Molecular characterization of a K+ channel blocker in the scorpionButhus martensii Karsch

K⁺ channel blockers of scorpion venoms are of important value in studying pharmacology and physiology of specific K⁺ channel of cells. Based on the amino acid sequences of BmP01 previously characterized as a small-conductance Ca²⁺-activated K⁺ channel blocker, two “back to back” degenarate primers have been designed and synthesized for inverse PCR strategy, its full-length cDNA has been cloned from the venom gland of the Chinese scorpionButhus martensii. The cDNA is composed of 3 parts: 5′ UTR, ORF and 3′ UTR. The flanking sequence of translation initiation codon ATG is AAAATGA, which is highly conserved in scorpion Na⁺ channel toxin and protozoan genes, suggesting that these genes may have followed a common mechanism for translation initiation. The 3′ UTR contains poly(A) signal AATAAA. The open reading frame encodes a precursor of 57 residues with a signal peptide of 28 residues and a mature peptide of 29 residues. The signal peptide is rich in hydrophobic amino acid residues and its length is significantly different from that of the determined scorpion Na⁺ channel toxin. The deduced amino acid sequence of mature peptide is completely consistent with BmP01 previously determined by primary structure analysis.     

Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying K<Stack><Subscript>in</Subscript><Superscript>+</Superscript></Stack> channels in <Emphasis Type="Italic">Arabidopsis</Emphasis> guard cells

We explore nitric oxide （NO） effect on K^＋in, channels in Arabidopsis guard cells. We observed NO inhibited K^＋in, currents when Ca^2＋ chelator EGTA （Ethylene glycol-bis（2-aminoethylether）-N,N,N′,N;tetraacetic acid） was not added in the pipette solution; K^＋in currents were not sensitive to NO when cytosolic Ca^2＋ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO elevates cytosolic Ca^2＋ by activating plasma membrane Ca^2＋ channels firstly, then inactivates K^＋in, chartnels, resulting in stomatal opening suppressed subsequently.     

Circular dichroism spectroscopic studies on structures formed by telomeric DNA sequencesin vitro

Telomere plays an important role in cellular processes, such as cell aging, death and carcinogenisis. Having special sequences, it can form quadruplex structurein vitro. Circular dichroism (CD) spectroscopic studies show that TTAGGG, (TTAGGG)₂ and (TTAGGG)₄ can all form quadruplexin vitro and exist mainly as parallel quadruplex without metal ions. Both K⁺ and Na⁺ can stabilize the tetrameric structure and facilitate the forming of anti-parallel conformation. Furthermore, the conformations of quadruplex can also be affected by sequence length, the nature and concentration of metal ions.     

Rearrangements of microtubule cytoskeleton in stomatal closure of <Emphasis Type="Italic">Arabidopsis</Emphasis> induced by nitric oxide

NO （nitric oxide）, known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows： （i） In vivo stomatsl aperture assays revealed that both vinblastine （microtubule-disrupUng drug） and SNP （exogenous NO donor） prevented stomatsl opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas tsxol （a microtubule-stsbilizing agent） was able to reduce this effect; （ii） microtubules in the opening Arabi-dopsis guard cells expressing GFP：a-tubulin-6 （AtGFP：a-tubulin-6） were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; （iii） under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and be- came oblique or exhibited a random pattern; （iv） furthermore, the involvement of cytosolic Ca^2＋ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM （a chelator of Ca^2＋） greatly suppressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of [Ca^2＋]cyt （free Ca^2＋ concentration in the cytoplasm）, finally leading to stomatsl closure.     

Reversible inactivation of K+ channels of Vicia stomatal guard cells following the photolysis of caged inositol 1,4,5-trisphosphate

RECENT investigations suggest that cytoplasmic D-myo-inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger in plants, as in animals, coupling environmental and other stimuli to intracellular Ca2+ release. Cytoplasmic levels of InsP3 and the turnover of several probable precursors in plants are affected by physiological stimuli--including light, osmotic stress and the phytohormone indoleacetic acid--and InsP3 activates Ca2+ channels and Ca2+ flux across plant vacuolar and microsomal membranes. Complementary data also link changes in cytoplasmic free Ca2+ to several physiological responses, notably in guard cells which regulate gas exchange through the stomatal pores of higher plant leaves. Recent evidence indicates that guard cell K+ channels and, hence, K+ flux for stomatal movements may be controlled by cytoplasmic Ca2+. So far, however, direct evidence of a role for InsP3 in signalling in plants has remained elusive. Here we report that InsP3 released from an inactive, photolabile precursor, the P5-1-(2-nitrophenyl)ethyl ester of InsP3 (caged InsP3) reversibly inactivates K+ channels thought to mediate K+ uptake by guard cells from Vicia faba L. while simultaneously activating an apparently time-independent, inward current to depolarize the membrane potential and promote K+ efflux through a second class of K+ channels. The data are consistent with a transient rise in cytoplasmic free Ca2+ and demonstrate that intact guard cells are competent to use InsP3 in signal cascades controlling ion flux through K+ channels.     

