Duration of competence and inducing capacity of blastomeres in notochord induction during ascidian embryogenesis

Notochord cells in ascidian embryos are formed by the inducing action of cells of presumptive endoderm, as well as neighboring presumptive notochord, at the 32-cell stage. Studies of the timing of induction using recombinations of isolated blastomeres have suggested that notochord induction must be initiated before the decompaction of blastomeres at the 32-cell stage and is completed by the 64-cell stage. However, it is not yet clear how the duration of notochord induction is strictly limited. In the present paper, the aim was to determine in detail when the presumptive notochord blastomeres lost their competence to respond, and when the presumptive endoderm blastomeres produced inducing signals for the notochord. Presumptive notochord blastomeres and presumptive endoderm blastomeres were isolated from early 32-cell embryos, and were heterochronously recombined at various stages ranging from the early 32-cell stage to the 64-cell stage. Presumptive notochord blastomeres could respond to inductive signals at the early 32-cell stage, and started to lose their responsiveness at the decompaction stage. By contrast, the presumptive endoderm blastomeres persisted in their inducing capacity even at the 64-cell stage. These observations suggest that the loss of competence in presumptive notochord blastomeres limits the duration of notochord induction in intact ascidian embryos.     

Brain-Derived Neurotrophic Factor Stimulates Interactions of Shp2 with Phosphatidylinositol 3-Kinase and Grb2 in Cultured Cerebral Cortical Neurons

Shp2, a protein tyrosine phosphatase possessing SH2 domains, is utilized in the intracellular signaling of various growth factors. Shp2 is highly expressed in the CNS. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, which also shows high levels of expression in the CNS, exerts neurotrophic and neuromodulatory effects in CNS neurons. We examined how BDNF utilizes Shp2 in its signaling pathway in cultured cerebral cortical neurons. We found that BDNF stimulated coprecipitation of several tyrosine-phosphorylated proteins with anti-Shp2 antibody and that Grb2 and phosphatidylinositol 3-kinase (PI3-K) were coprecipitated with anti-Shp2 antibody in response to BDNF. In addition, both anti-Grb2 and anti-PI3-K antibodies coprecipitated Shp2 in response to BDNF. The BDNF-stimulated coprecipitation of the tyrosine-phosphorylated proteins, Grb2, and PI3-K with anti-Shp2 antibody was completely inhibited by K252a, an inhibitor of TrkB receptor tyrosine kinase. This BDNF-stimulated Shp2 signaling was markedly sustained as well as BDNF-induced phosphorylation of TrkB and mitogen-activated protein kinases. In PC12 cells stably expressing TrkB, both BDNF and nerve growth factor stimulated Shp2 signaling similarly to that by BDNF in cultured cortical neurons. These results indicated that Shp2 shows cross-talk with various signaling molecules including Grb2 and PI3-K in BDNF-induced signaling and that Shp2 may be involved in the regulation of various actions of BDNF in CNS neurons.     

Cav2.1 Voltage-dependent Ca<Superscript>2+</Superscript> Channel Current is Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

To investigate the pathophysiological mechanisms of immune-mediated peripheral neuropathies, we studied the effects of sera from patients with Guillain-Barré syndrome (GBS) on the Cav2.1 voltage-dependent calcium channel (VDCC) current in Purkinje cells. Using the whole-cell recording technique, Cav2.1 VDCC current was measured in cerebellar Purkinje cells in the presence of serum from GBS patients with acute motor axonal neuropathy (AMAN) or acute inflammatory demyelinating polyneuropathy (AIDP). The AMAN patient sera significantly inhibited the Cav2.1 VDCC current compared with healthy volunteer sera, and this inhibition was fully reversible by washing out the AMAN serum. Similarly, IgG purified from AMAN sera also inhibited the Cav2.1 VDCC current. However, the activation and inactivation kinetics of the Cav2.1 VDCC currents were not affected by serum from an AMAN patient. Moreover, the VDCC current of Purkinje cells was also inhibited by IgG anti-GM1 monoclonal antibody (anti-GM1 mAb). In an immunocytochemical study using double fluorescence staining, Purkinje cells were stained by monoclonal IgG anti-GM1 mAb. In contrast, AIDP patient and healthy volunteer sera did not affect the Cav2.1 VDCC current. These results suggest that in some case of GBS, particularly of AMAN patients with IgG anti-GM1 mAb, muscle weakness may be induced by dysfunction of Cav2.1 VDCC functioning at the motor nerve terminals. Special issue article in honor of Dr. George DeVries.     

Involvement of Galectin-3 with Vascular Cell Adhesion Molecule-1 in Growth Regulation of Mouse BALB/3T3 Cells

β-Galactose residues on N-glycans have been implicated to be involved in growth regulation of cells. In the present study we compared the galactosylation of cell surface N-glycans of mouse Balb/3T3 cells between 30 and 100% densities and found the β-1,4-galactosylation of N-glycans increases predominantly in a 100-kDa protein band on lectin blot analysis in combination with digestions by diplococcal β-galactosidase and N-glycanase. When cells at 100% density were treated with jack bean β-galactosidase, the incorporation of 5-bromodeoxyuridine into the cells was stimulated in a dose-dependent manner, suggesting the involvement of the galactose residues in growth regulation of cells. A galactose-binding protein was isolated from the plasma membranes of cells at 100% density by affinity chromatography using an asialo-transferrin-Sepharose column and found to be galectin-3 as revealed by mass spectrometric analysis. The addition of recombinant galectin-3 into cells at 50% density inhibited the incorporation of 5-bromodeoxyuridine in a dose-dependent manner, but the inhibition was prevented with haptenic sugar. An immunocytochemical study showed that galectin-3 is present at the surface of cells at 100% density but not at 30% density where it locates inside the cells. Several glycoproteins bind to a galectin-3-immobilized column, a major of which was identified as vascular cell adhesion molecule (VCAM)-1. Immunocytochemical studies showed that some galectin-3 and VCAM-1 co-localize at the surface of cells at 100% density, indicating that the binding of galectin-3 secreted from cells to VCAM-1 is one of the pathways involved in the growth regulation of Balb/3T3 cells.     

Development of microsatellite markers for Quercus miyagii Koidz. (Fagaceae), an endemic species in the Ryukyu Islands, Japan

Quercus miyagii is an endemic tree species in the Ryukyu Islands, Japan. We isolated and characterized 15 microsatellite loci in this species. The number of alleles ranged from 2 to 16 and expected heterozygosities from 0.07 to 0.92. This set of markers is potentially useful to investigate the genetic structure, gene flow, and the biogeographic history of Q. miyagii in the Ryukyu Islands, Japan.     

Evaluation of mismatch-binding ligands as inhibitors for Rev-RRE interaction

Drugs targeting the stem-loop IIB of Rev responsible element (RRE) of HIV-1 mRNA are potential therapeutic agents for HIV-1 infection. The stem loop is characterized by an internal loop consist of consecutive G-G and G-A mismatches, which is the single binding site for Rev protein for nuclear export of viral mRNA. We report here that ligands binding to G-G and G-A mismatches in duplex DNA also bind to the internal loop in competition with Rev peptide and lead to the dissociation of pre-formed Rev-RRE complex in a model system.     

PimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria

Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.