Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein

The action of Cynara cardunculus L. protease on whole bovine K-casein, over a 3-h period at pH 6.4, was investigated. RpHPLC of the 3% trichloroacetic acid (TCA)-solublefraction of the K-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the PhelO5- Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled K-casein (FTC- K-casein). The values obtained for kat, k, and k were 1.04 s-l, 0.16 pM, and 6.5 pM-l s-l, respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the k, is significantly lower, which reflects the higher affinity of Cynara protease to K-casei

Escherichia coli expression, refolding and characterization of human laforin

Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5–7 mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.Fundação para a Ciência e a Tecnologia (FCT) – Programa Operacional “Ciência, Tecnologia, Inovação” (POCTI

Motivation and Anxiety in adult strength training practitioners during the Covid-19 pandemic

The objective of this study was to analyze which motivational factors influenced the search for the practice of strength training (ST) and its relationship with anxiety in adults during the new coronavirus (COVID-19) pandemic. Forty-three ST practitioners analyzed their motivational factors using the Exercise Motivations Inventory - 2 (EMI-2) and their anxiety level using the Depression and Stress Anxiety Scale (DASS-21). Friedman's tests were used to compare motivational factors, and Spearman's tests were used to relate the EMI-2 results with the DASS-21 anxiety subscale. The main motivational factors for the practice of ST were physical condition, disease prevention, fun and well-being, and stress control. Anxiety levels showed a negative relationship with the affiliation factor for older ST practitioners (≥26 years). It is concluded that health care is the biggest motivational aspect for the practice of ST during the Covid-19 Pandemic

Caninos y felinos en condición de calle: Relevamiento estadístico en la Ciudad Autónoma de Buenos Aires / Canines and Felines in Street Condition: Statistical Survey in the Autonomous City of Buenos Aires

In the city of Buenos Aires, Argentine Republic, not only the census but also the records in private veterinarians allow you to  estimate the animals with responsible holder, but to guarantee the success of health plans for the ethical control of urban fauna is necessary to estimate on a consistent basis. The presence of stray pets (dogs and cats) was registered in all the communes of the Autonomous City of Buenos Aires, using a systematic sampling, selecting a set of streets in each commune in the form of Greek guard. The results showed, based on 208 sightings, a total of dogs in street condition of 91,73 ± 33,12 animals every 1000 blocksEn la Ciudad de Buenos Aires, República Argentina, tanto los censos como los registros en veterinarias privadas permiten estimar los animales con tenedor responsable, pero para garantizar el éxito de los planes sanitarios destinados al control ético de la fauna urbana se requiere estimar sobre una base consistente. Se registró la presencia de animales domésticos vagabundos ( perros y gatos) en todas las comunas de la Ciudad Autónoma de Buenos Aires, utilizando un muestreo sistemático, seleccionando en forma de guarda griega una serie de calles en cada comuna. Los resultados demostraron, en base a 208 avistamientos, un total de perros en condición de calle del 91,73 ± 33,12 animales cada 1000 cuadra

Expression of the functional carbohydrate-binding module (CBM) of human laforin

Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin’s CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin’s CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.Fundação para a Ciência e a Tecnologia (FCT, Portugal)

Molecular analysis of the interaction between cardosin A and phospholipase Dα

Cardosin A is an RGD-containing aspartic proteinase from the stigmatic papillae of Cynara cardunculus L. A putative cardosin A-binding protein has previously been isolated from pollen suggesting its potential involvement in pollen2013pistil interaction [Faro C, Ramalho-Santos M, Vieira M, Mendes A, Simões I, Andrade R, Verissimo P, Lin X, Tang J & Pires E (1999) J Biol Chem274, 28724201328729]. Here we report the identification of phospholipase D03B1 as a cardosin A-binding protein. The interaction was confirmed by coimmunoprecipitation studies and pull-down assays. To investigate the structural and molecular determinants involved in the interaction, pull-down assays with cardosin A and various glutathione S-transferase-fused phospholipase D03B1 constructs were performed. Results revealed that the C2 domain of phospholipase D03B1 contains the cardosin A-binding activity. Further assays with mutated recombinant forms of cardosin A showed that the RGD motif as well as the unprecedented KGE motif, which is structurally and charge-wise very similar to RGD, are indispensable for the interaction. Taken together our results indicate that the C2 domain of plant phospholipase D03B1 can act as a cardosin A-binding domain and suggest that plant C2 domains may have an additional role as RGD/KGE-recognition domains