scholarly journals Partial characterization of proteolytic activity in Giardia duodenalis purified proteins

2007 ◽  
Vol 49 (6) ◽  
pp. 385-388 ◽  
Author(s):  
Erica Boarato David ◽  
Semíramis Guimarães ◽  
Paulo Eduardo Martins Ribolla ◽  
Silvana Torossian Coradi ◽  
Diego Peres Alonso
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
High Performance ◽  
Giardia Duodenalis ◽  
Protein Profiles ◽  
Aspartic Proteases ◽  
Sds Page ◽  
Host Parasite

This report describes a preliminary characterization of proteolytic activity of proteins isolated from lysate of Giardia trophozoites of an axenic Brazilian strain. Fractions obtained by high-performance liquid chromatography (FPLC) were tested in SDS-polyacrylamide gel for the protein profiles, and the proteases activity was analyzed using gelatin impregnated SDS-PAGE. The proteases characterization was based on inhibition assays employing synthetic inhibitors for cysteine (E-64, IAA), serine (PMSF, TPCK, TLCK, and elastatinal), metalo (EDTA) and aspartic (pepstatin) proteases. Among thirty eluted fractions, polypeptide bands were observed in eight of them, however, proteolytic activity was detected in four ones (F23, F24, F25 and F26). Protein profiles of these fractions showed a banding pattern composed by few bands distributed in the migration region of 45 to < 18 kDa. The zymograms revealed proteolytic activity in all the four fractions assayed, mainly distributed in the migration region of 62 to 35 kDa. Among the profiles, the main pronounced zones of proteolysis were distinguished at 62, 55, 53, 50, 46 and 40 kDa. In inhibition assays, the protease activities were significantly inhibited by cysteine (E-64) and serine proteases (TPCK, TLCK and elastatinal) inhibitors. Gels incubated with other cysteine and serine protease inhibitors, IAA and PMSF, respectively, showed a decrease in the intensity of hydrolysis zones. Indeed, in the assays with the inhibitors EDTA for metalloproteases and pepstatin for aspartic proteases, none inhibition was detected against the substrate. These observations are relevants, especially if we consider that to define the real role of the proteases in host-parasite interaction, the purification of these enzymes for detailed studies may be warranted.

scholarly journals Percepción de los entrenadores de alto rendimiento de atletismo: caracterización de su perfil polifacético (Perception of high performance track and field coaches: characterization of their multifaceted profile)

Retos ◽  
2015 ◽  
pp. 42-47
Author(s):  
Angeles Filgueira Perez
Keyword(s):  
High Performance ◽  
Target Population ◽  
Track And Field ◽  
Personal Qualities ◽  
High Level ◽  
Almost All ◽  
Methodological Aspects ◽  
The Ideal

En el presente estudio se ha tratado de obtener una aproximación al perfil ideal del entrenador de alto rendimiento en atletismo. En el mundo del deporte de alta competición, el entrenador debe desarrollar funciones de maestro, técnico y líder, lo cual hace difícil delimitar sus competencias (conocimientos, habilidades y cualidades personales). Por tanto, la principal motivación de este trabajo es establecer el papel que debe desempeñar el entrenador para la preparación física, técnica, táctica, psicológica y moral del deportista de alto nivel. Para ello, se ha considerado como población objeto de estudio al conjunto de entrenadores de alto rendimiento que en el momento de la investigación estuviesen activo, ya que interesaba realizar este estudio desde su propia perspectiva. Los datos recogidos forman parte de una investigación más amplia, llevada a cabo mediante encuesta, para la que se diseñó un cuestionario de 78 preguntas en el que se abarcaban tres temas: el perfil del entrenador y del deportista, así como la figura del formador de entrenadores en el Practicum. En este trabajo nos centramos en el primer tema y el análisis de la información obtenida nos permite concluir que la que práctica totalidad de los entrenadores de atletismo consideran necesario dominar con precisión los aspectos técnico-metodológicos. Además, consideran que su ética profesional debe estar regida por los principios de autonomía y beneficencia, de modo que valores como la honestidad y la justicia deben primar en el desarrollo de sus funciones.Abstract: In the current study, we have tried to get an approximation of the ideal profile of high performance coaches in athletics. In the high-level sports world, the coach must develop the role of teacher, technician and leader,  which makes it difficult to delimit  his/her competencies (knowledge, skills and personal qualities). Therefore, the main motivation of this work is to define the role that the coach plays in relation  to the physical, technical, tactical, psychological and moral preparation of elite athletes. Keeping this aim as an objective, we have considered as the target population the high performance coaches who are active at the time of the investigation, since we were interested  in knowing their own perspective. The data that was collected is a part of a wider investigation, conducted by a survey, for which we designed a questionnaire of 78 questions divided on three topics: the profile of the coach and the athlete, as well as the figure of the coach educator in the Practicum. In this work, we focus on the first topic and the analysis of the information that we obtained allows us to conclude that almost all the track and field coaches find it necessary to dominate the technical and methodological aspects. They also consider that professional ethics must be governed by the principles of autonomy and care that values   like honesty and justice must prevail in the performance of their work.

