Ploidy chimeras induced in haploid sporophytes of <Emphasis Type="Italic">Osmunda claytoniana</Emphasis> and <Emphasis Type="Italic">Osmunda japonica</Emphasis>

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.     

Crystal structure of the C-terminal domain of Mu phage central spike and functions of bound calcium ion

Bacteriophage Mu, which has a contractile tail, is one of the most famous genus of Myoviridae. It has a wide host range and is thought to contribute to horizontal gene transfer. The Myoviridae infection process is initiated by adhesion to the host surface. The phage then penetrates the host cell membrane using its tail to inject its genetic material into the host. In this penetration process, Myoviridae phages are proposed to puncture the membrane of the host cell using a central spike located beneath its baseplate. The central spike of the Mu phage is thought to be composed of gene 45 product (gp45), which has a significant sequence homology with the central spike of P2 phage (gpV). We determined the crystal structure of shortened Mu gp45Δ1-91 (Arg92–Gln197) at 1.5 Å resolution and showed that Mu gp45 is a needlelike structure that punctures the membrane. The apex of Mu gp45 and that of P2 gpV contained iron, chloride, and calcium ions. Although the C-terminal domain of Mu gp45 was sufficient for binding to the E. coli membrane, a mutant D188A, in which the Asp amino acid residue that coordinates the calcium ion was replaced by Ala, did not exhibit a propensity to bind to the membrane. Therefore, we concluded that calcium ion played an important role in interaction with the host cell membrane.     

Double-labeled donor probe can enhance the signal of fluorescence resonance energy transfer (FRET) in detection of nucleic acid hybridization

A set of fluorescently-labeled DNA probes that hybridize with the target RNA and produce fluorescence resonance energy transfer (FRET) signals can be utilized for the detection of specific RNA. We have developed probe sets to detect and discriminate single-strand RNA molecules of plant viral genome, and sought a method to improve the FRET signals to handle in vivo applications. Consequently, we found that a double-labeled donor probe labeled with Bodipy dye yielded a remarkable increase in fluorescence intensity compared to a single-labeled donor probe used in an ordinary FRET. This double-labeled donor system can be easily applied to improve various FRET probes since the dependence upon sequence and label position in enhancement is not as strict. Furthermore this method could be applied to other nucleic acid substances, such as oligo RNA and phosphorothioate oligonucleotides (S-oligos) to enhance FRET signal. Although the double-labeled donor probes labeled with a variety of fluorophores had unexpected properties (strange UV-visible absorption spectra, decrease of intensity and decay of donor fluorescence) compared with single-labeled ones, they had no relation to FRET enhancement. This signal amplification mechanism cannot be explained simply based on our current results and knowledge of FRET. Yet it is possible to utilize this double-labeled donor system in various applications of FRET as a simple signal-enhancement method.     

Enhancement of Glossiness Perception by Retinal-Image Motion: Additional Effect of Head-Yoked Motion Parallax

It has been argued that when an observer moves, a contingent retinal-image motion of a stimulus would strengthen the perceived glossiness. This would be attributed to the veridical perception of three-dimensional structure by motion parallax. However, it has not been investigated whether the effect of motion parallax is more than that of retinal-image motion of the stimulus. Using a magnitude estimation method, we examine in this paper whether cross-modal coordination of the stimulus change and the observer''s motion (i.e., motion parallax) is essential or the retinal-image motion alone is sufficient for enhancing the perceived glossiness. Our data show that a retinal-image motion simulating motion parallax without head motion strengthened the perceived glossiness but that its effect was weaker than that of motion parallax with head motion. These results suggest the existence of an additional effect of the cross-modal coordination between vision and proprioception on glossiness perception. That is, motion parallax enhances the perception of glossiness, in addition to retinal-image motions of specular surfaces.     

A novel 2,4-diaminopyrimidine derivative as selective inhibitor of protein kinase C theta prevents allograft rejection in a rat heart transplant model

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.