Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of L-glutamic acid diethyl ester

Papain polymerizes L-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (gamma-ethyl alpha-L-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 degrees C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 degrees C revealed that the accumulation of the initial dimerization product, L-glutamyl-L-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nalpha-benzoyl-L-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S'-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.     

Visualizing spatiotemporal dynamics of multicellular cell-cycle progression

The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in which every cell nucleus exhibits either red or green fluorescence. We performed time-lapse imaging to explore the spatiotemporal patterns of cell-cycle dynamics during the epithelial-mesenchymal transition of cultured cells, the migration and differentiation of neural progenitors in brain slices, and the development of tumors across blood vessels in live mice. These mice and cell lines will serve as model systems permitting unprecedented spatial and temporal resolution to help us better understand how the cell cycle is coordinated with various biological events.     

The flexible arm of tRNase Z is not essential for pre-tRNA binding but affects cleavage site selection

tRNase Z is an enzyme responsible for removing a 3′ trailer from pre-tRNA. Although most tRNase Zs cleave pre-tRNAs immediately after the discriminator nucleotide with the exception of Thermotoga maritima tRNase Z, which cleaves after the ₇₄CCA₇₆ sequence, our knowledge was limited about how the cleavage site in pre-tRNA is selected. Bacterial tRNase Zs contain a unique domain termed flexible arm, which extends from the core domain. Using various tRNase Z variants, here we examined how the flexible arm affects the cleavage site selection. T. maritima tRNase Z variants with modified flexible arms shifted the cleavage site and a Bacillus subtilis tRNase Z variant with no flexible arm showed an anomalous cleavage activity. Some of the T. maritima/B. subtilis chimeric enzymes had both properties: they recognized ₇₄CCA₇₆-containing pre-tRNA and cleaved it after the discriminator. Taken together, the present data indicate that the flexible arm is not essential for pre-tRNA binding but affects the cleavage site selection probably by pushing the distal region of the T arm in such a way that the discriminator nucleotide becomes closer to the catalytic site.     

Social isolation rearing-induced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice

Experiences during brain development may influence the pathogenesis of developmental disorders. Thus, social isolation (SI) rearing after weaning is a useful animal model for studying the pathological mechanisms of such psychiatric diseases. In this study, we examined the effect of SI on neurogenesis in the hippocampal dentate gyrus (DG) relating to memory and emotion-related behaviors. When newly divided cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before SI, the number of BrdU-positive cells and the rate of differentiation into neurons were significantly decreased after 4-week SI compared with those in group-housed mice. Repeated treatment of fluoxetine prevented the SI-induced impairment of survival of newly divided cells and ameliorated spatial memory impairment and part of aggression in SI mice. Furthermore, we investigated the changes in gene expression in the DG of SI mice by using DNA microarray and real-time PCR. We finally found that SI reduced the expression of development-related genes Nurr1 and Npas4 . These findings suggest that communication in juvenile is important in the survival and differentiation of newly divided cells, which may be associated with memory and aggression, and raise the possibility that the reduced expression of Nurr1 and/or Npas4 may contribute to the impairment of neurogenesis and memory and aggression induced by SI.     

Inhibitory effects of whisky congeners on melanogenesis in mouse B16 melanoma cells

We examined the effect of whisky congeners, substances other than ethanol in whisky, on melanogenesis in mouse B16 melanoma cells. Treatment with whisky congeners significantly blocked melanogenesis. Our results indicate that the inhibitory effects of whisky congeners on melanogenesis is due to direct inhibition of tyrosinase activity and to suppression of tyrosinase protein levels.     

Effects of elevated CO<Subscript>2</Subscript> and nitrogen on wood structure related to water transport in seedlings of two deciduous broad-leaved tree species

Wood structure might be altered through the physiological responses to atmospheric carbon dioxide concentration ([CO₂]) and nitrogen (N) deposition. We investigated growth, water relations and wood structure of 1-year-old seedlings of two deciduous broad-leaved tree species, Quercus mongolica (oak, a ring-porous species) and Alnus hirsuta (alder, a diffuse-porous species and N₂–fixer), grown under a factorial combination of two levels of [CO₂] (36 and 72 Pa) and nitrogen supply (N; low and high) for 141 days in phytotron chambers. In oak, there was no significant effect of [CO₂] on wood structure, although elevated [CO₂] tended to decrease stomatal conductance (g _s) and increased water use efficiency regardless of the N treatment. However, high N supply increased root biomass and induced wider earlywood and larger vessels in the secondary xylem in stems, leading to increased hydraulic conductance. In alder, there was significant interactive effect of [CO₂] and N on vessel density, and high N supply increased the mean vessel area. Our results suggest that wood structures related to water transport were not markedly altered, although elevated [CO₂] induced changes in physiological parameters such as g _s and biomass allocation, and that N fertilization had more pronounced effects on non-N₂-fixing oak than on N₂-fixing alder.     

