Highlights d STIM1B but not STIM1 localizes to presynaptic ER d STIM1B causes increased vesicle release at high demand d STIM1B alters slow Ca 2+ -dependent inactivation of I CRAC d Splicing alters frequency dependence of synapses and is decreased in AD

Background: Altered intracellular Ca 2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca 2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. Methods: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca 2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. Results: Altered thapsigargin-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor [TMEM66]) were found in neonatal platelet as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca 2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca 2+ –dependent inactivation, so reproducing the Ca 2+ -homeostasis pattern observed in neonatal platelets. Conclusions: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. S-STIM1 may avoid slow Ca 2+ –dependent inactivation and, subsequently, result in an enhanced thapsigargin-evoked SOCE as observed in neonatal platelets.

This study was designed to assess the production of nitric oxide (NO) by neutrophils in bronchial asthma.Thirty asthmatic patients (ten each of mild, moderate and severe asthma) and ten healthy controls were included in the study. Neutrophils from peripheral venous blood were stimulated with latex, and production of nitrite (an NO metabolise) and L-citrulline (a co-product of NO) was studied. It was postulated that peripheral blood neutrophils, being in a primed or activated state in asthma, would reflect the changes occurring in bronchial tree neutrophils.Nitrite and L-citrulline production by neutrophils was significantly higher in asthmatics (pv0.001) and increased with disease severity. A strong negative correlation was observed between peak expiratory flow and both nitrite (r~-0.87, pv0.001) and L-citrulline (r~-0.88, pv0.001) production.It is concluded that nitric oxide production by neutrophils is increased in bronchial asthma and can possibly contribute to airway narrowing and disease severity.

No abstract

No abstract