scholarly journals Characterization of the soluble, secreted form of urinary meprin

1996 ◽  
Vol 315 (2) ◽  
pp. 461-465 ◽  
Author(s):  
Robert J. BEYNON ◽  
Simon OLIVER ◽  
Duncan H. L. ROBERTSON
Keyword(s):  
Proteolytic Activity ◽  
Protein Band ◽  
Peptide Sequence ◽  
Soluble Form ◽  
Α Subunit ◽  
Alternative Processing ◽  
Sds Page ◽  
Processing Pathways ◽  
Membrane Bound

A soluble form of the kidney membrane metalloendopeptidase, meprin, is present in urine. Urinary meprin is expressed in BALB/C mice with the Mep-1a/a genotype (high meprin, expressing meprin-α and meprin-β) but not in BALB.K mice of the Mep-1b/b genotype (that only express meprin-β). Western blotting with antisera specific to the meprin-α and the meprin-β subunits established that the only form of meprin present in urine samples was derived from meprin-α. This form of meprin is partially active, and comprises at least three variants by non-reducing SDS/PAGE and by zymography and two protein bands on reducing SDS/PAGE. Sequencing of these two bands established that the N-terminus of the larger protein band begins with the pro-peptide sequence of the α-subunit (VSIKH..), whereas the smaller band possessed the mature meprin N-terminal sequence (NAMRDP..). Trypsin is able to remove the pro-peptide, with a concomitant activation in proteolytic activity. After deglycosylation, the size of the pro- and mature forms of urinary meprin are consistent with cleavage in the region of the X–I boundary. There is a pronounced sexual dimorphism in urinary meprin expression. Females secrete a slightly larger form, and its proteolytic activity is about 50% of that released by males. The urinary meprin is therefore a naturally occurring secreted form of this membrane-bound metalloendopeptidase and is more likely to be generated by alternative processing pathways than by specific release mechanisms.

Study of the Proteolytic Activity of the Tropical Legume Crotalaria spectabilis

2012 ◽  
Vol 67 (9-10) ◽  
pp. 495-509 ◽  
Author(s):  
Juliana da Silva Pacheco ◽  
Raquel Elisa da Silva-Lopez
Keyword(s):  
Proteolytic Activity ◽  
Serine Proteases ◽  
Ph Value ◽  
Cysteine Proteases ◽  
Leaf Extracts ◽  
Serine Protease Activity ◽  
Proteolytic Activities ◽  
Low Levels ◽  
Crotalaria Spectabilis

The characterization of legume proteases contributes to the understanding of the physiology of plants and their interaction with the environment. Thirteen extracts from various parts of Crotalaria spectabilis were made using different extraction systems. The highest protein content was found in seeds, and the most pronounced proteolytic activity was observed in leaf extracts, with an optimal pH value in the alkaline range. Proteases in extracts from roots, stems, and fl owers were active in various pH ranges. Proteases in all extracts were maximally active between 30 °C and 60 °C and were thermostable (24 h, 60 °C). Hemoglobin, bovine serum albumin, casein, and gelatin were hydrolyzed by C. spectabilis extracts in different ways. The highest serine protease activity was found in leaves. Seeds contained high levels of serine proteases and low levels of cysteine proteases. Flowers, roots, and stems contained different levels of serine, aspartic, and metalloproteases, respectively. The proteolytic activities in extracts were modulated by cations and oxidants to various degrees. C. spectabilis proteases are differentially expressed in distinctive organs, and their stability against heat and oxidants makes this plant an important source of stable proteases

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2455-2462 ◽  
Author(s):  
Masaru Nagai ◽  
Maki Kawata ◽  
Hisayuki Watanabe ◽  
Machiko Ogawa ◽  
Kumiko Saito ◽  
...  
Keyword(s):  
Lentinula Edodes ◽  
Veratryl Alcohol ◽  
Size Exclusion ◽  
Melanin Synthesis ◽  
Sds Page ◽  
Diammonium Salt ◽  
Exclusion Chromatography ◽  
Homogeneous Preparation