Mapping of a major locus controlling seed dormancy using backcrossed progenies in wheat (Triticum aestivum L.).

Seed dormancy is an important factor regulating preharvest sprouting (PHS) but is a complex trait for genetic analysis. We previously identified a major quantitative trait locus (QTL) controlling seed dormancy on the long arm of chromosome 4A (4AL) in common wheat. To transfer the QTL from the dormant lines 'OS21-5' and 'Leader' into the Japanese elite variety 'Haruyokoi', which has an insufficient level of seed dormancy, backcrossing was carried out through marker-assisted selection (MAS) using PCR-based codominant markers. Nineteen BC5F2 plants with homozygous alleles of 'OS21-5' or 'Haruyokoi' were developed and evaluated for seed dormancy under greenhouse conditions. The seeds harvested from plants with 'OS21-5' alleles showed a clearly high level of dormancy compared with seeds from plants with 'Haruyokoi' alleles. Additionally, the dormancy phenotype of BC3F3 seeds harvested from 128 BC3F2 plants with homozygous alleles of 'Leader' or 'Haruyokoi' showed a clear difference between these alleles. The QTL on 4AL confers a major gene, Phs1, which was mapped within a 2.6 cM region. The backcrossed lines developed in this study can be important sources for improving PHS resistance in Japanese wheat and for analyzing the mechanism of seed dormancy. MAS was useful for the development of near-isogenic lines in this complex trait, to facilitate the molecular dissection of genetic factors.     

Two structurally distinct inhibitors of glycogen synthase kinase 3 induced centromere positive micronuclei in human lymphoblastoid TK6 cells

Glycogen synthase kinase 3 (GSK3) is an attractive novel pharmacological target. Inhibition of GSK3 is recently regarded as one of the viable approaches to therapy for Alzheimer's disease, cancer, diabetes mellitus, osteoporosis, and bipolar mood disorder. Here, we have investigated the aneugenic potential of two potent and highly specific inhibitors of GSK3 by using an in vitro micronucleus test with human lymphoblastoid TK6 cells. One inhibitor was a newly synthesized maleimide derivative and the other was a previously known aminopyrimidine derivative. Both compounds elicited statistically significant and concentration-dependent increases in micronucleated cells. One hundred micronuclei (MN) of each were analyzed using centromeric DNA staining with fluorescence in situ hybridization. Both the two structurally distinct compounds induced centromere-positive micronuclei (CMN). Calculated from the frequency of MN cells and the percentage of CMN, CMN cell incidence after treatment with the maleimide compound at 1.2muM, 2.4muM, and 4.8muM was 11.6, 27.7, and 56.3 per 1000 cells, respectively; the negative control was 4.5. CMN cell incidence after the treatment with the aminopyrimidine compound at 1.8muM, 3.6muM, and 5.4muM was 6.7, 9.8 and 17.2 per 1000 cells, respectively. Both compounds exhibited concentration-dependent increase in the number of mitotic cells. The frequency of CMN cells correlated well with mitotic cell incidence after treatment with either compound. Furthermore, both inhibitors induced abnormal mitotic cells with asymmetric mitotic spindles and lagging anaphase chromosomes. These results lend further support to the hypothesis that the inhibition of GSK3 activity affects microtubule function and exhibits an aneugenic mode of action.     

Pregnane glycosides from Cynanchum atratum

Five new pregnane glycosides, cynanosides K-O (1-5) with a 14,15-seco-pregnane-type skeleton as the aglycon, together with five known compounds, cynascyroside C, sublanceoside E(1), sublanceoside I(1), atratoside A and atratoside B, were isolated from the roots of Cynanchum atratum. Their structures were determined on the basis of spectroscopic analysis and chemical evidence.