can be considered suitable for immuno-diagnosis of fasciolosis in these ruminants. PMID:16300897

  7. Vaccination of BALB/c mice with Leishmania major amastigote-specific cysteine proteinase.

    Science.gov (United States)

    Rafati, S; Baba, A A; Bakhshayesh, M; Vafa, M

    2000-04-01

    Cellular immune mechanisms resulting in interferon-gamma (IFN-gamma) production are essential for protection against cutaneous leishmaniasis. Antigens of the intracellular amastigote form of the parasite, found in mammalian hosts, are likely to be good candidates for the induction of T cell response and protection from development of leishmaniasis. We purified a stage-specific antigen from amastigote soluble antigen (A-SLA) of Leishmania major by immunoaffinity chromatography. The purified protein was characterized as a cysteine proteinase with enzymatic activity which is inhibited by E-64, and it was named the amastigote cysteine proteinase (ACP). BALB/c mice were immunized by two intraperitoneal injections, at a month interval, of 5 microg of ACP or A-SLA in Freund's complete adjuvant (FCA). Animals were challenged 4 weeks later with 106 L. major promastigotes and examined 4 months after the last injection. The immunized animals developed significantly smaller or no lesions compared with controls. Spleen cells from immunized mice showed a significant proliferative response and produced a high level of IFN-gamma in response to ACP, suggesting the induction of Th1 cells after immunization. These results make 24-kD ACP a possible component for an eventual cocktail vaccine against L. major infection. PMID:10759774

  8. Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

    Science.gov (United States)

    Martín, S; Molina, J M; Hernández, Y I; Ferrer, O; Muñoz, Ma C; López, A; Ortega, L; Ruiz, A

    2015-11-01

    The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small ruminants. PMID:26241655

  9. Immunoprotective effect of cysteine proteinase fractions from two Haemonchus contortus strains adapted to sheep and goats.

    Science.gov (United States)

    Molina, J M; Martín, S; Hernández, Y I; González, J F; Ferrer, O; Ruiz, A

    2012-08-13

    A preliminary analysis of the significance of genetic diversity in cysteine proteinase genes has been performed simultaneously in sheep and goats, with regard to the immunological control using these enzymes against haemonchosis. For this purpose, we have studied the cross-immunoprotective effect of cysteine protease-enriched protein fractions (CPFs) in adult worms of two Haemonchus contortus strains from North America and Spain that are adapted to sheep and goats, respectively. Previous genetic analysis of cysteine proteinase genes in both strains has shown that some of loci are polymorphic and these differences are translated into changes in the amino acid sequences. However, our results show that CPFs from H. contortus adult worms have a protective effect against the parasite in both sheep and goats. These results are similar regardless of whether they were obtained from sheep or goat-adapted H. contortus strains, which could be very important in case H. contortus CPFs were commercially used in different countries, as vaccines to prevent the negative effects of this parasite. Interestingly, this experimental inoculation of both species with a heterologous strain of H. contortus contributes to the idea shown in previous studies about how difficult is the interpretation and the comparison of vaccination where strains not adapted to a specific host are used. Therefore, the challenger of using heterologous strains could provide similar results to those observed in immunised animals. This study suggests the possibility of exploring the mechanisms involved in natural protection against non-adapted strains, in order to develop strategies to control haemonchosis. PMID:22487211

  10. Cloning and sequencing of cDNA encoding a rat salivary cysteine proteinase inhibitor inducible by beta-adrenergic agonists.

    Science.gov (United States)

    Shaw, P A; Cox, J L; Barka, T; Naito, Y

    1988-12-01

    The beta-adrenergic agonist isoproterenol induces a unique secretory protein (LM) in the salivary glands of developing and adult rats. In order to study the regulation of growth and gene expression by catecholamines, we have isolated and sequenced several cDNA clones encoding the LM protein. Each of the LM cDNA clones described identifies, by Northern blot analyses, a single mRNA species of approximately 900 bases in size. The mRNA encoding this secreted protein was not detected in submandibular glands or brains of untreated adult rats. Sequence analyses of the LM cDNA clones revealed a striking similarity to the family 2 of cysteine proteinase inhibitors. Furthermore, when purified LM protein was used to assay for inhibition of cysteine proteinases, the data demonstrated that it is indeed a type of cysteine proteinase inhibitor. This inhibitor, termed rat cystatin S, provides the first example of cysteine proteinase inhibitors that can be induced by beta-adrenergic agonists. PMID:3263967

  11. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity

    Science.gov (United States)

    Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  12. Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death

    Directory of Open Access Journals (Sweden)

    Gregor Kosec

    2006-12-01

    Full Text Available Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family, about 250 metallopeptidases (most leishmanolysin homologues, 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF. In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2, probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins, which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

  13. Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death

    Scientific Electronic Library Online (English)

    Gregor, Kosec; Vanina, Alvarez; Juan J., Cazzulo.

    2006-12-01

    Full Text Available Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 [...] metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF. In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

  14. Beta-adrenergic induction of a cysteine-proteinase-inhibitor mRNA in rat salivary glands.

    Science.gov (United States)

    Shaw, P A; Barka, T

    1989-02-01

    Transcripts encoding the cysteine-proteinase inhibitor rat cystatin S are induced in submandibular and parotid glands by the beta-adrenergic agonist isoproterenol (isoprenaline). High levels of cystatin S mRNA persist in glands of chronically treated animals for 6 days after discontinuation of the catecholamine, indicating a long half-life of the mRNA. Post-transcriptionally the size of the mRNA decreases, owing to a shortening of the poly(A) tail. PMID:2930478

  15. A Novel Trypsin Inhibitor-Like Cysteine-Rich Peptide from the Frog Lepidobatrachus laevis Containing Proteinase-Inhibiting Activity.

    Science.gov (United States)

    Wang, Yu-Wei; Tan, Ji-Min; Du, Can-Wei; Luan, Ning; Yan, Xiu-Wen; Lai, Ren; Lu, Qiu-Min

    2015-08-01

    Various bio-active substances in amphibian skins play important roles in survival of the amphibians. Many protease inhibitor peptides have been identified from amphibian skins, which are supposed to negatively modulate the activity of proteases to avoid premature degradation or release of skin peptides, or to inhibit extracellular proteases produced by invading bacteria. However, there is no information on the proteinase inhibitors from the frog Lepidobatrachus laevis which is unique in South America. In this work, a cDNA encoding a novel trypsin inhibitor-like (TIL) cysteine-rich peptide was identified from the skin cDNA library of L. laevis. The 240-bp coding region encodes an 80-amino acid residue precursor protein containing 10 half-cysteines. By sequence comparison and signal peptide prediction, the precursor was predicted to release a 55-amino acid mature peptide with amino acid sequence, IRCPKDKIYKFCGSPCPPSCKDLTPNCIAVCKKGCFCRDGTVDNNHGKCVKKENC. The mature peptide was named LL-TIL. LL-TIL shares significant domain similarity with the peptides from the TIL supper family. Antimicrobial and trypsin-inhibitory abilities of recombinant LL-TIL were tested. Recombinant LL-TIL showed no antimicrobial activity, while it had trypsin-inhibiting activity with a Ki of 16.5178 ?M. These results suggested there was TIL peptide with proteinase-inhibiting activity in the skin of frog L. laevis. To the best of our knowledge, this is the first report of TIL peptide from frog skin. PMID:26329591

  16. Production of acid and neutral cysteine-proteinase inhibitors by a cultured human skin epithelium cell line.

    Science.gov (United States)

    Hopsu-Havu, V K; Joronen, I; Rinne, A; Järvinen, M

    1985-01-01

    Human skin epithelial-like cells (NCTC-strain 2544) were grown in RPMI-1640 medium supplemented with foetal calf serum for up to 2 weeks. The culture medium and extracts made from the cells were subjected to gel-filtration chromatography in a Sephacryl S-200 column for fractionation of the proteins. The fractions were assayed for acid and neutral cysteine-proteinase inhibitor (ACPI, NCPI) using time-resolved fluoroimmunoassay and radioimmunoassay, and the cysteine-proteinase-inhibiting activities were assayed using papain. Free NCPI, i.e. a molecule with isoelectric variants at pHs 6.0 and 6.5, which has an Mr of around 12,000 and is capable of inhibiting papain, was detected both in the culture medium and in the cells. Immunodiffusion studies revealed its immunological identity with human spleen-derived NCPI. The amount of NCPI increased during the incubation period. ACPI--characterized as a molecule having an isoelectric point of 4.9, an Mr of about 12,000, papain-inhibiting capacity and antigenic reactivity with spleen-derived ACPI--was not detected in the culture medium. It was, however, detected in the cells after 2 weeks in culture. These data prove that ACPI and NCPI are synthesized by the NCTC-2544 cells under the present culture conditions. PMID:4051554

  17. Molecular karyotype and chromosomal localization of genes encoding ß-tubulin, cysteine proteinase, hsp 70 and actin in Trypanosoma rangeli

    Directory of Open Access Journals (Sweden)

    Toaldo CB

    2001-01-01

    Full Text Available The molecular karyotype of nine Trypanosoma rangeli strains was analyzed by contour-clamped homogeneous electric field electrophoresis, followed by the chromosomal localization of ß-tubulin, cysteine proteinase, 70 kDa heat shock protein (hsp 70 and actin genes. The T. rangeli strains were isolated from either insects or mammals from El Salvador, Honduras, Venezuela, Colombia, Panama and southern Brazil. Also, T. cruzi CL-Brener clone was included for comparison. Despite the great similarity observed among strains from Brazil, the molecular karyotype of all T. rangeli strains analyzed revealed extensive chromosome polymorphism. In addition, it was possible to distinguish T. rangeli from T. cruzi by the chromosomal DNA electrophoresis pattern. The localization of ß-tubulin genes revealed differences among T. rangeli strains and confirmed the similarity between the isolates from Brazil. Hybridization assays using probes directed to the cysteine proteinase, hsp 70 and actin genes discriminated T. rangeli from T. cruzi, proving that these genes are useful molecular markers for the differential diagnosis between these two species. Numerical analysis based on the molecular karyotype data revealed a high degree of polymorphism among T. rangeli strains isolated from southern Brazil and strains isolated from Central and the northern South America. The T. cruzi reference strain was not clustered with any T. rangeli strain.

  18. [Plant signaling peptides. Cysteine-rich peptides].

    Science.gov (United States)

    Ostrowski, Maciej; Kowalczyk, Stanis?aw

    2015-01-01

    Recent bioinformatic and genetic analyses of several model plant genomes have revealed the existence of a highly abundant group of signaling peptides that are defined as cysteine-rich peptides (CRPs). CRPs are usually in size between 50 and 90 amino acid residues, they are positively charged, and they contain 4-16 cysteine residues that are important for the correct conformational folding. Despite the structural differences among CRP classes, members from each class have striking similarities in their molecular properties and function. The present review presents the recent progress in research on signaling peptides from several families including: EPF/EPFL, SP11/SCR, PrsS, RALF, LURE, and some other peptides belonging to CRP group. There is convincing evidence indicating multiple roles for these CRPs as signaling molecules during the plant life cycle, ranging from stomata development and patterning, self-incompatibility, pollen tube growth and guidance, reproductive processes, and nodule formation. PMID:26281357

  19. Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

    International Nuclear Information System (INIS)

    The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. DQ066227). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and Mr of 11kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with Ki value of 0.1 and 0.6nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis

  20. Expressions of the genes for cysteine proteinase inhibitors cystatin C and cystatin S in rat submandibular salivary gland.

    Science.gov (United States)

    Barka, T; van der Noen, H

    1994-04-01

    Rat cystatin S and rat cystatin C are members of family 2 (cystatin) of the cystatin superfamily. All members of the cystatin family inhibit cysteine proteinases to varying degree. The expression of these two inhibitors, which have a 48% similarity at the nucleotide level, was studied in the submandibular gland using reverse transcriptase-polymerase chain reaction (RT-PCR). Northern blot hybridization and in situ hybridization with digoxigenin-labelled DNA probes. Both inhibitors were expressed in the serous acinar cells of the submandibular gland. In accord with previous findings, cystatin S mRNA was induced by the beta-adrenergic agonist isoproterenol. The level of cystatin S mRNA, which was very low in the glands of untreated rats and was demonstrable by RT-PCR but not by Northern blot hybridization, was not altered by acute inflammation produced by turpentine. Neither the administration of isoproterenol nor acute inflammation had any effect on the level of cystatin C mRNA, indicating beta-adrenoreceptors are not involved in the regulation of the cystatin C gene(s) in the submandibular gland. The data indicate that these two closely related genes, expressed in the same cells, are differently regulated. The consequence of this difference in gene regulation on the physiological and pathological roles of these inhibitors remains to be established. PMID:8024495

  1. "Purification and evaluation of somatic, excretory-secretory and Cysteine proteinase antigens of Fasciola Hepatica using IgG-ELISA in diagnosing Fascioliasis "

    Directory of Open Access Journals (Sweden)

    "Rokni MB

    2001-08-01

    Full Text Available Fasciolosis, or liver fluke disease, caused by parasites of the genus Fasciola is emerging as an important disease in man and animals, in the world and Iran, particularly in nortern parts. The economical losses in domestic animals are considerable. In the recent decade there were two major outbreaks of human fasciolosis in the Caspian region, northern part of Iran with 7000-10000 infected cases. Sicne it is impossible to diagnose fasciolosis in acute phase using coprological methods and even in chronic phases its sensitivity is low, evaluating and establishing a reliable and cost-effetive test is indispensable and notewortly.In the present survey, we produced and examined the sensitivity and specificity of liver fluke homogenate (LFH , excretory-secetory (ES and cysteine proteinase (CP antigens of F. hepatica using IgG-ELISA test. A 25-27 kilo Dalton coomassie blue-stained band was observed and using of specific inhibitors indicated that this antigen belongs to the class of cysteine proteinase. The sensitivity of LFH, ES and CP antigen in IgG-ELISa was 100% for each, while their specificity was 97.8%, 98.8% and 98.8% respectively. There was a significant difference in mean OD values between cases of proven fasciolosis and other true negative cases, including healthy control individuals and patients with other parasitic diseases.This present report is the first to demonstrate the purification and evaluation of F. hepatica cysteine proteinase antigen by IgG-ELISA test for the diagnosis of fasciolosis in Iran. In conclusion, the IgG-ELISa using ES and CP show high sensitivity and specificity and would be a valuable tool to diagnose human fasciolosis in Iran, particularly in endemic areas.

  2. A barley cysteine-protease inhibitor reduces teh performance of two aphid species in artificial diets and transgenic arabidopsis plants

    OpenAIRE

    Carrillo Gil, Laura; Martinez Muñoz, Manuel; Alvarez Alfageme, Fernando; Castañera, Pedro; Smagghe, Guy; Diaz Rodriguez, Isabel; Ortego, Felix

    2011-01-01

    Cystatins from plants have been implicated in plant defense towards insects, based on their role as inhibitors of heterologous cysteine-proteinases. We have previously characterized thirteen genes encoding cystatins (HvCPI-1 to HvCPI-13) from barley (Hordeum vulgare), but only HvCPI-1 C68 ? G, a variant generated by direct-mutagenesis, has been tested against insects. The aim of this study was to analyze the effects of the whole gene family members of barley cystatins against two aphids, Myzu...

  3. Differential gene expression in an actinorhizal symbiosis: evidence for a nodule-specific cysteine proteinase.

    OpenAIRE

    Goetting-Minesky, M P; Mullin, B C

    1994-01-01

    Nodules formed on the roots of actinorhizal plants as a consequence of nitrogen-fixing symbioses with the actinomycete Frankia appear to result from modification of the developmental pathway that leads to lateral root formation. Presently no information exists about factors that control this developmental switch or, until now, about genes that are differentially expressed as a result of an altered developmental pathway. Differential screening of an Alnus glutinosa nodule cDNA library revealed...

  4. Growth and development of Colorado potato beetle larvae, Leptinotarsa decemlineata, on potato plants expressing the oryzacystatin II proteinase inhibitor.

    Science.gov (United States)

    Cingel, Aleksandar; Savi?, Jelena; Vinterhalter, Branka; Vinterhalter, Dragan; Kosti?, Miroslav; Jovanovi?, Darka Šešlija; Smigocki, Ann; Ninkovi?, Slavica

    2015-08-01

    Plant proteinase inhibitors (PIs) are attractive tools for crop improvement and their heterologous expression can enhance insect resistance in transgenic plants. PI oryzacystatin II (OCII), isolated from rice, showed potential in controlling pests that utilize cysteine proteinases for protein digestion. To evaluate the applicability of the OCII gene in enhancing plant defence, OCII-transformed potatoes were bioassayed for resistance to Colorado potato beetle (Leptinotarsa decemlineata Say). Feeding on transformed leaves of potato cultivars Desiree and Jelica significantly affected larval growth and development, but did not change mortality rates. During the L2 and L3 developmental stages larvae consumed the OCII-transformed foliage faster as compared to the nontransformed control. Also these larvae reached the prepupal stage (end of L4 stage) 2 days earlier than those fed on control leaves. However, the total amounts of consumed OCII-transformed leaves were up to 23% lower than of control, and the maximal weights of prepupal larvae were reduced by up to 18% as compared to larvae fed on nontransformed leaves. The reduction in insect fitness reported in this study in combination with other control measures, could lead to improved CPB resistance management in potato. PMID:25820664

  5. Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor.

    Science.gov (United States)

    Nogueira, Fábio C S; Silva, Carlos P; Alexandre, Daniel; Samuels, Richard I; Soares, Emanoella L; Aragão, Francisco J L; Palmisano, Giuseppe; Domont, Gilberto B; Roepstorff, Peter; Campos, Francisco A P

    2012-08-01

    The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor. PMID:22833537

  6. Global proteome changes in larvae of Callosobruchus maculatus Coleoptera:Chrysomelidae:Bruchinae) following ingestion of a cysteine proteinase inhibitor

    DEFF Research Database (Denmark)

    Nogueira, Fábio C S; Silva, Carlos P

    2012-01-01

    The seed-feeding beetle Callosobruchus maculatus is an important cowpea pest (Vigna unguiculata) as well as an interesting model to study insect digestive physiology. The larvae of C. maculatus rely on cysteine and aspartic peptidases to digest proteins in their diet. In this work, the global proteomic changes induced in the intestinal tract of larval C. maculatus challenged by the ingestion of cystatin, a cysteine peptidase inhibitor, was investigated by a nanoLC-MS/MS approach. The ingestion of cystatin caused a delay in the development of the larvae, but the mortality was not high, indicating that C. maculatus is able to adapt to this inhibitor. This proteomic strategy resulted in the identification of 752 and 550 protein groups in the midgut epithelia and midgut contents, respectively, and quantitative analyses allowed us to establish relative differences of the identified proteins. Ingestion of cystatin led to significant changes in the proteome of both the midgut epithelia and midgut contents. We have observed that proteins related to plant cell wall degradation, particularly the key glycoside hydrolases of the families GH5 (endo-?-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, ?-amylases were downexpressed, indicating that an increase in hemicelluloses digestion helps the larvae to cope with the challenge of cystatin ingestion. Furthermore, a number of proteins associated with transcription/translation and antistress reactions were among the cystatin-responsive proteins, implying that a substantial rearrangement in the proteome occurred in C. maculatus exposed to the inhibitor.

  7. Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves.

    OpenAIRE

    Farmer, E.E.; Ryan, C. A.

    1990-01-01

    Inducible defensive responses in plants are known to be activated locally and systemically by signaling molecules that are produced at sites of pathogen or insect attacks, but only one chemical signal, ethylene, is known to travel through the atmosphere to activate plant defensive genes. Methyl jasmonate, a common plant secondary compound, when applied to surfaces of tomato plants, induces the synthesis of defensive proteinase inhibitor proteins in the treated plants and in nearby plants as w...

  8. Molecular insights into mechanisms of lepidopteran serine proteinase resistance to natural plant defenses.

    Science.gov (United States)

    Tamaki, Fábio K; Terra, Walter R

    2015-11-27

    Plants have a wide range of chemical defenses against predation, including substances that target digestive serine proteinases of herbivorous. Previous works demonstrated that lepidopteran insects have digestive serine proteinases resistant to plant proteinase inhibitors (PPIs) and ketone modifications, while coleopteran ones are sensitive to those plant defenses. This paper focuses on molecular aspects that lead lepidopteran serine proteinases to PPI and ketone modification resistance. Using biochemical experiments and computer 3D modeling we demonstrated that lepidopteran trypsins are more hydrophobic than coleopteran ones, a feature associated to trypsin oligomerization and decreased inhibition by PPI. Moreover, the determination of pKa values of chymotrypsin catalytic residues obtained by TPCK modification indicates that the environment around the active site of ketone-resistant and -sensitive chymotrypsins are different. Structural analysis using resistant and sensitive chymotrypsins data allowed us to point 2 hotspot regions around the active site that could explain the observed differences. Our set of results highlights features of serine proteinases important for understanding the resistance of insects to plant chemical defenses. PMID:26474705

  9. Human T cell responses against the major cysteine proteinase (cruzipain) of Trypanosoma cruzi: role of the multifunctional alpha 2-macroglobulin receptor in antigen presentation by monocytes.

    Science.gov (United States)

    Morrot, A; Strickland, D K; Higuchi, M de L; Reis, M; Pedrosa, R; Scharfstein, J

    1997-06-01

    Chagas' disease patients (CDP) develop both humoral and cellular immune responses against the major cysteine proteinase (cruzipain) from Trypanosoma cruzi. Here we demonstrate that complexes formed by cruzipain and alpha 2-macroglobulin (alpha 2M) are efficiently internalized by human monocytes, and that this process results in enhanced presentation of cruzipain peptides to CD4+ T cells from CDP. Purified or serum alpha 2M binds to polymorphic cruzipains, but only a fraction of the proteinases become covalently linked. Once bound to alpha 2M, fluorescein-labeled cruzipain (FITC-cruzipain) or [125I]cruzipain were more efficiently internalized by normal peripheral blood mononuclear cells (PBMC) or monocytes; this effect was abolished by (I) pre-treating the cells with receptor-associated protein (rRAP), a known antagonist the of alpha 2M receptor (alpha 2MR/LRP), and (II) inactivating [125I]cruzipain's active site prior to the reaction with alpha 2M, indicating that the exposure of receptor binding sites on alpha 2M complexes required bait region cleavage. We then sought to determine if the alpha 2MR/LRP-dependent uptake of alpha 2M:cruzipain by monocytes resulted in increased CD4+ T cell responses of PBMC-CDP (n = 13). These effects were only revealed after depletion of CD19+ B lymphocytes from PBMC-CDP; the threshold of T cell stimulation was far lower in cultures stimulated with alpha 2M:cruzipain, as compared to antigen alone. Myocardial specimens from CDP with chronic myocardiopathy (three necropsies) were analyzed by immunohistochemistry with mAb anti-cruzipain or anti-alpha 2MR/LRP (CD81+). Extracellular depots of cruzipain were localized amidst inflammatory mononuclear infiltrates, part of which contained CD91+ macrophage-like cells. Ongoing studies should clarify if T. cruzi cysteinyl proteinases play a role in the pathogenesis of Chagas' heart disease. PMID:9199965

  10. Characterization by rapid-kinetic and equilibrium methods of the interaction between N-terminally truncated forms of chicken cystatin and the cysteine proteinases papain and actinidin.

    Science.gov (United States)

    Lindahl, P; Nycander, M; Ylinenjärvi, K; Pol, E; Björk, I

    1992-08-15

    The interaction between five N-terminally truncated forms of chicken cystatin (starting at Leu-7, Leu-8, Gly-9, Ala-10 and Asp-15) and the cysteine proteinases papain and actinidin was studied by spectroscopic, kinetic and equilibrium methods. The u.v. absorption, near-u.v. c.d. and fluorescence emission difference spectra for the interactions with papain were all similar to the corresponding spectra for intact cystatin. The second-order association rate constants at 25 degrees C, pH 7.4, I 0.15, for the binding of the truncated forms to papain varied about 2-fold, from 6 x 10(6) to 1.5 x 10(7) M-1.s-1, and were comparable to the value of 9.9 x 10(6) M-1.s-1 for intact cystatin. In contrast, the rate constants for the dissociation of the complexes with papain increased markedly with increasing extent of truncation, from 7.5 x 10(-6)s-1 for Leu7 cystatin (a truncated form of cystatin having Leu-7 as its N-terminal amino acid) to 1.6s-1 for Ala10-cystatin, whereas the dissociation rate constants for the latter form and Asp15-cystatin were similar. Consequently, the binding affinities between the truncated cystatins and papain decreased in an analogous manner, as was also shown for the interaction with actinidin by equilibrium measurements. Studies of the binding of the truncated cystatins to inactivated papains indicated that small substituents on the active-site cysteine of the enzyme can be accommodated in the complex without any loss of affinity when the N-terminal segment of the inhibitor is removed. Taken together, the results suggest that in the N-terminal region of chicken cystatin only residues preceding Ala-10 participate in the interaction with proteinases. Of these residues, Leu-7 and Leu-8 together account for about two-thirds of the unitary free energy of binding contributed by the N-terminal region, the relative importance of the two residues being dependent on the target proteinase. Both Gly-9 and residues N-terminal of Leu-7 further stabilize the interaction but contribute substantially smaller binding energies than do the two leucine residues. PMID:1520264

  11. EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

    OpenAIRE

    Olsén Arne; Allhorn Maria; Collin Mattias

    2008-01-01

    Abstract Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of Endo...

  12. Evaluation of Live Recombinant Nonpathogenic Leishmania tarentolae Expressing Cysteine Proteinase and A2 Genes as a Candidate Vaccine against Experimental Canine Visceral Leishmaniasis.

    Science.gov (United States)

    Shahbazi, Mehdi; Zahedifard, Farnaz; Taheri, Tahereh; Taslimi, Yasaman; Jamshidi, Shahram; Shirian, Sadegh; Mahdavi, Niousha; Hassankhani, Mehdi; Daneshbod, Yahya; Zarkesh-Esfahani, Sayyed Hamid; Papadopoulou, Barbara; Rafati, Sima

    2015-01-01

    Canine Visceral Leishmaniasis (CVL) is a major veterinary and public health problem caused by Leishmania infantum (L. infantum) in many endemic countries. It is a severe chronic disease with generalized parasite spread to the reticuloendothelial system, such as spleen, liver and bone marrow and is often fatal when left untreated. Control of VL in dogs would dramatically decrease infection pressure of L. infantum for humans, since dogs are the main domestic reservoir. In the past decade, various subunits and DNA antigens have been identified as potential vaccine candidates in experimental animal models, but none has been approved for human use so far. In this study, we vaccinated outbreed dogs with a prime-boost regimen based on recombinant L. tarentolae expressing the L. donovani A2 antigen along with cysteine proteinase genes (CPA and CPB without its unusual C-terminal extension (CPB-CTE) and evaluated its immunogenicity and protective immunity against L. infantum infectious challenge. We showed that vaccinated animals produced significantly higher levels of IgG2, but not IgG1, and also IFN-? and TNF-?, but low IL-10 levels, before and after challenge as compared to control animals. Protection in dogs was also correlated with a strong DTH response and low parasite burden in the vaccinated group. Altogether, immunization with recombinant L. tarentolae A2-CPA-CPB-CTE was proven to be immunogenic and induced partial protection in dogs, hence representing a promising live vaccine candidate against CVL. PMID:26197085

  13. Antitumor Effects In Vitro and In Vivo and Mechanisms of Protection against Melanoma B16F10-Nex2 Cells By Fastuosain, a Cysteine Proteinase from Bromelia fastuosa

    Directory of Open Access Journals (Sweden)

    Carla A. Guimarães-Ferreira

    2007-09-01

    Full Text Available In the present work, the antitumor effect of fastuosain, a cysteine proteinase from Bromelia fastuosa, was investigated. In the intravenous model of lung colonization in C57BI/6 mice, fastuosain and bromelain injected intraperitoneally were protective, very few nodules of B16F10-Nex2 melanoma cells were detected. Tumor cells treated with fastuosain showed reduced expression of CD44 and decreased invasion through Matrigel, lost their cytoplasmic extensions and substrate adherence, became round and detached, forming strongly bound cell clusters in suspension. Peritoneal cells recruited and activated by fastuosain treatment (mainly monocytic cells and lymphocytes migrated to the lung, where pulmonary melanoma metastases grew. Adoptive transference of peritoneal cells recruited by fastuosain had no protective effect against lung metastases in recipient mice. Treatment of green fluorescent protein -chimeric animals with fastuosain did not change the number of cells that migrated to the lung, compared to PBSinjected control mice, but the number of positive major histocompatibility complex class II cells increased with fastuosain treatment. Murine antibodies against fastuosain, bromelain, cathepsins B and L crossreacted in ELISA and recognized surface and cytoplasmic components expressed on B16F10-Nex2 cells. Anti-fastuosain antibodies were cytotoxic/lytic to B16F10-Nex2 cells. Antitumor effects of fastuosain involve mainly the direct effect of the enzyme and elicitation of protective antibodies.

  14. EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase SpeB and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity

    Directory of Open Access Journals (Sweden)

    Olsén Arne

    2008-01-01

    Full Text Available Abstract Background The endoglycosidase EndoS and the cysteine proteinase SpeB from the human pathogen Streptococcus pyogenes are functionally related in that they both hydrolyze IgG leading to impairment of opsonizing antibodies and thus enhance bacterial survival in human blood. In this study, we further investigated the relationship between EndoS and SpeB by examining their in vitro temporal production and stability and activity of EndoS. Furthermore, theoretical structure modeling of EndoS combined with site-directed mutagenesis and chemical blocking of amino acids was used to identify amino acids required for the IgG glycan-hydrolyzing activity of EndoS. Results We could show that during growth in vitro S. pyogenes secretes the IgG glycan-hydrolyzing endoglycosidase EndoS prior to the cysteine proteinase SpeB. Upon maturation SpeB hydrolyzes EndoS that then loses its IgG glycan-hydrolyzing activity. Sequence analysis and structural homology modeling of EndoS provided a basis for further analysis of the prerequisites for IgG glycan-hydrolysis. Site-directed mutagenesis and chemical modification of amino acids revealed that glutamic acid 235 is an essential catalytic residue, and that tryptophan residues, but not the abundant lysine or the single cysteine residues, are important for EndoS activity. Conclusion We present novel information about the amino acid requirements for IgG glycan-hydrolyzing activity of the immunomodulating enzyme EndoS. Furthermore, we show that the cysteine proteinase SpeB processes/degrades EndoS and thus emphasize the importance of the SpeB as a degrading/processing enzyme of proteins from the bacterium itself.

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

    Science.gov (United States)

    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. PMID:24990521

  16. In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors.

    Science.gov (United States)

    Parde, Vinod D; Sharma, Hari C; Kachole, Manvendra S

    2010-09-01

    We evaluated 22 different host and non-host plant protease inhibitors (PIs) for in vivo inhibition of Helicoverpa armigera gut pro- and proteinases, and their biological activity against the pod borer, H. armigera, the most important pest of agriculture and horticultural crops worldwide. In vitro activation of H. armigera gut pro-proteinases (HaGPPs) in larvae fed on non-host plant PIs showed significant in vivo inhibition of HaGPPs activation in solution as well as in gel assays. The larvae fed on diet incorporated with Datura alba ness PIs showed highest inhibition of HaGPPs, followed by Psophocarpus tetragonolobus. Non-host plant PIs from Pongamia pinnata, Mucuna pruriens, Capsicum annuum, and Nigela sativa showed maximum inhibitory potential towards HaGPs in vivo, and also exhibited moderate level of inhibition of pro-proteinases. However, some of non-host plant PIs, such as those from Penganum harmala and Solanum nigrum, and the principal host plant PIs, viz., Cicer arietinum and Cajanus cajan did not inhibit HaGPP activity. Pro-proteinase level increased with the growth of the larvae, and maximum HaGPP activity was observed in the fifth-instars. Larvae fed on diets with D. alba ness PIs showed greater inhibition of HaGPPs as compared to the larvae fed on diets with P. tetragonolobus. Low concentrations of partially purified HaGPs treated with gut extract of larvae fed on D. alba ness showed that out of 10 proteinase isoforms, HaGPs 5 and 9 were activators of pro-proteinases. Larval growth and development were significantly reduced in the larvae fed on the non-host plant PIs, of which D. alba ness resulted in highest stunted growth of H. armigera larvae. The in vivo studies indicated that non-host plant PIs were good candidates as inhibitors of the HaGPs as well as HaGPPs. The PIs from the non-host plants can be expressed in genetically engineered plants to confer resistance to H. armigera. PMID:20416317

  17. A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants

    OpenAIRE

    Matsuda, Taiki; Kuramata, Masato; Takahashi, Yoshihiro; Kitagawa, Etsuko; Youssefian, Shohab; Kusano, Tomonobu

    2009-01-01

    We have identified a novel cDNA clone, termed DcCDT1, from Digitaria ciliaris, that confers cadmium (Cd)-tolerance to yeast (Saccharomyces cerevisiae). The gene encodes a predicted peptide of 55 amino acid residues of which 15 (27.3%) are cysteine residues. We found that monocotyledonous plants possess multiple DcCDT1 homologues, for example rice contains five DcCDT1 homologues (designated OsCDT1?5), whereas dicotyledonous plants, including Arabidopsis thaliana, Brassica rapa, poplar (Populus...

  18. Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules.

    Science.gov (United States)

    Maróti, Gergely; Downie, J Allan; Kondorosi, Éva

    2015-08-01

    Plants must co-exist with both pathogenic and beneficial microbes. Antimicrobial peptides with broad antimicrobial activities represent one of the first lines of defense against pathogens. Many plant cysteine-rich peptides with potential antimicrobial properties have been predicted. Amongst them, defensins and defensin-like peptides are the most abundant and plants can express several hundreds of them. In some rhizobial-legume symbioses special defensin-like peptides, the nodule-specific cysteine-rich (NCR) peptides have evolved in those legumes whose symbiotic partner terminally differentiates. In Medicago truncatula, >700 NCRs exist and collectively act as plant effectors inducing irreversible differentiation of rhizobia to nitrogen-fixing bacteroids. Cationic NCR peptides have a broad range of potent antimicrobial activities but do not kill the endosymbionts. PMID:26116977

  19. Expression of the cysteine proteinase inhibitor cystatin C gene in rat heart: use of digoxigenin-labeled probes generated by polymerase chain reaction directly for in situ and northern blot hybridizations.

    Science.gov (United States)

    Barka, T; van der Noen, H

    1993-12-01

    Cystatins represent a widely distributed superfamily of cysteine proteinase inhibitory proteins. We investigated the expression of the cystatin C gene, belonging to the family 2 of cystatins, in the hearts of female rats. Using a highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) we have detected cystatin C mRNA in the ventricule and atrium, as well as in liver and submandibular gland. A digoxigenin-labeled cystatin C probe, generated by PCR, hybridized to a single mRNA species of about 700 nucleotides on Northern blots. Northern blot hybridizations established that neither an acute inflammation produced by injection of turpentine nor administration of the beta-adrenergic agonist isoproterenol had an effect on the level of cystatin C mRNA in the heart. In situ hybridizations with digoxigenin-labeled probe localized the expression of the cystatin C gene to cardiac muscle fibers but not to other cardiac cellular elements. Cystatin C may be released by cardiac muscle fibers under physiological and pathological conditions and may modify inflammatory and necrobiotic processes. PMID:8245434

  20. Role for different cell proteinases in cancer invasion and cytolysis.

    OpenAIRE

    Zucker, S.; Beck, G.; DiStefano, J. F.; Lysik, R. M.

    1985-01-01

    The crucial role of non-plasminogen dependent serine proteinases is tissue invasive and cytolytic functions of Walker 256 cancer cells has been documented using a rat urinary bladder invasion and a 125I-labelled fibroblast cytolysis assay. The invasive capacity of these cancer cells was abrogated by non toxic concentrations of the serine proteinase inhibitors, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, but not by metallo or cysteine proteinase inhibitors. Although tumour cel...

  1. Multiple insect resistance in transgenic tomato plants over-expressing two families of plant proteinase inhibitors.

    Science.gov (United States)

    Abdeen, Ashraf; Virgós, Ariadna; Olivella, Elisenda; Villanueva, Josep; Avilés, Xavier; Gabarra, Rosa; Prat, Salomé

    2005-01-01

    Protease inhibitors have been proposed as potential defense molecules for increased insect resistance in crop plants. Compensatory over-production of insensitive proteases in the insect, however, has limited suitability of these proteins in plant protection, with very high levels of inhibitor required for increased plant resistance. In this study we have examined whether combined used of two inhibitors is effective to prevent this compensatory response. We show that leaf-specific over-expression of the potato PI-II and carboxypeptidase inhibitors (PCI) results in increased resistance to Heliothis obsoleta and Liriomyza trifolii larvae in homozygote tomato lines expressing high levels (>1% the total soluble proteins) of the transgenes. Leaf damage in hemizygous lines for these transformants was, however, more severe than in the controls, thus evidencing a compensation response of the larvae to the lower PI concentrations in these plants. Development of comparable adaptive responses in both insects suggests that insect adaptation does not entail specific recognition of the transgene, but rather represents a general adaptive mechanism triggered in response to the nutritional stress imposed by sub-lethal concentrations of the inhibitors. Combined expression of defense genes with different mechanisms of action rather than combinations of inhibitors may then offer a better strategy in pest management as it should be more effective in overcoming this general adaptive response in the insect. PMID:15821877

  2. Fasciola hepatica: secretion of a novel cathepsin L proteinase

    OpenAIRE

    Dowd, Andrew

    1994-01-01

    A 29 5 kDa cysteine proteinase was purified from medium m which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 amino acid residues) of the purified protein is homologous to known cathepsin L proteinases, including a 27 kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory (Smith e ta l , Mol Biochem Parasitol.,62,1-8,1993). The Nterminal sequences of the 29 5 and 27 kDa cathepsin L proteinases differ o...

  3. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  4. The toxic effects of l-Cysteine-capped cadmium sulfide nanoparticles on the aquatic plant Spirodela polyrrhiza

    Energy Technology Data Exchange (ETDEWEB)

    Khataee, Alireza, E-mail: ar_khataee@yahoo.com [University of Tabriz, Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry, Faculty of Chemistry (Iran, Islamic Republic of); Movafeghi, Ali [University of Tabriz, Department of Plant Biology, Faculty of Natural Sciences (Iran, Islamic Republic of); Nazari, Fatemeh [University of Tabriz, Research Laboratory of Advanced Water and Wastewater Treatment Processes, Department of Applied Chemistry, Faculty of Chemistry (Iran, Islamic Republic of); Vafaei, Fatemeh [University of Tabriz, Department of Plant Biology, Faculty of Natural Sciences (Iran, Islamic Republic of); Dadpour, Mohammad Reza [University of Tabriz, Department of Horticultural Science, Faculty of Agriculture (Iran, Islamic Republic of); Hanifehpour, Younes; Joo, Sang Woo, E-mail: swjoo@yu.ac.kr [Yeungnam University, School of Mechanical Engineering (Korea, Republic of)

    2014-12-15

    Plants play an important role in the fate of nanoparticles in the environment through their uptake, bioaccumulation, and transfer to trophic chains. However, the impacts of nanoparticles on plants as essential components of all ecosystems are not well documented. In the present study, the toxic effects of l-Cysteine-capped CdS nanoparticles on Spirodela polyrrhiza as an aquatic higher plant species were studied. l-Cysteine-capped CdS nanoparticles were synthesized using hydrothermal method and their characteristics were determined by XRD, SEM, HR-TEM, and FT-IR techniques. The diameter of majority of synthesized nanoparticles was about 15–20 nm. Subsequently, the uptake of l-Cysteine-capped CdS nanoparticles by the plant species was confirmed using epifluorescence microscopy. The activity of peroxidase and superoxide dismutase as antioxidant enzymes was assayed and the relative frond number was calculated in the presence of different concentrations of l-Cysteine-capped CdS nanoparticles. The obtained results revealed the toxic effects of the synthesized nanoparticles on S. polyrrhiza, leading to growth reduction and significant changes in antioxidant enzymes’ activity.Graphical Abstract.

  5. The toxic effects of l-Cysteine-capped cadmium sulfide nanoparticles on the aquatic plant Spirodela polyrrhiza

    International Nuclear Information System (INIS)

    Plants play an important role in the fate of nanoparticles in the environment through their uptake, bioaccumulation, and transfer to trophic chains. However, the impacts of nanoparticles on plants as essential components of all ecosystems are not well documented. In the present study, the toxic effects of l-Cysteine-capped CdS nanoparticles on Spirodela polyrrhiza as an aquatic higher plant species were studied. l-Cysteine-capped CdS nanoparticles were synthesized using hydrothermal method and their characteristics were determined by XRD, SEM, HR-TEM, and FT-IR techniques. The diameter of majority of synthesized nanoparticles was about 15–20 nm. Subsequently, the uptake of l-Cysteine-capped CdS nanoparticles by the plant species was confirmed using epifluorescence microscopy. The activity of peroxidase and superoxide dismutase as antioxidant enzymes was assayed and the relative frond number was calculated in the presence of different concentrations of l-Cysteine-capped CdS nanoparticles. The obtained results revealed the toxic effects of the synthesized nanoparticles on S. polyrrhiza, leading to growth reduction and significant changes in antioxidant enzymes’ activity.Graphical Abstract

  6. Proteinase activity in latex of three plants of the family Euphorbiaceae

    Scientific Electronic Library Online (English)

    Andréa Michel, Sobottka; Fabiana, Tonial; Sonja, Sytwala; Matthias, Melzig.

    2014-09-01

    Full Text Available Dentro da família Euphorbiaceae, os gêneros Euphorbia e Sapium são conhecidos por incluírem basicamente espécies produtoras de látex. No presente estudo, o látex das plantas Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil. e Sapium glandulosum (L.) Morong, espécies nativas [...] do Brasil, foi analisado em relação à atividade proteolítica. Todas as amostras analisadas possuem proteínas com significativa atividade, sendo que o látex da espécie E. papillosa apresenta a maior atividade específica. Com o objetivo de analisar quais os tipos de proteases responsáveis pela atividade proteolítica, realizaram-se ensaios com diferentes inibidores. Nas três plantas testadas a atividade foi inibida significativamente pelo cloridrato de 4-(fluoreto de 2-aminoetilbenzenossulfonil) (AEBSF), um inibidor de serino-proteases. Utilizando técnicas de eletroforese em gel de poliacrilamida (SDS-PAGE), as subunidades das proteínas foram separadas de acordo com sua massa molecular e, através da zimografia, a atividade proteolítica pode ser detectada visualmente. Abstract in english In the family of Euphorbiaceae, the genera Euphorbia and Sapium are known to contain essentially latex-bearing species. In the present study, the latex of Euphorbia selloi (Klotzsch & Garcke) Boiss., Euphorbia papillosa A.St.-Hil., and Sapium glandulosum (L.) Morong, plants native from Brazil, were [...] examined concerning proteolytic activity. All studied species have proteins with significant proteolytic activity and E. papillosa has the greatest specific activity. Aiming to verify the type of protease present, an assay with different inhibitors was performed. In the three tested plants, the proteolytic activity was significantly inhibited by a serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF). Using techniques of electrophoresis with polyacrylamide gels (SDS-PAGE), the subunits of proteins were separated according to their molecular masses, and the protein activity was visually detected by zymography.

  7. [Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants] [In Process Citation

    Science.gov (United States)

    Mu; Liu; Zhou; Wen; Zhang; Wei

    1999-01-01

    Arrowhead Proteinase Inhibitor(API), one kind of pure natural material, was derived from storage organ of Sagittaria trifolia. It belongs to serine proteinase inhibitor, and can inhibit trypsin, chemotrypsin and kallikrein. Furthermore, API is toxical to some species of insects such as lepidotera, Coleoptera and Diptrea etc. By means of pollen tube pathway, plasmid pBIAH-A(B) containing insect-resistant genes of API-A, API-B and selective marker gene of NPT-II were transferred into three lines of local winter wheat--JD-1, 8866, 866554. Then, Kanamycin-resistant screening and PCR analysis of genetic transformed plants showed that three of Kmr green plants (two from 866554, one from JD-1) were PCR positive with the positive rate of 0.29%. When the fragment of API gene was used as probe to hybrid with genomic DNA of Kmr green plants separately, all of three PCR positive ones displayed a single strong hybridizing band. Such results demonstrated that foreign target gene had been integrated into wheat genome already. Simultaneously, PCR analysis and Southern hybridization were carried out among selfiedoffsprings of transformed positive plant of the line 899554-3, some of them were PCR and Southern blotting positive, indicating that foreign gene integrated into wheat genome could stably transmitted into next generation. In addition, the expression level of NPT-II gene was checked via ELISA in our study, all of three PCR and Southern blot positive plants could yield high level of NPT-II. This data provided a more powerful evidence for integration of insecticide gene into wheat genome. PMID:10876664

  8. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    DEFF Research Database (Denmark)

    Semenova, Tatyana; Hughes, David Peter; Boomsma, Jacobus Jan; Schiøtt, Morten

    2011-01-01

    Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results: We determined proteinase activity profiles...

  9. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    OpenAIRE

    Hughes David P; Semenova Tatyana A; Boomsma Jacobus J; Schiøtt Morten

    2011-01-01

    Abstract Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activit...

  10. Influence of air temperature on proteinase activity and beverage quality in Coffea arabica

    Scientific Electronic Library Online (English)

    Hellen Marília Couto de, Abreu; Paula Macedo, Nobile; Milton Massao, Shimizu; Paula Yuri, Yamamoto; Emerson Alves, Silva; Carlos Augusto, Colombo; Paulo, Mazzafera.

    Full Text Available Fruits were collected from trees of Coffea arabica cv. Obatã grown at Mococa and Adamantina in São Paulo State, Brazil, which are regions with marked differences in air temperature that produce coffee with distinct qualities. Mococa is a cooler location that produces high-quality coffee, whereas cof [...] fee from Adamantina is of lower quality. The amino acid and protein contents, amino acid profile, and proteinase activity and type in endosperm protein extracts were analysed. Proteinase genes were identified, and their expression was assayed. All results indicate that temperature plays a role in controlling proteinase activity in coffee endosperm. Proteinase activity was higher in the endosperm of immature fruits from Adamantina, which was correlated with higher amino acid content, changes in the amino acid profile, and increased gene expression. Cysteine proteinases were the main class of proteinases in the protein extracts. These data suggest that temperature plays an important role in coffee quality by altering nitrogen compound composition.

  11. Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - From the field to the test tube and back

    DEFF Research Database (Denmark)

    Jutta, Papenbrock; Anja, Riemenschneider

    2007-01-01

    Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different Brassica napus genotypes it was shown that the genetic differ- ences among Brassica genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field ex- periment demonstrated that sulfur supply and infection with Pyrenopeziza brassica influenced L-cysteine desulfhydrase activity in Brassica napus. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant Arabi- dopsis thaliana. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)- lyase or enzymes not yet characterized. For the exact determi- nation of the H2S concentration in the cell a H2S-specific micro- sensor was used the first time for plant cells. The transfer of the results obtained for application back on Brassica was initiated.

  12. The Vaccinia Virus I7L Gene Product Is The Core Protein Proteinase

    OpenAIRE

    Byrd, Chelsea M; Bolken, Tove' C.; Dennis E. Hruby

    2002-01-01

    Maturation of vaccinia virus (VV) core proteins is required for the production of infectious virions. The VV G1L and I7L gene products are the leading candidates for the viral core protein proteinase (vCPP). Using transient-expression assays, data were obtained to demonstrate that the I7L gene product and its encoded cysteine proteinase activity are responsible for vCPP activity.

  13. Poliovirus 3C proteinase inhibition by organotelluranes.

    Science.gov (United States)

    Gouvea, Iuri E; Santos, Jorge A N; Burlandy, Fernanda M; Tersariol, Ivarne L S; da Silva, Edson E; Juliano, Maria A; Juliano, Luiz; Cunha, Rodrigo L O R

    2011-04-01

    The 3C proteinase, essential for human poliovirus (PV) replication, has unique characteristics as its three-dimensional structure resembles chymotrypsin, but its catalytic nucleophile is a cysteine SH group rather than the OH group of serine. Here, we describe the use of tellurium compounds as inhibitors of PV3C proteinase. A rapid, stoichiometric and covalent inactivation of PV3C was observed with both a chloro-telluroxetane and a bis-vinylic organotellurane. These compounds also inhibit human cathepsins B, L, S, and K with second order rate constants higher than those obtained for PV3C. Chloro-telluroxetane inhibits replication of PV in human embryonic rhabdomyosarcoma cells in the low micromolar range and below the toxic level for the host cells. Bis-vinylic organotellurane is more effective as antiviral agent but reduces the cell viability by 20% at 10 ?m, a concentration almost completely inhibiting virus growth. This is the first description of inhibition of viral 3C proteinase with antiviral property by this class of compounds. PMID:21521074

  14. Serine and cysteine protease-like genes in the genome of a gall midge and their interactions with host plant genotypes

    Science.gov (United States)

    For plant-feeding insects, digestive proteases are targets for engineering protease inhibitors for pest control. In this study, we identified 105 putative serine- and cysteine-protease genes from Hessian fly genome. Among the genes, 31 encode putative trypsins, 18 encode putative chymotrypsins, se...

  15. Functional Specialization and Evolution of Leader Proteinases in the Family Closteroviridae

    OpenAIRE

    Peng, Chih-Wen; Peremyslov, Valera V.; Mushegian, Arcady R; Dawson, William O.; Dolja, Valerian V

    2001-01-01

    Members of the Closteroviridae and Potyviridae families of the plant positive-strand RNA viruses encode one or two papain-like leader proteinases. In addition to a C-terminal proteolytic domain, each of these proteinases possesses a nonproteolytic N-terminal domain. We compared functions of the several leader proteinases using a gene swapping approach. The leader proteinase (L-Pro) of Beet yellows virus (BYV; a closterovirus) was replaced with L1 or L2 proteinases of Citrus tristeza virus (CT...

  16. Serpins in plants and green algae

    DEFF Research Database (Denmark)

    Roberts, Thomas Hugh; Hejgaard, JØrn

    2008-01-01

    Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary relationship between these serpins and those of plants remains unknown. Plant serpins are potent inhibitors of mammalian serine proteinases of the chymotrypsin family in vitro but, intriguingly, plants and green algae lack endogenous members of this proteinase family, the most common targets for animal serpins. An Arabidopsis serpin with a conserved reactive centre is now known to be capable of inhibiting an endogenous cysteine proteinase. Here, knowledge of plant serpins in terms of sequence diversity, inhibitory specificity, gene expression and function is reviewed. This was advanced through a phylogenetic analysis of amino acid sequences of expressed plant serpins, delineation of plant serpin gene structures and prediction of inhibitory specificities based on identification of reactive centres. The review is intended to encourage elucidation of plant serpin functions.

