STIM1 and STIM2 are located in the acidic Ca2+ stores and associates with Orai1 upon depletion of the acidic stores in human platelets

Mammalian cells accumulate Ca2+ into agonist-sensitive acidic organelles, vesicles that possess a vacuolar proton-ATPase. Acidic Ca2+ stores include secretory granules and lysosome-related organelles. Current evidence clearly indicates that acidic Ca2+ stores participate in cell signaling and function, including the activation of store-operated Ca2+ entry in human platelets upon depletion of the acidic stores, although the mechanism underlying the activation of store-operated Ca2+ entry controlled by the acidic stores remains unclear. STIM1 has been presented as the endoplasmic reticulum Ca2+ sensor, but its role sensing intraluminal Ca2+ concentration in the acidic stores has not been investigated. Here we report that STIM1 and STIM2 are expressed in the lysosome-related organelles and dense granules in human platelets isolated by immunomagnetic sorting. Depletion of the acidic Ca2+ stores using the specific vacuolar proton-ATPase inhibitor, bafilomycin A1, enhanced the association between STIM1 and STIM2 as well as between these proteins and the plasma membrane channel Orai1. Depletion of the acidic Ca2+ stores also induces time-dependent co-immunoprecipitation of STIM1 with the TRPC proteins hTRPC1 and hTRPC6, as well as between Orai1 and both TRPC proteins. In addition, bafilomycin A1 enhanced the association between STIM2 and SERCA3. These findings demonstrate the location of STIM1 and STIM2 in the acidic Ca2+ stores and their association with Ca2+ channels and ATPases upon acidic stores discharge.     

Controlling the intracellular localization of fluorescent polyamide analogues in cultured cells

The intracellular distribution of fluorescent-labeled polyamides was examined in live cells. We showed that BODIPY-labeled polyamides accumulate in acidic vesicles, mainly lysosomes, in the cytoplasm of HCT116 colon cancer cells and human rheumatoid synovial fibroblasts (RSF). Verapamil blocked vesicular accumulation and led to nuclear accumulation of the BODIPY-labeled polyamide in RSFs. We infer that the basic amine group commonly found at the end of synthetic polyamide chains is responsible for their accumulation in cytoplasmic vesicles in mammalian cells. Modifying the charge on a polyamide by replacing the BODIPY moiety with a fluorescein moiety on the amine tail allowed the polyamide to localize in the nucleus of the cell and bypass the cytoplasmic vesicles in HCT116 cells.     

A Novel Glucose 6-Phosphate Isomerase from Listeria monocytogenes

d-Arabinose 5-phosphate isomerases (APIs) catalyze the interconversion of d-ribulose 5-phosphate and d-arabinose 5-phosphate (A5P). A5P is an intermediate in the biosynthesis of 3-deoxy-d-manno-octulosonate (Kdo), an essential component of lipopolysaccharide, the lipopolysaccharide found in the outer membrane of Gram-negative bacteria. The genome of the Gram-positive pathogen Listeria monocytogenes contains a gene encoding a putative sugar isomerase domain API, Q723E8, with significant similarity to c3406, the only one of four APIs from Escherichia coli CFT073 that lacks a cystathionine-β-synthase domain. However, L. monocytogenes lacks genes encoding any of the other enzymes of the Kdo biosynthesis pathway. Realizing that the discovery of an API in a Gram-positive bacterium could provide insight into an alternate physiological role of A5P in the cell, we prepared and purified recombinant Q723E8. We found that Q723E8 does not possess API activity, but instead is a novel GPI (d-glucose 6-phosphate isomerase). However, the GPI activity of Q723E8 is weak compared with previously described GPIs. L. monocytogenes contains an ortholog of the well-studied two-domain bacterial GPI, so this maybe redundant. Based on this evidence glucose utilization is likely not the primary physiological role of Q723E8.     

Consequences of Cathodal Stimulation for Behavior: When Does It Help and When Does It Hurt Performance?

