Biochemical characterization of proteins in the crab Homalaspis plana Caracterización bioquímica de las proteínas de jaiba mora (Homalaspis plana).

Homalaspis plana is a crab found in the Pacific Ocean coasts from Guayaquil (Ecuador) all the way down to the Strait of Magellan, including all of the Chilean coast. This crustacean is an important resource because it provides jobs for artisanal fishermen. In Chile there is no product diversification from this crab and it has been poorly studied from the scientific point of view. The biochemical characterization of its proteins was initiated to be able to develop several products from its meat. We report the proteolytic activity from freshly extracted crab meat and the electrophoretical characterization of its proteins. No proteolytic activity was detected in freshly extracted crab meat, under any of the assay conditions used. SDS Polyacrylamide gel electrophoresis (PAGE) and native PAGE were carried out with proteins extracted from crab meat. Using extraction solutions at two different ionic strengths (0.05 and 0.5), myofibrillar and sarcoplasmic were separated, each showing differen

A DNA-modification methylase from Bacillus stearothermophilus V

A type II modification methylase (M BstVI) was partially purified from the thermophilic bacterium Bacillus stearothermophilus V. The methylase catalyses the transfer of methyl groups from S-adenosyl-L-methionine to unmodified double-stranded DNA. The product of methylation was identified by paper chromatography as N6-methyladenine. Since M BstVI protects DNA against cleavage by BstVI and XhoI restriction endonucleases, it follows that it methylates the adenine residue in the sequence 5'-C-T-C-G-A-G-3'

Molecular mechanisms of autophagy in the cardiovascular system

© 2015 American Heart Association, Inc. Autophagy is a catabolic recycling pathway triggered by various intra-or extracellular stimuli that is conserved from yeast to mammals. During autophagy, diverse cytosolic constituents are enveloped by double-membrane vesicles, autophagosomes, which later fuse with lysosomes or the vacuole to degrade their cargo. Dysregulation in autophagy is associated with a diverse range of pathologies including cardiovascular disease, the leading cause of death in the world. As such, there is great interest in identifying novel mechanisms that govern the cardiovascular response to disease-related stress. First described in failing hearts, autophagy within the cardiovascular system has been characterized widely in cardiomyocytes, cardiac fibroblasts, endothelial cells, and vascular smooth muscle cells. In all cases, a window of optimal autophagic activity seems to be critical to the maintenance of cardiovascular homeostasis and function; excessive or insuffic

Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase

1. 1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. 2. This enzyme, already described in plant tissues, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. 8. The mammary gland and salivary gland apyrases eluted as molecu

Autophagy as a therapeutic target in cardiovascular disease

The epidemic of heart failure continues apace, and development of novel therapies with clinical efficacy has lagged. Now, important insights into the molecular circuitry of cardiovascular autophagy have raised the prospect that this cellular pathway of protein quality control may be a target of clinical relevance. Whereas basal levels of autophagy are required for cell survival, excessive levels - or perhaps distinct forms of autophagic flux - contribute to disease pathogenesis. Our challenge will be to distinguish mechanisms that drive adaptive versus maladaptive autophagy and to manipulate those pathways for therapeutic gain. Recent evidence suggests this may be possible. Here, we review the fundamental biology of autophagy and its role in a variety of forms of cardiovascular disease. We discuss ways in which this evolutionarily conserved catabolic mechanism can be manipulated, discuss studies presently underway in heart disease, and provide our perspective on where this exciting fi

Octadecyl silica: A solid phase for protein purification by immunoadsorption

Immunoaffinity chromatography involves binding of an antigen or antibody to a solid matrix, usually agarose, frequently using the cyanogen bromide method. These methods are laborious, rather expensive, and their use has been mostly restricted to immunopurifications on the microscale. We propose here the use of octadecyl silica (SiCl8) beads, a matrix for HPLC, as an alternative solid phase for protein immunopurification and immunoadsorption. Antibodies or antigens are strongly bound to SiCl8 by a simple incubation; radiolabeled antibodies can only be eluted from SiCl8 by detergent-containing solutions. After the remaining free binding sites have been saturated with bovine serum albumin, SiCl8 is incubated with the antigen- or antibody-containing crude preparations and is then poured into a minicolumn. The nonspecifically bound proteins are removed by washing; specific proteins are eluted by disruption of the antigen-antibody complexes with a low pH buffer. With this methodology, we ha

Basal autophagy protects cardiomyocytes from doxorubicin-induced toxicity

Doxorubicin (Doxo) is one of the most effective anti-neoplastic agents but its cardiotoxicity has been an important clinical limitation. The major mechanism of Doxo-induced cardiotoxicity is associated to its oxidative capacity. However, other processes are also involved with significant consequences for the cardiomyocyte. In recent years, a number of studies have investigated the role of autophagy on Doxoinduced cardiotoxicity but to date it is not clear how Doxo alters that process and its consequence on cardiomyocytes viability. Here we investigated the effect of Doxo 1 uM for 24 h of stimulation on cultured neonatal rat cardiomyocytes. We showed that Doxo inhibits basal autophagy. This inhibition is due to both Akt/mTOR signaling pathway activation and Beclin 1 level decrease. To assess the role of autophagy on Doxo-induced cardiomyocyte death, we evaluated the effects 3-methyladenine (3-MA), bafilomycin A1 (BafA), siRNA Beclin 1 (siBeclin 1) and rapamycin (Rapa) on cell viability. Inhibition of autophagy with 3-MA, BafA and siBeclin 1 increased lactate dehydrogenase (LDH) release but, when autophagy was induced by Rapa, Doxo-induced cardiomyocyte death was decreased. These results suggest that Doxo inhibits basal autophagy and contributes to cardiomyocyte death. Activation of autophagy could be used as a strategy to protect the heart against Doxo toxicity.FONDECYT 1120212 11130285 1140329 CONICYT, Chile FONDAP 1513001

Recent insights and therapeutic perspectives of angiotensin-(1-9) in the cardiovascular system

Chronic RAS (renin-angiotensin system) activation by both AngII (angiotensin II) and aldosterone leads to hypertension and perpetuates a cascade of pro-hypertrophic, pro-inflammatory, pro-thrombotic and atherogenic effects associated with cardiovascular damage. In 2000, a new pathway consisting of ACE2 (angiotensin-converting enzyme2), Ang-(1-9) [angiotensin-(1-9)], Ang-(1-7) [angiotensin-(1-7)] and the Mas receptor was discovered. Activation of this novel pathway stimulates vasodilation, anti-hypertrophy and anti-hyperplasia. For some time, studies have focused mainly on ACE2, Ang-(1-7) and the Mas receptor, and their biological properties that counterbalance the ACE/AngII/AT1R (angiotensin type 1 receptor) axis. No previous information about Ang-(1-9) suggested that this peptide had biological properties. However, recent data suggest that Ang-(1-9) protects the heart and blood vessels (and possibly the kidney) from adverse cardiovascular remodelling in patients with hypertension and