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Cysteine proteinases and cystatins  

Directory of Open Access Journals (Sweden)

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.

Oliveira Adeliana S.

2003-01-01

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Cysteine proteinases and cystatins  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese 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 a [...] dição, 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. Abstract in english 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 th [...] e 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.

Adeliana S., Oliveira; José, Xavier-Filho; Maurício P., Sales.

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The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship  

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Abstract Background Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins. Results We have performed an evolutionary comparative analysis of cysteine proteinases C1...

Martinez Manuel; Diaz Isabel

2008-01-01

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The origin and evolution of plant cystatins and their target cysteine proteinases indicate a complex functional relationship  

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Full Text Available Abstract Background Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins. Results We have performed an evolutionary comparative analysis of cysteine proteinases C1A and C13 and their putative inhibitors in representative species of different taxonomic groups that appeared during the evolution of the Viridiplantae. The results indicate that whereas C1A cysteine proteinases are present in all taxonomic groups, cystatins and C13 cysteine proteinases are absent in some basal groups. Moreover, gene duplication events have been associated to the increasing structural and functional complexities acquired in land plants. Conclusion Comparative genomic analyses have provided us valuable insights into the conservation and evolution of the cystatin inhibitory family and their putative targets, the cysteine proteinases from families C1A and C13. Functionality of both families of proteins in plants must be the result of a coevolutionary process that might have occurred during the evolution of basal and land plants leading to a complex functional relationship among them.

Diaz Isabel

2008-07-01

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Investigations into the effects of plant derived cysteine proteinases on tapeworms (cestoda)  

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Gastrointestinal (GI) helminths pose a significant threat to the livestock industry and are a recognized cause of global morbidity in humans. Control relies principally on chemotherapy but in the case of nematodes is rapidly losing efficacy through widespread development and spread of resistance to conventional anthelmintics and hence the urgent need for novel classes of anthelmintics. Cysteine proteinases (CPs) from papaya latex have been shown to be effective against three murine nematodes ...

Mansur, Fadlul Azim Fauzi Bin

2013-01-01

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Developing a rapid throughput screen for detection of nematicidal activity of plant cysteine proteinases: the role of Caenorhabditis elegans cystatins.  

Science.gov (United States)

Plant cysteine proteinases (CPs) from papaya (Carica papaya) are capable of killing parasitic nematode worms in vitro and have been shown to possess anthelmintic effects in vivo. The acute damage reported in gastrointestinal parasites has not been found in free-living nematodes such as Caenorhabditis elegans nor among the free-living stages of parasitic nematodes. This apparent difference in susceptibility might be the result of active production of cysteine proteinase inhibitors (such as cystatins) by the free-living stages or species. To test this possibility, a supernatant extract of refined papaya latex (PLS) with known active enzyme content was used. The effect on wild-type (Bristol N2) and cystatin null mutant (cpi-1(-/-) and cpi-2(-/-)) C. elegans was concentration-, temperature- and time-dependent. Cysteine proteinases digested the worm cuticle leading to release of internal structures and consequent death. Both cystatin null mutant strains were highly susceptible to PLS attack irrespective of the temperature and concentration of exposure, whereas wild-type N2 worms were generally resistant but far more susceptible to attack at low temperatures. PLS was able to induce elevated cpi-1 and cpi-2 cystatin expression. We conclude that wild-type C. elegans deploy cystatins CPI-1 and CPI-2 to resist CP attack. The results suggest that the cpi-1 or cpi-2 null mutants (or a double mutant combination of the two) could provide a cheap and effective rapid throughput C. elegans-based assay for screening plant CP extracts for anthelmintic activity. PMID:24001183

Phiri, A M; De Pomerai, D; Buttle, D J; Behnke, J M B

2014-02-01

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Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination  

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Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP) and four cysteine proteinase ...

Lepelley Maud; Amor Mohamed; Martineau Nelly; Cheminade Gerald; Caillet Victoria; McCarthy James

2012-01-01

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Identification, classification and expression pattern analysis of sugarcane cysteine proteinases  

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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...

Gustavo Coelho Correa; Márcia Margis-Pinheiro; Rogério Margis

2001-01-01

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Identification, classification and expression pattern analysis of sugarcane cysteine proteinases  

Directory of Open Access Journals (Sweden)

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.

Gustavo Coelho Correa

2001-12-01

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The vagina has reducing environment sufficient for activation of Trichomonas vaginalis cysteine proteinases.  

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BACKGROUND: Trichomonas vaginalis, a worldwide distributed sexually transmitted protozoan, is remarkable for synthesis of numerous, distinct cysteine proteinases, the significance of which is evidenced by the presence in vivo of soluble proteinases in secretions and antiproteinase antibody in serum of patients with trichomonosis. These proteinases purportedly play a role in host parasitism and immune evasion. OBJECTIVE: It is known that for cysteine proteinases to be functional, they must be ...

Alderete, J. F.; Provenzano, D.

1997-01-01

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The nematicidal effect of cysteine proteinases on the root knot nematode Meloidogne incognita  

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Despite current control measures, plant parasitic nematodes are estimated to be responsible for > $100 billion of damage to worldwide crop production per annum. Current nematicides are highly toxic, and due to health and environmental safety concerns, many are being withdrawn from the market under directive 914/414/EEC. Alternative control strategies are urgently required. The cysteine proteinases papain, actinidain and recombinant endoproteinase B isoform 2 (R.EP-B2) have been demonstrate...

Gorny, Samuel Victor

2013-01-01

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Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa)  

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Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6 mu g/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the ...

Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-jankulovic, Marija

2013-01-01

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Primary structure of a cysteine proteinase inhibitor from the fruit of avocado (Persea americana Mill).  

Science.gov (United States)

The complete amino acid sequence of a proteinaceous cysteine proteinase inhibitor from the fruit of avocado (avocado cystatin) is presented. The protein consists of 100 amino acid residues and has a molecular mass of 11,300 Da. Comparison of this sequence with sequences of plant cysteine proteinase inhibitors (phytocystatins), including oryzacystatins I and II from rice seeds, cowpea cystatin, and corn cystatin, showed that the avocado cystatin molecule has 60% and 54% residues identical with the two forms of the rice seed proteins, oryzacystatins I and II, respectively, and 64% and 63% with the cowpea and corn proteins, respectively. The totally conserved sequence, Gln-Val-Val-Ala-Gly, among several of the animal cystatins as well as phytocystatins, is at positions 47-51 in the avocado cystatin molecule. PMID:8611758

Kimura, M; Ikeda, T; Fukumoto, D; Yamasaki, N; Yonekura, M

1995-12-01

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[Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor].  

Science.gov (United States)

The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes. A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE. The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR. Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions. Homology analysis showed that the identity was 77% between Dd-cpl-1 deduced protein Dd-CPL-1 and cathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus. Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1. Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases. PMID:21553491

Wang, Gaofeng; Peng, Deliang; Sun, Jianhua; Huang, Wenkun; Peng, Huan; Long, Haibo

2011-01-01

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[Behavior of cysteine proteinase inhibitors in autologous epidermis transplanted into oral mucosa for relative elevation of the alveolar process].  

Science.gov (United States)

The presence of cysteine proteinase inhibitors was investigated in skin autografts into the gingiva of denture carriers. Acid cysteine proteinase inhibitors was present both in the transplant and the surrounding gingival epithelium, while neutral cysteine proteinase inhibitors was seen only in the gingival epithelium. The results indicate that the normal expression of cysteine proteinase inhibitors is conserved during the adaptation of the skin autograft into the oral surroundings. PMID:3092550

Pernu, H; Rinne, A; Järvinen, M; Altonen, M; Hopsu-Havu, V K

1986-01-01

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A novel Glycine soja cysteine proteinase inhibitor GsCPI14, interacting with the calcium/calmodulin-binding receptor-like kinase GsCBRLK, regulated plant tolerance to alkali stress.  

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It has been well demonstrated that cystatins regulated plant stress tolerance through inhibiting the cysteine proteinase activity under environmental stress. However, there was limited information about the role of cystatins in plant alkali stress response, especially in wild soybean. Here, in this study, we focused on the biological characterization of a novel Glycine soja cystatin protein GsCPI14, which interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and positively regulated plant alkali stress tolerance. The protein-protein interaction between GsCBRLK and GsCPI14 was confirmed by using split-ubiquitin based membrane yeast two-hybrid analysis and bimolecular fluorescence complementation assay. Expression of GsCPI14 was greatly induced by salt, ABA and alkali stress in G. soja, and GsCBRLK overexpression (OX) in Glycine max promoted the stress induction of GmCPI14 expression under stress conditions. Furthermore, we found that GsCPI14-eGFP fusion protein localized in the entire Arabidopsis protoplast and onion epidermal cell, and GsCPI14 showed ubiquitous expression in different tissues of G. soja. In addition, we gave evidence that the GST-GsCPI14 fusion protein inhibited the proteolytic activity of papain in vitro. At last, we demonstrated that OX of GsCPI14 in Arabidopsis promoted the seed germination under alkali stress, as evidenced by higher germination rates. GsCPI14 transgenic Arabidopsis seedlings also displayed better growth performance and physiological index under alkali stress. Taken together, results presented in this study demonstrated that the G. soja cysteine proteinase inhibitor GsCPI14 interacted with the calcium/calmodulin-binding receptor-like kinase GsCBRLK and regulated plant tolerance to alkali stress. PMID:24407891

Sun, Xiaoli; Yang, Shanshan; Sun, Mingzhe; Wang, Sunting; Ding, Xiaodong; Zhu, Dan; Ji, Wei; Cai, Hua; Zhao, Chaoyue; Wang, Xuedong; Zhu, Yanming

2014-05-01

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Coffee cysteine proteinases and related inhibitors with high expression during grain maturation and germination  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Cysteine proteinases perform multiple functions in seeds, including participation in remodelling polypeptides and recycling amino acids during maturation and germination. Currently, few details exist concerning these genes and proteins in coffee. Furthermore, there is limited information on the cysteine proteinase inhibitors which influence the activities of these proteinases. Results Two cysteine proteinase (CP and four cysteine proteinase inhibitor (CPI gene sequences have been identified in coffee with significant expression during the maturation and germination of coffee grain. Detailed expression analysis of the cysteine proteinase genes CcCP1 and CcCP4 in Robusta using quantitative RT-PCR showed that these transcripts accumulate primarily during grain maturation and germination/post germination. The corresponding proteins were expressed in E. coli and purified, but only one, CcCP4, which has a KDDL/KDEL C-terminal sequence, was found to be active after a short acid treatment. QRT-PCR expression analysis of the four cysteine proteinase inhibitor genes in Robusta showed that CcCPI-1 is primarily expressed in developing and germinating grain and CcCPI-4 is very highly expressed during the late post germination period, as well as in mature, but not immature leaves. Transcripts corresponding to CcCPI-2 and CcCPI-3 were detected in most tissues examined at relatively similar, but generally low levels. Conclusions Several cysteine proteinase and cysteine proteinase inhibitor genes with strong, relatively specific expression during coffee grain maturation and germination are presented. The temporal expression of the CcCP1 gene suggests it is involved in modifying proteins during late grain maturation and germination. The expression pattern of CcCP4, and its close identity with KDEL containing CP proteins, implies this proteinase may play a role in protein and/or cell remodelling during late grain germination, and that it is likely to play a strong role in the programmed cell death associated with post-germination of the coffee grain. Expression analysis of the cysteine proteinase inhibitor genes suggests that CcCPI-1 could primarily be involved in modulating the activity of grain CP activity; while CcCPI-4 may play roles modulating grain CP activity and in the protection of the young coffee seedlings from insects and pathogens. CcCPI-2 and CcCPI-3, having lower and more widespread expression, could be more general "house-keeping" CPI genes.

Lepelley Maud

2012-03-01

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The resistance of cowpea seeds to bruchid beetles is not related to levels of cysteine proteinase inhibitors.  

Science.gov (United States)

A cDNA encoding a cysteine proteinase inhibitor was isolated from a cDNA library prepared from developing seeds of an insect-resistant line of cowpea. The sequence of the encoded protein was homologous with those of other plant cysteine endoproteinase inhibitors, and with Type 2 cystatins from animals. Southern blot analyses indicated that small gene families were present in both resistant and susceptible lines of cowpea, while northern blot analyses showed similar levels of expression. It is concluded that the levels of expression of the inhibitor do not account for the differences in insect resistance of the two lines. PMID:8219051

Fernandes, K V; Sabelli, P A; Barratt, D H; Richardson, M; Xavier-Filho, J; Shewry, P R

1993-10-01

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Partial isolation and characterization of a cysteine proteinase inhibitor from Lima bean (Phaseolus lunatus).  

Science.gov (United States)

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance. PMID:11262065

Lawrence, J C; Nielsen, S S

2001-02-01

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Th1 cell development induced by cysteine proteinases A and B in localized cutaneous leishmaniasis due to Leishmania guyanensis.  

Science.gov (United States)

The cysteine proteinases CPA and CPB from Leishmania major induced Th1 responses in patients with leishmaniasis due to Leishmania guyanensis. Furthermore, cysteine proteinases induced neither interleukin 4 (IL-4) nor IL-13 and low levels of IL-10 in controls and patients. The results suggest that CPs would be quite good candidates for a vaccine against different Leishmania species. PMID:12704171

Pascalis, Hervé; Lavergne, Anne; Bourreau, Eliane; Prévot-Linguet, Ghislaine; Kariminia, Amina; Pradinaud, Roger; Rafati, Sima; Launois, Pascal

2003-05-01

 
 
 
 
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Th1 Cell Development Induced by Cysteine Proteinases A and B in Localized Cutaneous Leishmaniasis Due to Leishmania guyanensis  

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The cysteine proteinases CPA and CPB from Leishmania major induced Th1 responses in patients with leishmaniasis due to Leishmania guyanensis. Furthermore, cysteine proteinases induced neither interleukin 4 (IL-4) nor IL-13 and low levels of IL-10 in controls and patients. The results suggest that CPs would be quite good candidates for a vaccine against different Leishmania species.

Pascalis, Herve?; Lavergne, Anne; Bourreau, Eliane; Pre?vot-linguet, Ghislaine; Kariminia, Amina; Pradinaud, Roger; Rafati, Sima; Launois, Pascal

2003-01-01

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Biochemical and immunological characterization of a recombinantly-produced antifungal cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa).  

Science.gov (United States)

Plant proteinase inhibitors are considered important defense molecules against insect and pathogen attack. The cysteine proteinase inhibitor (CPI) from green kiwifruit (Actinidia deliciosa) belongs to the cystatin family and shows potent antifungal activity (in vitro and in vivo). However, the low abundance of this molecule in fruit (6?g/g of fresh fruit) seems to limit further investigations on the interaction between phytocystatin and photopathogenic fungi. In this paper the cDNA of the kiwi CPI was expressed in Escherichia coli. Fifteen N-terminal amino acids were identified by Edman degradation, and 77% of the rCPI primary structure was confirmed by mass fingerprint. The structural homology of recombinant CPI (rCPI) to its natural counterpart has been clearly demonstrated in immunological assays (immunoblot and ELISA inhibition). Biological activity of rCPI was demonstrated in inhibition assay with cysteine proteinase papain (EC50 2.78nM). In addition, rCPI reveals antifungal properties toward pathogenic fungi (Alternaria radicina and Botrytis cinerea), which designates it as an interesting model protein for the exploration of plant phytocystatins - pathogen interactions. Understanding the molecular mechanisms of natural plant resistance could lead to the development of ecologically safe fungicides for controlling post-harvest diseases and maintaining food quality. PMID:23830694

Popovic, Milica; Andjelkovic, Uros; Burazer, Lidija; Lindner, Buko; Petersen, Arnd; Gavrovic-Jankulovic, Marija

2013-10-01

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The human anti-HIV antibodies 2F5, 2G12, and PG9 differ in their susceptibility to proteolytic degradation: down-regulation of endogenous serine and cysteine proteinase activities could improve antibody production in plant-based expression platforms.  

Science.gov (United States)

The tobacco-related species Nicotiana benthamiana has recently emerged as a promising host for the manufacturing of protein therapeutics. However, the production of recombinant proteins in N. benthamiana is frequently hampered by undesired proteolysis. Here, we show that the expression of the human anti-HIV antibodies 2F5, 2G12, and PG9 in N. benthamiana leaves leads to the accumulation of discrete heavy chain-derived degradation products of 30-40 kDa. Incubation of purified 2F5 with N. benthamiana intercellular fluid resulted in rapid conversion into the 40-kDa fragment, whereas 2G12 proved largely resistant to degradation. Such a differential susceptibility to proteolytic attack was also observed when these two antibodies were exposed to various types of proteinases in vitro. While serine and cysteine proteinases are both capable of generating the 40-kDa 2F5 fragment, the 30-kDa polypeptide is most readily obtained by treatment with the latter class of enzymes. The principal cleavage sites reside within the antigen-binding domain, the VH -CH 1 linker segment and the hinge region of the antibodies. Collectively, these results indicate that down-regulation of endogenous serine and cysteine proteinase activities could be used to improve the performance of plant-based expression platforms destined for the production of biopharmaceuticals. PMID:24478053

Niemer, Melanie; Mehofer, Ulrich; Torres Acosta, Juan Antonio; Verdianz, Maria; Henkel, Theresa; Loos, Andreas; Strasser, Richard; Maresch, Daniel; Rademacher, Thomas; Steinkellner, Herta; Mach, Lukas

2014-04-01

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Identification of a lymphocyte-produced cysteine proteinase based on its binding to ?-macroglobulin (?M)  

International Nuclear Information System (INIS)

Proteinases play a role in collagen vascular diseases. However, the role of lymphocytes in the production of these proteinases is poorly understood. Rabbit lymph node cells (LNC) were cultured with "3"5S-methionine or "1"4C-leucine. When culture supernatants were analyzed by radioimmunoelectrophoresis using an anti-rabbit ?M antiserum, radio-active ?M-precipitin arcs developed. LNC biosynthetically labeled in serum-free medium generated supernatants that, upon incubation with normal rabbit serum, yielded radio-active ?M precipitin arcs. Purified B and T cells as well as human T and B cell lines were labeled separately under identical conditions. Analysis revealed the presence of radiolabeled ?M molecules. T cells usually generated less intense radioactive ?M precipitin arcs than B cells. Thoracic duct lymphocytes also produced radioactive ?M precipitin arcs. They treated radiolabeled supernatants with a variety of proteinase inhibitors before reacting them with rabbit serum. Only inhibitors specific for cysteine or thiol proteinases were effective in abolishing radioactivity associated with ?M. It appears that lymphocytes actively produce a cysteine or thiol proteinase which, theoretically, is capable of degrading collagen

1986-03-01

25

Developing novel anthelmintics from plant cysteine proteinases  

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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 syn...

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

2008-01-01

26

Circadian rhythms of cysteine proteinases and cystatins, potential tumour markers, in normal sera  

International Nuclear Information System (INIS)

Circadian day/night variations have been evidenced in all major groups of organisms and at all levels of organisation of the organism. Circadian intra-individual variations are known for a number of analyses in serum including tumour-associated markers. It was suggested that the serum levels of cysteine proteinases and their inhibitors may be of clinical importance for prognosis and diagnosis in cancer. Since known circadian rhythms are important for choosing the best sampling time, interpretation of the results of a diagnostic test, patient monitoring, and timing of a therapy, our objective was to establish 24-h variations of cysteine proteinases, cathepsins B, H, L, and their low molecular weight inhibitors, stefin A, stefin B, and cystatin C, in sera from healthy subjects. (author)

2002-01-01

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Inhibition of Trypsin-Like Cysteine Proteinases (Gingipains) from Porphyromonas gingivalis by Tetracycline and Its Analogues  

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Extracellular cysteine proteinases, referred to as gingipains, are considered important virulence factors for Porphyromonas gingivalis, a bacterium recognized as a major etiologic agent of chronic periodontitis. We investigated the effect of tetracycline and its analogues, doxycycline and minocycline, on the enzymatic activities of gingipains. Tetracyclines at 100 ?M totally inhibited the amidolytic activity of arginine-specific gingipains (HRgpA and RgpB). In contrast, inhibition of Kgp was...

Imamura, Takahisa; Matsushita, Kenji; Travis, James; Potempa, Jan

2001-01-01

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

Directory of Open Access Journals (Sweden)

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.

Wei Wang

2014-02-01

29

Functional properties of a cysteine proteinase from pineapple fruit with improved resistance to fungal pathogens in Arabidopsis thaliana.  

Science.gov (United States)

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. PMID:24566309

Wang, Wei; Zhang, Lu; Guo, Ning; Zhang, Xiumei; Zhang, Chen; Sun, Guangming; Xie, Jianghui

2014-01-01

30

Analysis of the prtP gene encoding porphypain, a cysteine proteinase of Porphyromonas gingivalis.  

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The cloning and sequencing of the gene encoding porphypain, a cysteine proteinase previously isolated from detergent extracts of the Porphyromonas gingivalis W12 cell surface, are described. The prtP gene encoded a unique protein of 1,732 amino acids, including a putative signal sequence for protein secretion. The predicted molecular mass for the mature protein was 186 kDa, which was close to the observed molecular mass of 180 kDa. There was one copy of prtP in the genomes of seven P. gingiva...

Barkocy-gallagher, G. A.; Han, N.; Patti, J. M.; Whitlock, J.; Progulske-fox, A.; Lantz, M. S.

1996-01-01

31

Evaluation of Enzyme-linked Immunotransfer Blot for the Immunodiagnosis of Human Fascioliasis Using Cysteine Proteinase Antigen  

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The present study was targeted as examining sera obtaining from patients infected with Fasciola sp. by the Enzyme-linked Immunotransfer Blot (EITB) technique using the parasite`s Cysteine Proteinase (CE) antigen in order to evaluate the diagnostic potential of the assay. Altogether, a sort of sera including 80 cases of fasciolosis, 80 with other parasitosis other than fasciolosis and 30 normal control sera were enrolled in the trial. Hinge on the collected results, 78 fasciolosis serum...

2006-01-01

32

Roles of two conserved cysteine residues in the activation of human adenovirus proteinase.  

Science.gov (United States)

The roles of two conserved cysteine residues involved in the activation of the adenovirus proteinase (AVP) were investigated. AVP requires two cofactors for maximal activity, the 11-amino acid peptide pVIc (GVQSLKRRRCF) and the viral DNA. In the AVP-pVIc crystal structure, conserved Cys104 of AVP has formed a disulfide bond with conserved Cys10 of pVIc. In this work, pVIc formed a homodimer via disulfide bond formation with a second-order rate constant of 0.12 M(-1) s(-1), and half of the homodimer could covalently bind to AVP via thiol-disulfide exchange. Alternatively, monomeric pVIc could form a disulfide bond with AVP via oxidation. Regardless of the mechanism by which AVP becomes covalently bound to pVIc, the kinetic constants for substrate hydrolysis were the same. The equilibrium dissociation constant, K(d), for the reversible binding of pVIc to AVP was 4.4 microM. The K(d) for the binding of the mutant C10A-pVIc was at least 100-fold higher. Surprisingly, the K(d) for the binding of the C10A-pVIc mutant to AVP decreased at least 60-fold, to 6.93 microM, in the presence of 12mer ssDNA. Furthermore, once the mutant C10A-pVIc was bound to an AVP-DNA complex, the macroscopic kinetic constants for substrate hydrolysis were the same as those exhibited by wild-type pVIc. Although the cysteine in pVIc is important in the binding of pVIc to AVP, formation of a disulfide bond between pVIc and AVP was not required for maximal stimulation of enzyme activity by pVIc. PMID:11724559

McGrath, W J; Baniecki, M L; Peters, E; Green, D T; Mangel, W F

2001-12-01

33

[Proteinase inhibitors as antistress proteins in higher plants].  

Science.gov (United States)

Physicochemical and functional characteristics of plant protein proteinase inhibitors as antistress biopolymers were studied to determine the mechanisms for plant resistance to phytopathogens and to obtain disease-resistant cereal and leguminous cultures. The activity of trypsin, chymotrypsin, and subtilisin inhibitors varied in monocotyledonous and dicotyledonous cultures. Study varieties of leguminous and cereal cultures were shown to contain endogenous inhibitors specific to proteinases of phytopathogenic fungi Fusarium, Colletotrichum, Helminthosporium, and Botrytis. These inhibitors were characterized by species specificity and variety specificity. Protease inhibitors from buckwheat seeds inhibited proteases of fungal pathogens and suppressed germination of spores and growth of the fungal mycelium. Our results suggest that proteinaceous inhibitors of proteinases are involved in the protective reaction of plants under stress conditions. PMID:16212034

Dunaevski?, Ia E; Tsybina, T A; Beliakova, G A; Domash, V I; Shapno, T P; Zabre?ko, S A; Belozerski?, M A

2005-01-01

34

Characterisation of cysteine proteinases responsible for digestive proteolysis in guts of larval Western corn rootworm (Diabrotica virgifera) by expression in the yeast Pichia pastoris  

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Cysteine proteinases are the major class of enzymes responsible for digestive proteolysis in western corn rootworm (Diabrotica virgifera), a serious pest of maize. A larval gut extract hydrolysed typical cathepsin substrates, such as Z-phe-arg-AMC and Z-arg-arg-AMC, and hydrolysis was inhibited by Z-phe-tyr-DMK, specific for cathepsin L. A cDNA library representing larval gut tissue mRNA contained cysteine proteinase-encoding clones at high frequency. Sequence analysis of 11 cysteine proteina...

Bown, D. P.; Wilkinson, H. S.; Jongsma, M. A.; Gatehouse, J. A.

2004-01-01

35

Altered expression of cruzipain and a cathepsin B-like target in a Trypanosoma cruzi cell line displaying resistance to synthetic inhibitors of cysteine-proteinases.  

Science.gov (United States)

The therapeutic potential of synthetic inhibitors to the major cysteine-proteinase from Trypanosoma cruzi (cruzain or cruzipain) was recently demonstrated in animal models of Chagas' disease. A possible limitation of this strategy would be the emergence of parasite populations developing resistance to cysteine-proteinase inhibitors. Here, we describe the properties of a phenotypically stable T. cruzi cell line (R-Dm28) that displays increased resistance to Z-(SBz)Cys-Phe-CHN2, an irreversible cysteine-proteinase inhibitor which preferentially inactivates cathepsin L-like enzymes. Isolated from axenic cultures of the parental cells (IC50 1.5 microM), R-Dm28 epimastigotes exhibited 13-fold (IC50) 20 microM) higher resistance to this inhibitor and did not display cross-resistance to unrelated trypanocidal drugs, such as benznidazol and nifurtimox. Western blotting (with mAb), affinity labeling (with biotin-LVG-CHN2) and FACS analysis of R-Dm28 log-phase epimastigotes revealed that the cruzipain target was expressed at lower levels, as compared with Dm28c. Interestingly, this deficit was paralleled by increased expression of an unrelated Mr 30 000 cysteine-proteinase whose activity was somewhat refractory to inhibition by Z-(SBz)Cys-Phe-CHN,. N-terminal sequencing of the affinity-purified biotin-LVG-proteinase complex allowed its identification as a cathepsin B-like enzyme. Increased antigenic deposits of this proteinase were found in the grossly enlarged and electron dense reservosomes from R-Dm28 epimastigotes. Our data suggest that R-Dm28 resistance to toxic effects induced by the synthetic inhibitor may result from decreased availability of the most sensitive cysteine-proteinase target, cruzipain. The deficit in metabolic functions otherwise mediated by this cathepsin L-like proteinase is likely compensated by increased expression/accumulation of a cathepsin B-like target. PMID:10924756

Yong, V; Schmitz, V; Vannier-Santos, M A; de Lima, A P; Lalmanach, G; Juliano, L; Gauthier, F; Scharfstein, J

2000-06-01

36

Hemoglobin-binding protein purified from Porphyromonas gingivalis is identical to lysine-specific cysteine proteinase (Lys-gingipain).  

Science.gov (United States)

The functional protein that binds to human hemoglobin (hemoglobin-binding protein; HBP) was purified from Porphyromonas gingivalis cells. The analyses of the amino-terminal sequence and amino acid composition revealed that HBP is identical to lysine-specific cysteine proteinase (51 kDa Lys-gingipain; KGP) of P. gingivalis 381. It is a novel finding that KGP has binding affinity to hemoglobin. The binding activity of HBP was enhanced by acidic or anaerobic conditions. Arg-gingipain, a member of the gingipain family, of P. gingivalis exhibited no ability to bind to hemoglobin. The recombinant protein of KGP (r-KGP) generated in Escherichia coli showed both hemoglobin-binding and proteolytic activities. The treatment of r-KGP by protein disulfide isomerase effectively enhanced binding to hemoglobin, whereas the proteinase activity was decreased. The treated r-KGP significantly inhibited the binding of hemoglobin to the whole cell extracts in a dose-dependent manner. These results suggest that the hemoglobin binding of P. gingivalis is mediated by KGP through active domain(s) distinct from that for proteinase activity. PMID:9705827

Kuboniwa, M; Amano, A; Shizukuishi, S

1998-08-10

37

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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.

CB, Toaldo; M, Steindel; MA, Sousa; CC, Tavares.

38

Evaluation of Enzyme-linked Immunotransfer Blot for the Immunodiagnosis of Human Fascioliasis Using Cysteine Proteinase Antigen  

Directory of Open Access Journals (Sweden)

Full Text Available The present study was targeted as examining sera obtaining from patients infected with Fasciola sp. by the Enzyme-linked Immunotransfer Blot (EITB technique using the parasite`s Cysteine Proteinase (CE antigen in order to evaluate the diagnostic potential of the assay. Altogether, a sort of sera including 80 cases of fasciolosis, 80 with other parasitosis other than fasciolosis and 30 normal control sera were enrolled in the trial. Hinge on the collected results, 78 fasciolosis serum samples recognized two antigenic polypeptides of 27 and 29 kDa. The sensitivity, specificity and positive and negative predictive values for CP antigen were 97.5, 98.8, 98.7 and 97.7%, respectively. Utterly, one case of cross-reaction was verified with a toxocariasis case. Concluding remark suggests that the 27 and 29 kDa bands in EITB test could be imperative in the immunodiagnosis of human fascioliasis.

M.B. Rokni

2006-01-01

39

Enhanced Protective Efficacy of Nonpathogenic Recombinant Leishmania tarentolae Expressing Cysteine Proteinases Combined with a Sand Fly Salivary Antigen  

Science.gov (United States)

Background Novel vaccination approaches are needed to prevent leishmaniasis. Live attenuated vaccines are the gold standard for protection against intracellular pathogens such as Leishmania and there have been new developments in this field. The nonpathogenic to humans lizard protozoan parasite, Leishmania (L) tarentolae, has been used effectively as a vaccine platform against visceral leishmaniasis in experimental animal models. Correspondingly, pre-exposure to sand fly saliva or immunization with a salivary protein has been shown to protect mice against cutaneous leishmaniasis. Methodology/Principal Findings Here, we tested the efficacy of a novel combination of established protective parasite antigens expressed by L. tarentolae together with a sand fly salivary antigen as a vaccine strategy against L. major infection. The immunogenicity and protective efficacy of different DNA/Live and Live/Live prime-boost vaccination modalities with live recombinant L. tarentolae stably expressing cysteine proteinases (type I and II, CPA/CPB) and PpSP15, an immunogenic salivary protein from Phlebotomus papatasi, a natural vector of L. major, were tested both in susceptible BALB/c and resistant C57BL/6 mice. Both humoral and cellular immune responses were assessed before challenge and at 3 and 10 weeks after Leishmania infection. In both strains of mice, the strongest protective effect was observed when priming with PpSP15 DNA and boosting with PpSP15 DNA and live recombinant L. tarentolae stably expressing cysteine proteinase genes. Conclusion/Significance The present study is the first to use a combination of recombinant L. tarentolae with a sand fly salivary antigen (PpSP15) and represents a novel promising vaccination approach against leishmaniasis.

Taheri, Tahereh; Taslimi, Yasaman; Doustdari, Fatemeh; Seyed, Negar; Torkashvand, Fatemeh; Meneses, Claudio; Papadopoulou, Barbara; Kamhawi, Shaden; Valenzuela, Jesus G.; Rafati, Sima

2014-01-01

40

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 C...

Gregor Kosec; Vanina Alvarez; Cazzulo, Juan J.

2006-01-01

 
 
 
 
41

Analysis of human immunoglobulin-degrading cysteine proteinases of Trichomonas vaginalis.  

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Trichomonas vaginalis is a protozoan parasite that causes a widely distributed sexually transmitted disease (STD). Since immunoglobulin G (IgG) antibodies to specific trichomonad immunogens are found in serum and vaginal washes (VWs) from patients with trichomoniasis, a potential mechanism of immune evasion by this parasite might be the ability of T. vaginalis proteinases to degrade human immunoglobulins (Igs). Incubation of human IgG with lysates of T. vaginalis organisms resulted in time- a...

Provenzano, D.; Alderete, J. F.

1995-01-01

42

The involvement of a cysteine proteinase in the nodule development in Chinese milk vetch infected with Mesorhizobium huakuii subsp. rengei.  

Science.gov (United States)

Cys proteinases play important roles in plant cell development and senescence. A cDNA, AsNODf32, obtained by differential screening of a nodule cDNA library of the leguminous plant Chinese milk vetch (Astragalus sinicus), represents a nodule-specific Cys proteinase similar to that reported for the actinorhizal Alnus glutinosa-Flankia symbiosis. A characteristic feature of this proteinase is the presence of a putative vacuolar targetting signal, LQDA, within its propeptide. Expression of the AsNODf32 gene, which was studied on northern blots and in situ hybridization, showed good correlation with the onset of nodule senescence. In situ hybridization studies revealed that AsNODf32 was expressed in senescent-infected tissue at the base of the nodule, as well as in interzone II-III of the infected nodules. In addition to degrading old nodule tissues and bacteroids, AsNODf32 protein may be required as a component of tissue remodeling during nodule development. PMID:11080286

Naito, Y; Fujie, M; Usami, S; Murooka, Y; Yamada, T

2000-11-01

43

Wound-induced expression of a potato proteinase inhibitor II gene in transgenic tobacco plants  

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A potato proteinase inhibitor II gene was transferred into tobacco plants using Agrobacterium/Ti-plasmid-mediated gene transfer techniques. Whereas no or little expression of the proteinase inhibitor II gene could be detected in non-wounded leaves, high levels of proteinase inhibitor II mRNA were detected in leaves of several transgenic tobacco plants after mechanical wounding as well as after treatment of detached leaves with oligosaccharides. Wounding of a leaf also led to a systemic induct...

Sanchez-serrano, Jose J.; Keil, Michael; O Connor, Aileen; Schell, Jeff; Willmitzer, Lothar

1987-01-01

44

Identification of Old World Leishmania spp. by specific polymerase chain reaction amplification of cysteine proteinase B genes and rapid dipstick detection  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We used the cysteine proteinase B (cpb) gene family of the trypanosomatid genus Leishmania as a target to develop rapid, specific, and easy-to-use polymerase chain reaction (PCR) tests to discriminate Leishmania infantum, Leishmania donovani, Leishmania tropica, Leishmania aethiopica, and Leishmania major. Identification of all 5 Old World species and validation of intraspecies variability are features lacking in other species-specific PCRs. Amplicon analysis was done on agarose gels and was ...