  17. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W. (Stony Brook, NY); Mangel, Walter F. (Shoreham, NY)

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  18. Production of Plant Proteinase from Jack Fruit (Artocarpus integrifolis as a Source of Dairy Enzyme I. Isolation, Partial Purification and Some Properties

    Directory of Open Access Journals (Sweden)

    Al-Sayed Al-Tanboly

    2003-01-01

    Full Text Available The aim of the present work was to search for a novel plant proteinase enzyme from Jack fruit (Artocarpus integrifolis as a source of dairy enzymes that would be natural products which can be easily extracted at relatively low cost and no legal barriers. This enzyme was subjected to a purification scheme composed of ammonium sulfate fractionation followed by gel filtration on G-100 Sephadex column. The enzyme was purified 2.70-fold with a total yield of 23.77% of the original activity. There were relationship between temperature and incubation time, the enzyme activity increase was observed up to 55°C for 60 min reaction time and still constant thereafter. Proteinase was active over a broad temperature range retained about 37.4 and 24.9% of temperature activity at 35 and 80°C for 5 and 60 min. An energy of activation of 9.98 KJ mole?1 for the enzyme activity was derived from the Arrhenius plot of initial velocity (Vo across a temperature ranging from 40 to 55°C. The optimum pH was pH 7.5. The rate of thermal inactivation proceeded more rapidly at pH 7.0 and 8.0, when heating at 50°C for 60 min the enzyme activity lost about 95 and 92% its activity, respectively. Michaelis-constant of (Km values of 2.0 mg ml?1 and a maximum initial velocity (Vmax of 0.75 µ moles mg?1 when casein used as a substrate. A Molecular weight (MW determination of ~22 kDa was estimated by gel filtration methods using a Sephadex G-100. Cu2+, K2+ , Fe2+ and Zn2+ strongly inhibited the enzyme. However, Ca++ slightly stimulated. EDTA, sodium azide, Sodium citrate and urea among the chemical reagents inhibited the proteinase activity.

  19. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    Directory of Open Access Journals (Sweden)

    Hughes David P

    2011-01-01

    Full Text Available Abstract Background Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

  20. Evolutionary patterns of proteinase activity in attine ant fungus gardens

    DEFF Research Database (Denmark)

    Semenova, Tatyana; Hughes, David Peter

    2011-01-01

    Background: Attine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved. Results: We determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts. Conclusions: Proteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

  1. Analysis of the interaction between the helper component proteinase (HC-Pro) of Zucchini yellow mosaic virus (ZYMV) and the plant RNA methyltransferase Hua enhancer 1 (HEN1)

    OpenAIRE

    Jamoos, Rana

    2012-01-01

    The helper component-proteinase (HC-Pro) is a multifunctional protein found among potyviruses. It plays multiple roles in the viral infection cycle and some of these functions have been mapped to different regions of the protein. The subcellular localization of several viral RNA silencing suppressor (RSS) proteins was identified. In this study, we have shown that the Zucchini yellow mosaic virus (ZYMV) HC-Pro wild type (HC-ProFRNK) and its mutant, HC-ProFINK, had a diffuse cytoplasmic localiz...

  2. A Naturally Occurring Plant Cysteine Protease Possesses Remarkable Toxicity against Insect Pests and Synergizes Bacillus thuringiensis Toxin

    OpenAIRE

    Mohan, Srinidi; Ma, Peter W. K.; Williams, W Paul; Luthe, Dawn S

    2008-01-01

    When caterpillars feed on maize (Zea maize L.) lines with native resistance to several Lepidopteran pests, a defensive cysteine protease, Mir1-CP, rapidly accumulates at the wound site. Mir1-CP has been shown to inhibit caterpillar growth in vivo by attacking and permeabilizing the insect's peritrophic matrix (PM), a structure that surrounds the food bolus, assists in digestion and protects the midgut from microbes and toxins. PM permeabilization weakens the caterpillar defenses by facilitati...

  3. Purification of a cysteine protease inhibitor from larval hemolymph of the Tobacco Hornworm (Manduca sexta) and functional expression of the recombinant protein.

    Science.gov (United States)

    A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5 kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration of Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, ...

  4. Molecular Dissection of the Vaccinia Virus I7L Core Protein Proteinase

    OpenAIRE

    Byrd, Chelsea M; Bolken, Tove' C.; Dennis E. Hruby

    2003-01-01

    The vaccinia virus I7L gene product is predicted to be a cysteine proteinase and is demonstrated in this study to be responsible for cleavage of each of the three major core protein precursors (P4a, P4b, and P25K) in vivo. Mutagenesis of the putative catalytic triad of I7L or of the cleavage sites in the core protein precursors inhibits processing. A truncated protein lost the ability to cleave the core protein precursors.

  5. Thiol proteinase inhibitor - Oryzacystatin. Molecular cloning and expression in E. coli

    International Nuclear Information System (INIS)

    Insect depredation is a major reason for the reduction in crop yields world-wide. Promising results have already been achieved with transgenic plants expression cowpea trypsin inhibitor (CpTI) genes and modified delta-endotoxin genes. Insects, in general, hydrolyse ingested proteins with a variety of proteinase. The effect of the serine proteinase inhibitor against Lepidopteran insects is probably caused by the preponderance of serine type gut proteinase and a luminal pH in the neutral to alkaline range. On the other hand, the insect orders Coleoptera and Hemiptera have gut pHs in the mildly acidic range and commonly have thiol type gut proteinases. Plant transformation with a gene coding for a thiol proteinase inhibitor has been suggested as a strategy for interfering with the digestive physiology of Coleopteran and Hemipteran insects. Co-transformation of both the serine proteinase inhibitor and the thiol proteinase inhibitor genes might result in a broader spectrum of activity and increased durability of protection

  6. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Directory of Open Access Journals (Sweden)

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  7. Characterisation of functional and insecticidal properties of a cathepsin L-like proteinase from flesh fly (Sacrophaga peregrina) involved in differentiation of imaginal discs.

    Science.gov (United States)

    ScathL is a cathepsin L-like cysteine proteinase from Sacrophaga peregrina (flesh fly), which is involved in differentiation of imaginal discs. The protein has a potential insecticidal activity, since recombinant baculoviruses engineered to express ScathL have an enhanced ability to kill lepidoptera...

  8. Specificity of Binding of the Low Density Lipoprotein Receptor-related Protein to Different Conformational States of the Clade E Serpins Plasminogen Activator Inhibitor-1 and Proteinase Nexin-1*

    OpenAIRE

    Jensen, Jan K.; Dolmer, Klavs; Gettins, Peter G.W.

    2009-01-01

    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich ?40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins...

  9. Digestive proteinase activity of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae).

    Science.gov (United States)

    Naveh, V Hosseini; Bandani, A R; Azmayeshfard, P; Hosseinkhani, S

    2005-01-01

    The khapra beetle, Trogoderma granarium, is one of the most important stored product pests worldwide. A study of digestive proteinases in T. granarium was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. The pH of guts was determined by addition of pH indicator solutions to broken open gut regions. The last instar larvae were dissected in cold distilled water and the whole guts were cleaned from adhering unwanted tissues. The pooled gut homogenates were centrifuged and the supernatants were used in the subsequent enzyme assay. Total proteinases activity of the gut homogenates was determined using the protein substrate azocasein. Optimal azocasein hydrolysis by luminal proteinases of the larvae of T. granarium was highly alkaline in pH 10-10.5, although the pH of luminal contents was slightly acidic (pH 6.5). The extract showed the highest activity at 55 degrees C (pH 6.5), 45 degrees C (pH 8) and 30 degrees C (pH 10). The proteolytic activity was strongly inhibited in the presence of phenylmethylsulphonyl fluoride (82.33+/-4.37% inhibition). This inhibition was decreased with increasing of the pH of assay incubating medium. N-p-tosyl-L-lysine chloromethyl ketone (51.6+/-3.3% inhibition) and N-tosyl-L-phenylalanine chloromethyl ketone (27.23+/-4.37 % inhibition) showed inhibitory effect on proteolysis. Addition of thiol activators dithiothreitol and L-cysteine had not enhanced azocaseinolytic activity. The data suggest that protein digestion in the larvae of T. granarium is primarily dependent on serine proteinases; trypsin- and chymotrypsin-like proteinases. PMID:16628932

  10. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction

    OpenAIRE

    Chae, Keun; Gonong, Benedict J.; Kim, Seung-Chul; Kieslich, Chris A; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M

    2010-01-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranch...

  11. Candida guilliermondii isolated from HIV-infected human secretes a 50 kDa serine proteinase that cleaves a broad spectrum of proteinaceous substrates.

    Science.gov (United States)

    Dos Santos, André Luis Souza; Soares, Rosangela Maria de Araújo

    2005-01-01

    Non-albicans Candida species cause 35-65% of all candidemias in the general population, especially in immunosuppressed individuals. Here, we describe a case of a 19-year-old HIV-infected man with pneumonia due to a yeast-like organism. This clinical yeast isolate was identified as Candida guilliermondii through mycological tests. C. guilliermondii was cultivated in brain heart infusion medium for 48 h at 37 degrees C. After sequential centrifugation and concentration steps, the free-cell culture supernatant was obtained and extracellular proteolytic activity was assayed firstly using gelatin-SDS-PAGE. A 50 kDa proteolytic enzyme was detected with activity at physiological pH. This activity was completely blocked by 10 mM phenylmethylsulphonyl fluoride (PMSF), a serine proteinase inhibitor, suggesting that this extracellular proteinase belongs to the serine proteinase class. E-64, a strong cysteine proteinase inhibitor, and pepstatin A, a specific aspartic proteolytic inhibitor, did not interfere with the 50 kDa proteinase. Conversely, a zinc-metalloproteinase inhibitor (1,10-phenanthroline) restrained the proteinase activity released by C. guilliermondii by approximately 50%. Proteinases are a well-known class of enzymes that participate in a vast context of yeast-host interactions. In an effort to establish a functional implication for this extracellular serine-type enzyme, we investigated its capacity to hydrolyze some serum proteins and extracellular matrix components. We demonstrated that the 50 kDa exocellular serine proteinase cleaved human serum albumin, non-immune human immunoglobulin G, human fibronectin and human placental laminin, generating low molecular mass polypeptides. Collectively, these results showed for the first time the ability of an extracellular proteolytic enzyme other than aspartic-type proteinases in destroying a broad spectrum of relevant host proteins by a clinical species of non-albicans Candida. PMID:15607631

  12. Monoclonal antibodies to the two most basic papaya proteinases.

    Science.gov (United States)

    Goodenough, P W; Kilshaw, P J; McEwan, F; Owen, A J

    1986-08-01

    The proteinases from Carica papaya include papain, isoenzymes of chymopapain and two proteinases A and B distinguished by their unusually high pI. The identity of one of the most basic proteinases has been questioned. The present report describes the preparation and characterisation of two monoclonal antibodies that react specifically with papaya proteinases A and B respectively and a third that identifies a common structural feature found in papain and proteinase A. PMID:3545314

  13. PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW

    Directory of Open Access Journals (Sweden)

    Ezekiel Amri

    2012-01-01

    Full Text Available Papain is a plant proteolytic enzyme for the cysteine proteinase family cysteine protease enzyme in which enormous progress has been made to understand its functions. Papain is found naturally in papaya (Carica papaya L. manufactured from the latex of raw papaya fruits. The enzyme is able to break down organic molecules made of amino acids, known as polypeptides and thus plays a crucial role in diverse biological processes in physiological and pathological states, drug designs, industrial uses such as meat tenderizers and pharmaceutical preparations. The unique structure of papain gives it the functionality that helps elucidate how proteolytic enzymes work and also makes it valuable for a variety of purposes. In the present review, its biological importance, properties and structural features that are important to an understanding of their biological function are presented. Its potential for production and market opportunities are also discussed.

  14. Wound and methyl jasmonate induced pigeon pea defensive proteinase inhibitor has potency to inhibit insect digestive proteinases.

    Science.gov (United States)

    Lomate, Purushottam R; Hivrale, Vandana K

    2012-08-01

    Wounding of plants by chewing insects or other damage induces the synthesis of defensive proteinase inhibitors (PI) in both wounded and distal unwounded leaves. In the present paper we report the characterization of inducible defensive PI from pigeon pea (Cajanus cajan) and its in vitro interaction with Helicoverpa armigera gut proteinases (HGP). We found that PI activity was induced in local as well as systemic leaves of pigeon pea by the wounding and methyl jasmonate (MeJA) application. Consistent induction of PI was observed in two wild cultivars of pigeon pea at various growth stages. The estimated molecular weight of inducible PI was ~16.5 kDa. Electrophoretic analysis and enzyme assays revealed that the induced PI significantly inhibited total gut proteinase as well as trypsin-like activity from the midgut of H. armigera. The induced PI was found to be inhibitor of trypsin as well as chymotrypsin. Study could be important to know the further roles of defensive PIs. PMID:22721949

  15. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

    DEFF Research Database (Denmark)

    Roberts, T.H.; Marttila, S.; Rasmussen, S.K.; Hejgaard, Jørn

    2003-01-01

    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) ...

  16. Anthropogenic increase in carbon dioxide compromises plant defense against invasive insects

    Energy Technology Data Exchange (ETDEWEB)

    Zavala, J.; Casteel, C.; DeLucia, E.; Berenbaum, M. [University of Illinois at Urbana-Champaign, Urbana, IL (United States)

    2008-04-01

    Elevated levels of atmospheric carbon dioxide (CO{sub 2}), a consequence of anthropogenic global change, can profoundly affect the interactions between crop plants and insect pests and may promote yet another form of global change: the rapid establishment of invasive species. Elevated CO{sub 2} increased the susceptibility of soybean plants grown under field conditions to the invasive Japanese beetle (Popillia japonica) and to a variant of western corn rootworm (Diabrotica virgifera virgifera) resistant to crop rotation by down-regulating gene expression related to defense signaling [lipoxygenase 7 (lox7), lipoxygenase 8 (lox8), and 1-aminocyclopropane-1-carboxylate synthase (acc-s)]. The down-regulation of these genes, in turn, reduced the production of cysteine proteinase inhibitors (CystPIs), which are specific deterrents to coleopteran herbivores. Beetle herbivory increased CystPI activity to a greater degree in plants grown under ambient than under elevated CO{sub 2}. Gut cysteine proteinase activity was higher in beetles consuming foliage of soybeans grown under elevated CO{sub 2} than in beetles consuming soybeans grown in ambient CO{sub 2}, consistent with enhanced growth and development of these beetles on plants grown in elevated CO{sub 2}. These findings suggest that predicted increases in soybean productivity under projected elevated CO{sub 2} levels may be reduced by increased susceptibility to invasive crop pests.

  17. Improved methods for high-throughput extraction of green barley malt and assay of endoproteinase activity allow examination of the properties and putative functions of the proteinases in barley populations

    Science.gov (United States)

    We report efficient sample extraction and assay methods allowing determinations of activities from two mechanistic classes (cysteine and serine) of barley malt proteinase, and use the improved methods to assay > 2200 developmental lines of malting barley. The distributions of the resulting activitie...

  18. Stability of proteinase from Carica papaya latex in dense gases

    OpenAIRE

    Habulin, Maja; Primoži?, Mateja; Knez, Željko

    2012-01-01

    Proteinase from Carica papaya latex was tested on its thermal stability at atmospheric pressure and in supercritical carbon dioxide, near-critical propane and dimethyl-ether. In supercritical carbon dioxide at 300 bar thermalactivation of the examined proteinase was improved in the comparison toatmospheric pressure. In propane and dimethyl-ether (300 bar) activity of the examined proteinase decreased. Influence of compressionžexpansion cycles on residual activity of the same proteinase in sup...

  19. Formation of cysteine-S-conjugates in the Maillard reaction of cysteine and xylose.

    Science.gov (United States)

    Cerny, Christoph; Guntz-Dubini, Renée

    2013-11-15

    Cysteine-S-conjugates (CS-conjugates) occur in foods derived from plant sources like grape, passion fruit, onion, garlic, bell pepper and hops. During eating CS-conjugates are degraded into aroma-active thiols by ?-lyases that originate from oral microflora. The present study provides evidence for the formation of the CS-conjugates S-furfuryl-l-cysteine (FFT-S-Cys) and S-(2-methyl-3-furyl)-l-cysteine (MFT-S-Cys) in the Maillard reaction of xylose with cysteine at 100°C for 2h. The CS-conjugates were isolated using cationic exchange and reversed-phase chromatography and identified by (1)H NMR, (13)C NMR and LC-MS(2). Spectra and LC retention times matched those of authentic standards. To the best of our knowledge, this is the first time that CS-conjugates are described as Maillard reaction products. Furfuryl alcohol (FFA) is proposed as an intermediate which undergoes a nucleophilic substitution with cysteine. Both FFT-S-Cys and MFT-S-Cys are odourless but produce strong aroma when tasted in aqueous solutions, supposedly induced by ? -lyases from the oral microflora. The perceived aromas resemble those of the corresponding aroma-active thiols 2-furfurylthiol (FFT) and 2-methyl-3-furanthiol (MFT) which smell coffee-like and meaty, respectively. PMID:23790889

  20. Multiple pathways for vacuolar sorting of yeast proteinase A

    DEFF Research Database (Denmark)

    Westphal, V; Marcusson, E G

    1996-01-01

    The sorting of the yeast proteases proteinase A and carboxypeptidase Y to the vacuole is a saturable, receptor-mediated process. Information sufficient for vacuolar sorting of the normally secreted protein invertase has in fusion constructs previously been found to reside in the propeptide of proteinase A. We found that sorting of such a hybrid protein is dependent on the vacuolar protein-sorting receptor Vps10p. This was unexpected, as strains disrupted for VPS10 sort more than 85% of the proteinase A to the vacuole. Consistent with a role for Vps10p in sorting of proteinase A, we found that 1) overproduction of Vps10p suppressed the missorting phenotype associated with overproduction of proteinase A, 2) overproduction of proteinase A induced missorting of carboxypeptidase Y, 3) vacuolar sorting of proteinase A in a deltavps10 strain was readily saturated by modest overproduction of proteinase A, and 4) Vps10p and proteinase A interact directly and specifically as shown by chemical cross-linking. Interestingly, overexpression of two telomere-linked VPS10 homologues, VTH1 and VTH2 suppressed the missorting phenotypes of a deltavps10 strain. However, disruption of the VTH1 and VTH2 genes did not affect the sorting of proteinase A. We conclude that proteinase A utilizes at least two mechanisms for sorting, a Vps10p-dependent path and a Vth1p/Vth2p/Vps10p-independent path.

  1. Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten

    1992-01-01

    The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co-expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability to mediate its own activation. Elimination of the co-expressed PEP4 gene did not effectively stop the processing of the mutant zymogen, owing to a strong, proteinase-B-dependent, phenotypic lag. In a proteinase-B-negative strain, processing of pro-proteinase A led to an active form of a higher molecular mass than the normal mature form.

  2. Squash inhibitor family of serine proteinases

    International Nuclear Information System (INIS)

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor ?2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: Xa and XIIa, cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high kcat/Km index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

  3. Cloning and sequence analysis of serine proteinase of Gloydius ussuriensis venom gland

    International Nuclear Information System (INIS)

    Objective: To construct a cDNA library by using mRNA from Gloydius ussuriensis (G. Ussuriensis) venom gland, to clone and analyze serine proteinase gene from the cDNA library. Methods: Total RNA was isolated from venom gland of G. ussuriensis, mRNA was purified by using mRNA isolation Kit. The whole length cDNA was synthesized by means of smart cDNA synthesis strategy, and amplified by long distance PCR procedure, lately cDAN was cloned into vector pBluescrip-sk. The recombinant cDNA was transformed into E. coli DH5?. The cDNA of serine proteinase gene in the venom gland of G. ussuriensis was detected and amplified using the in situ hybridization. The cDNA fragment was inserted into pGEMT vector, cloned and its nucleotide sequence was determined. Results: The capacity of cDNA library of venom gland was above 2.3 x 106. Its open reading frame was composed of 702 nucleotides and coded a protein pre-zymogen of 234 amino acids. It contained 12 cysteine residues. The sequence analysis indicated that the deduced amino acid sequence of the cDNA fragment shared high identity with the thrombin-like enzyme genes of other snakes in the GenBank. the query sequence exhibited strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. Based on the amino acid sequences of other thrombin-like enzymes, the catalytic residues and disulfide bridges of this thrombin-like enzyme were deduced as follows: catalytic residues, His41, Asp86, Ser180; and six disulfide bridges Cys7-Cys139, Cys26-Cys42, Cys74-Cys232, Cys118-Cys186, Cys150-Cys165, Cys176-Cys201. Conclusion: The capacity of cDNA library of venom gland is above 2.3 x 106, overtop the level of 105 capicity. The constructed cDNA library of G. ussuriensis venom gland would be helpful platform to detect new target genes and further gene manipulate. The cloned serine proteinase gene exhibits strong amino acid sequence homology of 85% to the serine proteas of T. gramineus, thrombin-like serine proteinase I of D. acutus and serine protease catroxase II of C. atrox respectively. (authors)

  4. Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1

    DEFF Research Database (Denmark)

    Jensen, Jan Kristian; Dolmer, Klavs

    2009-01-01

    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

  5. A multifaceted study of stigma/style cysteine-rich adhesin (SCA)-like Arabidopsis lipid transfer proteins (LTPs) suggests diversified roles for these LTPs in plant growth and reproduction.

    Science.gov (United States)

    Chae, Keun; Gonong, Benedict J; Kim, Seung-Chul; Kieslich, Chris A; Morikis, Dimitrios; Balasubramanian, Shruthi; Lord, Elizabeth M

    2010-10-01

    Lily stigma/style cysteine-rich adhesin (SCA), a plant lipid transfer protein (LTP) which is secreted into the extracellular matrix, functions in pollen tube guidance in fertilization. A gain-of-function mutant (ltp5-1) for Arabidopsis LTP5, an SCA-like molecule, was recently shown to display defects in sexual reproduction. In the current study, it is reported that ltp5-1 plants have dwarfed primary shoots, delayed hypocotyl elongation, various abnormal tissue fusions, and display multibranching. These mutant phenotypes in vegetative growth are recessive. No abnormality was found in ltp5-1/+ plants. In a phylogenetic analysis of plant LTPs, SCA-like Arabidopsis LTPs were classified with conventional plant LTPs. Homology modelling-based electrostatic similarity index (ESI) clustering was used to show diversity in spatial distributions of electrostatic potentials of SCA-like LTPs, suggestive of their various roles in interaction in the extracellular matrix space. ?-Glucuronidase (GUS) analysis showed that SCA-like Arabidopsis LTP genes are diversely present in various tissues. LTP4 was found specifically in the guard cells and LTP6 in trichomes as well as in other tissues. LTP1 levels were specifically abundant in the stigma, and both LTP3 and LTP6 in the ovules. LTP2 and LTP4 gene levels were up-regulated in whole seedlings with 20% polyethylene glycol (PEG) and 300 mM NaCl treatments, respectively. LTP5 was up-regulated in the hypocotyl with 3?d dark growth conditions. LTP6 was specifically expressed in the tip of the cotyledon under drought stress conditions. The results suggest that SCA-like Arabidopsis LTPs are multifunctional, with diversified roles in plant growth and reproduction. PMID:20667964

  6. Seed-specific aspartic proteinase FeAP12 from buckwheat (Fagopyrum esculentum Moench

    Directory of Open Access Journals (Sweden)

    Timotijevi? Gordana S.

    2010-01-01

    Full Text Available Aspartic proteinase gene (FeAP12 has been isolated from the cDNA library of developing buckwheat seeds. Analysis of its deduced amino acid sequence showed that it resembled the structure and shared high homology with typical plant aspartic proteinases (AP characterized by the presence of a plant-specific insert (PSI, unique among APs. It was shown that FeAP12 mRNA was not present in the leaves, roots, steam and flowers, but was seed-specifically expressed. Moreover, the highest levels of FeAP12 expression were observed in the early stages of seed development, therefore suggesting its potential role in nucellar degradation.

  7. Use of fluorogenic proteinase assays to examine protein mobilization in barley varieties and across populations

    Science.gov (United States)

    Improved methods for efficient sample extraction and proteinase assay have simplified estimation of malt proteinase activity, making feasible examination of larger sample sets than was previously possible. The ability to characterize various proteinase activities across > 1000 lines enables use of ...

  8. First generation inhibitors of the adenovirus proteinase

    OpenAIRE

    McGrath, William J.; Graziano, Vito; Zabrocka, Katarzyna; Mangel, Walter F

    2013-01-01

    As there are more than 50 Adenovirus serotypes, the likelihood of developing an effective vaccine is low. Here we describe inhibitors of the adenovirus proteinase (AVP) with the ultimate objective of developing anti-adenovirus agents. Inhibitors were identified via structure-based drug design using as druggable sites the active site and a conserved cofactor pocket in the crystal structures of AVP. A lead compound was identified that had an IC50 of 18 ?M. One of eight structural derivatives of...

  9. Purification and characterization of an extracellular proteinase from Hendersonula toruloidea.

    OpenAIRE

    Rojanavanich, V; Yoshiike, T; Tsuboi, R; Takamori, K; Ogawa, H.

    1990-01-01

    An extracellular proteinase from a fast-growing strain of Hendersonula toruloidea was demonstrated when it was cultivated in liquid medium containing bovine serum albumin as the sole nitrogen source. Purification to homogeneity of the proteinase was performed by carboxymethyl cellulose, CM Sephadex G-50, and Sephacryl S-200 column chromatographies. The purified enzyme was a chymotrypsinlike serine proteinase, as indicated by the strong inhibitory activities of diisopropyl fluorophosphate, phe...

  10. Host genetic influences on the anthelmintic efficacy of papaya-derived cysteine proteinases in mice.

    Science.gov (United States)

    Luoga, Wenceslaus; Mansur, Fadlul; Stepek, Gillian; Lowe, Ann; Duce, Ian R; Buttle, David J; Behnke, Jerzy M

    2015-06-01

    Eight strains of mice, of contrasting genotypes, infected with Heligmosomoides bakeri were studied to determine whether the anthelmintic efficacy of papaya latex varied between inbred mouse strains and therefore whether there is an underlying genetic influence on the effectiveness of removing the intestinal nematode. Infected mice were treated with 330 nmol of crude papaya latex or with 240 nmol of papaya latex supernatant (PLS). Wide variation of response between different mouse strains was detected. Treatment was most effective in C3H (90·5-99·3% reduction in worm counts) and least effective in CD1 and BALB/c strains (36·0 and 40·5%, respectively). Cimetidine treatment did not improve anthelmintic efficacy of PLS in a poor drug responder mouse strain. Trypsin activity, pH and PLS activity did not differ significantly along the length of the gastro-intestinal (GI) tract between poor (BALB/c) and high (C3H) drug responder mouse strains. Our data indicate that there is a genetic component explaining between-mouse variation in the efficacy of a standard dose of PLS in removing worms, and therefore warrant some caution in developing this therapy for wider scale use in the livestock industry, and even in human medicine. PMID:25736575

  11. Channel catfish, Ictalurus punctatus, cysteine proteinases: Cloning, characterization and expression of cathepsin H and L

    Science.gov (United States)

    The antigen recognition by the host immune system is a complex biochemical process that requires a battery of enzymes. Cathepsins are one of the enzyme superfamilies involving in antigenic degradations. We observed the up-regulation of cathepsin H and L transcripts during the early stage of Edward...

  12. Keratinolytic proteinase from Bacillus thuringiensis AD-12.

    Science.gov (United States)

    Gegeckas, Audrius; Gudiukait?, Renata; Citavicius, Donaldas

    2014-08-01

    A new isolated strain noted to produce a novel detergent-stable serine keratinolytic proteinase and identified as Bacillus thuringiensis AD-12. Native keratinolytic proteinase from B. thuringiensis (BtKER) was purified and characterized. The purified BtKER enzyme is a monomer with a molecular mass of 39kDa. Biochemical characterization assays revealed that the BtKER attained optimal activity at pH 7 and 30°C. Residual activity after 1h incubation at 50°C was higher than 80%. The enzyme was activated and stabilized by Mn(2+) and Li(+) metal ions but inactivated by organic solvents. Purified BtKER showed the highest substrate specificity toward keratin from wool>sodium caseinate>collagen>BSA>gelatin in descending order. BtKER is the first reported keratinolytic proteinase from B. thuringiensis and obtained results suggested that new characterized enzyme can be a powerful biocatalyst in peptide production associated to hydrolysis of keratinous and/or keratin-like waste. PMID:24857878

  13. Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1992-01-01

    The Saccharomyces cerevisiae PEP4 gene encodes proteinase A, an aspartyl protease. pep4 mutants are defective in the activation of many vacuolar hydrolases, including proteinase B. We have expressed a pep4 mutation which directs the accumulation of pro-proteinase A with a defective active site. Co-expression with PEP4 leads to normal processing, i.e. the mutant zymogen is functional as a substrate for the maturation reaction in trans. We conclude that wild-type pro-proteinase A has the ability t...

  14. Characterization of proteinases from the midgut of Rhipicephalus (Boophilus microplus involved in the generation of antimicrobial peptides

    Directory of Open Access Journals (Sweden)

    Craik Charles S

    2010-07-01

    Full Text Available Abstract Background Hemoglobin is a rich source of biologically active peptides, some of which are potent antimicrobials (hemocidins. A few hemocidins have been purified from the midgut contents of ticks. Nonetheless, how antimicrobials are generated in the tick midgut and their role in immunity is still poorly understood. Here we report, for the first time, the contribution of two midgut proteinases to the generation of hemocidins. Results An aspartic proteinase, designated BmAP, was isolated from the midgut of Rhipicephalus (Boophilus microplus using three chromatographic steps. Reverse transcription-quantitative polymerase chain reaction revealed that BmAP is restricted to the midgut. The other enzyme is a previously characterized midgut cathepsin L-like cysteine proteinase designated BmCL1. Substrate specificities of native BmAP and recombinant BmCL1 were mapped using a synthetic combinatorial peptide library and bovine hemoglobin. BmCL1 preferred substrates containing non-polar residues at P2 subsite and polar residues at P1, whereas BmAP hydrolysed substrates containing non-polar amino acids at P1 and P1'. Conclusions BmAP and BmCL1 generate hemocidins from hemoglobin alpha and beta chains in vitro. We postulate that hemocidins may be important for the control of tick pathogens and midgut flora.

  15. Stress inducible proteinase inhibitor diversity in Capsicum annuum

    Directory of Open Access Journals (Sweden)

    Mishra Manasi

    2012-11-01

    Full Text Available Abstract Background Wound-inducible Pin-II Proteinase inhibitors (PIs are one of the important plant serine PIs which have been studied extensively for their structural and functional diversity and relevance in plant defense against insect pests. To explore the functional specialization of an array of Capsicum annuum (L. proteinase inhibitor (CanPIs genes, we studied their expression, processing and tissue-specific distribution under steady-state and induced conditions. Inductions were performed by subjecting C. annuum leaves to various treatments, namely aphid infestation or mechanical wounding followed by treatment with either oral secretion (OS of Helicoverpa armigera or water. Results The elicitation treatments regulated the accumulation of CanPIs corresponding to 4-, 3-, and 2-inhibitory repeat domains (IRDs. Fourty seven different CanPI genes composed of 28 unique IRDs were identified in total along with those reported earlier. The CanPI gene pool either from uninduced or induced leaves was dominated by 3-IRD PIs and trypsin inhibitory domains. Also a major contribution by 4-IRD CanPI genes possessing trypsin and chymotrypsin inhibitor domains was specifically revealed in wounded leaves treated with OS. Wounding displayed the highest number of unique CanPIs while wounding with OS treatment resulted in the high accumulation of specifically CanPI-4, -7 and ?10. Characterization of the PI protein activity through two dimensional gel electrophoresis revealed tissue and induction specific patterns. Consistent with transcript abundance, wound plus OS or water treated C. annuum leaves exhibited significantly higher PI activity and isoform diversity contributed by 3- and 4-IRD CanPIs. CanPI accumulation and activity was weakly elicited by aphid infestation yet resulted in the higher expression of CanPI-26, -41 and ?43. Conclusions Plants can differentially perceive various kinds of insect attacks and respond appropriately through activating plant defenses including regulation of PIs at transcriptional and post-translational levels. Based on the differentially elicited CanPI accumulation patterns, it is intriguing to speculate that generating sequence diversity in the form of multi-IRD PIs is a part of elaborative plant defense strategy to obtain a diverse pool of functional units to confine insect attack.

  16. Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer, Diatraea saccharalis.

    Science.gov (United States)

    Yang, Yunlong; Zhu, Yu Cheng; Ottea, James; Husseneder, Claudia; Leonard, B Rogers; Abel, Craig; Luttrell, Randall; Huang, Fangneng

    2013-08-01

    Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes. PMID:23955944

  17. Mechanism and ion-dependence of in vitro autoactivation of yeast proteinase A

    DEFF Research Database (Denmark)

    Van Den Hazel, H; Wolff, A M; Kielland-Brandt, Morten; Winther, Jakob R.

    1997-01-01

    Yeast proteinase A is synthesized as a zymogen which transits through the endoplasmic reticulum, the Golgi complex and the endosome to the vacuole. On arrival in the vacuole, activation takes place. It has previously been found that proteinase A can activate autocatalytically; however, the propeptide of proteinase A shows essentially no similarity to other known aspartic proteinase propeptides. To understand why proteinase A activation occurs rapidly in the vacuole but not at all in earlier comp...

  18. Serine proteinase inhibitors in the Compositae: distribution, polymorphism and properties.

    Science.gov (United States)

    Konarev, Alexander V; Anisimova, Irina N; Gavrilova, V A; Vachrusheva, T E; Konechnaya, G Yu; Lewis, Mervyn; Shewry, Peter R

    2002-02-01

    Multiple molecular forms of inhibitors of trypsin (TI) and chymotrypsin (CI), which are typical digestive enzymes of insects, mammals and micro-organisms, and subtilisin (SI), a proteinase of many bacteria and phytopathogenic fungi, were identified in seeds and vegetative organs of the majority of 128 wild and cultivated species representing 65 genera of three of the subfamilies of the Compositae. Inhibitors with M(r) ranging from 7450 to 7800 and combining activities towards subtilisin and trypsin and/or chymotrypsin (T/C/SI) had the widest distribution and may be involved in plant defense mechanisms. They were found in many species of the subfamilies Carduoideae (genera Carthamus, Centaurea, Cirsium), Cichorioideae (Lactuca, Taraxacum) and Asteroideae (Helianthus, Cosmos, Bidens). Partial amino acid sequencing showed that the safflower (Carthamus tinctorius) T/C/SI and Cosmos bipinnatus T/C/SI, T/SI and C/SI belonged to the potato I inhibitor family. The most active, variable and heterogeneous inhibitors were found in species of the tribe Heliantheae, which is placed in the evolutionary advanced subfamily Asteroideae. Seeds of Helianthus species, Eclipta prostrata, Gailardia aristata, Zinnia elegans and Silphium perfoliatum contained various TI with M(r) ranging from 1500 to 14,750, with some also containing SI. H. annuus seeds contain a unique cyclic TI of M(r) 1514 and similar TI were also present in other Helianthus spp. and the related species Tithonia diversifolia. Zinnia elegans contained a TI with M(r) 11,350 which appeared to represent a novel type of inhibitor distantly related to the cereal subgroup of Bowman-Birk inhibitors. TI and T/SI varied widely in H. annuus lines and wild Helianthus species in their presence or absence and composition. Similar T/SI components were found in the cultivated diploid H. annuus and annual diploid species with the B genome but not in perennials with the A genome. Some T/SI, SI and TI were detected in vegetative organs of sunflower and other Compositae. Studies of the polymorphism and distribution of proteinase inhibitors are relevant to the evolution of protective protein systems and the mechanisms of resistance to pathogenic organisms in the Compositae and other plants. PMID:11830136

  19. Proteinase 3. A distinct human polymorphonuclear leukocyte proteinase that produces emphysema in hamsters.

    OpenAIRE

    Kao, R C; Wehner, N G; Skubitz, K M; Gray, B. H.; Hoidal, J. R.

    1988-01-01

    Studies were designed to explore the possibility that human polymorphonuclear leukocyte granule constituents in addition to elastase (HLE) had the potential to cause emphysema. A two-step purification of three serine proteinases was developed. Granule extract proteins were initially separated by dye-ligand affinity chromatography. Fractions eluted were divided into four pools. Hamsters were given a single intratracheal instillation of saline +/- 0.1 mg protein of each pool. While pool 2 conta...

  20. Three distinct secreted aspartyl proteinases in Candida albicans.

    OpenAIRE

    White, T C; Miyasaki, S H; Agabian, N

    1993-01-01

    The secreted aspartyl proteinases of Candida albicans (products of the SAP genes) are thought to contribute to virulence through their effects on Candida adherence, invasion, and pathogenicity. From a single strain of C. albicans (WO-1) which expresses a phenotypic switching system, three secreted aspartyl proteinases have been identified as determined by molecular weight and N-terminal sequence. Each of the three identified proteins represents the mature form of one of three distinct protein...

  1. Protease Inhibitors from Plants with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Yoonkyung Park

    2009-06-01

    Full Text Available Antimicrobial proteins (peptides are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides. Plants produce a variety of proteins (peptides that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins. Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents.

  2. In praise of impurity: 30S ribosomal S15 protein-assisted crystallization of turnip yellow mosaic virus proteinase

    International Nuclear Information System (INIS)

    Diffraction-quality crystals of the turnip yellow mosaic virus proteinase/ubiquitin hydrolase could only be obtained from a protein preparation that was heavily contaminated with E. coli 30S ribosomal S15 protein. Crystal packing reveals the basis of this observation. Turnip yellow mosaic virus is an excellent model for eukaryotic positive-stranded RNA virus replication. Correct processing of the replication polyprotein is dependent on the virally encoded cysteine proteinase (PRO) domain. Crystalline needles obtained from highly pure preparations of the recombinant 17.6 kDa PRO did not diffract. In contrast, small hexagonal prisms that were obtained together with the needles under the same conditions but from a poorly purified preparation diffracted to 2 Å resolution and allowed structure determination by MIRAS. It turned out that the hexagonal crystals contained stoichiometric amounts of PRO and Escherichia coli 30S ribosomal S15, a 10.1 kDa protein commonly co-purified by immobilized metal-affinity chromatography. The solvent content is nearly 70%, with S15 bridging parallel infinite helices of PRO across large solvent channels. With hindsight, this spurious interaction not only yielded diffraction-quality crystals but would also have allowed structure determination by molecular replacement using S15 as a search model and subsequent automatic rebuilding of the asymmetric unit

  3. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

    DEFF Research Database (Denmark)

    Roberts, T.H.; Marttila, S.

    2003-01-01

    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (greater than or equal to15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.

  4. Proteinases of human epidermis; a possible mechanism for polymorphonuclear leukocyte chemotaxis

    International Nuclear Information System (INIS)

    Three neutral proteinases (EC 3.4.-,-) and cathepsin D have been identified in human epidermis utilizing a highly sensitive radioactive method. The proteinases were extracted in 1.0 M KCl and 0.1% Triton X-100 and separated by Sephadex G-75 chromatography. The neutral proteinase peaks were all inhibited by diisopropyl fluorophosphate and thus were serine proteinases. Incubation of the enzyme fractions with [3H]diisopropyl fluorophosphate followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated that the two larger molecular weight proteinases were enzyme mixtures. The small molecular weight [3H]diisopropyl fluorophosphate proteinase migrated as a single band. Injection of the small molecular weight neutral proteinase into rabbit skin produced a polymorphonuclear leukocyte infiltration and edema. The reaction was not observed with the diisopropul fluorophosphate-inhibited enzyme fraction. The release of neutral proteinases may be one of the signal events in the epidermal inflammatory response

  5. Neutrophil elastase and proteinase-3 trigger G protein-biased signaling through proteinase-activated receptor-1 (PAR1).

    Science.gov (United States)

    Mihara, Koichiro; Ramachandran, Rithwik; Renaux, Bernard; Saifeddine, Mahmoud; Hollenberg, Morley D

    2013-11-15

    Neutrophil proteinases released at sites of inflammation can affect tissue function by either activating or disarming signal transduction mediated by proteinase-activated receptors (PARs). Because PAR1 is expressed at sites where abundant neutrophil infiltration occurs, we hypothesized that neutrophil-derived enzymes might also regulate PAR1 signaling. We report here that both neutrophil elastase and proteinase-3 cleave the human PAR1 N terminus at sites distinct from the thrombin cleavage site. This cleavage results in a disarming of thrombin-activated calcium signaling through PAR1. However, the distinct non-canonical tethered ligands unmasked by neutrophil elastase and proteinase-3, as well as synthetic peptides with sequences derived from these novel exposed tethered ligands, selectively stimulated PAR1-mediated mitogen-activated protein kinase activation. This signaling was blocked by pertussis toxin, implicating a G?i-triggered signal pathway. We conclude that neutrophil proteinases trigger biased PAR1 signaling and we describe a novel set of tethered ligands that are distinct from the classical tethered ligand revealed by thrombin. We further demonstrate the function of this biased signaling in regulating endothelial cell barrier integrity. PMID:24052258

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

    OpenAIRE

    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....

  7. Regulatory Protein-Protein Interactions in Primary Metabolism: The Case of the Cysteine Synthase Complex

    Science.gov (United States)

    Sulfur is an essential nutrient for plant growth and development. In plant sulfur assimilation, cysteine biosynthesis plays a central role in fixing inorganic sulfur from the environment into the metabolic precursor for cellular thiol-containing compounds. A key regulatory feature of this process ...

  8. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

    Science.gov (United States)

    Lin, Chia-Wei; Su, Mei-Hsiu; Lin, Yu-Tsung; Chung, Chien-Hung; Ku, Hsin-Mei

    2015-07-01

    Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana. PMID:26184285

  9. Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI in Potyviruses Resistance

    Directory of Open Access Journals (Sweden)

    Chia-Wei Lin

    2015-07-01

    Full Text Available Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi with an inter-space hairpin RNA (ihpRNA construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

  10. Perspectives of digestive pest control with proteinase inhibitors that mainly affect the trypsin-like activity of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae

    Directory of Open Access Journals (Sweden)

    M.E. Pereira

    2005-11-01

    Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.

  11. Structure and mechanism of mouse cysteine dioxygenase

    OpenAIRE

    McCoy, Jason G.; Bailey, Lucas J.; Bitto, Eduard; Bingman, Craig A.; Aceti, David J.; Fox, Brian G.; Phillips, George N

    2006-01-01

    Cysteine dioxygenase (CDO) catalyzes the oxidation of l-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical ?-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal bindi...

  12. Combinatorial Cysteine Mutagenesis Reveals a Critical Intramonomer Role for Cysteines in Prestin Voltage Sensing

    OpenAIRE

    Bai, Jun-Ping; Surguchev, Alexei; Bian, Shumin; SONG, LEI; Santos-Sacchi, Joseph; Navaratnam, Dhasakumar

    2010-01-01

    Prestin is a member of the SLC26 family of anion transporters and is responsible for electromotility in outer hair cells, the basis of cochlear amplification in mammals. It is an anion transporting transmembrane protein, possessing nine cysteine residues, which generates voltage-dependent charge movement. We determine the role these cysteine residues play in the voltage sensing capabilities of prestin. Mutations of any single cysteine residue had little or no effect on charge movement. Howeve...

  13. Synthesis of macrocyclic trypanosomal cysteine protease inhibitors

    OpenAIRE

    Chen, Yen Ting; Lira, Ricardo; Hansell, Elizabeth; McKerrow, James H; ROUSH, WILLIAM R.

    2008-01-01

    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 r...

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

    OpenAIRE

    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; Staib, Peter

    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 ...

  15. Characterization of peptide proteinase inhibitors isolated from boar seminal plasma.

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Tichá, M.; Jonáková, V?ra

    Praha : UOCHB AV ?R, 2003 - (Slaninová, J.; Collinsová, M.; Klasová, L.), s. 1-57 [Biologicky aktivní peptidy /8./. Praha (CZ), 23.04.2003-25.04.2003] R&D Projects: GA ?R GA303/99/0357; GA ?R GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:MSM 113100001 Keywords : boar seminal plasma proteins * proteinase inhibitors Subject RIV: CE - Biochemistry

  16. Proteinase inhibitors in aggregated forms of boar seminal plasma proteins.

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Ma?ásková, Pavla; Tichá, M.; Jonáková, V?ra

    2003-01-01

    Ro?. 32, - (2003), s. 99-107. ISSN 0141-8130 R&D Projects: GA ?R GA303/02/0433; GA ?R GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915; CEZ:MSM 113100001 Keywords : proteinase inhibitors, aggregated forms, boar seminal plasma proteins Subject RIV: CE - Biochemistry Impact factor: 1.427, year: 2003

  17. Characterization of peptide proteinase inhibitors isolated from boar seminal plasma.

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Petra; Tichá, M.; Jonáková, V?ra

    2003-01-01

    Ro?. 6, - (2003), s. 35-37. ISSN 0010-0765. [Biologically Active Peptides /8./. Praha, 23.04.2003-25.04.2003] R&D Projects: GA ?R GA303/02/0433; GA ?R GP303/02/P069; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : proteinase inhibitors * aggregated forms * boar seminal plasma Subject RIV: CE - Biochemistry Impact factor: 1.041, year: 2003

  18. Proteinase K processing of rabbit muscle creatine kinase

    DEFF Research Database (Denmark)

    Leydier, C; Andersen, Jens S.; Couthon, F; Forest, E; Marcillat, O; Denoroy, L; Vial, C; Clottes, E

    1997-01-01

    Proteinase K cleaves selectively both cytosolic and mitochondrial isoforms of creatine kinase leading to the appearance of two fragments, a large N-terminal one (K1) and a small C-terminal peptide (K2) which remain associated together. The loss of enzymatic activity correlates with the extent of monomer cleavage. N-terminal sequencing of the K2 fragments from rabbit cytosolic and pig mitochondrial creatine kinase shows that these peptides begin with A328 and A324, respectively. Electrospray ioni...

  19. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    Directory of Open Access Journals (Sweden)

    Goldman Maria Helena S.

    1998-01-01

    Full Text Available The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

  20. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    OpenAIRE

    Goldman, Maria Helena S.; Goldman, Gustavo H.

    1998-01-01

    The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic intera...

  1. A structural comparison of 21 inhibitor complexes of the aspartic proteinase from Endothia parasitica.

    OpenAIRE

    Bailey, D.; Cooper, J. B.

    1994-01-01

    The aspartic proteinases are an important family of enzymes associated with several pathological conditions such as hypertension (renin), gastric ulcers (pepsin), neoplastic disease (cathepsins D and E), and AIDS (HIV proteinase). Studies of inhibitor binding are therefore of great importance for design of novel inhibitors for potential therapeutic applications. Numerous X-ray analyses have shown that transition-state isostere inhibitors of aspartic proteinases bind in similar extended confor...

  2. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    Science.gov (United States)

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno

    2014-10-01

    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor. PMID:24090421

  3. Multifunctional amaranth cystatin inhibits endogenous and digestive insect cysteine endopeptidases: A potential tool to prevent proteolysis and for the control of insect pests.

    Science.gov (United States)

    Valdés-Rodríguez, Silvia; Galván-Ramírez, Juan Pablo; Guerrero-Rangel, Armando; Cedro-Tanda, Alberto

    2015-09-01

    In a previous study, the amaranth cystatin was characterized. This cystatin is believed to provide protection from abiotic stress because its transcription is induced in response to heat, drought, and salinity. It has also been shown that recombinant amaranth cystatin inhibits bromelain, ficin, and cysteine endopeptidases from fungal sources and also inhibits the growth of phytopathogenic fungi. In the present study, evidence is presented regarding the potential function of amaranth cystatin as a regulator of endogenous proteinases and insect digestive proteinases. During amaranth germination and seedling growth, different proteolytic profiles were observed at different pH levels in gelatin-containing SDS-PAGE. Most of the proteolytic enzymes detected at pH 4.5 were mainly inhibited by trans-epoxysuccinyl-leucyl amido(4-guanidino)butane (E-64) and the purified recombinant amaranth cystatin. Furthermore, the recombinant amaranth cystatin was active against insect proteinases. In particular, the E-64-sensitive proteolytic digestive enzymes from Callosobruchus maculatus, Zabrotes subfasciatus, and Acanthoscelides obtectus were inhibited by the amaranth cystatin. Taken together, these results suggest multiple roles for cystatin in amaranth, specifically during germination and seedling growth and in the protection of A. hypochondriacus against insect predation. Amaranth cystatin represents a promising tool for diverse applications in the control of insect pest and for preventing undesirable proteolytic activity. PMID:25345487

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. A new crystal form of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    Science.gov (United States)

    Tanokura, M; Sasaki, H; Muramatsu, T; Iwata, S; Hamaya, T; Takizawa, T; Takahashi, K

    1993-10-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1), with unit cell dimensions of a = 69.75 +/- 0.06 A, b = 87.55 +/- 0.05 A, and c = 60.83 +/- 0.04 A. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 A3/Da. By assuming the specific volume to be 0.74 cm3/g, the solvent content (Vso1) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vso1 = 26%). Diffraction data were collected up to a resolution higher than 1.6 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. PMID:8276753

  6. Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus

    OpenAIRE

    1991-01-01

    Sera from patients with systemic lupus erythematosus contain specific autoantibodies directed against different polypeptide components of the multicatalytic proteinase (also known as proteasome or prosome). These human autoantibodies, in contrast to polyclonal antibodies obtained in rabbits against the purified enzyme, recognize highly conserved epitopes of the multicatalytic proteinase polypeptides from yeast to human.

  7. Collagenase inhibition in colonic mucosa by proteinase inhibitors.

    OpenAIRE

    Lewin, M. R.; Chowcat, N. L.; Jayaraj, A P; Boulos, P. B.

    1986-01-01

    The effect of proteinase inhibitors on collagenase activity in rabbit colonic mucosa, both in vitro and in vivo, has been measured by a tissue culture method. Aprotinin, when applied to colonic explants at doses of 20 and 40 KIU, significantly reduced the lytic activity by 97.3% and 99.1% respectively (P less than 0.001). At doses of 100, 200 and 400 KIUs, lysis was completely abolished. Rectal biopsies taken from rabbits at 4, 24, 48 and 72 h following either intramuscular, oral or rectal ad...

  8. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach; Brinch-Pedersen, Henrik

    2010-01-01

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and effici...

  9. Salicylic acid and cysteine contribute to arbutin-induced alleviation of angular leaf spot disease development in cucumber.

    Science.gov (United States)

    Ku?niak, El?bieta; Wielanek, Marzena; Chwatko, Gra?yna; G?owacki, Rafa?; Libik-Konieczny, Marta; Pi?tek, Milena; Gajewska, Ewa; Sk?odowska, Maria

    2015-06-01

    Arbutin induced suppression of angular leaf spot disease in cucumber resulting from lower populations of Pseudomonas syringae pv lachrymans in the infected tissues. This study provides insight into mechanisms that may potentially account for this effect. In the absence of the pathogen, exogenous arbutin-induced expression of PR1, the marker of salicylic acid signaling, increased the content of salicylic acid and modulated the cysteine pool. This suggested that arbutin promoted cucumber plants to a "primed" state. When challenged with the pathogen, the arbutin-treated plants showed strongly reduced infection symptoms 7 days after inoculation. At this time point, they were characterized by higher contents of free and protein-bound cysteine due to higher cysteine biosynthetic capacity related to increased activities of serine acetyltransferase and cysteine synthase when compared with plants infected without arbutin treatment. Moreover, in the arbutin-treated and infected plants the contents of free salicylic acid and its conjugates were also increased, partly owing to its biosynthesis via the phenylpropanoid pathway. We suggest that arbutin-induced abrogation of angular leaf spot disease in cucumber could be mediated by salicylic acid and cysteine-based signaling. PMID:25955697

  10. Septicemia Caused by Cysteine-Dependent Escherichia coli

    OpenAIRE

    1990-01-01

    A case of septicemia and urinary tract infection caused by cysteine-dependent Escherichia coli in a 70-year-old woman with bilateral staghorn calculi is described. This is the second report of a cysteine-dependent E. coli bacteremia. The bacterium was falsely susceptible to ampicillin and co-trimoxazole when tested on a medium without cysteine supplement.

