RESUMEN
The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.
Asunto(s)
Marcadores de Afinidad/análisis , Marcaje Isotópico/métodos , Espectrometría de Masas/métodos , Proteoma/análisis , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/metabolismo , Cisteína/análisis , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Lactalbúmina/química , Lactalbúmina/metabolismo , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteoma/química , Proteoma/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química , Dodecil Sulfato de Sodio/metabolismo , Factores de Tiempo , Tripsina/metabolismo , Urea/metabolismoRESUMEN
Four isolectins (TEL-I, TEL-II, TEL-III and TEL-IV) were isolated from seeds of Talisia esculenta by reverse-phase high-performance liquid chromatography. RP-HPLC was performed on a u-Bondapack C18 column (0.78 cm x 30 cm) (Waters 991-PDA system) at room temperature. Rechromatography of the four fractions on a C18 column under the same conditions yielded lectins with two dissimilar subunits (Mr 20 kDa and 40 kDa) bound noncovalently. The isolectins showed very similar characteristics, such as molecular masses, N-terminal sequences, and hemagglutinating activity, but differed in their isoelectric points and in inhibition by carbohydrates.
Asunto(s)
Lectinas/aislamiento & purificación , Sapindaceae/embriología , Semillas/química , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Pruebas de Hemaglutinación , Humanos , Focalización Isoeléctrica , Lectinas/química , Lectinas/farmacología , Datos de Secuencia Molecular , Peso Molecular , Lectinas de Plantas , RatasRESUMEN
Given the potential of reactive oxygen species to damage intracellular proteins during subsequent bouts of muscle contractions, it was suggested that, when this production exceeds the antioxidant capacity, the preexisting antioxidant pathways may be complemented by the synthesis of the defense mechanism represented by heat shock proteins (HSPs), stress proteins with the function of repair and maintaining protein folding. To test this hypothesis, we analyzed reactive carbonyl derivatives in plasma and the expression of HSP72 and activities of enzymes from the oxidative and antioxidant defense systems in the soleus muscle of sedentary rats and rats trained by two protocols: continuous and intermittent. We analyzed all three groups at rest and 2 h after acute exercise. After 8 wk of training, the animals from both groups clearly demonstrated higher resistance to exercise. Both trained groups showed significantly higher citrate synthase, catalase, and glutathione reductase activities than the control group (P < 0.01). After acute exercise, catalase and glutathione reductase activities significantly decreased (P < 0.01) and plasma reactive carbonyl derivatives significantly increased (P < 0.05) in the sedentary group, suggesting an oxidative-stress condition as responsible for exhaustion in this group. Finally, after acute exercise, the induction of HSP72 expression occurred only in the sedentary group, suggesting that HSP72 acts as a complementary protective mechanism in exercise-induced oxidative stress.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Músculo Esquelético/fisiología , Estrés Oxidativo , Esfuerzo Físico/fisiología , Animales , Catalasa/metabolismo , Citrato (si)-Sintasa/metabolismo , Glutatión Reductasa/metabolismo , Proteínas del Choque Térmico HSP72 , Masculino , Contracción Muscular , Ratas , Ratas WistarRESUMEN
A thrombin-like enzyme, balterobin, was purified from the venom of Bothrops alternatus. The purification steps included Sephadex G-75, heparin-sepharose and reverse phase HPLC C-18 column. Balterobin showed an apparent molecular weight of 30,000 in non-reduced conditions and displays a specific coagulant activity of 32.8 NIH units/mg over bovine fibrinogen. It also exhibits arginine amidase activity on DL-BAPNA. Like thrombin-like enzymes from other snakes, balterobin possesses valine as N-terminal residue, and is inhibited by PMSF.
Asunto(s)
Bothrops , Coagulantes/aislamiento & purificación , Venenos de Crotálidos/química , Trombina/aislamiento & purificación , Animales , Fibrinógeno/química , Peso MolecularRESUMEN
PIP: The data used were derived from the IPPUR/ITBI/IPTU archive, which contains approximately 2 million annual real estate transactions for the period 1968-88 and more for 1990 for the city of Rio de Janeiro. These registers are maintained for levying taxes and they describe the property, the objective of the transaction, the type, location, size, value as well as participation in the financial system of residency (SFH). This information allows the construction of intraurban mobility matrices, first between 96 neighborhoods of the city and then for 24 administrative regions (RAs) of Rio de Janeiro. Problems were abundant: only 1483 (29%) of 5089 transactions for 1985-88 were used, and 35% for 1990 because of poor data quality. The determinants of intraurban mobility were: 1) demographic (life cycle of families), 2) socioeconomic (changes of employment), and 3) environmental and cultural (dilapidation, violence, pollution, and life style). Mobility trends demonstrated that 46.2% of changes were downward moves and only 33.1% were upward moves. Among upward changes 16.8% involved the acquisition of a new apartment, while among downward moves this constituted only 8.9%. SFH financially assisted the purchase of 14% of upward moves vs. 12.9% of downward moves. Among upward deals in the 6 most favored residential areas, 45.3% of transactions occurred in the city. The moves did not indicate a strong segmentation of the market reaffirming the process of residential segregation between rich and poor people. More than half of real estate acquisitions were realized by families residing in the same RA or in the adjacent RA. More than 75% of transactions for residents of 6 RAs were carried out in the same RA or in adjacent ones. The 10 most important moves (1.74% of all potential moves) involved 21.17% of transactions in the city. The most important moves affected the 3 RAs of Barra da Tijuca of the southern zone, which represented 57.1% of all transactions that occurred in the RA, epitomizing upward mobility of the newly rich.^ieng
Asunto(s)
Vivienda , Dinámica Poblacional , Prejuicio , Movilidad Social , Población Urbana , Américas , Brasil , Demografía , Países en Desarrollo , Economía , Geografía , América Latina , Población , Características de la Población , Características de la Residencia , Clase Social , Factores Socioeconómicos , América del SurRESUMEN
"The paper explores the mediating role of [the] property market in generating residential segregation [in the city of Rio de Janeiro, Brazil].... It is shown that the market for apartments is still basically high-income oriented (in contrast with the situation in cities of the developed countries) and that the rapid (faster than family mobility) spatial deconcentration of this market...has played an important role in expelling...the poor, albeit through indirect means, from the areas subjected more intensively to new property developments." (SUMMARY IN ENG)