2009-01-01

45

Cationic solid lipid nanoparticles loaded by cysteine proteinase genes as a novel anti-leishmaniasis DNA vaccine delivery system: characterization and in vitro evaluations.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

ABSTRACT - Purpose. Leishmaniasis is a major health problem in many tropical and sub-tropical countries and development of a safe and easily-available vaccine has high priority. Although several antigens potentially capable of inducing protective immunity have been studied, in the absence of pharmaceutical industry interest they have remained as fine publications only. Amongst them, Cathepsin L-like cysteine proteinases (CPs) have received considerable attention and type I and II CPs have bee...

Doroud, Delaram; Vatanara, Alireza; Zahedifard, Farnaz; Gholami, Elham; Vahabpour, Rouholah; Rouholamini Najafabadi, Abdolhossein; Rafati, Sima

2010-01-01

46

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

2006-08-18

47

Cysteine proteinase inhibitor Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy.  

Science.gov (United States)

Kiwifruit has become a frequent cause of fruit allergy in the recent years. The molecular basis of type I hypersensitivity to kiwifruit is attributed to 11 IUIS allergens, with Act d 1, Act d 2 and Act d 5 characterized in extenso. Evaluation of the allergenic properties of Act d 4, a cysteine proteinase inhibitor from green kiwifruit (Actinidia deliciosa) was performed in this study. Identity of the purified glycoprotein was determined by Edman degradation and by mass fingerprint whereby more than 90% of the primary structure of the mature kiwifruit cystatin was confirmed. Using MALDI TOF analysis, molecular masses of 10902.5 and 11055.2 Da were detected for Act d 4, respectively. Positive skin prick reactivity with Act d 4 was induced in three kiwifruit allergic patients, as well as the upregulation of CD63 and CD203c molecules in the basophile activation assay. The IgE reactivity was detected in dot blot analysis while Western blot analysis was negative using sera from six kiwifruit patients, suggesting the presence of conformational IgE epitopes on the Act d 4 molecule. As activator of effector cells in type I hypersensitivity Act d 4 is a functional allergen contributing to the clinical symptoms of kiwifruit allergy. PMID:19885843

Popovic, Milica M; Milovanovic, Mina; Burazer, Lidija; Vuckovic, Olga; Hoffmann-Sommergruber, Karin; Knulst, Andre C; Lindner, Buko; Petersen, Arnd; Jankov, Ratko; Gavrovic-Jankulovic, Marija

2010-03-01

48

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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, My...

Carrillo Gil, Laura; Martinez Mun?oz, Manuel; Alvarez Alfageme, Fernando; Castan?era, Pedro; Smagghe, Guy; Diaz Rodriguez, Isabel; Ortego, Felix

2011-01-01

49

"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)

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.

"Rokni MB

2001-08-01

50

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

DEFF Research Database (Denmark)

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-β-1,4-mannanase) and GH 28 (polygalacturonase) were overexpressed. Conversely, α-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.

Nogueira, Fábio C S; Silva, Carlos P

2012-01-01

51

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

Science.gov (United States)

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

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

52

Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal Pathogen Porphyromonas gingivalis  

Science.gov (United States)

Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis. We therefore examined the potential role of the dominant P. gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 ?M). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 ?M), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P. gingivalis cells in a batch culture, in parallel with proteinase activity. Cysteine proteinases from P. gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition in P. gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention.

DeCarlo, Arthur A.; Paramaesvaran, Mayuri; Yun, Peter L. W.; Collyer, Charles; Hunter, Neil

1999-01-01

53

Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects.  

Science.gov (United States)

Proteinase inhibitors which act on the digestive enzymes of insect herbivores are a basic mechanism of plant defence. Attempts to exploit this defence mechanism in plant genetic engineering have used over-expression of both endogenous and exogenous inhibitors. While significant protection against insect pests has been routinely achieved, the engineered plants do not show levels of resistance considered commercially viable. As a result of selective pressures, insect herbivores have developed multiple mechanisms of adaptation to overcome the defensive effects of plant proteinase inhibitors. Common polyphagous crop pests are well adapted to deal with a range of different inhibitors, which have only limited effects on fitness as a result. A range of strategies have been attempted to improve effectiveness of proteinase inhibitors as antimetabolites towards insects, including selection for inhibitory activity against insect digestive enzymes, mutagenesis for novel inhibitory activity, and engineering inhibitors with multiple functions. However, proteinase inhibitor genes have only been used in transgenic crops in combination with other insecticidal genes. In Chinese genetically engineered cotton varieties which express Bt toxins as an insecticidal protein against lepidopteran larvae, the CpTI (cowpea trypsin inhibitor) gene has been employed as a second transgene to improve protection. This gene combination represents the only commercial deployment of a proteinase inhibitor transgene to date, with Bt/CpTI cotton grown on over 0.5 million hectares in 2005. Future prospects for using proteinase inhibitor genes to enhance insect resistance in transgenic crops will require reassessment of their mechanisms of action, particularly in affecting processes other than digestion, as exemplified by effects on sap-feeding hemipteran pests. PMID:21418023

Gatehouse, John A

2011-08-01

54

Polyamine depletion down-regulates expression of the Trichomonas vaginalis cytotoxic CP65, a 65-kDa cysteine proteinase involved in cellular damage.  

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Recently, we found that inhibition of putrescine synthesis by ornithine decarboxylase (ODC) significantly increased Trichomonas vaginalis adherence mediated by protein adhesins. Surprisingly and unexpectedly, trichomonal contact-dependent cytotoxicity was absent. Therefore, a role for polyamine depletion on regulation of T. vaginalis cytotoxicity mediated by the cysteine proteinase (CP) of 65-kDa, CP65, was investigated. We performed cytotoxicity and cell-binding assays followed by zymograms, as well as Western blot and indirect immunofluorescence assays using specific anti-CP65 antibodies to detect CP65. Trichomonads grown in the presence of the ODC inhibitor, 1-4-diamino-2-butanone (DAB) had lower levels of cytotoxicity that corresponded with diminished CP65 proteolytic activity when compared to untreated organisms handled identically. Likewise, semiquantitative and qRT-PCR as well as Western blot and immunofluorescence assays showed decreased amounts of tvcp65 mRNA and CP65 protein in DAB-treated parasites. These effects were reversed by addition of exogenous putrescine. These data show a direct link between polyamine metabolism and expression of the cytotoxic CP65 proteinase involved in trichomonal host cellular damage. PMID:18586550

Alvarez-Sánchez, María Elizbeth; Carvajal-Gamez, Bertha Isabel; Solano-González, Eduardo; Martínez-Benitez, Máximo; Garcia, Ana F; Alderete, John F; Arroyo, Rossana

2008-01-01

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Plants that express a potyvirus proteinase gene are resistant to virus infection.  

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Transgenic tobacco plants that express the genome-linked protein/proteinase-coding region of the potyvirus tobacco vein mottling virus (TVMV) were produced and tested for their reaction to inoculation with TVMV and two other potyviruses. These plants did not develop disease symptoms after being inoculated with large doses of TVMV but were as susceptible to infection by the other potyviruses as were control plants. Lines of tobacco that express the coat protein- or the nonstructural cylindrica...

Maiti, I. B.; Murphy, J. F.; Shaw, J. G.; Hunt, A. G.

1993-01-01

56

Hydrolysis of Interleukin-12 by Porphyromonas gingivalis Major Cysteine Proteinases May Affect Local Gamma Interferon Accumulation and the Th1 or Th2 T-Cell Phenotype in Periodontitis  

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Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-?) production. Here we demonstrate that in the presence or absence of serum,...

Yun, Peter L. W.; Decarlo, Arthur A.; Collyer, Charles; Hunter, Neil

2001-01-01

57

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

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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, the...

Allhorn Maria; Olsén Arne; Collin Mattias

2008-01-01

58

In silico predicted epitopes from the COOH-terminal extension of cysteine proteinase B inducing distinct immune responses during Leishmania (Leishmania amazonensis experimental murine infection  

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Full Text Available Abstract Background Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB is an important virulence factor in parasites from the Leishmania (Leishmania mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant. Results Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities. Conclusions Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

Pereira Bernardo AS

2011-08-01

59

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

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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.

Olsén Arne

2008-01-01

60

Recombinant cysteine proteinases-based vaccines against Leishmania major in BALB/c mice: the partial protection relies on interferon gamma producing CD8(+) T lymphocyte activation.  

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Together with poloxamer 407 as adjuvant the recombinant type I (rCPB) or type II (rCPA) cysteine proteinases of Leishmania major were screened as potential vaccines against L. major in a mouse model. The vaccines were delivered subcutaneously twice at 3 weeks intervals. Three weeks after booster injection, 5x10(5) stationary phase L. major promastigotes were inoculated subcutaneously in one footpad. Using the footpad thickness increase to monitor the clinical outcome/cutaneous lesion at site of L. major delivery, it was possible to document that rCPB but not rCPA allowed BALB/c mice to mount a partial protective response: indeed over the period under study (weeks 1-12) a clear delay was noticed after the immunization with rCPB. This partial protective effect was no more detectable if CD8 depleting antibody was given intravenously to rCPB-immunized mice, at the time of parasite challenge. Seven weeks after challenge, the draining lymph nodes were monitored for their frequencies of IFN-gamma positive CD4(+) and CD8(+) T lymphocytes using PMA and ionomycin as re-activating signals: interestingly the partial protection achieved in BALB/c mice immunized with rCPB together with poloxamer was correlated only to one immunological parameter, namely the higher frequency of IFN-gamma producing CD8(+) T lymphocytes. Of note also, in the lymph node draining the L. major-loaded footpad of C57BL/6 mice otherwise known to develop a transient lesion, the frequency of IFN-gamma producing CD8(+) T lymphocytes reach similar value 7 weeks after challenge and in absence of any prior immunization. Taken together, it was shown that the induced partial protection was mainly dependent on IFN-gamma producing CD8(+) T cells. PMID:12057598

Rafati, Sima; Kariminia, Amina; Seyde-Eslami, Shiva; Narimani, Manijeh; Taheri, Tahere; Lebbatard, Mai

2002-06-01

 
 
 
 
61

Crystallization and preliminary X-ray crystallographic studies of the plant aspartic proteinase cardosin A.  

Science.gov (United States)

The plant aspartic proteinase cardosin A was crystallized using vapour diffusion. Crystals belong to the monoclinic space group C2, cell dimensions a = 116.9 (2), b = 87.2 (8), c = 81.3 (1) A, beta = 104.4 (4) degrees, and contain two molecules in the asymmetric unit related by a non-crystallographic twofold axis. Diffraction data were collected at room temperature with radiation from a synchrotron source up to 2.85 A resolution. When the crystals were flash cooled to 110 K in a nitrogen stream the same resolution limit could also be obtained on a rotating-anode source. Recently, synchrotron radiation together with flash cooling led to an improvement of the diffraction data to 1.72 A resolution. PMID:9757116

Bento, I; Frazão, C; Coelho, R; Wilson, K; Dauter, Z; Carrondo, M A

1998-09-01

62

Interaction between potyvirus helper component-proteinase and capsid protein in infected plants.  

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Monoclonal antibodies were raised against helper component-proteinase (HcPro) purified from plants infected with the potyvirus Lettuce mosaic virus (LMV). These antibodies were used in a two-site triple antibody sandwich ELISA assay together with polyclonal antibodies directed against purified virions. An interaction between HcPro and the viral coat protein (CP) was demonstrated in extracts of LMV-infected leaves, as well as for two other potyviruses, Plum pox virus and Potato virus Y. The CP-HcPro interaction was not abolished in LMV derivatives with an HcPro GFP N-terminal fusion, or with a deletion from the CP of the amino acids involved in aphid transmission. Electron microscopy indicated that HcPro probably does not interact with the CP in the form of assembled virions or virus-like particles. Together, these results suggest that the interaction detected between CP and HcPro might be involved in a process of the potyvirus cycle different from aphid transmission. PMID:12075097

Roudet-Tavert, Geneviève; German-Retana, Sylvie; Delaunay, Thierry; Delécolle, Brigitte; Candresse, Thierry; Le Gall, Olivier

2002-07-01

63

Inhibitors of lysosomal cysteine proteases  

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Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

Lyanna O. L.

2011-04-01

64

Plant cysteine oxidases control the oxygen-dependent branch of the N-end-rule pathway  

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In plant and animal cells, amino-terminal cysteine oxidation controls selective proteolysis via an oxygen-dependent branch of the N-end rule pathway. It remains unknown how the N-terminal cysteine is specifically oxidized. Here we identify plant cysteine oxidase (PCO) enzymes that oxidize the penultimate cysteine of ERF-VII transcription factors by using oxygen as a co-substrate, thereby controlling the lifetime of these proteins. Consequently, ERF-VII proteins are stabilized under hypoxia and activate the molecular response to low oxygen while the expression of anaerobic genes is repressed in air. Members of the PCO family are themselves targets of ERF-VII transcription factors, generating a feedback loop that adapts the stress response according to the extent of the hypoxic condition. Our results reveal that PCOs act as sensor proteins for oxygen in plants and provide an example of how proactive regulation of the N-end rule pathway balances stress response to optimal growth and development in plants.

Weits, Daan A.; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Hubberten, Hans-Michael; Riegler, Heike; Hoefgen, Rainer; Perata, Pierdomenico; van Dongen, Joost T.; Licausi, Francesco

2014-01-01

65

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.  

Science.gov (United States)

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth. PMID:10497243

Faro, C; Ramalho-Santos, M; Vieira, M; Mendes, A; Simões, I; Andrade, R; Veríssimo, P; Lin, X; Tang, J; Pires, E

1999-10-01

66

Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells.  

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The role of cysteine proteinases in adherence of Trichomonas vaginalis NYH 286 to HeLa and human vaginal epithelial cells was evaluated. Only pretreatment of trichomonads, but not epithelial cells, with N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), an inhibitor of trichomonad cysteine proteinases, greatly diminished the ability of T. vaginalis to recognize and bind to epithelial cells. Leupeptin and L-1-tosylamide-2-phenylethyl chloromethyl ketone, other cysteine proteinase inhibitors,...

1989-01-01

67

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

Science.gov (United States)

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

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

2005-01-01

68

On the activity and specificity of cardosin B, a plant proteinase, on ovine caseins  

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The proteolytic activity of cardosin B, an aspartic proteinase from the thistle, Cynara cardunculus, on ovine ?s-caseins and ?-caseins (independently or present together in sodium caseinate) was followed by urea polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography. This enzyme degraded both types of caseins, but not to the same degree. In sodium-caseinate, by 10 h at 30°C, ?s-caseins were more susceptible to proteolysis by cardosin B than ?-casein ...

Silva, Sofia V.; Malcata, F. Xavier

1999-01-01

69

Hydrolysis of Interleukin-12 by Porphyromonas gingivalis Major Cysteine Proteinases May Affect Local Gamma Interferon Accumulation and the Th1 or Th2 T-Cell Phenotype in Periodontitis  

Science.gov (United States)

Porphyromonas gingivalis cysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-?) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-? production from CD4+ T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-? production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.

Yun, Peter L. W.; Decarlo, Arthur A.; Collyer, Charles; Hunter, Neil

2001-01-01

70

Modulation of an Interleukin-12 and Gamma Interferon Synergistic Feedback Regulatory Cycle of T-Cell and Monocyte Cocultures by Porphyromonas gingivalis Lipopolysaccharide in the Absence or Presence of Cysteine Proteinases  

Science.gov (United States)

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-?) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-? have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogen Porphyromonas gingivalis stimulated monocytes primed with IFN-? to release IL-12, thereby enhancing IFN-? accumulation in T-cell populations. P. gingivalis LPS was shown to enhance IL-12 induction of IFN-? in T cells in a manner independent from TNF-? contribution. The levels of T-cell IL-12 receptors were not affected by P. gingivalis LPS and played only a minor role in the magnitude of the IFN-? response. These data suggest that LPS from P. gingivalis establishes an activation loop with IL-12 and IFN-? with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-? accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-? levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) of P. gingivalis could lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.

Yun, Peter L. W.; DeCarlo, Arthur A.; Collyer, Charles; Hunter, Neil

2002-01-01

71

Chloroplast-like organelles were found in enucleate sieve elements of transgenic plants overexpressing a proteinase inhibitor.  

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SaPIN2a, a plant proteinase inhibitor from nightshade (Solanum americanum), was located to the enucleate sieve elements (SEs) of phloem. The expressed SaPIN2a in transgenic lettuce showed inhibition of plant endogenous trypsin- and chymotrypsin-like activities, suggesting that SaPIN2a can regulate proteolysis in plant cells. To further investigate the physiological role of SaPIN2a, we produced transgenic nightshade and lettuce plants overexpressing SaPIN2a from the cauliflower mosaic virus (CaMV) 35S promoter using Agrobacterium-mediated transformation. Overexpression of SaPIN2a in transgenic plants was demonstrated by northern blot and western blot analysis. SaPIN2a-overexpressing transgenic nightshade plants showed significantly lower height than wild-type plants. Transmission electron microscopy analysis showed that chloroplast-like organelles with thylakoids, which are not present in enucleate SEs of wild-type plants, were present in the enucleate SEs of SaPIN2a-overexpressing transgenic plants. This finding is discussed in terms of the possible role played by SaPIN2a in the regulation of proteolysis in SEs. PMID:17986772

Xie, Jun; Ouyang, Xue-Zhi; Xia, Kuai-Fei; Huang, Yu-Feng; Pan, Wen-Bi; Cai, Ying-Peng; Xu, Xinping; Li, Baojian; Xu, Zeng-Fu

2007-11-01

72

Cysteine peptidases and their inhibitors in breast and genital cancer.  

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Full Text Available Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.

Magdalena Milan

2010-11-01

73

Covalent binding of chloroacetamide herbicides to the active site cysteine of plant type III polyketide synthases.  

Science.gov (United States)

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1-2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides. PMID:14568070

Eckermann, Christian; Matthes, Bernd; Nimtz, Manfred; Reiser, Verena; Lederer, Barbara; Böger, Peter; Schröder, Joachim

2003-11-01

74

Fitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked.  

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Abstract Background The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs) expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores. Results In orde...

2004-01-01

75

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases.  

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Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied, mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulates in protein bodies and cell walls. This localisation in seeds is different from that previously described for cardoon flowers, suggesting a tissue-dependent targeting of the protein. It is known that procardosins are active and may have a role in proteolysis and processing of storage proteins. However, the presence of procardosin A in seeds could be related to the proposed role of the plant-specific insert in membrane lipid conversion during water uptake and solute leakage in actively growing tissues. This is in accordance with the recently proposed bifunctional role of aspartic proteinase precursor molecules that possess a membrane-destabilising domain in addition to a protease domain. Mature cardosin B, but not its mRNA, was detected in the first hours after seed imbibition and disappeared at the time of radicle emergence. This extracellular aspartic protease has already been implicated in cell wall loosening and remodelling, and its role in seed germination could be related to loosening tissue constraints for radicle protusion. The described pattern of cardosin A and B expression suggests a finely tuned developmental regulation and prompts an analysis of their possible roles in the physiology of postembryonic development. PMID:18767217

Pereira, Cláudia Sofia; da Costa, Diana Soares; Pereira, Susana; Nogueira, F de Moura; Albuquerque, P M; Teixeira, J; Faro, C; Pissarra, J

2008-01-01

76

Fitness benefits of trypsin proteinase inhibitor expression in Nicotiana attenuata are greater than their costs when plants are attacked.  

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Full Text Available Abstract Background The commonly invoked cost-benefit paradigm, central to most of functional biology, explains why one phenotype cannot be optimally fit in all environments; yet it is rarely tested. Trypsin proteinase inhibitors (TPIs expression in Nicotiana attenuata is known to decrease plant fitness when plants compete with unattacked conspecifics that do not produce TPIs and also to decrease the performance of attacking herbivores. Results In order to determine whether the putative benefits of TPI production outweigh its cost, we transformed N. attenuata to silence endogenous TPI production or restore it in a natural mutant that was unable to produce TPIs. We compared the lifetime seed production of N. attenuata genotypes of the same genetic background with low or no TPI to that of genotypes with high TPI levels on which M. sexta larvae were allowed to feed freely. Unattacked low TPI-producing genotypes produced more seed capsules than did plants with high TPI levels. Caterpillar attack reduced seed capsule production in all genotypes and reversed the pattern of seed capsule production among genotypes. M. sexta larvae attacking genotypes with high TPI activity consumed more TPI, less protein, and move later to the young leaves. Larval masses were negatively correlated (R2 = 0.56 with seed capsule production per plant. Conclusions Our results demonstrate that the fitness benefits of TPI production outweigh their costs in greenhouse conditions, when plants are attacked and that despite the ongoing evolutionary interactions between plant and herbivore, TPI-mediated decreases in M. sexta performance translates into a fitness benefit for the plant.

Baldwin Ian T

2004-08-01

77

Understanding and targeting a novel plant viral proteinase/substrate interaction. Final report, July 1, 1989--June 30, 1995  

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The past 3 years of funding have focused our efforts on trying to understand the molecular basis of a unique substrate interaction displayed by a viral proteinase. We have made good progress and during this funding period we have made four contributions to the scientific literature and have developed the application of the proteinase in the expression and purification of recombinant fusion proteins. A comprehensive review of virus-encoded proteinases, written during the funding period, emphazing the tremendous similarity of viral proteinases with their cellular counterparts and at the same time detail the unique characteristics which permit them to function in a cellular environment. The focus of the research effort was the tobacco etch virus (TEV) 27kDa NIa proteinase.

Dougherty, W.

1995-10-01

78

A trypsin-like proteinase in the midgut of Ectomyelois ceratoniae Zeller (Lepidoptera: Pyralidae): purification, characterization, and host plant inhibitors.  

Science.gov (United States)

A trypsin-like proteinase was purified and characterized in the midgut of Ectomyelois ceratoniae. A purification process that used Sepharyl G-100 and DEAE-cellulose fast flow chromatographies revealed a proteinase with specific activity of 66.7 ?mol/min/mg protein, recovery of 27.04 and purification fold of 23.35. Molecular weight of the purified protein was found to be 35.8 kDa. Optimal pH and temperature were obtained 9 and 20°C for the purified trypsin proteinase, respectively. The purified enzyme was significantly inhibited by PMSF, TLCK, and SBTI as specific inhibitors of trypsins in which TLCK showed the highest inhibitory effect. Trypsin proteinase inhibitors were extracted from four varieties of pomegranate including Brait, Torsh-Sabz, May-Khosh, and Shirin by ion exchange chromatography. It was found that fractions 17-20 of Brait; fractions 18 and 21-26 of Torsh-Sabz; fractions 1-7, 11-17, and 19-21 of May-Khosh and fraction 8 for Shirin showed presence of trypsin inhibitor in these host. Comparison of their inhibitory effects on the purified trypsin proteinase of E. ceratoniae demonstrated that fractions from May-khosh variety had the highest effect on the enzyme among other extracted fractions. Characterization of serine proteinases of insects mainly trypsins is one of the promising methods to decrease population and damages via extracting their inhibitors and providing resistant varieties. PMID:24338707

Ranjbar, Mina; Zibaee, Arash; Sendi, Jalal Jalali

2014-01-01

79

In-silico comparative study of inhibitory mechanism of Plant Serine Proteinase Inhibitors  

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The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use...

Siva Prasad, Chekkara Venkata Sathya; Gupta, Saurabh; Gaponenko, Alex; Dhar, Murli

2012-01-01

80

Tumor cell proteinase visualization and quantification using a fluorescent transition-state analog probe.  

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The fluorescent proteinase transition-state analog inhibitor, dansyl-L-argininal (DnsArgH), may be a selective probe of cysteine and serine-type proteinases in a fibrosarcoma tumor cell line (HSDM1C1). DnsArgH binds with high affinity to proteinases because of its transition-state analog properties, and on association it gives a dramatically increased fluorescent yield. The DnsArgH binding is inhibited by the serine proteinase inhibitor diisopropyl fluorophosphate and by the cysteine proteina...

Kozlowski, K. A.; Wezeman, F. H.; Schultz, R. M.

1984-01-01

 
 
 
 
81

The Internal Glycine-Rich Motif and Cysteine Suppress Several Effects of the HpaG(Xooc) Protein in Plants.  

Science.gov (United States)

ABSTRACT HpaG(Xooc), produced by Xanthomonas oryzae pv. oryzicola, is a member of harpin group of proteins that stimulate plant growth, hypersensitive cell death (HCD), and pathogen defense. The protein contains two copies of the glycine-rich motif (GRM), a characteristic of harpins, and a cysteine, which is absent in other harpins. Genetic modification generated the pro-tein mutants HpaG(Xooc)MG (MG) by deleting GRMs and HpaG(Xooc)C47T (C47T) by replacing cysteine with threonine. When applied to tobacco plants, C47T and MG were 1.2- and 1.7-fold stronger, respectively, than HpaG(Xooc) in inducing HCD, which occurred consistently with expression of the marker genes hin1 and hsr203. The proteins markedly alleviated infection of tobacco by Tobacco mosaic virus and Arabidopsis and tomato by Pseudomonas syringae. Treating tobacco plants with HpaG(Xooc), C47T, and MG decreased the viral infection by 58, 81, and 92%, respectively. In Arabidopsis and tomato plants treated with HpaG(Xooc), C47T, or MG, P. syringae multiplication was inhibited; bacterial population multiplied in 5 days in these plants were ca. 160-, 1,260-, or 15,860-fold smaller than that in control plants. So pathogen defense was induced in both plants. Defense-related genes Chia5, NPR1, and PR-1a were expressed consistently with resistance. In response to HpaG(Xooc), C47T, and MG, aerial parts and roots of tomato plants increased growth by 15 and 53%, 25 and 77%, and 46 and 106%, relative to controls. The expansin gene, EXP2, involved in the cell expansion and plant growth was expressed coordinately with plant growth promotion. These results suggest that the presence of GRM and cysteine in HpaG(Xooc) represses the effects of the protein in plants. PMID:18943492

Liu, Fengquan; Liu, Hongxia; Jia, Qin; Wu, Xiaojing; Guo, Xiaojing; Zhang, Shujian; Song, Feng; Dong, Hansong

2006-10-01

82

Plant serine proteinase inhibitors. Structure and biochemical applications on plasma kallikrein and related enzymes.  

Science.gov (United States)

The action of two Bowman-Birk and several plant Kunitz-type inhibitors were studied on trypsin, chymotrypsin, plasma kallikrein and factor XII. The primary structure of some of them was completely defined. The results showed that the Bowman-Birk type inhibitors, although potent inhibitors for trypsin (Ki in the range of 1-2 nM), are not able to inhibit plasma kallikrein. Factor XII (Ki = 1.4 microM) and chymotrypsin (Ki = 5.0 nM) are inhibited by Torresea cearensis trypsin inhibitor (TcTI) but not by Dioclea glabra trypsin inhibitor (DgTI). Both inhibitors reactive site regions are highly homologous, and the amino acid residues in P1 position are the same, Lys and His; major differences are in the charge of the C-terminal portion of the molecules. The studied Kunitz-type inhibitors were all able to inhibit plasma kallikrein (Ki between 4 and 80 nM), with the exception of Schizolobium parahyba chymotrypsin inhibitor (SpCI), that is specific for chymotrypsin. All Kunitz-type inhibitors inactivate chymotrypsin, but with a dissociation constant in the range of 0.1 to 0.6 microM. Factor XIIf is inhibited with Ki in the range of 0.1 microM. Bauhinia bauhinioides trypsin inhibitor (BbTI) did not promote factor XIIf inhibition. The Kunitz-type inhibitors are a highly homologous, sharing 60% identity in the N-terminal portion of the loop containing the reactive site, and 28.6% identity in the C-terminal portion of the same loop. PMID:8796268

Sampaio, C A; Oliva, M L; Sampaio, M U; Batista, I F; Bueno, N R; Tanaka, A S; Auerswald, E A; Fritz, H

1996-05-01

83

Plant defensins: Defense, development and application  

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Plant defensins are small, highly stable, cysteine-rich peptides that constitute a part of the innate immune system primarily directed against fungal pathogens. Biological activities reported for plant defensins include antifungal activity, antibacterial activity, proteinase inhibitory activity and insect amylase inhibitory activity. Plant defensins have been shown to inhibit infectious diseases of humans and to induce apoptosis in a human pathogen. Transgenic plants overexpressing defensins ...

Stotz, Henrik U.; Thomson, James G.; Wang, Yueju

2009-01-01

84

Comparison of two cysteine endopeptidases from latices of Morrenia brachystephana Griseb. and Morrenia odorata (Hook et Arn.) Lindley (Asclepiadaceae).  

Science.gov (United States)

The properties of morrenain b II, a proteinase isolated from the latex of Morrenia brachystephana, were compared with those of morrenain o II, a proteinase obtained from the latex of Morrenia odorata. Both peptidases were purified to homogeneity by acetone precipitation followed by cation exchange chromatography. The enzymes have pI values higher than 9.3 and similar molecular masses (close to 26 kDa) as determined by SDS-PAGE. They display maximum proteolytic activity within an alkaline pH range, and also exhibit esterolytic activity. The N-terminal sequences of morrenain o II and morrenain b II show a high degree of homology between each other and to other cysteine plant proteinases. PMID:11517946

Cavalli, S V; Cortadi, A; Arribére, M C; Conforti, P; Caffini, N O; Priolo, N

2001-05-01

85

Solution Structure of the Squash Aspartic Acid Proteinase Inhibitor (SQAPI) and Mutational Analysis of Pepsin Inhibition  

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The squash aspartic acid proteinase inhibitor (SQAPI), a proteinaceous proteinase inhibitor from squash, is an effective inhibitor of a range of aspartic proteinases. Proteinaceous aspartic proteinase inhibitors are rare in nature. The only other example in plants probably evolved from a precursor serine proteinase inhibitor. Earlier work based on sequence homology modeling suggested SQAPI evolved from an ancestral cystatin. In this work, we determined the solution structure of SQAPI using NM...

2010-01-01

86

PAPAIN, A PLANT ENZYME OF BIOLOGICAL IMPORTANCE: A REVIEW  

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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, indust...

Ezekiel Amri; Florence Mamboya

2012-01-01

87

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases  

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Summary. Following on from previous work, the temporal and spatial accumulation of the aspartic proteinases (EC 3.4.23) cardosin A and cardosin B during postembryonic seed development of cardoon (Cynara cardunculus) was studied. mRNA and protein analyses of both cardosins suggested that the proteins accumulate during seed maturation, and that cardosin A is later synthesised de novo at the time of radicle emergence. Immunocytochemistry revealed that the precursor form of cardosin A accumulate...

Pereira, Cla?udia Sofia; Costa, Diana Soares Da; Pereira, Susana; Nogueira, F. Moura; Albuquerque, P. M.; Teixeira, J.; Faro, C.; Pissarra, J.

2008-01-01

88

Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.  

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AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacill...

Abrahamson, M.; Wikstro?m, M.; Potempa, J.; Renvert, S.; Hall, A.

1997-01-01

89

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. Results One of the infestation specific Cuscuta genes encodes a cysteine protease. The protein and its intrinsic inhibitory peptide were heterologously expressed, purified and biochemically characterized. The haustoria specific enzyme was named cuscutain in accordance with similar proteins from other plants, e.g. papaya. The role of cuscutain and its inhibitor during the host parasite interaction was studied by external application of an inhibitor suspension, which induced a significant reduction of successful infection events. Conclusions The study provides new information about molecular events during the parasitic plant - host interaction. Inhibition of cuscutain cysteine proteinase could provide means for antagonizing parasitic plants.

Fuchsbauer Hans-Lothar

2010-10-01

90

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa).  

Science.gov (United States)

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 ?M CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules. PMID:24426085

Popovic, Milica; Andjelkovic, Uros; Grozdanovic, Milica; Aleksic, Ivana; Gavrovic-Jankulovic, Marija

2013-03-01

91

The squash aspartic proteinase inhibitor SQAPI is widely present in the cucurbitales, comprises a small multigene family, and is a member of the phytocystatin family.  

Science.gov (United States)

The squash (Cucurbita maxima) phloem exudate-expressed aspartic proteinase inhibitor (SQAPI) is a novel aspartic acid proteinase inhibitor, constituting a fifth family of aspartic proteinase inhibitors. However, a comparison of the SQAPI sequence to the phytocystatin (a cysteine proteinase inhibitor) family sequences showed approximately 30% identity. Modeling SQAPI onto the structure of oryzacystatin gave an excellent fit; regions identified as proteinase binding loops in cystatin coincided with regions of SQAPI identified as hypervariable, and tryptophan fluorescence changes were also consistent with a cystatin structure. We show that SQAPI exists as a small gene family. Characterization of mRNA and clone walking of genomic DNA (gDNA) produced 10 different but highly homologous SQAPI genes from Cucurbita maxima and the small family size was confirmed by Southern blotting, where evidence for at least five loci was obtained. Using primers designed from squash sequences, PCR of gDNA showed the presence of SQAPI genes in other members of the Cucurbitaceae and in representative members of Coriariaceae, Corynocarpaceae, and Begoniaceae. Thus, at least four of seven families of the order Cucurbitales possess member species with SQAPI genes, covering approximately 99% of the species in this order. A phylogenetic analysis of these Cucurbitales SQAPI genes indicated not only that SQAPI was present in the Cucurbitales ancestor but also that gene duplication has occurred during evolution of the order. Phytocystatins are widespread throughout the plant kingdom, suggesting that SQAPI has evolved recently from a phytocystatin ancestor. This appears to be the first instance of a cystatin being recruited as a proteinase inhibitor of another proteinase family. PMID:17103059

Christeller, John T; Farley, Peter C; Marshall, Richelle K; Anandan, Ananda; Wright, Michele M; Newcomb, Richard D; Laing, William A

2006-12-01

92

Involvement of papain and legumain proteinase in the senescence process of Medicago truncatula nodules.  

Science.gov (United States)

The symbiotic interaction between legumes and Rhizobiaceae leads to the formation of new root organs called nodules. Within the nodule, Rhizobiaceae differentiate into nitrogen-fixing bacteroids. However, this symbiotic interaction is time-limited as a result of the initiation of a senescence process, leading to a complete degradation of bacteroids and host plant cells. The increase in proteolytic activity is one of the key features of this process. In this study, we analysed the involvement of two different classes of cysteine proteinases, MtCP6 and MtVPE, in the senescence process of Medicago truncatula nodules. Spatiotemporal expression of MtCP6 and MtVPE was investigated using promoter- ?-glucuronidase fusions. Corresponding gene inductions were observed during both developmental and stress-induced nodule senescence. Both MtCP6 and MtVPE proteolytic activities were increased during stress-induced senescence. Down-regulation of both proteinases mediated by RNAi in the senescence zone delayed nodule senescence and increased nitrogen fixation, while their early expression promoted nodule senescence. Using green fluorescent protein fusions, in vivo confocal imaging showed that both proteinases accumulated in the vacuole of uninfected cells or the symbiosomes of infected cells. These data enlighten the crucial role of MtCP6 and MtVPE in the onset of nodule senescence. PMID:24527680

Pierre, Olivier; Hopkins, Julie; Combier, Maud; Baldacci, Fabien; Engler, Gilbert; Brouquisse, Renaud; Hérouart, Didier; Boncompagni, Eric

2014-05-01

93

Differential subcellular targeting of recombinant human ??-proteinase inhibitor influences yield, biological activity and in planta stability of the protein in transgenic tomato plants.  