  11. Factors supporting cysteine tolerance and sulfite production in Candida albicans.

    Science.gov (United States)

    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; Staib, Peter

    2013-04-01

    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. cdg1? and ssu1? 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, cdg1? 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

  12. Cysteine-Cysteine Contact Preference Leads to Target-Focusing in Protein Folding

    OpenAIRE

    Sardiu, Mihaela E.; Cheung, Margaret S.; Yu, Yi-Kuo

    2007-01-01

    We perform a statistical analysis of amino-acid contacts to investigate possible preferences of amino-acid interactions. We include in the analysis only tertiary contacts, because they are less constrained—compared to secondary contacts—by proteins' backbone rigidity. Using proteins from the protein data bank, our analysis reveals an unusually high frequency of cysteine pairings relative to that expected from random. To elucidate the possible effects of cysteine interactions in folding, we pe...

  13. Cysteine-cysteine contact preference leads to target-focusing in protein folding.

    Science.gov (United States)

    Sardiu, Mihaela E; Cheung, Margaret S; Yu, Yi-Kuo

    2007-08-01

    We perform a statistical analysis of amino-acid contacts to investigate possible preferences of amino-acid interactions. We include in the analysis only tertiary contacts, because they are less constrained--compared to secondary contacts--by proteins' backbone rigidity. Using proteins from the protein data bank, our analysis reveals an unusually high frequency of cysteine pairings relative to that expected from random. To elucidate the possible effects of cysteine interactions in folding, we perform molecular simulations on three cysteine-rich proteins. In particular, we investigate the difference in folding dynamics between a G?-like model (where attraction only occurs between amino acids forming a native contact) and a variant model (where attraction between any two cysteines is introduced to mimic the formation/dissociation of native/nonnative disulfide bonds). We find that when attraction among cysteines is nonspecific and comparable to a solvent-averaged interaction, they produce a target-focusing effect that expedites folding of cysteine-rich proteins as a result of a reduction of conformational search space. In addition, the target-focusing effect also helps reduce glassiness by lowering activation energy barriers and kinetic frustration in the system. The concept of target-focusing also provides a qualitative understanding of a correlation between the rates of protein folding and parameters such as contact order and total contact distance. PMID:17617551

  14. Helper component-proteinase (HC-Pro) protein of Papaya ringspot virus interacts with papaya calreticulin.

    Science.gov (United States)

    Shen, Wentao; Yan, Pu; Gao, Le; Pan, Xueying; Wu, Jinyan; Zhou, Peng

    2010-05-01

    Potyviral helper component-proteinase (HC-Pro) is a multifunctional protein involved in plant-virus interactions. In this study, we constructed a Carica papaya L. plant cDNA library to investigate the host factors interacting with Papaya ringspot virus (PRSV) HC-Pro using a Sos recruitment two-hybrid system (SRS). We confirmed that the full-length papaya calreticulin, designated PaCRT (GenBank accession no. FJ913889), interacts specifically with PRSV HC-Pro in yeast, in vitro and in plant cells using SRS, in vitro protein-binding assay and bimolecular fluorescent complementation assay, respectively. SRS analysis of the interaction between three PaCRT deletion mutants and PRSV HC-Pro demonstrated that the C-domain (residues 307-422), with a high Ca(2+)-binding capacity, was responsible for binding to PRSV HC-Pro. In addition, quantitative real-time reverse transcriptase-polymerase chain reaction assay showed that the expression of PaCRT mRNA was significantly upregulated in the primary stage of PRSV infection, and decreased to near-basal expression levels in noninoculated (healthy) papaya plants with virus accumulation inside host cells. PaCRT is a new calcium-binding protein that interacts with potyviral HC-Pro. It is proposed that the upregulated expression of PaCRT mRNA may be an early defence-related response to PRSV infection in the host plant, and that interaction between PRSV HC-Pro and PaCRT may be involved in plant calcium signalling pathways which could interfere with virus infection or host defence. PMID:20447282

  15. Effects of soybean proteinase inhibitors on development of the soil mite Scheloribates praeincisus (Acari: Oribatida).

    Science.gov (United States)

    Simões, R A; Silva-Filho, M C; Moura, D S; Delalibera, I

    2008-03-01

    Proteinase inhibitors (PI) are present in plant tissues, especially in seeds, and act as a defense mechanism against herbivores and pathogens. Serine PI from soybean such as Bowman-Birk (BBPI) and Kunitz have been used to enhance resistance of sugarcane varieties to the sugarcane borer Diatraea saccharalis (Fabricius) (Lepidoptera: Crambidae), the major pest of this crop. The use of these genetically-modified plants (GM) expressing PI requires knowledge of its sustainability and environmental safety, determining the stability of the introduced characteristic and its effects on non-target organisms. The objective of this study was to evaluate direct effects of ingestion of semi-purified and purified soybean PI and GM sugarcane plants on the soil-dwelling mite Scheloribates praeincisus (Berlese) (Acari: Oribatida). This mite is abundant in agricultural soils and participates in the process of organic matter decomposition; for this reason it will be exposed to PI by feeding on GM plant debris. Eggs of S. praeincisus were isolated and after larvae emerged, immatures were fed milled sugarcane leaves added to semi-purified or purified PI (Kunitz and BBPI) or immatures were fed GM sugarcane varieties expressing Kunitz and BBPI type PI or the untransformed near isogenic parental line variety as a control. Developmental time (larva-adult) and survival of S. praeincisus was evaluated. Neither Kunitz nor BBPI affected S. praeincisus survival. On the other hand, ingestion of semi-purified and purified Kunitz inhibitor diminished duration of S. praeincisus immature stages. Ingestion of GM senescent leaves did not have an effect on S. praeincisus immature developmental time and survival, compared to ingestion of leaves from the isogenic parental plants. These results indicate that cultivation of these transgenic sugarcane plants is safe for the non-target species S. praeincisus. PMID:18357504

  16. Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes.

    Science.gov (United States)

    Dutta, Tushar K; Papolu, Pradeep K; Banakar, Prakash; Choudhary, Divya; Sirohi, Anil; Rao, Uma

    2015-01-01

    Root-knot nematodes (Meloidogyne incognita) cause substantial yield losses in vegetables worldwide, and are difficult to manage. Continuous withdrawal of environmentally-harmful nematicides from the global market warrants the need for novel nematode management strategies. Utility of host-delivered RNAi has been demonstrated in several plants (Arabidopsis, tobacco, and soybean) that exhibited resistance against root-knot and cyst nematodes. Herein, a M. incognita-specific protease gene, cathepsin L cysteine proteinase (Mi-cpl-1), was targeted to generate tomato transgenic lines to evaluate the genetically modified nematode resistance. In vitro knockdown of Mi-cpl-1 gene led to the reduced attraction and penetration of M. incognita in tomato, suggesting the involvement of Mi-cpl-1 in nematode parasitism. Transgenic expression of the RNAi construct of Mi-cpl-1 gene resulted in 60-80% reduction in infection and multiplication of M. incognita in tomato. Evidence for in vitro and in vivo silencing of Mi-cpl-1 was confirmed by expression analysis using quantitative PCR. Our study demonstrates that Mi-cpl-1 plays crucial role during plant-nematode interaction and plant-mediated downregulation of this gene elicits detrimental effect on M. incognita development, reinforcing the potential of RNAi technology for management of phytonematodes in crop plants. PMID:25883594

  17. Tomato transgenic plants expressing hairpin construct of a nematode protease gene conferred enhanced resistance to root-knot nematodes

    Science.gov (United States)

    Dutta, Tushar K.; Papolu, Pradeep K.; Banakar, Prakash; Choudhary, Divya; Sirohi, Anil; Rao, Uma

    2015-01-01

    Root-knot nematodes (Meloidogyne incognita) cause substantial yield losses in vegetables worldwide, and are difficult to manage. Continuous withdrawal of environmentally-harmful nematicides from the global market warrants the need for novel nematode management strategies. Utility of host-delivered RNAi has been demonstrated in several plants (Arabidopsis, tobacco, and soybean) that exhibited resistance against root-knot and cyst nematodes. Herein, a M. incognita-specific protease gene, cathepsin L cysteine proteinase (Mi-cpl-1), was targeted to generate tomato transgenic lines to evaluate the genetically modified nematode resistance. In vitro knockdown of Mi-cpl-1 gene led to the reduced attraction and penetration of M. incognita in tomato, suggesting the involvement of Mi-cpl-1 in nematode parasitism. Transgenic expression of the RNAi construct of Mi-cpl-1 gene resulted in 60–80% reduction in infection and multiplication of M. incognita in tomato. Evidence for in vitro and in vivo silencing of Mi-cpl-1 was confirmed by expression analysis using quantitative PCR. Our study demonstrates that Mi-cpl-1 plays crucial role during plant-nematode interaction and plant-mediated downregulation of this gene elicits detrimental effect on M. incognita development, reinforcing the potential of RNAi technology for management of phytonematodes in crop plants. PMID:25883594

  18. Diisopropyl fluorophosphate labeling of sperm-associated proteinases

    International Nuclear Information System (INIS)

    Proteinase inhibitors have been shown to be capable of preventing various aspects of fertilization. Diisopropyl fluorophosphate (DFP) is an irreversible inhibitor of trypsin-like enzymes that is commercially available in a radiolabeled form. The experiments described herein were designed to determine if DFP would prevent sperm function in live, motile sperm and to identify the sperm proteins bound with DFP. DFP at 5 mM concentrations had no observable effect on sperm motility, but inhibited the penetration of zona-free hamster ova by human sperm (5.5%) compared to controls (33.5%). Acid extracts of motile sperm that had been incubated with radiolabeled DFP and collected by the swim-up procedure demonstrated the presence of radiolabeled DFP, and the autoradiography of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels of these extracts localized the uptake of radiolabeled DFP to proteins in the molecular weight region of the proacrosin-acrosin system. Acid-extracted proteinases from semen samples incubated with DFP demonstrated a concentration-dependent inhibition of both esterolytic hydrolysis of benzoyl-arginine ethyl ester on spectrophotometric analysis and proteolytic activity on gelatin SDS-PAGE zymography. DFP-labeled proteins were precipitated by highly specific antibodies to proacrosin. These results demonstrated that DFP is capable of inhibiting sperm function, and that it associates with the proacrosin-acrosin system in live motile sperm

  19. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida

    Directory of Open Access Journals (Sweden)

    Aurean D'Eça Júnior

    2011-06-01

    Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3% of isolates tested were phospholipase positive and 16 (44.4% were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%. Statistically significant differences were observed in relation to production of phospholipases among species (pINTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produção de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3% dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4% foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7%. Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2%. Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (?2=1.9 p=0.5901 (?2=1.9 p=0.5901. CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas.

  20. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida / Atividade diferencial in vitro de fosfolipases e proteinases ácidas de isolados clínicos de Candida

    Scientific Electronic Library Online (English)

    Aurean, D' Eça Júnior; Anderson França, Silva; Fernanda Costa, Rosa; Sílvio Gomes, Monteiro; Patrícia de Maria Silva, Figueiredo; Cristina de Andrade, Monteiro.

    2011-06-01

    Full Text Available INTRODUÇÃO: Candida são leveduras comensais, porém, se o equilíbrio da flora normal for interrompido ou as defesas imunitárias estiverem comprometidas, espécies de Candida podem causar manifestações de doença. Vários atributos contribuem na virulência e patogenicidade de Candida, inclusive a produçã [...] o de enzimas extracelulares hidrolíticas, especialmente fosfolipases e proteinases. O objetivo deste estudo foi verificar a atividade in vitro de fosfolipases e proteinases ácidas em isolados clínicos de Candida spp. MÉTODOS: Oitenta e dois isolados provenientes de pacientes hospitalizados coletados a partir de sítios de origem diversos foram analisados. A produção de fosfolipase foi verificada em meio egg yolk e a de proteinase em meio contendo soro albumina bovina. O estudo foi feito em triplicata. RESULTADOS: Cinquenta e seis (68,3%) dos isolados testados apresentaram atividade de fosfolipase positiva e 16 (44,4%) foram positivos para atividade de proteinase. C. tropicalis foi a espécie que apresentou o maior número de isolados positivos para fosfolipases (91,7%). Diferenças estatisticamente significantes em relação à produção de fosfolipases entre as espécies e entre as cepas provenientes de diferentes sítios de origem foram detectadas. Quanto à produção de proteinases ácidas, os isolados de C. parapsilosis testados foram os maiores produtores (69,2%). Entre as espécies analisadas, a porcentagem de produção de proteinase entre os isolados não diferiu estatisticamente (?2=1.9 p=0.5901 (?2=1.9 p=0.5901). CONCLUSÕES: A maioria dos isolados de C. não-albicans, assim como os de C. albicans, foram grandes produtores de enzimas hidrolíticas e, consequentemente, podem ser capazes de causar infecção em condições adequadas. Abstract in english INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extrace [...] llular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3%) of isolates tested were phospholipase positive and 16 (44.4%) were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%). Statistically significant differences were observed in relation to production of phospholipases among species (p

  1. In vivo and in vitro effect of Acacia nilotica seed proteinase inhibitors on Helicoverpa armigera (Hübner) larvae

    Indian Academy of Sciences (India)

    S Ramesh Babu; B Subrahmanyam; Srinivasan; I M Santha

    2012-06-01

    Acacia nilotica proteinase inhibitor (AnPI) was isolated by ammonium sulphate precipitation followed by chromatography on DEAE-Sephadex A-25 and resulted in a purification of 10.68-fold with a 19.5% yield. Electrophoretic analysis of purified AnPI protein resolved into a single band with molecular weight of approximately 18.6+1.00 kDa. AnPI had high stability at different pH values (2.0 to 10.0) except at pH 5.0 and are thermolabile beyond 80°C for 10 min. AnPI exhibited effective against total proteolytic activity and trypsin-like activity, but did not show any inhibitory effect on chymotrypsin activity of midgut of Helicoverpa armigera. The inhibition kinetics studies against H. armigera gut trypsin are of non-competitive type. AnPI had low affinity for H. armigera gut trypsin when compared to SBTI. The partially purified and purified PI proteins-incorporated test diets showed significant reduction in mean larval and pupal weight of H. armigera. The results provide important clues in designing strategies by using the proteinase inhibitors (PIs) from the A. nilotica that can be expressed in genetically engineered plants to confer resistance to H. armigera.

  2. Characterization of Peptides from Capsicum annuum Hybrid Seeds with Inhibitory Activity Against ?-Amylase, Serine Proteinases and Fungi.

    Science.gov (United States)

    Vieira Bard, Gabriela C; Nascimento, Viviane V; Ribeiro, Suzanna F F; Rodrigues, Rosana; Perales, Jonas; Teixeira-Ferreira, André; Carvalho, André O; Fernandes, Katia Valevski S; Gomes, Valdirene M

    2015-04-01

    Over the last several years, the activity of antimicrobial peptides (AMPs), isolated from plant species, against different microorganisms has been demonstrated. More recently, some of these AMPs have been described as potent inhibitors of ?-amylases and serine proteinases from insects and mammals. The aim of this work was to obtain AMPs from protein extracts of a hybrid Capsicum (Ikeda × UENF 1381) seeds and to evaluate their microbial and enzyme inhibitory activities. Initially, proteins were extracted from the Capsicum hybrid seeds in buffer (sodium phosphate pH 5.4,) and precipitated with ammonium sulfate (90% saturated). Extract of hybrid seeds was subjected to size exclusion chromatography, and three fractions were obtained: S1, S2 and S3. The amino acid sequence, obtained by mass spectrometry, of the 6 kDa peptide from the S3 fraction, named HyPep, showed 100% identity with PSI-1.2, a serine protease inhibitor isolated from C. annuum seeds, however the bifunctionality of this inhibitor against two enzymes is being shown for the first time in this work. The S3 fraction showed the highest antifungal activity, inhibiting all the yeast strains tested, and it also exhibited inhibitory activity against human salivary and Callosobruchus maculatus ?-amylases as well as serine proteinases. PMID:25750185

  3. The mitochondrial toxicity of cysteine-S-conjugates: Studies with pentachlorobutadienyl-L-cysteine

    International Nuclear Information System (INIS)

    Nephrotoxic cysteine conjugates, arising from mercapturate biosynthesis, can perturb the mitochondrial membrane potential and calcium homeostasis in renal epithelial cells. Activation of these cysteine conjugates to reactive species by mitochondrial ?-lyases results in covalent binding and mitochondrial damage. PCBC and related cysteine conjugates inhibit ADP-stimulated respiration in mitochondria respiring on alpha-ketoglutrate/malate and succinate indicating that both dehydrogenases may be targets. The respiratory inhibition is blocked by aminooxyacetic acid, an inhibitor of the ?-lyase. Hence, metabolic activation is required implying that covalent binding of reactive intermediates may be important to the mitochondrial injury. Binding of 35S-fragments has been found for 5 conjugates with varying degrees of mitochondrial toxicity. PCBC is more lipophilic and has a higher affinity for cellular membranes than other cysteine conjugates. PCBC rapidly depolarizes the inner membrane potential resulting in an inhibition of mitochondrial oxidative phosphorylation and calcium upon sequestration. Consequently, mitochondria and renal epithelial cells exposed to PCBC show a sudden release of calcium upon exposure to PCBC which is followed by a later increase in state 4 respiration leading to an inhibition of oxidative phosphorylation. The primary effect of other cysteine conjugates is an inhibition of the dehydrogenases, thus inhibiting state 3 respiration

  4. Functional analysis of a novel cysteine-rich antimicrobial peptide from the salivary glands of the tick Rhipicephalus haemaphysaloides.

    Science.gov (United States)

    Zhang, Houshuang; Yang, Siqi; Gong, Haiyan; Cao, Jie; Zhou, Yongzhi; Zhou, Jinlin

    2015-10-01

    Ticks encounter various microbes while sucking blood from an infected host and carrying these pathogens in themselves. Ticks can then transmit these pathogens to vertebrate hosts. The immune system of ticks can be stimulated to produce many bioactive molecules during feeding and pathogen invasion. Antimicrobial peptides (AMPs) are key effector molecules of a tick's immune response, as they can kill invading pathogenic microorganisms. In this study, we identified a novel cysteine-rich AMP, designated Rhamp1, in the salivary glands of unfed and fed female ticks (Rhipicephalus haemaphysaloides). Rhamp1 is encoded by a gene with an open reading frame of 333 bp, which in turn encodes a peptide of 12 kDa with a 22 amino acid residue signal peptide. The Rhamp1 protein had a pI of 8.6 and contained six conserved cysteine residues at the C-terminus. Rhamp1 shared 43% amino acid identity with a secreted cysteine-rich protein of another tick species, Ixodes scapularis. We cloned the Rhamp1 gene and attempted to express a recombinant protein using prokaryotic and eukaryotic systems, to determine its biological significance. Recombinant Rhamp1 was successfully expressed in both systems, yielding a glutathione S-transferase (GST)-tagged protein (36 kDa) from the prokaryotic system, and a polyhistidine-tagged Rhamp1 protein (14 kDa) from the eukaryotic system. Rhamp1 inhibited the activities of chymotrypsin (16%) and elastase (22%) and exerted low hemolytic activity. It also inhibited the growth of Gram-negative bacteria, including Pseudomonas aeruginosa (49%), Salmonella typhimurium (50%), and Escherichia coli (52%). Our findings suggest that Rhamp1 is a novel AMP in R. haemaphysaloides with the ability to inhibit proteinase activity. PMID:26152423

  5. Crystallization and preliminary X-ray investigation of proteinase A, a non-pepsin-type acid proteinase from Aspergillus niger var. macrosporus.

    Science.gov (United States)

    Tanokura, M; Matsuzaki, H; Iwata, S; Nakagawa, A; Hamaya, T; Takizawa, T; Takahashi, K

    1992-01-01

    Proteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase distinctly different in various properties from the family of pepsin-type aspartic proteinases, and so far it remains unknown which residues participate in the catalysis of the enzyme and how the mechanism operates. The acid proteinase A was crystallized from an ammonium sulfate solution by the hanging-drop vapor diffusion method. The space group of the crystals was P2(1)2(1)2(1) with unit cell dimensions of a = 54.7 A, b = 70.4 A and c = 38.0 A. On the assumption that there is one enzyme molecule in the asymmetric unit, the calculated ratio of volume to unit protein mass (Vm) was 1.64 A3 per dalton. Diffraction data were collected up to a resolution higher than 1.5 A, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation. The crystal of proteinase A is, therefore, suitable for the structural analysis with a high resolution. PMID:1731082

  6. Homology models of main proteinase from coronavirus associated with SARS

    Science.gov (United States)

    Liu, Hsuan-Liang; Lin, Jin-Chung; Ho, Yih; Chen, Chin-Wen

    2005-01-01

    In this study, two homology models of the main proteinase (M pro) from the novel coronavirus associated with severe acute respiratory syndrome (SARS-CoV) were constructed. These models reveal three distinct functional domains, in which an intervening loop connecting domains II and III as well as a catalytic cleft containing the substrate binding subsites S1 and S2 between domains I and II are observed. S2 exhibits structural variations more significantly than S1 during the 200 ps molecular dynamics simulations because it is located at the open mouth of the catalytic cleft and the amino acid residues lining up this subsite are least conserved. In addition, the higher structural variation of S2 makes it flexible enough to accommodate a bulky hydrophobic residue from the substrate.

  7. Meprin ? and meprin ?: Procollagen proteinases in health and disease.

    Science.gov (United States)

    Prox, Johannes; Arnold, Philipp; Becker-Pauly, Christoph

    2015-01-01

    Metalloproteases meprin ? and meprin ? were recently discovered as procollagen proteinases, capable of cleaving off the globular C- and N-terminal prodomains of fibrillar collagen type I and type III. This proteolytic process is indeed sufficient to induce collagen fibril assembly as visualized by transmission electron microscopy. The biological relevance was demonstrated with the help of meprin ? and meprin ? knock-out mice, which exhibit decreased collagen deposition in skin resulting in impaired tensile strength. On the other hand, overexpression of meprin metalloproteases was found under fibrotic conditions in the skin (keloids) and the lung (pulmonary hypertension). Thus, regulation of meprin activity by specific inhibition to reduce collagen maturation might be a suitable approach for the treatment of certain pathological conditions. PMID:25617491

  8. Altered Expression of Brain Proteinase-Activated Receptor-2, Trypsin-2 and Serpin Proteinase Inhibitors in Parkinson's Disease.

    Science.gov (United States)

    Hurley, Michael J; Durrenberger, Pascal F; Gentleman, Steve M; Walls, Andrew F; Dexter, David T

    2015-09-01

    Neuroinflammation is thought to contribute to cell death in neurodegenerative disorders, but the factors involved in the inflammatory process are not completely understood. Proteinase-activated receptor-2 (PAR2) expression in brain is increased in Alzheimer's disease and multiple sclerosis, but the status of PAR2 in Parkinson's disease is unknown. This study examined expression of PAR2 and endogenous proteinase activators (trypsin-2, mast cell tryptase) and proteinase inhibitors (serpin-A5, serpin-A13) in areas vulnerable and resistant to neurodegeneration in Parkinson's disease at different Braak ?-synuclein stages of the disease in post-mortem brain. In normal aged brain, expression of PAR-2, trypsin-2, and serpin-A5 and serpin-A13 was found in neurons and microglia, and alterations in the amount of immunoreactivity for these proteins were found in some brain regions. Namely, there was a decrease in neurons positive for serpin-A5 in the dorsal motor nucleus, and serpin-A13 expression was reduced in the locus coeruleus and primary motor cortex, while expression of PAR2, trypsin-2 and both serpins was reduced in neurons within the substantia nigra. There was an increased number of microglia that expressed serpin-A5 in the dorsal motor nucleus of vagus and elevated numbers of microglia that expressed serpin-A13 in the substantia nigra of late Parkinson's disease cases. The number of microglia that expressed trypsin-2 increased in primary motor cortex of incidental Lewy body disease cases. Analysis of Parkinson's disease cases alone indicated that serpin-A5 and serpin-A13, and trypsin-2 expression in midbrain and cerebral cortex was different in cases with a high incidence of L-DOPA-induced dyskinesia and psychosis compared to those with low levels of these treatment-induced side effects. This study showed that there was altered expression in brain of PAR2 and some proteins that can control its function in Parkinson's disease. Given the role of PAR2 in neuroinflammation, drugs that mitigate these changes may be neuroprotective when administered to patients with Parkinson's disease. PMID:25982926

  9. Inhibitory selectivity of canecystatin: a recombinant cysteine peptidase inhibitor from sugarcane

    International Nuclear Information System (INIS)

    The cDNA of a cystein peptidase inhibitor was isolated from sugarcane and expressed in Escherichia coli. The protein, named canecystatin, has previously been shown to exert antifungal activity on the filamentous fungus Trichoderma reesei. Herein, the inhibitory specificity of canecystatin was further characterized. It inhibits the cysteine peptidases from plant source papain (Ki=3.3 nM) and baupain (Ki=2.1x10-8 M), but no inhibitory effect was observed on ficin or bromelain. Canecystatin also inhibits lysosomal cysteine peptidases such as human cathepsin B (Ki=125 nM), cathepsin K (Ki=0.76 nM), cathepsin L (Ki=0.6 nM), and cathepsin V (Ki=1.0 nM), but not the aspartyl peptidase cathepsin D. The activity of serine peptidases such as trypsin, chymotrypsin, pancreatic, and neutrophil elastases, and human plasma kallikrein is not affected by the inhibitor, nor is the activity of the metallopeptidases angiotensin converting enzyme and neutral endopeptidase. This is the first report of inhibitory activity of a sugarcane cystatin on cysteine peptidases

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

    Science.gov (United States)

    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. PMID:26342866

  11. In Vivo Analysis of Secreted Aspartyl Proteinase Expression in Human Oral Candidiasis

    OpenAIRE

    Naglik, Julian R.; Newport, George; White, Theodore C.; Fernandes-Naglik, Lynette L.; Greenspan, John S.; Greenspan, Deborah; Sweet, Simon P.; Challacombe, Stephen J.; Agabian, Nina

    1999-01-01

    Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans ...

  12. Profile of Candida albicans-Secreted Aspartic Proteinase Elicited during Vaginal Infection

    OpenAIRE

    Taylor, Brad N.; Staib, Peter; Binder, Ayfer; Biesemeier, Antje; Sehnal, Miriam; Röllinghoff, Martin; Morschhäuser, Joachim; Schröppel, Klaus

    2005-01-01

    Vaginal infections caused by the opportunistic yeast Candida albicans are a significant problem in women of child-bearing age. Several factors are recognized as playing a crucial role in the pathogenesis of superficial candidiasis; these factors include hyphal formation, phenotypic switching, and the expression of virulence factors, including a 10-member family of secreted aspartic proteinases. In the present investigation, we analyzed the secreted aspartic proteinase gene (SAP) expression pr...

  13. Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

    OpenAIRE

    Bilinski, Carl A.; Russell, Inge; Stewart, Graham G.

    1987-01-01

    A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis m...

  14. An unusual specificity in the activation of neutrophil serine proteinase zymogens

    DEFF Research Database (Denmark)

    Salvesen, G; Enghild, J J

    1990-01-01

    The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusu...

  15. Oxidative regulation of neutrophil elastase-alpha-1-proteinase inhibitor interactions.

    OpenAIRE

    Ossanna, P J; Test, S T; Matheson, N R; Regiani, S; Weiss, S. J.

    1986-01-01

    Triggered human neutrophils were able to maintain released elastase in an active form in the presence of purified alpha-1-proteinase inhibitor (alpha-1-PI), serum or bronchoalveolar lavage fluid (BAL). The accumulation of free elastase activity was associated with a decrease in the ability of the alpha-1-PI to inhibit porcine pancreatic elastase, an increase in proteinase activity associated with alpha-2-macroglobulin, and the oxidation of alpha-1-PI to a molecule containing four methionine s...

  16. Isolation and sequencing of a genomic clone encoding aspartic proteinase of Rhizopus niveus.

    OpenAIRE

    Horiuchi, H.; Yanai, K; Okazaki, T; Takagi, M.; Yano, K.

    1988-01-01

    A gene encoding Rhizopus niveus aspartic proteinase was isolated from an R. niveus genomic library by using oligonucleotides probes corresponding to its partial amino acid sequence, and its nucleotide sequence was determined. By comparing its deduced amino acid sequence with the amino acid sequence of rhizopuspepsin (5, 26), we concluded that the R. niveus aspartic proteinase gene has an intron within its coding region and that it has a preproenzyme sequence of 66 amino acids upstream of the ...

  17. Neutrophil-derived Oxidants and Proteinases as Immunomodulatory Mediators in Inflammation

    OpenAIRE

    Witko-Sarsat, V; Descamps-Latscha, B.

    1994-01-01

    Neutrophils generate potent microbicidal molecules via the oxygen-dependent pathway, leading to the generation of reactive oxygen intermediates (ROI), and via the non-oxygen dependent pathway, consisting in the release of serine proteinases and metalloproteinases stored in granules. Over the past years, the concept has emerged that both ROI and proteinases can be viewed as mediators able to modulate neutrophil responses as well as the whole inflammatory process. This is w...

  18. Proteinase-Activated Receptor-1 Mediates Elastase-Induced Apoptosis of Human Lung Epithelial Cells

    OpenAIRE

    Suzuki, Tomoko; Moraes, Theo J.; Vachon, Eric; Ginzberg, Hedy H.; Huang, Tsun-Tsao; Matthay, Michael A.; Morley D. Hollenberg; Marshall, John; McCulloch, Christopher A.G.; Abreu, Maria Teresa Herrera; Chow, Chung-Wai; Downey, Gregory P

    2005-01-01

    Apoptosis of distal lung epithelial cells plays a pivotal role in the pathogenesis of acute lung injury. In this context, proteinases, either circulating or leukocyte-derived, may contribute to epithelial apoptosis and lung injury. We hypothesized that apoptosis of lung epithelial cells induced by leukocyte elastase is mediated via the proteinase activated receptor (PAR)-1. Leukocyte elastase, thrombin, and PAR-1–activating peptide, but not the control peptide, induced apoptosis in human airw...

  19. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    OpenAIRE

    Araki Hiromasa; Kawai Kenzo; Yagi Yasuhiro; Otani Hitomi; Ando Seijitsu; Fukuhara Shirou; Inagaki Chiyoko

    2007-01-01

    Abstract Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transit...

  20. The pep4 gene encoding proteinase A is involved in dimorphism and pathogenesis of Ustilago maydis.

    Science.gov (United States)

    Soberanes-Gutiérrez, Cinthia V; Juárez-Montiel, Margarita; Olguín-Rodríguez, Omar; Hernández-Rodríguez, César; Ruiz-Herrera, José; Villa-Tanaca, Lourdes

    2015-10-01

    Vacuole proteases have important functions in different physiological processes in fungi. Taking this aspect into consideration, and as a continuation of our studies on the analysis of the proteolytic system of Ustilago maydis, a phytopathogenic member of the Basidiomycota, we have analysed the role of the pep4 gene encoding the vacuolar acid proteinase PrA in the pathogenesis and morphogenesis of the fungus. After confirmation of the location of the protease in the vacuole using fluorescent probes, we obtained deletion mutants of the gene in sexually compatible strains of U.?maydis (FB1 and FB2), and analysed their phenotypes. It was observed that the yeast to mycelium dimorphic transition induced by a pH change in the medium, or the use of a fatty acid as sole carbon source, was severely reduced in ?pep4 mutants. In addition, the virulence of the mutants in maize seedlings was reduced, as revealed by the lower proportion of plants infected and the reduction in size of the tumours induced by the pathogen, when compared with wild-type strains. All of these phenotypic alterations were reversed by complementation of the mutant strains with the wild-type gene. These results provide evidence of the importance of the pep4 gene for the morphogenesis and virulence of U.?maydis. PMID:25597948

  1. Redundancy between Cysteine Cathepsins in Murine Experimental Autoimmune Encephalomyelitis

    OpenAIRE

    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 individ...

  2. Unfolding the fold of cyclic cysteine-rich peptides

    OpenAIRE

    Shehu, Amarda; Kavraki, Lydia E.; Clementi, Cecilia

    2008-01-01

    We propose a method to extensively characterize the native state ensemble of cyclic cysteine-rich peptides. The method uses minimal information, namely, amino acid sequence and cyclization, as a topological feature that characterizes the native state. The method does not assume a specific disulfide bond pairing for cysteines and allows the possibility of unpaired cysteines. A detailed view of the conformational space relevant for the native state is obtained through a hierarchic multi-resolut...

  3. Methionine-to-Cysteine Recycling in Klebsiella aerogenes

    OpenAIRE

    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 bacteriu...

  4. ?-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB

    OpenAIRE

    Steert, Koen; Berg, Maya; Mottram, Jeremy C.; Westrop, Gareth D; Coombs, Graham H.; Cos, Paul; Maes, Louis; Joossens, Jurgen; Van der Veken, Pieter; Haemers, Achiel; Augustyns, Koen

    2010-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of ?-ketoheterocycles 1-15 has been developed as reversible inhibitors of a r...

  5. Gamma irradiation or hydrocortisone treatment of rats increases the proteinase activity associated with histones of thymus nuclei

    International Nuclear Information System (INIS)

    An increase in the activity of histone-associated rat thymus nucleus proteinases specific for histones H2A, H2B and H1 was shown after ? irradiation or hydrocortisone treatment of animals. Histone H1-specific proteinase activity is dependent on DNA and increases in the presence of denatured DNA, whereas proteinases specific for core histones are inhibited in the presence of denatured DNA. The increase in the activity of histone-associated proteinases depends on the radiation dose and the time after irradiation or hydrocortisone injection. In the presence of dithiothreitol and sodium dodecyl sulfate, these proteinases dissociate from histones. It was found by gel electrophoresis that several proteinases of various molecular masses are closely associated with histones obtained from thymus nuclei of irradiated or hydrocortisone-treated rats. 43 refs., 7 figs

  6. Cysteine-containing peptides having antioxidant properties

    Science.gov (United States)

    Bielicki, John K. (Castro Valley, CA)

    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.

  7. pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro

    DEFF Research Database (Denmark)

    Sørensen, S O; van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1994-01-01

    Carboxypeptidase Y is a vacuolar enzyme from Saccharomyces cerevisiae. It enters the vacuole as a zymogen, procarboxypeptidase Y, which is immediately processed in a reaction involving two endoproteases, proteinase A and proteinase B. We have investigated the in vitro activation of purified procarboxypeptidase Y by purified proteinase A. This has identified two different processing intermediates; one active and one inactive. The intermediates define a 33 amino acid segment of the 91 amino acid p...

  8. Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

    OpenAIRE

    Birkeland, S E; Stepaniak, L; Sørhaug, T.

    1985-01-01

    Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detect...

  9. Role of cysteine-58 and cysteine-95 residues in the thiol di-sulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti.

    Science.gov (United States)

    Chauhan, Nikhil; Hoti, S L

    2016-01-01

    Macrophage Migration Inhibitory Factor (MIF) is the first human cytokine reported and was thought to have a central role in the regulation of inflammatory responses. Homologs of this molecule have been reported in bacteria, invertebrates and plants. Apart from cytokine activity, it also has two catalytic activities viz., tautomerase and di-sulfide oxidoreductase, which appear to be involved in immunological functions. The CXXC catalytic site is responsible for di-sulfide oxidoreductase activity of MIF. We have recently reported thiol-disulfide oxidoreductase activity of Macrophage Migration Inhibitory Factor-2 of Wuchereria bancrofti (Wba-MIF-2), although it lacks the CXXC motif. We hypothesized that three conserved cysteine residues might be involved in the formation of di-sulfide oxidoreductase catalytic site. Homology modeling of Wba-MIF-2 showed that among the three cysteine residues, Cys58 and Cys95 residues came in close proximity (3.23Å) in the tertiary structure with pKa value 9, indicating that these residues might play a role in the di-sulfide oxidoreductase catalytic activity. We carried out site directed mutagenesis of these residues (Cys58Ser & Cys95Ser) and expressed mutant proteins in Escherichia coli. The mutant proteins did not show any oxidoreductase activity in the insulin reduction assay, thus indicating that these two cysteine residues are vital for the catalytic activity of Wba-MIF-2. PMID:26432350

  10. Implication of Cysteine, Glutathione and Cysteine Synthase in Theobroma cacao L. Zygotic Embryogenesis

    OpenAIRE

    Minyaka Emile; Niemenak Nicolas; N.M.S. Soupi; Sangare Abdourahamane; Omokolo Ndoumou Denis

    2007-01-01

    An investigation on sulfur metabolism during cocoa zygotic embryogenesis was carried out by analysing total amino acids, cysteine, glutathione, cysteine synthase and proteins in the endosperm and in the embryos. Cacao clones SNK10 and Sca6 were used. As the embryo was getting mature, the endosperm became progressively cellularized from the mycropilar zone. Amino acid, cysteine, glutathione and protein contents were always higher in the embryos than in the endosperm in both genotypes. In the e...

  11. Isolation and characterization of a serine proteinase specific to human C3b from human erythrocyte membranes

    International Nuclear Information System (INIS)

    In a previous report, they have shown that human C3b bound through CR1 to human erythrocytes is cleaved by a membrane proteinase activity. Following the molecular analysis of this proteinase activity, they have purified it by a four step procedure: ammonium sulfate precipitation, biogel filtration, fluid phase electrophoresis and hydroxylapatite chromatography. The highly purified proteinase was labeled by 125I iodine or 3H-DFP and analyzed by gel electrophoresis: a single band membrane component was characterized by its apparent molecular weight of 57 K or 60 K, under non reducing or reducing conditions respectively and was called p 57. Its reactivity with 3H-DFP and the inhibition by PMSF of the proteinase activity indicate that p 57 is a serine proteinase. Moreover, it is sensitive to aprotinin and ?1-antitrypsin. This membrane proteinase presents a higher activity in the presence of detergent and cleaves both alpha and beta chains of human C3b. Polyclonal antibody prepared against this purified proteinase inhibits its activity. On the basis of its structure and its functions, i.e. molecular weight, antigenic properties, proteinase properties and proteinases inhibitors sensitivity, p57 is not related to CR1 or DAF, two others membrane components which react with human C3b and identified by others on human erythrocytes. These specific antibodies allow to analyze the presence of p57 on human cells

  12. An O-acetylserine (thiol) lyase from Leucaena leucocephala is a cysteine synthase but not a mimosine synthase.

    Science.gov (United States)

    Yafuso, Jannai T; Negi, Vishal Singh; Bingham, Jon-Paul; Borthakur, Dulal

    2014-07-01

    In plants, the final step of cysteine formation is catalyzed by O-acetylserine (thiol) lyase (OAS-TL). The purpose of this study was to isolate and characterize an OAS-TL from the tree legume Leucaena leucocephala (leucaena). Leucaena contains a toxic, nonprotein amino acid, mimosine, which is also formed by an OAS-TL, and characterization of this enzyme is essential for developing a mimosine-free leucaena for its use as a protein-rich fodder. The cDNA for a cytosolic leucaena OAS-TL isoform was obtained through interspecies suppression subtractive hybridization. A 40-kDa recombinant protein was purified from Escherichia coli and used in enzyme activity assays where it was found to synthesize only cysteine. The enzyme followed Michaelis-Menten kinetics, and the Km was calculated to be 1,850±414 ?M sulfide and the Vmax was 200.6±19.92 ?M cysteine min(-1). The N-terminal affinity His-tag was cleaved from the recombinant OAS-TL to eliminate its possible interference in binding with the substrate, 3-hydroxy-4-pyridone, for mimosine formation. The His-tag-cleaved OAS-TL was again observed to catalyze the formation of cysteine but not mimosine. Thus, the cytosolic OAS-TL from leucaena used in this study is specific for only cysteine synthesis and is different from previously reported OAS-TLs that also function as ?-substituted alanine synthases. PMID:24777760

  13. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTx{sub a}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Chul Won; Kim, Jim Il [Chonnam National Univ., Gwangju (Korea, Republic of); Sato, Kazuki [Fukuoka Women' s Univ., Fukuoka (Japan)

    2013-06-15

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTx{sub a}), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca{sup 2+} release from sarcoplasmic reticulum (SR). IpTx{sub a} increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states.

  14. Determination of Disulfide Bond Connectivity of Cysteine-rich Peptide IpTxa

    International Nuclear Information System (INIS)

    Cysteine-rich peptides stabilized by intramolecular disulfide bonds have often been isolated from venoms of microbes, animals and plants. These peptides typically have much higher stability and improved biopharmaceutical properties compared to their linear counterparts. Therefore the correct disulfide bond formation of small proteins and peptides has been extensively studied for a better understanding of their folding mechanism and achieving efficient generation of the naturally occurring biologically active product. Imperatoxin A (IpTxa), a peptide toxin containing 6 cysteine residues, was isolated from the venom of scorpion Pandinus imperator, selectively binds the ryanodine receptors and activates Ca2+ release from sarcoplasmic reticulum (SR). IpTxa increases the binding of ryanodine to ryanodine receptors (RyRs) and encourages reconstituted single channel to induce subconductance states

  15. Discovery of novel antimicrobial peptides with unusual cysteine motifs in dandelion Taraxacum officinale Wigg. flowers.

    Science.gov (United States)

    Astafieva, A A; Rogozhin, E A; Odintsova, T I; Khadeeva, N V; Grishin, E V; Egorov, Ts A

    2012-08-01

    Three novel antimicrobial peptides designated ToAMP1, ToAMP2 and ToAMP3 were purified from Taraxacum officinale flowers. Their amino acid sequences were determined. The peptides are cationic and cysteine-rich and consist of 38, 44 and 42 amino acid residues for ToAMP1, ToAMP2 and ToAMP3, respectively. Importantly, according to cysteine motifs, the peptides are representatives of two novel previously unknown families of plant antimicrobial peptides. ToAMP1 and ToAMP2 share high sequence identity and belong to 6-Cys-containing antimicrobial peptides, while ToAMP3 is a member of a distinct 8-Cys family. The peptides were shown to display high antimicrobial activity both against fungal and bacterial pathogens, and therefore represent new promising molecules for biotechnological and medicinal applications. PMID:22640720

  16. Modifications of chronic hepatotoxicity of pyrrolizidine (Senecio) alkaloids by butylated hydroxyanisole and cysteine.

    Science.gov (United States)

    Miranda, C L; Buhler, D R; Ramsdell, H S; Cheeke, P R; Schmitz, J A

    1982-02-01

    The chronic hepatotoxic effects of mixed pyrrolizidine alkaloids (PAs) from the poisonous plant tansy ragwort (Senecio jacobaea) and the ability of butylated hydroxyanisole (BHA) and cysteine to alter these hepatic effects were studied in male rats. In control animals, the i.p. administration of a single dose of mixed PAs, 160 mg/kg, produced marked fibrosis, biliary hyperplasia, megalocytosis, necrosis and calcification in liver 8 weeks post injection. In contrast, consumption of 0.75% BHA diet 10 days before and 14 days after PA administration reduced the incidence and/or completely prevented the occurrence of these pathological changes. Similar treatment with 1% cysteine, however, only reduced the severity of the hepatic lesions. PMID:7080083

  17. ? irradiation of Bowman--Birk soybean proteinase inhibitor

    International Nuclear Information System (INIS)

    Radiation damage to Bowman-Birk soybean proteinase inhibitor was studied in dilute aqueous solutions containing: (a) air, (b) N2O plus KBr, and (c) N2O plus KCNS. The degradation of tyrosines, monitored by changes in ultraviolet absorption and circular dichroism (CD) spectra of irradiated inhibitor, led to the loss of chymotrypsin-inhibitory activity without affecting antitrypsin activity. Of two tyrosyl residues in the inhibitor, Tyr 45, located next to the antichymotrypsin site, was shown to be essential to chymotrypsin-inhibitory activity. Radiation damage to Tyr 59 had no effect on either of the antiproteinase activities. The loss of trypsin-inhibitory activity paralleled decrease in disulfide Cd. The breakage of disulfide bonds was confirmed by the p-chloromercuribenzoate assay for sulfhydryl groups. However, the exact role of disulfide bonds in antitryptic activity, other than the maintenance of conformational integrity, is not apparent. It seems more likely that the loss of trypsin-inhibitory activity is due to damage in other amino acid(s), although the degradation of tyrosine (2 residues) and histidine (1 residue) did not affect antitryptic activity

  18. gamma. irradiation of Bowman--Birk soybean proteinase inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Wandell, J.L.; Kay, E.

    1977-12-01

    Radiation damage to Bowman-Birk soybean proteinase inhibitor was studied in dilute aqueous solutions containing: (a) air, (b) N/sub 2/O plus KBr, and (c) N/sub 2/O plus KCNS. The degradation of tyrosines, monitored by changes in ultraviolet absorption and circular dichroism (CD) spectra of irradiated inhibitor, led to the loss of chymotrypsin-inhibitory activity without affecting antitrypsin activity. Of two tyrosyl residues in the inhibitor, Tyr 45, located next to the antichymotrypsin site, was shown to be essential to chymotrypsin-inhibitory activity. Radiation damage to Tyr 59 had no effect on either of the antiproteinase activities. The loss of trypsin-inhibitory activity paralleled decrease in disulfide Cd. The breakage of disulfide bonds was confirmed by the p-chloromercuribenzoate assay for sulfhydryl groups. However, the exact role of disulfide bonds in antitryptic activity, other than the maintenance of conformational integrity, is not apparent. It seems more likely that the loss of trypsin-inhibitory activity is due to damage in other amino acid(s), although the degradation of tyrosine (2 residues) and histidine (1 residue) did not affect antitryptic activity.

  19. Functional role of aspartic proteinase cathepsin D in insect metamorphosis

    Directory of Open Access Journals (Sweden)

    Seo Sook

    2006-10-01

    Full Text Available Abstract Background Metamorphosis is a complex, highly conserved and strictly regulated development process that involves the programmed cell death of obsolete larval organs. Here we show a novel functional role for the aspartic proteinase cathepsin D during insect metamorphosis. Results Cathepsin D of the silkworm Bombyx mori (BmCatD was ecdysone-induced, differentially and spatially expressed in the larval fat body of the final instar and in the larval gut of pupal stage, and its expression led to programmed cell death. Furthermore, BmCatD was highly induced in the fat body of baculovirus-infected B. mori larvae, suggesting that this gene is involved in the induction of metamorphosis of host insects infected with baculovirus. RNA interference (RNAi-mediated BmCatD knock-down inhibited programmed cell death of the larval fat body, resulting in the arrest of larval-pupal transformation. BmCatD RNAi also inhibited the programmed cell death of larval gut during pupal stage. Conclusion Based on these results, we concluded that BmCatD is critically involved in the programmed cell death of the larval fat body and larval gut in silkworm metamorphosis.

  20. A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE

    Scientific Electronic Library Online (English)

    ENDER, BÇER; PAKZE, QETNKAYA.

    Full Text Available The interaction of novobiocin (NOV), an aminocoumarin antibiotic, with cysteine was studied by square-wave voltammetry technique on the hanging mercury drop electrode in different pH values. After the addition of NOV into the cysteine solution, the peak current of mercurous cysteine thiolate decreas [...] ed and its voltammetric peak potential shifted to more positive values. Voltammetric results showed that NOV binds with cysteine forming 1:1 nonelectroactive molecular complex by means of electrostatic and hydrogen-bonding interactions. The binding constants of NOV with cysteine at pHs 5, 7 and 10 were calculated to be 3.06x10³, 1.54x10(4) and 1.06x10(5) M-1, respectively. Furthermore, apossible mechanism of such interaction was also discussed.

  1. ?-Ketoheterocycles as inhibitors of Leishmania mexicana cysteine protease CPB

    Science.gov (United States)

    Steert, Koen; Berg, Maya; Mottram, Jeremy C.; Westrop, Gareth D.; Coombs, Graham H.; Cos, Paul; Maes, Louis; Joossens, Jurgen; Van der Veken, Pieter; Haemers, Achiel; Augustyns, Koen

    2011-01-01

    Cysteine proteases of the papain superfamily are present in nearly all eukaryotes and also play pivotal roles in the biology of parasites. Inhibition of cysteine proteases is emerging as an important strategy to combat parasitic diseases such as sleeping sickness, Chagas’ disease and leishmaniasis. Inspired by the in vivo antiparasitic activity of the vinyl sulfone based cysteine protease inhibitors (CPIs), a series of ?-ketoheterocycles 1-15 has been developed as reversible inhibitors of a recombinant L. mexicana cysteine protease CPB2.8. The isoxazoles 1-3 and especially the oxadiazole 15 are potent reversible inhibitors of CPB2.8, however, in vitro whole-organism screening against a panel of protozoan parasites did not fully correlate with the observed inhibition of the cysteine protease. PMID:20799311

  2. BINDING OF CHLOROFORM TO THE CYSTEINE OF HEMOGLOBIN

    Science.gov (United States)

    The products of the covalent binding of chloroform to rat hemoglobin during microsomal metabolism were isolated and identified by gas chromatography (GC) and mass spectroscopy (MS). After isolation by Proteinase K hydrolysis, amino acid analysis and cellulose thin-layer chromatog...

  3. Proteinase and phospholipase activity as virulence factors in Candida species isolated from blood.

    Science.gov (United States)

    Mohan das, Vinitha; Ballal, Mamatha

    2008-12-31

    The number of nosocomial blood stream infections due to Candida species has increased over the past few decades. In order to establish an infection, opportunistic pathogens have to evade the immune system, survive, divide in the host environment, and spread to other tissues. Proteinase and phospholipase secretion has been implicated as potential virulence factors for some Candida species responsible for catheter related candidemia in intensive care unit (ICU) patients with indwelling devices. We therefore have aimed at demonstrating the secretion of proteinase and phospholipase enzymes as virulent factors by Candida species isolated from blood samples collected from ICUs, dialysis units and oncology units. One hundred and fourteen isolates of Candida species were obtained from the blood samples and the isolates include 37 Candida albicans, 7 Candida glabrata, 5 Candida guilliermondii, 3 Candida kefyr, 45 Candida krusei, 5 Candida parapsilosis, and 12 Candida tropicalis. Proteinase assay was performed by using the Staib et al method. Phospholipase assay was performed by using the method of Samaranayake et al. Precipitation zone (Pz value) was determined. The percentage of isolates which produced detectable amounts of proteinase is 74.56% and 44.73% of isolates produced detectable amounts of phospholipase. We believe that production of both phospholipase and proteinase enzimes could be an important virulence factor for several Candida species. PMID:19071887

  4. Domain 15 of the serine proteinase inhibitor LEKTI blocks HIV infection in vitro

    Directory of Open Access Journals (Sweden)

    David Palesch

    2013-08-01

    Full Text Available Background: Lympho-epithelial Kazal-type-related inhibitor (LEKTI is a 15-domain serine proteinase inhibitor, parts of which have first been isolated from human blood filtrate. It is encoded by the gene SPINK5. In the past, different groups reported antiviral activities of certain serine proteinase inhibitors, such as mucous proteinase inhibitor and alpha1-proteinase inhibitor. The purpose of this study was to test two representative domains of the proteinase inhibitor LEKTI for anti-HIV activities.Methods: LEKTI domains 6 and 15 were recombinantly produced in E.coli. To test their inhibitory activity against HIV infection, the reporter cell line P4-R5 MAGI carrying an HIV-inducible reporter gene was infected by a CCR5-tropic HIV strain in the presence of different inhibitor concentrations. After three days, infection rates were determined by quantifying ß-galactosidase activities using the Galacto-Light Plus™ ß-Galactosidase Reporter Gene Assay.Results: In contrast to LEKTI domain 6, LEKTI domain 15 suppressed HIV-induced reporter gene activities with an IC50 value of approximately 29 µM.Conclusion: LEKTI domain 15 represents an inhibitor of HIV infection. (Med J Indones. 2013;22:131-5. doi: 10.13181/mji.v22i3.580Keywords: HIV, inhibition, LEKTI, P4-R5 MAGI

  5. Effect of pH on the production of alkaline proteinase by alkalophilic Bacillus sp

    International Nuclear Information System (INIS)

    The effect of the pH of the medium on the microbial growth and alkaline proteinase production, and on the uptake of various substances by alkalophilic Bacillus sp. No.8-1 were studied to investigate the physiological properties of alkalophilic bacteria. Both the microbial growth and alkaline proteinase production by replacement culture were maximum between pH 9 and 10. The alkaline proteinase production sources were also effective for the production. The uptake of various substances such as glucose, acetate, amino acids, and uracil, necessary for proteinase production by this strain, was maximum between pH 9 and 10. The uptake of ?-aminoisobutyric acid, a nonmetabolizable amino acid analogue, was also maximum at pH 10. The pH-dependence of these substance was not due to their ionic forms being affected by extracellular pH. It was concluded from above results that good production of alkaline proteinase in alkaline media was due to the active uptake of various nutrients in this culture condition. (auth.)

