Ca2+ transient induced by extracellular changes in osmotic pressure in Arabidopsis leaves: differential involvement of cell wall-plasma membrane adhesion

We investigated the mechanism underlying the perception of extracellular changes in osmotic pressure in Vallisneria gigantea Graebner and transgenic Arabidopsis thaliana (L.) Heynh. expressing cytoplasmic aequorin. Hypertonic and hypotonic treatments of A. thaliana leaves each rapidly induced a Ca2+ transient. Both responses were essentially dependent on the presence of extracellular Ca2+ and were sensitive to Gd3+ a potential blocker of stretch-activated Ca2+ channels. Immediately after plasmolysis caused by hypertonic treatment and subsequent deplasmolysis caused by hypotonic treatment, the cells did not respond to a second hypertonic treatment and exhibited an impaired adhesion of the plasma membrane (PM) to the cell wall (CW). Recovery of the responsiveness required about 6 h. By contrast, no refractory phenomenon was observed in response to hypotonic treatment. Pretreatment with cellulase completely inhibited the Ca2+ transient induced by hypertonic treatment, but it did not affect the response to hypotonic treatment. V. gigantea mesophyll cells pretreated with cellulase exhibited an impaired adhesion of the PM to the CW. The leaf cells of multicellular plants can respond to both hypertonic and hypotonic treatments through the stretch-activated Ca2+ channels, whereas cellulase-sensitive adhesion of the PM to the CW is involved only in the response to hypertonic treatment.     

G protein-coupled receptor for diapause hormone, an inducer of Bombyx embryonic diapause

Bombyx diapause hormone was the first chemical substance identified as a maternal control factor that arrests offspring development. However, the molecular mechanisms by which the hormone transduces the signal to the oocyte that induces embryonic diapause immediately after mesoderm segmentation are not fully understood. Here, we describe a cDNA for a G protein-coupled diapause hormone receptor with seven transmembrane domains. Its amino-acid sequence shows a high level of similarity to the receptors of mammalian neuromedin U and insect regulatory peptide, an FXPRL-amide C-terminus. When expressed in a Xenopus oocyte system, the receptor exhibited the highest affinity (EC(50), approximately 70nM) for diapause hormone, when compared with other Bombyx FXPR/KL-amide peptides. Diapause hormone without amidation at the C-terminus, which never induces embryonic diapause in vivo, had no effect in this heterologous expression system. The mRNA is expressed in the ovaries during Bombyx pupal-adult development. These results strongly indicate that the cDNA encodes the diapause hormone receptor.     

Design of Ca2+‐independent Staphylococcus aureus sortase A mutants

The catalytic activity of Staphylococcus aureus sortase A (SaSrtA) is dependent on Ca²⁺, because binding of Ca²⁺ to Glu residues distal to the active site stabilizes the substrate binding site. To obtain Ca²⁺‐independent SaSrtA, we substituted two Glu residues in the Ca²⁺‐binding pocket (Glu¹⁰⁵ and Glu¹⁰⁸). Although single mutations decreased SaSrtA activity, mutations of both Glu¹⁰⁵ and Glu¹⁰⁸ resulted in Ca²⁺‐independent activity. Kinetic analysis suggested that the double mutations affect the substrate binding site, without affecting substrate specificity. This approach will allow us to develop SaSrtA variants suitable for various applications, including in vivo site‐specific protein modification and labeling. Biotechnol. Bioeng. 2012; 109: 2955–2961. © 2012 Wiley Periodicals, Inc.     

健康早产儿24小时脑电图的定量评价

早产儿的生物学行为主要以睡眠为主,因此了解各个睡眠阶段的脑电图模型,对临床客观评价早产儿的大脑发育特点及健康状况具有重要的临床意义。本文旨在探讨健康早产儿不同睡眠阶段脑电图特点,进而为其睡眠阶段的自动判定提供标准。应用多导生理记录仪对24例健康早产儿进行了41次Fp1-C3双电极连续24小时脑电图记录,同时用两台摄像机同步记录眼球运动及肢体运动。依据呼吸、眼球及肢体运动视觉判定睡眠阶段。应用自回两台摄像机同步记录眼球运动最适赤池信息量准则（Min-AIC)、总功率（TP）、δ功率（δP）、θ功率（θP）、α功率（αP）、β功率（βP）及不连续性（Dis）进行分析。结果显示Min-AIC,TP,δP及Dis四个变量依睡眠阶段不同有显著性差异（P＜0．01）。多变量判别分析亦显示联合这四个变量可对睡眠阶段进行判定。联合Min-AIC、TP、δP及Dis四个变量适于对胎龄大于30周的健康早产儿的睡眠阶段进行判定。     

A comparative study on growth and morphology of wasabi plantlets under the influence of the micro-environment in shoot and root zones during photoautotrophic and photomixotrophic micropropagation