Science.gov (United States)

The response of protein accumulation site on yield, biological activity and in planta stability of therapeutic recombinant human proteinase inhibitor (??-PI) was analyzed via targeting to different subcellular locations, like endoplasmic reticulum (ER), apoplast, vacuole and cytosol in leaves of transgenic tomato plants. In situ localization of the recombinant ??-PI protein in transgenic plant cells was monitored by immunohistochemical staining. Maximum accumulation of recombinant ??-PI in T? and T? transgenic tomato plants was achieved from 1.5 to 3.2% of total soluble protein (TSP) by retention in ER lumen, followed by vacuole and apoplast, whereas cytosolic targeting resulted into degradation of the protein. The plant-derived recombinant ??-PI showed biological activity for elastase inhibition, as monitored by residual porcine pancreatic elastase (PPE) activity assay and band-shift assay. Recombinant ??-PI was purified from transgenic tomato plants with high yield, homogeneity and biological activity. Purified protein appeared as a single band of ?48-50 kDa on SDS-PAGE with pI value ranging between 5.1 and 5.3. Results of mass spectrometry and optical spectroscopy of purified recombinant ??-PI revealed the structural integrity of the recombinant protein comparable to native serum ??-PI. Enzymatic deglycosylation and lectin-binding assays with the purified recombinant ??-PI showed compartment-specific N-glycosylation of the protein targeted to ER, apoplast and vacuole. Conformational studies based on urea-induced denaturation and circular dichroism (CD) spectroscopy revealed relatively lower stability of the recombinant ??-PI protein, compared to its serum counterpart. Pharmacokinetic evaluation of plant derived recombinant and human plasma-purified ??-PI in rat, by intravenous route, revealed significantly faster plasma clearance and lower area under curve (AUC) of recombinant protein. Our data suggested significance of protein sorting sequences and feasibility to use transgenic plants for the production of stable, glycosylated and biologically active recombinant ??-PI for further therapeutic applications. PMID:23017899

Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Jain, G K; Amla, D V

2012-11-01

94

Characterization of the gene encoding an intracellular proteinase inhibitor of Bacillus subtilis and its role in regulation of the major intracellular proteinase.  

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The gene (ipi) for an intracellular proteinase inhibitor (BsuPI) from Bacillus subtilis was cloned and found to encode a polypeptide consisting of 119 amino acids with no cysteine residues. The deduced amino acid sequence contained the N-terminal amino acid sequence of the inhibitor, which was chemically determined previously, and showed no significant homology to any other proteinase inhibitors. Analysis of the transcription initiation site and mRNA showed that the ipi gene formed an operon ...

1993-01-01

95

Inibidores de proteases de hospedeiros nativos e exóticos e sua ação em intestinos de lagartas de Thyrinteina leucoceraea / Proteinase inhibitors of novel and native host plants and their action in midgut of Thyrinteina leucoceraea caterpillars  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: Portuguese Abstract in portuguese Os insetos podem causar perdas consideráveis aos seus hospedeiros, entretanto alguns deles habitam em plantas sem causar-lhes danos. Por exemplo, Thyrinteina leucoceraea, herbívoro da entomofauna brasileira, pode ser encontrado na goiabeira, hospedeiro nativo da família Myrtaceae, sem que cause dano [...] s severos a essa planta. Os Eucalyptus ssp., entretanto, são hospedeiros exóticos (também da família Myrtaceae) no Brasil, vindos da Austrália, os quais sofrem ataques das lagartas de T. leucoceraea, que se tornaram pragas severas dessas plantas. Sabe-se que as plantas podem se defender contra o ataque de herbívoros e que um dos seus mecanismos de defesa pode ser a produção de inibidores de proteases, que possuem a capacidade de diminuir o desenvolvimento dos insetos e podem levá-los à morte. Baseado no desempenho da lagarta de T. leucoceraea nesses dois hospedeiros e na possibilidade de defesa da planta, o objetivo deste trabalho foi verificar a produção de inibidores de proteases por plantas de eucalipto e de goiaba quando atacadas por essas lagartas, bem como observar a resposta bioquímica no intestino das lagartas a esses inibidores. Notou-se que as plantas de eucalipto produzem mais inibidores de proteases que as goiabeiras. O bom desenvolvimento de T. leucoceraea em plantas de eucalipto, apesar da alta concentração de inibidores de proteases, pode ser devido ao aumento da atividade enzimática nos intestinos das lagartas quando alimentadas com essa planta. Os dados evidenciaram que T. leucoceraea desenvolveu uma adaptação aos inibidores de proteases produzidos pelo eucalipto, por meio do aumento das atividades de serino-proteases e cisteíno-proteases. Abstract in english Insects may cause considerable losses to plants, but some insects inhabit plants without causing any damages. For example, Thyrinteina leucoceraea, found in the guava plants, and a native Myrtaceae family host, does not cause any serious damage. However, Eucalyptus ssp., novel hosts (also Myrtaceae) [...] in Brazil and introduced from Australia, suffer attacks by T. leucoceraea, which became a severe pest of this plant. Plants can defend themselves against herbivores using proteinase inhibitors which reduce insect development and lead them to death. Thus, based on studies on the development of T. leucoceraea caterpillars on these two hosts and plant defense, this work aimed to verify the production of proteinase inhibitors by guava and eucalyptus plants upon T. leucoceraea attack, and to observe the biochemical response of the midgut of the caterpillars to these inhibitors. Eucalyptus plants produced more proteinase inhibitors than guava plants. The good development of T. leucoceraea in eucalyptus plants despite the high concentration of proteinase inhibitors may be due to an increase of enzyme activity in the caterpillars' midgut. Our data suggest that T. leucoceraea developed an adaptation to the proteinase inhhibitor produced by eucalyptus plants, by increasing serine-proteinase and cys-proteinase activities.

Jeanne Scardini, Marinho; Maria Goreti Almeida, Oliveira; Raul Narciso Carvalho, Guedes; Angelo, Pallini; Claudinei Lima, Oliveira.

96

Plasma proteinase inhibitors.  

Science.gov (United States)

Plasma proteinase inhibitors account for about 10% of the total protein in plasma. They provide one mechanism for the control of proteinase activity, thus regulating many important biological reactions such as blood coagulation. Most plasma inhibitors are specific for one or a few related proteinases and control a particular biological event or pathway. Two other inhibitors, the alpha 2-macroglobulin (alpha 2M) and the alpha 1-proteinase (alpha 1PI = alpha 1-antitrypsin) have a broader specificity. The role of alpha 1PI, although theoretically able to inhibit a large number of enzymes, is the inhibition of leukocytic elastase. This function is particularly important in the lung where elastase may be released from neutrophils particularly in smokers. alpha 2-Macroglobulin reacts with a large number of very different proteinases by a mechanism quite different from those of the other inhibitors. The physiological role of this inhibitor is not clearly understood although it may act as a "back-up" inhibitor when levels of other inhibitors are low or if no specific inhibitor is available. PMID:6208604

Bodmer, J L; Schnebli, H P

1984-10-01

97

Antibody in sera of patients infected with Trichomonas vaginalis is to trichomonad proteinases.  

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BACKGROUND--A recent report demonstrated the immunogenic character of the cysteine proteinases of Trichomonas vaginalis. It was of interest, therefore, to examine for the presence of serum anti-proteinase antibody among patients with trichomoniasis. METHODS--An immunoprecipitation assay was used involving protein A-bearing Staphylococcus aureus first coated with the IgG fraction of goat anti-human Ig and then mixed with individual sera of patients to bind human antibody. These antibody-coated...

Alderete, J. F.; Newton, E.; Dennis, C.; Neale, K. A.

1991-01-01

98

Digestive duet: Midgut digestive proteinases of Manduca sexta ingesting Nicotiana attenuata with manipulated trypsin proteinase inhibitor expression  

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The defensive effect of endogenous trypsin proteinase inhibitors (NaTPIs) on the herbivore Manduca sexta was demonstrated by genetically altering NaTPI production in M. sexta's host plant, Nicotiana attenuata. To understand how this defense works, we studied the effects of NaTPI on M. sexta gut proteinase activity levels in different larval instars of caterpillars feeding freely on untransformed and transformed plants. Methodology/ Principal Findings Second and third instars larvae that fed o...

2008-01-01

99

Cationic inhibitors of serine proteinases from buckwheat seeds: study of their interaction with exogenous proteinases.  

Science.gov (United States)

The inhibition of exogenous serine proteinases of different origin by cationic protease inhibitors BWI-1c, -2c, -3c, and -4c from buckwheat (Fagopyrum esculentum Moench) seeds has been studied. High efficiency of the inhibitors in binding bovine trypsin and chymotrypsin as well as their broad antiprotease effect, including inhibition of proteinases secreted by fungi and bacteria, has been demonstrated. According to the data obtained, it is proposed that cationic inhibitors from buckwheat seeds may participate in the defense of plants against fungal and bacterial infection. PMID:15170382

Tsybina, T A; Dunaevsky, Y E; Popykina, N A; Larionova, N I; Belozersky, M A

2004-04-01

100

The expression pattern of a rice proteinase inhibitor gene OsPI8-1 implies its role in plant development.  

Science.gov (United States)

A rice proteinase inhibitor (PI) gene OsPI8-1 was identified. Belonging to the potato inhibitor I family, this gene contains a 201bp coding region with no introns and encodes a deduced protein of 66 amino acids which holds a PI domain. There are two uniform gene copies, OsPI8-1a and OsPI8-1b, with direct-repeat arrangement and an interval span of 13 kb on rice chromosome 8, corresponding to the site of BAC clone P0528B09 (Accession No. AP004703). Reverse transcription polymerase chain reaction (RT-PCR) assays showed that both OsPI8-1a and OsPI8-1b can be expressed in wild-type 'Zhonghua No.11'. To investigate the physiological functions of OsPI8-1 in plant development, we analyzed the expression patterns of the reporter gene beta-glucuronidase (GUS) driven by OsPI8-1 promoter at different developmental stages and tissues. It was demonstrated that no GUS signals were detected in the roots. Despite that very high GUS expression was examined in the shoot apical meristem, no detectable GUS activity in the developmental domains of leaf primordium was observed. OsPI8-1 promoter showed an obvious wound-induced response in mature leaves. Little GUS activity was detected in young nodes and internodes at the seedling stage, but active GUS expression was observed near the nodes on mature culms. In the developing stage of the anther, GUS signal was specifically located in the middle layer and the endothecium between the epidermis and tapetum. In the germinating seed, GUS expression was gradually accumulated in the side of scutellar epithelium close to the embryo. These tissue-specific accumulations suggested that OsPI8-1 has multiple endogenous roles on developmental regulation. In this report, the inhibitor function of OsPI8-1 to proteolytic enzymes and the potential influence of their poise on plant development (such as seed germination, tapetum degeneration, programmed cell death, etc.) were discussed. PMID:18022281

Wang, Jiang; Shi, Zhen-Ying; Wan, Xin-Shan; Shen, Ge-Zhi; Zhang, Jing-Liu

2008-09-29

 
 
 
 
101

Cysteine and cysteine-related signaling pathways in Arabidopsis thaliana.  

Science.gov (United States)

Cysteine occupies a central position in plant metabolism because it is a reduced sulfur donor molecule involved in the synthesis of essential biomolecules and defense compounds. Moreover, cysteine per se and its derivative molecules play roles in the redox signaling of processes occurring in various cellular compartments. Cysteine is synthesized during the sulfate assimilation pathway via the incorporation of sulfide to O-acetylserine, catalyzed by O-acetylserine(thiol)lyase (OASTL). Plant cells contain OASTLs in the mitochondria, chloroplasts, and cytosol, resulting in a complex array of isoforms and subcellular cysteine pools. In recent years, significant progress has been made in Arabidopsis, in determining the specific roles of the OASTLs and the metabolites produced by them. Thus, the discovery of novel enzymatic activities of the less-abundant, like DES1 with L-cysteine desulfhydrase activity and SCS with S-sulfocysteine synthase activity, has provided new perspectives on their roles, besides their metabolic functions. Thereby, the research has been demonstrated that cytosolic sulfide and chloroplastic S-sulfocysteine act as signaling molecules regulating autophagy and protecting the photosystems, respectively. In the cytosol, cysteine plays an essential role in plant immunity; in the mitochondria, this molecule plays a central role in the detoxification of cyanide, which is essential for root hair development and plant responses to pathogens. PMID:24285094

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

2014-02-01

102

Co-factor activated recombinant adenovirus proteinases  

Energy Technology Data Exchange (ETDEWEB)

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.

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

1996-08-06

103

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)

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.

Al-Sayed Al-Tanboly

2003-01-01

104

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

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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...

2008-01-01

105

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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.

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

106

Midgut proteinases of Sitotroga cerealella (Oliver) (Lepidoptera:Gelechiidae): Characterization and relationship to resistance in cereals  

International Nuclear Information System (INIS)

Midgut proteinases are vital to the insects which digest ingested food in the midgut. Insect midgut proteinases, therefore, have been considered as possible targets for the control of insect pests. Proteinaceous proteinase inhibitors are very attractive for their potential use in developing insect resistant plant varieties via genetic engineering. Sitotroga cerealella is one of the major storage pests of cereals, and no antibiotic resistance in wheat against this insect has been identified to date. A series of diagnostic inhibitors, thiol-reducing agents and a metal-ion chelator were used in the identification of proteinases in crude extracts from S. cerealella larval midguts with both protein and ester substrates. The partial inhibition of proteolytic activity in crude midgut extract toward [3H]-methemoglobin by pepstatin A suggested the presence of another proteinase which was sensitive to pepstatin A. The optimum pH range for the proteolytic activity, however, indicated that the major midgut proteinases were not carboxyl proteinases. Two proteinases were successfully purified by a combination of fractionation with ammonium sulfate, gel permeation and anion exchange chromatography. Characterization of the enzymes with the purified enzyme preparations confirmed that the two major proteinases were serine endoproteinases with trypsin-like and chymotrypsin-like specificities respectively. Bioassays were conducted using the artificial seeds to test naturally occurring proteinaceous proteinase inhibitors of potential value. Soybean trypsin inhibitor and the Bowman-Birk proteinase inhibitor had adverse effects on the development of the insect. A predictive model was constructed to evaluate effects of seed resistance in conjunction with other control methods on S. cerealella population dynamics

1989-01-01

107

Podborer (Helicoverpa armigera Hübn.) does not show specific adaptations in gut proteinases to dietary Cicer arietinum Kunitz proteinase inhibitor.  

Science.gov (United States)

We investigated the response of Helicoverpa armigera larvae towards ingestion of Cicer arietinum Kunitz proteinase inhibitor (CaKPI), which caused antagonistic effects on developing H. armigera larvae. CaKPI-degrading proteinases were not detectable in either control or sensitized larvae. There were negligible increases in total proteinase activity, as well as in trypsin-like and chymotrypsin-like activities of H. armigera gut proteinases (HGPs). Decrease in sensitivity of HGPs to inhibition by CaKPI was not observed when the inhibitor was fed suggesting that the insect had not shown a specific adaptive response to dietary CaKPI. Semi-quantitative reverse transcriptase polymerase chain reaction (Q RT-PCR) analysis showed a general up-regulation of proteases in larvae that ingested CaKPI and a specific regulation of individual transcripts was not observed. CaKPI had maximum inhibitory activity against HGP derived from fourth instar larvae. CaKPI was equally potent in inhibition of HGPs derived from larvae fed on different host plants, as well as various proteinase inhibitors (PIs) to which larval adaptation was previously reported. The lack of larval response to CaKPI was attributable to the atypical active site sequence and inhibitory activity of CaKPI and/or to the pre-adaptation of H. armigera larvae due to the constant exposure to basal levels of CaKPI in chickpea seeds or a chickpea seed-based diet. PMID:16140320

Srinivasan, Ajay; Chougule, Nanasaheb P; Giri, Ashok P; Gatehouse, John A; Gupta, Vidya S

2005-11-01

108

Processing of the lactococcal extracellular serine proteinase.  

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Activity of the lactococcal cell envelope-located serine proteinase depends on the presence of membrane-associated lipoprotein PrtM. To differentiate between the action of the proteinase and the action of PrtM in the process of proteinase maturation, an inactive form of the lactococcal proteinase was constructed. This was done by mutating one of the three amino acids thought to constitute the active site of the enzyme. The secreted form of this inactivated proteinase was the same size as the ...

Haandrikman, A. J.; Meesters, R.; Laan, H.; Konings, W. N.; Kok, J.; Venema, G.

1991-01-01

109

S-nitrosoglutathione reductases are low-copy number, cysteine-rich proteins in plants that control multiple developmental and defense responses in Arabidopsis  

Science.gov (United States)

S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO). We combined bioinformatics of plant GSNOR genes, localization of GSNOR in Arabidopsis thaliana, and microarray analysis of a GSNOR null mutant to gain insights into the function and regulation of this critical enzyme in nitric oxide (NO) homeostasis. GSNOR-encoding genes are known to have high homology across diverse eukaryotic taxa, but contributions of specific conserved residues have not been assessed. With bioinformatics and structural modeling, we show that plant GSNORs likely localize to the cytosol, contain conserved, solvent-accessible cysteines, and tend to be encoded by a single gene. Arabidopsis thaliana homozygous for GSNOR loss-of-function alleles exhibited defects in stem and trichome branching, and complementation with Green fluorescent protein (GFP) -tagged GSNOR under control of the native promoter quantitatively rescued these phenotypes. GSNOR-GFP showed fluorescence throughout Arabidopsis seedlings, consistent with ubiquitous expression of the protein, but with especially high fluorescence in the root tip, apical meristem, and flowers. At the cellular level we observed cytosolic and nuclear fluorescence, with exclusion from the nucleolus. Microarray analysis identified 99 up- and 170 down-regulated genes (?2-fold; p ? 0.01) in a GSNOR null mutant compared to wild type. Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism. One third of downregulated genes are linked to pathogen resistance, providing further basis for the reported pathogen sensitivity of GSNOR null mutants. Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

Xu, Shengbao; Guerra, Damian; Lee, Ung; Vierling, Elizabeth

2013-01-01

110

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

Directory of Open Access Journals (Sweden)

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.

Marian Dorcas Quain

2013-08-01

111

Endogenous inhibitors of lysosomal proteinases.  

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Specific inhibitors of three lysosomal proteinases are present in the cytosolic and lysosomal compartments of rabbit liver. The cytosolic inhibitors, purified by chromatography on DEAE-Trisacryl and Sephadex G-75, show specificities toward cathepsin M, cathepsins B and L, and fructose 1,6-bisphosphatase converting enzyme (CE), respectively, and are designated IM, IB/L, and ICE. Inhibitors with similar specificities have been isolated from the intralysosomal compartment. Two of these inhibitor...

Pontremoli, S.; Melloni, E.; Salamino, F.; Sparatore, B.; Michetti, M.; Horecker, B. L.

1983-01-01

112

Proteinases in Naegleria Fowleri (strain NF3), a pathogenic amoeba: a preliminary study.  

Science.gov (United States)

Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group. PMID:16493399

Mat Amin, Nakisah

2004-12-01

113

Proteinases secreted by Fasciola hepatica degrade extracellular matrix and basement membrane components.  

Science.gov (United States)

The invasive stages of the parasitic trematode Fasciola hepatica release proteinases into the medium in which they are maintained. In this study, we investigated the interaction of F. hepatica excretory/secretory (E/S) products and 2 cysteine proteinases (CL1 and CL2) purified from these products with extracellular matrix and basement membrane macromolecules. Fasciola hepatica E/S products contained collagenolytic activity on fibrillar types I and III collagen as well as basement membrane type IV collagen. CL1 and CL2 were capable of degrading acid-soluble type III and type IV collagen but not insoluble type I collagen. In contrast, neither the E/S products nor the purified CL1 and CL2 showed elastinolytic activity. Fibronectin and laminin were degraded by E/S products and by CL1 and CL2. Sequence analysis of fibronectin degradation products showed that the fragments obtained corresponded to complete biologically active domains. These results indicate that the cysteine proteinases secreted by F. hepatica may be involved in the process of tissue invasion by the parasite. PMID:9057688

Berasaín, P; Goñi, F; McGonigle, S; Dowd, A; Dalton, J P; Frangione, B; Carmona, C

1997-02-01

114

Cysteine Variants of Erythropoietin.  

Science.gov (United States)

The growth hormone supergene family comprises greater than 20 structurally related cytokines ad growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine ...

G. N. Cox

2004-01-01

115

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

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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 F...

Timotijevi? Gordana S.; Milisavljevi? Mira ?.; Radovi? Svetlana R.; Konstantinovi? M.M.; Maksimovi? Vesna R.

2010-01-01

116

Pest Protection Conferred by a Beta vulgaris Serine Proteinase Inhibitor Gene  

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Proteinase inhibitors provide a means of engineering plant resistance to insect pests. A Beta vulgaris serine proteinase inhibitor gene (BvSTI) was fused to the constitutive CaMV35S promoter for over-expression in Nicotiana benthamiana plants to study its effect on lepidopteran insect pests. Independently derived BvSTI transgenic tobacco T2 homozygous progeny were shown to have relatively high BvSTI gene transcript levels. BvSTI-specific polyclonal antibodies cross-reacted with the expected 3...

Smigocki, Ann C.; Ivic-haymes, Snezana; Li, Haiyan; Savic?, Jelena

2013-01-01

117

Inhibition of horse leucocyte proteinases by eglin, a proteinase inhibitor from leeches.  

Science.gov (United States)

Interaction of eglin c with three neutral proteinases (1, 2A and 2B) from horse leucocytes was investigated using synthetic and protein substrates. With N-tert-butyloxycarbonyl-L-alanine-p-nitrophenyl ester as substrate inhibition of proteinase 1 and 2A was practically complete at equimolar inhibitor concentrations (Ki below 1 nMol/l). The complex with proteinase 2B showed a dissociation constant of approximately 25 nMol/l. The latter proteinase was only partly inhibited also in the presence of azocasein, whereas almost linear inhibition was observed for all 3 proteinases with fibrinogen as substrate. The inhibition rate constants (kon) for horse leucocyte proteinases with eglin were in the range of 8 to 13 X 10(5) M-1 S-1. PMID:4004837

Potempa, J; Dubin, A; Seemüller, U; Schnebli, H P; Koj, A

1985-01-01

118

Proteinase inhibitors in Brazilian leguminosae  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Serine proteinase inhitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, a digestive enzyme, Factor XIIa and human plasma Kallikrein, two blood clotting factors. The inhibitors were purified from Enterolobium contortisiliquum (Mr=23,0 [...] 00), Torresea cearensis (Mr = 13,000), Bauhinia pentandra (Mr = 20,000) and Bauhinia bauhinioides (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XSSa, but does nor affect plasma kallikrein; both Bauhinia inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the Bpentandra inhibitor affects Factor XIIa. Ki values were calculated between 10 [raised to the power of] -7 and 10 [raised to the power of] -8 M.

C. A. M., Sampaio; M. L. V., Oliva; A. S., Tanaka; M. U., Sampaio.

119

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

Science.gov (United States)

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

Lomate, Purushottam R; Hivrale, Vandana K

2012-08-01

120

Differential antibiosis against Helicoverpa armigera exerted by distinct inhibitory repeat domains of Capsicum annuum proteinase inhibitors.  

Science.gov (United States)

Plant defensive serine proteinase inhibitors (PIs) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among PIs reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s). PMID:24559910

Joshi, Rakesh S; Gupta, Vidya S; Giri, Ashok P

2014-05-01

 
 
 
 
121

Isolation of a cDNA clone for the human lysosomal proteinase cathepsin B.  

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The cysteine proteinase cathepsin B is one member of the lysosomal acid hydrolases. Based on the peptide sequence of rat liver cathepsin B, an oligonucleotide mixture containing 128 different 17-mers was synthesized and used as a probe to screen adult and fetal human liver cDNA libraries. A recombinant clone with a 1540-nucleotide insert was identified from the fetal library, and DNA sequence analysis confirmed that this clone encodes human cathepsin B. The clone, designated pCB-1, has sequen...

Fong, D.; Calhoun, D. H.; Hsieh, W. T.; Lee, B.; Wells, R. D.

1986-01-01

122

Activities of the Porphyromonas gingivalis PrtP Proteinase Determined by Construction of prtP-Deficient Mutants and Expression of the Gene in Bacteroides Species  

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PrtP is a major cysteine proteinase of Porphyromonas gingivalis. The gene encoding this proteinase, prtP, was cloned into the Escherichia coli-Bacteroides shuttle vectors pFD288 and pFD340 and was expressed in Bacteroides cells, apparently under the control of its own promoter, when in pFD288, or a Bacteroides promoter present on pFD340. Proteolytically active PrtP was detected by fibrinogen zymography in cells or spent growth medium of several Bacteroides species harboring the recombinant pl...

Barkocy-gallagher, Genevieve A.; Foley, Joseph W.; Lantz, Marilyn S.

1999-01-01

123

Potato leafroll virus protein P1 contains a serine proteinase domain.  

Science.gov (United States)

The multi-domain potato leafroll virus replicase protein P1 was expressed in insect cells from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus. Using antisera raised against P1, it was shown that P1 was cleaved near the VPg in insect cells in a manner similar to that in plant cells, to produce a approximately 27 kDa C-terminal fragment. Furthermore, it was shown that the proposed serine proteinase-like domain within P1 is responsible for this processing and that this can occur in a trans (intermolecular) reaction. Four conserved residues within the serine proteinase domain that are essential for catalysis have been identified, consistent with the proposal that this domain comprises a serine proteinase. PMID:10859393

Li, X; Ryan, M D; Lamb, J W

2000-07-01

124

Allergic reactivity and IgG subclasses to a proteinase fraction of Setaria digitata in filariasis.  

Science.gov (United States)

A low molecular weight fraction (30 KDa) of the cattle filarial parasite Setaria digitata that was earlier demonstrated to have allergenic activity was characterized to be a zinc-dependent cysteine proteinase. Immediate type hypersensitivity (ITH) reaction to the proteinase was evaluated in lymphatic filariasis patients and in endemic controls from Orissa, India. The extent of ITH positivity to the proteinase in infected individuals ranged from 20% in chronic filariasis (CP) patients group to 56% in asymptomatic microfilaraemic carriers (AS). About 62% of endemic normals (EN) were also ITH positive. The serum levels of IgG subclasses were compared in ITH positive and ITH negative filarial patients (AS and CP) as well as in endemic normals (EN) respectively. IgG4 levels were found to be inversely dependent on ITH reaction only in AS groups. Asymptomatic patients (AS) with positive ITH reactivity had lower IgG4 than ITH negative individuals from the same group. The serum levels of other IgG subclasses except IgG2, did not correlate with ITH reactivity. IgG2 levels were higher in ITH negative EN and CP patients but not in the AS group. PMID:8522762

Beuria, M K; Bal, M; Das, M K

1995-09-01

125

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI and determination of hydrolysis sites of spectrin by Pf37 proteinase  

Directory of Open Access Journals (Sweden)

Full Text Available Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI, involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

I. Florent

1994-01-01

126

Plasmodium falciparum proteinases: cloning of the putative gene coding for the merozoite proteinase for erythrocyte invasion (MPEI) and determination of hydrolysis sites of spectrin by Pf37 proteinase  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Su [...] ch a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.

I., Florent; S., Le Bonniec; B., Carcy; P., Grellier; O., Mercereau-Puijalon; S., Bonnefoy; D., Dhermy; M., Monsigny; R., Mayer; J., Schrével.

127

Stability of proteinase from Carica papaya latex in dense gases  

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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 su...

Habulin, Maja; Primoz?ic?, Mateja; Knez, Z?eljko

2012-01-01

128

The glycosylation of the aspartic proteinases from barley (Hordeum vulgare L.) and cardoon (Cynara cardunculus L.).  

Science.gov (United States)

Plant aspartic proteinases characterised at the molecular level contain one or more consensus N-glycosylation sites [Runeberg-Roos, P., T?rmäkangas, K. & Ostman, A. (1991) Eur. J. Biochem. 202, 1021-1027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem, 232, 77-83; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 762-768]. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) aspartic proteinase (Asn333) and the cardoon (Cynara cardunculus L.) aspartic proteinase, cardosin A (Asn70 and Asn363). The oligosaccharides from each site were released from peptide pools by enzymatic hydrolysis with peptide-N-glycanase A or by hydrazinolysis and their structures were determined by exoglycosidase sequencing combined with matrix-assisted laser desorption/ionization time of flight mass spectrometry. It was observed that 6% of the oligosaccharides from the first glycosylation site of cardosin A are of the oligomannose type. Modified type glycans with proximal Fuc and without Xyl account for about 82%, 14% and 3% of the total oligosaccharides from the first and the second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. Oligosaccharides with Xyl but without proximal Fuc were only detected in the latter proteinase (4%). Glycans with proximal Fuc and Xyl account for 6%, 86% and 92% of total oligosaccharides from the first and second glycosylation sites of cardosin A and from H. vulgare aspartic proteinase, respectively. PMID:9057834

Costa, J; Ashford, D A; Nimtz, M; Bento, I; Frazäo, C; Esteves, C L; Faro, C J; Kervinen, J; Pires, E; Veríssimo, P; Wlodawer, A; Carrondo, M A

1997-02-01

129

Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms.  

Science.gov (United States)

Seeds of 32 species selected from two of the four major groups of gymnosperms, the ancient Cycadales and the economically important Coniferales, were analysed for inhibitors (I) of the serine proteinases trypsin (T), chymotrypsin (C), subtilisin (S) and elastase (E) using isoelectric focusing (IEF) combined with gelatin replicas. Subtilisin inhibitors were detected in 17 species, being particularly active in the Cycadales. Several species of the genera Cephalotaxus, Pseudotsuga and Cycas contained inhibitors active against elastase while strong CSTIs and CSIs were also present in Cycas pectinata and C. siamensis. No inhibitors were detected in seeds of Chamaecyparis, Thuja, Abies, Larix, Picea and Pinus spp. Serine proteinase inhibitors were purified from seeds of C. siamensis by affinity chromatography using trypsin and chymotrypsin, IEF and SDS-PAGE. Several CSTI components with M(r) ranging from 4000 to 18,000 were partially sequenced using Edman degradation and mass spectrometry. Most of the sequences were similar to a hypothetical protein encoded by an mRNA from sporophylls of C. rumphii which in turn was similar to Kunitz-type proteinase inhibitors from flowering plants. Analysis of expressed sequence tag (EST) databases confirmed the presence of mRNAs encoding Kunitz-type inhibitors in the Cycadales and Coniferales and also demonstrated their presence in a third major group of gymnosperms, the Ginkgoales. This is the first report of Kunitz-type serine proteinase inhibitors from plants other than Angiosperms. PMID:18755484

Konarev, Alexander V; Lovegrove, Alison; Shewry, Peter R

2008-10-01

130

Ixodidin, a novel antimicrobial peptide from the hemocytes of the cattle tick Boophilus microplus with inhibitory activity against serine proteinases.  

Science.gov (United States)

The presence of an effective immune response in the hemocoel of arthropods is essential for survival as it prevents the invasion of pathogens throughout the animal body. Antimicrobial peptides (AMPs) play an important role in this response by rapidly killing invading microorganisms. In this study, a novel cysteine-rich AMP has been isolated and characterized from the hemocytes of the cattle tick, Boophilus microplus. In addition to growth inhibition of Escherichia coli and Micrococcus luteus, the newly described AMP, designated ixodidin (derived from the Family Ixodidae), was found to exert proteolytic inhibitory activity against two exogenous serine proteinases, elastase and chymotrypsin. This is the first report of a molecule of an arachnid that has been shown to inhibit bacterial growth and proteinase activity. PMID:16191451

Fogaça, Andréa C; Almeida, Igor C; Eberlin, Marcos N; Tanaka, Aparecida S; Bulet, Philippe; Daffre, Sirlei

2006-04-01

131

Multiple pathways for vacuolar sorting of yeast proteinase A  

DEFF Research Database (Denmark)

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.

Westphal, V; Marcusson, E G

1996-01-01

132

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)

1996-01-01

133

Are proteinase inhibitors potentially useful in tumor therapy?  

Science.gov (United States)

Within the last 15 years a vast literature has arisen, which associates increased levels of proteinase activity with most in vitro transformed malignant cells and many tumor cells in vivo. As a consequence, proteinase inhibitors have been widely proposed as potential candidates for therapeutic use. The present review shows that in some studies proteinase inhibitors produced significant anti-tumor effects, while in most other studies only limited effects or no effects were observed. In some instances, opposite, or tumor enhancing, effects by proteinase inhibitors were observed. The reasons for the lack of a clear-cut success of proteinase inhibitors in tumor therapy may be: (1) proteinases may not be crucially involved in tumor growth and spread; (2) proteinases which may be crucially involved have not yet been identified; (3) lack of potent inhibitors with appropriate specificity, or use of inappropriate inhibitors or regimens. PMID:6203867

Nelles, L P; Schnebli, H P

1982-01-01

134

Subcellular distribution of glutathione and cysteine in cyanobacteria.  

Science.gov (United States)

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, glutamate, and glycine. Cysteine is the limiting factor for glutathione biosynthesis which can be especially crucial for cyanobacteria, which rely on both the sufficient sulfur supply from the growth media and on the protection of glutathione against ROS that are produced during photosynthesis. In this study, we report a method that allows detection and visualization of the subcellular distribution of glutathione in Synechocystis sp. This method is based on immunogold cytochemistry with glutathione and cysteine antisera and computer-supported transmission electron microscopy. Labeling of glutathione and cysteine was restricted to the cytosol and interthylakoidal spaces. Glutathione and cysteine could not be detected in carboxysomes, cyanophycin granules, cell walls, intrathylakoidal spaces, periplasm, and vacuoles. The accuracy of the glutathione and cysteine labeling is supported by two observations. First, preadsorption of the antiglutathione and anticysteine antisera with glutathione and cysteine, respectively, reduced the density of the gold particles to background levels. Second, labeling of glutathione and cysteine was strongly decreased by 98.5% and 100%, respectively, in Synechocystis sp. cells grown on media without sulfur. This study indicates a strong similarity of the subcellular distribution of glutathione and cysteine in cyanobacteria and plastids of plants and provides a deeper insight into glutathione metabolism in bacteria. PMID:20349253

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

2010-10-01

135

A cystatin-like cysteine proteinase inhibitor from venom of the African puff adder (Bitis arietans).  

Science.gov (United States)

Venoms from eight snakes have been screened for inhibitory activity against papain, strong activity being found in that of the African puff adder, Bitis arietans. The inhibitor from B. arietans venom has been purified by affinity chromatography on carboxymethyl-papain-Sepharose and ion-exchange chromatography. The inhibitor had an apparent Mr of 13,000 in SDS/polyacrylamide gel electrophoresis, and pI value of 6.5 (major component) or 6.3 (minor component). Values of Ki for the inhibition of papain, cathepsin B and dipeptidyl peptidase I were 0.10, 2.7 and 0.23 nM, respectively; chicken calpain was not inhibited. PMID:3500713

Evans, H J; Barrett, A J

1987-09-15

136

Levels of plasma cysteine-proteinase activity in bladder cancer patients.  