  6. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited ?-chymotrypsin to 9.4% residual activity and also inhibited ?-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  7. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Scientific Electronic Library Online (English)

    Leah Theresa, Sigle; Marcelo, Ramalho-Ortigao.

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae) are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibi [...] tors were identified (PpKzl1 and PpKzl2). Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells) that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited ?-chymotrypsin to 9.4% residual activity and also inhibited ?-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  8. Reaction mechanism of -acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    R Shankar; P Kolandaivel

    2007-09-01

    The gas-phase reaction mechanism of -acylhydroxamate with cysteine proteases has been investigated using ab initio and density functional theory. On the irreversible process, after breakdown of tetrahedral intermediate (INT1), small 1-2 anionotropic has been formed and rearranged to give stable by-products sulfenamide (P1) and thiocarbamate (P2) with considerable energy loss. While, on the reversible part of this reaction mechanism, intermediate (INT2) breaks down on oxidation, to form a stable product (P3). Topological and AIM analyses have been performed for hydrogen bonded complex in this reaction profile. Intrinsic reaction coordinates [IRC, minimum-energy path (MEP)] calculation connects the transition state between R-INT1, INT1-P1 and INT1-P2. The products P1, P2 and P3 are energetically more stable than the reactant and hence the reaction enthalpy is found to be exothermic.

  9. Mechanism and ion-dependence of in vitro autoactivation of yeast proteinase A : possible implications for compartmentalized activation in vivo

    DEFF Research Database (Denmark)

    Van Den Hazel, H; Wolff, A M

    1997-01-01

    Yeast proteinase A is synthesized as a zymogen which transits through the endoplasmic reticulum, the Golgi complex and the endosome to the vacuole. On arrival in the vacuole, activation takes place. It has previously been found that proteinase A can activate autocatalytically; however, the propeptide of proteinase A shows essentially no similarity to other known aspartic proteinase propeptides. To understand why proteinase A activation occurs rapidly in the vacuole but not at all in earlier compartments, we have purified the zymogen and investigated the conditions that trigger autoactivation and the mechanism of autoactivation. Autoactivation was triggered by acidic pH and its rate increased with increasing ionic strength. Kinetic evidence indicates that autoactivation mainly occurs via a bimolecular product-catalysed mechanism in which an active proteinase A molecule activates a zymogen molecule. Both the pH- and ionic-strength-dependence and the predominance of a product-catalysed mechanism are well adapted to the situation in vivo, since slow activation in the absence of active proteinase A helps to prevent activation in prevacuolar compartments, whereas, on delivery to the vacuole, lower pH, higher ionic strength and the presence of already active proteinases ensure rapid activation. Product-catalysed autoactivation may be a general mechanism by which cells ensure autoactivation of intracellular enzymes to be both rapid and compartmentalized.

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

    Science.gov (United States)

    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. PMID:26642319

  11. Neutrophil elastase and proteinase 3 trafficking routes in myelomonocytic cells

    International Nuclear Information System (INIS)

    Neutrophil elastase (NE) and proteinase 3 (PR3) differ in intracellular localization, which may reflect different trafficking mechanisms of the precursor forms when synthesized at immature stages of neutrophils. To shed further light on these mechanisms, we compared the trafficking of precursor NE (proNE) and precursor PR3 (proPR3). Like proNE [1], proPR3 interacted with CD63 upon heterologous co-expression in COS cells but endogenous interaction was not detected although cell surface proNE/proPR3/CD63 were co-endocytosed in myelomonocytic cells. Cell surface proNE/proPR3 turned over more rapidly than cell surface CD63 consistent with processing/degradation of the pro-proteases but recycling of CD63. Colocalization of proNE/proPR3/CD63 with clathrin and Rab 7 suggested trafficking through coated vesicles and late endosomes. Partial caveolar trafficking of proNE/CD63 but not proPR3 was suggested by colocalization with caveolin-1. Blocking the C-terminus of proNE/proPR3 by creating a fusion with FK506 binding protein inhibited endosomal re-uptake of proNE but not proPR3 indicating 'proC'-peptide-dependent structural/conformational requirements for proNE but not for proPR3 endocytosis. The NE aminoacid residue Y199 of a proposed NE sorting motif that interacts with AP-3 [2] was not required for proNE processing, sorting or endocytosis in rat basophilic leukemia (RBL) cells expressing heterologous Y199-deleted proNE; this suggests operation of another AP-3-link for proNE targeting. Our results show intracellular multi-step trafficking to be different between proNE and proPR3 consistent with their differential subcellular NE/PR3 localization in neutrophils.

  12. Specificity of proteinase K at P2 to P3' sub-sites and its comparison to other serine proteases.

    Science.gov (United States)

    Qasim, Mohammad A

    2014-01-01

    Specificity of the commercially important serine protease, proteinase K, has been investigated by measuring free energies of association of proteinase K with turkey ovomucoid third domain inhibitor variants at contact positions P2, P1, P1', P2', and P3'. Correlations of these values were run with similar values that have been obtained for six other serine proteases. Among the six proteases, subtilisin Carlsberg shows a near perfect correlation (Pearson Product correlation coefficient = 0.93 to 0.99) with proteinase K at all of these positions. Proteinase K has only 35% sequence identity with subtilisin Carlsberg, yet, the two enzymes are nearly identical in their specificity at P2 to P3' positions. With other serine proteases such as bovine chymotrypsin, human leukocyte elastase, porcine pancreatic elastase, Streptomyces griseus protease A and B, proteinase K showed relatively poor or no correlation. PMID:24050203

  13. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole

    2004-01-01

    Background: Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results: We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator ( CFTR), transcription factors CREB-RP and OCT-I, and components of the ubiquitin pathway. Conclusions: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses.

  14. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  15. Silver(I) Complex formation with Cysteine, Penicillamine and Glutathione

    OpenAIRE

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

    2013-01-01

    The complex formation between silver(I) and cysteine (H2Cys), penicillamine (H2Pen) or glutathione (H3Glu) in alkaline aqueous solution was examined using extended X-ray absorption fine structure (EXAFS) and 109Ag 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 2.45 ± 0....

  16. Cloning and characterization of a novel cysteine protease gene (HbCP1) from Hevea brasiliensis

    Indian Academy of Sciences (India)

    Shi-Qing Peng; Jia-Hong Zhu; Hui-Liang Li; Wei-Min Tian

    2008-12-01

    The full-length cDNA encoding a cysteine protease, designated HbCP1, was isolated for the first time from Hevea brasiliensis by the rapid amplification of cDNA ends (RACE) method. HbCP1 contained a 1371 bp open reading frame encoding 457 amino acids. The deduced HbCP1 protein, which showed high identity to cysteine proteases of other plant species, was predicted to possess a putative repeat in toxin (RTX) domain at the N-terminal and a granulin (GRAN) domain at the C-terminal. Southern blot analysis indicated that the HbCP1 gene is present as a single copy in the rubber tree. Transcription pattern analysis revealed that HbCP1 had high transcription in laticifer, and low transcription in bark and leaf. The transcription of HbCP1 in latex was induced by ethylene and tapping. Cloning of the HbCP1 gene will enable us to further understand the molecular characterization of cysteine protease and its possible function in the rubber tree.

  17. Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

    OpenAIRE

    Pederson, Jeffrey A.; Mileski, Gerald J.; Weimer, Bart C.; Steele, James L

    1999-01-01

    A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiologi...

  18. The aspartic proteinase from Saccharomyces cerevisiae folds its own inhibitor into a helix

    DEFF Research Database (Denmark)

    Li, M; Phylip, L H; Lees, W E; Winther, Jakob R.; Dunn, B M; Wlodawer, A; Kay, J; Gustchina, A

    2000-01-01

    Aspartic proteinase A from yeast is specifically and potently inhibited by a small protein called IA3 from Saccharomyces cerevisiae. Although this inhibitor consists of 68 residues, we show that the inhibitory activity resides within the N-terminal half of the molecule. Structures solved at 2.2 and 1.8 A, respectively, for complexes of proteinase A with full-length IA3 and with a truncated form consisting only of residues 2-34, reveal an unprecedented mode of inhibitor-enzyme interactions. Neith...

  19. The cell envelope subtilisin-like proteinase is a virulence determinant for Streptococcus suis

    Directory of Open Access Journals (Sweden)

    Gottschalk Marcelo

    2010-02-01

    Full Text Available Abstract Background Streptococcus suis is a major swine pathogen and zoonotic agent that mainly causes septicemia, meningitis, and endocarditis. It has recently been suggested that proteinases produced by S. suis (serotype 2 are potential virulence determinants. In the present study, we screened a S. suis mutant library created by the insertion of Tn917 transposon in order to isolate a mutant deficient in a cell surface proteinase. We characterized the gene and assessed the proteinase for its potential as a virulence factor. Results Two mutants (G6G and M3G possessing a single Tn917 insertion were isolated. The affected gene coded for a protein (SSU0757 that shared a high degree of identity with Streptococccus thermophilus PrtS (95.9% and, to a lesser extent, with Streptococcus agalactiae CspA (49.5%, which are cell surface serine proteinases. The SSU0757 protein had a calculated molecular mass of 169.6 kDa and contained the catalytic triad characteristic of subtilisin family proteinases: motif I (Asp200, motif II (His239, and motif III (Ser568. SSU0757 also had the Gram-positive cell wall anchoring motif (Leu-Pro-X-Thr-Gly at the carboxy-terminus, which was followed by a hydrophobic domain. All the S. suis isolates tested, which belonged to different serotypes, possessed the gene encoding the SSU0757 protein. The two mutants devoid of subtilisin-like proteinase activity had longer generation times and were more susceptible to killing by whole blood than the wild-type parent strain P1/7. The virulence of the G6G and M3G mutants was compared to the wild-type strain in the CD1 mouse model. Significant differences in mortality rates were noted between the P1/7 group and the M3G and G6G groups (p Conclusion In summary, we identified a gene coding for a cell surface subtilisin-like serine proteinase that is widely distributed in S. suis. Evidences were brought for the involvement of this proteinase in S. suis virulence.

  20. Organometallic palladium reagents for cysteine bioconjugation

    Science.gov (United States)

    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.

  1. Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

    Directory of Open Access Journals (Sweden)

    Larissa Ramos Chevreuil

    2011-03-01

    Full Text Available Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Amazônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10% p/v resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2% e 1,3 a 14,8%, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas.The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species studied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10% w/v, resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2% and 1.3 to 14.8%, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.

  2. Prospeção de inibidores de serinoproteinases em folhas de leguminosas arbóreas da floresta Amazônica / Prospecting serine proteinase inhibitors in leaves from leguminous trees of the Amazon forest

    Scientific Electronic Library Online (English)

    Larissa Ramos, Chevreuil; José Francisco de Carvalho, Gonçalves; Flávia Camila, SCHIMPL; Cristiane Santos do Carmo Ribeiro de, Souza; Luiz Augusto Gomes de, Souza; Silvana Cristina, Pando.

    2011-03-01

    Full Text Available Os inibidores de proteinases são proteínas extensivamente investigadas nos tecidos de estocagem, mas pouco prospectadas em outros tecidos vegetais. O objetivo deste estudo foi detectar a presença de inibidores de serinoproteinases em extratos foliares de quinze espécies de leguminosas arbóreas da Am [...] azônia. As espécies estudadas foram: Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata e S. polyphylla. Folhas foram coletadas, secas a 30ºC durante 48 h, trituradas e submetidas à extração com NaCl (0,15 M, 10% p/v) resultando no extrato total. Ensaios foram executados para determinar a concentração de proteínas e detectar a atividade inibitória contra a tripsina e quimotripsina bovina. Os teores de proteínas bruta e solúvel nos extratos foliares variaram de 7,9 a 31,2% e 1,3 a 14,8%, respectivamente. A atividade inibitória sobre a tripsina e quimotripsina foi observada em todos os extratos foliares. Contudo, nos extratos de E. maximum, L. leucocephala, P. pendula, S. corrugata e S. polyphylla a inibição foi maior sobre a tripsina, enquanto o extrato de P. multijuga foi mais efetivo contra a quimotripsina. Nós concluímos que nos extratos foliares de leguminosas arbóreas têm inibidores de serinoproteinases e exibem potencial aplicações biotecnológicas. Abstract in english The proteinase inhibitors are proteins extensively investigated in tissue storage, but few prospected in other plant tissues. The aim of this study was to detect the presence of serine proteinase inhibitors in leaf extracts from fifteen species of leguminous trees of the Amazon forest. The species s [...] tudied were Caesalpinia echinata, C. ferrea, Cedrelinga cateniformis, Copaifera multijuga, Dinizia excelsa, Enterolobium contortisiliquum, E. maximum, E. schomburgkii, Leucaena leucocephala, Ormosia paraensis, Parkia multijuga, P. pendula, P. platycephala, Swartzia corrugata and S. polyphylla. Leaves were collected, dried at 30ºC for 48 h, crushed and subjected to extraction with NaCl (0.15 M, 10% w/v), resulting in the total extract. Tests were performed to determine the concentration of proteins and to detect of inhibitory activity against bovine trypsin and chymotrypsin. The content of crude and soluble protein in leaf extracts varied from 7.9 to 31.2% and 1.3 to 14.8%, respectively. The inhibitory activity on trypsin and chymotrypsin was observed in all leaf extracts. However, in extracts of E. maximum, L. leucocephala, P. pendula, S. corrugata and S. polyphylla, the inhibition was greater on trypsin, while extract of P. multijuga was more effective against chymotrypsin. We conclude that leaf extracts of leguminous trees have serine proteinase inhibitors and show potential biotecnological applications.

  3. Antimicrobial Peptides from Plants.

    Science.gov (United States)

    Tam, James P; Wang, Shujing; Wong, Ka H; Tan, Wei Liang

    2015-01-01

    Plant antimicrobial peptides (AMPs) have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs) of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic), lipid transfer proteins, ?-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms. PMID:26580629

  4. Antimicrobial Peptides from Plants

    Directory of Open Access Journals (Sweden)

    James P. Tam

    2015-11-01

    Full Text Available Plant antimicrobial peptides (AMPs have evolved differently from AMPs from other life forms. They are generally rich in cysteine residues which form multiple disulfides. In turn, the disulfides cross-braced plant AMPs as cystine-rich peptides to confer them with extraordinary high chemical, thermal and proteolytic stability. The cystine-rich or commonly known as cysteine-rich peptides (CRPs of plant AMPs are classified into families based on their sequence similarity, cysteine motifs that determine their distinctive disulfide bond patterns and tertiary structure fold. Cystine-rich plant AMP families include thionins, defensins, hevein-like peptides, knottin-type peptides (linear and cyclic, lipid transfer proteins, ?-hairpinin and snakins family. In addition, there are AMPs which are rich in other amino acids. The ability of plant AMPs to organize into specific families with conserved structural folds that enable sequence variation of non-Cys residues encased in the same scaffold within a particular family to play multiple functions. Furthermore, the ability of plant AMPs to tolerate hypervariable sequences using a conserved scaffold provides diversity to recognize different targets by varying the sequence of the non-cysteine residues. These properties bode well for developing plant AMPs as potential therapeutics and for protection of crops through transgenic methods. This review provides an overview of the major families of plant AMPs, including their structures, functions, and putative mechanisms.

  5. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B; Brinch-Pedersen, Henrik

    2010-01-01

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced wit...

  6. Expression of human ?1-proteinase inhibitor in Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Punt Peter J

    2007-10-01

    Full Text Available Abstract Background Human ?1-proteinase inhibitor (?1-PI, also known as antitrypsin, is the most abundant serine protease inhibitor (serpin in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, ?1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant ?1-PI (r-?1-PI could provide an attractive alternative. Although r-?1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation. Results We have explored the possibility of expressing the gene for human ?1-PI in the filamentous fungus Aspergillus niger (A. niger, a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of ?1-PI with a strongly expressed, secreted leader protein (glucoamylase G2, separated by dibasic processing site (N-V-I-S-K-R that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and ?1-PI activity assays enabled us to select the transformant(s secreting a biologically active glycosylated r-?1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS analysis further confirmed that molecular mass of the r-?1-PI was similar to that of the pd-?1-PI. In vitro stability of the r-?1-PI from A. niger was tested in comparison with pd-?1-PI reference and non-glycosylated human r-?1-PI from E. coli. Conclusion We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for ?1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for ?1-PI in A. niger was successfully achieved to produce the secreted mature human r-?1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

  7. Cysteine inhibits mercury methylation by Geobacter sulfurreducens PCA

    Energy Technology Data Exchange (ETDEWEB)

    Lin, Hui [ORNL; Lu, Xia [ORNL; Liang, Liyuan [ORNL; Gu, Baohua [ORNL

    2015-01-01

    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, however, exhibited both time-dependent and cysteine concentration-dependent methylation. In 144 hour assay, nearly complete sorption of the Hg(II) by PCA-WT occurred in the presence of 1 mM cysteine, resulting in our highest observed methylmercury production. 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.

  8. Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases

    OpenAIRE

    Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

    2015-01-01

    Upon recognition of microbe-associated molecular patterns (MAMPs) such as the bacterial flagellin (or the derived peptide flg22) by pattern-recognition receptors (PRRs) such as the FLAGELLIN SENSING2 (FLS2), plants activate the pattern-triggered immunity (PTI) response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2) is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs) possess two copies of the C-X8-C-X2-C (DUF26) motif in their extracellular dom...

  9. The crystal structure of the cysteine protease Xylellain from Xylella fastidiosa reveals an intriguing activation mechanism.

    Science.gov (United States)

    Leite, Ney Ribeiro; Faro, Aline Regis; Dotta, Maria Amélia Oliva; Faim, Livia Maria; Gianotti, Andreia; Silva, Flavio Henrique; Oliva, Glaucius; Thiemann, Otavio Henrique

    2013-02-14

    Xylella fastidiosa is responsible for a wide range of economically important plant diseases. We report here the crystal structure and kinetic data of Xylellain, the first cysteine protease characterized from the genome of the pathogenic X. fastidiosa strain 9a5c. Xylellain has a papain-family fold, and part of the N-terminal sequence blocks the enzyme active site, thereby mediating protein activity. One novel feature identified in the structure is the presence of a ribonucleotide bound outside the active site. We show that this ribonucleotide plays an important regulatory role in Xylellain enzyme kinetics, possibly functioning as a physiological mediator. PMID:23333295

  10. Correlation of phospholipase and proteinase production of Candida with in vivo pathogenicity in Galleria mellonella

    Scientific Electronic Library Online (English)

    Rodnei Dennis, Rossoni; Júnia Oliveira, Barbosa; Simone Furgeri Godinho, Vilela; Jéssica Diane dos, Santos; Antonio Olavo Cardoso, Jorge; Juliana Campos, Junqueira.

    2013-09-01

    Full Text Available An essential factor to the virulence of the genus Candida is the ability to produce enzymes and this may be crucial in the establishment of fungal infections. AIM:This study investigated in vitro enzymatic activities of Candida species and their virulence in an in vivo Galleria mellonella experiment [...] al model. METHODS: Twenty-four clinical strains of Candida spp. isolated from the human oral cavity were evaluated, including the following species: C. albicans, C. dubliniensis, C. glabrata, C. tropicalis, C. krusei, C. parapsilosis, C. norvegensis, C. lusitaniae and C. guilliermondii. All Candida strains were tested in vitro for production of proteinase and phospholipase. The Candida strains were also injected into Galleria mellonella larvae to induce experimental candidiasis, and after 24 hours, the survival rate was assessed. RESULTS: Phospholipase and proteinase activity were observed in 100% of the C. albicans strains. In the non-albicans species, proteinase and phospholipase activity were observed in 25 and 43% of the studied strains, respectively. The most pathogenic Candida species in G. mellonella were C. albicans, C. dubliniensis and C. lusitaniae, whereas C. glabrata was the least virulent species. Furthermore, a positive significant correlation was found between both enzymatic activities with virulence in G. mellonella. CONCLUSIONS: The virulence of Candida strains in G. mellonella is related to the quantity of proteinases and phospholipases production of each strain.

  11. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis.

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Ro?. 90, ?. 3 (2008), s. 349-357. ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

  12. Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease.

    Science.gov (United States)

    Hollenberg, M D; Mihara, K; Polley, D; Suen, J Y; Han, A; Fairlie, D P; Ramachandran, R

    2014-03-01

    Although it has been known since the 1960s that trypsin and chymotrypsin can mimic hormone action in tissues, it took until the 1990s to discover that serine proteinases can regulate cells by cleaving and activating a unique four-member family of GPCRs known as proteinase-activated receptors (PARs). PAR activation involves the proteolytic exposure of its N-terminal receptor sequence that folds back to function as a 'tethered' receptor-activating ligand (TL). A key N-terminal arginine in each of PARs 1 to 4 has been singled out as a target for cleavage by thrombin (PARs 1, 3 and 4), trypsin (PARs 2 and 4) or other proteases to unmask the TL that activates signalling via Gq , Gi or G12 /13 . Similarly, synthetic receptor-activating peptides, corresponding to the exposed 'TL sequences' (e.g. SFLLRN-, for PAR1 or SLIGRL- for PAR2) can, like proteinase activation, also drive signalling via Gq , Gi and G12 /13 , without requiring receptor cleavage. Recent data show, however, that distinct proteinase-revealed 'non-canonical' PAR tethered-ligand sequences and PAR-activating agonist and antagonist peptide analogues can induce 'biased' PAR signalling, for example, via G12 /13 -MAPKinase instead of Gq -calcium. This overview summarizes implications of this 'biased' signalling by PAR agonists and antagonists for the recognized roles the PARs play in inflammatory settings. PMID:24354792

  13. Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

    International Nuclear Information System (INIS)

    This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

  14. Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

    Energy Technology Data Exchange (ETDEWEB)

    Vadas, Timothy M., E-mail: tvadas@umbc.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States); Ahner, Beth A., E-mail: baa7@cornell.ed [Department of Biological and Environmental Engineering, Cornell University, 320 Riley-Robb Hall, Ithaca, NY 14853 (United States)

    2009-08-15

    This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb-glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation. - Cysteine and glutathione mediate the transport of lead and cadmium into plant roots.

  15. Functional diversity of cysteine residues in proteins and unique features of catalytic redox-active cysteines in thiol oxidoreductases.

    Science.gov (United States)

    Fomenko, Dmitri E; Marino, Stefano M; Gladyshev, Vadim N

    2008-09-30

    Thiol-dependent redox systems are involved in regulation of diverse biological processes, such as response to stress, signal transduction, and protein folding. The thiol-based redox control is provided by mechanistically similar, but structurally distinct families of enzymes known as thiol oxidoreductases. Many such enzymes have been characterized, but identities and functions of the entire sets of thiol oxidoreductases in organisms are not known. Extreme sequence and structural divergence makes identification of these proteins difficult. Thiol oxidoreductases contain a redox-active cysteine residue, or its functional analog selenocysteine, in their active sites. Here, we describe computational methods for in silico prediction of thiol oxidoreductases in nucleotide and protein sequence databases and identification of their redox-active cysteines. We discuss different functional categories of cysteine residues, describe methods for discrimination between catalytic and noncatalytic and between redox and non-redox cysteine residues and highlight unique properties of the redox-active cysteines based on evolutionary conservation, secondary and three-dimensional structures, and sporadic replacement of cysteines with catalytically superior selenocysteine residues. PMID:18648218

  16. Presence of Active and Inactive Molecules of a Cell Wall-Associated Proteinase in Lactobacillus helveticus CP790

    OpenAIRE

    Yamamoto, N.; Akino, A.; Takano, T.; Shishido, K.

    1995-01-01

    Monoclonal antibodies against a cell wall-associated 45-kDa proteinase from Lactobacillus helveticus CP790 were prepared and used for an immunoblotting analysis of the cell wall extract of CP790. They were found to react with an unidentified 46-kDa protein as well as the 45-kDa proteinase. The 46-kDa protein was copurified with the 45-kDa proteinase by affinity column chromatography using antibody-fixed Sepharose and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then extracted...

  17. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte; Wassélius, Johan; Ekström, Ulf; Abrahamson, Magnus

    2010-01-01

    Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular prote...

  18. Measuring site occupancy : a new perspective on cysteine oxidation

    DEFF Research Database (Denmark)

    Rogowska-Wrzesinska, Adelina; Wojdyla, Katarzyna

    2014-01-01

    Site occupancy is an extremely important aspect of quantification of protein modifications. Knowing the degree of modification of each oxidised cysteine residue is critical to understanding the biological role of these modifications. Yet modification site occupancy is very often overlooked, in part because there are very few analytical tools that allow such measurements. Here we present a new strategy, which provides quantitative analysis of cysteine S-nitrosylation (SNO) and S-sulfenylation (SOH) simultaneously at the resolution of single cysteine and allows for determination of relative oxidation occupancy of the modification site. We show that, on one hand, heavily modified cysteines are not necessarily involved in the response to oxidative stress. On the other hand residues with low modification level can be dramatically affected by mild oxidative imbalance. We make use of high resolution mass spectrometry. The method relies on differential reduction of "total" cysteines, SNO cysteines and SOH cysteines with TCEP, sodium ascorbate and sodium arsenite respectively followed by iodoTMT(TM) alkylation. Enrichment of iodoTMT(TM)-containing peptides is performed using anti-TMT antibody. In vivo model of mild oxidative stress in Escherichia coli is used. To induce endogenous SNO bacteria were grown anaerobically in minimal media supplemented with fumarate or nitrate. Short-term treatment with submilimolar levels of hydrogen peroxide were used to induce SOH. We have quantified 114 SNO/SOH modified peptides corresponding to 90 proteins. Only 6 modified peptides changed significantly under mild oxidative stress. Quantitative information allowed us to determine relative modification site occupancy of each identified modified residue and pin point heavily modified ones. The method proved to be precise and sensitive enough to detect and quantify endogenous levels of oxidative stress on proteome-wide scale and brings a new perspective on the role of the modification site occupancy in cellular redox response.

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

    Science.gov (United States)

    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. PMID:26075905

  20. Hordeum vulgare cysteine protease heterologous expressed in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe

    During germination of barley seeds, the mobilization of protein is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. One of the key cysteine proteases in Barley, (Hordeum vulgare) endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH and the protein expression were monitered during induction by collecting 1 ml samples every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of functional, heterologous protein was produced and the protein production was highest after 4 days and the expression in the C-terminal mutant was slightly higher than for the full length protease.

  1. The Contribution of Proteinase-Activated Receptors to Intracellular Signaling, Transcellular Transport and Autophagy in Alzheimer´s Disease.

    Czech Academy of Sciences Publication Activity Database

    Mat?j, R.; Rohan, Z.; Holada, K.; Olejár, Tomáš

    2015-01-01

    Ro?. 12, ?. 1 (2015), s. 2-12. ISSN 1567-2050 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * autophagy * proteinase-activated receptors Subject RIV: EA - Cell Biology Impact factor: 3.889, year: 2014

  2. Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch.

    Czech Academy of Sciences Publication Activity Database

    Švec, Martin; Bauerová, Helena; Pichová, Iva; Konvalinka, Jan; St?íšovský, Kvido

    Praha : JPM, 2004 - (Hunter, E.; Ruml, T.; Pichová, I.; Rumlová, M.; Sakalian, M.). s. 75 ISBN 80-86313-13-1. [The Retrovirus Assembly Meeting. 02.10.2004-06.10.2004, Praha] Keywords : proteinases * betaretroviruses Subject RIV: CE - Biochemistry

  3. Secreted aspartic proteinases of .I.Candida parapsilosis./I..I.: activation and specificity of different Sap isoenzymes.

    Czech Academy of Sciences Publication Activity Database

    Dostál, Ji?í; Merkerová, Michaela; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    Oxford : The Biochemical Society, 2003. s. 9. [Harden Conference Proteinase Structure and Function /57./. 09.09.2003-13.09.2003, Oxford] Institutional research plan: CEZ:AV0Z4055905 Keywords : secreted aspartic proteases Subject RIV: CE - Biochemistry

  4. Purification, crystallization and preliminary crystallographic studies of a Kunitz-type proteinase inhibitor from tamarind (Tamarindus indica) seeds

    International Nuclear Information System (INIS)

    A 21 kDa Kunitz-type proteinase inhibitor was purified from tamarind (T. indica) seeds, crystallized and characterized by X-ray diffraction. A Kunitz-type proteinase inhibitor has been purified from tamarind (Tamarindus indica) seeds. SDS–PAGE analysis of a purified sample showed a homogeneous band corresponding to a molecular weight of 21 kDa. The protein was identified as a Kunitz-type proteinase inhibitor based on N-terminal amino-acid sequence analysis. It was crystallized by the vapour-diffusion method using PEG 6000. The crystals belonged to the orthorhombic space group C2221, with unit-cell parameters a = 37.2, b = 77.1, c = 129.1 Å. Diffraction data were collected to a resolution of 2.7 Å. Preliminary crystallographic analysis indicated the presence of one proteinase inhibitor molecule in the asymmetric unit, with a solvent content of 44%

  5. Candida albicans secreted aspartyl proteinases: isoenzyme pattern is determined by cell type, and levels are determined by environmental factors.

    OpenAIRE

    White, T C; Agabian, N

    1995-01-01

    For the pathogenic yeast Candida albicans, secreted aspartyl proteinase (Sap) activity has been correlated with virulence. A family consisting of at least eight SAP genes can be drawn upon to produce Sap enzymatic activity. In this study, the levels of Sap1, Sap2, and Sap3 isoenzymes were monitored under a variety of growth conditions for several strains, including strain WO-1, which alternates between two switch phenotypes, white (W) and opaque (O). When cultured under proteinase-inducing co...

  6. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    OpenAIRE

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Ji?í; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Pathogenic yeasts of the genus Candida produce secreted aspartic proteinases, which are known to enhance virulence. We focused on Sapp1p proteinase secreted by Candida parapsilosis and studied the final stage of its passage through the cell wall and release into the extracellular environment. We found that Sapp1p displays enzyme activity prior to secretion, and therefore, it is probably fully folded within the upper layer of the cell wall. The positioning of cell surface-associated Sapp1p was...

  7. Induction of protective immunity in cattle against infection with Fasciola hepatica by vaccination with cathepsin L proteinases and with hemoglobin.

    OpenAIRE

    Dalton, J. P.; McGonigle, S; Rolph, T P; Andrews, S. J.

    1996-01-01

    Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elic...

  8. The potency and specificity of the interaction between the IA3 inhibitor and its target aspartic proteinase from Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Phylip, L H; Lees, W E; Brownsey, B G; Bur, D; Dunn, B M; Winther, Jakob R.; Gustchina, A; Li, M; Copeland, T; Wlodawer, A; Kay, J

    2001-01-01

    The yeast IA3 polypeptide consists of only 68 residues, and the free inhibitor has little intrinsic secondary structure. IA3 showed subnanomolar potency toward its target, proteinase A from Saccharomyces cerevisiae, and did not inhibit any of a large number of aspartic proteinases with similar sequences/structures from a wide variety of other species. Systematic truncation and mutagenesis of the IA3 polypeptide revealed that the inhibitory activity is located in the N-terminal half of the sequen...

  9. Vaccination with Cathepsin L Proteinases and with Leucine Aminopeptidase Induces High Levels of Protection against Fascioliasis in Sheep

    OpenAIRE

    Piacenza, Lucía; Acosta, Daniel; Basmadjian, Isabel; Dalton, John P.; Carmona, Carlos

    1999-01-01

    The potential of different parasite proteinases for use as vaccine candidates against fascioliasis in sheep was studied by vaccinating animals with the cathepsin L proteinases CL1 and CL2 and with leucine aminopeptidase (LAP) purified from adult flukes. In the first trial, sheep were immunized with CL1 or CL2 and the mean protection levels obtained were 33 and 34%, respectively. Furthermore, a significant reduction in egg output was observed in sheep vaccinated either with CL1 (71%) or with C...

  10. Cathodic Behaviour of Cysteine at a Platinum Electrode

    Scientific Electronic Library Online (English)

    M. Fátima, Barroso; Teresa, Santos; M. Goreti F., Sales; Cristina, Delerue-Matos; M. Carmo V. F., Vaz.

    Full Text Available The electroreduction behaviour of cysteine was investigated using cyclic, square wave and differencial pulse voltammetric techniques at a platinum working electrode. The reduction of cysteine occurs at a potential of -0.36 V independent of pH. It is a reversible process, controlled mainly by diffusi [...] on and in the mechanism of reduction 1 electron per molecule is involved. Using the voltammetric techniques: Cyclic Voltammetry, Square Wave Voltammetry and Differencial Pulse Voltammetry, different parameters (pH, frequency, step potential, pulse amplitude, scan rate) were optimized in order to develop an electrochemical procedure for determination of cysteine in pharmaceutical products. The repeatability, reproducibility, precision and accuracy of the methods were studied. No electroactive interferences from the excipient were found in the pharmaceutical compounds.

  11. Cathodic Behaviour of Cysteine at a Platinum Electrode

    Directory of Open Access Journals (Sweden)

    M. Fátima Barroso

    2007-01-01

    Full Text Available The electroreduction behaviour of cysteine was investigated using cyclic, square wave and differencial pulse voltammetric techniques at a platinum working electrode. The reduction of cysteine occurs at a potential of -0.36 V independent of pH. It is a reversible process, controlled mainly by diffusion and in the mechanism of reduction 1 electron per molecule is involved. Using the voltammetric techniques: Cyclic Voltammetry, Square Wave Voltammetry and Differencial Pulse Voltammetry, different parameters (pH, frequency, step potential, pulse amplitude, scan rate were optimized in order to develop an electrochemical procedure for determination of cysteine in pharmaceutical products. The repeatability, reproducibility, precision and accuracy of the methods were studied. No electroactive interferences from the excipient were found in the pharmaceutical compounds.

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

    Science.gov (United States)

    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. PMID:26028748

  13. pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro

    DEFF Research Database (Denmark)

    SØrensen, S O; van den Hazel, H B

    1994-01-01

    Carboxypeptidase Y is a vacuolar enzyme from Saccharomyces cerevisiae. It enters the vacuole as a zymogen, procarboxypeptidase Y, which is immediately processed in a reaction involving two endoproteases, proteinase A and proteinase B. We have investigated the in vitro activation of purified procarboxypeptidase Y by purified proteinase A. This has identified two different processing intermediates; one active and one inactive. The intermediates define a 33 amino acid segment of the 91 amino acid propeptide as sufficient for maintaining the enzyme in an inactive state. The inactive intermediate was isolated from a processing reaction at neutral pH. In order to investigate the influence of vacuolar pH on processing in vivo, the autoactivation of proteinase A and its processing of procarboxypeptidase Y were studied in a vma2 prb1 mutant, which is deficient in vacuolar acidification and proteinase B activity. Efficient processing of procarboxypeptidase Y in the absence of proteinase B is dependent on acidic vacuolar pH, and the processing at neutral pH is slow and takes place in two steps similar to those identified in vitro.

  14. Repair of DNA methylphosphotriesters through a metalloactivated cysteine nucleophile.

    Science.gov (United States)

    Myers, L C; Terranova, M P; Ferentz, A E; Wagner, G; Verdine, G L

    1993-08-27

    The Escherichia coli Ada protein repairs methylphosphotriesters in DNA by direct, irreversible methyl transfer to one of its own cysteines. Upon methyl transfer, Ada acquires the ability to bind specific DNA sequences and thereby to induce genes that confer resistance to methylating agents. The amino-terminal domain of Ada, which comprises the methylphosphotriester repair and sequence-specific DNA binding elements, contains a tightly bound zinc ion. Analysis of the zinc binding site by cadmium-113 nuclear magnetic resonance and site-directed mutagenesis revealed that zinc participates in the autocatalytic activation of the active site cysteine and may also function as a conformational switch. PMID:8395079

  15. Human seminal proteinase and prostate-specific antigen are the same protein

    Indian Academy of Sciences (India)

    Abdul Waheed; Md Imtaiyaz Hassan; Robert L Van Etten; Faizan Ahmad

    2008-06-01

    Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and compared. Both are glycoproteins of 32–34 kDa with protease activities. Based on some physicochemical, enzymatic and immunological properties, it is concluded that these proteins are in fact identical. The protein exhibits properties similar to kallikrein-like serine protease, trypsin, chymotrypsin and thiol acid protease. Tests of the activity of the enzyme against some potential natural and synthetic substrates showed that bovine serum albumin was more readily hydrolysed than casein. The results of this study should be useful in purifying and assaying this protein. Based on published studies and the present results, the broad proteolytic specificity of human seminal proteinase suggests a role for this protein in several physiological functions.

  16. Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases

    International Nuclear Information System (INIS)

    Human cathepsin G is a serine proteinase with chymotrypsin-like specificity found in both polymorphonuclear leukocytes (neutrophils) and the U937 leukemic cell line. Utilizing RNA from the latter, the authors have constructed a cDNA library in ?gt11 and isolated a clone which apparently codes for the complete amino acid sequence of this enzyme. Analysis of the sequence reveals homology with rat mast cell proteinase II (47%) but a greater degree of identity (56%) with a product of activated mouse cytotoxic T lymphocytes. The close relationship between the three proteins indicates similarities in substrate specificity and in biosynthesis which they predict involves removal of a two amino acid activation peptide during or just before packaging to their respective storage granules

  17. Enhanced Response of a Proteinase K-Based Conductometric Biosensor Using Nanoparticles

    Science.gov (United States)

    Nouira, Wided; Maaref, Abderrazak; Elaissari, Abdelhamid; Vocanson, Francis; Siadat, Maryam; Jaffrezic-Renault, Nicole

    2014-01-01

    Proteinases are involved in a multitude of important physiological processes, such as protein metabolism. For this reason, a conductometric enzyme biosensor based on proteinase K was developed using two types of nanoparticles (gold and magnetic). The enzyme was directly adsorbed on negatively charged nanoparticles and then deposited and cross-linked on a planar interdigitated electrode (IDE). The biosensor was characterized with bovine serum albumin (BSA) as a standard protein. Higher sensitivity was obtained using gold nanoparticles. The linear range for BSA determination was then from 0.5 to 10 mg/L with a maximum response of 154 ?s. These results are greater than that found without any nanoparticles (maximum response of 10 ?s). The limit of detection (LOD) was 0.3 mg/L. An inter-sensor reproducibility of 3.5% was obtained. PMID:25057139

  18. Effect of cooking on proteinase inhibitors of Dolichos lablab bean (Dolichos lablab perpureus L.).

    Science.gov (United States)

    Devaraj, V R; Manjunath, N H

    1995-09-01

    Proteinase inhibitory activity in ten different varieties of Dolichos lablab perpureus. L. was determined. All the varieties tested exhibited appreciable level of proteinase inhibitory activity (PIA). The trypsin inhibitory activity (TIA) (Mean: 20170 TIU/g) was relatively higher than the chymotrypsin inhibitory activity (CIA) (Mean: 15380 CIU/g). Effect of temperature and cooking on PIA was studied. The nature of cooking medium and duration of cooking had profound effect on the PIA. The dry fried seeds lost their PIA very rapidly (91% in 20 min). Seeds cooked in slightly alkaline medium lost their PIA quickly (89% in 30 min) compared to those cooked in acidic (80% in 30 min) and neutral pH (83% in 30 min). The PIA in green pods was also determined and they had only one third of the PIA (8200 TIU/g and 8125 CIU/g) found in the dry seeds. PMID:8837868

  19. Structure of an inhibitor complex of the proteinase from feline immunodeficiency virus.

    Science.gov (United States)

    Wlodawer, A; Gustchina, A; Reshetnikova, L; Lubkowski, J; Zdanov, A; Hui, K Y; Angleton, E L; Farmerie, W G; Goodenow, M M; Bhatt, D

    1995-06-01

    The crystal structure of a recombinant form of the proteinase encoded by the feline immunodeficiency virus (FIV PR) has been solved at 2 A resolution and refined to an R-factor of 0.148. The refined structure includes a peptidomimetic, statine-based inhibitor, LP-149, which is an even more potent inhibitor of HIV PR. Kinetic parameters were obtained for the cleavage of five substrates by FIV PR, and inhibition constants were measured for four inhibitors. The structure of FIV PR resembles other related retroviral enzymes although few inhibitors of HIV PR are capable of inhibiting FIV PR. The structure of FIV PR will enhance our knowledge of this class of enzymes, and will direct testing of new proteinase inhibitors in a feline animal model. PMID:7664111

  20. Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.

    OpenAIRE

    Koloczek, H.; Banbula, A.; Salvesen, G. S.; Potempa, J.

    1996-01-01

    Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluore...

  1. In vitro differential activity of phospholipases and acid proteinases of clinical isolates of Candida

    Directory of Open Access Journals (Sweden)

    Aurean D'Eça Júnior

    2011-06-01

    Full Text Available INTRODUCTION: Candida yeasts are commensals; however, if the balance of normal flora is disrupted or the immune defenses are compromised, Candida species can cause disease manifestations. Several attributes contribute to the virulence and pathogenicity of Candida, including the production of extracellular hydrolytic enzymes, particularly phospholipase and proteinase. This study aimed to investigate the in vitro activity of phospholipases and acid proteinases in clinical isolates of Candida spp. METHODS: Eighty-two isolates from hospitalized patients collected from various sites of origin were analyzed. Phospholipase production was performed in egg yolk medium and the production of proteinase was verified in a medium containing bovine serum albumin. The study was performed in triplicate. RESULTS: Fifty-six (68.3% of isolates tested were phospholipase positive and 16 (44.4% were positive for proteinase activity. C. tropicalis was the species with the highest number of positive isolates for phospholipase (91.7%. Statistically significant differences were observed in relation to production of phospholipases among species (p<0,0001 and among the strains from different sites of origin (p=0.014. Regarding the production of acid protease, the isolates of C. parapsilosis tested presented a larger number of producers (69.2%. Among the species analyzed, the percentage of protease producing isolates did not differ statistically (?2=1.9 p=0.5901 (?2=1.9 p=0.5901. CONCLUSIONS: The majority of C. non-albicans and all C. albicans isolates were great producers of hydrolytic enzymes and, consequently, might be able to cause infection under favorable conditions.

  2. A Single Step Purification of Gastricsin-Like Proteinase from Atlantic Cod (Gadus morhua)

    OpenAIRE

    M. A. Amiza; R.K. Owusu Apenten

    2002-01-01

    A gastricsin-like proteinase was purified from gastric mucosa of Atlantic cod by a single step purification scheme on ion-exchange of Amberlite CG-50. The purification was very efficient as the recovery was 205% and the purification factor was 1796-fold. The enzyme preparation was homogeneous as observed by SDS-PAGE and isoelectric focusing. The enzyme has an estimated molecular weight of 34 kDa and the pI of 4.4.

  3. A Single Step Purification of Gastricsin-Like Proteinase from Atlantic Cod (Gadus morhua

    Directory of Open Access Journals (Sweden)

    M. A. Amiza

    2002-01-01

    Full Text Available A gastricsin-like proteinase was purified from gastric mucosa of Atlantic cod by a single step purification scheme on ion-exchange of Amberlite CG-50. The purification was very efficient as the recovery was 205% and the purification factor was 1796-fold. The enzyme preparation was homogeneous as observed by SDS-PAGE and isoelectric focusing. The enzyme has an estimated molecular weight of 34 kDa and the pI of 4.4.

  4. Effect of acute ozone exposure on the proteinase-antiproteinase balance in the rat lung

    International Nuclear Information System (INIS)

    Lung disease may result from a persisting proteinase excess or a depletion of antiproteinase in pulmonary parenchyma. We investigated the in vivo effect of a 48-hr exposure to ozone at 0.5, 1.0, or 1.5 ppm on proteinase and antiproteinase activity of rat lungs. Elastase inhibitory capacities of serum, lung tissue, and airway washings were measured as indicators of antielastase activity. Trypsin inhibitory capacity was measured using an esterolytic procedure. Proteinase was measured as radioactive release from a 14C-globin substrate. The 48-hr exposures to O3 at levels up to 1 ppm produced concentration-dependent decreases of 35-80% of antiproteinase activities in serum and in lung tissue. However, exposure to 1.5 ppm O3 resulted in no decrease in antiproteinase activities. Acid proteinase activities (pH 4.2) were increased 65-120% by exposure to 1 or 1.5 ppm O3, which correlated with inflammatory cells noted histologically. At 1.5 ppm O3, pulmonary edema and hemorrhage were noted in histologic sections. These changes led to a flooding of the alveoli with up to 40 times normal protein levels and a greater than fivefold increase in airway antiproteinase. These data suggest that serum and soluble lung tissue antiproteinase activity decreased upon exposure to low levels of ozone. However, if O3 exposure is high enough to produce pulmonary hemorrhage, antiproteinase may increase following serum exudation. These changes may be important in the development of ozone-induced lung diseases, especially emphysema

  5. Ixodes scapularis tick serine proteinase inhibitor (serpin) gene family; annotation and transcriptional analysis

    OpenAIRE

    Chalaire Katelyn C; Khumthong Rabuesak; Mulenga Albert

    2009-01-01

    Abstract Background Serine proteinase inhibitors (Serpins) are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. Results A sequence homology search strategy has allowed...

  6. SARS CoV Main Proteinase: The Monomer-Dimer Equilibrium Dissociation Constant

    Energy Technology Data Exchange (ETDEWEB)

    Graziano,V.; McGrath, W.; Yang, L.; Mangel, W.

    2006-01-01

    The SARS coronavirus main proteinase (SARS CoV main proteinase) is required for the replication of the severe acute respiratory syndrome coronavirus (SARS CoV), the virus that causes SARS. One function of the enzyme is to process viral polyproteins. The active form of the SARS CoV main proteinase is a homodimer. In the literature, estimates of the monomer-dimer equilibrium dissociation constant, K{sub D}, have varied more than 650000-fold, from <1 nM to more than 200 {mu}M. Because of these discrepancies and because compounds that interfere with activation of the enzyme by dimerization may be potential antiviral agents, we investigated the monomer-dimer equilibrium by three different techniques: small-angle X-ray scattering, chemical cross-linking, and enzyme kinetics. Analysis of small-angle X-ray scattering data from a series of measurements at different SARS CoV main proteinase concentrations yielded K{sub D} values of 5.8 {+-} 0.8 {mu}M (obtained from the entire scattering curve), 6.5 {+-} 2.2 {mu}M (obtained from the radii of gyration), and 6.8 {+-} 1.5 {mu}M (obtained from the forward scattering). The K{sub D} from chemical cross-linking was 12.7 {+-} 1.1 {mu}M, and from enzyme kinetics, it was 5.2 {+-} 0.4 {mu}M. While each of these three techniques can present different, potential limitations, they all yielded similar K{sub D} values.

  7. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole; Brunak, Søren; Blom, Nikolaj

    2004-01-01

    Background: Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS), efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested u...

  8. The proteinase activated receptor-2 (PAR-2) mediates mitogenic responses in human vascular endothelial cells.

    OpenAIRE

    Mirza, H; Yatsula, V; Bahou, W F

    1996-01-01

    Proteolytically cleaved receptors, typified by the functional thrombin receptor (TR), represent a novel class of receptors that mediate signaling events by functional coupling to G proteins. Northern blot analysis completed with a human proteinase activated receptor-2 (PAR-2) cDNA as probe demonstrated the approximately 3.5kb PAR-2 transcript in total cellular RNA from human umbilical vein endothelial cells (HUVEC). Microspectrofluorimetry using Fura2-loaded HUVEC demonstrated a dose-dependen...

  9. Elevated aspartic proteinase secretion and experimental pathogenicity of Candida albicans isolates from oral cavities of subjects infected with human immunodeficiency virus.

    OpenAIRE

    De Bernardis, F.; Chiani, P; Ciccozzi, M; PELLEGRINI, G; Ceddia, T; D'Offizzi, G; Quinti, I; Sullivan, P A; Cassone, A

    1996-01-01

    Isolates of Candida albicans from the oral cavities of subjects at different stages of human immunodeficiency virus (HIV) infection or uninfected controls were examined for (i) production of aspartic proteinase(s), a putative virulence-associated factor(s); (ii) the presence in the fungal genome of two major genes (SAP1 and SAP2) of the aspartic proteinase family; and (iii) experimental pathogenicity in a murine model of systemic infection. It was found that the fungal isolates from symptomat...

  10. Serpins in plants and green algae

    DEFF Research Database (Denmark)

    Roberts, Thomas Hugh; Hejgaard, Jørn

    2008-01-01

    Control of proteolysis is important for plant growth, development, responses to stress, and defence against insects and pathogens. Members of the serpin protein family are likely to play a critical role in this control through irreversible inhibition of endogenous and exogenous target proteinases. Serpins have been found in diverse species of the plant kingdom and represent a distinct clade among serpins in multicellular organisms. Serpins are also found in green algae, but the evolutionary rela...

  11. A Family of Bacterial Cysteine Protease Type III Effectors Utilizes Acylation-dependent and -independent Strategies to Localize to Plasma Membranes*

    OpenAIRE

    Dowen, Robert H.; Engel, James L.; Shao, Feng; Ecker, Joseph R; Dixon, Jack E.

    2009-01-01

    Bacterial phytopathogens employ a type III secretion system to deliver effector proteins into the plant cell to suppress defense pathways; however, the molecular mechanisms and subcellular localization strategies that drive effector function largely remain a mystery. Here, we demonstrate that the plant plasma membrane is the primary site for subcellular localization of the Pseudomonas syringae effector AvrPphB and five additional cysteine protease family members. AvrPphB and two AvrPphB-like ...

  12. Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation

    Directory of Open Access Journals (Sweden)

    Meng Guoliang

    2006-12-01

    Full Text Available Abstract Background We have recently reported the expression of murine Implantation Serine Proteinase genes in pre-implantation embryos (ISP1 and uterus (ISP1 and ISP2. These proteinases belong to the S1 proteinase family and are similar to mast cell tryptases, which function as multimers. Results Here, we report the purification and initial characterization of ISP1 and 2 with respect to their physico-chemical properties and physiological function. In addition to being co-expressed in uterus, we show that ISP1 and ISP2 are also co-expressed in the pre-implantation embryo. Together, they form a heterodimer with an approximate molecular weight of 63 kD. This complex is the active form of the enzyme, which we have further characterized as being trypsin-like, based on substrate and inhibitor specificities. In addition to having a role in embryo hatching and outgrowth, we demonstrate that ISP enzyme is localized to the site of embryo invasion during implantation and that its activity is important for successful implantation in vivo. Conclusion On the basis of similarities in structural, chemical, and functional properties, we suggest that this ISP enzyme complex represents the classical hatching enzyme, strypsin. Our results demonstrate a critical role for ISP in embryo hatching and implantation.

