In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites

We have found that two previously reported exonic mutations in the PINK1 and PARK7 genes affect pre-mRNA splicing. To develop an algorithm to predict underestimated splicing consequences of exonic mutations at the 5′ splice site, we constructed and analyzed 31 minigenes carrying exonic splicing mutations and their derivatives. We also examined 189 249 U2-dependent 5′ splice sites of the entire human genome and found that a new variable, the SD-Score, which represents a common logarithm of the frequency of a specific 5′ splice site, efficiently predicts the splicing consequences of these minigenes. We also employed the information contents (R_i) to improve the prediction accuracy. We validated our algorithm by analyzing 32 additional minigenes as well as 179 previously reported splicing mutations. The SD-Score algorithm predicted aberrant splicings in 198 of 204 sites (sensitivity = 97.1%) and normal splicings in 36 of 38 sites (specificity = 94.7%). Simulation of all possible exonic mutations at positions −3, −2 and −1 of the 189 249 sites predicts that 37.8, 88.8 and 96.8% of these mutations would affect pre-mRNA splicing, respectively. We propose that the SD-Score algorithm is a practical tool to predict splicing consequences of mutations affecting the 5′ splice site.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.     

Ligand-binding activity and expression profile of annexins in Caenorhabditis elegans

Mammalian annexins are implicated in several physiological mechanisms based on their calcium-dependent phospholipid/membrane binding and carbohydrate-binding activities. In this study, we investigated gene expression profiles of all four Caenorhabditis elegans annexins, nex-1, -2, -3 and -4, throughout the development, and compared phospholipid- and carbohydrate-binding properties of their protein products, NEX-1, -2, -3 and -4. We found that nex-1 and -3 are transcribed continuously during the developmental stages, while expression of nex-2 and -4 appeared to be temporal, peaking at the L1 stage followed by a gradual decrease toward the adult stage. NEX-1 and -3 were detected as single protein band in total worm extracts by immunoblotting, but NEX-2 was heterogenic in size. NEX-1, -2, and -3 showed the binding activities to phosphatidylserine, phosphatidylinositol and phosphatidylethanolamine, but not to phosphatidylcholine. In contrast to their uniform phospholipids-binding properties, their glycosaminoglycan-binding activities were distinctive. NEX-2 bound to heparan sulfate and chondroitin, NEX-3 bound only to heparan sulfate, and NEX-1 showed no lectin activities under tested conditions. NEX-4 had neither phospholipids- nor carbohydrate-binding properties. Differentiated expression profiles and ligand-binding properties of NEX-1, -2, -3 and -4, shown in our study, may represent distinctive roles for each C. elegans annexins.     

Enrichment of longevity phenotype in mtDNA haplogroups D4b2b, D4a, and D5 in the Japanese population

We report new results from the re-analysis of 672 complete mitochondrial (mtDNA) genomes of unrelated Japanese individuals stratified into seven equal sized groups by the phenotypes: diabetic patients, diabetic patients with severe angiopathy, healthy non-obese young males, obese young males, patients with Alzheimer’s disease, patients with Parkinson’s disease and centenarians. Each phenotype had 96 samples over 27 known haplogroups: A, B4a, B4b, B4c, B*, B5, D*, F1, F2, M*, M7a, M7b, M8, M9, D4a, D4b1, D4b2, D4d, D4e, D4g, D4h, D5, G, Z, M*, N9a, and N9b. A t-test comparing the fraction of samples in a haplogroup to healthy young males showed a significant enrichment of haplogroups D4a, D5, and D4b2 in centenarians. The D4b2 enrichment was limited to a subgroup of 40 of 61 samples which had the synonymous mutation 9296C > T. We identified this cluster as a distinct haplogroup and labeled it as D4b2b. Using an exhaustive procedure, we constructed the complete list of “mutation patterns” for centenarians and showed that the most significant patterns were in D4a, D5, and D4b2b. We argue that if a selection for longevity appeared only once, it was probably an autosomal event which could be dated to after the appearance of the D mega-group but before the coalescent time of D4a, D5, and D4b2b. Using a simple procedure, we estimated that this event occurred 24.4 ± 0.9 kYBP. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gabriela Alexe and Noriyuki Fuku are joint first authors.     

