A novel RNA-recognition-motif protein is required for premeiotic G1/S-phase transition in rice (Oryza sativa L.)

The molecular mechanism for meiotic entry remains largely elusive in flowering plants. Only Arabidopsis SWI1/DYAD and maize AM1, both of which are the coiled-coil protein, are known to be required for the initiation of plant meiosis. The mechanism underlying the synchrony of male meiosis, characteristic to flowering plants, has also been unclear in the plant kingdom. In other eukaryotes, RNA-recognition-motif (RRM) proteins are known to play essential roles in germ-cell development and meiosis progression. Rice MEL2 protein discovered in this study shows partial similarity with human proline-rich RRM protein, deleted in Azoospermia-Associated Protein1 (DAZAP1), though MEL2 also possesses ankyrin repeats and a RING finger motif. Expression analyses of several cell-cycle markers revealed that, in mel2 mutant anthers, most germ cells failed to enter premeiotic S-phase and meiosis, and a part escaped from the defect and underwent meiosis with a significant delay or continued mitotic cycles. Immunofluorescent detection revealed that T7 peptide-tagged MEL2 localized at cytoplasmic perinuclear region of germ cells during premeiotic interphase in transgenic rice plants. This study is the first report of the plant RRM protein, which is required for regulating the premeiotic G1/S-phase transition of male and female germ cells and also establishing synchrony of male meiosis. This study will contribute to elucidation of similarities and diversities in reproduction system between plants and other species.     

PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells

Translesion DNA synthesis (TLS) is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub) in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA) regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R) cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+) cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R) mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.     

TRIC-A channels in vascular smooth muscle contribute to blood pressure maintenance

TRIC channel subtypes, namely TRIC-A and TRIC-B, are intracellular monovalent cation channels postulated to mediate counter-ion movements facilitating physiological Ca(2+) release from internal stores. Tric-a-knockout mice developed hypertension during the daytime due to enhanced myogenic tone in resistance arteries. There are two Ca(2+) release mechanisms in vascular smooth muscle cells (VSMCs); incidental opening of ryanodine receptors (RyRs) generates local Ca(2+) sparks to induce hyperpolarization, while agonist-induced activation of inositol trisphosphate receptors (IP(3)Rs) evokes global Ca(2+) transients causing contraction. Tric-a gene ablation inhibited RyR-mediated hyperpolarization signaling to stimulate voltage-dependent Ca(2+) influx, and adversely enhanced IP(3)R-mediated Ca(2+) transients by overloading Ca(2+) stores in VSMCs. Moreover, association analysis identified single-nucleotide polymorphisms (SNPs) around the human TRIC-A gene that increase hypertension risk and restrict the efficiency of antihypertensive drugs. Therefore, TRIC-A channels contribute to maintaining blood pressure, while TRIC-A SNPs could provide biomarkers for constitutional diagnosis and personalized medical treatment of essential hypertension.     

The hMsh2-hMsh6 complex acts in concert with monoubiquitinated PCNA and Pol η in response to oxidative DNA damage in human cells

Posttranslational modification of PCNA by ubiquitin plays an important role in coordinating the processes of DNA damage tolerance during DNA replication. The monoubiquitination of PCNA was shown to facilitate the switch between the replicative DNA polymerase with the low-fidelity polymerase eta (η) to bypass UV-induced DNA lesions during replication. Here, we show that in response to oxidative stress, PCNA becomes transiently monoubiquitinated in an?S phase- and USP1-independent manner. Moreover, Polη interacts with mUb-PCNA at sites of oxidative DNA damage via its PCNA-binding and ubiquitin-binding motifs. Strikingly, while functional base excision repair is not required for this modification of PCNA or Polη recruitment to chromatin, the?presence of hMsh2-hMsh6 is indispensable. Our findings highlight an alternative pathway in response to oxidative DNA damage that may coordinate the removal of oxidatively induced clustered DNA lesions and could explain the high levels of oxidized DNA lesions in MSH2-deficient cells.     

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.     

Difference of pine ectomycorrhizal biomass in relation to forest conditions

In this study, two plots in a secondary and another two in planted Pinus densiflora stands were exposed to different forest treatments, and the ectomycorrhizal (EM) biomass and its ergosterol content was measured for a year. The unmanaged plot in the secondary stand had greater EM biomass than those in any other plots. Whereas understory cutting had less effect on EM biomass, litter and humus removal decreased pine EM biomass and its ergosterol content, suggesting that such forest treatment alters EM biomass and its structure.     

<Emphasis Type="Italic">Ophiocordyceps pulvinata</Emphasis> sp. nov., a pathogen of ants with a reduced stroma

Ophiocordyceps pulvinata, a pathogen of ants, is formally described as a new species. Genus level designation of this species is difficult due to several apparently conflicting morphological and ecological characters. Affinity with Ophiocordyceps is suggested by the dark color stroma and ascospore morphology. However, the species was included in a book of entomopathogenic fungi of Japan as Torrubiella sp. due to the production of perithecia on an astipitate stroma. Phylogenetic analyses of molecular data support a close relationship with O. unilateralis, a finding consistent with morphological characteristics of the color, asci and ascospores and ecological traits of host affiliation. Thus, O. pulvinata represents another example of the loss of stipe for the hypocrealean arthropod pathogenic fungi and highlights the utility of asci and ascospore morphology as taxonomically informative characters of closely related taxa.     

Silibinin induced-autophagic and apoptotic death is associated with an increase in reactive oxygen and nitrogen species in HeLa cells

Silibinin, as the major active constituent of silymarin, has its various biological effects. Here, we investigated the inhibitory effects of silibinin on HeLa cell growth in relation to autophagy and apoptosis induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation. Silibinin dose and time-dependently decreased cell growth cultured in medium containing 10% fetal bovine serum or in serum free media (SFM) with an IC(50) of approximately 80-100 and 40-60 μM at 24 h, respectively. Silibinin induced autophagy at 12 h, confirmed by monodansylcadervarine (MDC) staining and up-regulation of beclin-1, and induced apoptosis at 24 h, detected by observation of apoptotic bodies and activation of caspase-3. 3-methyladenine (3-MA) inhibited silibinin-induced autophagy and attenuated the silibinin's inhibitory effect on cell viability, suggesting that autophagy enhanced silibinin-induced cell death. Silibinin increased ROS levels at 12 h, and ROS scavenger, N-acetylcysteine (NAC), significantly reversed the cytotoxicity of silibinin through inhibiting both autophagy and apoptosis. Specific antioxidants were applied and results indicated that hydroxyl radical (·OH) was the major ROS induced by silibinin, and OH scavenger glutathione (GSH) inhibited apoptosis and autophagy. Silibinin also generated RNS production in the cells at 12 h. High concentration of N omega-nitro-l-arginine methyl ester (L-NAME) as nitric oxide synthase (NOS) inhibitor attenuated the cytotoxicity of silibinin by decreasing ROS levels, leading to down-regulation of apoptosis. Silibinin also could interrupt the respiring functions of mitochondria, leading to ROS production and oxidative damage.