Nutrient limitations alter cell division control and chromosome segregation through growth-related kinases and phosphatases

In dividing fission yeast Schizosaccharomyces pombe cells, the balance between Wee1 kinase and Cdc25 phosphatase which control the cyclin-dependent kinase (CDK) at the G2-M transition determines the rod-shaped cell length. Under nitrogen source starvation or glucose limitation, however, cell size determination is considerably modulated, and cell size shortening occurs for wild-type cells. For several mutants of kinases or phosphatases, including CDK, target of rapamycin complex (TORC) 1 and 2, stress-responsive mitogen-activated protein kinase (MAPK) Sty1/Spc1, MAPK kinase Wis1, calcium- and calmodulin-dependent protein kinase kinase-like Ssp1, and type 2A and 2A-related phosphatases inhibitor Sds23, this cell shortening does not normally occur. In tor1 and ssp1 mutants, cell elongation is observed. Sds23 that binds to and inhibits 2A and 2A-related phosphatases is synergistic with Ssp1 in the cell size determination and survival under low glucose and nitrogen source. Tor2 (TORC1) is required for growth, whereas Tor1 (TORC2) is needed for determining division size according to different nutrient conditions. Surprisingly, in growth-diminished tor2 mutant or rapamycin-treated cells, the requirement of separase/Cut1-securin/Cut2 essential for chromosome segregation is greatly alleviated. By contrast, defects of tor1 with secruin/cut2 or overproduction of Cut1 are additive. While Tor1 and Tor2 are opposite in their apparent functions, both may actually coordinate cell division with growth in response to the changes in nutrients.     

ACCUMULATION OF SULFURIC ACID IN DICTYOTALES (PHAEOPHYCEAE): TAXONOMIC DISTRIBUTION AND ION CHROMATOGRAPHY OF CELL EXTRACTS

Four species in the order Dictyotales ( Dictyopteris latiuscula (Okamura) Okamura, D. prolifera (Okamura) Okamura, D. repens (Okamura) Børgesen, and Spatoglossum crassum J. Tanaka) were found to be highly acidic as in some species of the order Desmarestiales (Phaeophyceae). The pH within their cells, presumably that of the vacuole, was estimated to be 0.5 to 0.9 by pH measurements of their cell extracts in distilled water. However, other species of these genera ( D. divaricata (Okamura) Okamura, D. undulata Holmes, and S. pacificum Yendo) did not show high acidity. Ion chromatography of the cell extracts showed that those species contained high concentrations of SO     within their cells, up to 10 times that in seawater but relatively low Cl⁻. The sum of cations examined (Na⁺, NH     , K⁺, Mg²⁺, Ca²⁺) was significantly lower than that of anions (Cl⁻, Br⁻, NO     , SO     ), and the difference is presumed to represent protons (H⁺), causing the extremely low cell sap pH. Estimated cellular proton concentrations calculated from the pH data roughly agreed with those calculated from differences between the sum of cations and anions and that of anions. Although certain other, nonacidic, dictyotalean species also contained high concentrations of SO     , these species contained high concentrations of Mg²⁺, and the sums of cations and anions were balanced.     

Mn2+ binding to factor VIII subunits and its effect on cofactor activity

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.     

Perinatal exposure to bisphenol A enhances contextual fear memory and affects the serotoninergic system in juvenile female mice

Perinatal exposure to bisphenol A (BPA), an endocrine-disrupting chemical, affects the central nervous system, including effects on emotional responses and neurotransmitter release. In this study, we investigated the effects of BPA (250 ng/kg/day, from gestational day 10 to postnatal day 20) on fear memory and serotonin (5-HT) metabolites in the brain using contextual fear conditioning (FC) and high-performance liquid chromatography (HPLC), respectively, in adult and juvenile mice of both sexes. Furthermore, we studied the effects of BPA on the gene expression of 5-HT metabolite-related enzymes and 5-HT receptors using quantitative real-time RT PCR in the brains of juvenile females. BPA enhanced fear memory and increased serotonin metabolite (5-HIAA) levels and 5-HIAA/5-HT in the hippocampus, the striatum, the midbrain, the pons, and the medulla oblongata of juvenile female mice. In contrast, alterations in those areas were much smaller in adult females and in both juvenile and adult males. Furthermore, BPA induced increases in the expression levels of Tph2, Slc6a4, and Maoa mRNA in the hippocampus of juvenile females, indicating that BPA induces hyper 5-HT turnover in the hippocampus.Our results suggest that perinatal exposure to a low dose of BPA enhances fear memory and the 5-HTergic system in juvenile mice.     

Impaired actin filaments decrease cisplatin sensitivity via dysfunction of volume-sensitive Cl− channels in human epidermoid carcinoma cells

Cisplatin is a widely used platinum-based anticancer drug in the chemotherapy of numerous human cancers. However, cancer cells acquire resistance to cisplatin. So far, functional loss of volume-sensitive outwardly rectifying (VSOR) Cl⁻ channels has been reported to contribute to cisplatin resistance of cancer cells. Here, we analyzed protein expression patterns of human epidermoid carcinoma KB cells and its cisplatin-resistant KCP-4 cells. Intriguingly, KB cells exhibited higher β-actin expression and clearer actin filaments than KCP-4 cells. The β-actin knockdown in KB cells decreased VSOR Cl⁻ currents and inhibited the regulatory volume decrease (RVD) process after cell swelling. Consistently, KB cells treated with cytochalasin D, which depolymerizes actin filaments, showed smaller VSOR Cl⁻ currents and slower RVD. Cytochalasin D also inhibited cisplatin-triggered apoptosis in KB cells. These results suggest that the disruption of actin filaments cause the dysfunction of VSOR Cl⁻ channels, which elicits resistance to cisplatin in human epidermoid carcinoma cells.     