Prolonged transient acidosis during early reperfusion contributes to the cardioprotective effects of postconditioning

We have previously reported that the prolonged transient acidosis during early reperfusion mediates the cardioprotective effects in canine hearts. Recently, postconditioning has been shown to be one of the novel strategies to mediate cardioprotection. We tested the contribution of the prolonged transient acidosis to the cardioprotection of postconditioning. Open-chest anesthetized dogs subjected to 90-min occlusion of the left anterior descending coronary artery and 6-h reperfusion were divided into four groups: 1) control group; no intervention after reperfusion (n = 6); 2) postconditioning (Postcon) group; four cycles of 1-min reperfusion and 1-min reocclusion (n = 7); 3) Postcon + sodium bicarbonate (NaHCO(3)) group; four cycles of 1-min reperfusion and 1-min reocclusion with the administration of NaHCO(3) (n = 8); and 4) NaHCO(3) group; administration of NaHCO(3) without postconditioning (n = 6). Infarct size, the area at risk (AAR), collateral blood flow during ischemia, and pH in coronary venous blood were measured. The phosphorylation of Akt and extracellular signal-regulated kinase (ERK) in ischemic myocardium was assessed by Western blot analysis. Systemic hemodynamic parameters, AAR, and collateral blood flow were not different among the four groups. Postconditioning induced prolonged transient acidosis during the early reperfusion phase. Administration of NaHCO(3) completely abolished the infarct size-limiting effects of postconditioning. Furthermore, the phosphorylation of Akt and ERK in ischemic myocardium induced by postconditioning was also blunted by the cotreatment of NaHCO(3). In conclusion, postconditioning mediates its cardioprotective effects possibly via prolonged transient acidosis during the early reperfusion phase with the activation of Akt and ERK.     

Production of hydrogen and methane from organic solid wastes by phase-separation of anaerobic process

Phase-separated two-stage anaerobic process was examined and evaluated using artificial organic solid waste in laboratory scale. Acidogenic process, which was combined with subsequent methanogenic process using packed-bed reactor, was operated emphasizing on either hydrogen production, or solublizing efficiency of solid materials. In either effluent from hydrogenogenic, or solublizing operation, maximum allowable OLR achieved at methanogenesis was higher than the single methanogenic process. Hydrogenogenic operation was more suitable to combine methanogenic process than solublizing operation, since retention time of hydrogenogenic operation was much shorter than the solublizing operation, obtaining almost the same levels of overall removal efficiency in both COD and VSS. The combination of hydrogenogenic operation in acidogenic process and methanogenic process produced approximately 442mmoll-reactor(1)days(-1) of methane and 199mmoll-reactor(1)days(-1) of hydrogen at 25h of total retention time indicating 82% of COD removal with 96% of VSS decomposition.     

Dopamine-selective potentiometric responses by new ditopic sensory elements based on a hexahomotrioxacalix[3]arene

New ditopic sensory elements 2 and 3 for catecholamines based on a hexahomotrioxacalix[3]arene, with a boronic acid substituent appended, were designed and synthesized. As an interesting mode of molecular recognition at membrane surfaces, the host, when incorporated into poly(vinyl chloride) (PVC) liquid membranes, displayed excellent potentiometric selectivity for dopamine over other catecholamines (noradrenaline and adrenaline) and inorganic cations (Na+, K+, and NH4+).     

Loss of calcineurin homologous protein-1 in chicken B lymphoma DT40 cells destabilizes Na+/H+ exchanger isoform-1 protein

NHE1/SLC9A1 is a ubiquitous isoform of vertebrate Na⁺/H⁺ exchangers (NHEs) functioning in maintaining intracellular concentrations of Na⁺ and H⁺ ions. Calcineurin homologous protein-1 (CHP1) binds to the hydrophilic region of NHE1 and regulates NHE1 activity but reportedly does not play a role in translocating NHE1 from the endoplasmic reticulum to the plasma membrane. However, an antiport function of NHE1 requiring CHP1 remains to be clarified. Here we established CHP1-deficient chicken B lymphoma DT40 cells by gene targeting to address CHP1 function. CHP1-deficient cells showed extensive decreases in Na⁺/H⁺ activities in intact cells. Although NHE1 mRNA levels were not affected, NHE1 protein levels were significantly reduced not only in the plasma membrane but in whole cells. The expression of a CHP1 transgene in CHP1-deficient cells rescued NHE1 protein expression. Expression of mutant forms of CHP1 defective in Ca²⁺ binding or myristoylation also partially decreased NHE1 protein levels. Knockdown of CHP1 also caused a moderate decrease in NHE1 protein in HeLa cells. These data indicate that CHP1 primarily plays an essential role in stabilization of NHE1 for reaching of NHE1 to the plasma membrane and its exchange activity. membrane protein; transporter; antiporter; quality control; degradation     

Antibacterial activity of a virgatusin-related compound

The relationship between antibacterial activity of tetra-substituted tetrahydrofuran lignans (1-4) and their absolute configurations was tested. Only compound 4 among two virgatusins and two related compounds exhibited growth inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus and Listeria denitrificans. Compound 4 affected the growth of B. subtilis in a bactericidic manner, and its ability to dissipate the cytoplasmic membrane potential was investigated using the fluorescence probe 3,3'-dipropylthiadicarbocyanine iodide. These results suggested that compound 4 damages cells by causing the loss of the proton motive force and disruption of the cellular integrity of the membrane, leading to cell death. In addition, it was shown that the antibacterial activity of a lignan was closely related to its absolute configuration and functional groups.