Glucose limitation and pka1 deletion rescue aberrant mitotic spindle formation induced by Mal3 overexpression in Schizosaccharomyces pombe

ABSTRACT

The cAMP-dependent protein kinase Pka1 is known as a regulator of glycogenesis, transition into meiosis, proper chromosome segregation, and stress responses in Schizosaccharomyces pombe. We demonstrated that both the cAMP/PKA pathway and glucose limitation play roles in appropriate spindle formation. Overexpression of Mal3 (1–308), an EB1 family protein, caused growth defects, increased 4C DNA content, and induced monopolar spindle formation. Overproduction of a high-affinity microtubule binding mutant (Q89R) and a recombinant protein possessing the CH and EB1 domains (1–241) both resulted in more severe phenotypes than Mal3 (1–308). Loss of functional Pka1 and glucose limitation rescued the phenotypes of Mal3-overexpressing cells, whereas deletion of Tor1 or Ssp2 did not. Growth defects and monopolar spindle formation in a kinesin-5 mutant, cut7-446, was partially rescued by pka1 deletion or glucose limitation. These findings suggest that Pka1 and glucose limitation regulate proper spindle formation in Mal3-overexpressing cells and the cut7-446 mutant.     

Possible Involvement of Extracellular ATP-P2Y Purinoceptor Signaling in Ischemia-induced Tolerance of Astrocytes in Culture

Extracellular adenosine 5′-triphosphate (ATP) activates specific G protein-coupled purinoceptors (P2Y), and ATP-P2Y signaling pathways induces intracellular Ca²⁺ mobilization resulting in changes in the gene expression of a variety of proteins in astrocytes. This study investigated whether the exposure of cultured astrocytes to sublethal ischemia produced resistance to subsequent lethal ischemic stress, and if so, whether the extracellular ATP-P2Y signaling pathways were responsible for the tolerance. Ischemia-like insults, sublethal oxygen-glucose deprivation (sOGD), produced tolerance to subsequent lethal OGD stress in cultured astrocytes. Early during reperfusion after sOGD, the amount of extracellular ATP and the expression of both P2Y₁ and P2Y₂ receptors were increased, leading to enhanced activation of the extracellular ATP-P2Y signaling pathways. The occurrence of intracellular spontaneous Ca²⁺ oscillations was also increased. In addition, sOGD treatment enhanced the expression of the phosphorylated form of extracellular signal-regulated protein kinases 1 and 2 (p-ERK 1/2), and treatment with an inhibitor of ERK significantly attenuated the sOGD-induced ischemic tolerance of astrocytes.     

Purification and Properties of Gitrus natsudaidai Pectinesterase

The level of glutamine synthetase in Micrococcus glutamicus ATCC 13032 varied in response to the nitrogen source in culture medium; it was 10?20 fold higher in glutamate-, peptone- or yeast extract-grown cells than in ammonia- or urea-grown cells. Ammonia (3 mM) reduced the enzyme level to 50% when added to glutamate medium. No difference between nitrogen sources was observed in extent of inhibition by Mg²⁺ of γ-glutamylhydroxamate-forming (transferring) reaction in crude extracts.

The optimum pH was 7.0 ? 8.0 for glutamine-forming (synthesizing) reaction and 7.0 for transferring reaction. The enzyme was stable to heating at 50°C for 10 min in 0.05 M potassium phosphate buffer (pH 6.0) containing 0.1 mM MnCl₂. Km values for glutamate, ammonia and ATP in synthesizing reaction were 7.9, 5.0 and 1.2 mM, respectively. GTP and hydroxylamine could be substituted for ATP and ammonia with about 10 and 30% reactivity. Mg²⁺ was effective as a cofactor in synthesizing reaction and Mn²⁺ showed 34% of the reactivity of Mg²⁺ at a concentration of 30 mM. Glutamine synthetase was inhibited by adenosine, AMP and ADP but not by amino acids other than D-threonine. The regulation system of glutamine synthetase in M. glutamicus is discussed.     

Salt-Sensitive growth of Staphylococcus aureus: stimulation of salt-induced autolysis by multiple environmental factors.

The growth of Staphylococcus aureus 209P became extremely sensitive to a high NaCl concentration following lowered temperature, reduced air-supply, and decreased Ca2+ concentration in the medium. Cells in high-NaCl and low-Ca2+ concentration media either autolyzed or transformed into protoplast-like forms during growth when grown standing below 37 degrees C. The abnormal growth, however, was invariably avoided by preliminary supplementation with polyanetholesulfonate (autolysin inhibitor) in the growth media. These results suggested that the autolytic activity of this organism was precisely controlled by multiple environmental factors such as ionic strength, temperature, air supply, and the concentration of Ca2+.     

钙对吸胀的绿豆种子脱水耐性的影响

以绿豆种子为材料,研究了预吸胀种子脱水耐性的变化,以及Ca^2 处理对种子脱水耐性的影响。结果表明：绿豆种子的脱水耐性随预吸胀时间的延长而下降;Ca^2 预吸胀处理能提高种子的脱水耐性,适宜的Ca^2 浓度为20mmol／L;Ca^2 能修复预吸胀种子的脱水伤害,适宜的Ca^2 浓度为2．5～5mmol／L。     

Ganglioside Function in Calcium Homeostasis and Signaling

Ganglioside function in eukaryotic cells encompasses a variety of modulatory interactions related to both development and mature cellular behavior. In relation to the nervous system this includes induction of neurite outgrowth and trophic/neuroprotective phenomena; more generally this applies to ganglioside effects on receptor function, adhesion reactions, and signal transduction mechanisms in neural and extraneural systems. Underlying many of these trophic effects are ganglioside-induced changes in cellular calcium, accomplished through modulation of Ca²⁺ influx channels, Ca²⁺ exchange proteins, and various Ca²⁺-dependent enzymes that are altered through association with gangliosides. A clear distinction needs to be drawn between intrinsic functions of gangliosides as naturally expressed by the cell and activities created by application of exogenous ganglioside(s) that may or may not reflect natural function. This review attempts to summarize findings in this area and point to possible future directions of research.     

