Hydrophobic Silicone Elastomer Chamber for Recording Trajectories of Motile Porcine Sperms without Adsorption

Motile porcine sperms adhere to hydrophilic materials such as glass and plastics. The adsorption of sperms to a hydrophobic poly(dimethylsiloxane) (PDMS) membrane is less compared with that to glass. We investigated the linear velocity (LV) and amplitude of lateral head displacement (ALHD) of motile porcine sperm on glass and PDMS preparations using computer-assisted sperm analysis (CASA). Significant decreases were observed in the 15-min LV (P<0.05) and ALHD (P<0.05) in motile porcine sperm on glass preparations compared with those on PDMS preparations. These differences were due to adsorption of the head and/or neck to hydrophilic substrates. Because of the elasticity of PDMS, we propose that a PDMS membrane should be used for CASA. To investigate the dynamics of motile porcine sperms with microfluidics, we do not recommend plasma treatment to bond PDMS and glass in the microchannel preparation; instead, we suggest that a PDMS molding process without plasma treatment be used for preparation of microfluidic channels

A semi-automated non-radiactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives

Nucleotide excision repair (NER) removes the major UV-photolesions from cellular DNA. In humans, compromised NER activity is the cause of several photosensitive diseases, one of which is the skin-cancer predisposition disorder, xeroderma pigmentosum (XP). Two assays commonly used in measurement of NER activity are ‘unscheduled DNA synthesis (UDS)’, and ‘recovery of RNA synthesis (RRS)’, the latter being a specific measure of the transcription-coupled repair sub-pathway of NER. Both assays are key techniques for research in NER as well as in diagnoses of NER-related disorders. Until very recently, reliable methods for these assays involved measurements of incorporation of radio-labeled nucleosides. We have established non-radioactive procedures for determining UDS and RRS levels by incorporation of recently developed alkyne-conjugated nucleoside analogues, 5-ethynyl-2′-deoxyuridine (EdU) and 5-ethynyuridine (EU). EdU and EU are respectively used as alternatives for 3H-thymidine in UDS and for 3H-uridine in RRS. Based on these alkyne-nucleosides and an integrated image analyser, we have developed a semi-automated assay system for NER-activity. We demonstrate the utility of this system for NER-activity assessments of lymphoblastoid samples as well as primary fibroblasts. Potential use of the system for large-scale siRNA-screening for novel NER defects as well as for routine XP diagnosis are also considered

Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established. We report that, in human fibroblasts, approximately half of the repair synthesis requires both polκ and polδ, and both polymerases can be recovered in the same repair complexes. Polκ is recruited to repair sites by ubiquitinated PCNA and XRCC1 and polδ by the classical replication factor complex RFC1-RFC, together with a polymerase accessory factor, p66, and unmodified PCNA. The remaining repair synthesis is dependent on polɛ, recruitment of which is dependent on the alternative clamp loader CTF18-RFC

Corneal Nociceptors and Tear Deficiency

Chronic tear deficiency enhances the excitability of corneal cold-sensitive nerves that detect ocular dryness, which can lead to discomfort in patients with dry eye disease (DED). However, changes in corneal nerve excitations through the polymodal nociceptor “transient receptor potential vanilloid 1” (TRPV1) and the potential link between this receptor and symptoms of DED remain unclear. In this study, we examined the firing properties of corneal cold-sensitive nerves expressing TRPV1 and possible contributions of chronic tear deficiency to corneal nerve excitability by TRPV1 activation. The bilateral excision of lacrimal glands in guinea pigs decreased the tear volume and increased the frequency of spontaneous eyeblinks 1–4 weeks after surgery. An analysis of the firing properties of the cold-sensitive nerves was performed by single-unit recordings of corneal preparations 4 weeks after surgery in both the sham-operated and gland-excised groups. Perfusion of the TRPV1 agonist, capsaicin (1 μM), transiently increased the firing frequency in approximately 46–48% of the cold-sensitive nerves characterized by low-background activity and high threshold (LB-HT) cold thermoreceptors in both groups. Gland excision significantly decreased the latency of capsaicin-induced firing in cold-sensitive nerves; however, its magnitude was unchanged. Calcium imaging of cultured trigeminal ganglion neurons from both groups showed that intracellular calcium elevation of corneal neurons induced by a low concentration of capsaicin (0.03 μM) was significantly larger in the gland excision group, regardless of responsiveness to cold. An immunohistochemical study of the trigeminal ganglion revealed that gland excision significantly increased the proportion of corneal neurons enclosed by glial fibrillary acidic protein (GFAP)-immunopositive satellite glial cells. Topical application of the TRPV1 antagonist, A784168 (30 μM), on the ocular surface attenuated eye-blink frequency after gland excision. Furthermore, gland excision enhanced blink behavior induced by a low concentration of capsaicin (0.1 μM). These results suggest that chronic tear deficiency sensitizes the TRPV1-mediated response in the corneal LB-HT cold thermoreceptors and cold-insensitive polymodal nociceptors, which may be linked to dry eye discomfort and hyperalgesia resulting from nociceptive stimuli in aqueous-deficient dry eyes