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58·0 kDa. The enzyme had an isoelectric point of around pH 6·9. The optimum pH for enzyme activity was around 3·0 against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 °C and stable up to 50 °C. The enzyme contained 8·6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. β-(3,4-Dihydroxyphenyl)alanine (l-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of l-dopa was identified as l-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

scholarly journals Identification and Characterization of a Well-Defined Series of Coronatine Biosynthetic Mutants of Pseudomonas syringae pv. tomato DC3000

2004 ◽  
Vol 17 (2) ◽  
pp. 162-174 ◽  
Author(s):  
David M. Brooks ◽  
Gustavo Hernández-Guzmán ◽  
Andrew P. Kloek ◽  
Francisco Alarcón-Chaidez ◽  
Aswathy Sreedharan ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
High Performance ◽  
Tomato Dc3000 ◽  
Disease Symptoms ◽  
Coronafacic Acid ◽  
Tn5 Mutants ◽  
Cor Genes ◽  
Identification And Characterization

To identify Pseudomonas syringae pv. tomato genes involved in pathogenesis, we carried out a screen for Tn5 mutants of P. syringae pv. tomato DC3000 with reduced virulence on Arabidopsis thaliana. Several mutants defining both known and novel virulence loci were identified. Six mutants contained insertions in biosynthetic genes for the phytotoxin coronatine (COR). The P. syringae pv. tomato DC3000 COR genes are chromosomally encoded and are arranged in two separate clusters, which encode enzymes responsible for the synthesis of coronafacic acid (CFA) or coronamic acid (CMA), the two defined intermediates in COR biosynthesis. High-performance liquid chromatography fractionation and exogenous feeding studies confirmed that Tn5 insertions in the cfa and cma genes disrupt CFA and CMA biosynthesis, respectively. All six COR biosynthetic mutants were significantly impaired in their ability to multiply to high levels and to elicit disease symptoms on A. thaliana plants. To assess the relative contributions of CFA, CMA, and COR in virulence, we constructed and characterized cfa6 cmaA double mutant strains. These exhibited virulence phenotypes on A. thalliana identical to those observed for the cmaA or cfa6 single mutants, suggesting that reduced virulence of these mutants on A. thaliana is caused by the absence of the intact COR toxin. This is the first study to use biochemically and genetically defined COR mutants to address the role of COR in pathogenesis.

Serotypes of Neisseria meningitidis associated with an increased incidence of meningitis cases in the Hamilton area, Ontario, during 1978 and 1979

1983 ◽  
Vol 29 (1) ◽  
pp. 129-136 ◽  
Author(s):  
Fraser E. Ashton ◽  
J. Alan Ryan ◽  
Colina Jones ◽  
Bernard R. Brodeur ◽  
Benito B. Diena
Keyword(s):  
Neisseria Meningitidis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Age Groups ◽  
Double Diffusion ◽  
Sds Page ◽  
Class 1 ◽  
Group B

The distribution of serotypes among strains of Neisseria meningitidis responsible for a marked increase of meningitis cases in the Hamilton area, Ontario, in 1978 and 1979 was determined. Twenty-six serogroup B and two serogroup W135 strains isolated from cerebrospinal fluid, blood, and skin of 28 patients were serotyped by agar gel double diffusion. Twenty-one (81 %) of the group B strains were serotype 2b as judged by the formation of characteristic serotype precipitin bands with the specific anti-2996 (type 2b) serum. Fourteen of the serotype 2b strains also reacted with anti-77252 serum, which suggested that one strain or several closely related strains were mainly responsible for the increase in meningitis during the 2-year period. Examination of the outer membrane complexes (OMC) of the strains by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS–PAGE) revealed that all 21 of the serotype 2b strains contained the class 2 protein (molecular weight 41 500) which is known to be the site of the serotype 2b determinant. Further characterization of the serotype 2b,77252 strains by enzyme-linked immunosorbent assays (ELISA) and SDS–PAGE suggested that the 77252 determinant was present in the class 1 proteins of these strains. The serotype 2b containing strains were isolated from 77.7 and 70% of males and females, respectively, from 81.8% of children less than 5 years of age, and from 75.0% of patients of all age groups. The study indicates the important role of serotype 2b meningococci in causing the increased incidence of meningitis and further substantiates the important association of the serotype 2b determinant with group B serotype 2 meningococcal disease in Canada.