Science.gov (United States)

Plasma activity of a Cathepsin B like (CB) cystine protease was evaluated employing a chromogenic assay in citrated plasma samples of 21 patients with transitional bladder cancer (TBC), and 36 healthy blood donors. The median value of total CB activity was significantly higher in the group of patients bearing bladder carcinoma (103.4 mU/ml) as compared to the control group (77.7 mU/ml). 57% of patients showed high values of total CB while only 25% of the controls showed values over the cut-off levels (97.0 mU/ml). When the proenzyme (pro-CB) was analyzed, the median value was also significantly higher in the TBC patients (42.2 mU/ml) as compared to the control group (20.6 mU/ml). While 62% of TBC group showed values over pro-CB cut-off level (35.0 mU/ml) only 31% of controls did so. Total CB median value from other 29 patients succesfully treated from TBC without evidence of disease at the moment of blood colection was near the cut-off (97.6 mU/ml) while pro-CB median values were lower than the cut-off levels (33.2 mU/ml). PMID:21590077

Eijan, A; Casabe, A; Puricelli, L; Pasik, L; Malagrino, H; Matos, E; Joffe, E

1997-01-01

137

Proteinases in bone resorption : obvious and less obvious roles  

DEFF Research Database (Denmark)

Bone resorption is critical for the development and the maintenance of the skeleton, and improper regulation of bone resorption leads to pathological situations. Proteinases are necessary for this process. In this review, we show that this need of proteinases is not only because they are required for the solubilization of bone matrix, but also because they are key components of the mechanism that determines where and when bone resorption will be initiated. Moreover, there are indications that proteinases may also determine whether resorption will be followed by bone formation. Some of the proteinases involved in these different steps of the resorption processes were recently identified, as for instance cathepsin K, MMP-9 (gelatinase B), and interstitial collagenase. However, there is also increasing evidence showing that the critical proteinase(s) may vary depending on the bone type or on other factors.

Delaissé, Jean-Marie; Engsig, M T

2000-01-01

138

Specificity of a wheat gluten aspartic proteinase.  

Science.gov (United States)

The substrate and peptide bond specificities of a purified wheat gluten aspartic proteinase (GlAP) are studied. GlAP shows maximum gluten hydrolysing activity at pH 3.0. At this pH, especially the wheat high molecular weight glutenin subunits (HMW-GS) and to a lesser extent the low molecular weight glutenin subunits and gliadins are hydrolysed. GlAP has no obvious effect on albumins and globulins. In its action on oxidised insulin B-chain, GlAP forms eight peptides and has high specificity for peptide bonds located between amino acid residues with large hydrophobic side chains (Leu, Phe, Tyr) but the peptide bond Glu13-Ala14 is also hydrolysed. Although structurally quite similar to a barley aspartic proteinase, the peptide bond specificity of GlAP towards oxidised insulin B-chain resembles slightly more that of a cardoon aspartic proteinase, cardosin B. HMW-GS 7, purified from cultivar Galahad-77, is rapidly hydrolysed by GlAP. N-Terminal amino acid sequence data show that GlAP cleaves at least one Met-Ile peptide bond at the end of the N-terminal domain and two Val-Leu peptide bonds in the repetitive domain of HMW-GS 7. PMID:9748641

Bleukx, W; Brijs, K; Torrekens, S; Van Leuven, F; Delcour, J A

1998-09-01

139

Keratinolytic proteinase from Bacillus thuringiensis AD-12.  

Science.gov (United States)

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

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

2014-08-01

140

Analysis of the proteinases of representative Trichomonas vaginalis isolates.  

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Isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with acrylamide copolymerized with gelatin (substrate-SDS-PAGE) were combined to evaluate the proteinases of both long-term-grown and fresh isolates of Trichomonas vaginalis. This two-dimensional substrate-SDS-PAGE resolved as many as 23 distinct proteinase activities in several isolates, and proteinases had relative molecular masses between 23 and 110 kilodaltons (kDa). Isoelectric points (pI) of pr...

Neale, K. A.; Alderete, J. F.

1990-01-01

 
 
 
 
141

Cysteine S-conjugate ?-lyases  

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Cysteine S-conjugate ?-lyases are pyridoxal 5?-phosphate (PLP)-containing enzymes that catalyze the conversion of cysteine S-conjugates [RSCH2CH(NH3+)CO2?] and selenium Se-conjugates [RSeCH2CH(NH3+)CO2?] that contain a leaving group in the ? position to pyruvate, ammonium and a sulfur-containing fragment (RSH) or selenium-containing fragment (RSeH), respectively. At least ten PLP enzymes catalyze ?-elimination reactions with such cysteine S-conjugates. All are enzymes involved in ami...

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

2010-01-01

142

Constitutive and inducible trypsin proteinase inhibitor production incurs large fitness costs in Nicotiana attenuata  

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Plant trypsin proteinase inhibitors (TPIs) are potent herbivore- and jasmonate (JA)-induced defenses, but support for the commonly invoked explanation for their inducible expression, namely their associated fitness costs, has been elusive. To determine whether the expression of TPIs incurs fitness costs, we expressed 175 bp of the seven-domain pi from Nicotiana attenuata in an antisense orientation in a TPI-producing genotype (WT) of N. attenuata to reduce TPI expression. Moreover, we express...

2004-01-01

143

The Glycosylation of the Aspartic Proteinases from Barley (Hordeum Vulgare L.) and Cardoon (Cynara Cardunculus L.)  

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Plant aspartic proteinases characterised at the molecular level contain one or more consensus N-glycosylation sites [Runeberg-Roos, P., Törmäkangas, K. & Östman, A. (1991) Eur. J. Biochem. 202, 102120131027; Asakura, T., Watanabe, H., Abe, K. & Arai, S. (1995) Eur. J. Biochem. 232, 77201383; Veríssimo, P., Faro, C., Moir, A. J. G., Lin, Y., Tang, J. & Pires, E. (1996) Eur. J. Biochem. 235, 76220137681. We found that the glycosylation sites are occupied for the barley (Hordeum vulgare L.) ...

Costa, Ju?lia; Ashford, David A.; Nimtz, Manfred; Bento, Isabel; Fraza?o, Carlos; Esteves, Cristina L.; Faro, Carlos J.; Kervinen, Jukka; Pires, Euclides; Veri?ssimo, Paula; Wlodawer, Alexander; Carrondo, Maria Arme?nia

1997-01-01

144

Cysteine Variants of Beta Interferon.  

Science.gov (United States)

The growth hormone supergene family comprises greater than 20 structurally related cytokines and growth factors. A general method is provided for creating site-specific, biologically active conjugates of these proteins. The method involves adding cysteine...

G. N. Cox

2005-01-01

145

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

Directory of Open Access Journals (Sweden)

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.

Craik Charles S

2010-07-01

146

Proteinase inhibitor-inducing factor activity in tomato leaves resides in oligosaccharides enzymically released from cell walls  

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The synthesis and accumulation of proteinase inhibitor I in excised tomato leaves can be induced with oligosaccharides obtained by fungal endo-?-1,4-polygalacturonase digestion of a pectic polysaccharide (Mr 5000-10,000) isolated from tomato leaves. Active oligosaccharides were also released from isolated tomato leaf cell walls by endopolygalacturonases partially purified from tomato plants. It is suggested that oligosaccharides, released from plant cell wall pectic polysaccharides by either...

1981-01-01

147

Purification and partial characterisation of a cathepsin L-like proteinase from sea cucumber (Stichopus japonicus) and its tissue distribution in body wall.  

Science.gov (United States)

A cathepsin L-like proteinase (CLP) with molecular weight of 30.9 kDa from the gut of sea cucumber (Stichopus japonicas, S. japonicus) was isolated and purified to homogeneity by several chromatographic procedures. The enzyme exhibited optimum activity at pH 5.0-5.5 and 50 °C, and showed thermostability up to 40 °C. The enzyme activity was completely inhibited by Zn(2+), strongly inhibited by Fe(2+) and Cu(2+), drastically reduced by cysteine proteinase inhibitors, but slightly enhanced by thiol-activating agents. The enzyme efficiently hydrolysed the specific substrate of cathepsin L, but hardly hydrolysed the specific substrates for cathepsin B, cathepsin H and cathepsin K. Immunohistochemical studies indicated that the CLP was more abundant in the epidermis rather than in the dermis of S. japonicus body wall. The distribution of CLP showed positive correlation with autolysis rate. Therefore, the relationship between CLP and autolysis deserved further study. PMID:24731331

Zhou, Da-Yong; Chang, Xian-Na; Bao, Sha-Sha; Song, Liang; Zhu, Bei-Wei; Dong, Xiu-Ping; Zong, Yuan; Li, Dong-Mei; Zhang, Mao-Mao; Liu, Yu-Xin; Murata, Yoshiyuki

2014-09-01

148

Isolation and structural analysis of a gene coding for a novel type of aspartic proteinase from buckwheat seed (Fagopyrum esculentum Moench)  

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A novel type of aspartic proteinase gene was isolated from the cDNA library of developing buckwheat seeds. This cDNA, FeAPL1, encoded an AP-like protein lacking the plant-specific insert (PSI) domain characteristic of typical plant aspartic proteinases. In addition the corresponding genomic fragment was isolated. It is demonstrated that this gene does not contain introns. Since bioinformatics analysis of the Arabidopsis genome showed that most potential AP genes are intronless and PSI-less, i...

Milisavljevi? Mira ?.; Timotijevi? Gordana S.; Radovi? Svetlana R.; Konstantinovi? M.M.; Maksimovi? Vesna R.

2007-01-01

149

Limited proteolysis by macrophage elastase inactivates human alpha 1- proteinase inhibitor  

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Inflammatory mouse peritoneal macrophages secrete a metalloproteinase that is not inhibited by alpha 1-proteinase inhibitor. This proteinase, macrophage elastase, recognizes alpha 1-proteinase inhibitor with macrophage elastase does not involve a stable proteinase-inhibitor complex and results in the proteolytic removal of a peptide of apparent molecular weight 4,000-5,000 from the inhibitor. After degradation by macrophage elastase, alpha 1-proteinase inhibitor is no longer able to inhibit h...

1980-01-01

150

Characterization of the Cell Wall-Bound Proteinase of Lactobacillus casei HN14  

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Lactobacillus casei HN14, which was isolated from homemade cheese, produces an extracellular, cell wall-bound proteinase. The HN14 proteinase can be removed from the cell envelope by washing the cells in a Ca2+-free buffer. The activity of the crude proteinase extract is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the HN14 proteinase is similar to the lactococcal PI-type enzyme, since it hydrolyzes ?-...

Kojic, M.; Fira, D.; Banina, A.; Topisirovic, L.

1991-01-01

151

Digestive proteinases from marine organisms and their applications  

Directory of Open Access Journals (Sweden)

Full Text Available Fish viscera have wide biotechnological potential as a source of digestive enzymes, especially proteinases. The biological diversity of fish species provides a wide array of enzymes with unique properties. Fish digestive proteolytic enzymes most commonly found include pepsin and trypsin. Those enzymes from fish viscera may have the advantages for the applications in the food industry since their temperature and other characteristics differ from homologous proteinases from warm-blooded animals. Therefore, digestive proteinases can be isolated as a value-added product from fish viscera and used as the processing aids in food industries to maximize the utilization of marine resources.

Sappasith Klomklao

2008-01-01

152

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)

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

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

2013-08-01

153

Oxidant Effects on Rat and Human Lung Proteinase Inhibitors.  

Science.gov (United States)

The activity of alpha-1-proteinase inhibitor (alpha-PI) in lung lavage fluids was measured by elastase inhibition and the immunological concentration by enzyme-linked immunoassay. The inhibitor's functional activity was defined as active: immunological co...

D. A. Johnson R. S. Winters K. R. Lee C. E. Smith

1990-01-01

154

Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema.  

Science.gov (United States)

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 ?M), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy. PMID:24140156

Cruz-Silva, Ilana; Neuhof, Christiane; Gozzo, Andrezza Justino; Nunes, Viviane Abreu; Hirata, Izaura Yoshico; Sampaio, Misako Uemura; Figueiredo-Ribeiro, Rita de Cássia; Neuhof, Heinz; Araújo, Mariana da Silva

2013-12-01

155

Identification and Characterization of Bacterial Cysteine Dioxygenases: a New Route of Cysteine Degradation for Eubacteria  

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In metazoa and fungi, the catabolic dissimilation of cysteine begins with its sulfoxidation to cysteine sulfinic acid by the enzyme cysteine dioxygenase (CDO). In these organisms, CDO plays an important role in the homeostatic regulation of steady-state cysteine levels and provides important oxidized metabolites of cysteine such as sulfate and taurine. To date, there has been no experimental evidence for the presence of CDO in prokaryotes. Using PSI-BLAST searches and crystallographic informa...

Dominy, John E.; Simmons, Chad R.; Karplus, P. Andrew; Gehring, Amy M.; Stipanuk, Martha H.

2006-01-01

156

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone.  

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A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation o...

Jakubowski, H.

1994-01-01

157

Three distinct secreted aspartyl proteinases in Candida albicans.  

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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...

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

1993-01-01

158

Multiplicity of aspartic proteinases from Cynara cardunculus L.  

Science.gov (United States)

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity. PMID:19488781

Sarmento, Ana Cristina; Lopes, Henrique; Oliveira, Cláudia S; Vitorino, Rui; Samyn, Bart; Sergeant, Kjell; Debyser, Griet; Van Beeumen, Jozef; Domingues, Pedro; Amado, Francisco; Pires, Euclides; Domingues, M Rosário M; Barros, Marlene T

2009-07-01

159

The induction of proteinases in corn and soybean by anoxia  

International Nuclear Information System (INIS)

This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with 3H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia

1989-01-01

160

Mechanisms of hepatitis C virus NS3 proteinase inhibitors.  

Science.gov (United States)

The NS3 serine proteinase is regarded as one of the preferred targets for the development of therapeutic agents against hepatitis C virus (HCV). Possible mechanisms of NS3 inhibitors include: (i) interference with the activation of the enzyme by its NS4A cofactor; (ii) binding to the structural zinc site; and (iii) binding to the active site. These mechanisms have been explored in detail by structural analysis of the enzyme. (i) The NS4A cofactor binds to the amino-terminal beta-barrel domain of the NS3 proteinase bringing about several conformational changes that result in enzyme activation. The interaction between NS3 and NS4A involves a very large surface area and therefore it is not a likely target for the development of inhibitors. (ii) The NS3 proteinase contains a structural zinc binding site. Spectroscopic studies have shown that changes in the conformation of this metal-binding site correlate with changes in the specific activity of the enzyme, and the NS3 proteinase is inhibited by compounds capable of extracting zinc from its native coordination sphere. (iii) Based on the observation that the NS3 proteinase undergoes inhibition by its cleavage products, potent, active site-directed inhibitors have been generated. Kinetic studies, site-directed mutagenesis, and molecular modelling have been used to characterize the interactions between the NS3 proteinase and its product inhibitors. PMID:10760031

De Francesco, R; Pessi, A; Steinkühler, C

1999-07-01

 
 
 
 
161

The induction of proteinases in corn and soybean by anoxia  

Energy Technology Data Exchange (ETDEWEB)

This study characterized the anaerobic changes in proteinase activities in corn and soybean roots and to investigate the possibility that these changes might contribute to the differential anaerobiosis tolerance of the two species. After 24 h of anoxia, crude protein extracts from H60 corn and Keller soybean root tips (10cm) were assayed for proteinase activities at pH range from 4.5 to 9.5. Turnover of aberrant proteins was studied in seedlings labelled with {sup 3}H-leucine for 12 h under: (a) puromycin (0.64 mM) in air, (b) ethanol (1%) in air, (c) nitrogen and (d) air. After the treatment, the labelled proteins remaining in roots were determined every 2 h for 6 h. In both corn and soybean, activities of alkali proteinases increased, and activities of acid proteinases declined under anoxia. Neutral proteinases increase in anoxic corn roots, but decline in anoxic soybean roots. The protein turnover rate in corn treated with puromycin, ethanol and nitrogen was much higher than in control roots. The protein turnover rate in soybean roots treated with puromycin, ethanol was similar to the rate of the control. The results indicated that: (a) anoxic corn can degrade aberrant proteins, but anoxic soybean cannot, (b) the degradation of aberrant proteins in anoxic corn is accomplished by neutral proteinases, and (c) the accumulation of aberrant proteins in soybean might contribute to the susceptibility of this species to anoxia.

VanToai, T.; Hwang, Shihying (Dept of Agriculture, Columbus, OH (USA))

1989-04-01

162

Detection of Homocysteine and Cysteine  

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At elevated levels, homocysteine (Hcy, 1) is a risk factor for cardiovascular diseases, Alzheimer’s disease, neural tube defects, and osteoporosis. Both 1 and cysteine (Cys, 3) are linked to neurotoxicity. The biochemical mechanisms by which 1 and 3 are involved in disease states are relatively unclear. Herein, we describe simple methods for detecting either Hcy or Cys in the visible spectral region with the highest selectivity reported to date without using biochemical techniques or prepar...

Wang, Weihua; Rusin, Oleksandr; Xu, Xiangyang; Kim, Kyu Kwang; Escobedo, Jorge O.; Fakayode, Sayo O.; Fletcher, Kristin A.; Lowry, Mark; Schowalter, Corin M.; Lawrence, Candace M.; Fronczek, Frank R.; Warner, Isiah M.; Strongin, Robert M.

2005-01-01

163

Neutrophils degrade subendothelial matrices in the presence of alpha-1-proteinase inhibitor. Cooperative use of lysosomal proteinases and oxygen metabolites.  

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Triggered neutrophils rapidly degraded labeled matrices secreted by cultured, venous endothelial cells via a process dependent on elastase but not oxygen metabolites. In the presence of high concentrations of alpha-1-proteinase inhibitor, the ability of the stimulated neutrophil to solubilize the matrix was impaired. However, at lower concentrations of alpha-1-proteinase inhibitor the neutrophil could enhance the degradative potential of its released elastase by a H2O2-dependent process. Coin...

Weiss, S. J.; Regiani, S.

1984-01-01

164

Comparative molecular model building of two serine proteinases from cytotoxic T lymphocytes.  

Science.gov (United States)

Two genes that are expressed when precursor cytotoxic T lymphocytes are transformed to T killer cells have been cloned and sequenced. The derived amino acid sequences, coding for cytotoxic cell protease 1 (CCP1) and Hannuka factor (HF) are highly homologous to members of the serine proteinase family. Comparative molecular model building using the known three-dimensional structures and the derived amino acid sequences of the lymphocyte enzymes has provided useful structural information, especially in predicting the conformations of the substrate binding sites. In applying this modelling procedure, we used the X-ray structures of four serine proteinases to provide a structurally based sequence alignment: alpha-chymotrypsin (CHT), bovine trypsin (BT), Streptomyces griseus trypsin (SGT), and rat mast cell protease 2 (RMCP2). The root mean square differences in alpha-carbon atom positions among these four structures when compared in a pairwise fashion range from 0.79 to 0.97 A for structurally equivalent residues. The sequences of the two lymphocyte enzymes were then aligned to these proteinases using chemical criteria and the superimposed X-ray structures as guides. The alignment showed that the sequence of CCP1 was most similar to RMCP2, whereas HF has regions of homology with both RMCP2 and BT. With RMCP2 as a template for CCP1 and the two enzymes RMCP2 and BT as templates for HF, the molecular models were constructed. Intramolecular steric clashes that resulted from the replacement of amino acid side chains of the templates by the aligned residues of CCP1 and HF were relieved by adjustment of the side chain conformational angles in an interactive computer graphics device. This process was followed by energy minimization of the enzyme model to optimize the stereochemical geometry and to relieve any remaining unacceptably close nonbonded contacts. The resulting model of CCP1 has an arginine residue at position 226 in the specificity pocket, thereby predicting a substrate preference for P1 aspartate or glutamate residues. The model also predicts favorable binding for a small hydrophobic residue at the P2 position of the substrate. The primary specificity pocket of HF resembles that of BT and therefore predicts a lysine or arginine preference for the P1 residue. The arginine at position 99 in the model of HF suggests a preference for aspartate or glutamate side chains in the P2 position of the substrate. Both CCP1 and HF have a free cysteine in the segment of polypeptide 88 to 93.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:3237717

Murphy, M E; Moult, J; Bleackley, R C; Gershenfeld, H; Weissman, I L; James, M N

1988-01-01

165

Label-free quantitative proteomics reveals differentially regulated proteins in the latex of sticky diseased Carica papaya L. plants.  

Science.gov (United States)

Papaya meleira virus (PMeV) is so far the only described laticifer-infecting virus, the causal agent of papaya (Carica papaya L.) sticky disease. The effects of PMeV on the laticifers' regulatory network were addressed here through the proteomic analysis of papaya latex. Using both 1-DE- and 1D-LC-ESI-MS/MS, 160 unique papaya latex proteins were identified, representing 122 new proteins in the latex of this plant. Quantitative analysis by normalized spectral counting revealed 10 down-regulated proteins in the latex of diseased plants, 9 cysteine proteases (chymopapain) and 1 latex serine proteinase inhibitor. A repression of papaya latex proteolytic activity during PMeV infection was hypothesized. This was further confirmed by enzymatic assays that showed a reduction of cysteine-protease-associated proteolytic activity in the diseased papaya latex. These findings are discussed in the context of plant responses against pathogens and may greatly contribute to understand the roles of laticifers in plant stress responses. PMID:22465191

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Choi, HyungWon; Sobreira, Tiago J P; Nohara, Lilian L; Wermelinger, Luciana S; Almeida, Igor C; Zingali, Russolina B; Fernandes, Patricia M B

2012-06-18

166

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

DEFF Research Database (Denmark)

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.

Hejgaard, Jørn

2003-01-01

167

Cysteine synthesis by Desulfovibrio vulgaris extracts.  

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Extracts of Desulfovibrio vulgaris were found to contain serine transacetylase and cysteine synthase activities. When extracts were incubated with bisulfite and o-acetylserine, or acetyl coenzyme A plus L-serine, under a hydrogen atmosphere, cysteine was formed. Pyruvate served as a reductant for bisulfite reduction to sulfide and concomitantly provided the acetyl moiety for acetyl coenzyme A formation. Consequently, when extracts were incubated with pyruvate, bisulfite, and L-serine, cystein...

Gevertz, D.; Amelunxen, R.; Akagi, J. M.

1980-01-01

168

Selective fluorescence detection of cysteine and N-terminal cysteine peptide residues†  

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A new fluorogenic fluorescein derivative containing an ?,?-unsaturated aldehyde moiety produces a selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions.

Lim, Soojin; Escobedo, Jorge O.; Lowry, Mark; Xu, Xiangyang; Strongin, Robert

2010-01-01

169

Cysteine Catabolism and Cysteine Desulfhydrase (CdsH/STM0458) in Salmonella enterica Serovar Typhimurium  

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Cysteine is potentially toxic and can affect diverse functions such as oxidative stress, antibiotic resistance, and swarming motility. The contribution of cysteine catabolism in modulating responses to cysteine has not been examined, in part because the genes have not been identified and mutants lacking these genes have not been isolated or characterized. We identified the gene for a previously described cysteine desulfhydrase, which we designated cdsH (formerly STM0458). We also identified a...

Oguri, Tamiko; Schneider, Barbara; Reitzer, Larry

2012-01-01

170

Inducible expression of a fusion gene encoding two proteinase inhibitors leads to insect and pathogen resistance in transgenic rice.  

Science.gov (United States)

Plant proteinase inhibitors (PIs) are considered as candidates for increased insect resistance in transgenic plants. Insect adaptation to PI ingestion might, however, compromise the benefits received by transgenic expression of PIs. In this study, the maize proteinase inhibitor (MPI), an inhibitor of insect serine proteinases, and the potato carboxypeptidase inhibitor (PCI) were fused into a single open reading frame and introduced into rice plants. The two PIs were linked using either the processing site of the Bacillus thuringiensis Cry1B precursor protein or the 2A sequence from the foot-and-mouth disease virus (FMDV). Expression of each fusion gene was driven by the wound- and pathogen-inducible mpi promoter. The mpi-pci fusion gene was stably inherited for at least three generations with no penalty on plant phenotype. An important reduction in larval weight of Chilo suppressalis fed on mpi-pci rice, compared with larvae fed on wild-type plants, was observed. Expression of the mpi-pci fusion gene confers resistance to C. suppressalis (striped stem borer), one of the most important insect pest of rice. The mpi-pci expression systems described may represent a suitable strategy for insect pest control, better than strategies based on the use of single PI genes, by preventing insect adaptive responses. The rice plants expressing the mpi-pci fusion gene also showed enhanced resistance to infection by the fungus Magnaporthe oryzae, the causal agent of the rice blast disease. Our results illustrate the usefulness of the inducible expression of the mpi-pci fusion gene for dual resistance against insects and pathogens in rice plants. PMID:24237606

Quilis, Jordi; López-García, Belén; Meynard, Donaldo; Guiderdoni, Emmanuel; San Segundo, Blanca

2014-04-01

171

Lactococcal proteinase maturation protein PrtM is a lipoprotein.  

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The production of enzymatically active proteinase by lactococci requires the joint presence of a proteinase gene, prtP, and a gene encoding a maturation protein, prtM. A 32-kDa protein produced by Escherichia coli upon expression of the prtM gene under the direction of the T7 RNA polymerase promoter was purified and used to obtain PrtM-specific antibodies. With these antibodies, immunogold labeling of lactococcal cells revealed that PrtM was associated with the lactococcal cell envelope. West...

Haandrikman, A. J.; Kok, J.; Venema, G.

1991-01-01

172

Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously  

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In the tobacco hornworm Manduca sexta, proteolytic activation of prophenoloxidase (proPO) is mediated by three proPO-activating proteinases (PAPs) and two serine proteinase homologs (SPHs) (Proceedings of the National Academy of Sciences, USA 95 (1998) 12220–12225; J. Biol. Chem. 278 (2003a) 3552–3561; Insect Biochem. Mol. Biol. 33 (2003b) 1049–1060). While our current data are consistent with the hypothesis that the SPHs serve as a cofactor/anchor for PAPs (Insect Biochemistry and Mole...

Gupta, Snehalata; Wang, Yang; Jiang, Haobo

2005-01-01

173

21 CFR 184.1272 - L-Cysteine monohydrochloride.  

Science.gov (United States)

...2010-01-01 2009-04-01 true L-Cysteine monohydrochloride. 184.1272 Section...Substances Affirmed as GRAS § 184.1272 L-Cysteine monohydrochloride. (a) L-Cysteine monohydrochloride is the chemical...

2010-01-01

174

Mutational analysis of the proteinase function of Potato leafroll virus.  

Science.gov (United States)

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513-515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif. PMID:11369899

Sadowy, E; Juszczuk, M; David, C; Gronenborn, B; Hulanicka, M D

2001-06-01

175

Evidence for cysteine sulfinate as a neurotransmitter  

International Nuclear Information System (INIS)

The Na"+-independent binding of L-["3H]cysteine sulfinate and L-["3H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding of L-["3H]cysteine sulfinate was saturable and occurred by a single high affinity process with a Ksub(b) of 100 nM +- 9 and a capacity (Bsub(max)) of 2.4 +- 0.22 pmol/mg protein. The regional distribution of the binding of L-["3H]cysteine sulfinate in the brain was found to be heterogeneous. The rate of L-["3H]cysteine sulfinate uptake shows a biphasic dependence on the concentration of L-cysteine sulfinate, corresponding to a high affinity (27.2 ?M) and a low affinity (398 ?M) transport system. The maximum L-["3H]cysteine sulfinate uptake is reached at 2min and the uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. (Auth.)

1982-05-06

176

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)

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.

Pereira M.E.

2005-01-01

177

The aspartic proteinase family of three Phytophthora species  

Directory of Open Access Journals (Sweden)

Full Text Available Abstract Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs are produced in a wide variety of species (from bacteria to humans and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the A? peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during their translocation into the host red blood cell. Since the translocation of Phytophthora effector proteins is currently a topic of intense research activity, the identification in Phytophthora of potential functional homologues of plasmepsin V would appear worthy of investigation. Indeed, elucidation of the physiological roles of the APs identified here offers areas for future study. The significant revision of gene models and detailed annotation presented here should significantly facilitate experimental design.

ten Have Arjen

2011-05-01

178

Cytolytic effects of neutrophils: role for a membrane-bound neutral proteinase  

Energy Technology Data Exchange (ETDEWEB)

A neutral serine proteinase, purified 250-fold from the plasma membrane fraction of human neutrophils, differs in its catalytic and molecular properties from the well-known neutral proteinases present in azurophil (primary) granules. Stimulation of neutrophils with low concentrations of phorbol 12-myristate 13-acetate (PMA) results in the release into the medium of the membrane-bound proteinase and the concomitant production of oxygen radicals. These concentrations of PMA also induce full cytolytic activity measured with /sup 51/Cr-labeled ox erythrocytes. A role for the neutral serine proteinase in the cytolytic activity of PMA-stimulated neutrophils is supported by the following observations: (i) the lytic activity of the stimulated neutrophils is correlated with the quantity of neutral proteinase present in the membranes; (ii) the extracellular medium from PMA-stimulated neutrophils causes the cytolysis of /sup 51/Cr-labeled erythrocytes that have been exposed to nonlytic concentrations of H/sub 2/O/sub 2/; (iii) cytolysis of H/sub 2/O/sub 2/-treated erythrocytes is also observed with the crude proteinase solubilized from neutrophil membranes or with the purified proteinase from the same source; and (iv) in each case the cytolytic activity is proportional to the proteinase activity present and is prevented by the addition of serine proteinase inhibitors. The authors conclude that cytolysis of target cells by PMA-activated neutrophils can result from the cooperative effects of oxygen radicals and the membrane-bound neutral serine proteinase.

Pontremoli, S.; Melloni, E.; Michetti, M.; Sacco, O.; Sparatore, B.; Salamino, F.; Damiani, G.; Horecker, B.L.

1986-03-01

179

Astrocytes and the regulation of cerebral cysteine/cystine redox potential: implications for cysteine neurotoxicity  

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The sulfur amino acid, cysteine plays an essential role in maintaining cellular redox potential and is a key constituent of the antioxidant, glutathione. Cysteine is highly reactive and readily oxidises to the disulfide form, cystine, producing oxygen radicals as a by-product. Extracellular oxidising conditions favour cystine, whereas cysteine is the dominant intracellular form of the amino acid. In the brain, astrocytes control the extracellular thiol redox potential by actively taking u...

Mcbean, Gethin J.

2012-01-01

180

Cysteine Dioxygenase: A Robust System for Regulation of Cellular Cysteine Levels  

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Cysteine catabolism in mammals is dependent upon cysteine dioxygenase (CDO), an enzyme that adds molecular oxygen to the sulfur of cysteine, converting the thiol to a sulfinic acid known as cysteinesulfinic acid (3-sulfinoalanine). CDO is one of the most highly regulated metabolic enzymes responding to diet that is known. It undergoes up to 45-fold changes in concentration and up to 10-fold changes in catalytic efficiency. This provides a remarkable responsiveness of the cell to changes in su...

Stipanuk, M. H.; Ueki, I.; Dominy, J. E.; Simmons, C. R.; Hirschberger, L. L.

2009-01-01

 
 
 
 
181

Structure of Leishmania major cysteine synthase  

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Cysteine biosynthesis is a potential target for drug development against parasitic Leishmania species; these protozoa are responsible for a range of serious diseases. To improve understanding of this aspect of Leishmania biology, a crystallographic and biochemical study of L. major cysteine synthase has been undertaken, seeking to understand its structure, enzyme activity and modes of inhibition. Active enzyme was purified, assayed and crystallized in an orthorhombic form with a dimer in the ...

2012-01-01

182

Selective conservation of the RSL-encoding, proteinase inhibitory-type, clade L serpins in Caenorhabditis species.  

Science.gov (United States)

Serpins are a highly conserved superfamily of serine and papain-like cysteine proteinase inhibitors that are divided phylogenetically into clades. Serpins also can be divided anatomically into those that reside predominately outside or inside cells. While the activities of the extracellular serpins are well understood, the biological functions, as well as the overall distribution of the intracellular (serpinIC) serpins is less well defined. Conceivably, the biological function of the serpinsIC might be revealed by analysis of species with genomes of lower complexity. To this end, we sought to define the clade L serpin repertoire of Caenorhabditis elegans and other nematode species. Analysis of the C. elegans genome revealed the presence of 9 serpin genes. Five genes encoded for full-length serpins with functional reactive site loops (RSL). By definition, these genes were designated proteinase inhibitory-type, RSL-encoding serpins. Four of the C. elegans genes encoded for proteins without an RSL or transcripts with premature termination codons. The high percentage of non-RSL encoding to RSL-encoding serpin genes suggested that the former served a unique biological function rather than residing in the genome as simple pseudogenes. If this hypothesis was correct, we expected these non-RSL encoding genes to be conserved precisely in other Caenorhabditis species. However, in contrast to the RSL-encoding serpins that were well conserved and segregated into 3 sub-clades, we failed to detect non-RSL encoding serpin orthologues in the genomes of Caenorhabditis briggsae and Caenorhabditis remanei. These data suggested that unlike their RSL-encoding paralogues, the relatively high percentage of non-RSL encoding serpins in C. elegans was a vestige of recent duplication events and these latter genes were unlikely to serve essential functions in Caenorhabditis species. PMID:16146754

Luke, Cliff J; Pak, Stephen C; Askew, David J; Askew, Yuko S; Smith, Justin E; Silverman, Gary A

2006-01-01

183

The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.  

Science.gov (United States)

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. PMID:22659439

Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

2012-09-01

184

Proteinase overproduction in Lactococcus lactis strains: regulation and effect on growth and acidification in milk.  

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Multicopy plasmids that contained the complete of 3'-deleted forms of the proteinase (prtP) gene of Lactococcus lactis subsp. cremoris SK11 under the control of different promoters were constructed and introduced into Prt- lactococcal strains. The production and location of the SK11 proteinase was determined in different hosts grown in industrial and laboratory media. In spite of the 10-fold-higher copy number of the prt genes, no overproduction of proteinase was observed in strain SK1128, a ...

Bruinenberg, P. G.; Vos, P.; Vos, W. M.

1992-01-01

185

Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa.  

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We traced an acid proteinase from Candida spp. in the initial stages of the pathogenesis of the mycosis. On infection of human buccal mucosa, proteinase antigens were detected by immuno-scanning electron microscopy on the surface of adhering blastoconidia and invading filamentous cells of C. albicans serotype A. Proteinase antigens were also present on blastoconidia of C. albicans serotype B, but were missing on filamentous cells of this serotype. Proteolytic isolates of C. tropicalis behaved...

Borg, M.; Ru?chel, R.

1988-01-01

186

Production, Characterization, and Epitope Mapping of a Monoclonal Antibody against Aspartic Proteinase of Candida albicans  

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A monoclonal antibody (MAb; MAb CAP1) that was reactive with extracellular aspartic proteinase of Candida albicans (CAP) was produced. The MAb showed strong sensitivity and reactivity to CAP but not to the aspartic proteinases of Candida parapsilosis, Candida tropicalis, and Aspergillus fumigatus or to human cathepsin D or porcine pepsin. The epitope of the CAP recognized by the MAb was the proteinaseous part of CAP and the putative epitope of the MAb was locat...

Na, Byoung-kuk; Chung, Gyung-tae; Song, Chul-yong

1999-01-01

187

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

2006-01-01

188

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

International Nuclear Information System (INIS)

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 ?-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

2006-01-01

189

The role of proteinase enzymes in the process of conversion of muscle to meat  

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Full Text Available Post mortem meat tenderization is a complex mechanism and unfortunately it has not been fully identified scientifically. It is known that endogenous proteinases have an important role in this mechanism. Detailed studies are being performed about the destructive effects of lysosomal proteinases and calcium dependent proteinases on the myofibrils and these are most common topics that are being investigated about meat tenderization processes by the scientists. The aim of this paper is to review the role of proteinase enzymes in the process of conversion of muscle to meat. .