  13. Trichoderma harzianum transformant has high extracellular alkaline proteinase expression during specific mycoparasitic interactions

    Scientific Electronic Library Online (English)

    Maria Helena S., Goldman; Gustavo H., Goldman.

    1998-09-01

    Full Text Available O micoparasita Trichoderma harzianum produz uma protease alcalina que pode estar especificamente envolvida em micoparasitismo. Foram construídas linhagens transgênicas deste fungo que super-expressam esta protease alcalina. A atividade de protease alcalina foi verificada em alguns destes transforman [...] tes e aqueles com maior atividade do que o tipo selvagem foram selecionados para estudos posteriores. Uma destas linhagens produziu um nível elevado e constitutivo de mRNA do gene que codifica a protease alcalina, prb1, durante interações micoparasíticas com o fitopatógeno Rhizoctonia solani. Abstract in english The mycoparasite Trichoderma harzianum produces an alkaline proteinase that may be specifically involved in mycoparasitism. We have constructed transformant strains of this fungus that overexpress this alkaline proteinase. Some of the transformants were assessed for alkaline proteinase activity, and [...] those with higher activity than the wild type were selected for further studies. One of these transformant strains produced an elevated and constitutive pbr1 mRNA level during mycoparasitic interactions with Rhizoctonia solani.

  14. Enabling olefin metathesis on proteins: chemical methods for installation of S-allyl cysteine.

    OpenAIRE

    Chalker, JM; LIN, Ya; Boutureira, O; Davis, BG

    2009-01-01

    Multiple, complementary methods are reported for the chemical conversion of cysteine to S-allyl cysteine on protein surfaces, a useful transformation for the exploration of olefin metathesis on proteins.

  15. The H1 histone-specific proteinase is associated with nuclear matrix and stimulated by DNA containing breaks of denatured sites

    International Nuclear Information System (INIS)

    Discovery of proteinase in nuclear matrix specific of H1 histone and dependent presence of breaks or denatured sites in DNA permits to assume that the given enzyme, obviously, participates in replication and DNA repair, in regulation of genes expression. Removal of H1 histone by proteinase is, probably, necessary for procedure of these processes, and, obviously, this proteinase suffers conformational changes in the composition of the DNA-histone complex. H1 histone disintegration in nucleohistone containing damaged sites of DNA by specific proteinase, probably, represents one of the mechanisms for providing DNA repair in cells of higher organisms

  16. L-cysteine, PIP3 and Insulin Signaling in Diabetes | Division of Cancer Prevention

    Science.gov (United States)

    Diabetes has become an epidemic and remains a major public health issue worldwide. The primary purpose of this application is to discover the mechanisms by which L-cysteine (LC) supplementation improves glucose homeostasis in diabetic patients. Diabetic patients have lower blood levels of L-cysteine (LC), hydrogen sulfide (H2S), and glutathione (GSH). Supplementation with cysteine-rich proteins (whey protein and ?-lactoalbumin), LC, or N-acetyl cysteine (NAC) has been shown to lower glycemia in diabetic animal studies.

  17. Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach; Brinch-Pedersen, Henrik

    2011-01-01

    During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient euka...

  18. Oxidative stress increases SNAT1 expression and stimulates cysteine uptake in freshly isolated rat cardiomyocytes

    OpenAIRE

    King, Nicola; LIN, HUA; Suleiman, M.-Saadeh

    2010-01-01

    Intracellular cysteine availability is an important rate-limiting factor governing glutathione synthesis in the heart. This is also dependent on the magnitude and rate of cysteine uptake into cardiomyocytes, which has been little studied. This study investigated the hypothesis that changes to cysteine transporter expression and activity during oxidative stress influence cardiomyocyte glutathione levels. The uptake of 0–3 mM l-[35S]cysteine into ventricular cardiomyocytes isolated from adult m...

  19. Cysteine protease inhibitors isolated from human malignant melanoma tissue.

    Science.gov (United States)

    Tsushima, H; Sumi, H; Hamanaka, K; Toki, N; Sato, H; Mihara, H

    1985-12-01

    Cysteine protease inhibitors that specifically reacted with several cysteine proteases were found in KSCN extract of human melanoma tissue. From 30 gm of the tissue, approximately 593.5 U inhibitor was obtained. The inhibitors were adsorbed on a papain-Sepharose column and could be eluted with 10 mmol/L phosphate buffer, pH 6.0, containing NaCl or KCl, or with 20 mmol/L acetate buffer, pH 4.0, containing KSCN. They revealed a strong inhibitory activity for cysteine proteases such as ficin, papain, and cathepsin B, but did not react with cysteine protease bromelain or serine protease trypsin. No immunologic relationship was confirmed between the inhibitor and other well-known plasma inhibitors such as alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, antithrombin III, C1-in-activator, and alpha 2-plasmin inhibitor. With Sephadex G-100, two main peaks of molecular weight 40,000 and 10,000 were detected in the KSCN extract of the human melanoma tissue. However, the inhibitors revealed three molecular weights of 10,000, 25,000, and 80,000 when estimated by Sephadex G-100 gel filtration after papain-Sepharose affinity chromatography. On the other hand, the molecular weights of the inhibitors changed to two peaks of 25,000 and 10,000 on rechromatography with a papain-Sepharose column. PMID:3934299

  20. Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes.

    Czech Academy of Sciences Publication Activity Database

    Kašperová, A.; Kunert, J.; Horynová, M.; Weigl, E.; Sebela, M.; Lenobel, René; Raška, M.

    2011-01-01

    Ro?. 54, ?. 5 (2011), E456-E462. ISSN 0933-7407 R&D Projects: GA ?R GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cysteine dioxygenase * dermatophytes * recombinant protein * keratinolytic fungi * cDNA Subject RIV: CE - Biochemistry Impact factor: 2.247, year: 2011

  1. Cysteine Enhances Bioavailability of Copper to Marine Phytoplankton.

    Science.gov (United States)

    Walsh, Michael J; Goodnow, Sarah D; Vezeau, Grace E; Richter, Lubna V; Ahner, Beth A

    2015-10-20

    Emiliania huxleyi, a ubiquitous marine algae, was cultured under replete and Cu-limiting conditions to investigate Cu uptake strategies involving thiols and associated redox reactions; comparisons to a model diatom, Thalassiosira pseudonana, were also drawn. Cu-limitation increased rates of cell surface reduction of Cu(II) to Cu(I) in E. huxleyi but not in T. pseudonana. Furthermore, Cu-limited E. huxleyi cells took up more Cu when cysteine was present compared to when no ligand was added, although a dependence on cysteine concentration was not observed. In contrast, Cu uptake by replete cells was dependent upon the relative abundance of inorganic species [Cu(I)']. We also show that cysteine can increase the bioavailability of Cu to Cu-limited cells, of both species, through the reductive release of Cu(I) from fairly strong Cu(II) ligands such as EDTA. Finally, support for a mechanism involving uptake of a Cys-Cu complex in E. huxleyi is drawn from the observation that Cu-limitation significantly enhances cysteine uptake by transporters that exhibit Michaelis-Menten kinetics. These Cu uptake strategies help explain the presence and distribution of dissolved thiols in surface seawater and have implications for the biogeochemical cycling of Cu in low Cu environments. PMID:26420592

  2. Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

    OpenAIRE

    Himelbloom, B H; Hassan, H. M. [??? ????? ???

    1986-01-01

    Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

  3. Phospholipase and Aspartyl Proteinase Activities of Candida Species Causing Vulvovaginal Candidiasis in Patients with Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Bassyouni, Rasha H; Wegdan, Ahmed Ashraf; Abdelmoneim, Abdelsamie; Said, Wessam; AboElnaga, Fatma

    2015-10-28

    Few research had investigated the secretion of phospholipase and aspartyl proteinase from Candida spp. causing infection in females with type 2 diabetes mellitus. This research aimed to investigate the prevalence of vulvovaginal candidiasis (VVC) in diabetic versus non-diabetic women and compare the ability of identified Candida isolates to secrete phospholipases and aspartyl proteinases with characterization of their genetic profile. The study included 80 females with type 2 diabetes mellitus and 100 non-diabetic females within the child-bearing period. Candida strains were isolated and identified by conventional microbiological methods and by API Candida. The isolates were screened for their extracellular phospholipase and proteinase activities by culturing them on egg yolk and bovine serum albumin media, respectively. Detection of aspartyl proteinase genes (SAP1 to SAP8) and phospholipase genes (PLB1, PLB2) were performed by multiplex polymerase chain reaction. Our results indicated that vaginal candidiasis was significantly higher among the diabetic group versus nondiabetic group (50% versus 20%, respectively) (p = 0.004). C. albicans was the most prevalent species followed by C. glabrata in both groups. No significant association between diabetes mellitus and phospholipase activities was detected (p = 0.262), whereas high significant proteinase activities exhibited by Candida isolated from diabetic females were found (82.5%) (p = 0.000). Non-significant associations between any of the tested proteinase or phospholipase genes and diabetes mellitus were detected (p > 0.05). In conclusion, it is noticed that the incidence of C. glabrata causing VVC is increased. The higher prevalence of vaginal candidiasis among diabetics could be related to the increased aspartyl proteinase production in this group of patients. PMID:26032358

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

    Science.gov (United States)

    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 ?(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 ?(2) -O,O-binding mode for synthetic as well as the natural enzyme. PMID:25823421

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

    International Nuclear Information System (INIS)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2003-01-15

    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. Plant protease inhibitors in control of phytophagous insects

    Scientific Electronic Library Online (English)

    Paulraj K., Lawrence; Kripa Ram, Koundal.

    2002-04-15

    Full Text Available Plant proteinase inhibitors (PIs) have been well established to play a potent defensive role against predators and pathogens. Although diverse endogenous functions for these proteins has been proposed, ranging from regulators of endogenous proteinases to act as storage proteins, evidence for many of [...] these roles is partial, or confined to isolated examples. On the other hand, many PIs have been shown to act as defensive compounds against pests by direct assay or by expression in transgenic crop plants, and a body of evidence for their role in plant defense has been accumulated consistently. The role and mechanism of action for most of these inhibitors are being studied in detail and their respective genes isolated. These genes have been used for the construction of transgenic crop plants to be incorporated in integrated pest management programmes. This article describes the classes of protease inhibitors, their regulation and genes used to construct transgenic plants against phytophagous insects.

  8. Additive In Vitro Antiplasmodial Effect of N-Alkyl and N-Benzyl-1,10-Phenanthroline Derivatives and Cysteine Protease Inhibitor E64

    Science.gov (United States)

    Wijayanti, Mahardika Agus; Sholikhah, Eti Nurwening; Hadanu, Ruslin; Jumina, Jumina; Supargiyono, Supargiyono; Mustofa, Mustofa

    2010-01-01

    Potential new targets for antimalarial chemotherapy include parasite proteases, which are required for several cellular functions during the Plasmodium falciparum life cycle. Four new derivatives of N-alkyl and N-benzyl-1,10-phenanthroline have been synthesized. Those are (1)-N-methyl-1,10-phenanthrolinium sulfate, (1)-N-ethyl-1,10-phenanthrolinium sulfate, (1)-N-benzyl-1,10-phenanthrolinium chloride, and (1)-N-benzyl-1,10-phenanthrolinium iodide. Those compounds had potential antiplasmodial activity with IC50 values from 260.42 to 465.38 nM. Cysteine proteinase inhibitor E64 was used to investigate the mechanism of action of N-alkyl and N-benzyl-1,10-phenanthroline derivatives. A modified fixed-ratio isobologram method was used to study the in vitro interactions between the new compounds with either E64 or chloroquine. The interaction between N-alkyl and N-benzyl-1,10-phenanthroline derivatives and E64 was additive as well as their interactions with chloroquine were also additive. Antimalarial mechanism of chloroquine is mainly on the inhibition of hemozoin formation. As the interaction of chloroquine and E64 was additive, the results indicated that these new compounds had a mechanism of action by inhibiting Plasmodium proteases. PMID:22332022

  9. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and without the 5 amino acid C-terminal sequence into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain SDM1163. Heterologous protein production was induced with 2% MeOH. To monitor the protein expression during induction, 1 ml samples was collected every hr for 24 hrs. After 4 days, the supernatant were harvested and analyzed by SDS-PAGE, activity assay and Western blot. A significant amount of heterologous protein was produced and the protein production was highest after 4 days and the expression in the C-terminal mutant was slightly higher than for the full length protease.

  10. Investigation of Serine-Proteinase-Catalyzed Peptide Splicing in Analogues of Sunflower Trypsin Inhibitor?1 (SFTI-1).

    Science.gov (United States)

    Karna, Natalia; ??gowska, Anna; Malicki, Stanis?aw; D?bowski, Dawid; Golik, Przemys?aw; Gitlin, Agata; Grudnik, Przemys?aw; Wladyka, Benedykt; Brzozowski, Krzysztof; Dubin, Grzegorz; Rolka, Krzysztof

    2015-09-01

    Serine-proteinase-catalyzed peptide splicing was demonstrated in analogues of the trypsin inhibitor SFTI-1: both single peptides and two-peptide chains (C- and N-terminal peptide chains linked by a disulfide bridge). In the second series, peptide splicing with catalytic amount of proteinase was observed only when formation of acyl-enzyme intermediate was preceded by hydrolysis of the substrate Lys-Ser peptide bond. Here we demonstrate that with an equimolar amount of the proteinase, splicing occurs in all the two-peptide-chain analogues. This conclusion was supported by high resolution crystal structures of selected analogues in complex with trypsin. We showed that the process followed a direct transpeptidation mechanism. Thus, the acyl-enzyme intermediate was formed and was immediately used for a new peptide bond formation; products associated with the hydrolysis of the acyl-enzyme were not observed. The peptide splicing was sequence- not structure-specific. PMID:26212347

  11. ROSICS: CHEMISTRY AND PROTEOMICS OF CYSTEINE MODIFICATIONS IN REDOX BIOLOGY

    OpenAIRE

    Kim, Hee-Jung; Ha, Sura; Lee, Hee Yoon; Lee, Kong-Joo

    2014-01-01

    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 ...

  12. Subcellular distribution of glutathione and cysteine in cyanobacteria

    OpenAIRE

    Zechmann, Bernd; Tomaši?, Ana; Horvat, Lucija; Fulgosi, Hrvoje

    2010-01-01

    Glutathione plays numerous important functions in eukaryotic and prokaryotic cells. Whereas it can be found in virtually all eukaryotic cells, its production in prokaryotes is restricted to cyanobacteria and proteobacteria and a few strains of gram-positive bacteria. In bacteria, it is involved in the protection against reactive oxygen species (ROS), osmotic shock, acidic conditions, toxic chemicals, and heavy metals. Glutathione synthesis in bacteria takes place in two steps out of cysteine,...

  13. Thiazolidine prodrugs of cysteamine and cysteine as radioprotective agents

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, J.C.; Koch, K.E.; Detrick, S.R. [Univ. of Utah, Salt Lake City, UT (United States)] [and others

    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.

  14. Viral proteinase requirements for the nucleocytoplasmic relocalization of cellular splicing factor SRp20 during picornavirus infections.

    Science.gov (United States)

    Fitzgerald, Kerry D; Chase, Amanda J; Cathcart, Andrea L; Tran, Genevieve P; Semler, Bert L

    2013-03-01

    Infection of mammalian cells by picornaviruses results in the nucleocytoplasmic redistribution of certain host cell proteins. These viruses interfere with import-export pathways, allowing for the cytoplasmic accumulation of nuclear proteins that are then available to function in viral processes. We recently described the cytoplasmic relocalization of cellular splicing factor SRp20 during poliovirus infection. SRp20 is an important internal ribosome entry site (IRES) trans-acting factor (ITAF) for poliovirus IRES-mediated translation; however, it is not known whether other picornaviruses utilize SRp20 as an ITAF and direct its cytoplasmic relocalization. Also, the mechanism by which poliovirus directs the accumulation of SRp20 in the cytoplasm of the infected cell is currently unknown. Work described in this report demonstrated that infection by another picornavirus (coxsackievirus B3) causes SRp20 to relocalize from the nucleus to the cytoplasm of HeLa cells, similar to poliovirus infection; however, SRp20 is relocalized to a somewhat lesser extent in the cytoplasm of HeLa cells during infection by yet another picornavirus (human rhinovirus 16). We show that expression of poliovirus 2A proteinase is sufficient to cause the nucleocytoplasmic redistribution of SRp20. Following expression of poliovirus 2A proteinase in HeLa cells, we detect cleavage of specific nuclear pore proteins known to be cleaved during poliovirus infection. We also find that expression of human rhinovirus 16 2A proteinase alone can cause efficient cytoplasmic relocalization of SRp20, despite the lower levels of SRp20 relocalization observed during rhinovirus infection compared to poliovirus. Taken together, these results further define the mechanism of SRp20 cellular redistribution during picornavirus infections, and they provide additional insight into some of the differences observed between human rhinovirus and other enterovirus infections. PMID:23255796

  15. Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism.

    Science.gov (United States)

    Coates, L; Erskine, P T; Crump, M P; Wood, S P; Cooper, J B

    2002-05-17

    Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered. PMID:12083527

  16. The effects of a plant proteinase inhibitor from Enterolobium contortisiliquum on human tumor cell lines

    OpenAIRE

    Nakahata, Adriana Miti; Mayer, Barbara; Ries, Christian; Andrade de Paula, Claudia Alessandra; Karow, Marisa; Neth, Peter; Sampaio, Misako U.; Jochum, Marianne; Oliva, Maria Luiza V.

    2011-01-01

    Supplementary to the efficient inhibition of trypsin, chymotrypsin, plasma kallikrein, and plasmin already described by the EcTI inhibitor from Enterolobium contortisiliquum, it also blocks human neutrophil elastase (K(iapp)=4.3 nM) and prevents phorbol ester (PMA)-stimulated activation of matrix metalloproteinase (MMP)-2 probably via interference with membrane-type 1 (MT1)-MMP. Moreover, plasminogen-induced activation of proMMP-9 and processing of active MMP-2 was also inhibited. Furthermore...

  17. Consequences of manganese replacement of copper for prion protein function and proteinase resistance

    OpenAIRE

    Brown, David R.; Hafiz, Farida; Glasssmith, Leslie L.; Wong, Boon-Seng; Jones, Ian M; Clive, Christine; Haswell, Stephen J

    2000-01-01

    The prion protein (PrP) binds copper and has antioxidant activity enhancing the survival of neurones in culture. The ability of the PrP to bind other cations was tested and it was found that only manganese could substitute for copper. Although initially manganese-loaded PrP exhibited similar structure and activity to copper-loaded PrP, after aging, manganese-loaded PrP became proteinase resistant and lost function. It was also found that manganese could be incorporated into PrP expressed by a...

  18. Secretory Aspartyl Proteinases Cause Vaginitis and Can Mediate Vaginitis Caused by Candida albicans in Mice

    OpenAIRE

    Pericolini, Eva; Gabrielli, Elena; Amacker, Mario; Kasper, Lydia; Roselletti, Elena; Luciano, Eugenio; Sabbatini, Samuele; Kaeser, Matthias; Moser, Christian; Hube, Bernhard; Vecchiarelli, Anna; Cassone, Antonio

    2015-01-01

    Vaginal inflammation (vaginitis) is the most common disease caused by the human-pathogenic fungus Candida albicans. Secretory aspartyl proteinases (Sap) are major virulence traits of C. albicans that have been suggested to play a role in vaginitis. To dissect the mechanisms by which Sap play this role, Sap2, a dominantly expressed member of the Sap family and a putative constituent of an anti-Candida vaccine, was used. Injection of full-length Sap2 into the mouse vagina caused local neutrophi...

  19. Characterization of HLA-DR-restricted T-cell epitopes derived from human proteinase 3

    DEFF Research Database (Denmark)

    Piesche, Matthias; Hildebrandt, York; Chapuy, Björn; Wulf, Gerald G; Trümper, Lorenz; Schroers, Roland

    2009-01-01

    Human proteinase 3 (PRTN3) is a leukemia-associated antigen specifically recognized by CD8+ cytotoxic T-lymphocytes (CTL). PRTN3 also has been shown to elicit both antibody responses and T-cell proliferation in patients with Wegener's granulomatosis. In order to improve current vaccines that aim to stimulate CTL without inducing harmful autoimmune disease, it is necessary to study the role of PRTN3-specific CD4+ T-helper (TH) and CD4+ T-regulatory (Treg) cells. Since both TH and Treg cells recog...

  20. Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530.

    OpenAIRE

    Böckle, B; Galunsky, B; Müller, R.

    1995-01-01

    A serine protease from the keratin-degrading Streptomyces pactum DSM 40530 was purified by casein agarose affinity chromatography. The enzyme had a molecular weight of 30,000 and an isoelectric point of 8.5. The proteinase was optimally active in the pH range from 7 to 10 and at temperatures from 40 to 75 degrees C. The enzyme was specific for arginine and lysine at the P1 site and for phenylalanine and arginine at the P1' site. It showed a high stereoselectivity and secondary specificity wit...

  1. Purification and Characterization of a Keratinolytic Serine Proteinase from Streptomyces albidoflavus

    OpenAIRE

    Bressollier, Philippe; Letourneau, François; Urdaci, Maria; Verneuil, Bernard

    1999-01-01

    Streptomyces strain K1-02, which was identified as a strain of Streptomyces albidoflavus, secreted at least six extracellular proteases when it was cultured on feather meal-based medium. The major keratinolytic serine proteinase was purified to homogeneity by a two-step procedure. This enzyme had a molecular weight of 18,000 and was optimally active at pH values ranging from 6 to 9.5 and at temperatures ranging from 40 to 70°C. Its sensitivity to protease inhibitors, its specificity on synthe...

  2. Random substitution of large parts of the propeptide of yeast proteinase A

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten; Winther, Jakob R.

    1995-01-01

    The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal...

  3. Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220.

    OpenAIRE

    Kräusslich, H G; Nicklin, M J; Toyoda, H.; Etchison, D; Wimmer, E.

    1987-01-01

    Poliovirus infection of HeLa cells induces rapid shutoff of host protein synthesis, whereas translation of poliovirus RNA is not inhibited. It is presumed that shutoff is the result of proteolytic cleavage of component p220 of eucaryotic initiation factor 4F. To study whether poliovirus proteinase 2A is involved in this cleavage, we translated synthetic RNAs that contained the coding region for poliovirus-specific polypeptides P1 and 2A in vitro and assayed for cleavage of p220. We report her...

  4. Cloning and molecular characterization of a human intracellular serine proteinase inhibitor.

    OpenAIRE

    Coughlin, P; J. Sun; Cerruti, L; H.H. Salem; Bird, P.

    1993-01-01

    We describe a cDNA encoding a serine proteinase inhibitor present in placental tissue and the cytosolic fraction of K562 cells. On the basis of its interaction with thrombin, through which it was discovered, the inhibitor has been operationally named the placental thrombin inhibitor (PTI). Amino acid sequence comparisons suggest that its reactive center is located at Arg-341 and Cys-342, that it lacks a classical N-terminal signal sequence, and that it has the highest degree of similarity to ...

  5. Direct observation by X-ray analysis of the tetrahedral "intermediate" of aspartic proteinases.

    OpenAIRE

    Veerapandian, B.; Cooper, J. B.; Sali, A; Blundell, T L; Rosati, R. L.; Dominy, B. W.; Damon, D. B.; Hoover, D. J.

    1992-01-01

    We report the X-ray analysis at 2.0 A resolution for crystals of the aspartic proteinase endothiapepsin (EC 3.4.23.6) complexed with a potent difluorostatone-containing tripeptide renin inhibitor (CP-81,282). The scissile bond surrogate, an electrophilic ketone, is hydrated in the complex. The pro-(R) (statine-like) hydroxyl of the tetrahedral carbonyl hydrate is hydrogen-bonded to both active-site aspartates 32 and 215 in the position occupied by a water in the native enzyme. The second hydr...

  6. Two distinct phases of apoptosis in mammary gland involution: proteinase-independent and -dependent pathways

    Energy Technology Data Exchange (ETDEWEB)

    Lund, Leif R; Romer, John; Thomasset, Nicole; Solberg, Helene; Pyke, Charles; Bissell, Mina J; Dano, Keld; Werb, Zena

    1996-01-01

    Postlactational involution of the mammary gland is characterized by two distinct physiological events: apoptosis of the secretory, epithelial cells undergoing programmed cell death, and proteolytic degradation of the mammary gland basement membrane. We examined the spatial and temporal patterns of apoptotic cells in relation to those of proteinases during involution of the BALB/c mouse mammary gland. Apoptosis was almost absent during lactation but became evident at day 2 of involution, when {beta}-casein gene expression was still high. Apoptotic cells were then seen at least up to day 8 of involution, when {beta}-casein gene expression was being extinguished. Expression of sulfated glycoprotein-2 (SGP-2), interleukin-1{beta} converting enzyme (ICE) and tissue inhibitor of metalloproteinases-1 was upregulated at day 2, when apoptotic cells were seen initially. Expression of the matrix metalloproteinases gelatinase A and stromelysin-1 and the serine proteinase urokinase-type plasminogen activator, which was low during lactation, was strongly upregulated in parallel starting at day 4 after weaning, coinciding with start of the collapse of the lobulo-alveolar structures and the intensive tissue remodeling in involution. The major sites of mRNA synthesis for these proteinases were fibroblast-like cells in the periductal stroma and stromal cells surrounding the collapsed alveoli, suggesting that the degradative phase of involution is due to a specialized mesenchymal-epithelial interaction. To elucidate the functional role of these proteinases during involution, at the onset of weaning we treated mice systemically with the glucocorticoid hydrocortisone, which is known to inhibit mammary gland involution. Although the initial wave of apoptotic cells appeared in the lumina of the gland, the dramatic regression and tissue remodeling usually evident by day 5 was substantially inhibited by systemic treatment with hydrocortisone. mRNA and protein for gelatinase A, stromelysin-1 and uPA were weakly induced, if at all, in hydrocortisonetreated mice. Furthermore, mRNA for membrane-type matrix metalloproteinase decreased after hydrocortisone treatment and paralleled the almost complete inhibition of activation of latent gelatinase A. Concomitantly, the gland filled with an overabundance of milk. Our data support the hypothesis that there are at least two distinct phases of involution: an initial phase, characterized by induction of the apoptosis-associated genes SGP-2 and ICE and apoptosis of fully differentiated mammary epithelial cells without visible degradation of the extracellular matrix, and a second phase, characterized by extracellular matrix remodeling and altered mesenchymal-epithelial interactions, followed by apoptosis of cells that are losing differentiated functions.

  7. Molecular cloning of the rat proteinase-activated receptor 4 (PAR4)

    OpenAIRE

    Zou Lei; Winston John H; Micci Maria; Hellmich Helen; Hoogerwerf Willemijntje A; Pasricha Pankaj J

    2002-01-01

    Abstract Background The proteinase-activated receptor 4 (PAR4) is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA. Results The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mo...

  8. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3.

    Science.gov (United States)

    do Nascimento, Rodrigo Pires; Junior, Nelson Alves; Coelho, Rosalie Reed Rodrigues

    2011-10-01

    Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates. PMID:24031767

  9. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Scientific Electronic Library Online (English)

    Rodrigo Pires do, Nascimento; Nelson, Alves Junior; Rosalie Reed Rodrigues, Coelho.

    2011-12-01

    Full Text Available Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands wer [...] e detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

  10. Brewer's spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Directory of Open Access Journals (Sweden)

    Rodrigo Pires do Nascimento

    2011-12-01

    Full Text Available Brewer's spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates.

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

    OpenAIRE

    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 specificit...

  12. Isolation and characterization of a gene encoding a polyethylene glycol-induced cysteine protease in common wheat

    Indian Academy of Sciences (India)

    Qing-Wei Zang; Cai-Xiang Wang; Xu-Yan Li; Zhi-Ai Guo; Rui-Lian Jing; Jun Zhao; Xiao-Ping Chang

    2010-09-01

    Plant cysteine protease (CP) genes are induced by abiotic stresses such as drought, yet their functions remain largely unknown. We isolated the full-length cDNA encoding a Triticum aestivum CP gene, designated TaCP, from wheat by the rapid amplification of cDNA ends (RACE) method. Sequence analysis revealed that TaCP contains an open reading frame encoding a protein of 362 amino acids, which is 96% identical to barley cysteine protease HvSF42. The TaCP transcript level in wheat seedlings was upregulated during polyethylene glycol (PEG) stress, with a peak appearing around 12 h after treatment. TaCP expression level increased rapidly with NaCl treatment at 48 h. TaCP responded strongly to low temperature (4°C) treatment from 1 h post-treatment and reached a peak of about 40-fold at 72 h. However, it showed only a very slight response to abscisic acid (ABA). More than one copy of TaCP was present in each of the three genomes of hexaploid wheat and its diploid donors. TaCP fused with green fluorescent protein (GFP) was located in the plasma membrane of onion epidermis cells. Transgenic Arabidopsis plants overexpressing TaCP showed stronger drought tolerance and higher CP activity under water-stressed conditions than wild-type Arabidopsis plants. The results suggest that TaCP plays a role in tolerance to water deficit.

  13. The Arabidopsis thaliana sulfiredoxin is a plastidic cysteine-sulfinic acid reductase involved in the photooxidative stress response.

    Science.gov (United States)

    Rey, Pascal; Bécuwe, Noëlle; Barrault, Marie-Bénédicte; Rumeau, Dominique; Havaux, Michel; Biteau, Benoît; Toledano, Michel B

    2007-02-01

    The 2-cysteine peroxiredoxins (2-Cys-Prxs) are antioxidants that reduce peroxides through a thiol-based mechanism. During catalysis, these ubiquitous enzymes are occasionally inactivated by the substrate-dependent oxidation of the catalytic cysteine to the sulfinic acid (-SO2H) form, and are reactivated by reduction by sulfiredoxin (Srx), an enzyme recently identified in yeast and in mammal cells. In plants, 2-Cys-Prxs constitute the most abundant Prxs and are located in chloroplasts. Here we have characterized the unique Srx gene in Arabidopsis thaliana (AtSrx) from a functional point of view, and analyzed the phenotype of two AtSrx knockout (AtSrx-) mutant lines. AtSrx is a chloroplastic enzyme displaying sulfinic acid reductase activity, as shown by the ability of the recombinant AtSrx to reduce the overoxidized 2-Cys-Prx form in vitro, and by the accumulation of the overoxidized Prx in mutant lines lacking Srx in vivo. Furthermore, AtSrx mutants exhibit an increased tolerance to photooxidative stress generated by high light combined with low temperature. These data establish that, as in yeast and in mammals, plant 2-Cys-Prxs are subject to substrate-mediated inactivation reversed by Srx, and suggest that the 2-Cys-Prx redox status and sulfiredoxin are parts of a signaling mechanism participating in plant responses to oxidative stress. PMID:17217469

  14. Identification and characterization of alpha-I-proteinase inhibitor from common carp sarcoplasmic proteins.

    Science.gov (United States)

    Siriangkanakun, Siriphon; Li-Chan, Eunice C Y; Yongsawadigul, Jirawat

    2016-02-01

    Purification of proteinase inhibitor from common carp (Cyprinus carpio) sarcoplasmic proteins resulted in 2.8% yield with purification fold of 111. Two inhibitors, namely inhibitor I and II, exhibited molecular mass of 47 and 52 kDa, respectively, based on non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both inhibitors I and II were identified to be alpha-1-proteinase inhibitor (?1-PI) based on LC-MS/MS. They were glycoproteins and molecular mass after peptide-N-glycosidase F treatment was 38 and 45 kDa, respectively. The N-glycosylation sites of both inhibitors were determined to be at N214 and N226. The inhibitors specifically inhibited trypsin. The common carp ?1-PI showed high thermal stability with denaturation temperatures of 65.43 and 73.31 °C, which were slightly less than those of ovomucoid. High stability toward NaCl was also evident up to 3M. The common carp ?1-PI effectively reduced autolytic degradation of bigeye snapper surimi at the concentration as low as 0.025%. PMID:26304452

  15. Random substitution of large parts of the propeptide of yeast proteinase A

    DEFF Research Database (Denmark)

    van den Hazel, H B; Kielland-Brandt, Morten

    1995-01-01

    The yeast aspartic protease, proteinase A, has a 54 amino-acid propeptide, which is removed during activation of the zymogen in the vacuole. Apart from being involved inhibition/activation, the propeptide has been shown to be essential for formation of a stable active enzyme (van den Hazel, H. B., Kielland-Brandt, M. C., and Winther, J. R. (1993) J. Biol. Chem. 268, 18002-18007). We have investigated the sequence requirements for function of the propeptide. The N-terminal half and the C-terminal half of the propeptide were replaced by random sequences at the genetic level, and collections of the mutants were subjected to a colony screen for ones exhibiting activity. A high frequency (around 1%) of active constructs was found, which indicates a very high tolerance for mutations in the propeptide. Thirty-nine functional mutant forms containing random sequence at either the N- or C-terminal half of the propeptide were characterized. Comparison of the propeptides of the active constructs suggests that a particular lysine residue is important for efficient biosynthesis of proteinase A.

  16. Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins.

    Science.gov (United States)

    Pescuma, Micaela; Espeche Turbay, María Beatriz; Mozzi, Fernanda; Font de Valdez, Graciela; Savoy de Giori, Graciela; Hebert, Elvira María

    2013-09-01

    Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and ?-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly ?-casein, while degradation of ?s-caseins was strain dependent. Contrariwise, ?-Casein was poorly degraded by the studied lactobacilli. ?-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products. PMID:23832109

  17. Intracellular localization of Treponema denticola chymotrypsin-like proteinase in chronic periodontitis

    Directory of Open Access Journals (Sweden)

    Emilia Marttila

    2014-07-01

    Full Text Available Treponema denticola is an important periodontal pathogen capable of tissue invasion. Its chymotrypsin-like proteinase (CTLP can degrade a number of basement membrane components in vitro, thus suggesting a contribution to tissue invasion by the spirochete. The aim of this study was to analyze the localization of CTLP in chronic periodontitis tissues ex vivo. A polyclonal antibody specific to T. denticola cell-bound CTLP was used to detect the spirochetes in the gingival tissues of patients with moderate to severe chronic periodontitis (n=25 by immunohistochemistry and periodic acid-Schiff staining (PAS. The presence of T. denticola in the periodontal tissue samples was analyzed by PCR. Periodontal tissue samples of 12 of the 25 patients were found to be positive for T. denticola by PCR. Moreover, CTLP could be detected in the periodontal tissues of all these patients by immunohistochemistry. In the epithelium, the CTLP was mostly intracellular. Typically, the positive staining could be seen throughout the whole depth of the epithelium. When detected extracellularly, CTLP was localized mainly as granular deposits. The connective tissue stained diffusely positive in four cases. The positive staining co-localized with the PAS stain in nine cases. T. denticola and its CTLP could be detected in diseased human periodontium both intra- and extracellularly. The granular staining pattern was suggestive of the presence of T. denticola bacteria, whereas the more diffused staining pattern was indicative of the recent presence of the bacterium and shedding of the cell-bound proteinase.

  18. Proteinase K-catalyzed synthesis of linear and star oligo(L-phenylalanine) conjugates.

    Science.gov (United States)

    Ageitos, Jose M; Baker, Peter J; Sugahara, Michihiro; Numata, Keiji

    2013-10-14

    Chemoenzymatic synthesis of peptides is a green and clean chemical reaction that offers high yields without using organic synthesis and serves as an alternative to traditional peptide synthesis methods. This report describes the chemoenzymatic synthesis of oligo(L-phenylalanine) mediated by proteinase K from Tritirachium album, which is one of the most widely used proteases in molecular biological studies. The synthesized linear oligo-phenylalanine showed a unique self-assembly in aqueous solutions. To further functionalize linear oligo(L-phenylalanine) as a low-molecular-weight gelator, it was cosynthesized with tris(2-aminoethyl)amine to obtain star-oligo(L-phenylalanine), which was bioconjugated to demonstrate its self-assembly into fluorescent fibers. The self-assembled fibers of star-oligo(L-phenylalanine) formed fibrous networks with various branching ratios, which depended on the molecular weights and molecular aspect ratios of star-oligo(L-phenylalanine). This is the first study to demonstrate that proteinase K is a suitable enzyme for chemoenzymatic cosynthesis of oligopeptides and star-shaped heteropeptides. PMID:24000943

  19. Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L.

    Science.gov (United States)

    Devaraj, V R; Manjunatha, N H

    1999-01-01

    A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS-PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an Mr of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI-trypsin/chymotrypsin complexes by difference spectral studies. Apparent Ka values of complexes of inhibitor with trypsin and chymotrypsin were 2.1x10(7) M(-1) and 3.1x10(7) M(-1), respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. PMID:10071928

  20. Structure of the SARS coronavirus main proteinase as an active C2 crystallographic dimer

    International Nuclear Information System (INIS)

    An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (Mpro) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV Mpro is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P21212 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies

  1. Structure of the SARS coronavirus main proteinase as an active C{sub 2} crystallographic dimer

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ting; Ooi, Amy; Lee, Hooi Chen; Wilmouth, Rupert [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore); Liu, Ding Xiang [Institute of Molecular and Cell Biology (Singapore); Lescar, Julien, E-mail: julien@ntu.edu.sg [School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 (Singapore)

    2005-11-01

    An orthorhombic crystal form of the SARS CoV main proteinase diffracting to a resolution of 1.9 Å is reported. The conformation of residues in the catalytic site indicates an active enzyme. The 34 kDa main proteinase (M{sup pro}) from the severe acute respiratory syndrome coronavirus (SARS-CoV) plays an important role in the virus life cycle through the specific processing of viral polyproteins. As such, SARS-CoV M{sup pro} is a key target for the identification of specific inhibitors directed against the SARS virus. With a view to facilitating the development of such compounds, crystals were obtained of the enzyme at pH 6.5 in the orthorhombic space group P2{sub 1}2{sub 1}2 that diffract to a resolution of 1.9 Å. These crystals contain one monomer per asymmetric unit and the biologically active dimer is generated via the crystallographic twofold axis. The conformation of the catalytic site indicates that the enzyme is active in the crystalline form and thus suitable for structure-based inhibition studies.

  2. Cysteine as a non toxic corrosion inhibitor for copper alloys in conservation

    DEFF Research Database (Denmark)

    Gravgaard, Mari; van Lanschot, Jettie

    2012-01-01

    The aim of this work is to examine cysteine as an alternative to benzotriazole (BTA) for the conservation of archaeological objects with bronze disease. Investigation of the two inhibitors involved the use of electrochemical techniques, measurements of weight change in high humidity and comparative studies of colour changes in the corrosion products. The results obtained in this article demonstrate that cysteine could be a non-toxic alternative to BTA. Cysteine performed as well as BTA on pre-co...

  3. Adsorption Dynamics and Self-Assembled L-cysteine on Au(100)

    DEFF Research Database (Denmark)

    Engelbrekt, Christian; Nazmutdinov, Renat R.; Yan, Jiawei; Zinkicheva, T. T.; Mao, Bingwei; Ulstrup, Jens; Zhang, Jingdong

    2014-01-01

    As the only amino acid with a functional thiol group, L - cysteine offers a strong perspective both for binding to gold and other metals, and for gentle immobilization of biomolecules. Binding to single - crystal, atomically planar surfaces offers the additional perspective that bound L - cysteine can be structurally mapped at the single - molecule level . In this work, we have followed the adsorption of L - cysteine on single - crystal Au(100) by measuring the electrode potential dynamics durin...

  4. Cysteine and obesity: consistency of the evidence across epidemiologic, animal and cellular studies.

    OpenAIRE

    Elshorbagy, AK; Kozich, V; Smith, AD; Refsum, H.

    2012-01-01

    PURPOSE OF REVIEW: The concentrations of several plasma amino acids increase in obesity. Notably, plasma total concentrations of the sulphur amino acid cysteine (tCys) are linearly associated with fat mass in large population studies. Animal and cellular experiments support the concept that cysteine may be obesogenic. Here we review experimental and epidemiologic findings linking cysteine and related compounds with fat regulation and obesity. RECENT FINDINGS: tCys, and to a lesser extent cyst...

  5. Differential induction of two procesing proteases controls the processing pattern of the trypsin proteinase inhibitor precursor in Nicotiana attenuata.

    Czech Academy of Sciences Publication Activity Database

    Horn, Martin; Patankar, A. G.; Zavala, J. A.; Wu, J.; Marešová, Lucie; V?jt?chová, Milana; Mareš, Michael; Baldwin, I. T.

    Ljubljana : -, 2005. s. 94. [International Symposium on Proteinase Inhibitors and Biological Control /9./. 25.06.2005-29.06.2005, Brdo Estate] R&D Projects: GA AV ?R(CZ) IAA4055303; GA ?R(CZ) GA522/04/1286 Institutional research plan: CEZ:AV0Z40550506 Keywords : posttranslational modifications * differential fragmentation * vacuolar processing enzyme Subject RIV: CE - Biochemistry

  6. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K

    Directory of Open Access Journals (Sweden)

    Marwan Mansoor Ali Mohammed

    2013-01-01

    Full Text Available Background: Biofilms are organized communities of microorganisms embedded in a self-produced extracellular polymeric matrix (EPM, often with great phylogenetic variety. Bacteria in the subgingival biofilm are key factors that cause periodontal diseases; among these are the Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis. The objectives of this study were to characterize the major components of the EPM and to test the effect of deoxyribonuclease I (DNase I and proteinase K. Methods: F. nucleatum and P. gingivalis bacterial cells were grown in dynamic and static biofilm models. The effects of DNase I and proteinase K enzymes on the major components of the EPM were tested during biofilm formation and on mature biofilm. Confocal laser scanning microscopy was used in observing biofilm structure. Results: Proteins and carbohydrates were the major components of the biofilm matrix, and extracellular DNA (eDNA was also present. DNase I and proteinase K enzymes had little effect on biofilms in the conditions used. In the flow cell, F. nucleatum was able to grow in partially oxygenated conditions while P. gingivalis failed to form biofilm alone in similar conditions. F. nucleatum supported the growth of P. gingivalis when they were grown together as dual species biofilm. Conclusion: DNase I and proteinase K had little effect on the biofilm matrix in the conditions used. F. nucleatum formed biofilm easily and supported the growth of P. gingivalis, which preferred anaerobic conditions.

  7. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall.

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Ji?í; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Ro?. 20, ?. 12 (2011), s. 2004-2012. ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ?R GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  8. Microplate fluorescence protease assays test the inhibition of select North American snake venoms' activities with an anti-proteinase library.

    Science.gov (United States)

    Price, Joseph A

    2015-09-01

    Snake envenomation is a relatively neglected significant world health problem, designated an orphan disease by the WHO. While often effective, antivenins are insufficient. Could another approach greatly aid inhibition of the venom toxins? New fluorescent substrates for measuring protease activity in microplate assays suitable for high throughput screening were tested and found reproducible with snake venom. Representative North American venoms showed relatively strong proteinase and collagenase, but weaker elastase activities. Caseinolytic activity is inhibited by the nonspecific proteinase inhibitor 1,10-phenanthroline and by EDTA, as is collagenase activity, consistent with the action of metalloproteinases. Both general protease and collagenase assays CV average 3%, and Km measured were above normal working conditions. Using a library of anti -proteinase compounds with multiple venoms revealed high inhibitor activity by three agents with known multiple metalloproteinase inhibitor activity (Actinonin, GM6001, and NNGH), which incidentally supports the concept that much of the degradative activity of certain venoms is due to metalloproteinases with collagenase activity. These results together support the use of microplate proteinase assays, particularly this collagenase assay, in future drug repurposing studies leading to the development of new treatments for those envenomations that have a major proteolytic component in their pathophysiology. PMID:26130521

  9. [The effect of exogenous protein--a proteinase inhibitor--on the development of experimental pancreatitis in dogs].

    Science.gov (United States)

    Matveev, N L; Valueva, T A; Emel'ianov, S I; Pisarevski?, G N; Titova, G P; Penin, V A; Mosolov, V V

    1990-01-01

    Small doses of an exogenous protein inhibitor of proteinases ovomucoid, isolated from duck egg-white, exhibited distinct therapeutic effects in acute pancreatitis of dogs. The inhibitor decreased the lethality rate and exceeded the base Kunitz trypsin inhibitor in its efficiency. PMID:2075722

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

    International Nuclear Information System (INIS)

    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

  11. Enhancement of cysteine catabolism into taurine impacts glutathione homeostasis in rats challenged with ethanol.

    Science.gov (United States)

    Ahn, Chul Won; Kwon, Do Young; Jun, Doo Sung; Lee, Yoo Min; Kim, Young Chul

    2015-06-01

    We determined the alterations in metabolic conversion of cysteine into glutathione and taurine in liver of rats treated with ethanol acutely. Ethanol treatment reduced cysteine as well as glutathione levels in liver for 24 h. However, cysteine dioxygenase was up-regulated rapidly, and hypotaurine/taurine levels were significantly higher than those found in the saline-treated rats. It is therefore suggested that enhancement of cysteine catabolism into taurine contributes to the depletion of hepatic glutathione, which could exacerbate the ethanol-induced oxidative liver injury. PMID:25833720

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

    Science.gov (United States)

    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. PMID:26308073

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

    Science.gov (United States)

    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.

  14. Functional analysis of cysteine residues of ECP elicitor proteins of the fungal tomato pathogen Cladosporium fulvum

    OpenAIRE

    Luderer, R.; Kock, M.J.D., de; Dees, R.H.L.; Wit, P.J.G.M., de; Joosten, M.H.A.J.

    2002-01-01

    A striking feature of all elicitor proteins of Cladosporium fulvum that are specifically recognized by tomato is that they contain an even number of cysteine residues. These cysteine residues are thought to be involved in disulphide bridges. In this study, a mutational analysis of the cysteine residues of ECP1, ECP2 and ECP5 was performed, to examine their role in stability and hypersensitive response-inducing activity of the proteins. We show that not all cysteine residues of the ECPs are cr...

  15. Crystallization and preliminary X-ray diffraction studies of the cysteine protease ervatamin A from Ervatamia coronaria

    International Nuclear Information System (INIS)

    Ervatamin A is a papain-family cysteine protease with high activity and stability. It has been isolated and purified from the latex of the medicinal flowering plant E. coronaria and crystallized by the vapour-diffusion technique. Crystals diffracted to 2.1 Å and the structure was solved by molecular replacement. The ervatamins are highly stable cysteine proteases that are present in the latex of the medicinal plant Ervatamia coronaria and belong to the papain family, members of which share similar amino-acid sequences and also a similar fold comprising two domains. Ervatamin A from this family, a highly active protease compared with others from the same source, has been purified to homogeneity by ion-exchange chromatography and crystallized by the vapour-diffusion method. Needle-shaped crystals of ervatamin A diffract to 2.1 Å resolution and belong to space group C2221, with unit-cell parameters a = 31.10, b = 144.17, c = 108.61 Å. The solvent content using an ervatamin A molecular weight of 27.6 kDa is 43.9%, with a VM value of 2.19 Å3 Da?1 assuming one protein molecule in the asymmetric unit. A molecular-replacement solution has been found using the structure of ervatamin C as a search model

  16. A new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine proteinase from Vipera lebetina snake venom.

    Science.gov (United States)

    Siigur, Ene; Tõnismägi, Külli; Trummal, Katrin; Samel, Mari; Vija, Heiki; Aaspõllu, Anu; Rönnholm, Gunilla; Subbi, Juhan; Kalkkinen, Nisse; Siigur, Jüri

    2011-02-01

    Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-L-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, A?-chain and more slowly B?-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms. PMID:20950666

  17. Cleavage of Poly(A)-Binding Protein by Poliovirus 3C Proteinase Inhibits Viral Internal Ribosome Entry Site-Mediated Translation?

    OpenAIRE

    Bonderoff, Jennifer M.; LaRey, Jennifer L.; Lloyd, Richard E.

    2008-01-01

    The two enteroviral proteinases, 2A proteinase (2Apro) and 3C proteinase (3Cpro), induce host cell translation shutoff in enterovirus-infected cells by cleaving canonical translation initiation factors. Cleavage of poly(A)-binding protein (PABP) by 3Cpro has been shown to be a necessary component for host translation shutoff. Here we show that 3Cpro inhibits cap-independent translation mediated by the poliovirus internal ribosome entry site (IRES) in a dose-dependent manner in HeLa translatio...

  18. Binding of amino acid side chains to preformed cavities: interaction of serine proteinases with turkey ovomucoid third domains with coded and noncoded P1 residues.

    OpenAIRE

    Bigler, T. L.; Lu, W; Park, S.J.; Tashiro, M; Wieczorek, M.; Wynn, R; Laskowski, M.

    1993-01-01

    In the association of serine proteinases with their cognate substrates and inhibitors an important interaction is the fitting of the P1 side chain of the substrate or inhibitor into a preformed cavity of the enzyme called the S1 pocket. In turkey ovomucoid third domain, which is a canonical protein proteinase inhibitor, the P1 residue is Leu18. Here we report the values of equilibrium constants, Ka, for turkey ovomucoid third domain and 13 additional Leu18X variants with six serine proteinase...

  19. Biospecific haemosorbents based on proteinase inhibitor. II. Efficiency of biospecific antiproteinase haemosorbent 'Ovosorb' in complex treatment of experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs.

    Science.gov (United States)

    Platé, N A; Kirkovsky, V V; Antiperovich, O F; Nicolaichik, V V; Valueva, T A; Sinilo, S B; Moin, V M; Lobacheva, G A

    1994-03-01

    The biospecific antiproteinase haemosorbent (BAH) 'Ovosorb' containing, in the bulk of polyacryamide gel, the ovomucoid from whites of duck eggs, was used for a complex treatment of the experimental generalized purulent peritonitis and acute destructive pancreatitis in dogs. The efficiency of BAH was manifested in the significant reduction of lethality of the experimental animals, a more rapid liquidation of proteinasaemia, normalization in plasma of alpha 1-proteinase inhibitor and protein metabolism. Thus, by eliminating proteinases from circulation, Ovosorb contributes to the cessation of imbalance in the proteinase-inhibitor system and is efficient in the therapy of pathological states related to this imbalance. PMID:8031989

  20. Serpin genes AtSRP2 and AtSRP3 are required for normal growth sensitivity to a DNA alkylating agent in Arabidopsis

    OpenAIRE

    Atwell Brian J; Ahn Joon-Woo; Roberts Thomas H

    2009-01-01

    Abstract Background The complex responses of plants to DNA damage are incompletely understood and the role of members of the serpin protein family has not been investigated. Serpins are functionally diverse but structurally conserved proteins found in all three domains of life. In animals, most serpins have regulatory functions through potent, irreversible inhibition of specific serine or cysteine proteinases via a unique suicide-substrate mechanism. Plant serpins are also potent proteinase i...

  1. Copper oxide assisted cysteine hierarchical structures for immunosensor application

    Energy Technology Data Exchange (ETDEWEB)

    Pandey, Chandra Mouli [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi 110012 (India); Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005 (India); Sumana, Gajjala, E-mail: sumanagajjala@gmail.com [Biomedical Instrumentation Section, CSIR-National Physical Laboratory, New Delhi 110012 (India); Tiwari, Ida [Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi 221005 (India)

    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??m 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.