Origins of the secondary plastids of Euglenophyta and Chlorarachniophyta as revealed by an analysis of the plastid‐targeting,nuclear‐encoded gene psbO1

Because the secondary plastids of the Euglenophyta and Chlorarachniophyta are very similar to green plant plastids in their pigment composition, it is generally considered that ancestral green algae were engulfed by other eukaryotic host cells to become the plastids of these two algal divisions. Recent molecular phylogenetic studies have attempted to resolve the phylogenetic positions of these plastids; however, almost all of the studies analyzed only plastid‐encoded genes. This limitation may affect the results of comparisons between genes from primary and secondary plastids, because genes in endosymbionts have a higher mutation rate than the genes of their host cells. Thus, the phylogeny of these secondary plastids must be elucidated using other molecular markers. Here, we compared the plastid‐targeting, nuclear‐encoded, oxygen‐evolving enhancer (psbO) genes from various green plants, the Euglenophyta and Chlorarachniophyta. A phylogenetic analysis based on the PsbO amino acid sequences indicated that the chlorarachniophyte plastids are positioned within the Chlorophyta (including Ulvophyceae, Chlorophyceae, and Prasinophyceae, but excluding Mesostigma). In contrast, plastids of the Euglenophyta and Mesostigma are positioned outside the Chlorophyta and Streptophyta. The relationship of these three phylogenetic groups was consistent with the grouping of the primary structures of the thylakoid‐targeting domain and its adjacent amino acids in the PsbO N‐terminal sequences. Furthermore, the serine‐X‐alanine (SXA) motif of PsbO was exactly the same in the Chlorarachniophyta and the prasinophycean Tetraselmis. Therefore, the chlorarachniophyte secondary plastids likely evolved from the ancestral Tetraselmis‐like alga within the Chlorophyta, whereas the Euglenophyte plastids may have originated from the unknown basal lineage of green plants.     

Prognostic Significance of Anti-Aminoacyl-tRNA Synthetase Antibodies in Polymyositis/Dermatomyositis-Associated Interstitial Lung Disease: A Retrospective Case Control Study

Background

In polymyositis/dermatomyositis (PM/DM), anti-aminoacyl-tRNA synthetase (ARS) antibodies are closely associated with interstitial lung disease (ILD), a frequent pulmonary complication. However, the clinical significance of anti-ARS antibodies is not well established.

Objective

We aimed to evaluate the clinical significance of anti-ARS antibodies in PM/DM-ILD patients.

Methods

Forty-eight consecutive PM/DM-ILD patients were studied retrospectively. Anti-ARS antibodies were screened by ELISA and confirmed by RNA immunoprecipitation test. Medical records, high-resolution computed tomography images, and surgical lung biopsy specimens were compared between ARS-positive (ARS group) and ARS-negative patients (non-ARS group).

Results

Anti-ARS antibodies were detected in 23 of 48 patients (48%). Radiologically, nonspecific interstitial pneumonia (NSIP) pattern was observed more frequently in the ARS group than in the non-ARS group (73.9% vs. 40%, P = 0.02). Pathologically, NSIP was the most frequent in both groups. Ten-year survival rate was also significantly higher in the ARS group than in the non-ARS group (91.6% vs. 58.7%, P = 0.02). Univariate Cox hazards analysis revealed that the presence of anti-ARS antibodies was associated with better prognosis (HR = 0.34, 95% CI 0.08–0.80; P = 0.01).

Conclusions

The presence of anti-ARS antibodies is a possible prognostic marker in patients with PM/DM-ILD.     

Localization of a pH-dependent, A2 subunit-interactive surface within the factor VIIIa A1 subunit

Factor VIIIa can be reconstituted from A2 subunit and A1/A3-C1-C2 dimer in a reaction that is facilitated by slightly acidic pH. We recently demonstrated that a truncated A1 (A1(37-336)) possessed markedly reduced affinity for A2 compared with intact A1, but retained 30% of native factor VIIIa activity in the presence of A3-C1-C2. We now identify A1-interactive regions for A2 using A1 fragments derived from a limited tryptic digest. Unfractionated trypsin-cleaved A1 inhibited reconstituted factor VIIIa activity. Two fragments, designated A1(37-121) and A1(221-336), markedly inhibited factor VIIIa reconstitution with either native A1 (K(i)=340 and 194 nM, respectively) or with A1(37-336) (K(i)=69 and 116 nM, respectively) at pH 6.0. A third fragment designated A1(122-206) did not possess inhibitory activity. At pH 7.2, the A1(221-336) partially inhibited reconstitution, whereas the A1(37-121) possessed little if any inhibitory activity. Both fragments inhibited factor VIIIa reconstitution as judged by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1 forms at pH 6.0. Furthermore, covalent cross-linking between A2 and A1(37-121) but not A1(221-336) was observed following reaction with a zero-length cross-linker. These findings demonstrate the presence of an extended, pH-dependent A2-interactive surface within regions 37-121 and 221-336 of A1. This interactive surface appears conformationally labile in the truncated A1 as judged by its apparent stabilization following association with A3-C1-C2.