STIM1 regulates store-operated Ca entry in oocytes

The single transmembrane-spanning Ca²⁺-binding protein, STIM1, has been proposed to function as a Ca²⁺ sensor that links the endoplasmic reticulum to the activation of store-operated Ca²⁺ channels. In this study, the presence, subcellular localization and function of STIM1 in store-operated Ca²⁺ entry in oocytes was investigated using the pig as a model. Cloning and sequence analysis revealed the presence of porcine STIM1 with a coding sequence of 2058 bp. In oocytes with full cytoplasmic Ca²⁺ stores, STIM1 was localized predominantly in the inner cytoplasm as indicated by immunocytochemistry or overexpression of human STIM1 conjugated to the yellow fluorescent protein. Depletion of the Ca²⁺ stores was associated with redistribution of STIM1 along the plasma membrane. Increasing STIM1 expression resulted in enhanced Ca²⁺ influx after store depletion and subsequent Ca²⁺ add-back; the influx was inhibited when the oocytes were pretreated with lanthanum, a specific inhibitor of store-operated Ca²⁺ channels. When STIM1 expression was suppressed using siRNAs, there was no change in cytosolic free Ca²⁺ levels in the store-depleted oocytes after Ca²⁺ add-back. The findings suggest that in oocytes, STIM1 serves as a sensor of Ca²⁺ store content that after store depletion moves to the plasma membrane to stimulate store-operated Ca²⁺ entry.     

Abscisic Acid Decreases Leaf Na+ Exclusion in Salt-Treated Phaseolus vulgaris L.

Previous results showed that in short-term NaCl-treated beans increased leaf abscisic acid (ABA) concentration was triggered by Na⁺ but not by Cl^-. In this work, the specificity of ABA signaling for Na⁺ homeostasis was studied by comparing the plant’s responses to solutions that modified accumulation of ABA and/or Na⁺ uptake and distribution, such as supplemental Ca²⁺, increased nutrient strength, different isosmotic composition, application of exogenous ABA, fluridone (an ABA inhibitor) and aminooxiacetic acid (AOA, an ethylene inhibitor). After fluridone pretreatment, salt-treated beans had lower Na⁺ uptake and higher leaf Na⁺ exclusion capacity than non-pretreated plants. Moreover, Na⁺ uptake was increased and leaf Na⁺ exclusion was decreased by AOA and ABA. NaCl and KCl similarly increased leaf ABA and decreased transpiration rates, whereas supplemental Ca²⁺ and increased strength nutrient solution decreased leaf ABA and leaf Na⁺. These results show (1) a non-ion-specific increase in ABA that probably signaled the osmotic component of salt, and (2) increased ABA levels that resulted in higher leaf Na⁺ concentrations due to lower Na⁺ exclusion or increased root-shoot Na⁺ translocation.     

Reassembly of flagellar B (alpha beta) tubulin into singlet microtubules: consequences for cytoplasmic microtubule structure and assembly

B(alpha beta) tubulin was obtained from a homogeneous class of microtubules, the incomplete B subfiber of sea urchin sperm flagellar doublet microtubules, by thermal fractionation. The thermally derived soluble B tubulin fraction (100, 000 g-h) repolymerizes in vitro, yielding microtubule-like structures. The microtubule-associated protein (MAP) composition and certain assembly parameters of thermally derived B tubulin are different from those reported for sonication- derived flageller tubulin and purified vertebrate tubulin. The "microtubules" reassembled from thermally prepared B tubulin are composed of 12-15 protofilaments (73% possess 14 protofilaments). A certain number possess a single "adlumenal component" applied to their inside walls, regardless of the number of protofilaments. Following the first cycle of polymerization, 81% of the B tubulin and essentially 100% of the MAPs remain cold insoluble. Evidence suggests that B tubulin assembles faithfully into a B lattice, creating a j seam between two protofilaments that are laterally bonded in a A-lattice configuration. The significance of these seams is discussed in relation to the mechanism of microtubule assembly, the stability of observed ribbons of protofilaments, and the three-dimensional organization of microtubule-associated components.     

Endogenous abscisic acid levels are linked to decreased growth of bush bean plants treated with NaCl

This paper studies the relative importance of endogenous ABA and ion toxicity in the leaf growth inhibition caused by NaCl in salt-adapted and unadapted bush beans. Adaptation to salt-stress was achieved by germination of seeds in 75 m M NaCl, while unadapted plants were germinated in tap water. The adaptation process caused a transitory increase in leaf ABA, Na⁺ and Cl⁻ concentrations, while leaf expansion was inhibited. However, when grown for 8 or 13 days in 75 m M NaCl-containing nutrient solution, primary and first trifoliolate leaves of salt-adapted plants had greater areas than those of unadapted plants. Concentrations of ABA, Na⁺ and Cl⁻ in these leaves were lower in adapted plants, and a strong negative correlation between leaf expansion growth and either leaf Na⁺, Cl⁻ or ABA concentrations could be established. However, in the second trifoliolate leaves only the ABA, but not the Na⁺ or Cl⁻, concentrations were significantly correlated with leaf expansion. Our results suggest that salt-induced inhibition of leaf expansion growth in bush beans is mediated by ABA rather than Na⁺ or Cl⁻ toxicity. Moreover, the increase of ABA, induced by the salt-pretreatment, seems to play an important role in limiting the accumulation of Na⁺ and Cl⁻ in the leaves, leading to adaptation of bush beans to salt-stress.     