  2. Assignment of the vibrational spectrum of L-cysteine

    Energy Technology Data Exchange (ETDEWEB)

    Parker, Stewart F., E-mail: stewart.parker@stfc.ac.uk

    2013-10-16

    Highlights: • Periodic density functional theory of the polymorphic forms of L-cysteine. • A weak dihydrogen bond in the gauche conformer of the monoclinic form was found. • Comparison of observed and calculated neutron spectra shows good agreement. - Abstract: Ab initio calculations of the complete unit cell of L-cysteine for both the orthorhombic and monoclinic polymorphs have been carried out. The results suggest the presence of a previously unrecognised, weak dihydrogen bond of a novel type: S–H···N–H in the gauche conformer of the monoclinic polymorph. Comparison of the calculated transition energies to those observed in the infrared, Raman and inelastic neutron scattering spectra of the orthorhombic form shows excellent agreement, as does the simulated INS spectra to that experimentally measured. The assignments are in general agreement with those in the literature but differ in detail. The strong intermolecular interactions present make the use of periodic-DFT essential in order to correctly assign the spectra. The need for, and the complementarity of, all three types of vibrational spectra: infrared, Raman and INS is clearly demonstrated.

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

    Science.gov (United States)

    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 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. PMID:10517767

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

    International Nuclear Information System (INIS)

    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-cysteine 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]t-cysteine showed that most of the sulfur in H2S was derived from sulfur in the L-cysteine supplied

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Nutritional Requirements and Nitrogen-Dependent Regulation of Proteinase Activity of Lactobacillus helveticus CRL 1062

    OpenAIRE

    Hebert, Elvira M.; Raya, Raul R; De Giori, Graciela S.

    2000-01-01

    The nutritional requirements of Lactobacillus helveticus CRL 1062 were determined with a simplified chemically defined medium (SCDM) and compared with those of L. helveticus CRL 974 (ATCC 15009). Both strains were found to be prototrophic for alanine, glycine, asparagine, glutamine, and cysteine. In addition, CRL 1062 also showed prototrophy for lysine and serine. The microorganisms also required riboflavin, calcium pantothenate, pyridoxal, nicotinic acid, and uracil for growth in liquid SCDM...

  7. A novel cysteine-rich antifungal peptide ToAMP4 from Taraxacum officinale Wigg. flowers.

    Science.gov (United States)

    Astafieva, A A; Rogozhin, Eugene A; Andreev, Yaroslav A; Odintsova, T I; Kozlov, S A; Grishin, Eugene V; Egorov, Tsezi A

    2013-09-01

    A novel peptide named ToAMP4 was isolated from Taraxacum officinale Wigg. flowers by a combination of acetic acid extraction and different types of chromatography: affinity, size-exclusion, and RP-HPLC. The amino acid sequence of ToAMP4 was determined by automated Edman degradation. The peptide is basic, consists of 41 amino acids, and incorporates three disulphide bonds. Due to the unusual cysteine spacing pattern, ToAMP4 does not belong to any known plant AMP family, but classifies together with two other antimicrobial peptides ToAMP1 and ToAMP2 previously isolated from the dandelion flowers. To study the biological activity of ToAMP4, it was successfully produced in a prokaryotic expression system as a fusion protein with thioredoxin. The recombinant peptide was shown to be identical to the native ToAMP4 by chromatographic behavior, molecular mass, and N-terminal amino acid sequence. The peptide displays broad-spectrum antifungal activity against important phytopathogens. Two ToAMP4-mediated inhibition strategies depending on the fungus were demonstrated. The results obtained add to our knowledge on the structural and functional diversity of AMPs in plants. PMID:23771034

  8. Antioxidant activity of bovine casein hydrolysates produced by Ficus carica L.-derived proteinase.

    Science.gov (United States)

    Di Pierro, Giovanna; O'Keeffe, Martina B; Poyarkov, Alexey; Lomolino, Giovanna; FitzGerald, Richard J

    2014-08-01

    A Ficus carica L. latex proteinase preparation was investigated for its ability to produce antioxidant hydrolysates/peptides from bovine casein (CN). The Oxygen Radical Absorbance Capacity (ORAC) values for NaCN and ?-CN hydrolysates ranged from 0.06 to 0.18, and from 0.51 to 1.19?mol Trolox equivalents/mg freeze-dried sample, respectively. Gel permeation HPLC showed that the ?-CN hydrolysate with a degree of hydrolysis of 21% had 65% of peptide material with a molecular mass <500Da. The RP-UPLC profiles also indicated that ?-CN was substantially hydrolysed during the early stages of hydrolysis. Analysis of the 4h ?-CN hydrolysate by LC-ESI-MS/MS allowed identification of 8 peptide sequences with potential antioxidant properties. PMID:24629973

  9. The effect of proteinases (keratinases) in the pathogenesis of Dermatophyte infection using scanning electron microscope

    International Nuclear Information System (INIS)

    Objective: To study the inter-relationship between the stratum corneum of host and the fungal micro-organisms using scanning electron microscopy for a complete understanding of the host parasite relationship. Material and Methods: Skin surface biopsies were obtained two patients suffering from tinea cruris infection. One patient was infected with trichophyton rubrum and the other with epidermophytom floccosum strains. Results: The scanning electron microphotographs obtained from two patients showed a large number of villi in the infected area. The fungal hyphae were seen to placed intercellularly as well seem to be traversing through the corneocytes in many places. Conclusion: From the results observed in this study it could be suggested that the secretion of proteinases from the fungal hyphae together with the mechanical force of the invading organisms in vivo might be playing part in the invasion of the organisms. (author)

  10. Production and administration to dogs of aerosols of alpha-1-proteinase inhibitor

    International Nuclear Information System (INIS)

    The feasibility of aerosol administration of alpha-1-proteinase inhibitor (human) (A1PI) was assessed. Of three different methods of aerosolizing A1PI that were evaluated, an ultrasonic nebulizer was found to be best suited to the present purpose, producing particles of a size that allowed them to reach the distal air spaces of the lung and that retained specific A1PI anti-elastase activity. Administration of 20 mg/kg of A1PI and 150 microCi of 131-iodine-A1PI to three dogs was accomplished without complications. Gamma camera scans documented a relatively homogenous distribution throughout the lungs. Bronchial lavage fluid that was recovered from the lungs of the dogs six hours after administration contained large amounts of human A1PI and showed a proportional elevation of anti-elastase activity. There was no evidence of acute toxicity

  11. Elimination of hydrogen sulphide and ? substitution in cystein, catalyzed by the cysteine-lyase of hens yolk-sac and yolk (1961)

    International Nuclear Information System (INIS)

    The yolk of incubated hen's eggs contains a pyridoxal phosphate activated enzyme, free of iron, copper, magnesium and calcium. This enzyme activates the ?-carbon atom of cysteine. Its reactivity is demonstrated by the ease with which this ?-carbon fixes various sulfur containing substances in which the sulfur has reducing properties: inorganic sulfide, sulfide or cysteine itself. In the absence of substances able to react with the ?-carbon atom, the active complex, consisting of the enzyme and the aminated tri-carbon chain, is hydrolysed to pyruvic acid and ammonia. The liberation of hydrogen sulfide thus appears to be the consequence either of the substitution of the ?-carbon atom of cysteine or of the decomposition of the complex which this aminoacid forms with the enzyme studied. The latter seems therefore to possess an activity which differs from the activity of the desulfhydrases as yet known. We suggest to call this enzyme cystein-lyase. (authors)

  12. Role of Candida albicans-Secreted Aspartyl Proteinases (Saps in Severe Early Childhood Caries

    Directory of Open Access Journals (Sweden)

    Wenqing Li

    2014-06-01

    Full Text Available Candida albicans is strongly associated with severe early childhood caries (S-ECC. However, the roles of secreted aspartyl proteinases (Saps, an important virulence factor of C. albicans, in the progress of S-ECC are not clear. In our study, the Saps activities were evaluated by the yeast nitrogen base–bovine serum albumi (YNB–BSA agar plate method and by the MTT method with bovine serum albumin (BSA as the substrate. Genotypes of C. albicans and gene expression of Sap1–5 were evaluated. The relationships of Saps activities and genotypes with S-ECC were analyzed. The results showed that enzyme activities of Saps in the S-ECC group were significantly higher than those in the caries free (CF group (p < 0.05. Genotypes A, B and C were detected in the S-ECC group, and genotypes A and C were detected in the CF group. In the genotype A group, Saps activity in the S-ECC group was significantly different from that in the CF group (p < 0.05. The gene expression level of Sap1 in the S-ECC group was significantly higher than that in the CF group (p = 0.001, while Sap4 expression was significantly lower than that in the CF group (p = 0.029. It can be concluded that Sap1–5 are the predominant proteinase genes expressed in C. albicans from dental biofilm and Sap1 may play an important role in the development of S-ECC.

  13. Anti-collagenase, anti-elastase and anti-oxidant activities of extracts from 21 plants

    OpenAIRE

    Hili Pauline; Thring Tamsyn SA; Naughton Declan P

    2009-01-01

    Abstract Background Owing to their roles in tissue remodelling in health and disease, several studies have reported investigations on plant extracts as inhibitors of proteinases and as anti-oxidants. Methods The anti-ageing and anti-oxidant properties of 23 plant extracts (from 21 plant species) were assessed as anti-elastase and anti-collagenase activities and in selected anti-oxidant assays along with phenolic content. Results Anti-elastase activities were observed for nine of the extracts ...

  14. Slime production and proteinase activity of Candida species isolated from blood samples and the comparison of these activities with minimum inhibitory concentration values of antifungal agents

    Scientific Electronic Library Online (English)

    Semiha, Ozkan; Fatma, Kaynak; Ayse, Kalkanci; Ufuk, Abbasoglu; Semra, Kustimur.

    2005-05-01

    Full Text Available Slime and proteinase activity of 54 strains consisting of 19 Candida parapsilosis and 35 C. albicans strains isolated from blood samples were investigated in this study. Ketoconazole, amphothericin B, and fluconazole susceptibility of Candida species were compared with slime production and proteinas [...] e activity of these species. For both Candida species, no correlation was detected between the slime activity and minimum inhibitory concentration (MIC) values of the three antifungal agents. For both Candida species no correlation was detected between the proteinase activity and the MIC values of amphothericin B, and fluconazole however, statistically significant difference, was determined between the proteinase activity and MIC values of ketoconazole (p = 0.007). Slime production was determined by using modified Christensen macrotube method and proteinase activity was measured by the method of Staib. Antifungal susceptibility was determined through the guidelines of National Committee for Laboratory Standards (NCCLS M27-A).

  15. [Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper].

    Science.gov (United States)

    Ramazanova, A S; Fil'kin, S Iu; Starkov, V G; Utkin, Iu N

    2011-01-01

    Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor. PMID:21899053

  16. Production of hydrogen sulfide from D-cysteine and its therapeutic potential

    Directory of Open Access Journals (Sweden)

    NorihiroShibuya

    2013-07-01

    Full Text Available Accumulating evidence shows that H2S has physiological functions in various tissues and organs. It includes regulation of neuronal activity, vascular tension, a release of insulin, and protection of the heart, kidney and brain from ischemic insult. H2S is produced by enzymes from L-cysteine; cystathionine ?-synthase (CBS, cystathionine ?-lyase (CSE, and 3-mercaptopyruvate sulfurtransferase (3MST along with cysteine aminotransferase (CAT. We recently discovered an additional pathway for the production of H2S from D-cysteine. D-Amino acid oxidase (DAO provides 3-mercaptopyruvate (3MP for 3MST to produce H2S. D-Cysteine protects cerebellar neurons from oxidative stress and attenuates ischemia-reperfusion injury caused in the kidney more effectively than L-cysteine. This review focuses on a novel pathway for the production of H2S and its therapeutic application especially to the renal diseases.

  17. Intramolecular synergistic effect of glutamic acid, cysteine and glycine against copper corrosion in hydrochloric acid solution

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Daquan, E-mail: zhdq@sh163.net [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Xie Bin; Gao Lixin; Cai Qirui [Department of Environmental Engineering, Shanghai University of Electric Power, Shanghai 200090 (China); Joo, Hyung Goun; Lee, Kang Yong [Stress Analysis and Failure Design Laboratory, School of Mechanical Engineering, Yonsei University, Seoul 120-749 (Korea, Republic of)

    2011-10-31

    The corrosion protection of copper by glutamic acid, cysteine, glycine and their derivative (glutathione) in 0.5 M hydrochloric acid solution has been studied by the electrochemical impedance spectroscopy and cyclic voltammetry. The inhibition efficiency of the organic inhibitors on copper corrosion increases in the order: glutathione > cysteine > cysteine + glutamic acid + glycine > glutamic acid > glycine. Maximum inhibition efficiency for cysteine reaches about 92.9% at 15 mM concentration level. The glutathione can give 96.4% inhibition efficiency at a concentration of 10 mM. The molecular structure parameters were obtained by PM3 (Parametric Method 3) semi-empirical calculation. The intramolecular synergistic effect of glutamic acid, cysteine and glycine moieties in glutathione is attributed to the lower energy of the lowest unoccupied molecular orbital (E{sub LUMO}) level and to the excess hetero-atom adsorption centers and the bigger coverage on the copper surface.

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

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

    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.

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

    International Nuclear Information System (INIS)

    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.

  20. Characterization of Amylase, Cellulase and Proteinase Enzyme in Stomach and Intestine of the Mekong Giant Catfish Fed with Various Diets Consisting of Spirulina

    Directory of Open Access Journals (Sweden)

    Sudaporn Tongsiri

    2010-07-01

    Full Text Available The amylase, cellulase and proteinase enzyme from the stomach and intestine of the Mekong GiantCatfish that had been fed with various diets consisting of Spirulina, were studied at pH 2-12 and at temperaturesbetween 25-80ºC. This study found that; amylase activities of the stomach were alkaline amylase and theoptimal temperatures to be 25 and 50ºC. Amylase activities of the intestine were neutral amylase as well asalkaline amylase and the optimal temperature range was at 25-30ºC. The cellulase activities were acidiccellulase and alkaline cellulase in both the stomach and the intestine. In the stomach, the acidic cellulaseshowed the optimal temperatures to be at 30, 40 and 50ºC, however alkaline cellulase were 30 and 50ºC. In theintestine, acidic cellulase showed the optimal temperatures to be at 25, 60 and 70ºC and the alkaline cellulaseto be 80ºC. For proteinase activity, the stomach was acidic proteinase and alkaline proteinase, but the intestinewas alkaline proteinase. The acidic proteinase activities of the stomach showed the optimal temperatures to beat 40, 50 and 60ºC and the alkaline proteinase activity proved to be at 40, 60 and 70ºC. The optimal conditionsfor amylase enzymes showed the higher specific activity in the intestine than in the stomach including theproteinase enzyme. At room temperature (25-30ºC, amylase and proteinase specific acitivity dominated in theintestine, while cellulase specific activity dominated in the stomach.

  1. The Secreted Aspartyl Proteinases Sap1 and Sap2 Cause Tissue Damage in an In Vitro Model of Vaginal Candidiasis Based on Reconstituted Human Vaginal Epithelium

    OpenAIRE

    Schaller, Martin; Bein, Matthias; Korting, Hans C.; Baur, Stefan; Hamm, Gerald; Monod, Michel; Beinhauer, Sabine; Hube, Bernhard

    2003-01-01

    Secreted aspartyl proteinases (Saps) contribute to the ability of Candida albicans to cause mucosal and disseminated infections. A model of vaginal candidiasis based on reconstituted human vaginal epithelium (RHVE) was used to study the expression and role of these C. albicans proteinases during infection and tissue damage of vaginal epithelium. Colonization of the RHVE by C. albicans SC5314 did not cause any visible epithelial damage 6 h after inoculation, although expression of SAP2, SAP9, ...

  2. Role of proteinase in the formation of inhibitory levels of hematin by group A streptococcus cultures on blood-containing media.

    OpenAIRE

    Hynes, W L; Tagg, J. R.

    1986-01-01

    Group A streptococci were tested for proteinase production and for the possible relationship of this production to the generation of bacteriocinlike inhibitor activity. Of 126 strains tested, 83% were positive for proteinase, and a similar distribution was found among strains isolated in association with rheumatic fever (89%) and nephritis (94%) and from uncomplicated acute infections (78%). Although application of an inhibitor production (P) typing scheme demonstrated a variety of P types, a...

  3. vfr, a Novel Locus Affecting Cysteine Protease Production in Streptococcus pyogenes?

    OpenAIRE

    Ma, Yongsheng; Bryant, Amy E.; Salmi, Dan B.; McIndoo, Eric; Stevens, Dennis L.

    2009-01-01

    A gene unique to Streptococcus pyogenes, called vfr, that negatively regulates speB, an important extracellular proteinase, has been identified. Disruption of vfr markedly increased SpeB production in a clinical strain of S. pyogenes and relieved its growth phase dependency. These findings may provide important insights into the pathogenesis of invasive S. pyogenes infections.

  4. Aspartic acid-promoted highly selective and sensitive colorimetric sensing of cysteine in rat brain.

    Science.gov (United States)

    Qian, Qin; Deng, Jingjing; Wang, Dalei; Yang, Lifen; Yu, Ping; Mao, Lanqun

    2012-11-01

    Direct selective determination of cysteine in the cerebral system is of great importance because of the crucial roles of cysteine in physiological and pathological processes. In this study, we report a sensitive and selective colorimetric assay for cysteine in the rat brain with gold nanoparticles (Au-NPs) as the signal readout. Initially, Au-NPs synthesized with citrate as the stabilizer are red in color and exhibit absorption at 520 nm. The addition of an aqueous solution (20 ?L) of cysteine or aspartic acid alone to a 200 ?L Au-NP dispersion causes no aggregation, while the addition of an aqueous solution of cysteine into a Au-NP dispersion containing aspartic acid (1.8 mM) causes the aggregation of Au-NPs and thus results in the color change of the colloid from wine red to blue. These changes are ascribed to the ion pair interaction between aspartic acid and cysteine on the interface between Au-NPs and solution. The concentration of cysteine can be visualized with the naked eye and determined by UV-vis spectroscopy. The signal output shows a linear relationship for cysteine within the concentration range from 0.166 to 1.67 ?M with a detection limit of 100 nM. The assay demonstrated here is highly selective and is free from the interference of other natural amino acids and other thiol-containing species as well as the species commonly existing in the brain such as lactate, ascorbic acid, and glucose. The basal dialysate level of cysteine in the microdialysate from the striatum of adult male Sprague-Dawley rats is determined to be around 9.6 ± 2.1 ?M. The method demonstrated here is facile but reliable and durable and is envisaged to be applicable to understanding the chemical essence involved in physiological and pathological events associated with cysteine. PMID:23025476

  5. Heterologous expression and purification of barley (Hordeum vulgare L.) cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach; Brinch-Pedersen, Henrik

    2011-01-01

    The mobilization of protein during germination of barley seeds is essential and Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins [1]. Cysteine proteases exist as pro-enzyme until activated through reduction of the active site cysteines and via removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficie...

  6. The role of lysosomal cysteine proteases in crustacean immune response

    Directory of Open Access Journals (Sweden)

    FL Garcia-Carreño

    2014-04-01

    Full Text Available Over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of intruders. The immune response represents a key component to optimize the biological fitness of individuals. Two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. Scientific contributions have improved our understanding of the crustacean immune response. Several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. This review summarizes the current information regarding the role of cysteine proteases in the immune system of Crustacea and points to aspects that are needed to provide a better integration of our knowledge.

  7. Utilização da fração semipurificada da proteinase do Trypanosoma cruzi no imunodiagnóstico da doença de Chagas The use of a semipurified fraction of Trypanosoma cruzi proteinase in immunodiagnosis of Chagas' disease

    OpenAIRE

    Ajax Mercês Atta; Angela Maria de Carvalho Pontes; Maria Luiza de Souza; Daria Repka; Rangel, Humberto A.

    1984-01-01

    Foram sensibilizadas hemácias humanas 0 Rh negativo com a fração semipurificada (Fp) da proteinase do Trypanosoma cruzi, e testadas quanto a antigenicidade com soros de pacientes portadores de tripanossomíase americana crônica e de outras doenças parasitárias não relacionadas. Reações de hemaglutinação positivas foram observadas com os soros de pacientes chagásicos e com alguns soros de indivíduos portadores de leishmaniose cutaneo-mucosa. Não foram observadas reações cruzadas com os soros de...

  8. Water molecules participate in proteinase-inhibitor interactions: crystal structures of Leu18, Ala18, and Gly18 variants of turkey ovomucoid inhibitor third domain complexed with Streptomyces griseus proteinase B.

    OpenAIRE

    Huang, K.; Lu, W; Anderson, S; Laskowski, M.; M. N. James

    1995-01-01

    Crystal structures of the complexes of Streptomyces griseus proteinase B (SGPB) with three P1 variants of turkey ovomucoid inhibitor third domain (OMTKY3), Leu18, Ala18, and Gly18, have been determined and refined to high resolution. Comparisons among these structures and of each with native, uncomplexed SGPB reveal that each complex features a unique solvent structure in the S1 binding pocket. The number and relative positions of water molecules bound in the S1 binding pocket vary according ...

  9. Functional Importance and Local Environments of the Cysteines in the Tetracycline Resistance Protein Encoded by Plasmid pBR322

    OpenAIRE

    Jewell, Jean E.; Orwick, Jill; Liu, Jun; Miller, Kurt W.

    1999-01-01

    The properties of the cysteines in the pBR322-encoded tetracycline resistance protein have been examined. Cysteines are important but not essential for tetracycline transport activity. None of the cysteines reacted with biotin maleimide, suggesting that they are shielded from the aqueous phase or reside in a negatively charged local environment.

  10. Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: Experiments and computer simulations

    DEFF Research Database (Denmark)

    GrØn, S.; Biedermann, K.

    1996-01-01

    Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Membrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that the proteinase A concentration decreased drastically at high dilution rates and the optimal volumetric productivities were at high dilution rates just below washout and at high ratios of recycling. Cell-recycling fermentation gave much higher volumetric productivities and stable product concentrations in contrast to simple continuous fermentation.

  11. Rapid turnover of antimicrobial-type cysteine-rich protein genes in closely related Oryza genomes.

    Science.gov (United States)

    Shenton, Matthew R; Ohyanagi, Hajime; Wang, Zi-Xuan; Toyoda, Atsushi; Fujiyama, Asao; Nagata, Toshifumi; Feng, Qi; Han, Bin; Kurata, Nori

    2015-10-01

    Defensive and reproductive protein genes undergo rapid evolution. Small, cysteine-rich secreted peptides (CRPs) act as antimicrobial agents and function in plant intercellular signaling and are over-represented among reproductively expressed proteins. Because of their roles in defense, reproduction and development and their presence in multigene families, CRP variation can have major consequences for plant phenotypic and functional diversification. We surveyed the CRP genes of six closely related Oryza genomes comprising Oryza sativa ssp. japonica and ssp. indica, Oryza glaberrima and three accessions of Oryza rufipogon to observe patterns of evolution in these gene families and the effects of variation on their gene expression. These Oryza genomes, like other plant genomes, have accumulated large reservoirs of CRP sequences, comprising 26 groups totaling between 676 and 843 genes, in contrast to antimicrobial CRPs in animal genomes. Despite the close evolutionary relationships between the genomes, we observed rapid changes in number and structure among CRP gene families. Many CRP sequences are in gene clusters generated by local duplications, have undergone rapid turnover and are more likely to be silent or specifically expressed. By contrast, conserved CRP genes are more likely to be highly and broadly expressed. Variable CRP genes created by repeated duplication, gene modification and inactivation can gain new functions and expression patterns in newly evolved gene copies. For the CRP proteins, the process of gain/loss by deletion or duplication at gene clusters seems to be an important mechanism in evolution of the gene families, which also contributes to their expression evolution. PMID:25842177

  12. "Comparison of Adult Somatic and Cysteine Proteinas Antigens of Fasciola gigantica in Enzyme Linked Immunosorbent Assay for Serodiagnosis of Human Fasciolosis"

    Directory of Open Access Journals (Sweden)

    MB Rokni

    2002-08-01

    Full Text Available Fasciolosis caused by Fasciola hepatica and F.gigantica is one of the major public health problems in the world and in Iran. Considering that stool examination for Fasciola eggs is not a sensitive method and only 25% of infected patients pass the eggs in the faeces , and immunodiagnosis methods are more applicable for this purpose, the present study was conducted to compare the somatic (S and cysteine proteinase (CP antigens of F.gigantica in IgG-ELISA to diagnose human fasciolosis. This has been the first report on this case so far in Iran. Serum samples obtained from 178 individuals collected during the fasciolosis outbreak in 1999 in the Gilan province, northern Iran, that were coprologically positive for fasciolosis, were analyzed by IgG-ELISA for total antibody responses against (S and CP antigens from Fasciola gigantica. The cut-off points for (S and CP were 0.38 and 0.33, respectively. All cases that showed clinical manifestations of fasciolosis, were also seropositive using both (S and CP antigens whereas all 25 non-infected controls were seronegative. Therefore, the sensitivity of the test was 100% for both antigens. On the other hand the specificity of (S and CP antigens were calculated as 96.4% and 98.1%, respectively. The positive and negative predictive values of the test regarding (S antigen were 97.8% and 100%, whereas these values as for CP antigen were 98.9% and 100% correspondingly. Two individuals with hydatidosis and two with toxocariasis had antibodies against (S antigen whereas concerning CP antigen, one individual with hydatidosis and another with toxocariasis showed cross reactivity against it. We have demonstrated that altogether CP antigen provide a more conclusive diagnosis as possessing lower cut-off and enabling better to discriminate between seronegative and seropositive subpopulations.This study may be useful to implement a reliable test to diagnose human fasciolosis and for seroepidmiological objectives.

  13. A Novel Aspartic Proteinase-Like Gene Expressed in Stratified Epithelia and Squamous Cell Carcinoma of the Skin

    OpenAIRE

    Rhiemeier, Verena; Breitenbach, Ute; Richter, Karl Hartmut; Gebhardt, Christoffer; Vogt, Ingeborg; Hartenstein, Bettina; Fürstenberger, Gerhard; Mauch, Cornelia; Hess, Jochen; Angel, Peter

    2006-01-01

    Homeostasis of stratified epithelia, such as the epidermis of the skin, is a sophisticated process that represents a tightly controlled balance between proliferation and differentiation. Alterations of this balance are associated with common human diseases including cancer. Here, we report the cloning of a novel cDNA sequence, from mouse back skin, that is induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and codes for a hitherto unknown aspartic proteinase-like protein ...

  14. prtH2, Not prtH, Is the Ubiquitous Cell Wall Proteinase Gene in Lactobacillus helveticus?

    OpenAIRE

    Genay, M.; Sadat, L.; Gagnaire, V.; Lortal, S.

    2009-01-01

    Lactobacillus helveticus strains possess an efficient proteolytic system that releases peptides which are essential for lactobacillus growth in various fermented dairy products and also affect textural properties or biological activities. Cell envelope proteinases (CEPs) are bacterial enzymes that hydrolyze milk proteins. In the case of L. helveticus, two CEPs with low percentages of amino acid identity have been described, i.e., PrtH and PrtH2. However, the distribution of the genes that enc...

  15. Proteinases of betaretroviruses bind single-stranded nucleic acids through a novel interaction module, the G-patch.

    Czech Academy of Sciences Publication Activity Database

    Švec, Martin; Bauerová, Helena; Pichová, Iva; Konvalinka, Jan; St?íšovský, Kvido

    2004-01-01

    Ro?. 576, 1/2 (2004), s. 271-276. ISSN 0014-5793 R&D Projects: GA AV ?R IAA4055304; GA MŠk LN00A032 Grant ostatní: 5th Framework(XE) QLK2-CT-2001-02360 Institutional research plan: CEZ:AV0Z4055905 Keywords : retrovirus * aspartic proteinase * maturation Subject RIV: CE - Biochemistry Impact factor: 3.843, year: 2004

  16. Production of proteinase A by Saccharomyces cerevisiae in a cell-recycling fermentation system: Experiments and computer simulations

    DEFF Research Database (Denmark)

    Grøn, S.; Biedermann, K.; Emborg, Claus

    1996-01-01

    Overproduction of proteinase A by recombinant Saccharomyces cerevisiae was investigated by cultivations in a cell-recycling bioreactor. Membrane filtration was used to separate cells from the broth. Recycling ratios and dilution rates were varied and the effect on enzyme production was studied both experimentally and by computer simulations. Experiments and simulations showed that cell mass and product concentration were enhanced by high ratios of recycling. Additional simulations showed that th...

  17. Saccharomyces cerevisiae can secrete Sapp1p proteinase of Candida parapsilosis but cannot use it for efficient nitrogen acquisition.

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Bauerová, Václava; Dostál, Ji?í; Sychrová, Hana; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2013-01-01

    Ro?. 51, ?. 3 (2013), s. 336-344. ISSN 1225-8873 R&D Projects: GA ?R GA310/09/1945; GA ?R GAP302/12/1151 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Candida parapsilosis * Saccharomyces cerevisiae * secreted aspartic proteinase * SAPP1 * nitrogen metabolism Subject RIV: EE - Microbiology, Virology; EE - Microbiology, Virology (FGU-C) Impact factor: 1.529, year: 2013

  18. Effect of sulphated polysaccharides on the alpha 1-antitrypsin inhibition of amidolysis catalysed by coagulation cascade proteinases.

    OpenAIRE

    Long, W. F.; Williamson, F. B.

    1981-01-01

    1 The ability of several sulphated polysaccharide anticoagulants to prevent alpha 1-antitrypsin inhibition of thrombin paralleled their ability to potentiate antithrombin III inhibition of thrombin. None of the compounds examined altered the ability of alpha 1-antitrypsin to inhibit activated coagulation factor X (Xa). 2 These results are consistent with the possibility that a direct polysaccharide-proteinase interaction may be involved in the sulphated polysaccharide-modulated inhibition of ...

  19. Proteinases in Excretory-Secretory Products of Toxocara canis Second-Stage Larvae: Zymography and Modeling Insights

    OpenAIRE

    Gonzalo Ernesto González-Páez; Fernando Alba-Hurtado; Carlos Gerardo García-Tovar; Raúl Argüello-García

    2014-01-01

    Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activi...

  20. Use of proteinase K for RT-PCR of cytokine mRNA in formalin fixed tissue

    DEFF Research Database (Denmark)

    Davies, G N; Bevan, I S; Banner, Jytte; Smith, H; Sweet, C

    1996-01-01

    Fresh tissue from cases of sudden infant death syndrome is becoming increasingly scarce and therefore researchers interesting in studying the aetiology of this syndrome have had to resort to archival tissue, usually in the form of paraffin wax sections. A simple method for isolating mRNA from formalin fixed, paraffin wax embedded material of sufficient purity for reverse transcription (RT)-PCR is described. Proteinase K treatment of formalin fixed, wax embedded tissue followed by RNA STAT-60 ext...

  1. Proteinase-activated receptors 1 and 4 counter-regulate endostatin and VEGF release from human platelets

    OpenAIRE

    Ma, Li; Perini, Rafael; McKnight, Webb; Dicay, Michael; Klein, Andre; Morley D. Hollenberg; Wallace, John L.

    2004-01-01

    The roles of proteinase-activated receptors (PARs) in platelet functions other than aggregation are not well understood. Among these is the release of factors that regulate the process of angiogenesis, such as endostatin and VEGF, which, respectively, inhibit and promote angiogenesis. PAR1 and PAR4 are expressed on the surface of human platelets and can be activated by thrombin. In the present study, we have attempted to determine the roles of PAR1 and PAR4 in regulating release of endostatin...

  2. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases

    OpenAIRE

    1989-01-01

    The role of basic fibroblast growth factor-(bFGF) induced proteinases in basement membrane (BM) invasion by bovine capillary endothelial (BCE) cells was studied using a quantitative in vitro assay previously described (Mignatti et al., 1986). 125I-iododeoxyuridine-labeled BCE cells were grown for 72 h on the human amnion BM, and cell invasion was determined by measuring the radioactivity associated with the tissue after removal of the noninvasive cell layer. BCE cells were noninvasive under n...

  3. Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

    OpenAIRE

    M. N. James; Sielecki, A; Salituro, F; Rich, D H; Hofmann, T.

    1982-01-01

    Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl-Val-Val-StaOEt, where StaOEt is the ethyl ester of statine [(4S,3S)-4-amino...

  4. Molecular cloning of the cDNA and gene for an elastinolytic aspartic proteinase from Aspergillus fumigatus and evidence of its secretion by the fungus during invasion of the host lung.

    OpenAIRE

    Lee, J.D.; Kolattukudy, P E

    1995-01-01

    Hydrolysis of structural proteins in the lung by extracellular proteinases secreted by Aspergillus fumigatus is thought to play a significant role in invasive aspergillosis. This fungus was found previously to secrete an elastinolytic serine proteinase and a metalloproteinase. We report that A. fumigatus also secretes an aspartic proteinase (aspergillopepsin F) that can catalyze hydrolysis of the major structural proteins of basement membrane, elastin, collagen, and laminin. The pH optimum fo...

  5. Differential expression of the putative Kex2 processed and secreted aspartic proteinase gene family of Cryphonectria parasitica.

    Science.gov (United States)

    Jacob-Wilk, Debora; Moretti, Marino; Turina, Massimo; Kazmierczak, Pam; Van Alfen, Neal K

    2012-03-01

    Kex2-silenced strains of Cryphonectria parasitica, the ascomycete causal agent of chestnut blight, show a significant reduction in virulence, reduced sexual and asexual sporulation and reductions in mating and fertility. Due to this and the known involvement of Kex2 in the processing of important proproteins in other systems, we searched the whole C. parasitica genome for putative Kex2 substrates. Out of 1299 open reading frames (ORFs) predicted to be secreted, 222 ORFs were identified as potential Kex2 substrates by this screen. Within the putative substrates we identified cell wall modifying proteins, putative proteinases, lipases, esterases, and oxidoreductases. This in silico screen also uncovered a family of nine secreted aspartic proteinases (SAPs) of C. parasitica. Northern blot analyses of this gene family showed differential expression when exposed to chestnut wood and Cryphonectria hypovirus 1 (CHV1). Due to the reduction in fungal virulence known to be caused upon hypoviral infection of C. parasitica, the differential gene expression observed, and the known involvement of SAPs in virulence in other systems, we conducted deletion analyses of four of these proteinases, representing different expression patterns. Deletion of each of the four SAPs did not affect growth rates, sporulation or virulence, suggesting that none of the considered SAPs is essential for the full development or virulence of C. parasitica under the conditions tested. PMID:22385619

  6. Structure-function relationships of proteinase from soybean (Bowman-Birk) and lima bean: modification by N-acetylimidazole

    Energy Technology Data Exchange (ETDEWEB)

    Kay, E.

    1978-01-01

    Contributions of tyrosyl residues to trypsin- and chymotrypsin-inhibitory activities in two homologous proteinase inhibitors were investigated by modifying them with N-acetylimidazole under various conditions. In Bowman-Birk soybean proteinase inhibitor, Tyr 55, immediately following the antichymotryptic site Leu 53-Ser 54, is relatively inaccessible to N-acetylimidazole and can only be acetylated in the presence of 6 M guanidine hydrochloride but not in 8 M urea. The acetylation of Tyr 55 is accompanied by 60% loss in antichymotryptic activity. Deacetylation with hydroxylamine restores the activity to the original level. Tyr 69, located in the antitrypsin portion of the inhibitor, is relatively exposed to N-acetylimidazole and can be acetylated without denaturing agent. The acetylation of Tyr 69 parallels decrease in antitryptic activity. The inhibitor acetylated at Tyr 69 is fully active toward chymotrypsin and has 30 to 40% antitryptic activity of the native. The original level of antitryptic activity is restored upon deacetylation. Tyr 69 of lima bean proteinase inhibitor is relatively inaccessible to N-acetylimidazole: 75% acetylation in the presence of 6 M guanidine hydrochloride and 17% without the denaturing agent. The acetylated inhibitor is fully active toward chymotrypsin but retains only 29% (acetylated without guanidine hydrochloride) and 17% (acetylated with guanidine hydrochloride) of the original antitryptic activity. Deacetylation partially restores the lost antitryptic activity in the inhibitor acetylated without the denaturing agent.

  7. Extracellular serine-proteinases isolated from Streptomyces alboniger: Partial characterization and effect of aprotinin on cellular structure

    Directory of Open Access Journals (Sweden)

    Lopes Andréa

    1999-01-01

    Full Text Available Streptomyces alboniger ATCC 12461 grown in brain heart infusion (BHI medium produced two extracellular serine-proteinases, denoted SP I and SP II, which were purified by ammonium sulfate precipitation and aprotinin-agarose affinity chromatography. SP I was purified 88,9-fold and SP II 66,7- fold, with 33.4% and 10.4% yield, respectively. The optimum pH for the proteinases activity, using a-N-p-tosyl-L-arginine-methyl ester (TAME as substrate, was 9-10 and the optimum temperature was 37ºC. The proteolytic activity of SP I and SP II was inhibited by aprotinin and SP I was partially inhibited by leupeptin, both serine-proteinase inhibitors. S. alboniger growth in BHI-liquid medium decreased when 5 mg/ml, 10 mg/ml of aprotinin was used, being completely inhibited with 20 mg/ml and 40 mg/ml. At the ultrastructural level, aprotinin-treated S. alboniger cells showed swelling of the bacterial body and condensation of the genetic material, probably related to the inhibition of its growth.

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

    Science.gov (United States)

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

    2015-04-01

    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 (? > 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. PMID:25781213

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

    Energy Technology Data Exchange (ETDEWEB)

    Bezerra, A. G. [Universidade Tecnologica Federal do Parana, Departamento Academico de Fisica (Brazil); Barison, A. [Universidade Federal do Parana, Departamento de Quimica (Brazil); Oliveira, V. S. [Universidade Federal do Parana, Departamento de Fisica (Brazil); Foti, L.; Krieger, M. A. [Fundacao Oswaldo Cruz, Instituto de Biologia Molecular do Parana (Brazil); Dhalia, R.; Viana, I. F. T. [Fundacao Oswaldo Cruz, Centro de Pesquisas Aggeu Magalhaes (Brazil); Schreiner, W. H., E-mail: wido@fisica.ufpr.br [Universidade Federal do Parana, Departamento de Fisica (Brazil)

    2012-09-15

    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 {mu}M 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 V{sub 2}O{sub 5} form.

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

    International Nuclear Information System (INIS)

    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 ?M 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.

  11. Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

    Directory of Open Access Journals (Sweden)

    Araki Hiromasa

    2007-04-01

    Full Text Available Abstract Background Proteinase-activated receptors (PARs; PAR1–4 that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, ?-smooth muscle actin (?-SMA for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells. Results Stimulation of PAR with thrombin (1 U/ml or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 ?M for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased ?-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-? (TGF-?. Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation.

  12. 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

    Energy Technology Data Exchange (ETDEWEB)

    Simmons, Chad R. [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States); Hao, Quan [MacCHESS at the Cornell High Energy Synchrotron Source, Cornell University, Ithaca, NY 14853-8001 (United States); Stipanuk, Martha H., E-mail: mhs6@cornell.edu [Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853-8001 (United States)

    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, ? = ? = ? = 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.

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

    Science.gov (United States)

    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

  14. SENSORY ANALYSIS OF A MODEL SYSTEM USING 5'-IMP AND CYSTEINE AT DIFFERENT pH

    Directory of Open Access Journals (Sweden)

    MADRUGA Marta Suely

    1998-01-01

    Full Text Available Sensory analysis was used to get an overall flavour description of a reaction mixtures containing 5'-IMP and Cysteine. Ribose/cysteine systems were used as reference systems. Results from triangle and aroma profiling show a clear correlation between the terms used and the volatile analysis described in literature for these model systems. For instance reactions at pH 3.0 and 4.5 for 5'-IMP/cysteine systems, which were described as "meaty" and "boiled meat" by panellists, presented, in the literature, the higher number of "meaty" compounds in volatile analysis (1, 7, 8, 20 .

  15. Antifungal defensins and their role in plant defense

    OpenAIRE

    PATRICIABARBOSAPELEGRINI; ArianeLacerda; Maria FatimaGrossi-de-Sa

    2014-01-01

    Since the beginning of the 90s lots of cationic plant, cysteine-rich antimicrobial peptides (AMP) have been studied. However, Broekaert et al. (1995) only coined the term “plant defensin,” after comparison of a new class of plant antifungal peptides with known insect defensins. From there, many plant defensins have been reported and studies on this class of peptides encompass its activity toward microorganisms and molecular features of the mechanism of action against bacteria and fungi. Plant...

  16. Soilborne wheat mosaic virus (SBWMV 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing

    Directory of Open Access Journals (Sweden)

    Howard Amanda

    2005-03-01

    Full Text Available Abstract Amino acid sequence analyses indicate that the Soilborne wheat mosaic virus (SBWMV 19K protein is a cysteine-rich protein (CRP and shares sequence homology with CRPs derived from furo-, hordei-, peclu- and tobraviruses. Since the hordei- and pecluvirus CRPs were shown to be pathogenesis factors and/or suppressors of RNA silencing, experiments were conducted to determine if the SBWMV 19K CRP has similar activities. The SBWMV 19K CRP was introduced into the Potato virus X (PVX viral vector and inoculated to tobacco plants. The SBWMV 19K CRP aggravated PVX-induced symptoms and restored green fluorescent protein (GFP expression to GFP silenced tissues. These observations indicate that the SBWMV 19K CRP is a pathogenicity determinant and a suppressor of RNA silencing.

  17. Susceptibility of Agrotis segetum (noctuidae) to Bacillus thuringiensis and analysis of midgut proteinases.

    Science.gov (United States)

    Ben Hamadou-Charfi, Dorra; Sauer, Annette Juliane; Abdelkefi-Mesrati, Lobna; Tounsi, Slim; Jaoua, Samir; Stephan, Dietrich

    2015-01-01

    Seventy-eight Bacillus thuringiensis isolates were selected for a screening against the Lepidoptera species Agrotis segetum to search the higher insecticidal activity. In a preliminary bioassay, the spore-crystal mixture of 78 B. thuringiensis isolates was tested against L1 larvae of A. segetum. Fifty-two isolates had more than 60% corrected mortality after 3 days. Seven isolates caused a corrected mortality of 100% on A. segetum. Twelve isolates were selected for a second bioassay investigating the effect of the vegetative insecticidal protein (Vip) against third-instar larvae. After 7 days, the weight gain and the larval stage of each larva were recorded. This bioassay showed an aberration in larval growth increases, morphology, and weight gain. After plasmid pattern analysis, the most active strains are most likely B. thuringiensis kurstaki strains expressing the Vip3A toxin. The absence of two proteinase activities observed in the case of Cry1Ac would be the consequence of the difference in susceptibility of A. segetum to the toxins used. PMID:24839868

  18. Proton NMR spectroscopy of the active site histidine of ?-lytic proteinase

    International Nuclear Information System (INIS)

    A histidine auxotroph of Lysobacter enzymogenes (ATC 29847) was grown on media containing either isotopically labeled [90% 13Cesup(epsilon)]L- or [90%15Nsup(delta), 90% 15Nsup(epsilon)]D,L-histidine. The enzyme, ?-lytic proteinase (EC 3.4.21.12), was isolated from these cultures as well as from cultures of wild-type bacteria grown on unlabeled medium. 1H NMR spectra at 360 MHz were obtained with all 3 purified enzymes. Presence of the adjacent 15N labels broadened the histidine Csup(epsilon)-H peak by about a factor of 2 by unresolved scalar coupling. Presence of a direcly bonded 13C led to disappearance of the histidine Csup(epsilon)-H peak by a combination of scalar coupling and dipolar broadening. These effects should be useful for the cross-assignment of 1H NMR peaks of 13C and 15N enriched proteins. The 13C and 15N labeled proteins were found to undergo the reversible a-b conformational transition which changes the pKsub(a)' of His57 from 6.5-5.9. (Auth.)

  19. Elastase-induced emphysema: retention of instilled proteinase in the rat

    International Nuclear Information System (INIS)

    Airway instillation of proteinases with the ability to degrade elastin has been used to produce disease in the rat analogous to human pulmonary emphysema. This study examined the retention, localization, and fate of endotracheally instilled elastase using 125I labeled enzyme and immunoperoxidase histochemistry. Porcine pancreatic elastase labeled with 125I was detected in rat lungs through 96 h after instillation; over half of the label was still present after 7 h. Similar results were obtained when elastase was reacted with a specific, catalytic site inactivator prior to instillation. Trypsin and denatured elastase, however, were cleared much more rapidly from the lung (less than half of the label present after 30 min). When lungs were homogenized after instillation of active elastase, the soluble fraction contained elastase bound to rat alpha1-antitrypsin. In addition, a small amount of label (less than 10%) appeared bound to insoluble components for extended periods of time. Using immunoperoxidase histochemistry, it was found that exogenous elastase was rapidly contained with pulmonary alveolar macrophages, as well as associated with alveolar septums and other parenchymal structures. Similar results were obtained with elastase from both porcine pancreas and human neutrophils. These results suggest that exogenous elastase in the rat, and perhaps endogenous elastolytic enzymes in humans, may have several fates in the lungs: complex formation with endogenous inhibitors, containment within the macrophage, and/or association with connective tissue targets

  20. Proteinase 3 carries small unusual carbohydrates and associates with ?lpha-defensins

    DEFF Research Database (Denmark)

    Zoega, Morten; Ravnsborg, Tina

    2012-01-01

    The neutrophil granulocyte is an important first line of defense against intruding pathogens and it contains a range of granules armed with antibacterial peptides and proteins. Proteinase 3 (PR3) is one among several serine proteases of the azurophilic granules in neutrophil granulocytes. Here, we characterize the glycosylation of PR3 and its association with antimicrobial human neutrophil peptides (HNPs, ?-defensins) and the effect of these on the mechanism of inhibition of the major plasma inhibitor of PR3, ?1-antitrypsin. The glycosylation of purified, mature PR3 showed some heterogeneity with carbohydrates at Asn 102 and 147 carrying unusual small moieties indicating heavy processing. Mass spectrometric analysis and immuno blotting revealed strong association of highly purified PR3 with ?-defensins and oligomers hereof. Irreversible inhibition of PR3 by ?1-antitrypsin did not affect its association with defensins. Other proteins from neutrophil granules were also found to be associated with defensins, whereas purified plasma proteins did not carry defensins. These results point to a role of defensins in controlling and targeting the activity of neutrophil granule proteins.

  1. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage.

    Science.gov (United States)

    Steinberger, Jutta; Grishkovskaya, Irina; Cencic, Regina; Juliano, Luiz; Juliano, Maria A; Skern, Tim

    2014-11-01

    Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pro) (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4 G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells. PMID:25240326

  2. Computational study of some benzamidine-based inhibitors of thrombin-like snake venom proteinases

    Science.gov (United States)

    Henriques, Elsa S.; Nascimento, Marco A. C.; Ramos, Maria João

    Pit viper venoms contain a number of serine proteinases that, despite their observed coagulant thrombin-like action in vitro, exhibit a paradoxical benign defibrinogenating (anticoagulant) action in vivo, with clinical applications in preventing thrombi and improved blood circulation. Considering that several benzamidine-based inhibitors, some highly selective to thrombin, also inhibit the enzymatic activity of such venombins, the modeling of their enzyme-inhibitor interactions could provide valuable information on the topological factors that determine the divergences in activity. The first step, and the object of the present study, was to derive the necessary set of parameters, consistent with the CHARMM force field, and to perform molecular dynamics (MD) simulations on a few selected representatives of the inhibitors in question under physiological conditions. Bonding and van der Waals parameters were derived by analogy to similar ones in the existing force field. Net atomic charges were obtained with a restrained fitting to the molecular electrostatic potential generated at B3LYP/6-31G(d) level. The parameters were refined to reproduce the available experimental geometries and crystal data, and the MD simulations of the free inhibitors in aqueous solution at 298 K provided an insightful description of their available conformational space.

  3. Proteinase activated-receptors-associated signaling in the control of gastric cancer.

    Science.gov (United States)

    Sedda, Silvia; Marafini, Irene; Caruso, Roberta; Pallone, Francesco; Monteleone, Giovanni

    2014-09-14

    Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior. PMID:25232234

  4. Conformational changes of ovine ?-1-proteinase inhibitor: The influence of heparin binding

    Science.gov (United States)

    Gupta, Vivek Kumar; Gowda, Lalitha R.

    2008-11-01

    ?-1-Proteinase inhibitor (?-1-PI), the archetypal serpin causes rapid, irreversible stoichiometric inhibition of redundant circulating serine proteases and is associated with emphysema, inflammatory response and maintenance of protease-inhibitor equilibrium in vascular and peri-vascular spaces. A homogenous preparation of heparin octasaccharide binds to ovine and human ?-1-PI and enhances their protease inhibitory activity phenomenally. Size-exclusion chromatography and dynamic light scattering experiments reveal that ovine ?-1-PI undergoes a decrease in the Stokes' radius upon heparin binding. A strong binding; characterizes this ?-1-PI-heparin interaction as revealed by the binding constant ( K?) 1.98 ± 0.2 × 10 -6 M and 2.1 ± 0.2 × 10 -6 M determined by fluorescence spectroscopy and equilibrium dialysis, respectively. The stoichiometry of heparin binding to ovine ?-1-PI was 1.1 ± 0.2:1. The Stern-Volmer constants ( Ksv) for heparin activated ovine and human ?-1-PI were found to be 5.13 × 10 -6 M and 5.67 × 10 -6 M, respectively, significantly higher than the native inhibitors. FTIR and CD spectroscopy project the systematic structural reorientations that ?-1-PI undergoes upon heparin binding characterized by a decrease in ?-helical content and a concomitant increase in ?-turn and random coil elements. It is likely that these conformational changes result in the movement of the ?-1-PI reactive site loop into an extended structure that is better poised to combat the cognate protease and accelerate the inhibition.

