Regulation of S-thiolation and S-nitrosylation in the thiol/nitric oxide system by radical scavengers.

Nitric oxide (NO) is a possible agent, which induces crosslinking among molecules containing sulfhydryl groups. However, the S-thiolation is essentially accompanied by S-nitrosylation. In the present study, we evaluated radical scavengers as a regulator for S-thiolation and S-nitrosylation by NO released from NO-generator, 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (P-NONOate). When glutathione was incubated with P-NONOate in 4% (vol/vol) O(2)-saturated buffer solution (pH 7.4) in the presence of nitrone spin-trapping agent, 5,5'-dimethyl-1-pyroline-N-oxide (DMPO), the prevention of S-thiolation and the promotion of S-nitrosylation were observed. The DMPO adduct was identified to be thiyl radical-DMPO adduct via ESR study. In contrast, nitroxyl radical, radical scavenger against oxygen-centered radicals, promoted the S-thiolation but prevented S-nitrosylation. Nitronyl nitroxide, radical scavenger against nitric oxide, can convert nitric oxide into nitrogen dioxide in the O(2)-independent manner. In the presence of nitronyl nitroxide in the thiol/P-NONOate system, S-thiolation was remarkably enhanced up to 60% (mol/mol) of sulfhydryl groups. However, nitronyl nitroxide at enough content (>or=1.0 mM) almost completely prevented S-nitrosylation, whereas nitronyl nitroxide at comparatively lower content (0.5 mM) contrarily enhanced the S-nitrosylation. Based on these facts, it appeared to be possible to consequently regulate S-thiolation and S-nitrosylation through controlling the thiyl radical chain reaction by radical scavengers.     

Amounts and patterns of nucleotide variation within and between two Japanese conifers, sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) (Cupressaceae sensu lato)

Sugi (Cryptomeria japonica) and hinoki (Chamaecyparis obtusa) are the most important timber species in Japan. To quantify and compare the level of nucleotide variation in these species, we investigated their variation at ten nuclear loci. Average values of nucleotide diversity at synonymous sites (π _SYN) found in sugi and hinoki were 0.0038 and 0.0069, respectively. However, although the average value of nucleotide diversity was higher in hinoki than in sugi, their average values of haplotype diversity were similar. Deviations from the standard neutral model were detected at two loci in hinoki using Tajima’s D, Fay and Wu’s H, and Strobeck’s S statistics, which seem to be due to its historical population structure. Levels of divergence between the two species at synonymous sites of the ten genes ranged from 0.121 to 0.566 (0.28 on average). These values positively correlated with their guanine + cytosine contents at third-codon positions of synonymous sites (%GC3s).     

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.     

Egg-jelly structure promotes efficiency of internal fertilization in the newt, Cynops pyrrhogaster

Egg-jelly is composed of a network of fibrous components and contains substances regulating the sperm-egg interaction. Many studies on the latter have been conducted, whereas the role of the egg-jelly structure in fertilization has not yet been fully assessed. In this study, we examined the fertilization efficiency in the presence and absence of the structure around the egg of the newt, Cynops pyrrhogaster, using a gelatin gel system. Although de-jellied eggs of C. pyrrhogaster can be fertilized with an adequate number of sperm, the fertilization rate was dramatically increased through the use of the gelatin gel. Sperm showed forward motility with straight morphology in the gel, whereas they swam in circles in solution. This result indicates that the gel structure is significant for sperm guidance to the egg surface, and its presence raises the fertilization efficiency in C. pyrrhogaster. When sperm were entangled in the gel structure, they were immediately folded and never showed any forward motility. Sperm with zigzag morphology were observed in the gelatin gel as well as in the egg-jelly, indicating the elimination of sperm by the gel structure. The effect of sperm elimination on successful fertilization was estimated using gelatin gels of different thickness. Though the variation did not affect the fertilization rate, the rate of normal development gradually increased in the thicker gels. This result indicates that sperm elimination in egg-jelly can function in the fertilization system. The roles of sperm guidance and sperm elimination under the physiological condition of internal fertilization of the newt are discussed.     

Development and characterization of microsatellite markers for Cryptomeria japonica D.Don

Thirty four microsatellite markers for Cryptomeria japonica D. Don were developed by searching three types of library: a database of C. japonica cDNA sequences, a standard non-enriched genomic DNA library and a microsatellite-enriched library using magnetic particles. The enrichment of microsatellite sequences using magnetic particles is very efficient compared to the other two methods both in terms of the numbers of markers generated, and in the polymorphism they detect. The microsatellites developed from the genomic DNA library generally have longer repeat sequences and are more polymorphic than those from cDNA. All the developed microsatellite markers in this study showed polymorphism among 28 plus trees selected from locations scattered throughout Japan. The mean number of alleles per locus (MNA) detected in the 28 plus trees ranged from 2 to 21 with an average of 7.5. The Polymorphism Information Content (PIC) ranged from 0.160 to 0.936 with an average of 0.666. Co-dominant segregation of alleles in a three-generation pedigree of C. japonica was demonstrated at 34 microsatellite loci, and the segregation was not distorted from Mendelian expectation for all loci. In 12 out of 34 loci, a null allele was detected. Key relationships between polymorphic parameters, such as MNA and PIC, and the characteristics of microsatellite sequences, such as the longest repeat number, total repeat number and total number of nucleotides, were investigated using rank correlation coefficients, Kendall's tau. A positive correlation was found between repeat lengths and polymorphisms. The markers provide sufficient resolution for investigating gene flow within forests and seed orchards, and for genome mapping.     