The growth of wasabi (Wasabia japonica Matsumura) plantlets under different micro-environments inside culture vessels in photoautotrophic micropropagation (PA) and photomixotrophic micropropagation (PM) conditions were compared. After 28 days of culture, dry weight, relative growth rate, leaf area, and leaf chlorophyll contents of plantlets in PA were greater than those in PM. The number of leaves did not differ significantly between PA and PM conditions. PA promoted root growth and development with a greater number of roots, root length, root diameter, root fresh weight, root dry weight, and root xylem vessel system. Dissolved oxygen concentration in PA culture medium sharply decreased after 7 days of culture and then recovered. In PM culture medium, no significant fluctuation of dissolved oxygen concentration was apparent. The net photosynthetic rates of plantlets in PA were much higher than those in PM and increased with culture time. In contrast, the net photosynthetic rates of wasabi plantlets in PM kept a low and constant value during the culture period. With the presence of gas exchange membranes attached to the vessel lids, the detected vapor pressure deficit was higher in PA than in PM conditions. Higher stomatal density and larger stomatal aperture on the abaxial and adaxial surfaces of the leaves in PM medium promoted leaf water loss following ex vitro conditions. Thus, PA is applicable for producing healthy wasabi transplants.     

Molecular cloning and preliminary expression analysis of banded dogfish (Triakis scyllia) TNF decoy receptor 3 (TNFRSF6B)

Decoy receptor 3 (DcR3), a member of TNF receptor superfamily, is a soluble receptor without death domain and cytoplasmic domain, and secreted by cells and binds with FasL, LIGHT and TL1A. The principal function of DcR3 is the inhibition of apoptosis by the binding cytotoxic ligands. Expression of DcR3 has been reported in a wide array of normal human tissues as well as tumors and tumor cell lines. Recently, DcR3 was reported to modulate a variety of immune responses in mammals. TNFR or DcR3 has been identified in some teleost fishes. However, DcR3 is not reported in cartilaginous fish which is the lowest vertebrate possessing the adaptive immune system. Here we identified DcR3 cDNA in shark (Trsc-DcR3) from an SSH library prepared from peripheral white blood cells stimulated with PMA. Four cysteine-rich domains (CRDs) in common with TNF receptor family members are present in the Trsc-DcR3 sequence. The deduced amino acid sequence of Trsc-DcR3 showed highest identity with the chicken (50.4%), followed by human (46.8%) and rainbow trout (36.5%) DcR3. In a phylogenetic tree of known TNFRSF sequences, the Trsc-DcR3 grouped with the chicken and human DcR3. Trsc-DcR3 mRNA was detected strongly in the gill, moderately in the brain, and weakly in the kidney, thymus and leydig. These data strongly suggest that the gene encoding Trsc-DcR3 in banded dogfish is a homolog of the human gene. mRNA expression of Trsc-DcR3 in the thymus and leydig suggests that DcR3 may act as a modulator in the immune system even at the phylogenetic level of cartilaginous fish.     

Vacuolar and cytoskeletal dynamics during elicitor-induced programmed cell death in tobacco BY-2 cells

Responses of plant cells to environmental stresses often involve morphological changes, differentiation and redistribution of various organelles and cytoskeletal network. Tobacco BY-2 cells provide excellent model system for in vivo imaging of these intracellular events. Treatment of the cell cycle-synchronized BY-2 cells with a proteinaceous oomycete elicitor, cryptogein, induces highly synchronous programmed cell death (PCD) and provide a model system to characterize vacuolar and cytoskeletal dynamics during the PCD. Sequential observation revealed dynamic reorganization of the vacuole and actin microfilaments during the execution of the PCD. We further characterized the effects cryptogein on mitotic microtubule organization in cell cycle-synchronized cells. Cryptogein treatment at S phase inhibited formation of the preprophase band, a cortical microtubule band that predicts the cell division site. Cortical microtubules kept their random orientation till their disruption that gradually occurred during the execution of the PCD twelve hours after the cryptogein treatment. Possible molecular mechanisms and physiological roles of the dynamic behavior of the organelles and cytoskeletal network in the pathogenic signal-induced PCD are discussed.Key words: actin microfilament, cell cycle, cryptogein, microtubules, nuclei, programmed cell death, tobacco BY-2 cells, vacuoles     

Oxidative renal tubular injuries induced by aminocarboxylate-type iron (III) coordination compounds as candidate renal carcinogens

Oxidative renal tubular injuries and carcinogenesis induced by Fe^III-nitrilotriacetate (NTA) and Fe^III–ethylenediamine-N,N′-diacetate (EDDA) have been reported in rodent kidneys, but the identity of iron coordination structure essential for renal carcinogenesis, remains to be clarified. We compared renal tubular injuries caused by various low molecular weight aminocarboxylate type chelators with injuries due to NTA and EDDA. We found that Fe^III-iminodiacetate (IDA), a novel iron-chelator, induced acute tubular injuries and lipid peroxidation to the same extent. We also prepared Fe^III-IDA solutions at different pHs, and studied resultant oxidative injuries and physicochemical properties. The use of Fe^III-IDA at pH 5.2, 6.2, and 7.2 resulted in renal tubular necrosis and apoptotic cell death, but neither tubular necrosis nor apoptosis was observed at pH 8.2. Spectrophotometric data suggested that Fe^III-IDA had the same dimer structure from pH 6.2 to 7.2 as Fe^III-NTA; but at a higher pH, iron polymerized and formed clusters. Fe^III-IDA was crystallized, and this was confirmed by X-ray analysis and magnetic susceptibility measurements. These data indicated that Fe^III-IDA possessed a linear μ-oxo bridged dinuclear iron (III) around neutral pH.