  5. Pulmonary penetration of alpha 1-proteinase inhibitor administered parenterally to dogs

    International Nuclear Information System (INIS)

    To study the penetration of alpha 1-proteinase inhibitor (A1Pl) into the lungs of healthy dogs, 83 mg/kg of active A1Pl was administered intravenously over 30 min followed by a bolus of 131I-A1Pl. Animals were lavaged 2 to 72 h after infusion, sequential gamma camera scans were acquired, and urine was analyzed for the excretion of desmosine. After a distribution phase, infused A1Pl left the bloodstream with a half-life of 103 +/- 24 h. Analysis of plasma antiprotease activity demonstrated preservation of function of the infused A1Pl. Lavage fluid A1Pl concentration and activity were significantly increased 24 h after infusion. Gamma camera scans demonstrated that lung, liver, and spleen acquired 131I-A1Pl similarly; radioactivities per gram of tissue of these organs were similar at autopsy. Excretion of desmosine did not decrease from a baseline of 157 +/- 59 nmol/24 h after A1Pl infusion, indicating no effect of A1Pl infusion on background elastolysis. These data suggest that intravenous administration of A1Pl can raise lung antiproteinase levels within 24 h despite the absence of preferential uptake by the lung of the infused protein

  6. Purification and partial characterization of ?1-proteinase inhibitor in the common marmoset (Callithrix jacchus).

    Science.gov (United States)

    Parambeth, Joseph Cyrus; Suchodolski, Jan S; Steiner, Jörg M

    2015-04-01

    Fecal alpha1-proteinase inhibitor (?1-PI) concentration has been to diagnose enteric protein loss in dogs and cats. Chronic lymphocytic enteritis is commonly seen in the marmoset (Callithrix jaccus) and is characterized by hypoalbuminemia. As a prelude to immunoassay development for detecting enteric protein loss, marmoset serum ?1-PI was purified using immunoaffinity chromatography and ceramic hydroxyapatite chromatography. Partial characterization was performed by reducing gel electrophoresis and enzyme inhibitory assays. Protein identity was confirmed with peptide mass fingerprinting and N-terminal amino acid sequencing. Molecular mass, relative molecular mass, and isoelectric point for marmoset ?1-PI were 54?kDa, 51,677, and 4.8-5.4, respectively. Trypsin, chymotrypsin, and elastase inhibitory activity were observed. N-terminal amino acid sequence for marmoset ?1-PI was EDPQGDAAQKMDTSHH. In conclusion, marmoset ?1-PI was successfully purified from serum with an overall yield of 12% using a rapid and efficient method. Purified marmoset ?1-PI has characteristics similar to those of ?1-PI reported for other species. PMID:25745866

  7. Molecular cloning of the rat proteinase-activated receptor 4 (PAR4

    Directory of Open Access Journals (Sweden)

    Zou Lei

    2002-02-01

    Full Text Available Abstract Background The proteinase-activated receptor 4 (PAR4 is a G-protein-coupled receptor activated by proteases such as thrombin and trypsin. Although activation of PAR4 has been shown to modulate rat gastrointestinal motility, the rat PAR4 sequence was unknown until now. This study aimed to identify the rat PAR4 cDNA. Results The cDNA coding for the rat PAR4 homologue was cloned from the duodenum. Northern blots demonstrated a 3.0 kb transcript in the duodenum. Protein homology with mouse and human counterparts was 90% and 75% respectively. PAR4 is expressed predominantly in the esophagus, stomach, duodenum and the spleen. When expressed in COS cells, PAR4 is activated by trypsin (1 nM, thrombin (50 nM, mouse PAR4 specific peptide (500 ?M and a putative rat PAR4 specific activating peptide (100 ?M, as measured by intracellular Ca2+-changes. Conclusions We have identified and characterized cDNA encoding the rat PAR4 homologue. PAR4 is expressed predominantly in the upper gastrointestinal tract. It is activated by trypsin, thrombin and its newly identified rat PAR4 specific activating peptide.

  8. Enhanced Arabidopsis pattern-triggered immunity by overexpression of cysteine-rich receptor-like kinases.

    Science.gov (United States)

    Yeh, Yu-Hung; Chang, Yu-Hsien; Huang, Pin-Yao; Huang, Jing-Bo; Zimmerli, Laurent

    2015-01-01

    Upon recognition of microbe-associated molecular patterns (MAMPs) such as the bacterial flagellin (or the derived peptide flg22) by pattern-recognition receptors (PRRs) such as the FLAGELLIN SENSING2 (FLS2), plants activate the pattern-triggered immunity (PTI) response. The L-type lectin receptor kinase-VI.2 (LecRK-VI.2) is a positive regulator of Arabidopsis thaliana PTI. Cysteine-rich receptor-like kinases (CRKs) possess two copies of the C-X8-C-X2-C (DUF26) motif in their extracellular domains and are thought to be involved in plant stress resistance, but data about CRK functions are scarce. Here, we show that Arabidopsis overexpressing the LecRK-VI.2-responsive CRK4, CRK6, and CRK36 demonstrated an enhanced PTI response and were resistant to virulent bacteria Pseudomonas syringae pv. tomato DC3000. Notably, the flg22-triggered oxidative burst was primed in CRK4, CRK6, and CRK36 transgenics and up-regulation of the PTI-responsive gene FLG22-INDUCED RECEPTOR-LIKE 1 (FRK1) was potentiated upon flg22 treatment in CRK4 and CRK6 overexpression lines or constitutively increased by CRK36 overexpression. PTI-mediated callose deposition was not affected by overexpression of CRK4 and CRK6, while CRK36 overexpression lines demonstrated constitutive accumulation of callose. In addition, Pst DC3000-mediated stomatal reopening was blocked in CRK4 and CRK36 overexpression lines, while overexpression of CRK6 induced constitutive stomatal closure suggesting a strengthening of stomatal immunity. Finally, bimolecular fluorescence complementation and co-immunoprecipitation analyses in Arabidopsis protoplasts suggested that the plasma membrane localized CRK4, CRK6, and CRK36 associate with the PRR FLS2. Association with FLS2 and the observation that overexpression of CRK4, CRK6, and CRK36 boosts specific PTI outputs and resistance to bacteria suggest a role for these CRKs in Arabidopsis innate immunity. PMID:26029224

  9. Controllable synthesis of TiO2 nanomaterials by assisting with l-cysteine and ethylenediamine

    KAUST Repository

    Tao, Yugui

    2013-11-21

    This paper reports a facile l-cysteine-assisted solvothermal synthesis of TiO2 nanomaterials using ethylenediamine (En) and distilled water as solvent. The influence of reaction time, temperature, l-cysteine and solvent was initially investigated. Results demonstrated the reaction temperature, l-cysteine and En significantly imposed impact on the phase and morphology of the particles. Amorphous nanosheets, mixed-crystal nanorods and pure anatase nanoparticles were controllably synthesized by varying reaction temperature. The formation of the amorphous nanosheets and mixed-crystal nanorods were directly affected by the presence of l-cysteine and En. And the presence of En distinctly affected the crystal phase of the products, which was rarely mentioned in other studies. Moreover, the photocatalytic activities of three typical samples were excellent. The possible formation mechanism of the sample was also discussed. © 2013 Springer Science+Business Media New York.

  10. Cysteine-Free Proteins in the Immunobiology of Arthropod-Borne Diseases

    Directory of Open Access Journals (Sweden)

    J. Santiago Mejia

    2010-01-01

    Full Text Available One approach to identify epitopes that could be used in the design of vaccines to control several arthropod-borne diseases simultaneously is to look for common structural features in the secretome of the pathogens that cause them. Using a novel bioinformatics technique, cysteine-abundance and distribution analysis, we found that many different proteins secreted by several arthropod-borne pathogens, including Plasmodium falciparum, Borrelia burgdorferi, and eight species of Proteobacteria, are devoid of cysteine residues. The identification of three cysteine-abundance and distribution patterns in several families of proteins secreted by pathogenic and nonpathogenic Proteobacteria, and not found when the amino acid analyzed was tryptophan, provides evidence of forces restricting the content of cysteine residues in microbial proteins during evolution. We discuss these findings in the context of protein structure and function, antigenicity and immunogenicity, and host-parasite relationships.

  11. Esophagitis Corrosive Treatment of N-acetyl Cysteine Preventing Early Stricture use development activity

    Directory of Open Access Journals (Sweden)

    Tülin Durgun Yetim

    2011-06-01

    Full Text Available Objective: Esophageal stricture early corrosive burns to investigate the effectiveness in preventing the use of N-acetyl-cysteine.Strictures of the esophagus due to caustic substances occur with the ingestion of solid or liquid corrosive materials. Materials and Methods: A prospective randomized study was performed to evaluate the effectiveness of conventional therapy alone and conventional therapy + N-acetyl cysteine. Fifty patients with grade 2b and grade 3 burnt were analyzed. Intensive n-acetyl cysteine therapy can reduce the incidence of stricture development in patients with advanced grade corrosive esophagitis.Results: N-acetyl cysteine group+ Konvansitonel used in 1 patient, stenosis developed in 7 patients in the group with conventional treatment. Conclusion: We believe that our findings will be better supported with the groups including larger number of patients or with the experimental studies.

  12. Synaptic dysfunctions and activity-dependent neurodegeneration in mice lacking cystein string protein-alpha

    OpenAIRE

    Rozas, José Luis; Gómez-Sánchez, L.; Linares-Clemente, P.; Vázquez, E; Luján, Rafael; Fernández-Chacón, R

    2011-01-01

    Cysteine string protein-alpha (CSP-?) is a synaptic vesicle protein that prevents presynaptic neurodegeneration. CSP-? KO mice suffer from a lethal neurological phenotype after the second postnatal week.

  13. Identification and preliminary characterization of protein-cysteine farnesyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Manne, V.; Roberts, D.; Tobin, A.; O' Rourke, E.; Barbacid, M.; De Virgilio, M.; Meyers, C. (Squibb Institute for Medical Research, Princeton, NJ (USA)); Ahmed, N.; Kurz, B.; Resh, M. (Princeton Univ., NJ (USA)); Kung, Hsiang-Fu (National Cancer Institute-Frederic Cancer Research Facility, MD (USA))

    1990-10-01

    Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus in order to exert their biological activity. Previous studies indicate that an intermediate in the mevalonate pathway, most likely farnesyl pyrophosphate, is the donor of this isoprenyl group. Inhibition of mevalonate synthesis reverts the abnormal phenotypes induced by the mutant RAS2{sup Val{endash}19} gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus oocytes induced by an onocogenic Ras p21 protein of human origin. These results have raised the possibility of using inhibitors of the mevalonate pathway to block the transforming properties of ras oncogenes. Unfortunately, mevalonate is a precursor of various end products essential to mammalian cells, such as dolichols, ubiquinones, heme A, and cholesterol. In this study, the authors describe an enzymatic activity(ies) capable of catalyzing the farnesylation of unprocessed Ras p21 proteins in vitro at the correct (Cys-186) residue. Gel filtration analysis of a partially purified preparation of protein farnesyltransferase revealed two peaks of activity at 250-350 kDa and 80-130 kDa. Availability of an in vitro protein farnesyltransferase assay should be useful in screening for potential inhibitors of ras oncogene function that will not interfere with other aspects of the mevalonate pathway.

  14. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin molecule were identified by N-terminal sequencing of the different proteolytic fragments. Desmin incubated with either m-calpain or mu-calpain was primarily cleaved in the head and tail region leaving the rod domain relatively intact even after prolonged incubation. Incubation with cathepsin B produces a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved in degradation of desmin early post-mortem, targeting the non-helical region of the desmin molecule and resulting in depolymerisation and initial disorganisation of the intermediate filament structures of the muscle cell.

  15. The oxidation of cysteine by aqueous ferricyanide: A kinetic study using boron doped diamond electrode voltammetry

    OpenAIRE

    Nekrassova, O; Allen, GD; Lawrence, NS; Jiang, L.; Jones, TGJ; Compton, RG

    2002-01-01

    The oxidation of cysteine by ferricyanide is well established. However, electrochemical studies of the mechanism have to date been underdeveloped due to the lack of resolution between the oxidation waves of the ferrocyanide and cysteine on most electrode materials. It is shown that on boron doped diamond electrode, unlike for example platinum electrodes, the voltammetric responses of the ferrocyanide wave and the targets are sufficiently different, with the former at a lower oxidizing potenti...

  16. Micronutrients, N-acetyl cysteine, probiotics and prebiotics, a review of effectiveness in reducing HIV progression

    OpenAIRE

    Ruben Hummelen; Jaimie Hemsworth; Gregor Reid

    2010-01-01

    Low serum concentrations of micronutrients, intestinal abnormalities, and an inflammatory state have been associated with HIV progression. These may be ameliorated by micronutrients, N-acetyl cysteine, probiotics, and prebiotics. This review aims to integrate the evidence from clinical trials of these interventions on the progression of HIV. Vitamin B, C, E, and folic acid have been shown to delay the progression of HIV. Supplementation with selenium, N-acetyl cysteine, probiotics, and prebio...

  17. Myeloid-derived Suppressor Cells Inhibit T Cell Activation by Depleting Cystine and Cysteine

    OpenAIRE

    Srivastava, Minu K.; Sinha, Pratima; Clements, Virginia K.; RODRIGUEZ, PAULO; Ostrand-Rosenberg, Suzanne

    2009-01-01

    Myeloid-derived suppressor cells (MDSC) are present in most cancer patients and are potent inhibitors of T-cell-mediated anti-tumor immunity. Their inhibitory activity is attributed to production of arginase, reactive oxygen species, inducible nitric oxide synthase, and IL-10. We now report that MDSC also block T cell activation by sequestering cystine and limiting the availability of cysteine. Cysteine is an essential amino acid for T cell activation because T cells lack cystathionase, which...

  18. Immunoprotection in goats against Haemonchus contortus after immunization with cysteine protease enriched protein fractions

    OpenAIRE

    Ruiz, Antonio; Molina, José; González, Jorge; Conde, Magnolia; Martín, Sergio; Hernández, Yeray

    2004-01-01

    Haemonchus cysteine proteases, because of their apparent critical function in worm physiology, are considered important candidates in the immunological control of haemonchosis in sheep. Only limited information is, however, available on the immunoprotective properties of these molecules in goats. In the present study cysteine proteases of Haemonchus contortus adult worms isolated from a goat strain (Gran Canaria, Spain) were enriched by affinity chromatography and evaluated as immunoprotectiv...

  19. Axial and equatorial ligand effects on biomimetic cysteine dioxygenase model complexes

    OpenAIRE

    Gonzalez-Ovalle, Luis E.; Quesne, Matthew G; Kumar, Devesh; Goldberg, David P.; de Visser, Sam P.

    2012-01-01

    Density functional theory (DFT) calculations are presented on biomimetic model complexes of cysteine dioxygenase and focus on the effect of axial and equatorial ligand placement. Recent studies of one of us [Y. M. Badiei, M. A. Siegler and D. P. Goldberg, J. Am. Chem. Soc. 2011, 133, 1274] gave evidence of a nonheme iron biomimetic model of cysteine dioxygenase using an i-propyl-bis(imino)pyridine, equatorial tridentate ligand. Addition of thiophenol, an anion – either chloride or triflate – ...

  20. Topography of the cysteine residues in DNA-dependent RNA polymerase

    International Nuclear Information System (INIS)

    Chemical modification of DNA-dependent RNA polymerase with radioactive monoiodo[14C]acetic acid and N-[14C]ethylmaleamide has been performed. The positions of the exposed and functionally important cysteine residues in the enzyme have been determined. The topography of the cysteine residues in the ? subunit of RNA polymerase has been studied in detail. The results obtained are summarized in the form of a model

  1. Reaction of acetaldehyde with cysteine and its potential significance in alcoholic liver disease

    International Nuclear Information System (INIS)

    Cysteine is important as a precursor for the synthesis of glutathione and in regeneration of glutathione during the ?-glutamyl cycle. The reaction of ?-aminothiols like cysteine with aldehydes to form thiazolidines is well known. Cysteine reacts rapidly with acetaldehyde, the first oxidation product of ingested ethanol, in PBS at 37deg C, pH 7.4 to form 2-thiazolidine-4-carboxylic acid (TC) (k2 = 5.8 M-1 sec-1), TC is stable under the same conditions: kobs = 4.6 x 10-2 h-1 (t1/2 = 15 hours) for ring opening. These rate constants suggest that appreciable amounts of TC may form in liver during ethanol metabolism, potentially decreasing the available concentration of cysteine for glutathione synthesis. Using [2-13C] ethanol, NAD+ and an NAD+ regenerating system, alcohol dehydrogenase and cysteine, TC labelled with 13C in the methyl carbon can be produced quantitatively. Use of a polarization transfer sequence allows observation of only the methyl protons by proton NMR. The effect of rat liver homogenate on the formation and stability of TC will be described. Similarly the effect of replacement of cysteine by its ?,? -dimethyl analog, penicillamine, will be discussed

  2. Pyridoxal-phosphate dependent mycobacterial cysteine synthases: Structure, mechanism and potential as drug targets.

    Science.gov (United States)

    Schnell, Robert; Sriram, Dharmarajan; Schneider, Gunter

    2015-09-01

    The alarming increase of drug resistance in Mycobacterium tuberculosis strains poses a severe threat to human health. Chemotherapy is particularly challenging because M. tuberculosis can persist in the lungs of infected individuals; estimates of the WHO indicate that about 1/3 of the world population is infected with latent tuberculosis providing a large reservoir for relapse and subsequent spread of the disease. Persistent M. tuberculosis shows considerable tolerance towards conventional antibiotics making treatment particularly difficult. In this phase the bacilli are exposed to oxygen and nitrogen radicals generated as part of the host response and redox-defense mechanisms are thus vital for the survival of the pathogen. Sulfur metabolism and de novo cysteine biosynthesis have been shown to be important for the redox homeostasis in persistent M. tuberculosis and these pathways could provide promising targets for novel antibiotics for the treatment of the latent form of the disease. Recent research has provided evidence for three de novo metabolic routes of cysteine biosynthesis in M. tuberculosis, each with a specific PLP dependent cysteine synthase with distinct substrate specificities. In this review we summarize our present understanding of these pathways, with a focus on the advances on functional and mechanistic characterization of mycobacterial PLP dependent cysteine synthases, their role in the various pathways to cysteine, and first attempts to develop specific inhibitors of mycobacterial cysteine biosynthesis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. PMID:25484279

  3. Electrochemical behaviour of dopamine at covalent modified glassy carbon electrode with l-cysteine: preliminary results

    Scientific Electronic Library Online (English)

    Carlos Alberto, Martínez-Huitle; Monica, Cerro-Lopez; Marco Antonio, Quiroz.

    Full Text Available The surface of glassy carbon (GC) electrode has been modified by oxidation of L-cysteine. The covalent modified GC electrode with L-Cysteine has been studied, according the supporting electrolyte used. Favourable interactions between the L-cysteine film and DA enhance the current response compared t [...] o that at the Nafion GC and bare GC electrodes, achieving better performances than those other electrodes. This behaviour was as result of the adsorption of the cysteine layer film, compact and uniform formation; depending on L-cysteine solution (phosphate buffer or chloridric acid supporting electrolyte) used for modifying GC surface. In cyclic voltammetric measurements, modified electrodes can successfully separate the oxidation/reduction DA peaks in different buffer solutions, but an evident dependence in the response was obtained as function of pH and modified electrode. The modified electrode prepared with L-cysteine/HCl solution was used to obtain the calibration curve and it exhibited a stable and sensitive response to DA. The results are described and discussed in the light of the existing literature.

  4. Landmark mapping: A general method for localizing cysteine residues within a protein

    International Nuclear Information System (INIS)

    The authors describe a general method to locate the positions of cysteine residues relative to the amino terminus of a protein, using a modified chemical cleavage of the polypeptide backbone at cysteine. The cleavage reaction introduces the carbon atom of 14CN into the carboxyl-terminal fragment produced at each cleavage of the polypeptide chain. Peptides containing the amino terminus of the intact protein are not labeled; all other peptides are labeled at their amino termini. Partial cleavage of a protein followed by gel electrophoresis and autoradiography identifies a ladder of unlabeled peptides that maps positions of the cysteine residues relative to the protein amino terminus. To map individual proteins present in a complex mixture, the polypeptides are cyanolated in solution with 14CN, and the modified proteins are separated by discontinuous SDS/PAGE. The gel is stained, and the desired protein is excised, cleaved at cysteine within the gel slice, and mapped in the second dimension by gel electrophoresis. These techniques are demonstrated with proteins of known sequence containing from zero to five cysteine residues. The cysteine landmark map should be particularly useful in locating protein modifications, in questions of protein similarity, and in mapping functional domains. A strategy is also presented for locating other residues in the polypeptide, for which specific cleavage methods exist

  5. The Tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1

    Science.gov (United States)

    Bar-Ziv, Amalia; Levy, Yael; Citovsky, Vitaly; Gafni, Yedidya

    2015-01-01

    The viral V2 protein is one of the key factors that Tomato yellow leaf curl geminivirus (TYLCV), a major tomato pathogen worldwide, utilizes to combat the host defense. Besides suppressing the plant RNA silencing defense by targeting the host SGS3 component of the silencing machinery, V2 also interacts with the host CYP1 protein, a papain-like cysteine protease likely involved in hypersensitive response reactions. The biological effects of the V2-CYP1 interaction, however, remain unknown. We addressed this question by demonstrating that V2 inhibits the enzymatic activity of CYP1, but does not interfere with post-translational maturation of this protein. PMID:25797621

  6. The Tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1.

    Science.gov (United States)

    Bar-Ziv, Amalia; Levy, Yael; Citovsky, Vitaly; Gafni, Yedidya

    2015-05-01

    The viral V2 protein is one of the key factors that Tomato yellow leaf curl geminivirus (TYLCV), a major tomato pathogen worldwide, utilizes to combat the host defense. Besides suppressing the plant RNA silencing defense by targeting the host SGS3 component of the silencing machinery, V2 also interacts with the host CYP1 protein, a papain-like cysteine protease likely involved in hypersensitive response reactions. The biological effects of the V2-CYP1 interaction, however, remain unknown. We addressed this question by demonstrating that V2 inhibits the enzymatic activity of CYP1, but does not interfere with post-translational maturation of this protein. PMID:25797621

  7. Will transgenic plants adversely affect the environment?

    Indian Academy of Sciences (India)

    Vassili V Velkov; Alexander B Medvinsky; Mikhail S Sokolov; Anatoly I Marchenko

    2005-09-01

    Transgenic insecticidal plants based on Bacillus thuringiensis (Bt) endotoxins, on proteinase inhibitors and on lectins, and transgenic herbicide tolerant plants are widely used in modern agriculture. The results of the studies on likelihood and non-likelihood of adverse effects of transgenic plants on the environment including: (i) effects on nontarget species; (ii) invasiveness; (iii) potential for transgenes to ‘escape’ into the environment by horizontal gene transfer; and (iv) adverse effects on soil biota are reviewed. In general, it seems that large-scale implementation of transgenic insecticidal and herbicide tolerant plants do not display considerable negative effects on the environments and, moreover, at least some transgenic plants can improve the corresponding environments and human health because their production considerably reduces the load of chemical insecticides and herbicides.

  8. Role of L2 cysteines in papillomavirus infection and neutralization

    Directory of Open Access Journals (Sweden)

    Karanam Balasubramanyam

    2009-10-01

    Full Text Available Abstract Vaccination of mice with minor capsid protein L2 or passive transfer with the L2-specific neutralizing monoclonal antibody RG-1 protects against human papillomavirus type 16 (HPV16 challenge. Here we explored the nature of the RG-1 epitope and its contribution to viral infectivity. RG-1 bound equivalently HPV16 L2 residues 17-36 with or without an intact C22-C28 disulphide bridge. HPV16 L2 mutations K20A, C22A, C22S, C28A, C28S, or P29A prevented RG-1 binding, whereas Y19A, K23A or Q24A had no impact. Mutation of either C22 or C28 to alanine or serine compromises HPV16 pseudoviral infectivity both in vitro and in the murine vaginal tract, but does not impact pseudovirion assembly. Despite their lack of infectivity, HPV16 pseudovirions containing C22S or C28S mutant L2 bind to cell surfaces, are taken up, and expose the 17-36 region on the virion surface as for wild type HPV16 pseudovirions suggesting normal furin cleavage of L2. Mutation of the second cysteine residue in Bovine papillomavirus type 1 (BPV1 L2 to serine (C25S dramatically reduced the infectivity of BPV1 pseudovirions. Surprisingly, in contrast to the double mutation in HPV16 L2, the BPV1 L2 C19S, C25S double mutation reduced BPV1 pseudovirion infectivity of 293TT cells by only half.

  9. 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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis.

    Science.gov (United States)

    Millet, Arnaud; Martin, Katherine R; Bonnefoy, Francis; Saas, Philippe; Mocek, Julie; Alkan, Manal; Terrier, Benjamin; Kerstein, Anja; Tamassia, Nicola; Satyanarayanan, Senthil Kumaran; Ariel, Amiram; Ribeil, Jean-Antoine; Guillevin, Loïc; Cassatella, Marco A; Mueller, Antje; Thieblemont, Nathalie; Lamprecht, Peter; Mouthon, Luc; Perruche, Sylvain; Witko-Sarsat, Véronique

    2015-11-01

    Granulomatosis with polyangiitis (GPA) is a systemic necrotizing vasculitis that is associated with granulomatous inflammation and the presence of anti-neutrophil cytoplasmic antibodies (ANCAs) directed against proteinase 3 (PR3). We previously determined that PR3 on the surface of apoptotic neutrophils interferes with induction of antiinflammatory mechanisms following phagocytosis of these cells by macrophages. Here, we demonstrate that enzymatically active membrane-associated PR3 on apoptotic cells triggered secretion of inflammatory cytokines, including granulocyte CSF (G-CSF) and chemokines. This response required the IL-1R1/MyD88 signaling pathway and was dependent on the synthesis of NO, as macrophages from animals lacking these pathways did not exhibit a PR3-associated proinflammatory response. The PR3-induced microenvironment facilitated recruitment of inflammatory cells, such as macrophages, plasmacytoid DCs (pDCs), and neutrophils, which were observed in close proximity within granulomatous lesions in the lungs of GPA patients. In different murine models of apoptotic cell injection, the PR3-induced microenvironment instructed pDC-driven Th9/Th2 cell generation. Concomitant injection of anti-PR3 ANCAs with PR3-expressing apoptotic cells induced a Th17 response, revealing a GPA-specific mechanism of immune polarization. Accordingly, circulating CD4+ T cells from GPA patients had a skewed distribution of Th9/Th2/Th17. These results reveal that PR3 disrupts immune silencing associated with clearance of apoptotic neutrophils and provide insight into how PR3 and PR3-targeting ANCAs promote GPA pathophysiology. PMID:26436651

  11. Regulation of neutrophilic inflammation by proteinase-activated receptor 1 during bacterial pulmonary infection.

    Science.gov (United States)

    José, Ricardo J; Williams, Andrew E; Mercer, Paul F; Sulikowski, Michal G; Brown, Jeremy S; Chambers, Rachel C

    2015-06-15

    Neutrophils are key effector cells of the innate immune response to pathogenic bacteria, but excessive neutrophilic inflammation can be associated with bystander tissue damage. The mechanisms responsible for neutrophil recruitment to the lungs during bacterial pneumonia are poorly defined. In this study, we focus on the potential role of the major high-affinity thrombin receptor, proteinase-activated receptor 1 (PAR-1), during the development of pneumonia to the common lung pathogen Streptococcus pneumoniae. Our studies demonstrate that neutrophils were indispensable for controlling S. pneumoniae outgrowth but contributed to alveolar barrier disruption. We further report that intra-alveolar coagulation (bronchoalveolar lavage fluid thrombin-antithrombin complex levels) and PAR-1 immunostaining were increased in this model of bacterial lung infection. Functional studies using the most clinically advanced PAR-1 antagonist, SCH530348, revealed a key contribution for PAR-1 signaling in influencing neutrophil recruitment to lung airspaces in response to both an invasive and noninvasive strain of S. pneumoniae (D39 and EF3030) but that PAR-1 antagonism did not impair the ability of the host to control bacterial outgrowth. PAR-1 antagonist treatment significantly decreased pulmonary levels of IL-1?, CXCL1, CCL2, and CCL7 and attenuated alveolar leak. Ab neutralization studies further demonstrated a nonredundant role for IL-1?, CXCL1, and CCL7 in mediating neutrophil recruitment in response to S. pneumoniae infection. Taken together, these data demonstrate a key role for PAR-1 during S. pneumoniae lung infection that is mediated, at least in part, by influencing multiple downstream inflammatory mediators. PMID:25948816

  12. Bowman-Birk proteinase inhibitor from Clitoria fairchildiana seeds: Isolation, biochemical properties and insecticidal potential.

    Science.gov (United States)

    Dantzger, Miriam; Vasconcelos, Ilka Maria; Scorsato, Valéria; Aparicio, Ricardo; Marangoni, Sergio; Macedo, Maria Lígia Rodrigues

    2015-10-01

    Herein described is the biochemical characterisation, including in vitro and in vivo assays, for a proteinase inhibitor purified from Clitoria fairchildiana seeds (CFPI). Purification was performed by hydrophobic interaction and gel filtration chromatography. Kinetic studies of the purified inhibitor showed a competitive-type inhibitory activity against bovine trypsin and chymotrypsin, with an inhibition stoichiometry of 1:1 for both enzymes. The inhibition constants against trypsin and chymotrypsin were 3.3×10(-10) and 1.5×10(-10)M, respectively, displaying a tight binding property. SDS-PAGE showed that CFPI has a single polypeptide chain with an apparent molecular mass of 15kDa under non-reducing conditions. However, MALDI-TOF analysis demonstrated a molecular mass of 7.973kDa, suggesting that CFPI is dimeric in solution. The N-terminal sequence of CFPI showed homology with members of the Bowman-Birk inhibitor family. CFPI remained stable to progressive heating for 30min to each temperature range of 37 up to 100°C and CD analysis exhibited no changes in spectra at 207nm after heating at 90°C and subsequent cooling. Moreover, CFPI was active over a wide pH range (2-10). In contrast, reduction with DTT resulted in a loss of inhibitory activity against trypsin and chymotrypsin. CFPI also exhibited significant inhibitory activity against larval midgut trypsin enzymes from Anagasta kuehniella (76%), Diatraea saccharalis (59%) and Heliothis virescens (49%). Its insecticidal properties were further analysed by bioassays and confirmed by negative impact on A. kuehniella development. PMID:26330217

  13. Expression of serine proteinase P186 of Arthrobotrys oligospora and analysis of its nematode-degrading activity.

    Science.gov (United States)

    Zhao, Hailong; Qiao, Jun; Meng, Qingling; Gong, Shasha; Chen, Cheng; Liu, Tianli; Tian, Lulu; Cai, Xuepeng; Luo, Jianxun; Chen, Chuangfu

    2015-12-01

    The nematode-trapping fungi possess a unique capability of predating and invading nematodes. As a representative nematode-trapping fungus, Arthrobotrys oligospora has been widely used to study the interactions between nematode-trapping fungi and their hosts. Serine proteinase is one of the important virulence factors during process of invasion of the nematode-trapping fungi into nematodes. In this study, using reverse transcription polymerase chain reaction, we amplified the gene sequence of serine proteinase 186 from A. oligospora, cloned it into pPIC9K vector and expressed it in the yeast Pichia pastoris. The expressed recombinant serine proteinase186 (reP186) was purified via Ni-affinity chromatography. The in vitro nematode-degrading activity of reP186 was analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis revealed that reP186 with molecular weight of 33 kDa was successfully obtained. ReP186 was capable of degrading a series of protein substrates including casein, gelatin, bovine serum albumin, denatured collagen and nematode cortical layer. The reP186 exhibited the maximal activity at pH 8.0 and 55 °C and was highly sensitive to the inhibitor, phenylmethanesulfonylfluoride. Treatment of Caenorhabditis elegans and Haemonchus contortus with reP186 for 12, 24 and 36 h, respectively, resulted in 62, 88 and 100 % of killing rates for C. elegans, and 52, 65 and 84 % of killing rates for H. contortus, respectively, indicating a relatively strong nematode-degrading bioactivity of reP186. PMID:26419902

  14. VaSP1, catalytically active serine proteinase from Vipera ammodytes ammodytes venom with unconventional active site triad.

    Science.gov (United States)

    Kurtovi?, Tihana; Brgles, Marija; Leonardi, Adrijana; Lang Balija, Maja; Sajevic, Tamara; Križaj, Igor; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2014-01-01

    VaSP1, a serine proteinase from Vipera ammodytes ammodytes venom, is a glycosylated monomer of 31.5 kDa, as determined by MALDI mass spectrometry, showing multiple isoelectric points between pH 6.5 and pH 8.5. Partial amino acid sequencing of VaSP1 by Edman degradation and MS/MS analysis identified sequences which allowed its classification among the so-called snake venom serine proteinase homologues, members of the peptidase S1 family, however being devoid of the canonical catalytic triad. Only few representatives of this group have been identified so far with just two of them characterised in detail at the protein level. Despite substitution of His57 with Arg, VaSP1 possesses proteolytic activity which can be inhibited by Pefabloc, benzamidine, Zn²? ions, DTT and trypsin inhibitor II, a Kunitz/BPTI group member. It hydrolyses N(?)-benzoyl-Phe-Val-Arg-p-NA, exhibiting Michaelis-Menten behaviour with K(m) = 48.2 ?M and V(m) = 0.019 nM s?¹. The pH for optimal activity on tested substrate is around 9.0. VaSP1 also cleaves insulin B-chain, digesting it at positions His¹?-Leu¹¹, Ala¹?-Leu¹? and Tyr¹?-Leu¹?. Furthermore, the novel serine proteinase is active towards wide array of proteins involved in haemostasis where its degradation of fibrinogen, fibrin, prothrombin, factor X and plasminogen in vivo probably results in depletion of coagulation factors in blood circulation. The possibility that VaSP1 possesses anticoagulant properties has been further indicated by its ability to prolong prothrombin time and activated partial thromboplastin time. PMID:24269689

  15. Differential labelling of cysteines for simultaneous identification of thioredoxin h-reducible disulphides in native protein extracts: insight into recognition and regulation of proteins in barley seeds by thioredoxin h

    DEFF Research Database (Denmark)

    Maeda, Kenji; Finnie, Christine; Svensson, Birte

    2005-01-01

    Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridin...

  16. Arabidopsis cysteine-rich receptor-like kinase 45 functions in the responses to abscisic acid and abiotic stresses

    KAUST Repository

    Zhang, Xiujuan

    2013-06-01

    The phytohormone abscisic acid (ABA) regulates seed germination, plant growth and development, and response to abiotic stresses such as drought and salt stresses. Receptor-like kinases are well known signaling components that mediate plant responses to developmental and environmental stimuli. Here, we characterized the biological function of an ABA and stress-inducible cysteine-rich receptor-like protein kinase, CRK45, in ABA signaling in Arabidopsis thaliana. The crk45 mutant was less sensitive to ABA than the wild type during seed germination and early seedling development, whereas CRK45 overexpression plants were more sensitive to ABA compared to the wild type. Furthermore, overexpression of CRK45 led to hypersensitivity to salt and glucose inhibition of seed germination, whereas the crk45 mutant showed the opposite phenotypes. In addition, CRK45 overexpression plants had enhanced tolerance to drought. Gene expression analyses revealed that the expression of representative stress-responsive genes was significantly enhanced in CRK45 overexpression plants in response to salt stress. ABA biosynthetic genes such as NCED3,. 22NCED3, 9-Cis-Epoxycarotenoid Dioxygenase 3.NCED5,. 33NCED5, 9-Cis-Epoxycarotenoid Dioxygenase 5.ABA2,. 44ABA2, Abscisic Acid Deficient 2. and AAO355AAO3, Abscisic Aldehyde Oxidase 3. were also constitutively elevated in the CRK45 overexpression plants. We concluded that CRK45 plays an important role in ABA signaling that regulates Arabidopsis seeds germination, early seedling development and abiotic stresses response, by positively regulating ABA responses in these processes. © 2013 Elsevier Masson SAS.

  17. N-terminal of Papaya ringspot virus type-W (PRSV-W) helper component proteinase (HC-Pro) is essential for PRSV systemic infection in zucchini.

    Science.gov (United States)

    Yap, Yun-Kiam; Duangjit, Janejira; Panyim, Sakol

    2009-06-01

    The Papaya ringspot virus (PRSV) is one of the limiting factors affecting papaya and cucurbits production worldwide. PRSV belongs to the potyvirus genus which consists of 30% of known plant viruses. Two serological closely related strains, namely type-P and -W, have been reported. PRSV type-P infects both papaya and cucurbits, while type-W infects only cucurbits. The genome of PRSV Thailand isolate consists of a (+) RNA molecule of 10323 nucleotides, which is first translated into a single polypeptide and further cleaved by three viral encoded proteases into ten gene products. Helper-component proteinase (HC-Pro), which is encoded by the 2nd cistron of the potyviral genome, has been implicated in aphid transmission, viral movement, viral replication and suppression of host viral defense system. Studies of the Tobacco etch virus (TEV), Lettuce mosaic virus (LMV), Onion yellow dwarf virus (OYDV) and Wheat streak mosaic virus (WSMV) indicate that the N-terminal of HC-Pro is dispensable for systemic infection in their respective hosts. However, deletion analysis of the Tobacco vein mottling virus (TVMV) indicates otherwise. In this study, we examined whether HC-Pro is essential for PRSV systemic infection in cucurbits and the role of its N-terminal in systemic infection. Our results indicated that HC-Pro is indispensable for PRSV infection in zucchini. Deletion analysis of PRSV HC-Pro showed that deletion of as few as 54 amino acids at the N-terminal of HC-Pro completely abolished the infectivity of the corresponding cDNA clone. Therefore, it is proposed that the N-terminal of HC-Pro is involved in systemic infection of PRSV, in addition to its conserved function in aphid transmission. PMID:19322647

  18. Neutrophil elastase reduces secretion of secretory leukoproteinase inhibitor (SLPI by lung epithelial cells: role of charge of the proteinase-inhibitor complex

    Directory of Open Access Journals (Sweden)

    Hiemstra Pieter S

    2008-08-01

    Full Text Available Abstract Background Secretory leukoproteinase inhibitor (SLPI is an important inhibitor of neutrophil elastase (NE, a proteinase implicated in the pathogenesis of lung diseases such as COPD. SLPI also has antimicrobial and anti-inflammatory properties, but the concentration of SLPI in lung secretions in COPD varies inversely with infection and the concentration of NE. A fall in SLPI concentration is also seen in culture supernatants of respiratory cells exposed to NE, for unknown reasons. We investigated the hypothesis that SLPI complexed with NE associates with cell membranes in vitro. Methods Respiratory epithelial cells were cultured in the presence of SLPI, varying doses of proteinases over time, and in different experimental conditions. The likely predicted charge of the complex between SLPI and proteinases was assessed by theoretical molecular modelling. Results We observed a rapid, linear decrease in SLPI concentration in culture supernatants with increasing concentration of NE and cathepsin G, but not with other serine proteinases. The effect of NE was inhibited fully by a synthetic NE inhibitor only when added at the same time as NE. Direct contact between NE and SLPI was required for a fall in SLPI concentration. Passive binding to cell culture plate materials was able to remove a substantial amount of SLPI both with and without NE. Theoretical molecular modelling of the structure of SLPI in complex with various proteinases showed a greater positive charge for the complex with NE and cathepsin G than for other proteinases, such as trypsin and mast cell tryptase, that also bind SLPI but without reducing its concentration. Conclusion These data suggest that NE-mediated decrease in SLPI is a passive, charge-dependent phenomenon in vitro, which may correlate with changes observed in vivo.

  19. Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.

    OpenAIRE

    Nicklin, M J; Harris, K. S.; Pallai, P V; Wimmer, E.

    1988-01-01

    Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D...

  20. Chromosomal mapping of the human proteinase inhibitor 6 (PI6) gene to 6p25 by fluorescence in situ hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Coughlin, P.; Sun, J.; Salem, H. [Monash Medical School, Box Hill (Australia)] [and others

    1995-03-20

    Proteinase inhibitor (PI6), also known as placental thrombin inhibitor, is a recently identified protein originally purified from human placentas that belongs to the serpin super-family of proteins. The amino acid sequence of PI6 suggests that it is a member of a subfamily known as {open_quotes}oval-bumin serpins{close_quotes}. Characteristic features of this subfamily are: (a) they lack N-terminal and C-terminal sequences common in other serpins, (b) they do not have a classical N-terminal signal sequence for secretion, and (c) they display a similar gene organization, with seven exons and eight introns. 9 refs., 1 fig.

  1. SDZ PRI 053, an orally bioavailable human immunodeficiency virus type 1 proteinase inhibitor containing the 2-aminobenzylstatine moiety.

    OpenAIRE

    Billich, A.; Fricker, G; Müller, I.; Donatsch, P.; Ettmayer, P; Gstach, H; Lehr, P; Peichl, P; D. Scholz; Rosenwirth, B

    1995-01-01

    A series of inhibitors of human immunodeficiency virus type 1 (HIV-1) proteinase containing the 2-aralkyl-amino-substituted statine moiety as a novel transition-state analog was synthesized, with the aim to obtain compounds which combine anti-HIV potency with oral bioavailability. The reduced-size 2-aminobenzylstatine derivative SDZ PRI 053, which contains 2-(S)-amino-3-(R)-hydroxyindane in place of an amino acid amide, is a potent and orally bioavailable inhibitor of HIV-1 replication. The a...

  2. Synthesis of the proteinase inhibitor LEKTI domain 6 by the fragment condensation method and regioselective disulfide bond formation.

    Science.gov (United States)

    Vasileiou, Zoe; Barlos, Kostas K; Gatos, Dimitrios; Adermann, Knut; Deraison, Celine; Barlos, Kleomenis

    2010-01-01

    Proteinase inhibitors are of high pharmaceutical interest and are drug candidates for a variety of indications. Specific kallikrein inhibitors are important for their antitumor activity and their potential application to the treatment of skin diseases. In this study we describe the synthesis of domain 6 of the kallikrein inhibitor Lympho-Epithilial Kazal-Type Inhibitor (LEKTI) by the fragment condensation method and site-directed cystine bridge formation. To obtain the linear LEKTI precursor, the condensation was best performed in solution, coupling the protected fragment 1-22 to 23-68. This method yielded LEKTI domain 6 of high purity and equipotent to the recombinantly produced peptide. PMID:20069636

  3. Brewer’s spent grain and corn steep liquor as alternative culture medium substrates for proteinase production by Streptomyces malaysiensis AMT-3

    Science.gov (United States)

    do Nascimento, Rodrigo Pires; Junior, Nelson Alves; Coelho, Rosalie Reed Rodrigues

    2011-01-01

    Brewer’s spent grain and corn steep liquor or yeast extract were used as the sole organic forms for proteinase production by Streptomyces malaysiensis in submerged fermentation. The influence of the C and N concentrations, as well as the incubation periods, were assessed. Eight proteolytic bands were detected through gelatin-gel-electrophoresis in the various extracts obtained from the different media and after different incubation periods, with apparent molecular masses of 20, 35, 43, 50, 70, 100, 116 and 212 kDa. The results obtained suggest an opportunity for exploring this alternative strategy for proteinases production by actinomycetes, using BSG and CSL as economically feasible substrates. PMID:24031767

  4. Improved identification of wheat gluten proteins through alkylation of cysteine residues and peptide-based mass spectrometry

    OpenAIRE

    Rombouts, Ine; Lagrain, Bert; Brunnbauer, Markus; Delcour, Jan; Koehler, Peter

    2013-01-01

    The concentration and composition of wheat gluten proteins and the presence, concentration and location of cysteine residues therein are important for wheat flour quality. However, it is difficult to identify gluten proteins, as they are an extremely polymorphic mixture of prolamins. We here present methods for cysteine labeling of wheat prolamins with 4-vinylpyridine (4-VP) and iodoacetamide (IDAM) which, as compared to label-free analysis, substantially improve identification of cysteine-co...

  5. Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate ?-lyase

    OpenAIRE

    Cooper, Arthur J. L.; Pinto, John T.; Krasnikov, Boris F.; Niatsetskaya, Zoya V.; HAN, Qian; Li, Jianyong; Vauzour, David; Spencer, Jeremy P.E.

    2008-01-01

    Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate ?-lyase. The ?-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate ?-lyase. S-(1,1,2,2-Tetrafluoroethyl)-L-cysteine is an excellent aminotransferase and ?-lyase substrate of rhGTK. Moderate aminotran...

  6. Utilização da fração semipurificada da proteinase do Trypanosoma cruzi no imunodiagnóstico da doença de Chagas / The use of a semipurified fraction of Trypanosoma cruzi proteinase in immunodiagnosis of Chagas' disease

    Scientific Electronic Library Online (English)

    Ajax Mercês, Atta; Angela Maria de Carvalho, Pontes; Maria Luiza de, Souza; Daria, Repka; Humberto A., Rangel.

    1984-12-01

    Full Text Available Foram sensibilizadas hemácias humanas 0 Rh negativo com a fração semipurificada (Fp) da proteinase do Trypanosoma cruzi, e testadas quanto a antigenicidade com soros de pacientes portadores de tripanossomíase americana crônica e de outras doenças parasitárias não relacionadas. Reações de hemaglutina [...] ção positivas foram observadas com os soros de pacientes chagásicos e com alguns soros de indivíduos portadores de leishmaniose cutaneo-mucosa. Não foram observadas reações cruzadas com os soros de pacientes portadores de leishmaniose visceral, malária, toxoplasmose, sífilis, esquistossomose e mononucleose. Os resultados obtidos são favoráveis ao emprego desta fração antigênica em testes de imunodiagnóstico da tripanossomíase americana. Abstract in english Group 0 Rh negative human erytrocytes were coated with the semipurified fraction of T. cruzi proteinase and tested with sera both from patients with chagas' disease and from others with unrelated parasitic diseases. Positive haemagglutination reactions were only observed with the sera from the forme [...] r and with that from two patients with mucocutaneous leishmaniasis. No crossed reactions were observed with visceral leishmaniasis, malaria, toxoplasmosis syphilis, schistosomiasis or mononucleosis sera. Results suggest that this purified fraction can be used in immunodiagnosis of American Trypanosomiasis.

  7. Utilização da fração semipurificada da proteinase do Trypanosoma cruzi no imunodiagnóstico da doença de Chagas The use of a semipurified fraction of Trypanosoma cruzi proteinase in immunodiagnosis of Chagas' disease

    Directory of Open Access Journals (Sweden)

    Ajax Mercês Atta

    1984-12-01

    Full Text Available Foram sensibilizadas hemácias humanas 0 Rh negativo com a fração semipurificada (Fp da proteinase do Trypanosoma cruzi, e testadas quanto a antigenicidade com soros de pacientes portadores de tripanossomíase americana crônica e de outras doenças parasitárias não relacionadas. Reações de hemaglutinação positivas foram observadas com os soros de pacientes chagásicos e com alguns soros de indivíduos portadores de leishmaniose cutaneo-mucosa. Não foram observadas reações cruzadas com os soros de pacientes portadores de leishmaniose visceral, malária, toxoplasmose, sífilis, esquistossomose e mononucleose. Os resultados obtidos são favoráveis ao emprego desta fração antigênica em testes de imunodiagnóstico da tripanossomíase americana.Group 0 Rh negative human erytrocytes were coated with the semipurified fraction of T. cruzi proteinase and tested with sera both from patients with chagas' disease and from others with unrelated parasitic diseases. Positive haemagglutination reactions were only observed with the sera from the former and with that from two patients with mucocutaneous leishmaniasis. No crossed reactions were observed with visceral leishmaniasis, malaria, toxoplasmosis syphilis, schistosomiasis or mononucleosis sera. Results suggest that this purified fraction can be used in immunodiagnosis of American Trypanosomiasis.

  8. Acetaminophen-cysteine adducts during therapeutic dosing and following overdose

    Directory of Open Access Journals (Sweden)

    Judge Bryan S

    2011-03-01

    Full Text Available Abstract 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 a non-acetaminophen hepatotoxin. Conclusions Lower concentrations of APAP-CYS are detectable after exposure to therapeutic doses of acetaminophen and higher concentrations are detected after acute acetaminophen overdose and in patients with acetaminophen toxicity following repeated exposure.

  9. Influence of cysteine solutions on the stability of CdS photoanodes

    Energy Technology Data Exchange (ETDEWEB)

    Kirsch-de Mesmaeker, A.; Decoster, A.M.; Nasielski, J.

    1981-01-01

    The influence of cysteine in aqueous alkaline solution as a reducing electrolyte is tested on the photoanodic behavior of polycrystalline CdS films. It is found that, compared to NaOH alone, cysteine increases the photo-oxidation currents, and shifts the zero photocurrent potential cathodically. At pH 11.5 at the beginning of the illumination, the photocurrents slowly increase by factors up to 4, thanks to some modification of the CdS surface, with some photocorrosion. At pH 9.2, the photocorrosion and the electrode modification are less important. However, even at this pH, cysteine does not seem to stabilize the electrode better than the sulfide. The advantage of the cysteine-cystine electrolyte is, unlike polysulfides, to be transparent in the CdS band gap region. Cystine alone has properties similar to those of cysteine at the very beginning of the illumination, but afterwards the photocurrent drops very quickly, until the CdS film is completely dissolved.

  10. Sulfate and taurine excretion in rats after L-cysteine administration.

    Directory of Open Access Journals (Sweden)

    Yoshida,Shigeko

    1989-10-01

    Full Text Available Excretion of sulfate and taurine, two major metabolites of sulfur, was examined in rats to study the nutritional status of sulfur metabolism in the mammals. Rats maintained on a conventional laboratory diet excreted 1.83 +/- 0.14 mmol of free sulfate and 229.0 +/- 75.3 mumol of taurine/kg of body weight per day. When the diet was changed to a synthetic 25% casein diet, the taurine excretion decreased to 15% of the previous daily excretion, but sulfate excretion decreased only slightly. These decreased levels returned to the original levels when 5 mmol of L-cysteine/kg of body weight was administered into the stomach through a catheter. One week after the first L-cysteine administration, when sulfate and taurine excretion had returned to the original levels, 5 mmol of L-cysteine/kg of body weight was administered likewise. The rats excreted sulfur corresponding to about 95% of L-cysteine administered in the form of free sulfate and taurine within a few days following L-cysteine administration, and sulfate excretion was 3.5 times more than taurine excretion. These results seem to suggest that, in rats, sulfur metabolism is in a state of equilibrium and that sulfate is formed preferentially to taurine.

  11. An FITC-BODIPY FRET couple: application to selective, ratiometric detection and bioimaging of cysteine.

    Science.gov (United States)

    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. PMID:25346180

  12. Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe

    2011-01-01

    During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease B2 (HvEPB2) was ligated into the Pichia pastoris expression vector pPICZ A? and electrotransformed into Pichia pastoris strain KM71H. Heterologous protein production was induced with 2% MeOH and maximum yield were obtained after 4 days where the supernatant was harvested. Purification of HvEPB2 from the supernatant were performed by Ni2+-affininty chromatography. The purified fractions were analyzed via SDS-PAGE, western blotting for confirming the presence of HvEPB2 and via activity assaying. Incubation of purified HvEPB2 with Osborne fractionated barley seed storage proteins for 12 hrs revealed after SDS-PAGE a significant degradation of the storage proteins.

  13. Spectroscopic characterization of cysteine and methionine using density functional theory method

    Science.gov (United States)

    Naganathappa, Mahadevappa; Chaudhari, Ajay

    2015-05-01

    The present study reports theoretical infrared and electronic absorption spectra of neutral cysteine and methionine molecules in gas phase, their ions and in water ice. We also report infrared and electronic absorption spectra of nitrogen-substituted (in place of sulfur atom) cysteine and methionine. The geometrical parameters, dipole moments, rotational and centrifugal distortional constants for these molecules are reported at B3LYP/6-311++g(d,p) level of theory. A large change in vibrational and electronic absorption spectra has been observed upon ionization of cysteine and methionine. Calculated vibrational frequencies are compared with the available experimental frequencies for the neutral cysteine and methionine in gas phase. An influence of water ice on vibrational frequencies of neutral cysteine and methionine is studied using integral equation formalism polarizable continuum model (IEFPCM) at the same level of theory. Time Dependent Density Functional Theory (TDDFT) approach has been adapted to calculate the electronic absorption spectra of these molecules. The intense lines are suggested in order to detect these molecules in space.