Dümen Emek

2006-01-01

190

Evidence for cysteine sulfinate as a neurotransmitter.  

Science.gov (United States)

The Na+-independent binding of L-[3H]cysteine sulfinate and L-[3H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding of L-[3H]cysteine sulfinate was saturable and occurred by a single high affinity process with a Kb of 100 nM +/- 9 and a capacity (Bmax) of 2.4 +/- 0.22 pmol/mg protein. Sodium ions were found to have a biphasic effect; low concentrations (in the range of 0.1-3 mM) induced a marked inhibition of the binding whereas higher concentrations (10-300 mM) resulted in a dose-dependent stimulation of binding. The inhibition potency, expressed as the Ki values of a wide range of compounds with known pharmacological activities was tested. L-Cysteine sulfinate was the most potent inhibitor being 3-fold more potent than L-glutamate and 80 times more potent than L-aspartate. The regional distribution of the binding of L-[3H]cysteine sulfinate in the brain was found to be heterogeneous. These results provide the first evidence for an interaction of cysteine sulfinate with specific receptor sites on the synaptic membrane. The rate of L-[3H]cysteine sulfinate uptake shows a biphasic dependence on the concentration of L-cysteine sulfinate, corresponding to a high affinity (27.2 microM) and a low affinity (398 microM) transport system. The maximum L-[3H]cysteine sulfinate uptake is reached at 2 min. The reversibility of this transport was demonstrated. The L-[3H]cysteine sulfinate uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. The decrease or increase in the electrical membrane potential (delta psi) caused by replacing the chloride ions by the sulfate or sulfocyanate ions respectively leads to a decrease or increase in the rate of uptake. Increase in the extravesicular osmolarity leads to a decrease in the extent of L-[3H]cysteine sulfinate accumulation. Amino acids with an acidic group in position omega were found to be potent inhibitors (the most potent being L-aspartate). The length of the carbon chain also has a bearing on the inhibitory effect. The regional distribution of L-[3H]cysteine sulfinate uptake in the brain was heterogeneous. These results demonstrate the existence of a high affinity system which may correspond to the transmitter inactivation. Binding and uptake sites are distinguishable as evidenced by the affinity constants, the ionic and pharmacological effects and the different regional distributions in the brain. Finally, these results give further evidence for a neurotransmitter role of L-cysteine sulfinate. PMID:6124301

Recasens, M; Varga, V; Nanopoulos, D; Saadoun, F; Vincendon, G; Benavides, J

1982-05-01

191

Aminotransferase, L-amino acid oxidase and beta-lyase reactions involving L-cysteine S-conjugates found in allium extracts. Relevance to biological activity?  

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Several cysteine S-conjugates that occur in extracts of garlic and other plants of the allium family possess anti-oxidant properties, and many, including S-allyl-L-cysteine (SAC) and S-allylmercapto-L-cysteine (SAMC), are promising anti-cancer agents. To understand possible biochemical mechanisms contributing to the protective effects, the ability of selected allium-derived L-cysteine S-conjugates to undergo various enzyme-catalyzed transformations was investigated. SAC, SAMC, S-propylmercapto-L-cysteine and S-penta-1,3-dienylmercapto-L-cysteine were shown to be substrates of: (a) highly purified rat kidney glutamine transaminase K (GTK); (b) purified snake venom L-amino acid oxidase; and (c) a cysteine S-conjugate beta-lyase present in rat liver cytosol. S-Methylmercapto-L-cysteine was shown to be a substrate of GTK and L-amino acid oxidase, but not of the cysteine S-conjugate beta-lyase. Evidence is presented that a major enzyme responsible for the cysteine S-conjugate beta-lyase reactions in the rat liver cytosol is gamma-cystathionase. The possible role of gamma-cystathionase in generating sulfane sulfur from the disulfide-containing cysteine S-conjugates present in allium extracts, and the possible role of this sulfane sulfur in enzyme regulation, targeting of cancer cells and detoxification reactions is discussed. An interesting side finding of the present work is that rat liver mitochondria are more active than rat liver cytosol in catalyzing a cysteine S-conjugate beta-lyase reaction with the mitochondrial protoxicant S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) at physiological pH and at low substrate concentration. PMID:15627473

Cooper, Arthur J L; Pinto, John T

2005-01-15

192

cDNA Cloning and Molecular Modeling of Procerain B, a Novel Cysteine Endopeptidase Isolated from Calotropis procera  

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Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimen...

Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

2013-01-01

193

Metabolism of L-cysteine in guinea pig liver.  

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The metabolism of L-cysteine in guinea pig liver was studied. Guinea pig liver contained 0.45 +/- 0.05 (mean +/- SD) mumol of cysteine, 0.180 +/- 0.080 mumol of 3-mercaptolactate-cysteine disulfide [S-(2-hydroxy-2-carboxyethylthio)cysteine, HCETC], and 8.082 +/- 0.516 mumol of reduced glutathione per g of fresh tissue. The taurine content was 0.912 +/- 0.158 mumol per g of fresh liver. Cysteine dioxygenase (EC 1.13.11.20) activity was several-fold lower than cysteine aminotransferase (EC 2...

Hosaki, Yasuhiro; Nishina, Hideo; Ubuka, Toshihiko

1986-01-01

194

Benzoquinone reveals a cysteine-dependent desensitization mechanism of TRPA1.  

Science.gov (United States)

The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel. PMID:23478802

Ibarra, Yessenia; Blair, Nathaniel T

2013-05-01

195

Benzoquinone Reveals a Cysteine-Dependent Desensitization Mechanism of TRPA1  

Science.gov (United States)

The transient receptor potential ankyrin 1 (TRPA1) nonselective cation channel has a conserved function as a noxious chemical sensor throughout much of Metazoa. Electrophilic chemicals activate both insect and vertebrate TRPA1 via covalent modification of cysteine residues in the amino-terminal region. Although naturally occurring electrophilic plant compounds, such as mustard oil and cinnamaldehyde, are TRPA1 agonists, it is unknown whether arthropod-produced electrophiles activate mammalian TRPA1. We characterized the effects of the electrophilic arthropod defensive compound para-benzoquinone (pBQN) on the human TRPA1 channel. We used whole-cell recordings of human embryonic kidney cells heterologously expressing either wild-type TRPA1 or TRPA1 with three serine-substituted cysteines crucial for electrophile activation (C621S, C641S, C665S). We found that pBQN activates TRPA1 starting at 10 nM and peaking at 300 nM; higher concentrations caused rapid activation followed by a fast decline. Activation by pBQN required reactivity with cysteine residues, but ones that are distinct from those previously reported to be the key targets of electrophiles. The current reduction we found at higher pBQN concentrations was a cysteine-dependent desensitization of TRPA1, and did not require prior activation. The cysteines required for desensitization are not accessible to all electrophiles as iodoacetamide and internally applied 2-(trimethylammonium)ethyl methanesulfonate failed to cause desensitization (despite large activation). Interestingly, following pBQN desensitization, wild-type TRPA1 had dramatically reduced response to the nonelectrophile agonist carvacrol, whereas the triple cysteine mutant TRPA1 retained its full response. Our results suggest that modification of multiple cysteine residues by electrophilic compounds can generate both activation and desensitization of the TRPA1 channel.

Ibarra, Yessenia

2013-01-01

196

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

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Full Text Available 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 embryo, the contents of these molecules were higher in the earlier stages while in the endosperm, their contents were almost constant during maturation. There was a negative correlation (r = -0.623; p<0.01 between cysteine content in the embryo and glutathione content in the endosperm. Meanwhile cysteine content was positively correlated to amino acids (r = 0.883; p<0.01 and protein (r = 0.866; p<0.01 in the embryo. Our findings suggest that cysteine might be mainly provided by the endosperm for embryo development. In the embryo, two cysteine synthase isoforms (A and B were revealed from stage 5+ to stage 8+ but were not detected from stage 0+ to stage 4+. Reversely, in the endosperm, both isoforms were present only from stage 0+ to stage 3+. Similarity in protein distribution in the endosperm at different embryo stages suggests that embryogenesis takes place through seven steps characterized by their protein patterns.

Minyaka Emile

2007-01-01

197

Role of D-cysteine desulfhydrase in the adaptation of Escherichia coli to D-cysteine.  

Science.gov (United States)

D-cysteine, a powerful inhibitor of Escherichia coli growth, is decomposed in vitro into pyruvate, H2S, and NH3 by D-cysteine desulfhydrase. To assess the role of this reaction in the adaptation of the bacterium to growth on D-cysteine, the gene of the desulfhydrase was cloned. It corresponds to the open reading frame yedO at 43.03 min on the genetic map of E. coli. The amino acid sequence deduced from this gene is homologous to those of several 1-aminocyclopropane-carboxylate deaminases. However, the E. coli desulfhydrase does not use 1-aminocyclopropane-1-carboxylate as substrate. Various mutants in which the yedO gene was inactivated or overexpressed were constructed. They exhibited hypersensitivity or resistance, respectively, to the presence of d-cysteine in the culture medium. Growth protection against D-cysteine in minimal medium was conferred by the simultaneous addition of isoleucine, leucine, and valine. In agreement with this behavior, D-cysteine inhibited the activity of threonine deaminase, a key enzyme of the isoleucine, leucine, and valine pathway. Finally, in the presence of the intact yedO gene, E. coli growth was improved by addition of D-cysteine as the sole sulfur source. In agreement with a role of the desulfhydrase in sulfur metabolism, yedO expression was induced under conditions of sulfate limitation. PMID:11527960

Soutourina, J; Blanquet, S; Plateau, P

2001-11-01

198

Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.  

Science.gov (United States)

RNA silencing limits transient expression of heterologous proteins in plants. Co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. Little is known whether silencing suppression could be improved without harm to plant tissues. This study reports development of enhanced silencing suppressors by engineering the helper component proteinase (HCpro) of Potato virus A (PVA). Mutations were introduced to a short region of HCpro (positions 330-335 in PVA HCpro), which is hypervariable among potyviruses. Three out of the four HCpro mutants suppressed RNA silencing more efficiently and sustained expression of co-expressed jellyfish green fluorescent protein for a longer time than wild-type HCpro in agroinfiltrated leaves of Nicotiana benthamiana. Leaf tissues remained healthy-looking without any visible signs of stress. PMID:23933077

Haikonen, T; Rajamäki, M-L; Valkonen, J P T

2013-11-01

199

Interaction between proteolytic strains of Lactococcus lactis influenced by different types of proteinase during growth in milk.  

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The influence of the type of cell envelope-located proteinase (PI versus PIII) on the associative growth of Lactococcus lactis in milk was studied. Two genetically engineered strains, differing only by the type of proteinase, were first used as a model study. An interaction occurred during the second exponential growth phase of the mixed culture and resulted in a decrease in growth rate of the PI-type proteinase strain, whereas that of the PIII-type proteinase strain remained unaffected. The ...

Flambard, B.; Richard, J.; Juillard, V.

1997-01-01

200

S-alk(en)yl-L-cysteine sulfoxides, alliinase and aroma in Leucocoryne.  

Science.gov (United States)

Levels of S-alk(en)yl-L-cysteine sulfoxides, alliinase and enzymatically generated pyruvic acid were determined in the bulb, leaf and scape of five species and a natural hybrid of Leucocoryne (Liliaceae), a genus of ornamental geophytes indigenous to Chile. (+)-S-Methyl-L-cysteine sulfoxide (MCSO) was present in all plant parts of all species at levels between 0.09 and 1.41 mg g(-1) fr. wt. Trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide (PRENCSO) was present in plant parts of three species only (L. angustipetala, L. oadorata and L. purpurea) at levels between 0.12 and 1.82 mg g(-1) fr. wt. No other S-alk(en)yl-L-cysteine sulfoxides were detected. Alliinase (EC 4.4.1.4) was detected in the leaf, bulb and scape of L. angustipetala and L. purpurea, only in the leaves of L. coquimbensis and L. purpurea x L. coquimbensis, and only in the bulb of L. odorata. Enzymatically generated pyruvic acid was detected in all plant parts of all species at levels between trace amounts and 5.33 micromol g(-1) fr. wt. As PRENCSO is produced only in Leucocoryne species exhibiting a strong and unpleasant onion-like aroma, it is probable that the enzymatic degradation of PRENCSO is the main cause of that aroma. Consequently, Leucocoryne cultivars should be selected in species and hybrids that lack the ability to synthesise PRENCSO. PMID:11065288

Lancaster, J E; Shaw, M L; Walton, E F

2000-09-01

 
 
 
 
201

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

Science.gov (United States)

This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. PMID:24388866

Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

2014-03-15

202

Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus  

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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.

1991-01-01

203

Chromosomal stabilization of the proteinase genes in Lactococcus lactis.  

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The plasmid-encoded proteinase genes prtP and prtM of Lactococcus lactis subsp. cremoris Wg2 were integrated by a Campbell-like mechanism into the L. lactis subsp. lactis MG1363 chromosome by using the insertion vector pKLG610. Two transformants were obtained that differed in the number of amplified pKLG610 copies in head-to-tail arrangements on their chromosomes; MG610 contained approximately two copies, and MG611 contained about eight copies. The amplifications were stably maintained during...

Leenhouts, K. J.; Gietema, J.; Kok, J.; Venema, G.

1991-01-01

204

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin  

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Proteinase-activated receptor 1 (PAR1) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR1 by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR1 (hPAR1) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten ...

2011-01-01

205

Measurement of Cysteine Dioxygenase Activity and Protein Abundance  

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Cysteine dioxygenase is an iron (Fe2+)-dependent thiol dioxygenase that uses molecular oxygen to oxidize the sulfhydryl group of cysteine to generate 3-sulfinoalanine (commonly called cysteinesulfinic acid). Cysteine dioxygenase activity is routinely assayed by measuring cysteinesulfinate formation from substrate L-cysteine at pH 6.1 in the presence of ferrous ions to saturate the enzyme with metal cofactor, a copper chelator to diminish substrate oxidation, and hydroxylamine to inhibit pyrid...

Stipanuk, Martha H.; Dominy, John E.; Ueki, Iori; Hirschberger, Lawrence L.

2008-01-01

206

CSCI/RCPSC Henry Friesen Lecture. Mentors and the butterfly effect: triggers for discovering signalling by proteinases via proteinase-activated receptors (PARs) and more.  

Science.gov (United States)

The essential role of proteinases as regulatory digestive enzymes, recognized since the late 1800s, has been underscored by the discovery that more than 2% of the genome codes for proteinases and their inhibitors. Further, by the early 1970s it was appreciated that in addition to their digestive actions, proteinases can affect cell function: (1) by the generation or degradation of peptide hormones and (2) by the direct regulation of signalling by receptors like the one for insulin. It was the discovery in the 1990s of the novel G-protein-coupled 'proteinase-activated receptor' (PAR) family that has caused a paradigm shift in the understanding of the way that proteinases can regulate cell signalling. This overview provides a perspective for the discovery of the PARs and my laboratory's role in (1) understanding the molecular pharmacology of these fascinating receptors and (2) identifying the potential pathophysiological roles that the PAR family can play in inflammatory disease. In this context, the overview also portrays the essential impact that seemingly minor comments/insights provided by my lifelong mentors have had on kindling my intense interest in proteinase-mediated signalling. The 'butterfly effect' of those comments has led to an unexpectedly large impact on my own research directions. Hopefully my own 'butterfly comments' will also be heard by my trainees and other colleagues with whom I am currently working and will promote future discoveries that will be directly relevant to the treatment of inflammatory disease. PMID:23217564

Hollenberg, Morley D

2012-01-01

207

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

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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, w...

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

2007-01-01

208

Septicemia caused by cysteine-dependent Escherichia coli.  

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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.

Yuen, K. Y.; Seto, W. H.; Tsui, K. H.; Hui, W. T.

1990-01-01

209

Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.  

Science.gov (United States)

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis. PMID:8258639

Betageri, R; Hopkins, J L; Thibeault, D; Emmanuel, M J; Chow, G C; Skoog, M T; de Dreu, P; Cohen, K A

1993-10-01

210

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

1990-01-01

211

Diisopropyl fluorophosphate labeling of sperm-associated proteinases  

Energy Technology Data Exchange (ETDEWEB)

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.

Odem, R.R.; Willand, J.L.; Polakoski, K.L. (Washington Univ. School of Medicine, St. Louis, MO (USA))

1990-02-01

212

Polymorphism of the membrane proteinases of the mitochondria  

International Nuclear Information System (INIS)

Three protein fractions capable of catalyzing the proteolysis of cytochrome c and three other fractions catalyzing the hydrolysis of N-?-benzoyl-L-arginine-p-nitroanilide (BAPA) and N-?-benzoyl-L-arginine-?-naphthylamide (BANA) were separated by electrophoresis in polyacrylamide gel in the absence of SDS detergent extracts from ultrasonic submitochondrial particles (SMP). The indicated fractions were isolated from gel and studied according to a series of parameters. It was shown that cytochrome c hydrolases have the same molecular weight (17,000) but different isoelectric points (4.0, 4.2, and 4.4). The total cytochrome c hydrolase activity of these enzymes was inhibited by phenylmethylsulfonyl fluoride but was insensitive to ethylenediaminetetraacetate and o-phenanthroline. The three BANA (BAPA) hydrolases also had similar molecular weights (? 17,500) and different isoelectric points (4.2, 4.3, and 4.7). In addition to the indicated hydrolases, minor components, possessing the same activities but differing in strength of the bond to the inner mitochondrial membrane, molecular weight, and sensitivity to proteinase inhibitors, were detected in the detergent extracts of the SMP. It was concluded that there is a polymorphism of the proteinases associated with the inner mitochondrial membrane

1985-09-20

213

Study of growth requirements other than cysteine of naturally occurring Escherichia coli and Klebsiella spp. auxotrophic for cysteine.  

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Cysteine remains the preferred supplement for cultivation of Cys- auxotrophs in vitro. Methionine, which reduced cysteine requirements, and branched-chain amino acids, which decreased cysteine toxicity, were identified as the components of casein hydrolysate responsible for growth enhancement by this additive. Glutathione and DL-homocysteine can be substituted for cysteine. Accumulation of these compounds in patients with renal impairment may favor selection of Cys- strains in vivo.

Mciver, C. J.; Tapsall, J. W.

1993-01-01

214

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)

Full Text Available SciELO Brazil | Language: English Abstract in portuguese 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

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

215

Inhibitory effects of cysteine and cysteine derivatives on germination of sporangiospores and hyphal growth of different Zygomycetes.  

Science.gov (United States)

The in vitro antifungal activity of cysteine (D- and L-cysteine) and its four derivatives (L-cysteine-methyl-ester, N-acetyl-cysteine, N-isobutyryl-D-cysteine, and N-isobutyryl-L-cysteine) were investigated on 20 fungal isolates representing 16 genera (Absidia, Actinomucor, Backusella, Gilbertella, Micromucor, Mortierella, Mucor, Mycotypha, Phycomyces, Rhizomucor, Rhizopus, Saksenaea, Syncephalastrum, Thamnostylum, Umbellopsis, and Zygorynchus). The inhibitory potential of different concentrations of these compounds, ranging from 0.625 to 10 mM, were investigated on the germination of sporangiospores as well as on hyphal extension, using broth microdilution method and agar plate test. Treatment with cysteine and its derivatives resulted in a strong inhibition in most studied strains. At 10 mM of compounds, complete blockage of growth was observed for some isolates. Sensitive species exhibited severe changes in colony morphology in the presence of 10 mM L-cysteine, N-acetyl-cysteine, and N-isobutyryl-L-cysteine. Microscopic observations revealed that 10 mM N-acetyl-cysteine induced dramatic modifications in the structural organization of the hyphae. Results suggest that cysteine and its derivatives have a therapeutic potential against fungal infections caused by Zygomycetes species. PMID:19381868

Galgóczy, László; Kovács, Laura; Krizsán, Krisztina; Papp, Tamás; Vágvölgyi, Csaba

2009-09-01

216

Are proteinase 3 and cathepsin C enzymes related to pathogenesis of periodontitis?  

Science.gov (United States)

Aim. Cathepsin C is the activator of the polymorphonuclear leukocyte-derived proteinase 3, which contributes to inflammatory processes. The aim of the present study was to investigate gingival crevicular fluid (GCF) proteinase 3 and cathepsin C levels in periodontal diseases. Design. Eighteen patients with chronic periodontitis (CP), 20 patients with generalized aggressive periodontitis (G-AgP), 20 patients with gingivitis, and 18 healthy subjects were included in the study. Periodontal parameters including probing depth, clinical attachment level, papilla bleeding index, and plaque index were assessed in all study subjects. GCF proteinase 3 and cathepsin C levels were analyzed by ELISA. Results. GCF proteinase 3 total amount was significantly higher in diseased groups compared to control group, after adjusting age (P 0.05). Periodontal parameters of sampling sites were positively correlated with GCF proteinase 3 total amounts (P 0.05). Conclusions. Elevated levels of GCF proteinase 3 in CP, G-AgP, and gingivitis might suggest that proteinase 3 plays a role during inflammatory periodontal events in host response. However, cathepsin C in GCF does not seem to have an effect on the pathogenesis of periodontal diseases. PMID:24949444

Türko?lu, Oya; Azars?z, Elif; Emingil, Gülnur; Kütükçüler, Necil; Atilla, Gül

2014-01-01

217

Limited proteolysis by macrophage elastase inactivities human. cap alpha. /sub 1/-proteinase inhibitor  

Energy Technology Data Exchange (ETDEWEB)

Ever since the initial description of ..cap alpha../sub 1/-proteinase inhibitor (..cap alpha../sub 1/PI), the role of this plasma glycoprotein and its allelic polymorphism in disease and in healthy physiology has been the subject of much investigation, ..cap alpha../sub 1/PI inactivates a number of serine proteinases, including granulocyte elastase, and thus affords protection from the connective tissue degradation mediated by this class of proteinases. Because an imbalance in the ratio between ..cap alpha../sub 1/PI and proteinase may contribute to the development of destructive lung diseases, proteinases have been implicated in the pathogenesis of pulmonary emphysema. Both macrophages and polymorphonuclear leukocytes have been implicated in disruption of the ..cap alpha../sub 1/PI-proteinase balance. In this report, a new mechanism for alteration of the ..cap alpha../sub 1/PI-proteinase balance is demonstrated. It was found that the purified form of macrophage elastase catalytically degrades and inactivates ..cap alpha../sub 1/PI so that it no longer inhibits the elastinolytic activity of granulocyte elastase.

Banda, M.J.; Clark, E.J.; Werb, Z.

1980-12-01

218

Zinc-Binding Cysteines: Diverse Functions and Structural Motifs  

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Full Text Available Cysteine residues are known to perform essential functions within proteins, including binding to various metal ions. In particular, cysteine residues can display high affinity toward zinc ions (Zn2+, and these resulting Zn2+-cysteine complexes are critical mediators of protein structure, catalysis and regulation. Recent advances in both experimental and theoretical platforms have accelerated the identification and functional characterization of Zn2+-bound cysteines. Zn2+-cysteine complexes have been observed across diverse protein classes and are known to facilitate a variety of cellular processes. Here, we highlight the structural characteristics and diverse functional roles of Zn2+-cysteine complexes in proteins and describe structural, computational and chemical proteomic technologies that have enabled the global discovery of novel Zn2+-binding cysteines.

Nicholas J. Pace

2014-04-01

219

Binding of the recombinant proteinase inhibitor eglin c, of the soybean Bowman-Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves: thermodynamic study.  

Science.gov (United States)

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c (eglin c), of the soybean Bowman-Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F-C and F-T, respectively) to Leu-proteinase, the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves, has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka (at 21 degrees C) for complex formation decrease thus reflecting the acidic pK-shift of the hystidyl catalytic residue from approximately 6.9, in the free Leu-proteinase, to approximately 5.1, in the enzyme: inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for the proteinase:inhibitor complex formation are: Leu-proteinase:eglin c-Ka = 2.2 x 10(11) M-1, delta G degree = -64 kJ/mol, delta H degree = +5.9 kJ/mol, and delta S degree = +240 kJ/molK; Leu-proteinase:BBI-Ka = 3.2 x 10(10) M-1, delta G degree = -59 kJ/mol, delta H degree = +8.8 kJ/mol, and delta S degree = +230 J/molK; and Leu-proteinase:F-C-Ka = 1.1 x 10(6) M-1, delta G degree = -34 kJ/mol, delta H degree = +18 J/mol, and delta S degree = +180 J/molK (values of Ka, delta G degree and delta S degree were obtained at 21.0 degrees C; values of delta H degree were temperature-independent over the range explored, i.e. between 10.0 degrees C and 40.0 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1669829

Ascenzi, P; Amiconi, G; Ballio, A; Bolognesi, M; Menegatti, E; Schnebli, H P; Aducci, P

1991-01-01

220

Primary radiation products in cysteine hydrochloride  

International Nuclear Information System (INIS)

ESR and ENDOR spectroscopy were used to identify three primary radicals in single crystals of cysteine hydrochloride x irradiated at 4.2 degreeK. The reduction product is an anion formed by addition of an electron to the carboxyl group. One oxidation product results from the loss of the sulfhydryl hydrogen atom. Another oxidation product is an electron vacancy localized mainly on atomic chlorine

1976-04-01

 
 
 
 
221

Characterization of a Cell Envelope-Associated Proteinase Activity from Streptococcus thermophilus H-Strains  

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The production and biochemical properties of cell envelope-associated proteinases from two strains of Streptococcus thermophilus (strains CNRZ 385 and CNRZ 703) were compared. No significant difference in proteinase activity was found for strain CNRZ 385 when cells were grown in skim milk medium and M17 broth. Strain CNRZ 703 exhibited a threefold-higher proteinase activity when cells were grown in low-heat skim milk medium than when grown in M17 broth. Forty-one percent of the total activity...

Shahbal, Samaha; Hemme, Denis; Renault, Pierre

1993-01-01

222

Partial characterization of hepatopancreatic and extracellular digestive proteinases of wild and cultivated Octopus maya  

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Proteinases from hepatopancreas (HP) and gastric juice (GJ) from wild and cultured red octopus (Octopus maya) were characterized. Hepatopancreas assays revealed optimal activity at pH 4, 9-10 and 10 for wild and pH 3, 8, and 9, for cultured octopuses, for total proteinases, trypsin and chymotrypsin, respectively. In the gastric juice, maximum activity was recorded at pH 6, 8, and 7 for total proteinases, trypsin, and chymotrypsin, respectively for both wild and cultured octopus. A reduction o...

Martinez, Romain; Santos, R.; Alvarez, A.; Cuzon, Gerard; Arena, L.; Mascaro, M.; Pascual, C.; Rosas, C.

2011-01-01

223

Homology models of main proteinase from coronavirus associated with SARS  

Science.gov (United States)

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.

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

2005-01-01

224

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin  

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Proteinase-activated receptor 1 (PAR1) induces activation of platelet and vascular cells after proteolytic cleavage of its extracellular N terminus by thrombin. In pathological situations, other proteinases may be generated in the circulation and might modify the responses of PAR1 by cleaving extracellular domains. In this study, epitope-tagged wild-type human PAR1 (hPAR1) and a panel of N-linked glycosylation-deficient mutant receptors were permanently expressed in epithelial cells (Kirsten murine sarcoma virus-transformed rat kidney cells and CHO cells). We have analyzed the role of N-linked glycosylation in regulating proteinase activation/disarming and cell global expression of hPAR1. We reported for the first time that glycosylation in the N terminus of hPAR1 downstream of the tethered ligand (especially Asn75) governs receptor disarming to trypsin, thermolysin, and the neutrophil proteinases elastase and proteinase 3 but not cathepsin G. In addition, hPAR1 is heavily N-linked glycosylated and sialylated in epithelial cell lines, and glycosylation occurs at all five consensus sites, namely, Asn35, Asn62, Asn75, Asn250, and Asn259. Removing these N-linked glycosylation sequons affected hPAR1 cell surface expression to varying degrees, and N-linked glycosylation at extracellular loop 2 (especially Asn250) of hPAR1 is essential for optimal receptor cell surface expression and receptor stability.

Xiao, Yu Pei; Morice, Alyn H.; Compton, Steven J.; Sadofsky, Laura

2011-01-01

225

Comparative cleavage sites within the reactive-site loop of native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.  

Science.gov (United States)

Human alpha1-proteinase inhibitor (alpha1-PI) is responsible for the tight control of neutrophil elastase activity which, if down regulated, may cause local excessive tissue degradation. Many bacterial proteinases can inactivate alpha1-PI by hydrolytic cleavage within its reactive site, resulting in the down regulation of elastase, and this mechanism is likely to contribute to the connective tissue damage often associated with bacterial infections. Another pathway of the inactivation of alpha1-PI is reversible and involves oxidation of a critical active-site methionine residue that may influence inhibitor susceptibility to proteolytic inactivation. Hence, the aim of this work was to determine whether this oxidation event might affectthe rate and pattern of the cleavage of the alpha1-PI reactive-site loop by selected bacterial proteinases, including thermolysin, aureolysin, serralysin, pseudolysin, Staphylococcus aureus serine proteinase, streptopain, and periodontain. A shift of cleavage specificity was observed after alpha1-PI oxidation, with a preference for the Glu354-Ala355 bond by most of the proteinases tested. Only aureolysin and serralysin cleave the oxidized form of alpha1-PI faster than the native inhibitor, suggesting that bacteria which secrete these metalloproteinases may specifically take advantage of the host defense oxidative mechanism to accelerate elimination of alpha1-PI and, consequently, tissue degradation by neutrophil elastase. PMID:10595584

Rapala-Kozik, M; Potempa, J; Nelson, D; Kozik, A; Travis, J

1999-10-01

226

Oxidant effects on rat and human lung proteinase inhibitors  

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This project tested the hypothesis that inhaled oxidants could cause lung damage by inactivating the proteinase inhibitors that normally protect the lung from proteolysis. Rat alpha-1-proteinase inhibitor (alpha 1-PI)2 was purified from blood plasma, and antibodies to this inhibitor were prepared. The activity of alpha 1-PI in lung lavage fluids from rats was measured by elastase inhibition, and the immunological concentration of alpha 1-PI was quantified in an enzyme-linked immunoassay. The ratio of the amount of active alpha 1-PI relative to its immunological concentration was examined as a measure of the inhibitor's functional activity. This ratio and the ratio of the immunological concentration of alpha 1-PI to the total protein concentration were determined in lung lavage fluids from rats exposed to air, 10 parts per million (ppm) nitrogen dioxide, and diesel emissions (3.5 mg/m3 particulates) for 12, 18, and 24 months. Only diesel exposures resulted in a statistically significant reduction in the functional activity of alpha 1-PI of 30 percent (p less than 0.05). Similar studies were performed on rats exposed to nitrogen dioxide and ozone for 12 and 18 months. No statistically significant effects were observed in the functional activity of alpha 1-PI or its immunological concentration. In other protocols, rats were acutely exposed to 0.8 ppm or 1.2 ppm ozone for two, four, or eight hours, and to 0.5 ppm or 0.8 ppm ozone in conjunction with 8 percent carbon dioxide for two or seven hours. Although these acute exposure conditions did not reduce the functional activity of alpha 1-PI, the immunological concentration of alpha 1-PI and the elastase inhibitory activity, relative to other proteins, were significantly increased in relation to the total amount of ozone inhaled.

Johnson, D.A.; Winters, R.S.; Lee, K.R.; Smith, C.E. (East Tennessee State Univ., Johnson City (USA))

1990-12-01

227

Isolation and analysis of cDNAs encoding tomato cysteine proteases expressed during leaf senescence.  

Science.gov (United States)

Several cDNAs for mRNAs that change in abundance during tomato leaf senescence were isolated. In this paper we report molecular cloning and expression analysis of two cysteine proteases. SENU2 is identical to the cDNA C14 which encodes a cysteine protease previously shown to be expressed in response to extremes of temperature in tomato fruit [43]. SENU3 cDNA clone was 1.2 kb in length and hybridized to a transcript of 1.4 kb which suggested that the clone was not full-length. The missing 5' end was isolated using rapid amplification of cDNA ends (RACE). Southern blot analysis of tomato genomic DNA indicates that SENU3 is encoded by a single or low copy gene. SENU3 was also shown to have significant homology with known cysteine proteases. These two senescence-associated cysteine proteases are also expressed during other developmental processes, including seed germination, consistent with a role in protein turnover. SENU2 and SENU3 mRNAs were detectable in young fully expanded leaves and increased in abundance with leaf age, reaching a maximum during the later stages of visible leaf senescence. Such a pattern of expression suggests that the onset of leaf senescence is a gradual event. Analysis of senescence in transgenic plants deficient in ethylene biosynthesis, in which leaf senescence is delayed, indicated that enhanced accumulation of SENU2 and SENU3 mRNA was similarly delayed but not prevented. PMID:8624407

Drake, R; John, I; Farrell, A; Cooper, W; Schuch, W; Grierson, D

1996-02-01

228

Expression and localisation of a senescence-associated KDEL-cysteine protease from Lilium longiflorum tepals.  

Science.gov (United States)

Senescence is a tightly regulated process and both compartmentalisation and regulated activation of degradative enzymes is critical to avoid premature cellular destruction. Proteolysis is a key process in senescent tissues, linked to disassembly of cellular contents and nutrient remobilisation. Cysteine proteases are responsible for most proteolytic activity in senescent petals, encoded by a gene family comprising both senescence-specific and senescence up-regulated genes. KDEL cysteine proteases are present in senescent petals of several species. Isoforms from endosperm tissue localise to ricinosomes: cytosol acidification following vacuole rupture results in ricinosome rupture and activation of the KDEL proteases from an inactive proform. Here data show that a Lilium longiflorum KDEL protease gene (LlCYP) is transcriptionally up-regulated, and a KDEL cysteine protease antibody reveals post-translational processing in senescent petals. Plants over-expressing LlCYP lacking the KDEL sequence show reduced growth and early senescence. Immunogold staining and confocal analyses indicate that in young tissues the protein is retained in the ER, while during floral senescence it is localised to the vacuole. Our data therefore suggest that the vacuole may be the site of action for at least this KDEL cysteine protease during tepal senescence. PMID:24268162

Battelli, Riccardo; Lombardi, Lara; Picciarelli, Piero; Lorenzi, Roberto; Frigerio, Lorenzo; Rogers, Hilary J

2014-01-01

229

Purification and characterization of cysteine protease from germinating cotyledons of horse gram  

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Abstract Background Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. To study the mechanism of protein mobilization, undertaken the task of purifying and characterizing proteases, which occur transiently in germinating seeds of horse gram. Results Cysteine protease (CPRHG) was purified to homogeneity with 118 fold by four step procedure comprising Crude...

Jinka Rajeswari; Ramakrishna Vadde; Rao Sridhar K; Rao Ramakrishna P

2009-01-01

230

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

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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 ...

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

231

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

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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...

Taylor, Brad N.; Staib, Peter; Binder, Ayfer; Biesemeier, Antje; Sehnal, Miriam; Ro?llinghoff, Martin; Morschha?user, Joachim; Schro?ppel, Klaus

2005-01-01

232

Pseudomonas aeruginosa elastase does not inactivate alpha 1-proteinase inhibitor in the presence of leukocyte elastase.  

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Pseudomonas aeruginosa elastase rapidly inactivates alpha 1-proteinase inhibitor by splitting its Pro-357-Met-358 peptide bond. The present study was aimed at testing whether this reaction takes place in the presence of leukocyte elastase. To this end was added alpha 1-proteinase inhibitor to a mixture of the two elastases, and we performed the following assays: (i) measurement of the residual leukocyte elastase activity, (ii) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (ii...