Prediction of Solid-Liquid Equilibrium for KCI in Mixed Water-Ethanol Solutions Using the LIQUAC Model

The LIQUAC model is often used to predict vapor-liquid equilibria, osmotic coefficients, and mean ion activity coefficients for electrolyte systems. This paper describes a thermodynamic method to analyze solid-liquid equilibrium for electrolytes in mixed solvents solutions using the LIQUAC model. The KCI solubilities in mixed water-ethanol solutions are predicted with the LIQUAC model and its original interaction parameters. This method is also used to obtain new K^＋-ethanol interaction parameters in the LIQUAC model from the solubility data. The new interaction parameters accurately predict the vapor-liquid equilibrium data of K^＋ salts （including KCI, KBr, and KCOOCH3） in mixed water-ethanol solutions. The results illustrate the flexibility of the LIQUAC model which can predict not only vapor-liquid equilibrium but also solid-liquid equilibrium in mixed solvent systems.     

Effect of fatty acids on polyamine contents and Na+/H+ antiport activity in PM vesicles isolated from roots of barley under salt stress

With 200 mmol/L NaCl treatment on barley cultivar “Jian 4” (Hordeum vulgare L. cv. J4) seedlings for 6 d, the contents of covalently and noncovalently conjugated polyamines (PAs) and activities of H⁺-ATPase in plasma membrane (PM) vesicles isolated from the roots decreased remarkably. Moreover, the activity of Na⁺/H⁺ antiport was detected first in PM vesicles. The results showed that the decrease in the contents of membrane phospholipid, noncovalently conjugated PAs and activity of H⁺-ATPase caused by NaCl could be restored partially by application of 1 mmol/L stearie acid (C_16:0) and linoleic acid (C_18:2), and C_18:2 was more effective than C_16:0 In addition, a reduction in the contents of covalently conjugated PAs was only reversed partially in the presence of C_18:2 Furthermore, Na⁺/H⁺ antiport activity was strengthened by exogenous C_16:0 and C_18:2 and C_18:2 was more effective than C_16:0. The correlative analysis suggested that, after application of C_16:0 and C_18:2 under salt stress, there was a significant positive correlation existing among phospholipid content, noncovalently conjugated PA levels, H⁺-ATPase activities and Na⁺/H⁺ antiport activities, indicating that one of the mitigative mechanisms of exogenous fatty acids on salt injury was to improve membrane phospholipid and PA contents, leading to an enhance in membrane integrity and a change in charge status of PM vesicles, so the activity of membrane-associated enzyme H⁺-ATPase was increased and synthesis of Na⁺/H⁺ antiport protein was activated.     

Construction and expression of retroviruses encoding dual drug resistance genes in human umbilical cord blood CD34+ cells

A series of retro viral vectors encoding humanmdr1 gene alone as wetl as in combination with either humanmgmt gene or human mutantSer ³¹-dhfr gene are engineered. The resultant retroviruses are used to transduce human umbilical cord blood CD34⁺ cetls. It has been shown that expression of dual drug resistance genes in transduced cetls confers a broad range of resistance to both kinds of corresponding drugs. These data suggest a rationale for the use of such double chemoresistance gene constructs in anin vivo model in which transduced hematopoietic cetls will acquire multiple protection against the cytotoxic side effects of combination chemotherapy and may have future application in chemoprotection of normal tissues, thus killing tumor cetls more effectivety.     

Effects of La3+ and Gd3+ on Ca2+ influx in rat hepatoma H-35 cells

Effects of La³⁺ and Gd³⁺ on Ca²⁺ influx were investigated in rat hepatoma H-35 cells by measuring the initial rate of⁴⁵Ca²⁺ uptake. It was found that the maximum initial rate of Ca²⁺ uptake was increased six-to ten-fold at low concentrations of La³⁺ and Gd³⁺. Kinetic analyses by measuring the initial rate of Ca²⁺ influx at different external Ca²⁺ concentrations indicated the existence of two intracellular exchangeable components in the basal Ca²⁺ system, with low and high affinities for Ca²⁺, and only one class of Ca²⁺ binding sites was observed in the La³⁺-or Gd³⁺-treated cells. For high affinity, La³⁺ and Gd³⁺ increased both kinetic parametersK _m andV _max of basai Ca²⁺ influx. La³⁺ and Gd³⁺ compete directly with Ca²⁺ for Ca²⁺ binding site for low affinity. The kinetics is competitive.     

Effects of the disaggregation of high—polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture（stomata opening）

Observation under an electron microscope reveals that in closed and open stomata of V.faba,the average volume of particles in guard cell vacuoles(GCV) reduces about 3 orders in magnitude,while the distribution density of the particles increases about 2 orders of magnitude.By using the method of the ration of fluorescent emissions with laser scanning confocal microscopy,the monitoring to stomata opening shows that during 10 to 30 s before the first distinguishable aperture of stomata,there is a change of pH in GCV about-0.5 units.A quick stomatal opening immediately follows the changes of pH in GCV to reach a steady aperture about 12μm in 100-200s.This work proposes a model for the osmoregulation in GCV for stomatal opening.The proposed osmoregulation is related to the disaggregation of some polymerized particles inside GCV,which is probably induced by a-ΔpH in the vacuole.This model describes a process of osmoregulation that avoids the massive energy consuming transportation across cell membranes,which is a foundation of the current chemiosmotic hypothesis.This model is a supplement to the multiple controlling hypothesis for the stomatal movement,which widens research principle ideas for other quick movements in plants.     

