The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content.

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker® Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.

Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavß2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gßγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Gißγ-AKT-Cavß2 pathway, suggesting a new mechanism for regulation of cardiac function.

Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

Cardiomyocyte death: mechanisms and translational implications.

Cardiovascular disease (CVD) is the leading cause of morbidity and mortality worldwide. Although treatments have improved, development of novel therapies for patients with CVD remains a major research goal. Apoptosis, necrosis, and autophagy occur in cardiac myocytes, and both gradual and acute cell death are hallmarks of cardiac pathology, including heart failure, myocardial infarction, and ischemia/reperfusion. Pharmacological and genetic inhibition of autophagy, apoptosis, or necrosis diminishes infarct size and improves cardiac function in these disorders. Here, we review recent progress in the fields of autophagy, apoptosis, and necrosis. In addition, we highlight the involvement of these mechanisms in cardiac pathology and discuss potential translational implications.

An inositol 1,4,5-triphosphate (IP3)-IP3 receptor pathway is required for insulin-stimulated glucose transporter 4 translocation and glucose uptake in cardiomyocytes.

Intracellular calcium levels ([Ca2+]i) and glucose uptake are central to cardiomyocyte physiology, yet connections between them have not been studied. We investigated whether insulin regulates [Ca2+]i in cultured cardiomyocytes, the participating mechanisms, and their influence on glucose uptake via SLC2 family of facilitative glucose transporter 4 (GLUT4). Primary neonatal rat cardiomyocytes were preloaded with the Ca2+ fluorescent dye fluo3-acetoxymethyl ester compound (AM) and visualized by confocal microscopy. Ca2+ transport pathways were selectively targeted by chemical and molecular inhibition. Glucose uptake was assessed using [3H]2-deoxyglucose, and surface GLUT4 levels were quantified in nonpermeabilized cardiomyocytes transfected with GLUT4-myc-enhanced green fluorescent protein. Insulin elicited a fast, two-component, transient increase in [Ca2+]i. Nifedipine and ryanodine prevented only the first component. The second one was reduced by inositol-1,4,5-trisphosphate (IP3)-receptor-selective inhibitors (xestospongin C, 2 amino-ethoxydiphenylborate), by type 2 IP3 receptor knockdown via small interfering RNA or by transfected Gßγ peptidic inhibitor ßARKct. Insulin-stimulated glucose uptake was prevented by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM, 2-amino-ethoxydiphenylborate, and ßARK-ct but not by nifedipine or ryanodine. Similarly, insulin-dependent exofacial exposure of GLUT4-myc-enhanced green fluorescent protein was inhibited by bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid-AM and xestospongin C but not by nifedipine. Phosphatidylinositol 3-kinase and Akt were also required for the second phase of Ca2+ release and GLUT4 translocation. Transfected dominant-negative phosphatidylinositol 3-kinase γ inhibited the latter. In conclusion, in primary neonatal cardiomyocytes, insulin induces an important component of Ca2+ release via IP3 receptor. This component signals to glucose uptake via GLUT4, revealing a so-far unrealized contribution of IP3-sensitive Ca2+ stores to insulin action. This pathway may influence cardiac metabolism in conditions yet to be explored in adult myocardium.

Parallel activation of Ca(2+)-induced survival and death pathways in cardiomyocytes by sorbitol-induced hyperosmotic stress.

Hyperosmotic stress promotes rapid and pronounced apoptosis in cultured cardiomyocytes. Here, we investigated if Ca(2+) signals contribute to this response. Exposure of cardiomyocytes to sorbitol [600 mosmol (kg water)(-1)] elicited large and oscillatory intracellular Ca(2+) concentration increases. These Ca(2+) signals were inhibited by nifedipine, Cd(2+), U73122, xestospongin C and ryanodine, suggesting contributions from both Ca(2+) influx through voltage dependent L-type Ca(2+) channels plus Ca(2+) release from intracellular stores mediated by IP(3) receptors and ryanodine receptors. Hyperosmotic stress also increased mitochondrial Ca(2+) levels, promoted mitochondrial depolarization, reduced intracellular ATP content, and activated the transcriptional factor cyclic AMP responsive element binding protein (CREB), determined by increased CREB phosphorylation and electrophoretic mobility shift assays. Incubation with 1 mM EGTA to decrease extracellular [Ca(2+)] prevented cardiomyocyte apoptosis induced by hyperosmotic stress, while overexpression of an adenoviral dominant negative form of CREB abolished the cardioprotection provided by 1 mM EGTA. These results suggest that hyperosmotic stress induced by sorbitol, by increasing Ca(2+) influx and raising intracellular Ca(2+) concentration, activates Ca(2+) release from stores and causes cell death through mitochondrial function collapse. In addition, the present results suggest that the Ca(2+) increase induced by hyperosmotic stress promotes cell survival by recruiting CREB-mediated signaling. Thus, the fate of cardiomyocytes under hyperosmotic stress will depend on the balance between Ca(2+)-induced survival and death pathways.

Maple syrup urine disease (MSUD)--clinical profile of 47 Filipino patients.