Improvement in soft magnetic properties of thin bilayer ribbons using magnetoelastic effect

We have prepared Fe-Ni-system bilayer ribbons with different magnetostriction (compositions) and investigated the improvement of soft magnetic properties using the magnetoelastic effect. A toroidal core with D = 10 mm was made from the Fe6Ni94/Fe56Ni44 bilayer ribbon, and the B-H loop of the core was measured. The shape of the hysteresis loop dramatically changed depending on the inner layer (inner magnetic phase). This result indicates that the direction of the anisotropy induced by bending stress was changed depending on the inner layer. The slope of the B-H loop and coercivity reduced when the Fe56Ni44 layer was on the inner side. From the experimental results, we found that domain rotation was dominant for the magnetization process. Consequently, the increase in the coercivity over frequency could be suppressed by controlling the magnetization process. From these results, we found that a thin bilayer ribbon with positive and negative magnetostriction constant is an attractive material for reducing iron losses under high frequency

Crustal structure and growth of the Forearc region of Izu-Ogasawara arc

（独）海洋研究開発機構(JAMSTEC)では、2002年から伊豆・小笠原・マリアナ島弧において、大陸地殻の生成過程を明らかにすることを目的に構造調査を進め、現在の島弧地殻のボリュームより多くの玄武岩マグマが必要で島弧地殻を生成する過程でマフィックな島弧地殻の一部をマントル内に戻していること（Takahashi et al., 2007, 2008; Tatsumi et al., 2008）、火山フロントと背弧側の地殻の厚さ分布には相関があり過去のリフティングが検出されたこと（Kodaira et al., 2009）などがわかってきた。前弧域の地殻に関しては、厚い地殻と薄い地殻が存在すること（Takahashi et al., 2011）、地磁気異常から島弧的な構造があること（Yamazaki and Yuasa, 1998）がわかっているが、地殻構造から実証されていなかった。前弧域の地殻構造を求め、地殻進化の影響をどの程度受けているのか、前弧域の島弧成長を明らかにするために、（独）海洋研究開発機構の深海調査船「かいれい」を用いて人工地震探査を行った。 地震探査の測線は、新黒瀬からスミス海脚、第二東鳥島海丘、大町海山を通って、小笠原トラフに至る。得られた速度構造から前弧域は25km程度の地殻の厚い部分と10～15km程度の薄い部分があることが明らかになった。厚い地殻は、北緯32.5度付近、スミス海脚、第二東鳥島海丘、大町海山の下に分布する。新黒瀬側は厚い地殻を持たない。大町海山の内部には異常に厚い下部地殻が分布する。薄い地殻が分布するところでは、堆積層が厚く地殻の厚さの半分近くを占める。大町海山以外の地殻が厚く分布するところでは、P波速度6km/sの速度コンターが上に凸、7km/sの速度コンターが下に凸の形状を示す。火山フロントに沿った地殻構造では、むしろ6km/s以下の速度を持つ層が厚いことが示されている（Kodaira et al., 2007）。これは、前弧域下の島弧地殻は、火山フロント下と比較して未分化な物質を多く含むことを示唆おり、過去の掘削結果とも整合する（e.g., Taylor, 1992）。前弧海盆下の島弧地殻の分布は、地磁気異常の空間分布（Yamazaki and Yuasa, 1998）とよく合致する。新黒瀬周辺で見られる地磁気異常は、本研究から明らかになった地殻が薄く地殻全体が盛り上がっている形状と合致する。伊豆小笠原島弧の本州弧への衝突が新黒瀬の浅海部を作っているものと示唆される。SCG66-03発表要旨 / 日本地球惑星科学連合2012年大会（2012年5月20日～5月25日, 幕張メッセ国際会議場） / 日本惑星科学連合の許諾に基づき本文ファイルを掲

Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in types I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of types I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes

Phyllosphere yeasts rapidly break down biodegradable plastics

The use of biodegradable plastics can reduce the accumulation of environmentally persistent plastic wastes. The rate of degradation of biodegradable plastics depends on environmental conditions and is highly variable. Techniques for achieving more consistent degradation are needed. However, only a few microorganisms involved in the degradation process have been isolated so far from the environment. Here, we show that Pseudozyma spp. yeasts, which are common in the phyllosphere and are easily isolated from plant surfaces, displayed strong degradation activity on films made from poly-butylene succinate or poly-butylene succinate-co-adipate. Strains of P. antarctica isolated from leaves and husks of paddy rice displayed strong degradation activity on these films at 30°C. The type strain, P. antarctica JCM 10317, and Pseudozyma spp. strains from phyllosphere secreted a biodegradable plastic-degrading enzyme with a molecular mass of about 22 kDa. Reliable source of biodegradable plastic-degrading microorganisms are now in our hands