Ef7 encodes an ELF3-like protein and promotes rice flowering by negatively regulating the floral repressor gene Ghd7 under both short- and long-day conditions

Much progress has been made in our understanding of photoperiodic flowering of rice and the mechanisms underlying short-day (SD) promotion and long-day (LD) repression of floral induction. In this study, we identified and characterized the Ef7 gene, one of the rice orthologs of Arabidopsis EARLY FLOWERING 3 (ELF3). The ef7 mutant HS276, which was induced by γ-irradiation of the japonica rice cultivar 'Gimbozu', flowers late under both SD and LD conditions. Expression analyses of flowering time-related genes demonstrated that Ef7 negatively regulates the expression of Ghd7, which is a repressor of the photoperiodic control of rice flowering, and consequently up-regulates the expression of the downstream Ehd1 and FT-like genes under both SD and LD conditions. Genetic analyses with a non-functional Ghd7 allele provided further evidence that the delayed flowering of ef7 is mediated through the Ghd7 pathway. The analysis of light-induced expression of Ghd7 revealed that the ef7 mutant was more sensitive to red light than the wild-type plant, but the gate of Ghd7 expression was unchanged. Thus, our results show that Ef7 functions as a floral promoter by repressing Ghd7 expression under both SD and LD conditions.     

Expression and functional analysis of two lycopene β-cyclases from citrus fruits

In the present study, two LCYb genes (CitLCYb1 and CitLCYb2) were isolated from Satsuma mandarin (Citrus unshiu Marc.), Valencia orange (Citrus sinensis Osbeck) and Lisbon lemon (Citrus limon Burm.f.) and their functions were analyzed by the color complementation assay in lycopene-accumulating E. coli cells. The results showed that CitLCYb1 and CitLCYb2 shared high identity at the amino acid level among the three citrus varieties. The N-terminal region of the two proteins encoded by CitLCYb1 and CitLCYb2 was predicted to contain a 51-residue chloroplastic transit peptide, which shared low similarity. In Satsuma mandarin, the secondary structures of the CitLCYb1 and CitLCYb2 encoding proteins without the transit peptide were quite similar. Moreover, functional analysis showed that both enzymes of CitLCYb1 and CitLCYb2 participated in the formation of β-carotene, and when they were co-expressed with CitLCYe, α-carotene could be produced from lycopene in E. coli cells. However, although CitLCYb2 could convert lycopene to α-carotene in E. coli cells, its extremely low level of expression indicated that CitLCYb2 did not participate in the formation of α-carotene during the green stage in the flavedo. In addition, the high expression levels of CitLCYb1 and CitLCYb2 during the orange stage played an important role in the accumulation of β,β-xanthophylls in citrus fruits. The results presented in this study might contribute to elucidate the mechanism of carotenoid accumulation in citrus fruits.     

Screening of PC and PMMA-binding peptides for site-specific immobilization of proteins

In the present study, we used proteomic research technology to develop a method for the screening and evaluation of material-binding peptides for protein immobilization. Using this screening method, soluble Escherichia coli proteins that preferentially adsorbed onto polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model plastic materials were first isolated and identified by 2-dimensional electrophoresis (2DE) combined with peptide mass fingerprinting (PMF). The genes of identified protein candidates (ELN, MLT, OMP, and BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity chromatography. The candidates for PC and PMMA-binding peptides were isolated from peptide fragments from affinity protein candidates, which were digested with trypsin and chymotrypsin. Consequently, 5 candidates for the PC-binding peptide and 2 candidates for the PMMA-binding peptide were successfully identified by MALDI-TOF MS. All of the peptides identified were introduced to the C-terminus of glutathione S-transferase (GST) as a model protein for immobilization. Adsorption of peptide-fused and wild-type GSTs onto the plastic surfaces was directly monitored using a quartz crystal microbalance (QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6 as PC-binders and PM-OMP25 as a PMMA-binder significantly enhanced the adsorption rates of GST, achieving an adsorption density that was more than 10 times higher than that of wild-type GST. Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-OMP25 in the adsorption state were 2 times higher than that of wild-type GST. Thus, the PC and PMMA-binding peptides identified in this study, namely PC-OMP6 and PM-OMP25, were considerably useful for site-specific immobilization of proteins, while maintaining a higher adsorption density and residual activity levels. The method demonstrated in this study will be applicable to the isolation of a variety of material-binding peptides against the surfaces of unique materials.     