Maple syrup urine disease (MSUD) is a very rare disorder of branched-chain amino acid metabolism. However, it is the most common inborn error of metabolism in the Philippines. We present a retrospective review of 21 patients diagnosed with MSUD between 1999 and 2004. The patients presented clinically between 2 and 14 days of life (mean 5 days) and the diagnosis of MSUD was established between 6 days and 11 months of age (mean 39 days). The classical burnt sugar odour was noted in the majority of patients (81%). The diagnosis of MSUD was initially based on clinical suspicion and confirmed biochemically by measurement of leucine/isoleucine levels by thin-layer chromatography. The acute management included removal of accumulated branched-chain amino acids by peritoneal dialysis in 62% of the patients. Mortality rate of this group of patients was 24% and follow-up rate was 87%. We compared this series with a previously reported series of 26 patients to determine whether diagnosis and the management of MSUD improved over the two periods. Four cases have been diagnosed early since 1992, the majority of whom had the classic form of MSUD with the onset of symptoms in the first two weeks of life. A small subset of patients with early nonspecific symptoms was diagnosed much later owing to a low-level clinical suspicion among clinicians. Overall, however, there appears to be a small but general trend towards earlier diagnosis, reduced mortality and long-term follow up in the later series. Although we are able to diagnose and manage MSUD in the Philippines, we recognize that the clinical outcome remains poor and is due mainly to late referral of cases and inadequate long-term management. In the Philippines, we recommend that all newborns who are considered to be septic, have feeding difficulties, fail to regain their birth weight or present with any other symptoms suggestive of MSUD be evaluated in the first instance by analysis of urine for ketones and if they are positive have blood collected and sent to our laboratory for leucine/isoleucine measurement.

Transient multiple acyl-CoA dehydrogenation deficiency in a newborn female caused by maternal riboflavin deficiency.

A newborn female presented on the first day of life with clinical and biochemical findings consistent with multiple acyl-CoA dehydrogenase deficiency (MADD). Riboflavin supplementation corrected the biochemical abnormalities 24 h after commencing the vitamin. In vitro acylcarnitine profiling in intact fibroblasts both in normal and riboflavin depleted media showed normal oxidation of fatty acids excluding defects in electron transfer flavoprotein (ETF), or ETF ubiquinone oxidoreductase (ETF:QO), or a genetic abnormality in flavin metabolism. In addition, sequencing of the genes encoding ETF and ETF:QO in the proband did not reveal any pathogenic mutations. Determination of the maternal riboflavin status after delivery showed that the mother was riboflavin deficient. Repeat testing done two years after the infant's birth and while on a normal diet showed that the mother was persistently riboflavin deficient and showed a typical MADD profile on plasma acylcarnitine testing. A possible genetic defect in riboflavin transport of metabolism in the mother is postulated to be the cause of the transient MADD seen in the infant. Sequencing of the SLC16A12, RFK and FLAD1 genes encoding key enzymes in riboflavin transport of metabolism in the mother did not identify any pathogenic mutations. The underlying molecular basis of the mother's defect in riboflavin metabolism remains to be established.

Low citrulline may not be diagnostic of ornithine transcarbamylase deficiency: a case report.

A newborn boy with family history of severe ornithine transcarbamylase (OTC) deficiency was investigated prospectively and managed aggressively at birth based on an existing protocol for at risk neonates. Undetectable citrulline levels at birth suggested that the infant was affected; however, normal plasma glutamine and urine orotic acid levels confused the diagnosis to some extent. Mutation testing confirmed that the patient did not have OTC deficiency. Thus the low plasma citrulline level did not validate our initial biochemical suspicion of OTC deficiency, and this highlights the importance of considering all available clinical, biochemical and molecular evidence in determining disease status.

Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype.

Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004-2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.

Lesch-Nyhan disease in a 20-year- old man incorrectly described as developing 'cerebral palsy' after general anaesthesia in infancy.

Lesch-Nyhan disease (LND) is a rare X-linked recessive genetic disorder caused by a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) enzyme. The classic clinical condition is characterized by cognitive impairment, hypotonia at rest, choreoathetosis, hyperuricaemia and the hallmark symptom of severe and involuntary self-mutilation. We describe a man with LND who was initially thought to have suffered from a dyskinetic cerebral palsy after an uncomplicated inguinal herniorrhaphy under general anaesthesia at 5 1/2 months of age. In the absence of overt self-injurious behaviour, the diagnosis was not considered for nearly two decades. The diagnosis of LND was established at 20 years of age through clinical review, biochemical examinations and molecular analysis. HPRT haemolysate activity was 7.6% of the normal control, suggesting that he had a milder variant of the disease. Mutation analysis of the HPRT gene revealed a novel missense mutation, c.449T > G in exon 6 (p.V150G). Cascade testing of family members revealed that the mother was heterozygous for the mutation but two siblings (a brother and a sister) did not carry the sequence mutation. Whether the onset of neurological abnormalities in this particular case can be attributed to the general anaesthesia is discussed.

