Reelin regulates differentiation of neural stem cells by activation of notch signaling through Disabled-1 tyrosine phosphorylation

We have previously reported the cross-talk between Reelin and Notch-1 signaling pathways, which are 2 major pathways that regulate brain development. We found that Reelin activated Notch-1 signaling, leading to the expression of brain lipid binding protein (BLBP) and the formation of radial glial cells in human neural progenitor cells (hNPCs). In the current study, we investigated the molecular mechanisms by which Reelin activates Notch-1. We show that Reelin-stimulated Notch-1 activation is dependent on Reelin signaling. The induction of Disabled-1 (Dab-1) tyrosine phosphorylation, and the subsequent activation of Src family kinases, were found to be essential steps for the activation of Notch-1 signaling by Reelin. Reelin treatment increased the interaction between Dab-1 and Notch-1 intracellular domain (NICD), and enhanced NICD translocation to the nucleus. This study advances our knowledge of the regulation of Notch-1 activation by Reelin signaling in hNPCs, as an approach to understanding cell fate determination, differentiation, and neurogenesis during brain development.     

Expression of Arabidopsis plastidial phosphoglucomutase in tobacco stimulates photosynthetic carbon flow into starch synthesis

Phosphoglucomutase (PGM, EC 2.7.5.1) is one of the enzymes constituting the carbohydrate synthesis pathway in higher plants. It catalyzes the reversible conversion of glucose 6-phosphate (Glc6P) to glucose 1-phosphate (Glc1P). Previously, metabolic turnover analysis using (13)CO(2) in tobacco leaves demonstrated that conversion of Glc6P to Glc1P may limit carbon flow into carbohydrate synthesis. In order to assess the effects of PGM, Arabidopsis thaliana cytosolic or plastidial PGM was expressed under the control of cauliflower mosaic virus 35S promoter in tobacco plants (Nicotiana tabacum cv. Xanthi) and phenotypic analysis was performed. The transgenic plants expressing Arabidopsis plastidial PGM showed 3.5-8.2-fold higher PGM activity than that of wild-type, and leaf starch and sucrose contents increased 2.3-3.2-fold and 1.3-1.4-fold, respectively over wild-type levels. In vivo(13)C-labeling experiments indicated that photosynthetically fixed carbon in the transgenic plants could be converted faster to Glc1P and adenosine 5'-diphosphate glucose than in wild-type, suggesting that elevation of plastidial PGM activity should accelerate conversion of Glc6P to Glc1P in chloroplasts and increase carbon flow into starch. On the other hand, transgenic plants expressing Arabidopsis cytosolic PGM showed a 2.1-3.4-fold increase in PGM activity over wild-type and a decrease of leaf starch content, but no change in sucrose content. These results suggest that plastidial PGM limits photosynthetic carbon flow into starch.     

Quantitative connection between polyglutamine aggregation kinetics and neurodegenerative process in patients with Huntington’s disease

Background

Despite enormous progress in elucidating the biophysics of aggregation, no cause-and-effect relationship between protein aggregation and neurodegenerative disease has been unequivocally established. Here, we derived several risk-based stochastic kinetic models that assess genotype/phenotype correlations in patients with Huntington??s disease (HD) caused by the expansion of a CAG repeat. Fascinating disease-specific aspects of HD include the polyglutamine (polyQ)-length dependence of both age at symptoms onset and the propensity of the expanded polyQ protein to aggregate. In vitro, aggregation of polyQ peptides follows a simple nucleated growth polymerization pathway. Our models that reflect polyQ aggregation kinetics in a nucleated growth polymerization divided aggregate process into the length-dependent nucleation and the nucleation-dependent elongation. In contrast to the repeat-length dependent variability of age at onset, recent studies have shown that the extent of expansion has only a subtle effect on the rate of disease progression, suggesting possible differences in the mechanisms underlying the neurodegenerative process.

Results

Using polyQ-length as an index, these procedures enabled us for the first time to establish a quantitative connection between aggregation kinetics and disease process, including onset and the rate of progression. Although the complexity of disease process in HD, the time course of striatal neurodegeneration can be precisely predicted by the mathematical model in which neurodegeneration occurs by different mechanisms for the initiation and progression of disease processes. Nucleation is sufficient to initiate neuronal loss as a series of random events in time. The stochastic appearance of nucleation in a cell population acts as the constant risk of neuronal cell damage over time, while elongation reduces the risk by nucleation in proportion to the increased extent of the aggregates during disease progression.

Conclusions

Our findings suggest that nucleation is a critical step in gaining toxic effects to the cell, and provide a new insight into the relationship between polyQ aggregation and neurodegenerative process in HD.     

