Biphasic functions of the kinase-defective Ephb6 receptor in cell adhesion and migration

EphB6 is a unique member in the Eph family of receptor tyrosine kinases in that its kinase domain contains several alterations in conserved amino acids and is catalytically inactive. Although EphB6 is expressed both in a variety of embryonic and adult tissues, biological functions of this receptor are largely unknown. In the present study, we examined the function of EphB6 in cell adhesion and migration. We demonstrated that EphB6 exerted biphasic effects in response to different concentrations of the ephrin-B2 ligand; EphB6 promoted cell adhesion and migration when stimulated with low concentrations of ephrin-B2, whereas it induced repulsion and inhibited migration upon stimulation with high concentrations of ephrin-B2. A truncated EphB6 receptor lacking the cytoplasmic domain showed monophasic-positive effects on cell adhesion and migration, indicating that the cytoplasmic domain is essential for the negative effects. EphB6 is constitutively associated with the Src family kinase Fyn. High concentrations of ephrin-B2 induced tyrosine phosphorylation of EphB6 through an Src family kinase activity. These results indicate that EphB6 can both positively and negatively regulate cell adhesion and migration, and suggest that tyrosine phosphorylation of the receptor by an Src family kinase acts as the molecular switch for the functional transition.     

Intake of dried bonito broth flavored with dextrin solution induced conditioned place preference in mice

We investigated to determine whether dried bonito broth flavor induces a reinforcing effect using the conditioned place preference (CPP) test. Only dried bonito broth did not induce CPP. Sucrose induced CPP in 20% solution. A 21.86% dextrin solution, with the same calorie content as the 20% sucrose solution, did not induce CPP, but a dextrin solution flavored with dried bonito broth (BD) induced CPP. An AD solution containing the same concentrations of dextrin, NaCl, IMP, GMP, and amino acids as found in BD tended to increase the time spent in the conditioned box but did not significantly. Aromatic compounds, such as citral, vanillin, and menthol flavored AD solutions did not induce CPP, whereas an AD solution supplemented with dried bonito flavoring agent induced CPP. In mice with transected olfactory nerves, CPP was not induced by voluntary intake of BD. These results suggest that the aromatic profile of the dried bonito broth plays an important role in BD-induced CPP.     

The influence of gender, athletic events, and athletic experience on the subjective dominant hand and the determination of the dominant hand based on the laterality quotient (LQ) and the validity of the LQ

This study aimed to reveal the influence of gender, athletic events and athletic experience on the subjective dominant hand and the dominant hand based on the laterality quotient (LQ). It also aimed to examine the validity of the Edinburgh Inventory (Oldfield, 1971). Males and females (n=3,726) living in 7 prefectures in Japan (age: 16-45 yrs) participated in this survey. Analysis was performed on 3,557 separate datasets with high reliability. The reliability of the survey was examined using a test-retest method consisting of 100 people selected randomly from all participants. All participants provided the same answers for each question. The influence of gender, event and experience was examined for the subjective and LQ-based dominant hands. In addition, concordance rates of the subjective dominant hand and the LQ-based dominant hand and both dominant hands were examined. Differences of concordance rates between hands used in the 10 movement questions of the Inventory and the subjective dominant hand were tested using the chi(2) test. The frequency differences among items were tested using Ryan's method (multiple comparisons). Significant gender differences were found between rates of the LQ-based dominant hand (males: 94.4%; females: 96.6%) and the subjective dominant hand (males: 91.6%; females: 94.0%), but the degree was only 2.0-4.0%. Insignificant differences were found among athletic events, two groups of different athletic experience, and gender according to each athletic event. The subjective dominant hand almost always agreed with the LQ-based dominant hand (complete concordance rate=0.96, kappa=0.67). Of the 10 question items, inexperienced answers were found only in the item "Knife (without fork)". The "Toothbrush", "Broom (upper hand)", and "Opening box (lid)" items had significantly lower correspondence with the subjective dominant hand (79.7-87.0%) than the other items (92.1-95.7%).In conclusion, athletic experience appears to have little influence on handedness, although there is a slight gender difference. The subjective dominant hand almost always agrees with the dominant hand based on the Inventory. A more efficient handedness inventory may be constructed by excluding the above 4 items.     

Roles of heparan sulfate sulfation in dentinogenesis

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix.     

Lipid composition of mangrove and its relevance to salt tolerance

Lipid compositions of mangrove trees were studied in relation to the salt-tolerance mechanism. Leaves and roots were obtained from seven mature mangrove trees on Iriomote Island, Okinawa: Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia candel, Lumnitzera racemosa, Avicennia marina, Pemphis acidula and Sonneratia alba. Lipids of mangrove leaves mainly consisted of 11 lipid classes: polar lipids, unknown (UK) 1–6, sterols, triacyl glycerols, wax ester and sterol ester (UK 3 and 4 were found to be tri-terpenoid alcohol in this study). Of these lipid classes, sterol ester was the main lipid in all species comprising 17.6–33.7% of total lipids. Analysis of the chemical structure found that the sterol esters mainly consisted of fatty acid esters of tri-terpenoid alcohols. One major tri-terpenoid alcohol was identified to be lupeol by interpretation of infrared resonance, nuclear magnetic resonance and mass spectrometry. Because of the unique anatomy of the mangrove root, lipid analyses were made separately for epidermis, cortex and innermost stele, respectively. The concentration of free tri-terpenoid alcohols showed a higher tendency in the outside part than in the inside portion of the roots, suggesting their protective roles. Relevance of lipid composition to salt tolerance was studied with propagules of K. candel and B. gymnorrhiza planted with varied salt concentrations. The proportions of free tri-terpenoids increased with salinity in both leaves and roots of K. candel, and only in roots of B. gymnorrhiza. No salt-dependent changes were noted in the phospholipid and fatty acid compositions in both species. These findings suggested that salt stress specifically modulated the terpenoid concentrations in mangroves. Electronic Publication     