Padrines, M.; Bieth, J. G.

1989-01-01

233

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

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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...

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

1986-01-01

234

Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.  

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The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium do...

Loomes, L. M.; Senior, B. W.; Kerr, M. A.

1992-01-01

235

Expression of a microbial serine proteinase inhibitor gene enhances the tobacco defense against oomycete pathogens  

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In order to identify Nicotiana megalosiphon genes for novel inhibitors of microbial serine proteinase involved in resistance to the oomycete Phytophthora parasitica var. nicotianae, SuperSAGE technology combined with next-generation sequencing were used to generate libraries in order to identify transcripts that are differentially up-regulated. We identified a N. megalosiphon inhibitor of microbial serine proteinase gene (NmIMSP) rapidly induced during the interaction. Silencing of NmIMSP gen...

Silva, Yussuan; Portieles, Roxana; Pujol, Merardo; Terauchi, Ryohei; Matsumura, Hideo; Serrano, Mario; Borra?s-hidalgo, Orlando

2014-01-01

236

Purification of a novel serine proteinase inhibitor from the skeletal muscle of white croaker (Argyrosomus argentatus).  

Science.gov (United States)

A novel serine proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Argyrosomus argentatus). The purification was carried out by ammonium sulfate fractionation, DEAE-Sephacel, heating treatment followed by column chromatographies on SP-Sepharose, Sephadex G-150 and gel-filtration high performance liquid chromatography. The molecular mass of the inhibitor was 55 kDa as estimated by SDS-PAGE and gel filtration. It specifically inhibited a myofibril-bound serine proteinase (MBSP) isolated from the skeletal muscle of lizard fish (Saurida wanieso). No inhibition, however, was detected toward other serine proteinases such as bovine trypsin, bovine chymotrypsin and a myofibril-bound serine proteinase from carp (Cyprinus carpio) muscle. Interestingly, the sequences of tryptic digested peptide fragments of MBSPI revealed high identity to that of porcine phosphoglucose isomerase (PGI) (76%) and other PGIs. Furthermore, purified MBSPI exhibits PGI activity, suggesting the inhibitor is a protein closely related to PGI. When rabbit muscle PGI was investigated, it also specifically suppressed the activity of MBSP. It thus strongly suggests that MBSPI is actually PGI and conversely, PGI is a specific inhibitor toward myofibril-bound serine proteinase(s). PMID:10833440

Cao, M J; Osatomi, K; Matsuda, R; Ohkubo, M; Hara, K; Ishihara, T

2000-06-01

237

[Purification and immobilization of the proteinase from mung bean burgeon inactivating soybean trypsin inhibitor].  

Science.gov (United States)

By 30%-60% s(NH4)2SO4 fractional precipitation, anion-exchange chromatographs on DEAE-Sepharose CL-6B, gel filtration on Sephacryl S-200 and anion-exchange chromatographs on Waters AP-1 column(ProteinTm-Pak DEAE 15HR), a proteinase which can inactivated STI was purified from mung bean(Phaseolus aureus) burgeon. It was stable at temperatures lower than 50 degrees C and pH7.5-8.5, and the Km and Vmax of the proteinase for STI was 769.2 alpha-N-benzoyl-L-arginine ethyl ester(BAEE)/mL and 115.3BAEE/min/mL respectively. The molecular weight of the proteinase was estimated to be 29.8kD by SDS-PAGE. The proteinase immobilized by polyacrylamide was stable at temperatures lower than 60 degrees C and pH7.0-9.0, and the apparent Km* and Vmax* of the immobilized proteinase for STI was 1303.8 (BAEE)/mL and 94.34(BAEE)/min/mL respectively. The half-life of the immobilized proteinase was about 12 days at 4 degrees C. PMID:11411234

Chen, Z; Yang, X Q; Zhao, M M

2001-03-01

238

Modulation of Ion Transport Across Rat Distal Colon by Cysteine  

Science.gov (United States)

The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionine-?-synthase and cystathionine-?-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and ?-cyano-L-alanine, i.e., inhibitors of H2S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e., an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl? and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl? secretion, whereas Na cysteinate – after a transient inhibitory phase – activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K+ channels. Indeed, after preinhibition of basolateral K+ channels with tetrapentylammonium or Ba2+, the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H2S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H2S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H2S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.

Pouokam, Ervice; Diener, Martin

2012-01-01

239

Unfolding the fold of cyclic cysteine-rich peptides  

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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...

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

2008-01-01

240

Modulation of ion transport across rat distal colon by cysteine  

Directory of Open Access Journals (Sweden)

Full Text Available The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionin-beta-synthase and cystathionin-gamma-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and beta-cyano-L-alanine, i.e. inhibitors of H2S-producing enzymes. In contrast, Na cysteinate evoked a biphasic change in Isc, i.e. an initial fall followed by a secondary increase, which was also reduced by these enzyme inhibitors. All responses were dependent on the presence of Cl- and inhibited by bumetanide, suggesting that free cysteine induces an inhibition of transcellular Cl- secretion, whereas Na cysteinate – after a transient inhibitory phase – activates anion secretion. The assumed reason for this discrepancy is a fall in the cytosolic pH induced by free cysteine, but not by Na cysteinate, as observed in isolated colonic crypts loaded with the pH-sensitive dye, BCECF. Intracellular acidification is known to inhibit epithelial K+ channels. Indeed, after preinhibition of basolateral K+ channels with tetrapentylammonium or Ba2+, the negative Isc induced by free cysteine was reduced significantly. In consequence, stimulation of endogenous H2S production by Na cysteinate causes, after a short inhibitory response, a delayed activation of anion secretion, which is missing in the case of free cysteine, probably due to the cytosolic acidification. In contrast, diallyl trisulfide, which is intracellularly converted to H2S, only evoked a monophasic increase in Isc without the initial fall observed with Na cysteinate. Consequently, time course and amount of produced H2S seem to strongly influence the functional response of the colonic epithelium evoked by this gasotransmitter.

MartinDiener

2012-03-01

 
 
 
 
241

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles  

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Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins’ intrinsi...

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

2012-01-01

242

Significance of Cuscutain, a cysteine protease from Cuscuta reflexa, in host-parasite interactions  

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Abstract Background Plant infestation with parasitic weeds like Cuscuta reflexa induces morphological as well as biochemical changes in the host and the parasite. These modifications could be caused by a change in protein or gene activity. Using a comparative macroarray approach Cuscuta genes specifically upregulated at the host attachment site were identified. Results One of the infestation specific Cuscuta genes encodes a cysteine pr...

Bleischwitz Marc; Albert Markus; Fuchsbauer Hans-Lothar; Kaldenhoff Ralf

2010-01-01

243

Peptide-formation on cysteine-containing peptide scaffolds  

Science.gov (United States)

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

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

1999-01-01

244

The local electrostatic environment determines cysteine reactivity of tubulin.  

Science.gov (United States)

Of the 20 cysteines of rat brain tubulin, some react rapidly with sulfhydryl reagents, and some react slowly. The fast reacting cysteines cannot be distinguished with [14C]iodoacetamide, N-[(14)C]ethylmaleimide, or IAEDANS ([5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid]), since modification to mole ratios 1 cysteine/dimer always leads to labeling of 6-7 cysteine residues. These have been identified as Cys-305alpha, Cys-315alpha, Cys-316alpha, Cys-347alpha, Cys-376alpha, Cys-241beta, and Cys-356beta by mass spectroscopy and sequencing. This lack of specificity can be ascribed to reagents that are too reactive; only with the relatively inactive chloroacetamide could we identify Cys-347alpha as the most reactive cysteine of tubulin. Using the 3.5-A electron diffraction structure, it could be shown that the reactive cysteines were within 6.5 A of positively charged arginines and lysines or the positive edges of aromatic rings, presumably promoting dissociation of the thiol to the thiolate anion. By the same reasoning the inactivity of a number of less reactive cysteines could be ascribed to inhibition of modification by negatively charged local environments, even with some surface-exposed cysteines. We conclude that the local electrostatic environment of cysteine is an important, although not necessarily the only, determinant of its reactivity. PMID:12023292

Britto, P J; Knipling, Leslie; Wolff, J

2002-08-01

245

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

1994-11-01

246

Reactive cysteine is protonated in the triplet excited state of the LOV2 domain in Adiantum phytochrome3.  

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Phototropin is a plant blue-light sensor protein that possesses a flavin mononucleotide (FMN) as the chromophore in LOV domains. Its photoreaction is an adduct formation between FMN and a nearby cysteine that takes place in the triplet excited state of FMN. In this communication, we revealed that the reactive cysteine is protonated in the triplet excited state of the LOV2 domain of Adiantum phytochrome3 by means of low-temperature FTIR spectroscopy. Its hydrogen-bonding interaction is strengthened in the triplet excited state, presumably with the FMN chromophore. Such strong interaction drives adduct formation on a microsecond time scale. PMID:15669833

Sato, Yoshiaki; Iwata, Tatsuya; Tokutomi, Satoru; Kandori, Hideki

2005-02-01

247

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 c

1986-03-05

248

Subunit structure of the rat alpha-macroglobulin proteinase inhibitors.  

Science.gov (United States)

Rats produce 2 alpha-macroglobulin (alpha M) proteinase inhibitors, the alpha 1 M, normally found in the plasma, and the alpha 2 M, an acute phase protein. The alpha-macroglobulins were purified from the plasma of rats with adjuvant arthritis by polyethylene glycol precipitation, chromatography on a Zn2+ affinity column, and filtration on Sephacryl S-300 superfine. Comparison of the purified proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis following reduction reveals a 185 000 Da subunit for rat alpha 2 M identical to the human alpha 2 M, but a 167 000 plus a 38 000 Da subunit for rat alpha 1 M. Heat/alkali treatment (pH 11, 37 degrees C for 45 min) prior to reduction results in the appearance of 125 000 Da and 60 000 Da components from rat alpha 2 M analogous to the pattern of human alpha 2 M. In contrast, alpha 1 M showed in addition to the 125 000 Da band (and the unaltered 38 000 Da band), two bands of approx. 25 000 Da. Incubation with trypsin (approximately 1 mol/mol alpha M) prior to reduction causes formation of approximately 90 000 Da components from both rat inhibitors and the human alpha 2 M. The data suggests that only rat alpha 2 M and not rat alpha 1 M is structurally homologous to human alpha 2 M. PMID:6182084

Nelles, L P; Schnebli, H P

1982-07-01

249

Describing some characters of serine proteinase using fractal analysis  

International Nuclear Information System (INIS)

In this paper we calculated the fractal dimensions of four proteins, chymotrypsin, elastase, trypsin and subtilisin, which are made up of about 220–275 amino acids and belong to the family of serine proteinase by using three definitions of fractal dimension i.e. the chain fractal dimension (DL), the mass fractal dimension (Dm) and the correlation fractal dimension (Dc). We also analyzed the relationship between fractal dimension and space structure or secondary structure contents of proteins. The results showed that the values of fractal dimensions are almost same for the global mammalian enzymes (chymotrypsin, elastase and trypsin), but different for the global subtilisin. This demonstrated that the more similar structures, the more equal fractal dimensions, and if the fractal dimensions of proteins are different from each other, the three dimensional structures should not be similar. On the other hand, the detailed structures and fractal dimensions of the active sites of four enzymes are extraordinarily similar. Therefore, the fractal method can be applied to the elucidation of the proteins evolution.

2012-07-01

250

Procerain, a stable cysteine protease from the latex of Calotropis procera.  

Science.gov (United States)

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue. PMID:12591258

Dubey, Vikash Kumar; Jagannadham, M V

2003-04-01

251

cDNA cloning and molecular modeling of procerain B, a novel cysteine endopeptidase isolated from Calotropis procera.  

Science.gov (United States)

Procerain B, a novel cysteine protease (endopeptidase) isolated from Calotropis procera belongs to Asclepiadaceae family. Purification of the enzyme, biochemical characterization and potential applications are already published by our group. Here, we report cDNA cloning, complete amino acid sequencing and molecular modeling of procerain B. The derived amino acid sequence showed high sequence homology with other papain like plant cysteine proteases of peptidase C1A superfamily. The three dimensional structure of active procerain B was modeled by homology modeling using X-ray crystal structure of actinidin (PDB ID: 3P5U), a cysteine protease from the fruits of Actinidia arguta. The structural aspect of the enzyme is also discussed. PMID:23527269

Singh, Abhay Narayan; Yadav, Prity; Dubey, Vikash Kumar

2013-01-01

252

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases  

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Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the...

Carrillo Gil, Laura; Martinez Mun?oz, Manuel; Ramessar, Koreen; Cambra Marin, Ines; Castan?era, Pedro; Ortego, Felix; Diaz Rodriguez, Isabel

2011-01-01

253

Cysteine protease inhibitor from pearl millet: a new class of antifungal protein.  

Science.gov (United States)

A cysteine protease inhibitor exhibiting antifungal activity from pearl millet seeds has been purified to homogeneity by ammonium sulphate precipitation and chromatographic procedures involving CM- sephadex and SP-sepharose cation exchange columns. The molecular characterization has revealed its molecular mass as 24 kD and isoelectric point 9.8. The amino acid composition data shows presence of high content of serine and glycine (34 residues/mole) and absence of tryptophan. The inhibitor exhibits potent antifungal activity against Trichoderma reesei, a dead wood fungus with minimum inhibitory dose to inhibit mycelial growth or spore germination is as low as 1 microgram/ml (250 ng/disc). In addition to Trichoderma reesei, the antifungal activity is observed against some important phytopathogenic fungi, namely, Claviceps, Helminthosporium, Curvularia, Alternaria and Fusarium species. To the best of our knowledge, a cysteine protease inhibitor as an antifungal protein is reported for the first time from a plant system. PMID:9610368

Joshi, B N; Sainani, M N; Bastawade, K B; Gupta, V S; Ranjekar, P K

1998-05-19

254

A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE  

Scientific Electronic Library Online (English)

Full Text Available SciELO Chile | Language: English Abstract in english 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.

ENDER, BÇER; PAKZE, QETNKAYA.

255

A VOLTAMMETRIC STUDY ON THE INTERACTION OF NOVOBIOCIN WITH CYSTEINE  

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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 decreased 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.

ENDER BÇER

2009-01-01

256

Characterization and functional analysis of serine proteinase and serine proteinase homologue from the swimming crab Portunus trituberculatus.  

Science.gov (United States)

Serine proteases (SPs), with their homologues (SPHs), a family of multifunctional proteins, play a crucial role in innate immune system. In our present study, we made an appropriate correction: serine protease homologue PtcSPH (Li et al., [1]) obtained from the swimming crab Portunus trituberculatus was actually a serine protease and re-designated as PtcSP. Sequence analysis revealed PtcSP and PtSP (Li et al., [2]) might be encoded by the same genomic locus and generated by alternative splicing of the pre-mRNA. Eight exons were identified in genomic DNA sequence of PtcSP. A comprehensive phylogenetic analysis was made combined with our previous reports (Cui et al., [3]; Li et al., [1,2]). The result showed SPs and SPHs of P. trituberculatus had different origins in gene evolution. To further characterize the function(s) of proteins, the recombinant serine proteases or homologues were assayed for various biological functions: proteinase activity, antimicrobial activity and microorganisms binding activity. The recombinant protein PtcSP exhibited trypsin-like protease activity and antibacterial activity. PtSPH1 (Li et al., [2]) lacked proteolytic activity but displayed binding activity to yeast and the crab pathogenic bacterium, Vibrio alginolyticus. Further, the N-terminal clip domain of PtcSP had antibacterial activity and the C-terminal SP-like domain had trypsin-like protease activity. PMID:23664866

Song, Chengwen; Cui, Zhaoxia; Liu, Yuan; Li, Qianqian; Li, Xihong; Shi, Guohui; Wang, Chunlin

2013-08-01

257

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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.

Leah Theresa, Sigle; Marcelo, Ramalho-Ortigao.

258

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

Science.gov (United States)

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. PMID:24037187

Sigle, Leah Theresa; Ramalho-Ortigão, Marcelo

2013-09-01

259

Determination of germ tube, phospholipase, and proteinase production by bloodstream isolates of Candida albicans  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english Introduction Candida albicans is a commensal and opportunistic agent that causes infection in immunocompromised individuals. Several attributes contribute to the virulence and pathogenicity of this yeast, including the production of germ tubes (GTs) and extracellular hydrolytic enzymes, particularl [...] y phospholipase and proteinase. This study aimed to investigate GT production and phospholipase and proteinase activities in bloodstream isolates of C. albicans. Methods One hundred fifty-three C. albicans isolates were obtained from blood samples and analyzed for GT, phospholipase, and proteinase production. The assays were performed in duplicate in egg yolk medium containing bovine serum albumin and human serum. Results Detectable amounts of proteinase were produced by 97% of the isolates, and 78% of the isolates produced phospholipase. GTs were produced by 95% of the isolates. A majority of the isolates exhibited low levels of phospholipase production and high levels of proteinase production. Conclusions Bloodstream isolates of C. albicans produce virulence factors such as GT and hydrolytic enzymes that enable them to cause infection under favorable conditions.

Mattei, Antonella Souza; Alves, Sydney Hartz; Severo, Cecilia Bittencourt; Guazzelli, Luciana da Silva; Oliveira, Flavio de Mattos; Severo, Luiz Carlos.

260

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

Science.gov (United States)

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

 
 
 
 
261

Effect of Drug Transporter Genes on Cysteine Export and Overproduction in Escherichia coli  

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l-Cysteine is an important amino acid in terms of its industrial applications. We previously found a marked production of l-cysteine from glucose in recombinant Escherichia coli cells expressing an altered cysE gene encoding feedback inhibition-insensitive serine acetyltransferase. Also, a lower level of cysteine desulfhydrase (CD) activity, which is involved in l-cysteine degradation, increased l-cysteine productivity in E. coli. The use of an l-cysteine efflux system could be promising for ...

Yamada, Satoshi; Awano, Naoki; Inubushi, Kyoko; Maeda, Eri; Nakamori, Shigeru; Nishino, Kunihiko; Yamaguchi, Akihito; Takagi, Hiroshi

2006-01-01

262

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.

2010-11-15

263

Human placental extract mediated inhibition of proteinase K: implications of heparin and glycoproteins in wound physiology.  

Science.gov (United States)

Efficient debridement of the wound bed following the removal of microbial load prevents its progression into a chronic wound. Bacterial infection and excessive proteolysis characterize impaired healing and therefore, their inhibition might restore the disturbed equilibrium in the healing process. Human placental extract exhibits reversible, non-competitive inhibition towards Proteinase K, a microbial protease, by stabilizing it against auto-digestion. Scattering and fluorescence studies followed by biochemical analysis indicated the involvement of a glycan moiety. Surface plasmon resonance demonstrated specific interaction of heparin with Proteinase K having Kd in ?M range. Further, Proteinase K contains sequence motifs similar to other heparin-binding proteins. Molecular docking revealed presence of clefts suitable for binding of heparin-derived oligosaccharides. Comprehensive analysis of this inhibitory property of placental extract partly explains its efficacy in curing wounds with common bacterial infections. PMID:24435659

Sharma, Kanika; Mukherjee, Chaitali; Roy, Siddhartha; De, Debashree; Bhattacharyya, Debasish

2014-09-01

264

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

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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.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 transformantes 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.

Maria Helena S. Goldman

1998-09-01

265

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

DEFF Research Database (Denmark)

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. Neither form of the free inhibitor has detectable intrinsic secondary structure in solution. However, upon contact with the enzyme, residues 2-32 become ordered and adopt a near-perfect alpha-helical conformation. Thus, the proteinase acts as a folding template, stabilizing the helical conformation in the inhibitor, which results in the potent and specific blockage of the proteolytic activity.

Li, M; Phylip, L H

2000-01-01

266

Cysteine and cystine transport at the blood-brain barrier  

Energy Technology Data Exchange (ETDEWEB)

The nature of cysteine and cystine uptake from the cerebral capillary lumen was studied in the rat using the carotid injection technique, (/sup 35/S)-Cysteine uptake was readily inhibited by the synthetic amino acid 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH), the defining substrate for the leucine-preferring (L) system in the Ehrlich ascites cell. The addition of nonradioactive alanine or serine, representatives of the alanine, serine, and cysteine-preferring (ASC) system, produced no significant decrease in the uptake of cysteine after cysteine transport by the L system was blocked with BCH. This indicated that the major component of cysteine's transport from the brain capillary lumen was by the L system with no detectable uptake of cysteine by the ASC system. No carrier-mediated transport of cystine, the disulfide form of the amino acid, was detected, nor was there any inhibition by cystine of the transport of the neutral amino acid methionine or the basic amino acid arginine. These results suggest that the ASC system, if present, is not quantitatively important for the transport of neutral amino acids from the brain capillary lumen.

Wade, L.A.; Brady, H.M.

1981-09-01

267

Metabolism of L-cysteine in guinea pig liver.  

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Full Text Available The metabolism of L-cysteine in guinea pig liver was studied. Guinea pig liver contained 0.45 +/- 0.05 (mean +/- SD mumol of cysteine, 0.180 +/- 0.080 mumol of 3-mercaptolactate-cysteine disulfide [S-(2-hydroxy-2-carboxyethylthiocysteine, HCETC], and 8.082 +/- 0.516 mumol of reduced glutathione per g of fresh tissue. The taurine content was 0.912 +/- 0.158 mumol per g of fresh liver. Cysteine dioxygenase (EC 1.13.11.20 activity was several-fold lower than cysteine aminotransferase (EC 2.6.1.3 activity. Lactate dehydrogenase (EC 1.1.1.27 activity was about 10-fold higher than 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2 activity. These results indicate that the oxidative metabolism of L-cysteine in the guinea pig liver is not as active as in the rat liver and that L-cysteine, at least in part, is metabolized via the transaminative pathway, in which 3-mercaptopyruvate is partly reduced to 3-mercaptolactate and is utilized to form HCETC.

Hosaki,Yasuhiro

1986-02-01

268

Purification and characterization of cysteine aminotransferase from rat liver cytosol.  

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Full Text Available Cysteine aminotransferase (L-cysteine: 2-oxoglutarate aminotransferase, EC 2.6.1.3 was purified over 400-fold from the high-speed supernatant fraction of rat liver. The purified enzyme was homogeneous as judged by gel filtration, isoelectric focusing and disc electrophoresis. The molecular weight of the enzyme was about 74,000 by gel filtration and the isoelectric point was 6.2 (4 degrees C. The enzyme catalyzed transamination between L-cysteine and 2-oxoglutarate and the reverse reaction. The optimum pH was 9.7. The Km value for L-cysteine was 22.2 mM, and that for 2-oxoglutaric acid was 0.06 mM. L-Aspartate was a potent inhibitor of the cysteine aminotransferase reaction. The enzyme was very active toward L-alanine 3-sulfinic acid at pH 8.0, and was also very active toward L-aspartic acid (Km = 1.6 mM. Ratios of activities for L-aspartic acid and L-cysteine were essentially constant during the purification of the enzyme. Evidence based on substrate specificity, enzyme inhibition, and physicochemical properties indicates that cytosolic cysteine aminotransferase is identical with cytosolic aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1.

Akagi,Reiko

1982-06-01

269

Expression of a pathogen-induced cysteine protease (AdCP) in tapetum results in male sterility in transgenic tobacco.  

Science.gov (United States)

Usable male sterility systems have immense potential in developing hybrid varieties in crop plants, which can also be used as a biological safety containment to prevent horizontal transgene flow. Barnase-Barstar system developed earlier was the first approach to engineer male sterility in plants. In an analogous situation, we have evolved a system of inducing pollen abortion and male sterility in transgenic tobacco by expressing a plant gene coding for a protein with known developmental function in contrast to the Barnase-Barstar system, which deploys genes of prokaryotic origin, i.e., from Bacillus amyloliquefaciens. We have used a plant pathogen-induced gene, cysteine protease for inducing male sterility. This gene was identified in the wild peanut, Arachis diogoi differentially expressed when it was challenged with the late leaf spot pathogen, Phaeoisariopsis personata. Arachis diogoi cysteine protease (AdCP) was expressed under the strong tapetum-specific promoter (TA29) and tobacco transformants were generated. Morphological and histological analysis of AdCP transgenic plants showed ablated tapetum and complete pollen abortion in three transgenic lines. Furthermore, transcript analysis displayed the expression of cysteine protease in these male sterile lines and the expression of the protein was identified in western blot analysis using its polyclonal antibody raised in the rabbit system. PMID:24615687

Shukla, Pawan; Singh, Naveen Kumar; Kumar, Dilip; Vijayan, Sambasivam; Ahmed, Israr; Kirti, Pulugurtha Bharadwaja

2014-06-01

270

Effect of (L)-cysteine on acetaldehyde self-administration.  

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Acetaldehyde (ACD), the first metabolite of ethanol, has been implicated in several behavioural actions of alcohol, including its reinforcing effects. Recently, we reported that l-cysteine, a sequestrating agent of ACD, reduced oral ethanol self-administration and that ACD was orally self-administered. This study examined the effects of l-cysteine pre-treatment during the acquisition and maintenance phases of ACD (0.2%) self-administration as well as on the deprivation effect after ACD extinction and on a progressive ratio (PR) schedule of reinforcement. In a separate PR schedule of reinforcement, the effect of l-cysteine was assessed on the break-point produced by ethanol (10%). Furthermore, we tested the effect of l-cysteine on saccharin (0.2%) reinforcement. Wistar rats were trained to self-administer ACD by nose poking on a fixed ratio (FR1) schedule in 30-min daily sessions. Responses on an active nose-poke caused delivery of ACD solution, whereas responses on an inactive nose-poke had no consequences. l-cysteine reduced the acquisition (40 mg/kg), the maintenance and the deprivation effect (100 mg/kg) of ACD self-administration. Furthermore, at the same dose, l-cysteine (120 mg/kg) decreased both ACD and ethanol break point. In addition, l-cysteine was unable to suppress the different responses for saccharin, suggesting that its effect did not relate to an unspecific decrease in a general motivational state. Compared to saline, l-cysteine did not modify responses on inactive nose-pokes, suggesting an absence of a non-specific behavioural activation. Taken together, these results could support the hypotheses that ACD possesses reinforcing properties and l-cysteine reduces motivation to self-administer ACD. PMID:22440691

Peana, Alessandra T; Muggironi, Giulia; Fois, Giulia R; Zinellu, Manuel; Sirca, Donatella; Diana, Marco

2012-08-01

271

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

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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/.

Blom Nikolaj

2004-06-01

272

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

DEFF Research Database (Denmark)

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.

Kiemer, Lars; Lund, Ole

2004-01-01

273

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.  

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Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF. PMID:1686554

Sponer, M; Nick, H P; Schnebli, H P

1991-11-01

274

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

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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

Qasim, Mohammad A

2014-01-01

275

Modulation of Ion Transport Across Rat Distal Colon by Cysteine  

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The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionine-?-synthase and cystathionine-?-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and ?-cyano-L-alanine, i.e., inhibito...

Pouokam, Ervice; Diener, Martin

2012-01-01

276

Modulation of ion transport across rat distal colon by cysteine  

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The aim of this study was to identify the actions of stimulation of endogenous production of H2S by cysteine, the substrate for the two H2S-producing enzymes, cystathionine-ß-synthase and cystathionine-ß-lyase, on ion transport across rat distal colon. Changes in short-circuit current (Isc) induced by cysteine were measured in Ussing chambers. Free cysteine caused a concentration-dependent, transient fall in Isc, which was sensitive to amino-oxyacetate and ß-cyano-L-alanine, i.e., inhibito...

Pouokam, Ervice; Diener, Martin

2012-01-01

277

Purification and characterization of cysteine aminotransferase from rat liver cytosol.  

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Cysteine aminotransferase (L-cysteine: 2-oxoglutarate aminotransferase, EC 2.6.1.3) was purified over 400-fold from the high-speed supernatant fraction of rat liver. The purified enzyme was homogeneous as judged by gel filtration, isoelectric focusing and disc electrophoresis. The molecular weight of the enzyme was about 74,000 by gel filtration and the isoelectric point was 6.2 (4 degrees C). The enzyme catalyzed transamination between L-cysteine and 2-oxoglutarate and the reverse reactio...

Akagi, Reiko

1982-01-01

278

Cysteine mutants as chemical sensors for ligand-receptor interactions.  

Science.gov (United States)

The incorporation of cysteine residues into membrane receptors by mutagenesis has enabled the development of engineered proteins. Chemical modification of the mutant receptor using a wide range of biochemical and biophysical probes has facilitated functional studies of ligand-receptor interactions. In particular, the substituted-cysteine accessibility method (SCAM) represents a successful example of how to probe transmembrane receptor domains after chemical modification of the mutants with sulfydryl-reacting molecules. We propose an extension of this methodology using site-specific affinity probes that react with cysteine mutants to gain reliable structural information on the binding of a ligand in its receptor site. PMID:11282416

Foucaud, B; Perret, P; Grutter, T; Goeldner, M

2001-04-01

279

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

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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...

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

1999-01-01

280

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell.  

Science.gov (United States)

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures. PMID:2212656

Schechter, N M; Irani, A M; Sprows, J L; Abernethy, J; Wintroub, B; Schwartz, L B

1990-10-15

 
 
 
 
281

Method Comparison of Two Test Kits for the Determination of Elastase/?1-Proteinase-Inhibitor Complex  

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A method comparsion of the 5h-elastase/?1-proteinase-inhibitor test kit with a newly developed 2 h-version was performed. The within-run and between-days coefficients of variation as well as the recovery of the assigned values were satisfactory for both test kits. Good agreement was found between the 2 h- and 4 h-test kits using specimens from 1600 patients. In conclusion, the 2 h-test kis can be used as an alternative for the determination of elastase/?1-proteinase-inhibitor complex.

Suin Boutemard, C.; Fink, P. C.; Haeckel, R.

1988-01-01

282

On the modeling of snake venom serine proteinase interactions with benzamidine-based thrombin inhibitors  

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Pit viper venoms contain a number of serine proteinases that exhibit one or more thrombin-like activities on fibrinogen and platelets, this being the case for the kinin-releasing and fibrinogen-clotting KN-BJ from the venom of Bothrops jararaca. A three-dimensional structural model of the KN-BJ2 serine proteinase was built by homology modeling using the snake venom plasminogen activator TSV-PA as a major template and porcine kallikrein as additional structural support. A set of intrinsic buri...

Henriques, Elsa S.; Fonseca, Nelson; Ramos, Maria Joa?o

2004-01-01

283

[Properties of Bacillus pumilus subtilisin like proteinase secreted from recombinant strain on different growth stages].  

Science.gov (United States)

Bacillus pumilus 3-19 glutamylendopeptidase has been isolated from culture liquid of Bacillus subtilis recombinant strain on different growth stages: growth retardation (early enzyme) and stationary phase (late enzyme). The effect of purified proteinase of different growth stages on insulin beta-chain, protein and oligopeptide substrates has been studied. Comparative study of physicochemical properties of early and late proteinases was carried out. Two protein fractions were different in catalytic characteristics and demonstrated different sensitivity to the presence of metal cations. PMID:23844506

Balaban, N P; Danilova, Iu V; Shamsutdinov, T R; Mardanova, A M; Cheremin, A M; Rudenskaia, G N; Sharipova, M R

2013-01-01

284

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

Science.gov (United States)

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. PMID:19179756

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

2008-12-01

285

Hydration effects in L-Cysteine I  

Science.gov (United States)

Two special dynamical transitions of universal character have recently been observed in macromolecules at T* ˜ 100 K and TD ˜ 220 K. The underlying mechanisms governing these transitions have been the subject of debate. In the present work, a survey is reported on the temperature and hydration dependence of structural, vibrational and thermodynamical properties of amino acid (orthorhombic polymorph of the amino acid L-cysteine at different hydration levels). The temperature dependence of x-ray powder diffraction patterns, Raman spectra and specific heat revealed these two transitions at T* ˜ 70 K and TD ˜ 230 K for this sample. The data were analyzed considering amino acid--amino acid, amino acid--water, water--water phonon--phonon interactions and molecular rotor activation. DFT calculations were performed on L-Cys considering interactions with adsorbed water molecules. Our results indicated that the two referred temperatures define the triggering of very simple and particular events that govern all the interactions of the biomolecular: as well specific relaxation mechanisms.

Lima, Thamires; Ishikawa, Mariana; Martinho, Herculano

2013-03-01

286

Expression of human ?1-proteinase inhibitor in Aspergillus niger  

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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.

Punt Peter J

2007-10-01

287

Reversible targeting of noncatalytic cysteines with chemically tuned electrophiles  

DEFF Research Database (Denmark)

Targeting noncatalytic cysteine residues with irreversible acrylamide-based inhibitors is a powerful approach for enhancing pharmacological potency and selectivity. Nevertheless, concerns about off-target modification motivate the development of reversible cysteine-targeting strategies. Here we show that electron-deficient olefins, including acrylamides, can be tuned to react with cysteine thiols in a rapidly reversible manner. Installation of a nitrile group increased the olefins' intrinsic reactivity, but, paradoxically, eliminated the formation of irreversible adducts. Incorporation of these electrophiles into a noncovalent kinase-recognition scaffold produced slowly dissociating, covalent inhibitors of the p90 ribosomal protein S6 kinase RSK2. A cocrystal structure revealed specific noncovalent interactions that stabilize the complex by positioning the electrophilic carbon near the targeted cysteine. Disruption of these interactions by protein unfolding or proteolysis promoted instantaneous cleavage of the covalent bond. Our results establish a chemistry-based framework for engineering sustained covalent inhibition without accumulating permanently modified proteins and peptides.

Serafimova, Iana M; Pufall, Miles A

2012-01-01

288

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  

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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.

Larissa Ramos Chevreuil

2011-03-01

289

Modification of Keap1 Cysteine Residues by Sulforaphane  

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Activation of the transcription factor NF-E2-related factor-2 (Nrf2) through modification of Kelch-like ECH-associated protein 1 (Keap1) cysteines, leading to up-regulation of the antioxidant response element (ARE), is an important mechanism of cellular defense against reactive oxygen species and xenobiotic electrophiles. Sulforaphane, occurring in cruciferous vegetables such as broccoli, is a potent natural ARE activator that functions by modifying Keap1 cysteine residues, but there are conf...

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

2011-01-01

290

Cathodic Behaviour of Cysteine at a Platinum Electrode  

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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, differen...

2007-01-01

291

Effects of cysteine on the pharmacokinetics of paclitaxel in rats.  

Science.gov (United States)

Paclitaxel is a P-gp substrate and metabolized via CYP2C and 3A subfamily in rats. It has been reported that cysteine causes the changes in expression of CYP isozymes and intestinal P-gp mediated efflux activity in rats. Thus, the effects of cysteine on the pharmacokinetics of intravenous and oral paclitaxel were investigated in rats. After intravenous administration of paclitaxel (30 mg/kg) to control (CON), single cysteine treatment (ST) and cysteine treatment for a week (CT) rats, the pharmacokinetic parameters were comparable among three groups of rats. Also the pharmacokinetic parameters between CON and ST rats were comparable after oral administration of paclitaxel (30 mg/kg) to rats. These results are consistent with that oral cysteine supplement on a single day did not considerably inhibit the metabolism of paclitaxel via hepatic and/or intestinal CYP3A subfamily and P-gp mediated efflux of paclitaxel in the liver and/or intestine both after intravenous and oral administration to rats. After oral administration of paclitaxel (30 mg/kg) to rats, the greater AUC(06 h) in CT rats was mainly due to that oral cysteine supplement for seven consecutive days enhanced the gastrointestinal absorption of paclitaxel compared with those in CON and ST rats. PMID:22477198

Lee, Yu Kyung; Han, Seung Yon; Chin, Young-Won; Choi, Young Hee

2012-03-01

292

Activation of inactive renin during the selective destruction of proteinase inhibitors in human plasma by a metalloproteinase in Bitis arietans venom.  

