Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1.

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

TRP channel function in platelets and megakaryocytes: basic mechanisms and pathophysiological impact.

Transient receptor potential (TRP) proteins form a superfamily of cation channels that are expressed in a wide range of tissues and cell types. During the last years, great progress has been made in understanding the molecular complexity and the functions of TRP channels in diverse cellular processes, including cell proliferation, migration, adhesion and activation. The diversity of functions depends on multiple regulatory mechanisms by which TRP channels regulate Ca2+ entry mechanisms and intracellular Ca2+ dynamics, either through membrane depolarization involving cation influx or store- and receptor-operated mechanisms. Abnormal function or expression of TRP channels results in vascular pathologies, including hypertension, ischemic stroke and inflammatory disorders through effects on vascular cells, including the components of blood vessels and platelets. Moreover, some TRP family members also regulate megakaryopoiesis and platelet production, indicating a complex role of TRP channels in pathophysiological conditions. In this review, we describe potential roles of TRP channels in megakaryocytes and platelets, as well as their contribution to diseases such as thrombocytopenia, thrombosis and stroke. We also critically discuss the potential of TRP channels as possible targets for disease prevention and treatment.

Genetic Background Behind the Amino Acid Profiles of Fermented Soybeans Produced by Four Bacillus spp.

Strains of four Bacillus spp. were respectively inoculated into sterilized soybeans and the free amino acid profiles of the resulting cultures were analyzed to discern their metabolic traits. After 30 days of culture, B. licheniformis showed the highest production of serine, threonine, and glutamic acid; B. subtilis exhibited the highest production of alanine, asparagine, glycine, leucine, proline, tryptophan, and lysine. B. velezensis increased the γ-aminobutyric acid (GABA) concentration to >200% of that in the control samples. B. sonorensis produced a somewhat similar amino acid profile with B. licheniformis. Comparative genomic analysis of the four Bacillus strains and the genetic profiles of the produced free amino acids revealed that genes involved in glutamate and arginine metabolism were not common to the four strains. The genes gadA/B (encoding a glutamate decarboxylase), rocE (amino acid permease), and puuD (γ-glutamyl-γ-aminobutyrate hydrolase) determined GABA production, and their presence was species-specific. Taken together, B. licheniformis and B. velezensis were respectively shown to have high potential to increase concentrations of glutamic acid and GABA, while B. subtilis has the ability to increase essential amino acid concentrations in fermented soybean foods.

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response.

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 µM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 µM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

The power metric: a new statistically robust enrichment-type metric for virtual screening applications with early recovery capability.

A new metric for the evaluation of model performance in the field of virtual screening and quantitative structure-activity relationship applications is described. This metric has been termed the power metric and is defined as the fraction of the true positive rate divided by the sum of the true positive and false positive rates, for a given cutoff threshold. The performance of this metric is compared with alternative metrics such as the enrichment factor, the relative enrichment factor, the receiver operating curve enrichment factor, the correct classification rate, Matthews correlation coefficient and Cohen's kappa coefficient. The performance of this new metric is found to be quite robust with respect to variations in the applied cutoff threshold and ratio of the number of active compounds to the total number of compounds, and at the same time being sensitive to variations in model quality. It possesses the correct characteristics for its application in early-recognition virtual screening problems.

Mechanism of dopamine D2 receptor-induced Ca(2+) release in PC-12 cells.