Regulatory function of whey acidic protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro

Although possible biological functions of whey acidic protein (WAP) have been suggested, few studies have focused on investigating the function of WAP. This paper describes evidence for WAP function in lobulo-alveolar development in mammary glands in vivo and in the cell cycle progression of mammary epithelial cells in vitro. Ubiquitous overexpression of WAP transgene impaired only lobulo-alveolar development in the mammary glands of transgenic female mice but not other physiological functions, indicating that the inhibitory function of WAP is specific to mammary alveolar cells. The forced expression of WAP significantly inhibited the proliferation of mouse mammary epithelial cells (HC11 cells and EpH4/K6 cells), whereas it did not affect that of NIH3T3 cells. Co-culturing of WAP-clonal cells and control cells using a transwell insert demonstrated that WAP inhibited the proliferation of HC11 cells through a paracrine action but not that of NIH3T3 cells, and that WAP was able to bind to HC11 cells but not to NIH3T3 cells. Apoptosis was not enhanced in the HC11 cells with stable WAP expression (WAP-clonal HC11 cells). BrdU incorporation and FACScan analyses revealed that cell cycle progression from the G0/G1 to the S phase was inhibited in the WAP-clonal HC11 cells. Among G1 cyclins, the expression of cyclin D1 and D3 was significantly decreased in the WAP-clonal HC11 cells. The present results provide the first documented evidence that WAP plays a negative regulatory role in the cell cycle progression of mammary epithelial cells through an autocrine or paracrine mechanism in vivo.     

Cellular stresses induce the nuclear accumulation of importin alpha and cause a conventional nuclear import block

We report here that importin alpha accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin alpha appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin alpha. In addition, nuclear retention and the importin beta/Ran-independent import of importin alpha also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin alpha from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin alpha is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin alpha can be involved in a common physiological response to various stress conditions.     

Dynamics of homologous chromosome pairing during meiotic prophase in fission yeast

Pairing of homologous chromosomes is important for homologous recombination and correct chromosome segregation during meiosis. It has been proposed that telomere clustering, nuclear oscillation, and recombination during meiotic prophase facilitate homologous chromosome pairing in fission yeast. Here we examined the contributions of these chromosomal events to homologous chromosome pairing, by directly observing the dynamics of chromosomal loci in living cells of fission yeast. Homologous loci exhibited a dynamic process of association and dissociation during the time course of meiotic prophase. Lack of nuclear oscillation reduced association frequency for both centromeric and arm regions of the chromosome. Lack of telomere clustering or recombination reduced association frequency at arm regions, but not significantly at centromeric regions. Our results indicate that homologous chromosomes are spatially aligned by oscillation of telomere-bundled chromosomes and physically linked by recombination at chromosome arm regions; this recombination is not required for association of homologous centromeres.     

Development of nuclear microsatellite markers for the Japanese conifers Tsuga diversifolia and T. sieboldii (Pinaceae)

Nuclear microsatellite markers were developed for the two Tsuga species native to the Japanese Archipelago, Tsuga diversifolia and T. sieboldii, and a population with genetic affinities to T. diversifolia on Ulleung Island, Korea. Tsuga diversifolia and T. sieboldii are widespread dominant trees of temperate and subalpine forests in Japan but to date no genetic markers have been developed for these species. Fifteen polymorphic loci were developed and characterized, of which 14 are reliably amplified in each taxon. Across both species and the Ulleung Island population, the number of alleles per locus ranged from 3 to 26 (average = 13.93) and observed heterozygosity ranged from 0.005 to 0.935 (average = 0.535). In addition, all 15 loci were successfully amplified in a single accession of the Chinese species, T. chinensis. These markers will be useful for investigating the species’ biogeography, range‐wide genetic diversity, conservation genetic issues and potential for hybridisation.     

H3K27 Demethylase,JMJD3, Regulates Fragmentation of Spermatogonial Cysts

The spermatogonial stem cell (SSC) compartment is maintained by self-renewal of stem cells as well as fragmentation of differentiating spermatogonia through abscission of intercellular bridges in a random and stochastic manner. The molecular mechanisms that regulate this reversible developmental lineage remain to be elucidated. Here, we show that histone H3K27 demethylase, JMJD3 (KDM6B), regulates the fragmentation of spermatogonial cysts. Down-regulation of Jmjd3 in SSCs promotes an increase in undifferentiated spermatogonia but does not affect their differentiation. Germ cell-specific Jmjd3 null male mice have larger testes and sire offspring for a longer period compared to controls, likely secondary to increased and prolonged maintenance of the spermatogonial compartment. Moreover, JMJD3 deficiency induces frequent fragmentation of spermatogonial cysts by abscission of intercellular bridges. These results suggest that JMJD3 controls the spermatogonial compartment through the regulation of fragmentation of spermatogonial cysts and this mechanism may be involved in maintenance of diverse stem cell niches.