Modulation of corticosterone availability to white adipose tissue of lean and obese Zucker rats by corticosteroid-binding globulin.

Corticosterone-binding (CB) capacity was determined in periovarian and subcutaneous white adipose tissue (WAT), as well as in plasma of lean and obese Zucker rats. In lean rats, plasma CB was twice the level of obese rats. In lean rat WAT, dexamethasone binding accounted for only 0.05-0.09% of corticosterone binding, and aldosterone bound even less; in the obese rats, dexamethasone accounted for 0.2 - 0.3 % of corticosterone binding. Scatchard plots showed that KD for corticosterone was 3.1 nM (WAT) or 3.4 nM (plasma) in lean rats and 1.8 nM (WAT) or 1.5 nM (plasma) in obese rats. The total CB capacity in WAT was lower in the obese than in lean rats (47-50%). Plasma non-esterified fatty acid levels were higher in obese rats. The results suggest that CBG may limit the access of glucocorticoids to adipocytes more weakly in obese rats because of the lower CBG. Fatty acids may increase the affinity of CBG for corticosterone, which would make WAT cells less accessible to circulating glucocorticoids. The modulation of CBG by fatty acids may protect fat reserves by decreasing the sensitivity of WAT to glucocorticoids.     

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3- [Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin- 3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta- strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, - 2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc- alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin- oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.      

Growth retardation of inner cell mass cells in polyspermic porcine embryos produced in vitro

The in vitro viability of polyspermic pig eggs was investigated. Immature oocytes were matured and fertilized in vitro. Approximately 10 h after insemination, the eggs were centrifuged at 12 000 x g for 10 min and individually classified into two (2PN)- and poly-pronuclear (PPN, 3 or 4 pronuclei) eggs. The classified eggs were cultured in vitro or in vivo. Nuclei numbers of inner cell mass (ICM) and trophectoderm (TE) were compared between 2PN- and PPN-derived blastocysts. The frequency of development in vitro of 2PN and PPN eggs to the blastocyst stage was 53.6% and 40.7%, respectively. The mean number (8.2 +/- 0.7, n = 48) of ICM nuclei of 2PN-derived blastocysts was higher than that (4.2 +/- 0.8, n = 37) of PPN-derived blastocysts (p < 0.001), whereas there was no difference (p > 0.05) in mean numbers of total (46.7 +/- 3.4 vs. 39. 9 +/- 3.9) and TE nuclei (38.5 +/- 2.9 vs. 35.7 +/- 3.3) between the two groups. Development of 2PN and PPN eggs cultured in vivo to the blastocyst stage was 33.3% and 27.4%, respectively. The numbers of ICM and TE nuclei of these embryos cultured in vivo showed a pattern similar to that for the in vitro-produced blastocysts. Additionally, fetuses were obtained on Day 21 from both the 2PN and the PPN groups. This suggests that polyspermic pig embryos develop to the blastocyst stage and beyond, although showing a smaller ICM cell number as compared to normal embryos.     

Daily Oral Oleoyl‐Estrone Gavage Induces a Dose‐Dependent Loss of Fat in Wistar Rats

Objective: To establish whether single daily oral doses of oleoyl‐estrone result in dose‐dependent slimming effects on normal weight rats, and to determine the changes in energy parameters induced by this treatment. Research Methods and Procedures: The effects of a daily oral gavage of oleoyl‐estrone (0, 0.2, 0.5, 1, 2, 5, 10, and 20 μmol/kg per day) in 0.2 ml of sunflower oil given over a 10‐day period were studied in groups, each of which contained six adult female Wistar rats initially weighing 190 to 230 g. A group of intact control rats receiving no gavage was included for comparison. Body weight and food intake were measured daily. Rats were killed on day 10 of treatment, and body composition (protein nitrogen, lipids, and water), liver lipids, and plasma parameters (glucose, triacylglycerols, total cholesterol, free fatty acids, 3‐hydroxybutyrate, urea, aspartate, alanine transaminases, insulin, leptin, and free and acyl‐estrone) were measured. Results: The administration of oleoyl‐estrone resulted in a dose‐dependent loss of body fat, because of a partly maintained energy expenditure combined with decreased food intake. The differences in the energy budget were met by internal fat pools. The changes recorded did not affect the levels of the main plasma energy homeostasis indicators: unaltered glucose, triacylglycerols, free fatty acids, 3hydroxybutyrate, and urea. Protein was accrued even under conditions of severe lipid store drainage. There were no changes in transaminases. No lipid accumulation was recorded in the liver. Plasma insulin and leptin levels decreased with increased oleoyl‐estrone doses, whereas the levels of free and esterified estrone increased with treatment, although not in proportion to the dose received. Discussion: Oral treatment with oleoyl‐estrone resulted in the specific dose‐related loss of fat reserves with little change to other metabolic parameters. These results agree with the postulated role of oleoyl‐estrone as a ponderostat signal.