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

The proteolytic activity of cardosin B, an aspartic proteinase from the thistle, Cynara cardunculus, on ovine αs-caseins and β-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, αs-caseins were more susceptible to proteolysis by cardosin B than β-casein whereas, in isolated form, the reverse was observed. Sequencing of the peptides produced by hydrolysis of Na-caseinate showed that the major cleavage sites in αs1-casein were Leu156-Asp157 and Trp164-Tyr165 whereas, in β-casein, they were Leu127-Thr128, Leu165-Ser166 and Leu90-Tyr191. The bonds Trpl64-Tyr165 and Leu165-Ser166 were the most susceptible to cardosin B when this enzyme acted upon isolated αs1- and β-casein, respectively

Studies pertaining to coagulant and proteolytic activities of plant proteases from cynara cardunculus

Studies encompassing variation of milk clotting time with the concentration of proteases extracted from Cynara cardunculus were performed; milk clotting time dependence is not linear, and a model was postulated that fits well the experimental data obtained, and hence may be useful in predicting changes during the cheesemaking process. Parallel studies were also conducted pertaining to proteolysis of a mixture of ovine and caprine caseins, in attempts to investigate the stability of the aforementioned coagulating enzymes in crude or in pure form, with or without previous incubation for a certain time under typical ripening conditions. The enzymes exhibited an increase in activity whenever previous incubation had taken place. Moreover, the extent of enzyme-mediated proteolysis was always higher on caprine than on ovine milk caseins

Proteolysis of ovine caseins by cardosin A, an aspartic acid proteinase from Cynara cardunculus L.

The breakdown of as -caseins and ~-caseins (in the form of as -caseins, the form of ~-caseins, and the form of a mixture of as- and ~-caseins in Na-caseinate) by cardosin A, one of the major two proteinases present in the flowers of Cynara cardunculus L., was experimentally studied via urea polyacrylamide gel electrophoresis. In Na-caseinate, as- and ~-caseins were degraded up to 46 and 76 %, respectively, by 10 h of hydrolysis. In separated form, as-caseins reached a leveI of degradationup to 67 %while ~-caseins were quickly and extensively degraded up to 76 %. Ingeneral, ~-caseins seemed to be more susceptible to proteolysis than as-caseins

Caseins as source of bioactive peptides

Biologically active peptides are of particular interest in food science and nutrition because they have been shown to play physiological roles, including opioid-like features, as well as immunostimulating and anti-hypertensive activities, and ability to enhance calcium absorption. Hidden or inactive in the amino-acid sequence of dairy proteins, they can be released or activated in vivo during gastrointestinal digestion, or upstream during food processing via specific, enzyme-mediated proteolysis. Caseins, in either milk or dairy products (e.g. cheese), are important sources of those peptides; their biological significance, their impact on human health and the manufacture of novel functional food ingredients therefrom have been subject to intensive research, which will be briefly presented and critically discussed in this review

Comparative catalytic activity of two plant proteinases upon caprine caseins in solution

The proteolytic activities of cardosins A and B, two (plant) proteinases from Cynara cardunculus, toward caprine caseins, independently, or in the presence of each other as Na-caseinate, were studied in a comparative fashion using polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography. The electrophoretic degradation patterns of both αs- and β-casein, brought about by the cardosins, were similar to one another. In what concerns the specificity of these two enzymes upon caseinate, the major cleavage sites were Leu127-Thr128 and Leu190-Tyr191, both in β-casein. When caseins were tested independently, both cardosins cleaved Phe153-Tyr154 in αs1-casein, as well as Leu127-Thr128 and Leu190-Tyr191 in β-casein

Comparative studies on the gelling properties of cardosins extracted from Cynara cardunculus and chymosin on cow's skim milk

A comparative study was developed on the clottingactivities and gelling properties of cardosins A and B, extracted from dried flowers of Cynara cardunculus, and chymosin on cow’s skim milk, at various pH values. The determination of the total milk-clotting activity was performed followingan international standard, whereas a rheometer was employed to measure the viscoelastic properties of the gels subsequently formed: the evolution of the complex modulus (G ) and the phase angle (d) was monitored with time. The G values of the milk gels were higher for cardosins than for chymosin at pH 6.6, but the reverse held at pH 6.4 and 6.2. The d values were identical for all three enzymes tested. Chymosin exhibited the highest specific milk clotting activity, followed by cardosin B. The clottingactivity of chymosin seems to be more influenced than that of cardosins by the pH of milk

Avaliação da exposição ocupacional a vibrações transmitidas ao sistema corpo inteiro: estudo preliminar em motoristas de pesados de mercadorias