Science.gov (United States)

Puff adder venom, which has been pretreated with phenylmethylsulphonyl fluoride and extensively dialysed, is capable of destroying selectively proteinase inhibitory activity in human plasma by an action of an EDTA-sensitive venom proteinase. We found that incubation of 1/5 vol. of such venom with human plasma at 25 degrees C leads to a concomitant increase in renin to 4.4 times control by 5 h. The activation of inactive renin was abolished by 10 mM EDTA and the rate of activation was reduced by 50% in the presence of 5 mM phenylmethylsulphonyl fluoride and by 90% when 0.32 mg/ml soybean trypsin inhibitor and 5 mM N-ethylmaleimide were added as well. The venom proteinase thus appears to activate inactive renin via an activation of endogenous plasma proteinases. This may be accomplished either by activation of proteinase precursors or action on proteinase inhibitor-proteinase complexes. By destroying proteinase inhibitors at the same time as it activates endogenous proteinases, Bitis arietans metalloproteinase would appear to be particularly useful for studies of the activation of inactive renin in human plasma, since endogenous proteinases are then free to activate inactive renin without subsequent inhibition by endogenous proteinase inhibitors. PMID:6988010

Morris, B J; Lawrence, C H

1980-03-14

293

Revised sequence of the Porphyromonas gingivalis prtT cysteine protease/hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase.  

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The prtT gene from Porphyromonas gingivalis ATCC 53977 was previously isolated from an Escherichia coli clone possessing trypsinlike protease activity upstream of a region encoding hemagglutinin activity (J. Otogoto and H. Kuramitsu, Infect. Immun. 61;117-123, 1993). Subsequent molecular analysis of this gene has revealed that the PrtT protein is larger than originally reported, encompassing the hemagglutination region. Results of primer extension experiments indicate that the translation sta...

Madden, T. E.; Clark, V. L.; Kuramitsu, H. K.

1995-01-01

294

Isolation and possible relevance of Thermoactinomyces candidus proteinases in farmer's lung disease.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The thermophilic actinomycetes are the most common etiological agents causing hypersensitivity pneumonitis. Antigen preparations of these organisms contain proteolytic activity. Further investigation of the proteinases of the thermophilic actinomycetes was undertaken to determine whether this activity may contribute directly to the pathogenesis of hypersensitivity pneumonitis and pulmonary mycotoxicosis. The presence of proteolytic activity in aerosolized dust from moldy silage was demonstrat...

Roberts, R. C.; Nelles, L. P.; Treuhaft, M. W.; Marx, J. J.

1983-01-01

295

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

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english 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.

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

296

Concurrent occurrence of insect proteinases and their inhibitors in insect midgut.  

Czech Academy of Sciences Publication Activity Database

. Izmir : Entomological Society of Turkey, 2006. s. 134-134.[European Congress of Entomology /8./. 17.09.2006-22.09.2006, Izmir]Grant CEP: GA ?R(CZ) GA522/06/1591Výzkumný zám?r: CEZ:AV0Z50070508Klí?ová slova: serin proteinasesKód oboru RIV: ED - Fyziologie

Taranushenko, J.; Sehnal, František

297

Aorsin, a novel serine proteinase with trypsin-like specificity at acidic pH.  

Science.gov (United States)

A proteinase that hydrolyses clupeine and salmine at acidic pH, called aorsin, was found in the fungus Aspergillus oryzae. Purified aorsin also hydrolysed benzyloxycarbonyl-Arg-Arg-4-methylcoumaryl-7-amide optimally at pH 4.0. The specificity of aorsin appeared to require a basic residue at the P(1) position and to prefer paired basic residues. Aorsin activated plasminogen and converted trypsinogen to trypsin. The trypsin-like activity was inhibited strongly by antipain or leupeptin, but was not inhibited by any other standard inhibitors of peptidases. To identify the catalytic residues of aorsin, a gene was cloned and an expression system was established. The predicted mature protein of aorsin was 35% identical with the classical late-infantile neuronal ceroid lipofuscinosis protein CLN2p and was 24% identical with Pseudomonas serine-carboxyl proteinase, both of which are pepstatin-insensitive carboxyl proteinases. Several putative catalytic residues were mutated. The k (cat)/ K(m) values of the mutant enzymes Glu(86)-->Gln, Asp(211)-->Asn and Ser(354)-->Thr were 3-4 orders of magnitude lower and Asp(90)-->Asn was 21-fold lower than that of wild-type aorsin, indicating that the positions are important for catalysis. Aorsin is another of the S53 family serine-carboxyl proteinases that are not inhibited by pepstatin. PMID:12519073

Lee, Byung Rho; Furukawa, Masato; Yamashita, Koichiro; Kanasugi, Yurie; Kawabata, Choko; Hirano, Kenichi; Ando, Kenichi; Ichishima, Eiji

2003-04-15

298

Analysis of the subproteomes of proteinases and heparin-binding toxins of eight Bothrops venoms.  

Science.gov (United States)

Viperid snakes show the most complex snake-venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2-DE, and their subproteomes of proteinases were explored by 2-D immunostaining and 2-D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti-coagulant and anti-inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high-affinity for heparin as composed of mainly serine proteinases and C-type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin-binding toxins. Only the non-bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin-bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity. PMID:19137556

Paes Leme, Adriana F; Kitano, Eduardo S; Furtado, Maria F; Valente, Richard H; Camargo, Antonio C M; Ho, Paulo L; Fox, Jay W; Serrano, Solange M T

2009-02-01

299

Impact of N-Chlorotaurine on Viability and Production of Secreted Aspartyl Proteinases of Candida spp.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

N-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against Candida spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of N-chlorotaurine as an antimicrobial agent in yeast infections.

Nagl, Markus; Gruber, Andreas; Fuchs, Anita; Lell, Claudia P.; Lemberger, Eva-maria; Borg-von Zepelin, Margarete; Wu?rzner, Reinhard

2002-01-01

300

OZONE EFFECTS ON ALPHA-1-PROTEINASE INHIBITOR IN VIVO: BLOOD PLASMA INHIBITORY ACTIVITY IS UNCHANGED  

Science.gov (United States)

The possible oxidative inactivation of human blood plasma alpha-1-proteinase inhibitor (PI) by inhaled ozone was assessed. Eleven male volunteers (non-smokers) were exposed to 0.5 ppm ozone for four hours on two consecutive days and ten control subjects were exposed to air under ...

 
 
 
 
301

Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).  

Science.gov (United States)

The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands. PMID:15612815

Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

2004-12-29

302

Virtual screening of novel noncovalent inhibitors for SARS-CoV 3C-like proteinase.  

Science.gov (United States)

The SARS coronavirus 3C-like proteinase is considered as a potential drug design target for the treatment of severe acute respiratory syndrome (SARS). Owing to the lack of available drugs for the treatment of SARS, the discovery of inhibitors for SARS coronavirus 3C-like proteinase that can potentially be optimized as drugs appears to be highly desirable. We have built a "flexible" three-dimensional model for SARS 3C-like proteinase by homology modeling and multicanonical molecular dynamics method and used the model for virtual screening of chemical databases. After Dock procedures, strategies including pharmocophore model, consensus scoring, and "drug-like" filters were applied in order to accelerate the process and improve the success rate of virtual docking screening hit lists. Forty compounds were purchased and tested by HPLC and colorimetric assay against SARS 3C-like proteinase. Three of them including calmidazolium, a well-known antagonist of calmodulin, were found to inhibit the enzyme with an apparent K(i) from 61 to 178 microM. These active compounds and their binding modes provide useful information for understanding the binding sites and for further selective drug design against SARS and other coronavirus. PMID:15667124

Liu, Zhenming; Huang, Changkang; Fan, Keqiang; Wei, Ping; Chen, Hao; Liu, Shiyong; Pei, Jianfeng; Shi, Lei; Li, Bo; Yang, Kun; Liu, Ying; Lai, Luhua

2005-01-01

303

Isolation, acid proteinase secretion, and experimental pathogenicity of Candida parapsilosis from outpatients with vaginitis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Candida parapsilosis was isolated from the vaginas of several nonpregnant, nondiabetic, mostly premenopausal outpatients who presented the characteristic signs and symptoms of a frank vulvovaginal candidiasis (heavy discharge with cottage cheese appearance and intense itching, with or without vulvar erythema and dyspareunia). All isolates conformed morphologically, biochemically, and serologically to the standard description of the species. They showed high acid proteinase-secretory activity ...

Bernardis, F.; Lorenzini, R.; Verticchio, R.; Agatensi, L.; Cassone, A.

1989-01-01

304

Comparison of specificity of human and horse leucocyte proteinases with synthetic peptide substrates.  

Science.gov (United States)

Highly purified horse leucocyte proteinases 1, 2A and 2B hydrolyze synthetic substrates which are decomposed also by human leucocyte elastase but they are unable to hydrolyze typical substrates of cathepsin G. Thus in distinction to other mammalian species horse leucocytes are devoid of cathepsin G and contain only elastases. PMID:3639831

Dubin, A; Potempa, J; Schnebli, H P; Koj, A

1986-01-01

305

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

Science.gov (United States)

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

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

2014-03-01

306

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2*  

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Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for c...

2010-01-01

307

Irreversible Oxidation of the Active-site Cysteine of Peroxiredoxin to Cysteine Sulfonic Acid for Enhanced Molecular Chaperone Activity*  

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The thiol (–SH) of the active cysteine residue in peroxiredoxin (Prx) is known to be reversibly hyperoxidized to cysteine sulfinic acid (–SO2H), which can be reduced back to thiol by sulfiredoxin/sestrin. However, hyperoxidized Prx of an irreversible nature has not been reported yet. Using an antibody developed against the sulfonylated (–SO3H) yeast Prx (Tsa1p) active-site peptide (AFTFVCPTEI), we observed an increase in the immunoblot intensity in proportion to ...

Lim, Jung Chae; Choi, Hoon-in; Park, Yu Sun; Nam, Hyung Wook; Woo, Hyun Ae; Kwon, Ki-sun; Kim, Yu Sam; Rhee, Sue Goo; Kim, Kanghwa; Chae, Ho Zoon

2008-01-01

308

Loss of expression of neutrophil proteinase-3: a factor contributing to thrombotic risk in paroxysmal nocturnal hemoglobinuria  

Science.gov (United States)

Background A deficiency of specific glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria may be responsible for most of the clinical features of this disease, but some functional consequences may be indirect. For example, the absence of certain glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria cells may influence expression of other membrane proteins. Membrane-bound proteinase 3 co-localizes with glycosylphosphatidyl inositol-linked neutrophil antigen 2a, which is absent in patients with paroxysmal nocturnal hemoglobinuria. Design and Methods We compared expression of proteinase 3 and neutrophil antigen 2a by flow cytometry and western blotting in normal and paroxysmal nocturnal hemoglobinuria cells and measured cytoplasmic and soluble levels of proteinase 3 by enzyme-linked immunosorbent assays in controls and patients with paroxysmal nocturnal hemoglobinuria. Finally, we studied the effects of proteinase 3 on platelet activation using an in vitro aggregometry assay and flow cytometry. Results We showed that membrane-bound proteinase 3 is deficient in patients’ cells, but invariantly present in the cytoplasm regardless of disease phenotype. When we isolated lipid rafts from patients, both molecules were detected only in the rafts from normal cells, but not diseased ones. Membrane-bound proteinase 3 was associated with a decrease in plasma proteinase 3 levels, clone size and history of thrombosis. In addition, we found that treating platelets ex vivo with proteinase 3, but not other agonists, decreased the exposure of an epitope on protease activated receptor-1 needed for thrombin activation. Conversely, treatment of whole blood with serine protease inhibitor enhanced expression of this epitope on protease activated receptor-1 located C-terminal to the thrombin cleavage site on platelets. Conclusions We demonstrated that deficiency of glycosylphosphatidyl inositol-anchored proteins in paroxysmal nocturnal hemoglobinuria results in decreased membrane-bound and soluble proteinase 3 levels. This phenomenon may constitute another mechanism contributing to a prothrombotic propensity in patients with paroxysmal nocturnal hemoglobinuria.

Jankowska, Anna M.; Szpurka, Hadrian; Calabro, Mark; Mohan, Sanjay; Schade, Andrew E.; Clemente, Michael; Silverstein, Roy L.; Maciejewski, Jaroslaw P.

2011-01-01

309

Partial Purification and Characterization of a Cysteine Protease Inhibitor from the Plerocercoid of Spirometra erinacei  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargan...

Chung, Young-bae; Yang, Hyun-jong

2008-01-01

310

Conservation of L and 3C proteinase activities across distantly related aphthoviruses  

DEFF Research Database (Denmark)

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV. and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV X also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.

Hinton, T.; Ross-Smith, N.

2002-01-01

311

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

Energy Technology Data Exchange (ETDEWEB)

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.

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

312

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.

2009-01-01

313

A new cell surface proteinase: sequencing and analysis of the prtB gene from Lactobacillus delbruekii subsp. bulgaricus.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Investigation of the chromosomal region downstream of the lacZ gene from Lactobacillus delbrueckii subsp. bulgaricus revealed the presence of a gene (prtB) encoding a proteinase of 1,946 residues with a predicted molecular mass of 212 kDa. The deduced amino acid sequence showed that PrtB proteinase displays significant homology with the N termini and catalytic domains of lactococcal PrtP cell surface proteinases and is probably synthesized as a preproprotein. However, the presence of a cystei...

Gilbert, C.; Atlan, D.; Blanc, B.; Portailer, R.; Germond, J. E.; Lapierre, L.; Mollet, B.

1996-01-01

314

Purification, Characterization and Partial Amino Acid Sequencing of Two New Aspartic Proteinases from Fresh Flowers of Cynara cardunculus L.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Two new aspartic proteinases have been isolated from stigmas of the cardoon Cynara cardunculus L. by a two-step purification procedure including extraction at low pH, gel filtration on Superdex 200, and ion-exchange chromatography on Mono Q. To follow the conventional nomenclature for aspartic proteinases, we have named these proteinases cardosin A and cardosin B. On SDS/PAGE, cardosin A migrated as two bands with apparent molecular masses of 31 000 Da and 15000 Da where as the chains of card...

Veri?ssimo, Paula; Faro, Carlos; Moir, Arthur J. G.; Lin, Yingzhang; Tang, Jordan; Pires, Euclides

1996-01-01

315

Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a mo...

Verchot, J.; Carrington, J. C.

1995-01-01

316

Reconstitution of a branch of the Manduca sexta prophenoloxidase activation cascade in vitro: Snake-like hemolymph proteinase 21 (HP21) cleaved by HP14 activates prophenoloxidase-activating proteinase-2 precursor  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Upon wounding or infection, a serine proteinase cascade in insect hemolymph leads to prophenoloxidase (proPO) activation and melanization, a defense response against invading microbes. In the tobacco hornworm Manduca sexta, this response is initiated via hemolymph proteinase 14 (HP14), a mosaic protein that interacts with bacterial peptidoglycan or fungal ?-1,3-glucan to autoactivate. In this paper, we report the expression, purification, and functional analysis of M. sexta HP21 precursor, a...

Wang, Yang; Jiang, Haobo

2007-01-01

317

Purification of a novel myofibril-bound serine proteinase inhibitor (MBSPI) from the skeletal muscle of lizard fish.  

Science.gov (United States)

A novel myofibril-bound serine proteinase inhibitor (MBSPI) was purified to homogeneity from the skeletal muscle of lizard fish (Saurida wanieso). Purification was carried out by ammonium sulfate fractionation, followed by column chromatographies on DEAE-Sephacel, SP-Sepharose and Sephadex G-150. MBSPI was purified 7.7-fold starting from the DEAE-Sephacel fraction, with a yield of 0.2%. It is a monomeric protein with the molecular mass of 50 kDa as estimated by SDS-PAGE and gel filtration. MBSPI reveals high inhibition specificity toward a myofibril-bound serine proteinase (MBSP) purified from lizard fish muscle. No inhibition is detected toward bovine trypsin, bovine chymotrypsin, two trypsins from carp hepatopancreas and a serine proteinase isolated from the sarcoplasmic fraction of white croaker muscle. It does not exert any inhibitory activity toward a myofibril-bound serine proteinase from carp muscle. PMID:11163300

Cao, M J; Osatomi, K; Hara, K; Ishihara, T

2001-01-01

318

Comparative studies of the Spi1 proteins of three equine alpha-1-proteinase inhibitor haplotypes following isolation by affinity chromatography.  

Science.gov (United States)

1. Antiproteinase deficiency can result in excessive proteinase-induced tissue damage. The major anti-elastase (Spi1) protein of equine alpha 1-proteinase inhibitor (alpha 1-PI) was isolated from the plasma/serum of three common haplotypes (I, L and U). 2. The N-terminal amino acid sequences of the three inhibitors were identical, but were only approx 65-77% homologous with two other published equine Spi1 sequences. 3. All three inhibitors complexed quickly and irreversibly with equine leucocyte proteinase 2A (kass = 2 x 10(7) M-1 sec-1). They were also efficient inhibitors of chymase (rat mast cell proteinase-II; kass = 2 x 10(5) M-1 sec-1; Ki = 2 x 10(-10) M). There was therefore no evidence of deficient inhibition in the Spi1 variants of the I,L and U haplotypes. PMID:8224372

Pemberton, A D; Miller, H R; John, H A; Scudamore, C L

1993-09-01

319

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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...

White, T. C.; Agabian, N.

1995-01-01

320

Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of the enzyme during infection in vitro.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The opportunistic yeastlike fungi of the genus Candida comprise three species which are proteolytic in vitro. Among them, C. albicans and C. tropicalis are of foremost medical importance. However, a strict correlation between extracellular proteolytic activity and virulence is opposed by the low virulence of the third proteolytic species, C. parapsilosis. We purified the secretory acid proteinase of C. parapsilosis (clinical isolate 265). The enzyme is a carboxyl proteinase (EC 3.4.23) like a...

Ru?chel, R.; Bo?ning, B.; Borg, M.

1986-01-01

 
 
 
 
321

Purification and characterization of a T cell specific serine proteinase (TSP-1) from cloned cytolytic T lymphocytes.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

We describe the purification of a T cell specific serine proteinase derived from a cloned murine cytolytic T lymphocyte line. Analysis of the enzyme by sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a mol. wt of approximately 60 kd under non-reducing conditions and of approximately 30 kd under reducing conditions. The proteinase cleaves the model peptide substrate H-D-Pro-Phe-Arg-NA, at the 4 nitroanilide (NA) group with high efficiency. Much lower or no activity of the en...

Simon, M. M.; Hoschu?tzky, H.; Fruth, U.; Simon, H. G.; Kramer, M. D.

1986-01-01

322

Identification of a Serine Proteinase Homolog (Sp-SPH) Involved in Immune Defense in the Mud Crab Scylla paramamosain  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Clip domain serine proteinase homologs are involved in many biological processes including immune response. To identify the immune function of a serine proteinase homolog (Sp-SPH), originally isolated from hemocytes of the mud crab, Scylla paramamosain, the Sp-SPH was expressed recombinantly and purified for further studies. It was found that the Sp-SPH protein could bind to a number of bacteria (including Aeromonas hydrophila, Escherichia coli, Staphylococcus aureus, Vibrio fluvialis, Vibrio...

2013-01-01

323

Binding properties of the regulatory domains in Manduca sexta hemolymph proteinase-14, an initiation enzyme of the prophenoloxidase activation system  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Pathogen recognition and rapid initiation of defense responses are essential for the survival of host insects. In Manduca sexta, hemolymph proteinase-14 precursor (proHP14) senses non-self presence and triggers a branched serine proteinase pathway which leads to prophenoloxidase activation and melanin formation around the invading organisms. To understand functions of individual domains in HP14, we have produced a series of HP14 domains and truncation mutants and studied their interactions wi...

Wang, Yang; Jiang, Haobo

2010-01-01

324

Kinetic modelling of enzyme inactivation Kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F.  

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The kinetics of heat inactivation of the extracellular proteinase from Pseudomonas fluorescens 22F was studied. It was established, by making use of kinetic modelling, that heat inactivation in the temperature range 35 - 70 °C was most likely caused by intermolecular autoproteolysis, where unfolded proteinase molecules are attacked by still active species. Kinetic modelling also showed that sodium caseinate acted as a competitive inhibitor against autoproteolysis. Autoproteolysis experiments...

Schokker, E. P.

1997-01-01

325

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

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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...

Piacenza, Luci?a; Acosta, Daniel; Basmadjian, Isabel; Dalton, John P.; Carmona, Carlos

1999-01-01

326

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser,...

Strisovsky, K.; Tessmer, U.; Langner, J.; Konvalinka, J.; Kra?usslich, H. G.

2000-01-01

327

Clinical effects of L-cystein combined with radiotherapy  

International Nuclear Information System (INIS)

Twenty-eight patients with cancer of the cervix uteri, etc., who had had radiotherapy were administered L-cystein. L-cystein's ability to protect against leukopenia was studied. Teletherapy using 60Co was performed. A dose of 200 R (total dose, 6,000 R) was administered as a rule. Treatment was remarkably effective in 1 of 28 patients (no leukopenia was noted after irradiation), effective in 23 (leukopenia occurred but radiotherapy could be continued), unremarkable in 2 (marked leukopenia was observed but radiotherapy using other agents could be continued), and ineffective in 2 (radiotherapy was discontinued due to marked leukopenia). The rate of effectiveness was 85.7%, and L-cystein was found to have the ability to protect against leukopenia. (Nishio, M.)

1980-01-01

328

Cathodic Behaviour of Cysteine at a Platinum Electrode  

Directory of Open Access Journals (Sweden)

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.

M. Fátima Barroso

2007-01-01

329

Use of plants in novel approaches for control of gastrointestinal helminths in livestock with emphasis on small ruminants.  

Science.gov (United States)

Helminth infections are a major cause for reduced productivity in livestock, particularly those owned by the poor worldwide. Phytomedicine has been used for eons by farmers and traditional healers to treat parasitism and improve performance of livestock, and many modern commercial medicines are derived from plants. However, scientific evidence on the anti-parasitic efficacy of most plant products is limited, regardless of their wide ethnoveterinary usage. Scientific validation of the anti-parasitic effects and possible side-effects of plant products in ruminants is necessary prior to their adoption as a novel method for parasite control. A variety of methods has been explored to validate the anthelmintic properties of such plant remedies, both in vivo and in vitro. In vitro assays are useful as pre-screens of activity and are mainly performed with the free-living rather than parasitic stages of nematodes. Concentrations of potentially active substances used in vitro do not always correspond to in vivo bioavailability. Therefore, in vitro assays should always be accompanied by in vivo studies when used to validate the anthelmintic properties of plant remedies. In vivo controlled studies have shown that plant remedies have in most instances resulted in reductions in the level of parasitism much lower than those observed with anthelmintic drugs. Whether it is necessary or not to achieve very high efficacy in order for plant remedies to have a role in the control of parasitism depends on the determination of biologically important levels of reduction of parasitism and it will be required prior to the wide-scale use of plant products for parasite control. Similarly, standardisation of validation studies in reference to the numbers of animals required for in vivo studies to measure direct anthelmintic effects of a plant needs to be established. Although in many cases the active compounds in the herbal remedies have not been fully identified, plant enzymes, such as cysteine proteinases, or secondary metabolites, such as alkaloids, glycosides and tannins have shown dose-dependent anti-parasitic properties. However, as some of the active compounds may also have anti-nutritional effects, such as reduced food intake and performance, it is essential to validate the anti-parasitic effects of plant products in relation to their potential anti-nutritional and other side effects. A concerted effort on isolation, development, and validation of the effects of these herbal remedies will have to be undertaken before their wider acceptance. PMID:16725262

Githiori, John B; Athanasiadou, Spiridoula; Thamsborg, Stig M

2006-07-31

330

Seven cysteine-deficient mutants depict interplay between thermal and chemical stabilities of individual cysteine residues in MAP kinase JNK1  

International Nuclear Information System (INIS)

To characterize the role of cysteine residues on the structure, function and stability of JNK1, we prepared and evaluated the wild-type JNK1 and seven cysteine-deficient JNK1 proteins. The solvent exposed cysteine residues did not influence biological function and mutating these residues raised the thermal stability because of newly formed hydrogen bonds and of higher hydration as speculated by the mutant structures. The surface cysteine involved in the molecular-surface hydrophobic pocket did not affect biological function; although a moderate thermal destabilization was observed. Cysteines in the loosely-assembled hydrophobic environment moderately contributed to thermal stability and the mutations of these cysteines had negligible effect on enzyme activity. The other cysteines are involved in the tightly-filled hydrophobic core and mutation of these residues conferred the adverse effects on the thermal stability and enzyme activity. (author)

2013-06-01

331

Plants  

Science.gov (United States)

In this logic activity, students must determine how to represent three quantities using a fixed amount of space (Venn diagram) and objects. The goal is to represent the siblingsâ ages, 5,6, and 7, using only ten plants. This resource includes teacher notes with extension suggestions and possible support options.

Team, Nrich

2012-01-01

332

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

DEFF Research Database (Denmark)

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.

Sørensen, S O; van den Hazel, H B

1994-01-01

333

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

Science.gov (United States)

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. PMID:8119286

Sørensen, S O; van den Hazel, H B; Kielland-Brandt, M C; Winther, J R

1994-02-15

334

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

DEFF Research Database (Denmark)

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 sequence. Crystal structures of different forms of IA3 complexed with proteinase A showed that residues in the N-terminal half of the IA3 sequence became ordered and formed an almost perfect alpha-helix in the active site of the enzyme. This potent, specific interaction was directed primarily by hydrophobic interactions made by three key features in the inhibitory sequence. Whereas IA3 was cut as a substrate by the nontarget aspartic proteinases, it was not cleaved by proteinase A. The random coil IA3 polypeptide escapes cleavage by being stabilized in a helical conformation upon interaction with the active site of proteinase A. This results, paradoxically, in potent selective inhibition of the target enzyme.

Phylip, L H; Lees, W E

2001-01-01

335

Retinoic acid modulation of glutathione and cysteine metabolism in chondrocytes.  

Science.gov (United States)

The major objective of this investigation was to determine the thiol status of chondrocytes and to relate changes in the level of glutathione and cysteine to maturation of the cells as they undergo terminal differentiation. Chondrocytes were isolated from the cephalic portion of chick embryo sterna and treated with all-trans retinoic acid for one week. We found that the addition of 100 nM retinoic acid to the cultures decreased the intracellular levels of glutathione and cysteine from 6.1 to 1.6 and 0.07 to 0.01 nmol/microgram DNA respectively; retinoic acid also caused a decrease in the extracellular concentration of cysteine. The decrease in chondrocyte thiols was dose and time dependent. To characterize other antioxidant systems of the sternal cell culture, the activities of catalase, glutathione reductase and superoxide dismutase were determined. Activities of all of those enzymes were high in the retinoic acid-treated cells; the conditioned medium also contained these enzymes and the cytosolic isoenzyme of superoxide dismutase. We probed the specificity of the thiol response by using immature caudal chondrocytes. Unlike the cephalic cells, retinoic acid did not change intracellular glutathione and extracellular cysteine levels, although the retinoid caused a reduction in the intracellular cysteine concentration. Finally, we explored the effect of medium components on chondrocyte thiol status. We noted that while ascorbate alone did not change cell thiol levels, it did cause a 4-fold decrease in the extracellular cysteine concentration. When retinoic acid and ascorbic acid were both present in the medium, there was a marked decrease in the level of glutathione. In contrast, the phosphate concentration of the culture medium served as a powerful modulator of both glutathione and cysteine. Results of the study clearly showed that there is a profound decrease in intracellular levels of both cysteine and glutathione and that thiol levels are responsive to ascorbic acid and the medium phosphate concentration. These findings point to a critical role for thiols in modulating events linked to chondrocyte maturation and cartilage matrix synthesis and mineralization. PMID:8660285

Teixeira, C C; Shapiro, I M; Hatori, M; Rajpurohit, R; Koch, C

1996-02-15

336

The tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1.  

Science.gov (United States)

The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred both in vitro and in vivo, within living plant cells. The V2 binding site within mCYP1 was identified in the direct proximity to the papain-like cysteine protease active site. PMID:22827939

Bar-Ziv, Amalia; Levy, Yael; Hak, Hagit; Mett, Anahit; Belausov, Eduard; Citovsky, Vitaly; Gafni, Yedidya

2012-08-01

337

The Tomato yellow leaf curl virus (TYLCV) V2 protein interacts with the host papain-like cysteine protease CYP1  

Science.gov (United States)

The V2 protein of Tomato yellow leaf curl geminivirus (TYLCV) is an RNA-silencing suppressor that counteracts the innate immune response of the host plant. However, this anti-host defense function of V2 may include targeting of other defensive mechanisms of the plant. Specifically, we show that V2 recognizes and directly binds the tomato CYP1 protein, a member of the family of papain-like cysteine proteases which are involved in plant defense against diverse pathogens. This binding occurred both in vitro and in vivo, within living plant cells. The V2 binding site within mCYP1 was identified in the direct proximity to the papain-like cysteine protease active site.

Bar-Ziv, Amalia; Levy, Yael; Hak, Hagit; Mett, Anahit; Belausov, Eduard; Citovsky, Vitaly; Gafni, Yedidya

2012-01-01

338

A Kunitz proteinase inhibitor from corms of Xanthosoma blandum with bactericidal activity.  

Science.gov (United States)

Bacterial infections directly affect the world's population, and this situation has been aggravated by indiscriminate use of antimicrobial agents, which can generate resistant microorganisms. In this report, an initial screening of proteins with antibacterial activity from corms of 15 species of the Xanthosoma genus was conducted. Since Xanthosoma blandum corms showed enhanced activity toward bacteria, a novel protein with bactericidal activity was isolated from this particular species. Edman degradation was used for protein N-termini determination; the primary structure showed similarities with Kunitz inhibitors, and this protein was named Xb-KTI. This protein was further challenged against serine proteinases from different sources, showing clear inhibitory activities. Otherwise, no hemolytic activity was observed for Xb-KTI. The results demonstrate the biotechnological potential of Xb-KTI, the first proteinase inhibitor with antimicrobial activity described in the Xanthosoma genus. PMID:21520894

Lima, Thaís B; Silva, Osmar N; Migliolo, Ludovico; Souza-Filho, Carlos R; Gonçalves, Eduardo G; Vasconcelos, Ilka M; Oliveira, José T A; Amaral, André C; Franco, Octávio L

2011-05-27

339

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

Energy Technology Data Exchange (ETDEWEB)

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 lambdagt11 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.

Salvesen, G.; Farley, D.; Shuman, J.; Przybyla, A.; Reilly, C.; Travis, J.

1987-04-21

340

Luminal proteinases from Plodia interpunctella and the hydrolysis of Bacillus thuringiensis CryIA(c) protoxin.  

Science.gov (United States)

The ability of proteinases in gut extracts of the Indianmeal moth, Plodia interpunctella, to hydrolyze Bacillus thuringiensis (Bt) protoxin, casein, and rho-nitroanilide substrates was investigated. A polyclonal antiserum to protoxin CryIA(c) was used in Western blots to demonstrate slower protoxin processing by gut enzymes from Bt subspecies entomocidus-resistant larvae than enzymes from susceptible or kurstaki-resistant strains. Enzymes from all three strains hydrolyzed N-alpha-benzoyl-L-arginine rho-nitroanilide, N-succinyl-ala-ala-pro-phenylalanine rho-nitroanilide, and N-succinyl-ala-ala-pro-leucine rho-nitroanilide. Zymograms and activity blots were used to estimate the apparent molecular masses, number of enzymes, and relative activities in each strain. Several serine proteinase inhibitors reduced gut enzyme activities, with two soybean trypsin inhibitors, two potato inhibitors, and chymostatin the most effective in preventing protoxin hydrolysis. PMID:8969468

Oppert, B; Kramer, K J; Johnson, D; Upton, S J; Mcgaughey, W H

1996-06-01

 
 
 
 
341

[Trypsin-like proteinases and trypsin inhibitors in fruiting bodies of higher fungi].  

Science.gov (United States)

The activity of trypsin-like proteinases and trypsin inhibitors was measured in fruiting bodies of various species of basidial fungi (Basidiomycetes). Fruiting bodies of all fungi contained these enzymes, with the exceptions of polypore (Coriolus versicolor (Fr.) Karst) and hedgehog fungus (Hericium erinaceus (Fr.) Quel), belonging to the families Polyporaceae and Hericiaceae, respectively, in which the enzyme activities were barely detectable. The activity of trypsin-like proteinases was the highest in fruiting bodies of Boletaceae and Agaricaceae. Fruiting bodies of all fungi contained trypsin inhibitors. The highest activity of trypsin inhibitors was detected in basidiomycetes of the families Boletaceae, Agaricaceae, and Pleurotaceae, including Boletus castanus (Fr.) Karst, orange-cap boletus (Leccinum aurantiacum (Fr.) Sing), and brown-cap boletus (Leccinum melanum (Fr.) Karst). PMID:16358748

Gzogian, L A; Proskuriakov, M T; Ievleva, E V; Valueva, T A

2005-01-01

342

Candida proteinases in the degradation of oral mucosal tissue components associated with Candida invasion  

Digital Repository Infrastructure Vision for European Research (DRIVER)

The aim of this thesis was to compare the degradation of human oral epithelial proteins by proteinases of different Candida yeast species. We focused on proteins associated with Candida invasion in the cell-to-cell junction, the basement membrane zone, the extracellular matrix, and local tissue inflammatory regulators. Another main objective was to evaluate the effect of the yeast/hyphal transition and pH on the degradative capability of Candida. The enzymatic activity of the Candida pro...

Pa?rna?nen, Pirjo

2010-01-01

343

Characterization of two cDNAs encoding serine proteinases from the hard tick Haemaphysalis longicornis.  

Science.gov (United States)

Host vaccination against tick infestation is at present the most practical and sustainable alternative tick control method to the current acaricide use which has serious limitations. However the success of this approach to control ticks depends upon the identification of target vaccine antigens. Members of the serine proteinase gene family may represent an interesting group of proteins to target as candidate antigens because of their involvement in regulation of many physiological functions and development processes in a wide range of organisms. We used RT-PCR with the 3' and 5' RACE to clone two cDNAs encoding full-length serine proteinases from the hard tick, Haemaphysalis longicornis. RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His(57) and Ser(195) conserved among most known serine proteinase. Gene specific primers designed from nucleotide sequences of the RT-PCR products were used to prime the 3' and 5' RACE. Southern blotting analysis showed that both HLSG-1 and -2 are single copy. The 2 cDNAs, HLSG-1 and -2 are 1.2 and 1.0 kb long in size with open reading frames encoding polypeptides with 37.7 and 31.2 kDa predicted molecular mass respectively. Northern blotting analysis of total RNA from unfed and partially fed whole ticks showed that the expression of mRNAs for both HLSG-1 and -2 was induced by blood feeding. Expression analysis by RT-PCR showed that both HLSG-1 and -2 are expressed in other tick organs in addition to salivary glands and midguts. The 6 serine proteinase consensus cyteine residues are well conserved in both HLSG-1 and -2. We have discussed our findings with respect to tick vaccine development research. PMID:11378417

Mulenga, A; Sugimoto, C; Ingram, G; Ohashi, K; Misao, O

2001-06-22

344

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. Bulgaricus  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Aims:?To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. Methods and Results:?HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with ?s1-casein (f 1–23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward ?s1-...