A possible configuration of H 5 + cluster

On the basis of our previous work, a configuration of H ₅ ⁺ has been presented that is a square center structure, and the energy for the structure of H ₅ ⁺ has been obtained by IACQM. The result showed that there is an extreme energy E_e: −2.45981 [2 Ry] atR = 2.50a ₀. It indicates that the square center structure of H ₅ ⁺ system has the certain stability, which is a possible configuration of H ₅ ⁺ .     

Measurement of the absorption spectrum of H2O+ in the visible region

Together with the 74 lines belonging to (0,9,0)- (0,0,0) band, the high-resolution absorption spectrum of H₂O⁺ A²A₁-X²B₁ system was observed in the visible region of 16680 — 17300 cm^-1 using optical heterodyne magnetic rotation enhanced velocity modulation spectroscopy for the first time, which verifies the high sensitivity and high signal to noise ratio (S/N) of this technique.     

Relationship between stomatal density and the changes of atmospheric CO2 concentrations

The relationship between the stomatal density of five woody plants endemic to China, i. e.Eucommia ulmoides, Quercus liaotungensis, Q. glandulifera var.brevipetiolata, Cyclocarya paliurus andFicus heteromorpha, and the atmospheric CO₂ concentrations was studied by observations on leaves of the herbarium-stored specimms(1920s–1990s). The results showed that the stomatal density inEucommia ulmoides, Quercus liaotungensis andQ. glandulifera var.brevipetiolata decreased significantly in response to the elevated atmospheric CO₂ concentrations, while inCyclocarya paliurus it decreased slightly and inFicus heteromorpha there were no responses.     

H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

Localization of muscarinic acetylcholine receptor in plant guard cells

Acetylcholine (ACh), as an important neurotransmitter in animals, also plays a significant role in various kinds of physiological functions in plants. But relatively little is known about its receptors in plants. A green fluorescence BODIPY FL-labeled ABT, which is a high affinity ligand of muscarinic acetylcholine receptor (mAChR), was used to localize mAChR in plant guard cells. InVicia faba L. andPisum sativum L., mAChR was found both on the plasma membrane of guard cells. mAChR may also be distributed on guard cell chloroplast membrane ofVicia faba L. The evidence that mAChR localizes in the guard cells provides a new possible signal transduction pathway in ACh mediated stomata movement.     

Identification of integrin-like in guard cells ofVicia faba

We addressed the existence and localization of integrin-like in guard cells ofVicia faba by using a probe of polyclonal antibody against the human integrin (α_vβ₃/β₅). Western blot results showed that three integrins-like of about 47.3, 43.7 and 41.1 ku were detected from the preparation of membrane fragments of purified guard cell protoplasts. Further research with immunofluorescent scanning microscopy indicated that those integrins-like were localized on plasma membrane of guard cells, most nearing the dorsal wall, which is consistent with the reception of signals from epidermal cells to guard cells. Thus our results indicate, for the first time, that integrins-like are present at guard cell plasma membrane ofVicia faba.     

Na+-permeable channels of human sperm membrane reassembled into giant liposome

Previous data showed that a Na⁺-transmembrane flux was accompanied with acrosome reaction of sperm. However, the electrophysiological recording and characterization of Na⁺ current in human sperm membrane have not been yet reported. In the present investigation, membrane proteins extracted from human sperms were reassembled into liposome bilayer, and then the liposomes were fused by dehydration-rehydration into giant liposomes with the diameter of more than 10 ώm. By patch clamping the giant liposomes two kinds of single channel currents were recorded in a NaCl solution system. Both of them were Na⁺-carried, TTX-sensitive and strongly rectifying, but with different unit conductance and open probability. Moreover, bursting activity and channel-substates as well as two open time constants were observed in the larger channel.     

黑鲷NKCC1分子特征及其对急性盐度胁迫的表达响应

为了解黑鲷(Acanthopagrus schlegelii)在盐度胁迫过程中的适应机制,本研究利用基因扩增、聚类分析、荧光定量PCR等技术对NKCC1基因进行生物信息学分析以及在不同组织中的表达特征研究,并探讨其在急性盐度胁迫下的表达机制.结果表明NKCC1基因的开放阅读框(Open Reading Frame,OR...     

XTR+公钥体制中核心算法的改进

在XTR⁺公钥体制中提出无矩阵的核心算法, 显著提高了核心算法的运算效率, 从而提高了应用XTR⁺公钥体制的可证明IND CCA2安全的密码系统、 可证明安全的数字签名、 可证明安全的盲签名协议和零知识证明协议的运算效率.