Chromosome aberration frequencies and chromosome instability in mice after long-term exposure to low-dose-rate gamma-irradiation

Chronological changes of chromosome aberration rates related to accumulated doses in chronically exposed humans and animals at a low-dose-rate have not been well studied. C3H female specific pathogen-free mice (8 weeks of age) were chronically irradiated. Chromosome aberration rate in mouse splenocytes after long-term exposure to low-dose-rate (LDR) gamma-rays was serially determined by conventional Giemsa method. Incidence of dicentrics and centric rings increased almost linearly up to 8000 mGy following irradiation for about 400 days at a LDR of 20 mGy/day. Clear dose-rate effects were observed in the chromosome aberration frequencies between dose rates of 20 mGy/day and 200 Gy/day. Furthermore, the frequencies of complex aberrations increased as accumulated doses increased in LDR irradiation. This trend was also observed for the incidences of micronuclei and trisomies of chromosomes 5, 13 and 18 in splenocytes, detected by micronucleus assay and metaphase fluorescence in situ hybridization (FISH) method, respectively. Incidences of 2-4 micronuclei and trisomy increased in mouse splenocytes after irradiation of 8000 mGy at a LDR of 20 mGy/day. These complex chromosome aberrations and numerical chromosome aberrations seem to be induced indirectly after radiation exposure and thus the results indicate that continuous gamma-ray irradiation for 400 days at LDR of 20 mGy/day induced chromosomal instability in mice. These results are important to evaluate the biological effects of long-term exposure to LDR radiation in humans.     

Variable region of betanodavirus RNA2 is sufficient to determine host specificity

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The viruses have been classified into 4 distinct types based on nucleotide sequence similarities in the variable region (the so-called T4 region) of the smaller genomic segment RNA2 (1.4 kb). Betanodaviruses have marked host specificity, although the primary structures of the viral RNAs and encoded proteins are similar among the viruses. We have previously demonstrated, using reassortants between striped jack nervous necrosis virus (SJNNV) and redspotted grouper nervous necrosis virus (RGNNV), that RNA2, which encodes the coat protein, strictly controls host specificity. However, because RNA2 is large, we were unable to propose a mechanism underlying this RNA2-based host specificity. To identify the RNA2 region that controls host specificity, we constructed RNA2 chimeric viruses from SJNNV and RGNNV and tested their infectivity in the original host fish, striped jack Pseudocaranx dentex and sevenband grouper Epinephelus septemfasciatus. Among these chimeric viruses, SJNNV mutants containing the variable region of RGNNV RNA2 infected sevenband grouper larvae in a manner similar to RGNNV, while RGNNV mutants containing the variable region of SJNNV RNA2 infected striped jack larvae in a manner similar to SJNNV. Immunofluorescence microscopic studies using anti-SJNNV polyclonal antibodies revealed that these chimeric viruses multiplied in the brains, spinal cords and retinas of the infected fish, as in infections by the parental viruses. These results indicate that the variable region of RNA2 is sufficient to control host specificity in SJNNV and RGNNV.     

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

6-O-sulfation of heparan sulfate differentially regulates various fibroblast growth factor-dependent signalings in culture

Heparan sulfate (HS) interacts with diverse heparin-binding growth factors and thereby regulates their bioactivities. These interactions depend on the structures characterized by the sulfation pattern and isomer of uronic acid residues. One of the biosynthetic modifications of HS, namely 6-O-sulfation, is catalyzed by three isoforms of HS6-O-sulfotransferase. We generated HS6ST-1- and/or HS6ST-2-deficient mice (6ST1-KO, 6ST2-KO, and double knock-out (dKO)) that exhibited different phenotypes. We examined the effects of HS 6-O-sulfation in heparin-binding growth factor signaling using fibroblasts derived from these mutant mice. Mouse embryonic fibroblasts (MEF) prepared from E14.5 dKO mice produced HS with little 6-O-sulfate, whereas 2-O-sulfation in HS from dKO-MEF (dKO-HS) was increased by 1.9-fold. HS6-O-sulfotransferase activity in the dKO-MEF was hardly detected, and HS2-O-sulfotransferase activity was 1.5-fold higher than that in wild type (WT)-MEFs. The response of dKO-MEFs to fibroblast growth factors (FGFs) was distinct from that of WT-MEFs; in dKO-MEFs, FGF-4- and FGF-2-dependent signalings were reduced to approximately 30 and 60% of WT-MEFs, respectively, and FGF-1-dependent signaling was moderately reduced compared with that of WT-MEFs but only at the lower FGF-1 concentrations. Analysis with a surface plasmon resonance biosensor demonstrated that the apparent affinity of dKO-HS for FGF-4 was markedly reduced and was also reduced for FGF-1. In contrast, the affinity of dKO-HS for FGF-2 was 2.5-fold higher than that of HS from WT-MEFs. Thus, 6-O-sulfate in HS may regulate the signalings of some of HB-GFs, including FGFs, by inducing different interactions between ligands and their receptors.     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.     

Population dynamics of Propsilocerus akamusi and Chironomus plumosus (Diptera: Chironomidae) in Lake Suwa in relation to changes in the lake's environment

Hydrobiologia - Lake Suwa is a shallow eutrophic lake in central Japan. We have investigated the long-term population dynamics of chironomids in this lake. The objective of this study was...