Intracellular Ca(2+) levels are tightly regulated in the neuronal system. The loss of Ca(2+) homeostasis is associated with many neurological diseases and neuropsychiatric disorders such as Parkinson's, Alzheimer's, and schizophrenia. We investigated the mechanisms involved in intracellular Ca(2+) signaling in PC-12 cells. The stimulation of NGF-differentiated PC-12 cells with 3µM ATP caused an early Ca(2+) release followed by a delayed Ca(2+) release. The delayed Ca(2+) release was dependent on prior ATP priming and on dopamine secretion by PC-12 cells. Delayed Ca(2+) release was abolished in the presence of spiperone, suggesting that it is due to the activation of D2 dopamine receptors (D2R) by dopamine secreted by PC-12 cells. This was shown to be independent of PKA activation but dependent on PLC activity. An endocytosis step was required for inducing the delayed Ca(2+) release. Given the importance of calcyon in clathrin-mediated endocytosis, we verified the role of this protein in the delayed Ca(2+) release phenomenon. siRNA targeting of calcyon blocked the delayed Ca(2+) release, decreased ATP-evoked IP3R-mediated Ca(2+) release, and impaired subsequent Ca(2+) oscillations. Our results suggested that calcyon is involved in an unknown mechanism that causes a delayed IP3R-mediated Ca(2+) release in PC-12 cells. In schizophrenia, Ca(2+) dysregulation may depend on the upregulation of calcyon, which maintains elevated Ca(2+) levels as well as dopamine signaling.

Tratamiento quirúrgico del síndrome de Roce Escápulo-torácico / Surgical treatment of the scapulothoracic friction syndrome

INTRODUCCIËN: El Síndrome de Roce Escápulo-torácico es una causa de dolor en la región posterior del hombro y que puede ser causado por una disfunción de esta articulación. Cuando falla el tratamiento conservador o cuando se debe a lesiones tumorales, está indicado el tratamiento quirúrgico. Con el presente trabajo nos propusimos evaluar los resultados obtenidos con este tratamiento en nuestro grupo de estudio. MÉTODOS: Se realizó un estudio de intervención en 17 pacientes portadores de un Síndrome de Roce Escápulo - torácico, que fueron atendidos quirúrgicamente en el Servicio de Miembro Superior del Complejo Científico Ortopédico Internacional Frank País entre marzo 2002 y marzo 2008. La técnica quirúrgica estuvo en correspondencia con la causa desencadenante del Roce Escápulo- torácico. La valoración funcional de los resultados se efectuó a los 6 meses de la operación, se tuvo como referencia tres de las categorías del Test de valoración funcional del hombro de la Universidad de los Ángeles, California (dolor, función y grado de satisfacción del paciente); además se le agregaron a este test, tres categorías para nuestro estudio (flexión, abducción y antepulsión). RESULTADOS: La edad promedio en nuestra serie fue de 31,8 años; 15 pacientes (89,4 por ciento) correspondieron al sexo femenino, los grupos de edades más afectados los menores de 20 años y entre 31 a 40 con 6 casos cada uno, predominaron las lesiones en el miembro dominante 10 pacientes (58,8 por ciento) y aparecieron con mayor frecuencia en los intelectuales. La evaluación del dolor, la función, la flexión, la abducción y la antepulsión mostró una tendencia significativa a la mejoría del puntaje. El 82 por ciento de los pacientes tuvieron resultados excelente o bueno

INTRODUCTION: The scapulothoracic friction syndrome is a pain cause in the posterior area of the shoulder and that may be provoked by a dysfunction of this joint. When conservative treatment fails or when it is due to tumor injuries, surgical treatment is prescribed. The aim of present paper is to assess the results obtained with this type of treatment in our study group. METHODS: An intervention study was conducted in 17 patients with a scapulothoracic friction syndrome operated on the Upper Member Service of the Frank País International Orthopedics Scientific Complex from March, 2002 to March, 2008. Surgical technique was in correspondence with the triggering cause of this syndrome. Functional assessment of results was made at 6 months after surgery taking into account three categories of the Shoulder Functional Assessment Test of the Los Angeles University, California (pain, function and the patient's satisfaction grade), also for our study, three categories more were added to this test (flexion, abduction and antepulsion). RESULTS: Mean age in our series was of 31,8 years; 15 patients (89,4 percent were women, the more involved age groups were those under 20 years and between 31 to 40 years each, with predominance of the dominant limb in 10 patients (58,8 percent) with greater frequency in the intellectual ones. Assessment of pain, function, flexion, abduction and antepulsion showed a significant trend to improve of score. The 82 percent of patients showed excellent or good results