Excreted/secreted products of developing Taenia saginata metacestodes

Parasitology ◽  
1988 ◽  
Vol 97 (3) ◽  
pp. 477-487 ◽  
Author(s):  
G. W. P. Joshua ◽  
L. J. S. Harrison ◽  
M. M. H. Sewell
Keyword(s):  
Taenia Saginata ◽  
Cellular Infiltrate ◽  
Diagnostic Assays ◽  
Antigenic Characterization ◽  
Sds Page ◽  
Host Parasite Relationship ◽  
Parasite Relationship ◽  
Host Parasite

SummaryExcretions and secretions (ES) and somatic components of 4, 8, 12 and 16-week-old Taenia saginata metacestodes were biosynthetically radio-isotope labelled by incubating the larvae in the presence of [35S]methionine. Despite their small size, 4-week-old metacestodes produced as much isotope-labelled ES/parasite as older metacestodes, indicating a proportionately greater metabolic activity of the parasite at this age. In situ the 4-week-old metacestodes were surrounded by a marked granulomatous cellular infiltrate which had largely resolved around 8-week-old metacestodes. Examination of the isotope-labelled ES by SDS-PAGE revealed distinct age-specific components from 4- and 12-week-old metacestodes and other ES components which were produced by all the ages of metacestodes examined. In comparison the labelled somatic components were conserved. Antigenic characterization of the ES by immunoprecipitation against a panel of clinically defined bovine sera combined with SDS-PAGE analysis, identified some highly immunogenic parasite products and others which did not elicit an antibody response demonstrable by immunoprecipitation. These components are of interest in relation to the host/parasite relationship, to the construction of diagnostic assays for the detection of T. saginata cysticercosis, and to the immunity that cattle develop against this parasite.

Isolation and Characterization of Viridin, a New 65 kDa Antifungal Protein from the Mould Trichoderma viride

1999 ◽  
Vol 380 (10) ◽  
pp. 1243-1245 ◽  
Author(s):  
Jian-Jiang Hao ◽  
Chuan-dong Geng ◽  
Wei-jun Xie ◽  
Zhen-zhen Gong ◽  
Wang-Yi Liu ◽  
...  
Keyword(s):  
High Performance ◽  
Culture Medium ◽  
Basic Protein ◽  
Trichoderma Viride ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Cation Exchange Chromatography ◽  
Isolation And Characterization ◽  
Sds Page

AbstractA new extracellular antifungal protein with a yield of 10 mg per liter was isolated from the culture medium of the mouldTrichoderma viride. The protein, which we named viridin, was purified by carboxymethyl-cellulose cation-exchange chromatography and Superose 12 HR 10/30 high-performance liquid chromatography. Viridin, a basic protein of approximately 65 kDa as determined by SDS-PAGE, inhibits the growth of the cotton pathogenVerticillum dahliae, the IC50being 6 ΜM.

scholarly journals Purification and Characterization of a Prominent Polygalacturonase Isozyme Produced by Phomopsis cucurbitae in Decayed Muskmelon Fruit

HortScience ◽  
1997 ◽  
Vol 32 (3) ◽  
pp. 488F-489
Author(s):  
J.X. Zhang ◽  
B.D. Bruton ◽  
C.L. Biles
Keyword(s):  
Latent Infection ◽  
Gel Filtration ◽  
Central American ◽  
Purification And Characterization ◽  
Fruit Storage ◽  
Sds Page ◽  
Fruit Decay ◽  
Isozyme Activity

Phomopsis cucurbitae is a latent infecting pathogen that infects unripe muskmelon fruit, but causes decay after harvest. This fungus causes severe losses during muskmelon fruit storage and marketing in the U.S., Japan, and some Central American countries. Previous studies showed that the fungus produced the cell wall-degrading enzyme polygalacturonase (PG) in both culture and muskmelon fruit tissue. The role of P. cucurbitae PG in the fruit decay process and its relation to latent infection is not well-understood. A prominent PG isozyme produced by the fungus in decayed fruit was purified to homogeneity by a sequence of extraction, ultrafiltration, preparative isoelectric focusing, anion exchange, and gel filtration chromatography. This isozyme exhibited endo-activity, a molecular weight of 54 kDa according to SDS-PAGE, and a pI of 4.2 based on IEF-PAGE. Isozyme activity was optimal at 40–45°C and pH 5.0. It had a Km of 44.7 g/ml and a Vmax of 0.313. The purified isozyme also effectively macerated mature muskmelon fruit tissues. This isozyme was the most prominent of the PG isozymes produced by P. cucurbitae in decaying fruit, and may play an important role in postharvest decay.

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