2002-01-01

345

Interaction of mouse macrophage elastase with native and oxidized human alpha 1-proteinase inhibitor.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Native and oxidized alpha 1-proteinase inhibitor (alpha 1-PI) were compared as substrates for the metalloproteinase macrophage elastase. At substrate concentrations at which native alpha 1-PI was readily degraded by macrophage elastase, oxidized alpha 1-PI was hardly degraded at all. Incubation of macrophage elastase with oxidized alpha 1-PI before the addition of native alpha 1-PI showed that oxidized alpha 1-PI was not an inhibitor of macrophage elastase. Competition experiments with up to ...

Banda, M. J.; Clark, E. J.; Sinha, S.; Travis, J.

1987-01-01

346

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 "1"4C-globin substrate. The 48-hr exposures to O_3 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 O_3 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 O_3, which correlated with inflammatory cells noted histologically. At 1.5 ppm O_3, 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 O_3 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

1987-01-01

347

Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus aureus-Derived Proteinases  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by Staphylococcus aureus, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time- and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that t...

Sieprawska-lupa, Magdalena; Mydel, Piotr; Krawczyk, Katarzyna; Wo?jcik, Kinga; Puklo, Magdalena; Lupa, Boguslaw; Suder, Piotr; Silberring, Jerzy; Reed, Matthew; Pohl, Jan; Shafer, William; Mcaleese, Fionnuala; Foster, Timothy; Travis, Jim; Potempa, Jan

2004-01-01

348

Conformational and functional alterations on an aspartic proteinase promoted by trifluoroethanol  

Digital Repository Infrastructure Vision for European Research (DRIVER)

O estudo das proteinases aspárticas tem vindo a ganhar interesse devido à importância desta classe de enzimas na etiologia e evolução de doenças humanas que são hoje fonte duma preocupação crescente, como são os casos da doença de Alzheimer, do cancro da mama ou da SIDA. A base molecular de algumas destas doenças está associada a erros de folding (enrolamento), que impossibilitam a sua função. Estudos de estabilidade sobre esta classe de enzimas são de ...

Almeida, Ana Sofia Fraga

2010-01-01

349

Mast cell tryptase regulates rat colonic myocytes through proteinase-activated receptor 2.  

Digital Repository Infrastructure Vision for European Research (DRIVER)

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved and activated by trypsin-like enzymes. PAR-2 is highly expressed by small intestinal enterocytes where it is activated by luminal trypsin. The location, mechanism of activation, and biological functions of PAR-2 in the colon, however, are unknown. We localized PAR-2 to the muscularis externa of the rat colon by immunofluorescence. Myocytes in primary culture also expressed PAR-2, assessed by immunofluoresc...

Corvera, C. U.; De?ry, O.; Mcconalogue, K.; Bo?hm, S. K.; Khitin, L. M.; Caughey, G. H.; Payan, D. G.; Bunnett, N. W.

1997-01-01

350

Polymorphonuclear leukocyte mediated oxidative inactivation of alpha-1-proteinase inhibitor: Modulation by nitric oxide  

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Alpha-1-proteinase inhibitor activity was studied in presence of resting and activated polymorphonuclear leucocytes. Four different agonists; phorbol myristic acetate, N-formyl-methionyl-leucyl-phenylalanine, opsonised zymosan and arachidonic acid decreased the inhibitor activity by 23.3%, 20%, 12% and 16.6^ respectively. The inhibitor activity was protected by using various free radical scavengers. Catalase and superoxide dismutase both restored activity by about 18%, mannitol by 13% and sod...

Mir, Mohammad Muzaffar; Khan, Abdul Rashid; Dar, Nazir Ahmad; Salahuddin, Mohammad

2005-01-01

351

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

Digital Repository Infrastructure Vision for European Research (DRIVER)

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 ...

2009-01-01

352

Comparison of self-processing of foot-and-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1?.  

Science.gov (United States)

The foot-and-mouth disease virus leader proteinase (Lb(pro)) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lb(pro) L200F provide structural evidence for intramolecular self-processing. (15)N-HSQC measurements of Lb(pro) L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLb(pro), lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1?, with a papain-like fold like Lb(pro), stably binds its own CTE. Parts of the ?-sheet domains but none of the ?-helical domains of Lb(pro) and nsp1? superimpose; consequently, the ?-helical domain of nsp1? is oriented differently relative to its ?-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1? but not Lb(pro). PMID:23756127

Steinberger, Jutta; Kontaxis, Georg; Rancan, Chiara; Skern, Tim

2013-09-01

353

Screening of yeasts from Brazilian Amazon rain forest for extracellular proteinases production.  

Science.gov (United States)

Eighty seven yeast strains representing 34 species isolated from Parahancornia amapa fruit and associated Drosophila flies collected in the Brazilian Amazon rain forest, were screened for proteinase production. Proteolytic activity was tested through casein hydrolysis in solid medium supplemented with 0.5% casein and glucose. Among 23 strains, 18 from genus Candida and 5 from Pichia were caseinolytic and produced proteinases in yeast carbon base liquid medium supplemented with casein 0.01%. The proteolytic activity was tested on pH ranging from 2.0 to 9.0 in correspondence to the pH of the cultures media in which the yeasts were grown. Six highly proteolytic strains: Candida parapsilosis AP153A, C. krusei AP176, C. sorbosa DR215, C. sorbosa AP259, C. valida AP209A and C. sorboxylosa AP287 were selected and the pH optima of production and the proteolytic activity were determined. In general the secretion of proteinase was maximum throughout the exponential and the stationary phases. Greater production occurred in acidic culture and high activity was observed at pH 3.0, 4.0 and 5.0. PMID:9779603

Braga, A A; de Morais, P B; Linardi, V R

1998-08-01

354

In vitro evaluation of proteinase, phospholipase and haemolysin activities of Candida species isolated from clinical specimens  

Directory of Open Access Journals (Sweden)

Full Text Available Background: Virulence attributes of Candida species include adherence to host tissues, morphological changes and secretion of extracellular hydrolytic enzymes. These enzymes play pivotal roles in pathogenicity of candida infection. Aim: The present study aimed to determine phospholipase, proteinase and haemolysin activities in Candida species isolated from various clinical samples. Material and Method: A total of 110 Candida species isolated from various clinical specimens were identified up to species level by standard mycological techniques and were tested for extracellular hydrolytic enzyme activity. Results: Phospholipase activity was in 60.9% of isolates, 59.1% produced proteinase and haemolysin activity was demonstrated seen in 51.8% of Candida isolates. Maximum strains of Candida albicans produced extracellular hydrolytic enzymes. Among Non-albicans Candida (NAC species, phospholipase and proteinase activity was higher in C.tropicalis whereas, haemolysin production was more in C.dubliniensis. Conclusion: From the present study it can be concluded, that both C. albicans and NAC species produce of extracellular hydrolytic enzymes. Since these enzymes are important to understand the co-relation between the species and infection their detection is extremely important.

Sachin C.D

2012-01-01

355

Proteinase inhibitors and dendrotoxins. Sequence classification, structural prediction and structure/activity.  

Science.gov (United States)

The amino acid sequences of four presynaptically active toxins from mamba snake venom (termed 'dendrotoxins') were compared systematically with homologous sequences of members of the proteinase inhibitor family (Kunitz). A comparison based on the complete sequences revealed that relatively few amino acid changes are necessary to abolish antiprotease activity and convert a proteinase inhibitor into a dendrotoxin. When comparison centred only on the sequence segments known to comprise the antiprotease site of bovine pancreatic trypsin inhibitor, the dendrotoxins were clearly classified apart from all the known inhibitors. Since the mode of action of the bovine pancreatic trypsin/kallikrein inhibitor involves beta sheet formation with the enzyme, predictions were obtained for this secondary structure in the region of the 'antiprotease site' throughout the homologues. Again, the dendrotoxins were clearly distinguished from the inhibitors. Structure/activity analyses, based on the crystal structures of inhibitor/enzyme complexes, suggest that unlike proteinase inhibitors, dendrotoxins might specifically co-ordinate the active-site 'catalytic' histidine residues of serine proteases. Although the significance of this remains to be studied, the presynaptic target is expected to involve an as yet uncharacterised member of the serine protease family. PMID:4076193

Dufton, M J

1985-12-16

356

Implantation Serine Proteinases heterodimerize and are critical in hatching and implantation  

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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.

Meng Guoliang

2006-12-01

357

Control of exogenous proteinases and their inhibitors at the macrophage cell surface.  

Science.gov (United States)

The actions and availability of human neutrophil elastase and its protein inhibitor, Eglin, when co-incubated with macrophages were investigated. Eglin did not induce radical production by mouse peritoneal macrophages; nor were specific binding sites for Eglin detected on these cells. Mouse peritoneal macrophages could inactivate both elastase and Eglin extensively, when these targets were used at concentrations appropriate to the extravascular fluids. Two methods were used for assessing such inactivation: one, as in previous literature, only took account of molecules remaining in the supernatant after interaction with the cells; the other (lacking from most previous studies) took into account all target molecules, including those associated with the cells. From an analysis of both types of experiment, it was shown that the cell-derived inactivators were stable products, whose quantity was not significantly influenced by the induction of a macrophage oxidative burst and its associated free radicals. They were probably mainly proteinases and proteinase inhibitors. Thus, mouse peritoneal macrophages restrict the activity of proteinases and inhibitors by means of stable molecules, such as proteins. Other mononuclear phagocytes may use free radicals and oxidants more extensively in this respect. PMID:2758063

Dean, R T; Schnebli, H P

1989-08-18

358

Identification of neutrophil elastase as the proteinase in burn wound fluid responsible for degradation of fibronectin.  

Science.gov (United States)

To identify proteinases responsible for fibronectin degradation in the wound environment we studied wound fluid obtained from burn patients. Immunoblotting experiments showed that extensive degradation of fibronectin had occurred in some burn wound fluid samples, in which case intact fibronectin molecules were undetectable, and the largest fibronectin fragment was 116 kDa. The 116-kDa fragment as well as a smaller 90-kDa fragment contained the fibronectin cell binding domain. These burn-fluid samples degraded freshly added fibronectin. Activity of the fibronectin-degrading enzyme was blocked by a broad-spectrum serine proteinase inhibitor or by specific neutrophil elastase inhibitors but not by metalloproteinase inhibitors or inhibitors of trypsin-like or chymotrypsin-like serine proteinases. Enzyme activity also was neutralized by antibodies against human neutrophil elastase. Incubation of fibronectin with burn wound fluid or purified human neutrophil elastase generated similar fibronectin-degradation products. Finally, direct assay of burn-wound-fluid samples with a synthetic elastase substrate showed a correlation between fluid-phase elastase activity and fibronectin degradation. Based on these findings, we conclude that burn-wound-fluid elastase is responsible for extensive fibronectin degradation. Acute elevation of elastase did not appear to hinder normal wound repair. PMID:8040604

Grinnell, F; Zhu, M

1994-08-01

359

Epithelial effects of proteinase-activated receptors in the gastrointestinal tract  

Scientific Electronic Library Online (English)

Full Text Available SciELO Brazil | Language: English Abstract in english The intestinal epithelium plays a crucial role in providing a barrier between the external environment and the internal milieu of the body. A compromised mucosal barrier is characteristic of mucosal inflammation and is a key determinant of the development of intestinal diseases such as Crohn's disea [...] se and ulcerative colitis. The intestinal epithelium is regularly exposed to serine proteinases and this exposure is enhanced in numerous disease states. Thus, it is important to understand how proteinase-activated receptors (PARs), which are activated by serine proteinases, can affect intestinal epithelial function. This review surveys the data which demonstrate the wide distribution of PARs, particularly PAR-1 and PAR-2, in the gastrointestinal tract and accessory organs, focusing on the epithelium and those cells which communicate with the epithelium to affect its function. PARs have a role in regulating secretion by epithelia of the salivary glands, stomach, pancreas and intestine. In addition, PARs located on subepithelial nerves, fibroblasts and mast cells have important implications for epithelial function. Recent data outline the importance of the cellular site of PAR expression, as PARs expressed on epithelia may have effects that are countered by PARs expressed on other cell types. Finally, PARs and their ability to promote epithelial cell proliferation are discussed in terms of colon cancer.

MacNaughton, Wallace K.

360

Aggregation properties of whey protein hydrolysates generated with Bacillus licheniformis proteinase activities.  

Science.gov (United States)

Hydrolysis of whey protein concentrate (WPC) with Alcalase 2.4 L, a Bacillus licheniformis proteinase preparation, induces gelation. The aggregation behavior of WPC hydrolysates generated with Alcalase and Prolyve 1000, a Bacillus licheniformis proteinase that did not induce gelation, were studied by turbidity and particle size analysis. With the use of synthetic peptide substrates, it was shown that Alcalase contains a glutamyl endopeptidase (GE) activity not present in Prolyve. Comparison of the aggregation behavior of WPC hydrolysates generated with Alcalase, Prolyve, and combinations of Prolyve with a GE activity isolated from Alcalase showed that GE was responsible for the observed enzyme-induced peptide aggregation in Alcalase hydrolysates. Hydrolysates generated with Prolyve, having a degree of hydrolysis (DH) of 11.8% and 10.4% of peptide material greater than 10 kDa, could be induced to aggregate by the addition of GE. These results emphasize the contribution of enzyme specificity to the physicochemical and functional characteristics of proteinase hydrolysates of WPC. PMID:15713050

Spellman, David; Kenny, Patricia; O'Cuinn, Gerard; FitzGerald, Richard J

2005-02-23

 
 
 
 
361

Effect of Hen`s Age on the Level of Cystatin in the Chicken Egg White  

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Cystatins, protein-type cysteine proteinase inhibitors are widely distributed in animals and plants. It is generally assumed that these inhibitors are involved in the regulation of physiological and pathological processes caused by cysteine proteinases Cystatins. The study aimed to isolate and characterize the cystatin from various chicken in different ages between 20-80 weeks. We used acid treatment, affinity chromatography Sepharose-Papain-4B, and desalting G-25 column, followed gel chromat...

Tadeusz Trziszka; Yousif Saleh; Wieslaw Kopeae; Maciej Siewinski; Ewelina Wesierska

2004-01-01

362

Chitosan in Plant Protection  

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Full Text Available Chitin and chitosan are naturally-occurring compounds that have potential in agriculture with regard to controlling plant diseases. These molecules were shown to display toxicity and inhibit fungal growth and development. They were reported to be active against viruses, bacteria and other pests. Fragments from chitin and chitosan are known to have eliciting activities leading to a variety of defense responses in host plants in response to microbial infections, including the accumulation of phytoalexins, pathogen-related (PR proteins and proteinase inhibitors, lignin synthesis, and callose formation. Based on these and other proprieties that help strengthen host plant defenses, interest has been growing in using them in agricultural systems to reduce the negative impact of diseases on yield and quality of crops. This review recapitulates the properties and uses of chitin, chitosan, and their derivatives, and will focus on their applications and mechanisms of action during plant-pathogen interactions.

Abdelbasset El Hadrami

2010-03-01

363

Interaction of ?-1,3-Glucan with Its Recognition Protein Activates Hemolymph Proteinase 14, an Initiation Enzyme of the Prophenoloxidase Activation System in Manduca sexta*  

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A serine proteinase pathway in insect hemolymph leads to prophenoloxidase activation, an innate immune response against pathogen infection. In the tobacco hornworm Manduca sexta, recombinant hemolymph proteinase 14 precursor (pro-HP14) interacts with peptidoglycan, autoactivates, and initiates the proteinase cascade (Ji, C., Wang, Y., Guo, X., Hartson, S., and Jiang, H. (2004) J. Biol. Chem. 279, 34101–34106). Here, we report the purification and characterization of pro-HP14 from the hemoly...

Wang, Yang; Jiang, Haobo

2006-01-01

364

75 FR 31790 - Determination That Cysteine Hydrochloride Injection, USP, 7.25%, Was Not Withdrawn From Sale for...  

Science.gov (United States)

...FDA-2008-P-0278] Determination That Cysteine Hydrochloride Injection, USP, 7.25...FDA) is announcing its determination that Cysteine Hydrochloride Injection, USP, 7.25% (Cysteine HCl), was not withdrawn from sale...

2010-06-04

365

Cysteine functionalized copper organosol: synthesis, characterization and catalytic application  

Energy Technology Data Exchange (ETDEWEB)

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

Panigrahi, Sudipa [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Kundu, Subrata [Department of Chemical Engineering, University of Nebraska, Lincoln (United States); Basu, Soumen [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Praharaj, Snigdhamayee [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Jana, Subhra [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Pande, Surojit [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Ghosh, Sujit Kumar [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India); Pal, Anjali [Department of Civil Engineering, Indian Institute of Technology, Kharagpur-721302 (India); Pal, Tarasankar [Department of Chemistry, Indian Institute of Technology, Kharagpur-721302 (India)

2006-11-14

366

Cysteine functionalized copper organosol: synthesis, characterization and catalytic application  

International Nuclear Information System (INIS)

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

2006-11-14

367

Cysteine functionalized copper organosol: synthesis, characterization and catalytic application  

Science.gov (United States)

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

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

2006-11-01

368

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

Science.gov (United States)

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

369

Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases.  

Science.gov (United States)

Phytocystatins are inhibitors of cysteine-proteases from plants putatively involved in plant defence based on their capability of inhibit heterologous enzymes. We have previously characterised the whole cystatin gene family members from barley (HvCPI-1 to HvCPI-13). The aim of this study was to assess the effects of barley cystatins on two phytophagous spider mites, Tetranychus urticae and Brevipalpus chilensis. The determination of proteolytic activity profile in both mite species showed the presence of the cysteine-proteases, putative targets of cystatins, among other enzymatic activities. All barley cystatins, except HvCPI-1 and HvCPI-7, inhibited in vitro mite cathepsin L- and/or cathepsin B-like activities, HvCPI-6 being the strongest inhibitor for both mite species. Transgenic maize plants expressing HvCPI-6 protein were generated and the functional integrity of the cystatin transgene was confirmed by in vitro inhibitory effect observed against T. urticae and B. chilensis protein extracts. Feeding experiments impaired on transgenic lines performed with T. urticae impaired mite development and reproductive performance. Besides, a significant reduction of cathepsin L-like and/or cathepsin B-like activities was observed when the spider mite fed on maize plants expressing HvCPI-6 cystatin. These findings reveal the potential of barley cystatins as acaricide proteins to protect plants against two important mite pests. PMID:21082183

Carrillo, Laura; Martinez, Manuel; Ramessar, Koreen; Cambra, Inés; Castañera, Pedro; Ortego, Felix; Díaz, Isabel

2011-01-01

370

A seminaphthofluorescein-based fluorescent chemodosimeter for the highly selective detection of cysteine  

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A fluorescent chemodosimeter for cysteine detection was developed based on a tandem conjugate addition and intramolecular cyclization reaction. The method exhibited an excellent selectivity for cysteine over other biothiols such as homocysteine and glutathione.

Yang, Xiaofeng; Guo, Yixing; Strongin, Robert M.

2012-01-01

371

The plasmid-encoded lactococcal envelope-associated proteinase is encoded by a chromosomal gene in Lactococcus lactis subsp. cremoris BC101.  

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The plasmid-free strain Lactococcus lactis subsp. cremoris BC101 produced an extracellular proteinase physicochemically similar to the proteinase encoded by the plasmid-linked prtP gene of other lactococcal strains. The absence of detectable plasmids in strain BC101 indicated that the prtP proteinase gene may be chromosomally located. The chromosomal linkage of the prtP proteinase gene in BC101 was confirmed by pulsed-field electrophoresis of chromosomal DNA and hybridization, using as a prob...

Nissen-meyer, J.; Lillehaug, D.; Nes, I. F.

1992-01-01

372

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway.  

Science.gov (United States)

Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2(a )mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells. PMID:18273641

Duarte, Patrícia; Pissarra, José; Moore, Ian

2008-05-01

373

The transsulfuration pathway : a source of cysteine for glutathione in astrocytes  

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Astrocyte cells require cysteine as a substrate for glutamate cysteine ligase (?-glutamylcysteine synthase; EC 6.3.2.2) catalyst of the rate-limiting step of the ?-glutamylcycle leading to formation of glutathione (l-?-glutamyl-l-cysteinyl-glycine; GSH). In both astrocytes and glioblastoma/astrocytoma cells, the majority of cysteine originates from reduction of cystine imported by the x c ? cystine-glutamate exchanger. However, the transsulfuration pathway, which supplies cysteine from ...

Mcbean, Gethin J.

2012-01-01

374

Functional significance of conserved cysteines in the human organic cation transporter 2  

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The significance of conserved cysteines in the human organic cation transporter 2 (hOCT2), namely the six cysteines in the long extracellular loop (loop cysteines) and C474 in transmembrane helix 11, was examined. Uptake of tetraethylammonium (TEA) and 1-methyl-4-phenypyridinium (MPP) into Chinese hamster ovary cells was stimulated >20-fold by hOCT2 expression. Both cell surface expression and transport activity were reduced considerably following mutation of individual loop cysteines (C51, C...

Pelis, Ryan M.; Dangprapai, Yodying; Cheng, Yaofeng; Zhang, Xiaohong; Terpstra, Jennifer; Wright, Stephen H.

2012-01-01

375

Electrochemical behaviour of dopamine at covalent modified glassy carbon electrode with l-cysteine: preliminary results  

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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 to 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 uni...

Carlos Alberto Martínez-Huitle; Monica Cerro-Lopez; Marco Antonio Quiroz

2009-01-01

376

Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase  

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Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (cont...

King, Nicola; Lin, Hua; Suleiman, M. -saadeh

2010-01-01

377

Cysteine-Mediated Reductive Dissolution of Poorly Crystalline Iron(III)Oxides by Geobacter sulfurreducens  

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The reductive dissolution of poorly crystalline ferric oxides in the presence of cysteine was investigated to evaluate the potential of cysteine as a possible electron carrier to stimulate the reduction of iron(III) oxides by Geobacter sulfurreducens. The extent and rate of biotic and abiotic reduction of iron(III) oxides in the presence of cysteine at various concentrations were compared. Iron(III) oxides were reduced abiotically by cysteine. The initial rate and extent of iron(III) oxide re...

Doong, Ruey-an; Schink, Bernhard

2002-01-01

378

Desulfurization of Cysteine-Containing Peptides Resulting from Sample Preparation for Protein Characterization by MS  

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In this paper, we have examined two cysteine modifications resulting from sample preparation for protein characterization by MS: (1) a previously observed conversion of cysteine to dehydroalanine, now found in the case of disulfide mapping and (2) a novel modification corresponding to conversion of cysteine to alanine. Using model peptides, the conversion of cysteine to dehydroalanine via ?-elimination of a disulfide bond was seen to result from the conditions of typical tryptic digestion (3...

Wang, Zhouxi; Rejtar, Tomas; Zhou, Zhaohui Sunny; Karger, Barry L.

2010-01-01

379

Transaminative metabolism of L-cysteine in guinea pig liver and kidney.  

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Transaminative metabolism of L-cysteine was investigated using homogenates of guinea pig liver and kidney. L-Cysteine was transaminated in the presence of 2-oxoglutarate and the homogenate of either liver or kidney. S-(2-Hydroxy-2-carboxyethylthio)cysteine (HCETC) (3-mercaptolactate-cysteine disulfide) was formed by liver homogenate, but the amount was very small. On the other hand, a relatively large amount of HCETC was formed in the presence of kidney homogenate. Transamination between 3...

Taniguchi, Miyabi; Hosaki, Yasuhiro; Ubuka, Toshihiko

1984-01-01

380

Dealing with methionine/homocysteine sulfur: cysteine metabolism to taurine and inorganic sulfur  

Science.gov (United States)

Synthesis of cysteine as a product of the transsulfuration pathway can be viewed as part of methionine or homocysteine degradation, with cysteine being the vehicle for sulfur conversion to end products (sulfate, taurine) that can be excreted in the urine. Transsulfuration is regulated by stimulation of cystathionine ?-synthase and inhibition of methylene tetrahydrofolate reductase in response to changes in the level of S-adenosylmethionine, and this promotes homocysteine degradation when methionine availability is high. Cysteine is catabolized by several desulfuration reactions that release sulfur in a reduced oxidation state, generating sulfane sulfur or hydrogen sulfide (H2S), which can be further oxidized to sulfate. Cysteine desulfuration is accomplished by alternate reactions catalyzed by cystathionine ?-synthase and cystathionine ?-lyase. Cysteine is also catabolized by pathways that require the initial oxidation of the cysteine thiol by cysteine dioxygenase to form cysteinesulfinate. The oxidative pathway leads to production of taurine and sulfate in a ratio of approximately 2:1. Relative metabolism of cysteine by desulfuration versus oxidative pathways is influenced by cysteine dioxygenase activity, which is low in animals fed low-protein diets and high in animals fed excess sulfur amino acids. Thus, desulfuration reactions dominate when cysteine is deficient, whereas oxidative catabolism dominates when cysteine is in excess. In rats consuming a diet with an adequate level of sulfur amino acids, about two thirds of cysteine catabolism occurs by oxidative pathways and one third by desulfuration pathways. Cysteine dioxygenase is robustly regulated in response to cysteine availability and may function to provide a pathway to siphon cysteine to less toxic metabolites than those produced by cysteine desulfuration reactions.

Ueki, Iori

2010-01-01

 
 
 
 
381

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

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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...

King, Nicola; Lin, Hua; Suleiman, M. -saadeh

2011-01-01

382

Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli.  

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Cysteine, in concentrations down to 0.04 micrograms/ml, induces transient amino acid starvation in Escherichia coli growing in minimal medium. The duration depends on the concentration and is 5 min at 2 micrograms of cysteine per ml. At low cysteine concentrations, threonine and isoleucine almost completely abolish the starvation.

Sørensen, M. A.; Pedersen, S.

1991-01-01

383

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

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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.

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

1986-01-01

384

A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities.  

Science.gov (United States)

ABSTRACT The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL(134)H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL(134)H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro. PMID:18944411

González-Jara, Pablo; Atencio, Felix A; Martínez-García, Belén; Barajas, Daniel; Tenllado, Francisco; Díaz-Ruíz, José Ramón

2005-08-01

385

Plant caspase-like proteases in plant programmed cell death  

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Programmed cell death (PCD) is a genetically-controlled disassembly of the cell. In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (Cysteine-containing Aspartate-specific proteases). Accumulating evidence in recent years suggests the existence of caspase-like activity in plants and its functional involvement in various types of plant PCD, although no functional homologs of animal caspases were identified in plant genome. In this mini-review, ...

2009-01-01

386

Preparation of cysteine-34-nitroxide spin labeled human ??-microglobulin.  

Science.gov (United States)

?(1)-Microglobulin (?(1)m) is a protein of yet unresolved function occurring in blood plasma and urine. It consists of a lipocaline type of fold with two cysteine residues forming a disulfide bridge and the third cysteine-34 remaining a free, somewhat reactive thiol. A number of investigations point to an interaction with heme and we have recently reported, that heme binding triggers the formation of a stable ?(1)m trimer upon modification of cysteine-34 with 2-iodoacetamide, i.e., [?(1)m(heme)(2)](3) [J.F. Siebel, R.L. Kosinsky, B. ?kerström, M. Knipp, Insertion of heme b into the structure of the Cys34-carbamidomethylated human lipocalin ?(1)-microglobulin-formation of a [(heme)(2)(?(1)-microglobulin)](3) complex, ChemBioChem 13 (2012) 879-887]. For further structural and functional investigations, an improved purification protocol for ?(1)m was sought, in particular yielding an untagged amino acid sequence. The method reported herein improves the speed and the yield of the protein production even when an expression plasmid without tag was applied. Furthermore, for the purpose of future structural studies using electron paramagnetic resonance (EPR) techniques, in accordance to the modification with 2-iodoacetamide (?(1)m(AM)), the protein was modified with 3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (3-(2-iodoacetamido)-PROXYL) yielding the nitroxide spin labeled ?(1)m(N-O). The extinction coefficient of the protein was calibrated using magnetic circular dichroism (MCD) spectroscopy of tryptophan (?(280nm)=40,625M(-1)cm(-1)). The parallel quantification by absorbance spectroscopy (protein) and cw-EPR spectroscopy (radical spin) determined the degree of spin labeling to 90%. Characterization of the protein by circular dichroism (CD) spectroscopy and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) upon tryptic digestion further demonstrated the similar fold of ?(1)m(AM) and ?(1)m(N-O), but also established the modification of cystein-34 as well as the formation of the cysteine-72-cysteine-169 disulfide bond. PMID:23201281

Nalepa, Anna I; Taing, Johanna J; Savitsky, Anton; Knipp, Markus

2013-03-01

387

Structure of the proteinase inhibitor eglin c with hydrolysed reactive centre at 2.0 A resolution.  

Science.gov (United States)

The inhibition of serine proteinases by both synthetic and natural inhibitors has been widely studied. Eglin c is a small thermostable protein isolated from the leech, Hirudo medicinalis. Eglin c is a potent serine proteinase inhibitor. The three-dimensional structure of native eglin and of its complexes with a number of proteinases are known. We here describe the crystal structure of hydrolysed eglin not bound to a proteinase. The body of the eglin has a conformation remarkably similar to that in the known complexes with proteinases. However, the peptide chain has been cut at the 'scissile' bond between residues 45 and 46, presumed to result from the presence of subtilisin DY in the crystallisation sample. The residues usually making up the inhibiting loop of eglin take up a quite different conformation in the nicked inhibitor leading to stabilising contacts between neighbouring molecules in the crystal. The structure was solved by molecular replacement techniques and refined to a final R-factor of 14.5%. PMID:8425603

Betzel, C; Dauter, Z; Genov, N; Lamzin, V; Navaza, J; Schnebli, H P; Visanji, M; Wilson, K S

1993-02-15

388

Induction of release and up-regulated gene expression of interleukin (IL-8 in A549 cells by serine proteinases  

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Full Text Available Abstract Background Hypersecretion of cytokines and serine proteinases has been observed in asthma. Since protease-activated receptors (PARs are receptors of several serine proteinases and airway epithelial cells are a major source of cytokines, the influence of serine proteinases and PARs on interleukin (IL-8 secretion and gene expression in cultured A549 cells was examined. Results A549 cells express all four PARs at both protein and mRNA levels as assessed by flow cytometry, immunofluorescence microscopy and reverse transcription polymerase chain reaction (PCR. Thrombin, tryptase, elastase and trypsin induce a up to 8, 4.3, 4.4 and 5.1 fold increase in IL-8 release from A549 cells, respectively following 16 h incubation period. The thrombin, elastase and trypsin induced secretion of IL-8 can be abolished by their specific inhibitors. Agonist peptides of PAR-1, PAR-2 and PAR-4 stimulate up to 15.6, 6.6 and 3.5 fold increase in IL-8 secretion, respectively. Real time PCR shows that IL-8 mRNA is up-regulated by the serine proteinases tested and by agonist peptides of PAR-1 and PAR-2. Conclusion The proteinases, possibly through activation of PARs can stimulate IL-8 release from A549 cells, suggesting that they are likely to contribute to IL-8 related airway inflammatory disorders in man.

He Shaoheng

2006-05-01

389

Biochemical characterization, cDNA cloning, and molecular modeling of araujiain aII, a papain-like cysteine protease from Araujia angustifolia latex.  

Science.gov (United States)

Araujiain aII, the protease with highest specific activity purified from latex of Araujia angustifolia (Apocynaceae), shows optimum proteolytic activity at alkaline pH, and it is completely inhibited by the irreversible inhibitor of cysteine proteases trans-epoxysucciny-L: -leucyl-amido(4-guanidino) butane. It exhibits esterolytic activity on several N-?-Cbz-amino acid p-nitrophenyl esters with a preference for Gln, Ala, and Gly derivatives. Kinetic enzymatic assays were performed with the thiol proteinase substrate p-Glu-Phe-Leu-p-nitroanilide (K (m) = 0.18 ± 0.03 mM, k (cat) = 1.078 ± 0.055 s(-1), k (cat)/K (m) = 5.99 ± 0.57 s(-1) mM(-l)). The enzyme has a pI value above 9.3 and a molecular mass of 23.528 kDa determined by mass spectrometry. cDNA of the peptidase was obtained by reverse transcription-PCR starting from total RNA isolated from latex. The deduced amino acid sequence was confirmed by peptide mass fingerprinting analysis. The N-terminus of the mature protein was determined by automated sequencing using Edman's degradation and compared with the sequence deduced from cDNA. The full araujiain aII sequence was thus obtained with a total of 213 amino acid residues. The peptidase, as well as other Apocynaceae latex peptidases, is a member of the subfamily C1A of cysteine proteases. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was suggested by molecular modeling. PMID:21424535

Obregón, Walter D; Lufrano, Daniela; Liggieri, Constanza S; Trejo, Sebastián A; Vairo-Cavalli, Sandra E; Avilés, Francesc X; Priolo, Nora S

2011-08-01

390

Reduction of selenite by cysteine in ionic media  

Science.gov (United States)

This paper focuses on the abiotic reduction of selenite (Se(IV)) by cysteine (Cys, NH3+CH(CH2SH)COOH), which is a representative thiol produced by aquatic organism under oxidative stress. The rates of reduction of Se(IV) by cysteine were measured in deaerated NaCl solutions and natural waters as a function of pH (4.0-9.0), temperature (10-40 °C), and ionic strength (0.01-1.0 M). The rates showed a complex dependence on pH with similar values from pH 4.0-5.0, increasing values from pH 5.0-7.0 and then decreasing values at pH higher than 7.0. An apparent energy of activation obtained was 31 ± 6 kJ mol-1, which was independent of ionic strength.

Gennari, Francesca; Sharma, Virender K.; Pettine, Maurizio; Campanella, Luigi; Millero, Frank J.

2014-01-01

391

Isolation and characterization of a cysteine protease of freesia corms.  

Science.gov (United States)

A protease, freesia protease (FP)-A, was purified to electrophoretic homogeneity from regular freesia (Freesia reflacta) corms in harvest time. The Mr of FP-A was estimated to be 24 k by SDS-PAGE. The optimum pH of the enzyme was 8.0 using a casein substrate. These enzymes were strongly inhibited by p-chloromercuribenzoic acid but not by phenylmethane-sulfonylfluoride and EDTA. These results indicate that FP-A belongs to the cysteine proteases. The amino terminal sequence of FP-A was similar to that of papain, and the sequences was regarded to the conservative residues of cysteine protease. From the hydrolysis of peptidyl-p-NAs, the specificity of FP-A was found to be broad. It was thought that FP-A was a new protease from freesia corms. PMID:11999426

Uchikoba, Tetsuya; Okubo, Michiko; Arima, Kazunari; Yonezawa, Hiroo

2002-02-01

392

Identification and preliminary characterization of protein-cysteine farnesyltransferase.  

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Ras proteins must be isoprenylated at a conserved cysteine residue near the carboxyl terminus (Cys-186 in mammalian Ras p21 proteins) 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 RAS2Val-19 gene in Saccharomyces cerevisiae and blocks the maturation of Xenopus ...

1990-01-01