Intermolecular functional coupling between phosphoinositides and the potassium channel KcsA

Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K⁺ channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology. It has been reported that KcsA activity is regulated by membrane phospholipids, such as phosphatidylglycerol. However, there has been no quantitative analysis of the correlation between direct lipid binding and the functional modification of KcsA, and it is unknown whether PIPns modulate KcsA function. Here, using contact bubble bilayer recording, we observed that the open probability of KcsA increased significantly (from about 10% to 90%) when the membrane inner leaflet contained only a small percentage of PIPns. In addition, we found an increase in the electrophysiological activity of KcsA correlated with a larger number of negative charges on PIPns. We further analyzed the affinity of the direct interaction between PIPns and KcsA using microscale thermophoresis and observed a strong correlation between direct lipid binding and the functional modification of KcsA. In conclusion, our approach was able to reconstruct the direct modification of KcsA by PIPns, and we propose that it can also be applied to elucidate the mechanism of modification of other ion channels by PIPns.     

Structural proteins in the egg-shell of the oriental garden cricket, Gryllus mitratus

1. The egg-shell of the oriental garden cricket, Gryllus mitratus, contained at least two different types of structural protein in an approximate ratio of 5:1. The major fraction was extracted in a solvent containing dithiothreitol, EDTA and 8m-urea, and was purified to apparent homogeneity as judged by free-boundary electrophoresis and ultracentrifugation. This was designated SH-fraction and its S-carboxymethyl derivative (CM-fraction) was also prepared. The minor fraction, insoluble in the solvent, was designated insoluble residue. 2. The major fraction was a phosphoprotein, rich in serine (29.8mol% of the total amino acids) and phosphate (nearly equimolar to serine), and O-phosphoserine was identified in its partial acid hydrolysate. The content of cystine was rather low (0.9mol%) in spite of the importance of this amino acid residue in the native form of the protein. The insoluble residue contained only a small amount of phosphorus, and its amino acid composition was clearly different from the major fraction. 3. CM-fraction, a fibrous protein with an average molecular weight of 57500, behaved as a typical polyanion owing to the high content of phosphate. SH-fraction and CM-fraction were precipitable from their aqueous solutions by the addition of bivalent metal cations, and the precipitation of CM-fraction by Ca(2+) and Mg(2+) was studied in detail. 4. When SH-fraction was exposed to air, intermolecular disulphide linkages were formed, yielding a net-like gel that changed its volume with changes in Ca(2+), Mg(2+) and Na(+). 5. The possible role of this protein fraction in maintaining the integrity of the egg-shell, and a comparison of its composition and properties with other egg-shell proteins and other phosphoproteins, are discussed.     

On experiences of i2b2 (Informatics for integrating biology and the bedside) database with Japanese clinical patients' data

Informatics for Integrating Biology and the Bedside (i2b2) is a database system to facilitate sharing and reuse of clinical patients' data collected in individual hospitals. The i2b2 provides an ontology based object-oriented database system with highly simple and flexible database schema which enables us to integrate clinical patients' data from different laboratories and different hospitals. 392 patients' data including carcinoma and non-carcinoma specimens from cancer patients are transported from the Integrated Clinical Omics Database (iCOD) to the i2b2 database for a feasibility study to check applicability of i2b2 ontology and database schema on Japanese clinical patients' data. No modification is required for the i2b2 data model to deal with Japanese characters. Some modification of ontology is required to integrate biomedical information extracted from the cancer patients' data. We believe that the i2b2 system will be practical infrastructure to integrate Japanese clinical databases if appropriate disease ontology for Japanese patients is provided.     

Draft Sequences of the Radish (Raphanus sativus L.) Genome

Radish (Raphanus sativus L., n = 9) is one of the major vegetables in Asia. Since the genomes of Brassica and related species including radish underwent genome rearrangement, it is quite difficult to perform functional analysis based on the reported genomic sequence of Brassica rapa. Therefore, we performed genome sequencing of radish. Short reads of genomic sequences of 191.1 Gb were obtained by next-generation sequencing (NGS) for a radish inbred line, and 76,592 scaffolds of ≥300 bp were constructed along with the bacterial artificial chromosome-end sequences. Finally, the whole draft genomic sequence of 402 Mb spanning 75.9% of the estimated genomic size and containing 61,572 predicted genes was obtained. Subsequently, 221 single nucleotide polymorphism markers and 768 PCR-RFLP markers were used together with the 746 markers produced in our previous study for the construction of a linkage map. The map was combined further with another radish linkage map constructed mainly with expressed sequence tag-simple sequence repeat markers into a high-density integrated map of 1,166 cM with 2,553 DNA markers. A total of 1,345 scaffolds were assigned to the linkage map, spanning 116.0 Mb. Bulked PCR products amplified by 2,880 primer pairs were sequenced by NGS, and SNPs in eight inbred lines were identified.