As vibrações transmitidas ao corpo inteiro (VCI) são um agente físico que afecta os condutores de camiões e apresenta consequências para a sua saúde, estando fortemente associada à dor lombar. No presente estudo seleccionou-se um motorista que conduz um veículo pesado de mercadorias com classificação N3 e que efectua rotas de pequeno curso no Norte de Portugal. As medições realizaram-se em dois dias representativos da actividade semanal, em duas rotas distintas (Rota 1 e 2), de acordo com a metodologia definida no Decreto-Lei n.º 46/2006 de 24 de Fevereiro e na NP ISO 2631-1 de 2007. Foi utilizado um analisador de vibrações, sendo o acelerómetro triaxial fixado ao assento do veículo, de forma a quantificar a aceleração eficaz nos três eixos ortogonais x, y e z. Complementarmente ao processo de amostragem, aplicou-se um questionário a sete motoristas. Os resultados demonstram a dominância da direcção vertical (z) com picos de aceleração na frequência de 1,6Hz. A degradação do piso e a ausência de carga do veículo foram identificados como factores responsáveis pelo aumento dos níveis vibracionais. Relativamente à exposição do trabalhador, obteve-se valores de A(8) Rota 1 de 0,22 m/s2 e A(8) Rota 2 de 0,14 m/s2, sendo o A(8) semanal de 0,19 m/s2, verificando-se que os valores obtidos são inferiores ao valor de acção de exposição preconizado na legislação nacional (0,5m/s2). Quanto aos sintomas manifestados pelos motoristas destacam-se: dor lombar (43%), fadiga (71%), irritabilidade (57%) e dores de cabeça (57%). Apesar dos valores obtidos evidenciarem que o trabalhador não se encontra exposto, segundo o preconizados na legislação, devem ser implementadas medidas que visem a manutenção e conservação dos pisos das vias, manutenção dos veículos e elaboração de planos de formação que abordem o tipo de condução e as posturas a adoptar para salvaguarda do bem-estar e conforto dos trabalhadores.info:eu-repo/semantics/publishedVersio

Sustained gene expression in the retina by improved episomal vectors

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.Fundacao para a Ciencia e Tecnologia, Portugal [PEst/OE/EQB-LA 0023/2013, SFRH/BD/76873/2011, SFRH/BD/70318/2010, PTDC/SAU/BEB/098475/2008]; European Union [PIRG-GA-2009-249314

Quantitative studies on the enzymatic hydrolysis of milk proteins brought about by cardosins precipitated by ammonium sulfate

Hydrolysis of whey proteins may produce peptide mixtures with better functional properties than the original protein mixture, viz. higher solubilites and lower allergenic effects. Cynara cardunculus is a wild plant that possesses (aspartic) proteases in its flower cells; those enzymes exhibit general proteolytic and specific milk clotting activities, which are rather useful in traditional cheesemaking. This study was thus aimed at characterizing the enzymatic action of crude extracts of said plant after preliminary purification by salting out with ammonium sulfate at two different concentration levels, viz. 30% and 70% saturation. The coagulant activity on milk, and the proteolytic activity using casein and azocasein as substrates, of the crude extract and of each precipitated fraction were measured at 37°C and pH 5.2. The profile of hydrolysis of the major whey proteins, i.e. α-lactalbumin (α-La), β-lactoglobulin (β-Lg) and bovine serum albumin (BSA) was characterized by gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate. The 30% and 70% saturation fractions exhibited lower coagulant and proteolytic specific activities than the crude extract. However, the relative ratio of coagulant to proteolytic activity, which is a useful indicator of appropriateness for cheesemaking, was higher for the partially purified fractions. The extents of hydrolysis of whey proteins brought about by the partially purified extracts were above those by their crude counterpart, but qualitative hydrolysis patterns were essentially identical to each other; by 24 h, α-La was substantially depleted, whereas β-Lg was very poorly hydrolyzed and BSA was only slightly hydrolyzed. The native proteins were converted to lower and lower molecular weight peptides

Hydrolysis of caprine and ovine milk proteins, brought about by aspartic peptidases from Silybum marianum flowers

The flowers of cardoon (Asteraceae) are a rich source of aspartic peptidases which possess milk clotting activity – and are thus used in traditional cheesemaking in the Iberian Peninsula. This study was aimed at characterizing the enzymatic action of the aspartic peptidases present in flowers of Silybum marianum (L.) Gaertn. (Asteraceae), specifically upon degradation of caseins. The proteolytic activities toward Na-caseinates previously prepared from caprine and ovine milks were studied, in a comparative fashion, using urea-PAGE, tricine-SDS-PAGE, densitometry, electroblotting and sequencing. Caprine as1- and b-caseins were degraded up to 68% and 40%, respectively, during 24 h of incubation. Only one important and well-defined band corresponding to a molecular weight of 14.4 kDa – i.e. a fragment of b-casein, was observed by 12 h of hydrolysis. By 24 h of incubation, ovine as- and b-caseins were degraded up to 76% and 19%, respectively. In what concerns specificity, the major cleavage site in ovine caseinate was Leu99-Arg100 in as1-casei