Polyclad turbellarians collected on the Osaka University Expedition to Viti Levu,Fiji, in 1985, with remarks on distribution and phylogeny of the genus Discoplana

We report the first records from Viti Levu, Fiji, for four species of polyclad turbellarians: Discoplana gigas (Schmarda), Paraplanocera oligoglena (Schmarda), Cestoplana cuneata Sopott-Ehlers et Schmidt, and Pericelis byerleyana (Collingwood). D. gigas has the widest distribution in the Indo-West Pacific among the six described species of Discoplana, and shows a wider range of color variation than has been attributed to it before. Analysis of morphological features in these species reveals that Discoplana can be divided hierarchically into several sister-species groups based upon differences in structure of the vagina and of the penis. Four species, occurring mainly in the Pacific, share an apomorphic feature of the vagina, a feature not seen in the other two from the Indian Ocean. This suggests that Discoplana originated in the Indo-West Pacific.     

CENP-32 is required to maintain centrosomal dominance in bipolar spindle assembly

Centrosomes nucleate spindle formation, direct spindle pole positioning, and are important for proper chromosome segregation during mitosis in most animal cells. We previously reported that centromere protein 32 (CENP-32) is required for centrosome association with spindle poles during metaphase. In this study, we show that CENP-32 depletion seems to release centrosomes from bipolar spindles whose assembly they had previously initiated. Remarkably, the resulting anastral spindles function normally, aligning the chromosomes to a metaphase plate and entering anaphase without detectable interference from the free centrosomes, which appear to behave as free asters in these cells. The free asters, which contain reduced but significant levels of CDK5RAP2, show weak interactions with spindle microtubules but do not seem to make productive attachments to kinetochores. Thus CENP-32 appears to be required for centrosomes to integrate into a fully functional spindle that not only nucleates astral microtubules, but also is able to nucleate and bind to kinetochore and central spindle microtubules. Additional data suggest that NuMA tethers microtubules at the anastral spindle poles and that augmin is required for centrosome detachment after CENP-32 depletion, possibly due to an imbalance of forces within the spindle.     

Neonatal repetitive maternal separation causes long-lasting alterations in various neurotrophic factor expression in the cerebral cortex of rats

AimsThis study was carried out to examine the effects of early postnatal maternal separation stress on the development of the cerebral cortex with respect to time-dependent fluctuations of neurotrophic factor ligand and receptor expression.Main methodsWistar rats were separated from their mothers for 3 h per day during postnatal days (PND) 10 to 15. The cerebral cortex was analyzed by real-time RT-PCR for the evaluation of the expression of mRNA for brain-derived neurotrophic factor (BDNF), TrkB, insulin-like growth factor-1 (IGF-1), and type 1 IGF receptor (IGF-1R) on PND16, 20, 30, and 60.Key findingsThe expression of these neurotrophic factor ligands and receptors in the cerebral cortex was enhanced on PND16 and PND20, and then it returned to baseline levels on PND30. By PND60, however, the expression levels were attenuated.SignificanceThe important implication of this study is the persistent abnormal fluctuation of neurotrophic factor expression for a prolonged period, triggered even after the brain growth spurt. Given that neurotrophic factors play important roles in brain development, it can be speculated that the altered expression of these factors induced by maternal separation may interrupt normal brain development and ultimately lead to functional disruption. However, the possibility of such changes leading to various functional disruptions and the underlying mechanisms involved require further study.     

Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, β-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 μM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and β-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 μM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.     

Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergent-resistant microdomain-associated membrane proteins Tat2p and Fur4p

In eukaryotic cells many cell surface proteins are attached to the membrane via the glycosylphosphatidylinositol (GPI) moiety. In yeast, GPI also plays important roles in the production of mannoprotein in the cell wall. We previously isolated gwt1 mutants and found that GWT1 is required for inositol acylation in the GPI biosynthetic pathway. In this study we isolated a new gwt1 mutant allele, gwt1-10, that shows not only high temperature sensitivity but also low temperature sensitivity. The gwt1-10 cells show impaired acyltransferase activity and attachment of GPI to proteins even at the permissive temperature. We identified TAT2, which encodes a high affinity tryptophan permease, as a multicopy suppressor of cold sensitivity in gwt1-10 cells. The gwt1-10 cells were also defective in the import of tryptophan, and a lack of tryptophan caused low temperature sensitivity. Microscopic observation revealed that Tat2p is not transported to the plasma membrane but is retained in the endoplasmic reticulum in gwt1-10 cells grown under tryptophan-poor conditions. We found that Tat2p was not associated with detergent-resistant membranes (DRMs), which are required for the recruitment of Tat2p to the plasma membrane. A similar result was obtained for Fur4p, a uracil permease localized in the DRMs of the plasma membrane. These results indicate that GPI-anchored proteins are required for the recruitment of membrane proteins Tat2p and Fur4p to the plasma membrane via DRMs, suggesting that some membrane proteins are redistributed in the cell in response to environmental and nutritional conditions due to an association with DRMs that is dependent on GPI-anchored proteins.     

Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin

Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)(S/T)P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.     

A novel method for in vitro radiolabeling and testing enveloped viruses by phosphatidylethanolamine N-methyltransferase and host cell-specific binding

The present study developed a novel virus labeling and testing method, referred to as an envelope-labeled virus assay (ELVA), in which virus envelope is labeled in vitro by the action of phosphatidylethanolamine N-methyltransferase (PEMT) and tested through a host cell-specific binding. A recombinant strain (vGFPuv) of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and Spodoptera frugiperda (Sf-9) insect cells were used as a model of viruses and host cells, respectively. The labeling mixture, which contained PEMT, [methyl-3H]S-adenosylmethionine (SAM), and a trace amount of detergent Triton X-100, brought about little change in virus titer of vGFPuv on a 1-h incubation, but was so toxic to Sf-9 cells as to immediately cause cell death. After being incubated with vGFPuv, therefore, the labeling mixture was neutralized by adsorptive removal of PEMT and Triton X-100 before Sf-9 cells were contacted with the mixture to extract the virus. The Sf-9 cells were then washed with a phosphate buffered saline (PBS), and lipid extracts with a 1% SDS solution were subjected to a liquid scintillation analysis for the determination of labeling efficiency. As a result, a significant amount of radioactivity was determined in the extracts, demonstrating the validity of ELVA for labeling and testing enveloped viruses. The conditions for the PEMT reaction and cell-virus binding were examined, and the lower detection limit of AcMNPV by ELVA was found to lie in the order of 10(3) plaque forming unit (pfu) per milliliter. Since the labeling reaction and detection of virus are based on neither immunological nor genetic characteristics of virus, ELVA is also expected to be a convenient and comprehensive test of other enveloped viruses.     

SSEA-1 marks regionally restricted immature subpopulations of embryonic retinal progenitor cells that are regulated by the Wnt signaling pathway

Identification and expansion of retinal progenitor cells are critical issues from both scientific and clinical aspects. Here, we identified SSEA-1 (CD15) as a novel surface antigen that can be used to define immature retinal progenitor cells. SSEA-1-expressing retinal cells were found in the peripheral region of the early embryonic mouse retina, and then their number dramatically disappeared along with retinal development. FACS analysis showed that the cells strongly positive for SSEA-1 co-expressed Ki67 proliferation antigen in all the developmental stages examined. The SSEA-1-expressing cells formed larger colonies than the non-expressing ones in retinal re-aggregation cultures. Moreover, late onset of rhodopsin expression was observed in SSEA-1-positive progenitor cells, supporting the idea that these cells have an intrinsically immature character. Differential expression of Wnt signal-related genes between SSEA-1-positive and -negative subpopulations of retina cells was revealed, and the expression of constitutively active forms of Wnt signaling molecules resulted in a greater number of SSEA-1-positive cells. In light of all of the data taken together, we propose SSEA-1 to be a surface marker to define a regionally restricted immature subset of progenitor cells of mouse neural retina, with SSEA-1 expression by them positively regulated by Wnt signals.     

Transformation of poplar (Populus alba) plastids and expression of foreign proteins in tree chloroplasts

Plastid transformation offers several unique advantages compared with nuclear genome transformation, such as high level of transgene expression within plastids, expressing multiple transgenes as operons, lack of position effect due to site-specific transgene integration, and reducing risks of gene flow via pollen due to maternal inheritance of the plastid genome. Plastid transformation has been applied to several herbal species, but as yet there are no applications to tree species. We report here the first successful plastid transformation in a tree species, Populus alba. A vector for plastid transformation of poplar (Populus alba) was constructed, which carried the spectinomycin resistance gene and the green fluorescence protein gene as marker genes. In the regenerated shoots, the site-specific integration of foreign genes and the establishment of a high homoplastomic state were confirmed. Immunoblot analysis and histological observations corroborated the accumulation of green fluorescence protein in chloroplasts. The establishment of a plastid transformation system in poplar provides a novel tool for tree biotechnology.