Ca2+ Influx through Store-operated Calcium Channels Replenishes the Functional Phosphatidylinositol 4,5-Bisphosphate Pool Used by Cysteinyl Leukotriene Type I Receptors

Oscillations in cytoplasmic Ca²⁺ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca²⁺ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), the source of the Ca²⁺-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca²⁺ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li⁺, an inhibitor of inositol monophosphatases that prevents PIP₂ resynthesis. In Li⁺-treated cells, cytoplasmic Ca²⁺ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca²⁺ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca²⁺ oscillations, and these could not be rescued by inositol or PI4P. In Li⁺-treated cells, recovery of the cytoplasmic Ca²⁺ oscillations in the presence of inositol or PI4P was suppressed when Ca²⁺ influx through store-operated Ca²⁺ channels was inhibited. After rundown of the Ca²⁺ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca²⁺ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP₂ pools. Our findings show that store-operated Ca²⁺ entry is needed to sustain cytoplasmic Ca²⁺ signaling following leukotriene receptor activation both by refilling the Ca²⁺ stores and by helping to replenish the PIP₂ pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.     

Aggregation of STIM1 underneath the plasma membrane induces clustering of Orai1

STIM1 and Orai1 have recently been identified to be crucial in the regulation of store-operated Ca(2+) entry. However, it remains to be established how STIM1 couples store depletion to the functioning of Orai1 in the plasma membrane. Using quantitative measurement, we find little STIM1 on the surface membrane which is not increased by store depletion. We further demonstrate that Orai1 assembles into clusters that co-localize with STIM1 aggregations upon store depletion. The clustering of Orai1 is only seen when Oari1 are co-expressed with STIM1, but not when expressed alone. Moreover, ER retreat from cell periphery leads to mismatching of Orai1 and STIM1 puncta. Therefore, we propose that store depletion causes aggregation and translocation of STIM1 in close apposition to the plasma membrane, which in turn recruits Orai1 in the plasma membrane to the sites of STIM1 aggregates to assemble functional units of CRAC channels in a stoichiometric manner.     

The structure of the Arabidopsis thaliana SOS3: molecular mechanism of sensing calcium for salt stress response

The Arabidopsis thaliana SOS3 gene encodes a calcium sensor that is required for plant salt tolerance. The SOS3 protein binds to and activates the self-inhibited SOS2 protein kinase, which mediates the expression and activities of various transporters important for ion homeostasis under salt stress. SOS3 belongs to a unique family of calcium-binding proteins that contain two pairs of EF hand motifs with four putative metal-binding sites. We report the crystal structure of a dimeric SOS3 protein in complex with calcium, and with calcium and manganese. Analytical ultracentrifugation experiments and circular dichroism measurements show that calcium binding is responsible for the dimerization of SOS3. This leads to a change in the global shape and surface properties of the protein that may be sufficient to transmit the Ca(2+) signal elicited during salt stress.     

Supplemental calcium does not improve growth of salt-stressed Brassicas

Whole plant and callus cultures of different rapid-cycling Brassica species were treated with salinity (8 dS m^-1) and/or supplemental Ca (up to 10 mM total concentration). None of these cultures responded to supplemental Ca with improved growth indicating that the salt tolerance of these genotypes was not dependent upon Ca.     

Indo-1 can simultaneously detect Ba2+ entry and Ca2+ blockade at a plasma membrane calcium channel

The fluorescent chelator Indo-1 can make simultaneous determinations of two intracellular ion concentrations, such as [Ca²⁺] and [Cd²⁺], or [Ca²⁺] and [Ba²⁺], in a normal cell suspension. The second ion can be detected even if its spectrum when bound to Indo-1 is same as for the calcium-bound or the ion-free Indo-1, as long as there is a change in height. This is because the mathematical analysis uses not only the spectral shape, but also takes into account increases in total signal intensity. For maximum accuracy, whole spectra were analyzed. When 3 mM [Ba²⁺] was added to a B cell line that had been stimulated with anti-immunoglobulin to open receptor operated calcium channels, there was a sudden drop in 400 nm Indo-1 fluorescence. Spectral analysis showed that this was due to a drop in intracellular [Ca²⁺], which was consistent with blockage of the receptor-operated calcium current by extracellular Ba²⁺. The conductance for Ba²⁺ was also observable as a slow rise in total fluorescence. There was also a slow increase in intracellular [Ca²⁺] as barium accumulated in the cell, which was tentatively attributed to blockage of the plasma membrane calcium pump by intracellular Ba²⁺.     

Implications of calcium nutrition on the response of Phaseolus vulgaris L. to salinity

The effect of Ca²⁺ level in the growth medium on the response of germination and early seedling growth of Phaseolus vulgaris to NaCl salinity was investigated. When NaCl concentration was increased germination and early seedling growth was decreased. The addition of Ca²⁺ to the media increased both germination percentage and seedling growth. Chloride concentrations were not affected by the level of Ca²⁺. Potassium and Ca²⁺ concentrations and transport from roots to shoots were decreased by NaCl, but were restored by increasing Ca²⁺ in the medium. The opposite was true for Na⁺. Leakage of NO₃ ^- and H₂PO₄ ^- was increased by salinity and reduced by high Ca²⁺ in the medium. The results are discussed in terms of the beneficial effects of calcium for plant growth under saline conditions.