Purification and characterization of an iron-nickel hydrogenase from Thermococcus celer.

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H2. T. celer hydrogenase had an apparent MW of 155,000+/-30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000+/-2,000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent Mr 84,000+/-3,000. Measurements of iron and acid-labile sulfide in different preparations of T. celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.

Direct electrochemical characterization of hyperthermophilic Thermococcus celer metalloenzymes involved in hydrogen production from pyruvate.

The reduction potentials of the metalloproteins pyruvate ferredoxin oxidoreductase (POR), ferredoxin, and hydrogenase isolated from hyperthermophilic Thermococcus celer (Topt = 88 degrees C) were determined as a function of temperature from 10 to 85 degrees C. Square-wave voltammetry experiments were carried out on 15 microL samples directly at an unmodified "edge-polished" pyrolytic graphite electrode using MgCl2 as an electrode promoter. POR exhibited two voltammetric waves with peaks at -280 and -403 mV at room temperature, indicating multiple redox centers, and a single wave at -420 mV at 85 degrees C. These waves displayed different temperature-dependent peak positions and peak heights, indicating that these redox centers have different thermodynamic and kinetic properties. Ferredoxin displayed a single linear temperature-dependent voltammetric wave at -280 mV at room temperature and -327 mV at 85 degrees C. Hydrogenase displayed a single biphasic temperature-dependent voltammetric wave at -197 mV at room temperature and -211 mV at 85 degrees C. Thermodynamic parameters associated with electron transfer, namely standard enthalpies and entropies for the redox centers in the various proteins, are reported.

Optimization of the growth conditions of the extremely thermophilic microorganisms Thermococcus celer and Pyrococcus woesei.

Growth medium components and cultivation conditions for the extremely thermophilic Archaea Thermococcus celer and Pyrococcus woesei were optimized. A culture media based in marine water was formulated. Both Archaea demonstrated to be strictly anaerobic with optimal growth temperature of 85 degrees and 95 degrees C, respectively. Sodium sulfide, but not cysteine, was used as a sulfur and reductive capacity source. It was observed that hydrogen sulfide could be replaced by 30 microM titanium (III) nitrile acetate. The addition of elemental S(o) enhanced growth of both microorganisms, with T. celer far more sensitive than P. woesei to the absence of S(o). P. woesei utilized maltose as a carbon source, while T. celer was able to use only peptides from yeast extract, peptone and tryptone as its carbon source. Optimum carbon source concentrations were 1.25 g/L for T. celer and 5 g/L for P. woesei. Although both Archaea required peptides as a nitrogen source, the addition of ammonia chloride to a nitrogen-limited media did not stimulate growth, which suggests that neither Archaea appear to metabolize ammonia. The growth of P. woesei, but not T. celer, was stimulated considerably in the presence of iron. Co, Ni, Zn, Mo. Mn and Mg were essential trace elements needed for optimal growth of both bacteria.

Omeprazole, a specific gastric secretion inhibitor on oxynticopeptic cells, reduces gizzard erosion in broiler chicks fed with toxic fish meals.

The relation between gizzard erosion-black vomit (GE-BV) and gastric secretion is not completely understood. A pharmacological approach to reduce the presence of GE-BV in chicks due to fish meal in diets is also unknown. In this study the use of omeprazole, a H+/K+ ATPase inhibitor, and fish meals of different biotoxicological characteristics, showed that: 1) Omeprazole decreased total gastric acid content, GE scores and severe GE (SGE) cases, in a dose-dependent manner. This reduction was significant at levels higher than 20 mg omeprazole/Kg body weight (BW)/day (p < 0.01). The addition of 50 mg omeprazole/kg BW/day almost completely prevented the incidence of SGE cases and reduced in 50% GE score in chicks (p < 0.01). 2) A significant reduction in specific mortality, near 90%, was also seen with all toxic fish meals when omeprazole (50 mg/Kg BW/day) was added to experimental diets in comparison to control groups. However, no mortality was observed when omeprazole was added to diets containing non-toxic fish meals. 3) In chicks fed with toxic fish meals, addition of different amounts of omeprazole to diets changed the relative weight of proventriculus (p < 0.01) and gizzard (p < 0.05). Maximum effect was obtained with omeprazole concentration higher than 50 mg/Kg BW/day. 4) Omeprazole did not change feed intake in chicks fed with toxic fish meal. However, in some fish meal a reduction on weight gain was observed with the addition of omeprazole.

Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli.

Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.

[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 different electrophoretical patterns in SDS-PAGE.

Human placental ATP-diphosphohydrolase: biochemical characterization, regulation and function.

1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M(r) and pI values of both ATPase-ADPase activities. 2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher. 3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DES and slightly by DCCD. 4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A. 5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of -SH, COO-, -OH, and probably also Tyr and Trp. 6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes. 7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

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 have obtained high purity preparations of proteins in single steps, even when these proteins are present in trace amounts (picograms) in a complex mixture such as human serum. Similarly, specific antibodies against an intracellular parasite (Trypanosoma cruzi) were completely absorbed from human serum with SiCl8 coated with parasite antigens.