Cathodal Transcranial Direct Current Stimulation (C-tDCS) has been reported, across different studies, to facilitate or hinder performance, or simply to have no tangible effect on behavior. This discrepancy is most prominent when C-tDCS is used to alter a cognitive function, questioning the assumption that cathodal stimulation always compromises performance. In this study, we aimed to study the effect of two variables on performance in a simple cognitive task (letter Flanker), when C-tDCS was applied to the left prefrontal cortex (PFC): (1) the time of testing relative to stimulation (during or after), and (2) the nature of the cognitive activity during stimulation in case of post-tDCS testing. In three experiments, we had participants either perform the Flanker task during C-tDCS (Experiment 1), or after C-tDCS. When the Flanker task was administered after C-tDCS, we varied whether during stimulation subjects were engaged in activities that posed low (Experiment 2) or high (Experiment 3) demands on the PFC. Our findings show that the nature of the task during C-tDCS has a systematic influence on the outcome, while timing per se does not.     

Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Recovery of bovine lysozyme from transgenic sugarcane stalks: extraction, membrane filtration, and purification

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20–30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.     

Bayesian Estimation of Hunting Success Rate and Harvest for Spatially Correlated Post‐stratified Data

We consider the estimation of success rate and harvest under post survey stratification at the sub‐domain (county) level. Often in this situation, the population size for the sub‐domain is unknown and the random mechanism that dictates the sample size for sub‐domains is ignored. Finding good estimators of success rate and harvest is very important for wildlife abundance. A Bayesian hierarchical model is developed to estimate both success rate and harvest simultaneously. The model includes a random sub‐domain sample size correlated with the number of successes in the sub‐domain, fixed week effects, random geographic effects, and spatial correlations between neighboring sub‐domains. The computation is done by Gibbs sampling and adaptive rejection sampling techniques. The method developed is illustrated using data from the Missouri Turkey Hunting Survey. The estimation of success rate is improved by treating the the sub‐domain sample size as a random variable instead of a fixed constant. The Bayesian model yields a reasonable harvest estimation. The spatial pattern of the estimated harvest matches the pattern of the check station data.     

Differential responsiveness of human neutrophils to the autocrine actions of 1-O-alkyl-homologs and 1-acyl analogs of platelet-activating factor.

The phlogistic actions of six molecular species of platelet-activating factor (PAF) (1-O-alkyl-PAF homologs, 16:0-, 18:0- and 18:1-alkyl-PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) and their respective 1-acyl-PAF analog counterparts, 16:0-, 18:0- and 18:1-acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (AGPC)) were assessed relative to five human neutrophilic polymorphonuclear leukocyte (PMN) functional responses: 1) lysosomal enzyme secretion; 2) specific desensitization to 16:0-AGEPC-induced lysosomal enzyme secretion; 3) O2- production; 4) chemotaxis; and 5) priming for enhanced O2- production. With respect to inducing lysozyme secretion, 18:0-AGEPC was 30- and 75-fold less potent than 16:0-AGEPC and 18:1-AGEPC, respectively, and was 25- and 40-fold less potent for inducing beta-glucuronidase secretion. 18:0-AGEPC was also 10-fold less active than 18:1- and 16:0-AGEPC for inducing O2- production. Thus, the rank order of potency of the alkyl-PAF homologs for inducing both lysosomal enzyme secretion and O2- production was 18:1- greater than or equal to 16:0- much greater than 18:0-AGEPC. In contrast, these three alkyl-PAF homologs had the same potency for desensitizing PMN to subsequent 16:0-AGEPC-induced lysosomal enzyme secretion and for priming PMN for augmented O2- production in response to FMLP or human recombinant C5a. Paradoxically, however, the rank order of potency of the alkyl-PAF homologs for effecting PMN chemotaxis was 18:0- greater than 18:1- much greater than 16:0-AGEPC. At concentrations as high as 1.0 microM, the acyl-PAF analogs did not initiate PMN lysosomal enzyme secretion, O2- production, or chemotaxis. However, the acyl-PAF analogs induced partial PMN desensitization to 16:0-AGEPC. A novel finding of potential (patho)-physiologic significance was the ability of acyl-PAF at nM concentrations to prime PMN for significantly enhanced O2- production after stimulation with FMLP or human recombinant C5a. The priming action of acyl-PAF was due to an increase in the rate as opposed to a prolongation of O2- production. The differing rank orders of potency of the alkyl-PAF homologs and acyl-PAF analogs for stimulating several physiologic responses of the same target cell, the human PMN, support the premise that there may be more than one PAF receptor subtype on the PMN and/or that differences in the biophysical properties of the various molecular species of PAF modulate their interaction with PAF receptor(s) linked to stimulus-response coupling.