Histological study of the spermatheca in three thelytokous parthenogenetic ant species,Pristomyrmex punctatus,Pyramica membranifera and Monomorium triviale (Hymenoptera: Formicidae)

Gotoh, A., Billen, J., Tsuji, K., Sasaki, T. and Ito, F. 2011. Histological study of the spermatheca in three thelytokous parthenogenetic ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale (Hymenoptera: Formicidae). —Acta Zoologica (Stockholm) 00 :1–8. The evolution of obligate parthenogenesis may induce the degeneration of female mating ability and subsequently affect the morphology of the female reproductive organs related to mating and/or sperm storage. Here, we investigated the size and structure of the sperm storage organ, the spermatheca, in three thelytokous parthenogenetic myrmicine ant species, Pristomyrmex punctatus, Pyramica membranifera and Monomorium triviale, and compared it with that of their related sexually reproducing species. So far, mated individuals have never been found in these three species, which appears to be in line with their parthenogenetic status. Although the spermatheca appears to be useless in these species, we could not find any evidence on the degeneration in size and morphology of their spermathecae. The spermathecal reservoir still has the columnar hilar epithelium, which is one of the major features for a functional spermatheca in ants.     

Fragmented CagA protein is highly immunoreactive in Japanese patients

Background: High‐molecular‐weight cell‐associated proteins (HM‐CAP) assay is the most popular serological immunoassay worldwide and has been developed from US isolates as the antigens. The accuracy is reduced when the sera are from adults and children in East Asia including Japan. To overcome the reduced accuracy, an enzyme immunoassay using Japanese strain–derived HM‐CAP (JHM‐CAP) was developed, in which the antigens were prepared by exactly the same procedure as HM‐CAP. The performance of JHM‐CAP was better than that of HM‐CAP in Japanese adults as well as in children. The higher sensitivity was because of the presence of 100‐kDa protein that was absent in the preparation of HM‐CAP antigen. Materials and Methods: Immunoblot analysis and peptide mass fingerprinting methods were used to identify the distinctive 100‐kDa protein present in JHM‐CAP antigens. The peptide sequence and identification were analyzed by Mascot Search on the database of Helicobacter pylori. The identified protein was confirmed by immunoblot with a specific antibody and inhibition assay by the sera. Results: The distinctive 100‐kDa protein was a fragment of CagA derived from Japanese clinical isolates, and the sera of Japanese patients had strongly reacted to the protein, probably to the exposed epitope on the fragmented CagA. The fragmentation of CagA had occurred in the process of antigen preparation in Japanese isolates, not in US isolates even under the same preparation. Conclusion: The distinctive 100‐kDa protein was a fragment of CagA protein of H. pylori derived from Japanese clinical isolates, and Japanese patients including children are likely to react strongly to the exposed epitopes on fragmented CagA.     

Some characteristics of membrane Cd<Superscript>2+</Superscript> transport in rat thymocytes: an analysis using Fluo-3

Although cadmium-induced apoptosis of lymphocytes is one of common features in the immunotoxicity of cadmium, the membrane pathway for intracellular cadmium accumulation is not fully elucidated. To characterize membrane Cd²⁺ transport of rat thymocytes, the change in intracellular Cd²⁺ concentration under various conditions was examined by the use of Fluo-3, a fluorescent probe for monitoring the change in intracellular concentration of divalent metal cations. The membrane Cd²⁺ transport was estimated by the augmentation of Fluo-3 fluorescence induced by bath application of CdCl₂. Lowering temperature strongly suppressed the augmentation of Fluo-3 fluorescence by CdCl₂, suggesting that the metabolic process can be involved in membrane Cd²⁺ transport. External acidification (decreasing pH) and membrane depolarization by adding KCl attenuated the augmentation, indicating the requirement of electrochemical driving force for membrane Cd²⁺ transport into the cells. Bath application of CaCl₂ and ZnCl₂ equally decreased the augmentation, suggesting their competition with Cd²⁺ at the membrane transport. The augmentation by CdCl₂ was lesser in the cells treated with N-ethylmaleimide inducing chemical depletion of cellular thiols. The result suggests the contribution of sulfhydryl groups to membrane Cd²⁺ transport. Taken together, it is suggested that the cells possess a temperature-sensitive membrane Cd²⁺ pathway, driven by electrochemical gradient of Cd²⁺ and transmembrane potential, with competitive binding site. Based on the characteristics described above, it is unlikely that the membrane Cd²⁺ transport in rat thymocytes is attributed to a single transport system although it has characteristics that are similar to those of divalent cation transporter 1.     

Kinetochore stretching inactivates the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors the attachment of microtubules to the kinetochore and inhibits anaphase when microtubule binding is incomplete. The SAC might also respond to tension; however, how cells can sense tension and whether its detection is important to satisfy the SAC remain controversial. We generated a HeLa cell line in which two components of the kinetochore, centromere protein A and Mis12, are labeled with green and red fluorophores, respectively. Live cell imaging of these cells reveals repetitive cycles of kinetochore extension and recoiling after biorientation. Under conditions in which kinetochore stretching is suppressed, cells fail to silence the SAC and enter anaphase after a delay, regardless of centromere stretching. Monitoring cyclin B levels as a readout for anaphase-promoting complex/cyclosome activity, we find that suppression of kinetochore stretching delays and decelerates cyclin B degradation. These observations suggest that the SAC monitors stretching of kinetochores rather than centromeres and that kinetochore stretching promotes silencing of the SAC signal.