  14. Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Dharmendra; Subramanian, Kartik; Bisaria, Virendra S.; Sreekrishnan, T.R.; Gomes, James [Indian Inst. of Technology, Dept. of Biochemical Engineering and Biotechnology, New Delhi (India)

    2005-02-01

    The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l{sup -1} of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l{sup -1} h{sup -1}) every 2 h to the production medium increased the production of methionine to 3.39 g l{sup -1}. A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers. (Author)

  15. Protein-catalyzed isotopic exchange reaction between cysteine and sulfide in spinach leaves

    International Nuclear Information System (INIS)

    A protein has been isolated from spinach leaves which catalyzes the following isotopic exchange reaction: Cys-SH + H235S reversible Cys-35SH + H2S. This enzyme has an pH-optimum above 9.0; its molecular weight has been estimated on a Sephadex-G-100 column to be around 64,000 daltons. During purification this exchange reaction activity follows cysteine synthase activity on DEAE-cellulose and Sephadex-G-100 column chromatography; however, this enzyme fraction has not been purified to homogeneity to prove that both activities are catalyzed by the same protein. The apparent Ksub(m) for 1) H2S has been determined to be 0.86 mM using cysteine as substrate and 0.6 mM using O-acetylserine as substrate; 2) for cysteine was found to be 3.3 mM; and 3) for O-acetylserine to be 3.3 mM. For catalysis no metal ion is required and the reaction proceeds without addition of pyridoxalphosphate. This exchange reaction might prove to be a simple method to prepare labelled cysteine from non-labelled cysteine and labelled H2S. The exchange reaction was found too in Chlorella pyrenoidosa and in Rhodospirillum rubrum. (orig.)

  16. Inhibition of cysteine proteases in acute and chronic spinal cord injury.

    Science.gov (United States)

    Ray, Swapan K; Samantaray, Supriti; Smith, Joshua A; Matzelle, Denise D; Das, Arabinda; Banik, Naren L

    2011-04-01

    Spinal cord injury (SCI) is a serious neurological disorder that debilitates mostly young people. Unfortunately, we still do not have suitable therapeutic agents for treatment of SCI and prevention of its devastating consequences. However, we have gained a good understanding of pathological mechanisms that cause neurodegeneration leading to paralysis or even death following SCI. Primary injury to the spinal cord initiates the secondary injury process that includes various deleterious factors for ultimate activation of different cysteine proteases for degradation of cellular key cytoskeleton and other crucial proteins for delayed death of neurons and glial cells at the site of SCI and its penumbra in different animal models. An important aspect of SCI is the increase in intracellular free Ca(2+) concentration within a short time of primary injury. Various studies in different laboratories demonstrate that the most important cysteine protease for neurodegeneration in SCI is calpain, which absolutely requires intracellular free Ca(2+) for its activation. Furthermore, other cysteine proteases, such as caspases and cathepsin B also make a contribution to neurodegeneration in SCI. Therefore, inhibition of cysteine proteases is an important goal in prevention of neurodegeneration in SCI. Studies showed that individual inhibitors of cysteine proteases provided significant neuroprotection in animal models of SCI. Recent studies suggest that physiological hormones, such as estrogen and melatonin, can be successfully used for prevention of neurodegeneration and preservation of motor function in acute SCI as well as in chronic SCI in rats. PMID:21373949

  17. Ixodes scapularis tick serine proteinase inhibitor (serpin gene family; annotation and transcriptional analysis

    Directory of Open Access Journals (Sweden)

    Chalaire Katelyn C

    2009-05-01

    Full Text Available Abstract Background Serine proteinase inhibitors (Serpins are a large superfamily of structurally related, but functionally diverse proteins that control essential proteolytic pathways in most branches of life. Given their importance in the biology of many organisms, the concept that ticks might utilize serpins to evade host defenses and immunizing against or disrupting their functions as targets for tick control is an appealing option. Results A sequence homology search strategy has allowed us to identify at least 45 tick serpin genes in the Ixodes scapularis genome that are structurally segregated into 32 intronless and 13 intron-containing genes. Nine of the intron-containing serpins occur in a cluster of 11 genes that span 170 kb of DNA sequence. Based on consensus amino acid residues in the reactive center loop (RCL and signal peptide scanning, 93% are putatively inhibitory while 82% are putatively extracellular. Among the 11 different amino acid residues that are predicted at the P1 sites, 16 sequences possess basic amino acid (R/K residues. Temporal and spatial expression analyses revealed that 40 of the 45 serpins are differentially expressed in salivary glands (SG and/or midguts (MG of unfed and partially fed ticks. Ten of the 38 serpin genes were expressed from six to 24 hrs of feeding while six and fives genes each are predominantly or exclusively expressed in either MG and SG respectively. Conclusion Given the diversity among tick species, sizes of tick serpin families are likely to be variable. However this study provides insight on the potential sizes of serpin protein families in ticks. Ticks must overcome inflammation, complement activation and blood coagulation to complete feeding. Since these pathways are regulated by serpins that have basic residues at their P1 sites, we speculate that I. scapularis may utilize some of the serpins reported in this study to manipulate host defense. We have discussed our data in the context of advances on the molecular physiology of I. scapularis. Although the paper is descriptive, this study provides the first step toward a comprehensive understanding of serpins in tick physiology.

  18. Electronic Structure of Transition Metal-Cysteine Complexes From X-Ray Absorption Spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Leung, B.O.; Jalilehvand, F.; Szilagyi, R.K.

    2009-05-19

    The electronic structures of Hg{sup II}, Ni{sup II}, Cr{sup III}, and Mo{sup V} complexes with cysteine were investigated by sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy and density functional theory. The covalency in the metal-sulfur bond was determined by analyzing the intensities of the electric-dipole allowed pre-edge features appearing in the XANES spectra below the ionization threshold. Because of the well-defined structures of the selected cysteine complexes, the current work provides a reference set for further sulfur K-edge XAS studies of bioinorganic active sites with transition metal-sulfur bonds from cysteine residues as well as more complex coordination compounds with thiolate ligands.

  19. A Critical Role for Cysteine 57 in the Biological Functions of Selenium Binding Protein-1.

    Science.gov (United States)

    Ying, Qi; Ansong, Emmanuel; Diamond, Alan M; Yang, Wancai

    2015-01-01

    The concentration of selenium-binding protein1 (SBP1) is often lower in tumors than in the corresponding tissue and lower levels have been associated with poor clinical outcomes. SBP1 binds tightly selenium although what role selenium plays in its biological functions remains unknown. Previous studies indicated that cysteine 57 is the most likely candidate amino acid for selenium binding. In order to investigate the role of cysteine 57 in SBP1, this amino acid was altered to a glycine and the mutated protein was expressed in human cancer cells. The SBP1 half-life, as well as the cellular response to selenite cytotoxicity, was altered by this change. The ectopic expression of SBP1(GLY) also caused mitochondrial damage in HCT116 cells. Taken together, these results indicated that cysteine 57 is a critical determinant of SBP1 function and may play a significant role in mitochondrial function. PMID:26593911

  20. Adsorption Dynamics and Self-Assembled L-cysteine on Au(100)

    DEFF Research Database (Denmark)

    Engelbrekt, Christian; Nazmutdinov, Renat R.

    As the only amino acid with a functional thiol group, L - cysteine offers a strong perspective both for binding to gold and other metals, and for gentle immobilization of biomolecules. Binding to single - crystal, atomically planar surfaces offers the additional perspective that bound L - cysteine can be structurally mapped at the single - molecule level . In this work, we have followed the adsorption of L - cysteine on single - crystal Au(100) by measuring the electrode potential dynamics during the adsorption process. In situ STM revealed the structure of the self - assembled ordered layers. The molecular assemblies were studied through simulated STM image contrast based on density functional theory (DFT) including solvation effects. The adsorption kinetics showed clearly a complex pattern with at least one intermediate state. The modelling disclosed details of the interaction of all functional groups with the Au(100) - substrate.

  1. Influence of cysteine and selenodicysteine on the uptake of zinc by Chlorella vulgaris Beijerinck

    International Nuclear Information System (INIS)

    The uptake of zinc labelled with radioactive 65Zn in the presence of cysteine and selenodicysteine by Chlorella vulgaris was examined. The concentration of zinc ions in the medium was 20 mg per 1. The uptake yield was found to be enhanced by selenodicysteine. At concentration of 10-7-10-6 M the growth rate of Chlorella vulgaris was accelerated by the latter, provided that the specific activity of 65Zn was 3.7 MBq/1. At this specific zinc activity cysteine increased the uptake yield during the initial 50 h of the incubation process. At specific 65Zn-activity of 55.5 MBq/1 selenodicysteine and cysteine only slightly influenced the zinc uptake by Chlorella vulgaris. No increment in the biomass was observed at this specific zinc radioactivity. (author)

  2. Comparative study of action of cell wall proteinases from various strains of Streptococcus cremoris on bovine ?/sub s1-/, ?-, and kappa-casein

    International Nuclear Information System (INIS)

    Experiments are described in which partially purified cell wall proteinases of eight strains of S. cremoris, including strain HP, were compared in their action on ?/sub s1-/, ?-, and kappa-casein, as visualized by starch gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and TLC, and also in their action on methyl-14C-labeled ?-casein

  3. The association of plasma cysteine and gamma-glutamyltransferase with BMI and obesity.

    OpenAIRE

    Elshorbagy, AK; Refsum, H.; Smith, AD; Graham, IM

    2009-01-01

    We recently reported a strong positive association of plasma total cysteine (tCys) with fat mass in over 5,000 subjects. As gamma-glutamyltransferase (GGT) enzyme increases cysteine availability by catalyzing glutathione breakdown and is positively associated with BMI and adiposity, we hypothesized that GGT might explain the association of tCys with adiposity. To study whether the associations of tCys and serum GGT with BMI and obesity were interrelated we conducted a cross-sectional study us...

  4. Theoretical simulation of the ROA spectra of neutral cysteine and serine

    Science.gov (United States)

    Pecul, Magdalena

    2006-08-01

    Vibrational Raman Optical Activity (ROA) spectra have been calculated for neutral conformations of amino acids cysteine and serine. The purpose of the study was to investigate the conformational dependence of the ROA differential intensity, paying particular attention to the conformation of the polar side chains. In cysteine, the ROA intensities of side chain vibrations tend to vary fairly regularly with the value of the ?(NCCS) dihedral angle, while for serine the formation of hydrogen bonds by the side chain hydroxyl group seems to be the deciding factor determining the ROA intensities of the side chain vibrations.

  5. Do cysteine residues regulate transient receptor potential canonical type 6 (TRPC6) channel protein expression?

    DEFF Research Database (Denmark)

    Thilo, Florian; Liu, Ying

    2012-01-01

    The regulation of calcium influx through transient receptor potential canonical type 6 channel is mandatory for the activity of human monocytes. We submit the first evidence that cysteine residues of homocysteine or acetylcysteine affect TRPC6 expression in human monocytes. We observed that patients with chronic renal failure had significantly elevated homocysteine levels and TRPC6 mRNA expression levels in monocytes compared to control subjects. We further observed that administration of homocysteine or acetylcysteine significantly increased TRPC6 channel protein expression compared to control conditions. We therefore hypothesize that cysteine residues increase TRPC6 channel protein expression in humans.

  6. DFT study on cysteine adsorption mechanism on Au(111) and Au(110)

    Energy Technology Data Exchange (ETDEWEB)

    Buimaga-Iarinca, Luiza; Floare, Calin G.; Calborean, Adrian; Turcu, Ioan [National Institute for Research and Development of Isotopic and Molecular Technologies, 65-103 Donath, 400293 Cluj-Napoca (Romania)

    2013-11-13

    Periodic density functional theory calculations were used to investigate relevant aspects of adsorption mechanisms of cysteine dimers in protonated form on Au(111) and Au(110) surfaces. The projected densities of states are explicitly discussed for all main chemical groups of cysteine, i.e. the amino group (NH2), the thiol group (SH) and the carboxylic group (COOH) to identify differences in adsorption mechanism. Special emphasis is put on the analysis of changes in the electronic structure of molecules adsorbed on Au(111) and Au(110) surfaces as well as the accompanying charge transfer mechanisms at molecule-substrate interaction.

  7. An investigation of the metabolism of S-carboxymethyl-L-cysteine in man using a novel HPLC-ECD method.

    Science.gov (United States)

    Karim, E F; Millership, J S; Temple, D J; Woolfson, A D

    1988-01-01

    The metabolism of an oral dose of S-carboxymethyl-L-cysteine (SCMC) in man has been studied; the quantitative determination of SCMC, S-methyl-L-cysteine (SMC) and their sulphoxide metabolites (SCMCO and SMCO), in urine, was carried out using high performance liquid chromatography (HPLC) with electrochemical detection (ECD); the possibility of stereospecific sulphoxidation was investigated. PMID:3243319

  8. Diabetes-induced alterations in tissue collagen and carboxymethyllysine in rat kidneys: Association with increased collagen-degrading proteinases and amelioration by Cu(II)-selective chelation.

    Science.gov (United States)

    Brings, Sebastian; Zhang, Shaoping; Choong, Yee S; Hogl, Sebastian; Middleditch, Martin; Kamalov, Meder; Brimble, Margaret A; Gong, Deming; Cooper, Garth J S

    2015-08-01

    Advanced glycation end-products (AGEs) comprise a group of non-enzymatic post-translational modifications of proteins and are elevated in diabetic tissues. AGE-modification impairs the digestibility of collagen in vitro but little is known about its relation to collagen-degrading proteinases in vivo. N(?)-carboxymethyllysine (CML) is a stable AGE that forms on lysyl side-chains in the presence of glucose, probably via a transition metal-catalysed mechanism. Here, rats with streptozotocin-induced diabetes and non-diabetic controls were treated for 8weeks with placebo or the Cu(II)-selective chelator, triethylenetetramine (TETA), commencing 8weeks after disease induction. Actions of diabetes and drug treatment were measured on collagen and collagen-degrading proteinases in kidney tissue. The digestibility and CML content of collagen, and corresponding levels of mRNAs and collagen, were related to changes in collagen-degrading-proteinases. Collagen-degrading proteinases, cathepsin L (CTSL) and matrix metalloproteinase-2 (MMP-2) were increased in diabetic rats. CTSL-levels correlated strongly and positively with increased collagen-CML levels and inversely with decreased collagen digestibility in diabetes. The collagen-rich mesangium displayed a strong increase of CTSL in diabetes. TETA treatment normalised kidney collagen content and partially normalised levels of CML and CTSL. These data provide evidence for an adaptive proteinase response in diabetic kidneys, affected by excessive collagen-CML formation and decreased collagen digestibility. The normalisation of collagen and partial normalisation of CML- and CTSL-levels by TETA treatment supports the involvement of Cu(II) in CML formation and altered collagen metabolism in diabetic kidneys. Cu(II)-chelation by TETA may represent a treatment option to rectify collagen metabolism in diabetes independent of alterations in blood glucose levels. PMID:25900786

  9. Dietary L-cysteine improves the antioxidative potential and lipid metabolism in rats fed a normal diet.

    Science.gov (United States)

    Lee, Seulki; Han, Kyu-Ho; Nakamura, Yumi; Kawakami, Sakura; Shimada, Ken-ichiro; Hayakawa, Touru; Onoue, Hirotake; Fukushima, Michihiro

    2013-01-01

    L-cysteine works as a precursor of the antioxidant, glutathione. We investigated the effects of L-cysteine (1% and 2%) on lipid metabolism and the antioxidative system in rats fed a normal diet. Administering L-cysteine dependently decreased the food intake, fat mass weight and body weight dose. Dietary L-cysteine also decreased the triglyceride levels in the serum and liver. However, there were no significant differences in the hepatic TBARS and glutathione (GSH) levels among the groups. The activities of catalase and glutathione reductase in the rats receiving 2% L-cysteine were significantly higher (paffected the antioxidative enzyme activities, and the lipid levels in the serum and liver which might be related to the reduced food intake. PMID:23832363

  10. Synthesis and structural studies of cyclic bioactive cysteine rich peptides

    OpenAIRE

    Sørum, Lisbeth

    2009-01-01

    Naturally occurring cyclic peptides have great therapeutic potential. Cyclotides are circular proteins found in the plants of the Rubiaceae, Violaceae and Cucurbitaceae families. They have an exceptional stability against chemical, enzymatic and thermal conditions due to their cyclic cystine knot (CCK) motif, which is the combination of a cyclic backbone and multiple disulfide bonds. In this thesis, kalata B5, a cyclotide from the bracelet subfamily, was synthesized and oxidized. The outcome ...

  11. Mining the active proteome in plant science and biotechnology.

    OpenAIRE

    Ko?odziejek, I; Van der Hoorn, RA

    2010-01-01

    Protein activity is essential functional information, yet difficult to predict from transcript or protein data. Activity-based protein profiling (ABPP) displays active proteins in proteomes using small molecule probes that irreversibly label proteins in their active state. Here, we review proof-of-concept ABPP studies in plant science. These studies displayed activities of dozens of plant cysteine proteases, lipases, methylesterases and the proteasome. ABPP in plants revealed differential pro...

  12. Lifespan extension and increased resistance to environmental stressors by N-Acetyl-L-Cysteine in Caenorhabditis elegans

    Scientific Electronic Library Online (English)

    Seung-Il, Oh; Jin-Kook, Park; Sang-Kyu, Park.

    2015-05-01

    Full Text Available OBJECTIVE: This study was performed to determine the effect of N-acetyl-L-cysteine, a modified sulfur-containing amino acid that acts as a strong cellular antioxidant, on the response to environmental stressors and on aging in C. elegans. METHOD: The survival of worms under oxidative stress conditi [...] ons induced by paraquat was evaluated with and without in vivo N-acetyl-L-cysteine treatment. The effect of N-acetyl-L-cysteine on the response to other environmental stressors, including heat stress and ultraviolet irradiation (UV), was also monitored. To investigate the effect on aging, we examined changes in lifespan, fertility, and expression of age-related biomarkers in C. elegans after N-acetyl-L-cysteine treatment. RESULTS: Dietary N-acetyl-L-cysteine supplementation significantly increased resistance to oxidative stress, heat stress, and UV irradiation in C. elegans. In addition, N-acetyl-L-cysteine supplementation significantly extended both the mean and maximum lifespan of C. elegans. The mean lifespan was extended by up to 30.5% with 5 mM N-acetyl-L-cysteine treatment, and the maximum lifespan was increased by 8 days. N-acetyl-L-cysteine supplementation also increased the total number of progeny produced and extended the gravid period of C. elegans. The green fluorescent protein reporter assay revealed that expression of the stress-responsive genes, sod-3 and hsp-16.2, increased significantly following N-acetyl-L-cysteine treatment. CONCLUSION: N-acetyl-L-cysteine supplementation confers a longevity phenotype in C. elegans, possibly through increased resistance to environmental stressors.

  13. Single Residue Mutation in Active Site of Serine Acetyltransferase Isoform 3 from Entamoeba histolytica Assists in Partial Regaining of Feedback Inhibition by Cysteine

    OpenAIRE

    Kumar, Sudhir; Mazumder, Mohit; Dharavath, Sudhaker; Gourinath , S.

    2013-01-01

    The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site res...

  14. Improvement of Drosophila acetylcholinesterase stability by elimination of a free cysteine

    Directory of Open Access Journals (Sweden)

    Ladurantie Caroline

    2002-07-01

    Full Text Available Abstract Background Acetylcholinesterase is irreversibly inhibited by organophosphate and carbamate insecticides allowing its use for residue detection with biosensors. Drosophila acetylcholinesterase is the most sensitive enzyme known and has been improved by in vitro mutagenesis. However, it is not sufficiently stable for extensive utilization. It is a homodimer in which both subunits contain 8 cysteine residues. Six are involved in conserved intramolecular disulfide bridges and one is involved in an interchain disulfide bridge. The 8th cysteine is not conserved and is present at position 290 as a free thiol pointing toward the center of the protein. Results The free cysteine has been mutated to valine and the resulting protein has been assayed for stability using various denaturing agents: temperature, urea, acetonitrile, freezing, proteases and spontaneous-denaturation at room temperature. It was found that the C290V mutation rendered the protein 1.1 to 2.7 fold more stable depending on the denaturing agent. Conclusion It seems that stabilization resulting from the cysteine to valine mutation originates from a decrease of thiol-disulfide interchanges and from an increase in the hydrophobicity of the buried side chain.

  15. Ultrasonic-assisted synthesis of magnetite based MRI contrast agent using cysteine as the biocapping coating

    International Nuclear Information System (INIS)

    Highlights: ? We used cysteine as surfactant to synthesize stable magnetite-based ferrofluids. ? pH increase from 11 to 12 led to particle size decrease from 19.58 to 10.02 nm. ? Cytotoxicity assay showed that synthesized particles were biocompatible. ? MRI results showed that magnetite particles were accumulated in lymph nodes. - Abstract: Magnetite nanoparticles (mean particle size ranging from 10 to 20 nm) were prepared by a biomolecule-assisted solution-phase approach under ultrasonic irradiation. Cysteine was used as the capping agent in the solution. The results show that cysteine could be an efficient biocapping agent in producing Fe3O4 nanoparticles. The crystal structure and magnetic properties of the nanoparticles were characterized by XRD and VSM techniques, respectively. FT-IR was used to investigate the presence of cysteine on the nanoparticles surface. The influence of pH value of the solution on the size distribution and hydrodynamic size of nanoparticles were studied by TEM and DLS methods, respectively. The MTT assay performed by incubation of L929 cells, showed the good biocompability of synthesized ferrofluids. In vitro T1 and T2 relaxivity measurements along with in vivo studies, which were conducted on rats, demonstrate that synthesized nanoparticles are applicable as the contrast agents, especially for imaging of the lymphatic system.

  16. Bioluminescence of the Ca2+-binding photoprotein aequorin after cysteine modification.

    Science.gov (United States)

    Kurose, K; Inouye, S; Sakaki, Y; Tsuji, F I

    1989-01-01

    Aequorin is a monomeric Ca2+-binding protein (Mr, 21,400) that emits light upon reacting with Ca2+. The protein has three Ca2+-binding sites, three cysteine residues, and a noncovalently bound chromophore that consists of coelenterazine and molecular oxygen. Light is emitted via an intramolecular reaction in which coelenterazine is oxidized by the bound oxygen. After light emission, aequorin may be regenerated by incubating the protein with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol. To understand structure-function relationships in this protein, we used the technique of site-specific mutagenesis to replace the three cysteine residues with serine. Six of the seven modified aequorins had reduced luminescence activity, whereas the seventh with all three cysteines replaced by serine had luminescence activity equal to or greater than that of the wild-type aequorin. Further, the time required for the regeneration of the triply substituted aequorin was substantially increased compared to the time required for the regeneration of the wild-type aequorin. The results suggest that cysteine plays an important role in the regeneration of aequorin but not in its catalytic activity. PMID:2643108

  17. Cysteine: A Novel Neural Inducer for Rat Bone Marrow Mesenchymal Stem Cells

    Directory of Open Access Journals (Sweden)

    Malek Soleimani Mehranjani

    2014-06-01

    Full Text Available Objective: Mesenchymal stem cells (MSCs can differentiate into various cell types. Since cysteine has structural similarities to neuronal inducers ?-mercaptoethanol and glutathione, we examined its effect on neural induction of rat bone marrow MSCs. Materials and Methods: In this experimental study, cells were treated in a medium containing 1mM cysteine for 24 hours prior to treatment with neuron inducing medium containing 10 mM cysteine for 1, 2 and 3 hours. Cell viability and morphology were assessed by 3-(4,5-dimethylthiazol-2-Yl-2,5-diphenyltetrazolium bromide (MTT assay and, Hoechst, propidium iodide and acridine orange staining respectively. Expression of nestin and ?-Tubulin III genes, as neural cell-specific markers, was studied reverse transcription polymerase chain reaction (RT-PCR. The data was statistically analyzed using One-Way ANOVA and Tukey’s test and p<0.05 was considered significant. Results: After 3 hours of treatment, neuron like morphology with a considerable expression of nestin and ?-Tubulin III genes was apparent. The mean cell viability was not significantly different at 1, 2 and 3 hours following induction, compared with the control cells. Conclusion: Cysteine can induce neural features in rat bone marrow MSCs without reducing cell viability. Therefore, it can be considered as a safer alternative to toxic neural inducer agents such as ?-mercaptoethanol.

  18. SULFUR ASSIMILATION IN SOYBEAN: MOLECULAR CLONING AND CHARACTERIZATION OF O-ACETYLSERINE (THIOL) LYASE (CYSTEINE SYNTHASE)

    Science.gov (United States)

    Soybean (Glycine max [L.] Merr.) is a good protein source for both humans and livestock. However, soybean seed proteins are deficient in the sulfur-containing amino acids cysteine and methionine. This deficiency has stimulated efforts to improve the amino acid composition of soybean seed proteins. ...

  19. Chemical introduction of the green fluorescence: imaging of cysteine cathepsins by an irreversibly locked GFP fluorophore.

    Science.gov (United States)

    Frizler, Maxim; Yampolsky, Ilia V; Baranov, Mikhail S; Stirnberg, Marit; Gütschow, Michael

    2013-09-21

    An activity-based probe, containing an irreversibly locked GFP-like fluorophore, was synthesized and evaluated as an inhibitor of human cathepsins and, as exemplified with cathepsin K, it proved to be suitable for ex vivo imaging and quantification of cysteine cathepsins by SDS-PAGE. PMID:23912233

  20. Sperm nuclei glutathione peroxidases and their occurrence in animal species with cysteine-containing protamines.

    Science.gov (United States)

    Bertelsmann, Holger; Kuehbacher, Markus; Weseloh, Gundolf; Kyriakopoulos, Antonios; Behne, Dietrich

    2007-10-01

    The selenoenzyme sperm nuclei glutathione peroxidase (snGPx), also called the nuclear form of phospholipid hydroperoxide glutathione peroxidase (n-PHGPx), was found to be involved in the stabilization of condensed sperm chromatin, most likely by thiol to disulfide oxidation of the cysteine residues of the mammalian protamines, small nuclear basic proteins in the nuclei of sperm cells. By applying Acidic Urea-PAGE in combination with SDS-PAGE, snGPx with an apparent molecular mass of 34 kDa and a 24-kDa protein were purified from rat sperm nuclei. The 24-kDa protein was identified by means of mass spectrometry as a truncated form of snGPx produced by cleavage at the N-terminal end. After defined processing of spermatozoa and detergent treatment of the sperm nuclei fraction, snGPx and its truncated form were shown to be the only selenoproteins present in mature mammalian sperm nuclei. Both forms were found in mature rat and horse sperm nuclei but in man only snGPx was detected. In trout and chicken, species with sperm cells which likewise undergo chromatin condensation but do not contain cysteine in their protamines, the snGPx proteins were missing. This can be taken as an indirect proof of the function of snGPx to act as protamine cysteine thiol peroxidase in the mammalian species with cysteine-containing protamines. PMID:17714875

  1. Improved method for synthesis of cysteine modified hyaluronic acid for in situ hydrogel formation.

    Science.gov (United States)

    Zhang, Xin; Sun, Pengcheng; Huangshan, Lingzi; Hu, Bi-Huang; Messersmith, Phillip B

    2015-06-14

    We developed a new strategy for the functionalization of hyaluronic acid by chemical modification of its C-6 hydroxyl groups through an ether bond to obtain a cysteine-hyaluronic acid conjugate. This conjugate is suitable to prepare injectable and in situ formed hydrogels cross-linked by native chemical ligation and Michael addition under mild conditions. PMID:25977950

  2. Mutations at the cysteine codons of the recA gene of Escherichia coli

    International Nuclear Information System (INIS)

    Each of the three cysteine residues in the Escherichia coli RecA protein was replaced with a number of other amino acids. To do this, each cysteine codon was first converted to a chain-terminating amber codon by oligonucleotide-directed mutagenesis. These amber mutants were then either assayed for function in different suppressor strains or reverted by a second round of mutagenesis with oligonucleotides that had random sequences at the amber codon. Thirty-three different amino acid substitutions were obtained. Mutants were tested for three functions of RecA: survival following UV irradiation, homologous recombination, and induction of the SOS response. It was found that although none of the cysteines is essential for activity, mutations at each of these positions can affect one or more of the activities of RecA, depending on the particular amino acid substitution. In addition, the cysteine at position 116 appears to be involved in the RecA-promoted cleavage of the LexA protein

  3. Evidence of both extra- and intracellular cysteine targets of protein modification for activation of RET kinase.

    Science.gov (United States)

    Akhand, Anwarul A; Ikeyama, Takanari; Akazawa, Satoru; Kato, Masashi; Hossain, Khaled; Takeda, Kozue; Suzuki, Haruhiko; Takahashi, Masahide; Nakashima, Izumi

    2002-04-12

    By use of a specifically sulfhydryl group-reactive chemical, 1,4-butanediyl-bismethanethiosulfonate (BMTS), we studied the localization of oxidative stress-responsive target cysteines for activation of a receptor-type protein tyrosine kinase, c-RET. The chemical, which reacted with RET proteins on the cell surface for sulfhydryl-linked aggregation, induced autophosphorylation and activation of RET kinase. When extracellular domain-deleted RET mutant (RET-PTC-1) cells were exposed to BMTS, neither the molecular status nor the activity of the enzyme was affected, suggesting that the target cysteines of BMTS to which cells were exposed for reaction are located in the cysteine-rich region of the extracellular domain of RET kinase. Despite this result, the exposure of a subcellular form of c-RET or RET-PTC-1 kinase isolated by immunoprecipitation to BMTS did induce activation of the enzyme. These results suggest that cysteines in both the extracellular and the intracellular domains of RET can work as target sites of accessible BMTS and possibly other oxidative elements for structural modification and activation of RET kinase. PMID:11944888

  4. Lysosomotropic Agents and Cysteine Protease Inhibitors Inhibit Scrapie-Associated Prion Protein Accumulation

    OpenAIRE

    Doh-ura, Katsumi; Iwaki, Toru; Caughey, Byron

    2000-01-01

    We report that lysosomotropic agents and cysteine protease inhibitors inhibited protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells. The inhibition occurred without either apparent effects on normal prion protein biosynthesis or turnover or direct interactions with prion protein molecules. The findings introduce two new classes of inhibitors of the formation of protease-resistant prion protein.

  5. Changes in brain cysteinic cathepsin L activity at exposure to x-rays

    International Nuclear Information System (INIS)

    The influence of different doses of x-rays on the activity and localization of lysosomal cysteinic cathepsin L of the gray matter of large brain hemispheres in rats at acute radiation lesion was studied. Cathepsin L activity has phasic character. Irradiation changes intracellular compartmentalization of cathepsin L considerably increases non-sedimented activity

  6. NEW EMBO MEMBER’S REVIEW: Lysosomal cysteine proteases: facts and opportunities

    OpenAIRE

    Turk, Vito; Turk, Boris; Turk, Dušan

    2001-01-01

    From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non-selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.

  7. Synthesis and characterization of arsenic-doped cysteine-capped thoria-based nanoparticles

    International Nuclear Information System (INIS)

    Thoria materials have been largely used in the nuclear industry. Nonetheless, fluorescent thoria-based nanoparticles provide additional properties to be applied in other fields. Thoria-based nanoparticles, with and without arsenic and cysteine, were prepared in 1,2-ethanediol aqueous solutions by a simple precipitation procedure. The synthesized thoria-based nanoparticles were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (ED-XRS), Raman spectroscopy, Fourier transform infrared (FT-IR) spectroscopy and fluorescence microscopy. The presence of arsenic and cysteine, as well as the use of a thermal treatment facilitated fluorescence emission of the thoria-based nanoparticles. Arsenic-doped and cysteine-capped thoria-based nanoparticles prepared in 2.5 M 1,2-ethanediol solutions and treated at 348 K showed small crystallite sizes and strong fluorescence. However, thoria nanoparticles subjected to a thermal treatment at 873 K also produced strong fluorescence with a very narrow size distribution and much smaller crystallite sizes, 5 nm being the average size as shown by XRD and TEM. The XRD data indicated that, even after doping of arsenic in the crystal lattice of ThO2, the samples treated at 873 K were phase pure with the fluorite cubic structure. The Raman and FT-IR spectra shown the most characteristics vibrational peaks of cysteine together with other peaks related to the bonds of this molecule to thoria and arsenic when present

  8. Direct radiolabelling of proteins at cysteine using [18F]-fluorosugars.

    OpenAIRE

    Boutureira, O; Bernardes, GJ; D'Hooge, F; Davis, BG

    2011-01-01

    A strategy for the site-specific attachment of 2-deoxy-2-fluorosugars to cysteine and dehydroalanine tagged proteins is reported. When combined with thionation of fluorosugars, such as the widely available (18)F probe 2-deoxy-2-[(18)F]fluoroglucose ([(18)F]FDG), this methodology allows fast and direct access to site-specific [(18)F]FDG-labelled proteins.

  9. Ultrasonic-assisted synthesis of magnetite based MRI contrast agent using cysteine as the biocapping coating

    Energy Technology Data Exchange (ETDEWEB)

    Ahmadi, Reza [Department of Materials Science and Engineering, Sharif University of Technology, P.O. Box 11155-9466, Tehran (Iran, Islamic Republic of); Malek, Mahrooz [Medical Image Centre, Imam Khomeini Hospital, Tehran Medical University, Tehran (Iran, Islamic Republic of); Hosseini, Hamid Reza Madaah [Department of Materials Science and Engineering, Sharif University of Technology, P.O. Box 11155-9466, Tehran (Iran, Islamic Republic of); Shokrgozar, Mohammad Ali [National Cell Bank of Iran, Pasteur Institute of Iran, Tehran (Iran, Islamic Republic of); Oghabian, Mohammad Ali [Research Centre for Sciences and Technology in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Masoudi, Afshin [Department of Materials Science and Engineering, Sharif University of Technology, P.O. Box 11155-9466, Tehran (Iran, Islamic Republic of); Gu Ning; Zhang Yu [Jiangsu Key Laboratory of Biomaterials and Devices, Nanjing (China)

    2011-12-15

    Highlights: Black-Right-Pointing-Pointer We used cysteine as surfactant to synthesize stable magnetite-based ferrofluids. Black-Right-Pointing-Pointer pH increase from 11 to 12 led to particle size decrease from 19.58 to 10.02 nm. Black-Right-Pointing-Pointer Cytotoxicity assay showed that synthesized particles were biocompatible. Black-Right-Pointing-Pointer MRI results showed that magnetite particles were accumulated in lymph nodes. - Abstract: Magnetite nanoparticles (mean particle size ranging from 10 to 20 nm) were prepared by a biomolecule-assisted solution-phase approach under ultrasonic irradiation. Cysteine was used as the capping agent in the solution. The results show that cysteine could be an efficient biocapping agent in producing Fe{sub 3}O{sub 4} nanoparticles. The crystal structure and magnetic properties of the nanoparticles were characterized by XRD and VSM techniques, respectively. FT-IR was used to investigate the presence of cysteine on the nanoparticles surface. The influence of pH value of the solution on the size distribution and hydrodynamic size of nanoparticles were studied by TEM and DLS methods, respectively. The MTT assay performed by incubation of L929 cells, showed the good biocompability of synthesized ferrofluids. In vitro T1 and T2 relaxivity measurements along with in vivo studies, which were conducted on rats, demonstrate that synthesized nanoparticles are applicable as the contrast agents, especially for imaging of the lymphatic system.

  10. A novel cysteine-sparing NOTCH3 mutation in a Chinese family with CADASIL.

    Science.gov (United States)

    Ge, Wei; Kuang, Hanzhe; Wei, Bin; Bo, Le; Xu, Zhice; Xu, Xingshun; Geng, Deqin; Sun, Miao

    2014-01-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an adult onset cerebral small vessel disorder caused by the mutations of the neurogenic locus notch homolog protein 3 (NOTCH3) gene. The extracellular part of NOTCH3 is composed of 34 epidermal growth factor-like (EGF-like) repeat domains. Each EGF-like domain is rich of cysteine and glycine to produce three loops that are essential for high-affinity binding to its ligand. Nearly all reported CADASIL-associated mutations result in gain or loss of a cysteine residue within the EGF-like domains. Only a few cysteine-sparing NOTCH3 mutations have been documented in the patients with CADASIL to date. Here, we reported a Chinese CADASIL family with a cysteine-sparing NOTCH3 mutation. In this family, affected patients had dizziness, memory loss, gait instability, or hemiplegia. Brain magnetic resonance imaging (MRI) showed diffuse leukoencephalopathy with confluent signal abnormalities in the periventricular white matter, basal ganglia, and centrum semiovale bilaterally. By screening the entire coding region of NOTCH3, a novel missense mutation p.G149V (c.446G>T) was found. This mutation was not detected in 400 normal controls. Considering the critical position of glycine within the C-loop of EGF-like domain and its high conservation through evolution, p.G149V mutation could be a potential pathogenic cause for CADASIL. PMID:25098330

  11. Formation of elemental sulfur by Chlorella fusca during growth on L-cysteine ethylester

    International Nuclear Information System (INIS)

    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 ?max 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. 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

  12. Helper component-proteinase enhances the activity of 1-deoxy-D -xylulose-5-phosphate synthase and promotes the biosynthesis of plastidic isoprenoids in Potato virus Y-infected tobacco.

    Science.gov (United States)

    Li, Heng; Ma, Dongyuan; Jin, Yongsheng; Tu, Yayi; Liu, Liping; Leng, Chunxu; Dong, Jiangli; Wang, Tao

    2015-10-01

    Virus-infected plants show strong morphological and physiological alterations. Many physiological processes in chloroplast are affected, including the plastidic isoprenoid biosynthetic pathway [the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway]; indeed, isoprenoid contents have been demonstrated to be altered in virus-infected plants. In this study, we found that the levels of photosynthetic pigments and abscisic acid (ABA) were altered in Potato virus Y (PVY)-infected tobacco. Using yeast two-hybrid assays, we demonstrated an interaction between virus protein PVY helper component-proteinase (HC-Pro) and tobacco chloroplast protein 1-deoxy-D-xylulose-5-phosphate synthase (NtDXS). This interaction was confirmed using bimolecular fluorescence complementation (BiFC) assays and pull-down assays. The Transket_pyr domain (residues 394-561) of NtDXS was required for interaction with HC-Pro, while the N-terminal region of HC-Pro (residues 1-97) was necessary for interaction with NtDXS. Using in vitro enzyme activity assays, PVY HC-Pro was found to promote the synthase activity of NtDXS. We observed increases in photosynthetic pigment contents and ABA levels in transgenic plants with HC-Pro accumulating in the chloroplasts. During virus infection, the enhancement of plastidic isoprenoid biosynthesis was attributed to the enhancement of DXS activity by HC-Pro. Our study reveals a new role of HC-Pro in the host plant metabolic system and will contribute to the study of host-virus relationships. PMID:25736930

  13. Studies on the presence and release of proteolytic enzymes (proteinases) in gastro-intestinal nematodes of ruminants.

    Science.gov (United States)

    Knox, D P; Jones, D G

    1990-04-01

    The proteolytic activity of worm homogenates prepared from the third stage larval (L3) and adult stages of the ovine gastro-intestinal nematodes, Nematodirus battus, Ostertagia circumcincta, Trichostrongylus colubriformis, Trichostrongylus vitrinus and Haemonchus contortus, and the rodent intestinal nematode, Nippostrongylus brasiliensis, has been examined using the protein substrates azocasein, azocoll and elastin-orcein. Activity detected in third stage larvae was usually higher than that observed in the adult. Species and stage differences were demonstrated. The in vitro release of proteolytic activity, detected using protein substrates and specific low molecular weight peptide substrates, was, similarly, shown to exhibit a degree of species and stage specificity. Acetylcholinesterase and lactate dehydrogenase activities were determined as reference 'secretory' and 'non-secretory' enzymes, respectively. Three separate peaks of 'tryptic' activity were detected following anion-exchange chromatography of culture fluids derived from adult Ostertagia circumcincta. These peaks could be ascribed to different proteinase classes on the basis of inhibitor sensitivity. PMID:2185170

  14. Domain 2 of a Kazal serine proteinase inhibitor SPIPm2 from Penaeus monodon possesses antiviral activity against WSSV.

    Science.gov (United States)

    Visetnan, Suwattana; Donpudsa, Suchao; Supungul, Premruethai; Tassanakajon, Anchalee; Rimphanitchayakit, Vichien

    2014-12-01

    A 5-domain Kazal type serine proteinase inhibitor SPIPm2 from Penaeus monodon is involved in innate immune defense against white spot syndrome virus (WSSV). To test which domains were involved, the 5 domains of SPIPm2 were over-expressed and tested against WSSV infection. By using hemocyte primary cell culture treated with each recombinant SPIPm2 domain along with WSSV, the expression of WSSV early genes ie1, WSV477 and late gene VP28 were substantially reduced as compared to other domains when the recombinant domain 2, rSPIPm2D2, was used. Injecting the WSSV along with rSPIPm2D2 but not with other domains caused delay in mortality rate of the infected shrimp. The results indicate that the SPIPm2D2 possesses strong antiviral activity and, hence, contributes predominantly to the antiviral activity of SPIPm2. PMID:25301720

  15. A barley cystatin stably expressed in rice exhibits strong in vitro inhibitory activity against gut proteinases of rice water weevil / La expresión en arroz de una cistatina de cebada inhibe significativamente la actividad proteinasa digestiva del picudo acuático del arroz in vitro

    Scientific Electronic Library Online (English)

    Raúl, Armas; Carlos, Hernández; Daymi, Abreu; Maylin, Pérez; Yeosvany, Cabrera; Merardo, Pujol; Julio, Alfonso-Rubi.

    2009-12-01

    Full Text Available El picudo acuático del arroz, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), es una de las principales plagas del cultivo en Cuba. En este trabajo describimos la obtención de plantas transgénicas de arroz que expresan la cistatina de cebada HvCPI-1 (gene Icy1) como un modelo para e [...] xplorar la potencialidad de esta proteína en el control del picudo acuático. Se transformó arroz (Oryza sativa L. cv IACuba-28) vía Agrobacterium tumefaciens conteniendo un plásmido que porta el gen Icy1 fusionado al promotor 35S del CaMV y al primer exón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas Texón/intrón/exón del gen Act-1 de arroz. De 65 líneas transgénicas independientes, 62 resultaron positivas al análisis por PCR-Southern blot. El transgen se expresó correctamente según se pudo apreciar por western y dot blot con un nivel de expresión superior al 2% de las proteínas totales extraídas en plantas T1. La integridad funcional fue confirmada por la reducción hasta un 90% de la actividad cisteino proteinasa en el extracto digestivo de larvas del picudo acuático por extractos de hojas de arroz. Además, extractos de raices de plantas transgénicas de generación T2 produjeron una inhibición significativa, 70% a pH 4.5 y 45% a pH 6.0, de la actividad tipo catepsina B en extractos del intestino de larvas de L. brevirostris. Estos resultados demuestran el potencial de la cistatina de cebada como un efectivo componente para ser usado en combinación con otras estrategias para el control de esta plaga como una alternativa contra el desarrollo de insecto resistencia Abstract in english Rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), is one of the most important rice pests in Cuba. Here, we describe the production of transgenic rice plants expressing barley cystatin HvCPI-1 (Icy1 gene) to explore the potential of this protein for the control of r [...] ice water weevil. Rice plants (Oryza sativa L. cv IACuba-28) were transformed via Agrobacterium tumefaciens with a plasmid carrying the Icy1 gene fused to the 35S promoter and the first exon/intron/exon from rice actin-1 gene. From 65 independent transgenic lines, 62 were positive in the PCR-Southern blot analyses. The transgene was correctly translated as indicated by western- and dot-blot assays with level of expression in T¹ plants of up to 2% of the total extracted protein. The functional integrity of the protein was confirmed in vitro by a reduction of up to 90% of the cysteine-proteinase activity in the gut of rice water weevils exposed to rice leaf extracts. Moreover, proteins extracted from T² transgenic rice roots showed a significant inhibition of up to 70% at pH 4.5 and 45% at pH 6.0 of the cathepsin B-like activity in the L. brevirostris larvae gut. These results

  16. Flexibility of cold- and heat-adapted subtilisin-like serine proteinases evaluated with fluorescence quenching and molecular dynamics

    DEFF Research Database (Denmark)

    Sigtryggsdóttir, Asta Rós; Papaleo, Elena

    2014-01-01

    The subtilisin-like serine proteinases, VPR, from a psychrotrophic Vibrio species and aqualysin I (AQUI) from the thermophile Thermus aquaticus, are structural homologues, but differ significantly with respect to stability and catalytic properties. It has been postulated that the higher catalytic activity of cold adapted enzymes when compared to homologues from thermophiles, reflects their higher molecular flexibility. To assess a potential difference in molecular flexibility between the two homologous proteinases, we have measured their Trp fluorescence quenching by acrylamide at different temperatures. We also investigated protein dynamics of VPR and AQUI at an atomic level by molecular dynamics simulations. VPR contains four Trp residues, three of which are at corresponding sites in the structure of AQUI. To aid in the comparison, a Tyr at the fourth corresponding site in AQUI was mutated to Trp (Y191W). A lower quenching effect of acrylamide on the intrinsic fluorescence of the thermophilic AQUI_Y191W was observed at all temperatures measured (10-55°C), suggesting that it possesses a more rigid structure than VPR. The MD analysis (C? rmsf profiles) showed that even though VPR and AQUI have similar flexibility profiles, the cold adapted VPR displays higher flexibility in most regions of the protein structure. Some of these regions contain or are in proximity to some of the Trp residues (Trp6, Trp114 and Trp208) in the proteins. Thus, we observe an overall agreement between the fluorescence quenching data and the flexibility profiles obtained from the MD simulations to different flexibilities of specific regions in the proteins.

  17. Kallikrein-related peptidase 14 acts on proteinase-activated receptor 2 to induce signaling pathway in colon cancer cells.

    Science.gov (United States)

    Gratio, Valérie; Loriot, Céline; Virca, G Duke; Oikonomopoulou, Katerina; Walker, Francine; Diamandis, Eleftherios P; Hollenberg, Morley D; Darmoul, Dalila

    2011-11-01

    Serine proteinases participate in tumor growth and invasion by cleaving and activating proteinase-activated receptors (PARs). Recent studies have implicated PAR-1 and PAR-4 (activated by thrombin) and PAR-2 (activated by trypsin but not by thrombin) in human colon cancer growth. The endogenous activators of PARs in colon tumors, however, are still unknown. We hypothesize that the kallikrein-related peptidase (KLK) family member KLK14, a known tumor biomarker, is produced by colonic tumors and signals to human colon cancer cells by activating PARs. We found that i) KLK14 mRNA was present in 16 human colon cancer cell lines, ii) KLK14 protein was expressed and secreted in colon cancer cell lines, and iii) KLK14 (0.1 ?mol/L) induced increases in intracellular calcium in HT29, a human colon cancer-derived cell line. KLK14-induced calcium flux was associated with internalization of KLK14-mediated activation of PAR-2. Furthermore, KLK14 induced significant extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and HT29 cell proliferation, presumably by activating PAR-2. A PAR-2 cleavage and activation-blocking antibody dramatically reduced KLK14-induced ERK1/2 signaling. Finally, ectopic expression of KLK14 in human colon adenocarcinomas and its absence in normal epithelia was demonstrated by IHC analysis. These results demonstrate, for the first time, the aberrant expression of KLK14 in colon cancer and its involvement in PAR-2 receptor signaling. Thus, KLK14 and its receptor, PAR-2, may represent therapeutic targets for colon tumorigenesis. PMID:21907696

  18. SDZ PRI 053, an orally bioavailable human immunodeficiency virus type 1 proteinase inhibitor containing the 2-aminobenzylstatine moiety.

    Science.gov (United States)

    Billich, A; Fricker, G; Müller, I; Donatsch, P; Ettmayer, P; Gstach, H; Lehr, P; Peichl, P; Scholz, D; Rosenwirth, B

    1995-07-01

    A series of inhibitors of human immunodeficiency virus type 1 (HIV-1) proteinase containing the 2-aralkyl-amino-substituted statine moiety as a novel transition-state analog was synthesized, with the aim to obtain compounds which combine anti-HIV potency with oral bioavailability. The reduced-size 2-aminobenzylstatine derivative SDZ PRI 053, which contains 2-(S)-amino-3-(R)-hydroxyindane in place of an amino acid amide, is a potent and orally bioavailable inhibitor of HIV-1 replication. The antiviral activity of SDZ PRI 053 was demonstrated in various cell lines, in primary lymphocytes, and in primary monocytes, against laboratory strains as well as clinical HIV-1 isolates (50% effective dose = 0.028 to 0.15 microM). Cell proliferation was impaired only at 100- to 300-fold-higher concentrations. The mechanism of antiviral action of the proteinase inhibitor SDZ PRI 0.53 was demonstrated to be inhibition of gag precursor protein processing. The finding that the inhibitory potency of SDZ PRI 053 in chronic virus infection, determined by p24 release, was considerably lower than that in de novo infection may be explained by the fact that the virus particles produced in the presence of SDZ PRI 053 are about 50-fold less infectious than those from untreated cultures. Upon intravenous administration, half-lives in blood of 100 and 32 min in mice and rats, respectively, were measured. Oral bioavailability of SDZ PRI 053 in rodents was 20 to 60%, depending on the dose. In mice, rats, and dogs, the inhibitor levels after oral administration remained far above the concentrations needed to efficiently block HIV replication in vitro for a prolonged period. This compound is thus a promising candidate for clinical use in HIV disease. PMID:7492076

  19. Cysteine-Functionalized Nanostructured Lipid Carriers for Oral Delivery of Docetaxel: A Permeability and Pharmacokinetic Study.

    Science.gov (United States)

    Fang, Guihua; Tang, Bo; Chao, Yanhui; Xu, Helin; Gou, Jingxin; Zhang, Yu; Xu, Hui; Tang, Xing

    2015-07-01

    Here we report the development and evaluation of cysteine-modified nanostructured lipid carriers (NLCs) for oral delivery of docetaxel (DTX). The NLCs ensure high encapsulation efficiency of docetaxel, while the cysteine bound the NLCs with PEG2000-monostearate (PEG2000-MSA) as a linker, and allowed a specific interaction with mucin of the intestinal mucus layer and facilitated the intestinal transport of docetaxel. The cysteine-modified NLCs (cNLCs) had a small particle size (NLCs (uNLCs) (-6.39 ± 0.07 mV). This correlates well with the location of the cysteine group on the surface of the NLCs obtained by X-ray photoelectron spectroscopy (XPS). The cNLCs significantly improved the mucoadhesion properties compared with uNLCs. The intestinal absorption of cNLCs in total intestinal segments was greatly improved in comparison with uNLCs and docetaxel solution (DTX-Sol), and the in vivo imaging system captured pictures also showed not only increased intestinal absorption but also improved accumulation in blood. The cNLCs could be absorbed into the enterocytes via both endocytosis and passive transport. The results of the in vivo pharmacokinetic study indicated that the AUC0-t of cNLCs (1533.00 ng/mL·h) was markedly increased 12.3-fold, and 1.64-fold compared with docetaxel solution and uNLCs, respectively. Overall, the cysteine modification makes nanostructured lipid carriers more suitable as nanocarriers for oral delivery of docetaxel. PMID:25974386

  20. Glutamine transaminase K and cysteine S-conjugate beta-lyase activity stains.

    Science.gov (United States)

    Abraham, D G; Cooper, A J

    1991-09-01

    An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing L-phenyl-alanine, alpha-keto-gamma-methiolbutyrate (alpha KMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and alpha KMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate beta-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing alpha KMB (to ensure maintenance of the enzyme in the pyridoxal 5'-phosphate form), S-(1,2-dichlorovinyl)-L-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate beta-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent Mr approximately 95,000). This band coincides with both the cysteine S-conjugate beta-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate beta-lyase activity with an Rf value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1723851