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The solution culture method was used to study the effects of sodium nitroprusside (SNP) on plant growth, nutrient uptake and antioxidant enzyme activities of tomato seedlings under iron deficiency and NO采用营养液培养方法,研究外源NO供体(硝普钠,SNP)对缺铁和硝酸盐胁迫番茄幼苗生长、养分吸收及抗氧化酶活性的影响.结果表明: 处理7 d后,缺铁使番茄幼苗生长受到抑制,叶绿素a、b、类萝卜素含量显著降低,出现明显失绿症状;降低叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性,电解质渗漏率、丙二醛含量明显增加,脯氨酸和可溶性糖含量变化不显著,幼苗叶片和根中N、P、K、Ca、Mg、Fe含量比对照处理有不同程度的减少.硝酸盐和缺铁双重胁迫对番茄幼苗生长抑制加剧,叶绿素a、b、类萝卜素含量、SOD、POD和CAT活性显著降低,电解质渗漏率、脯氨酸、可溶性糖和丙二醛含量明显增加;番茄幼苗叶片和根中N、P、Mg、Fe含量显著减少,而K、Ca含量显著增加. 与不添加处理相比,添加0.1 mmol·L

Collaborative Filtering recommendation is one of the most widely used recommendation systems in e-commerce recommendation systems. However, because of the growing number of users and goods, the recommended quality of traditional collaborative filtering technology is getting lower and lower. In this paper, we propose a method of Similarity Measurement Based on User - Attribute and User - Score to improve the accuracy of similarity calculation between users. First, this method calculates the similarity of user attribute and the similarity of user rating. Next, we combine the two similarities into the new similarity measure based on weighted fusion. Finally, we incorporate this similarity measure into the traditional collaborative filtering algorithm and improve the recommended quality. The experimental results show that the presented algorithm can improve the recommendation accuracy and produce a better recommendation results.

The migration and distribution characteristics of the COD in the enriching process of Coking Desulfurization Waste Solution (CDWS) were researched in this paper. The results show that COD in the early and late condensate are all up to 10000mg/L and it is decreased at first and then increased in CDWS evaporating concentration process. This result is caused by substances containing organic at prophase and inorganic in the late. Therefore, removing the organic matter in the wastewater and controlling the temperature in CDWS concentrating process is the key to reduce the COD in condensate.

As a classical method for sulfide determination in wastewater, iodimetry is adopted by many countries. But the existing of high concentration of reducing materials such as thiosulfate, sulfite and dissolved organic, which cant be eliminated by the pretreatment of zinc acetate-precipitation filtration, have an important influence on determination. The study on elimination method is carried out in this paper. It is indicated that the interference of S2O32-and SO32-can be eliminated by washing sedimentation with KOH solution of 10%. Iodometric titration with the pretreatment of zinc acetate-precipitation filtration and washing precipitate with 10% KOH is an accurate method to determine the sulfide content.

Affects of thiosulfate (S2O32-) and sulfate (SO42-) on determination of thiocyanate (SCN-) in desulfurization waste solution (DWS) by spectrophotometry were investigated. The experiment results suggested that S2O32-and Fe3+ generated purple compound [Fe(S2O3)2]-which has a strong absorption at 450nm, but the purple soon fades in this process. As long as the reacting time is longer than 120s, the impact of S2O32-can be eliminated. In addition, sulfate has no effect. At 450nm wavelength, linear relationship between concentration of SCN-and absorbance was C=19.7272A-0.2774, which conformed to the Beer Law in the range of 2.00 ~ 19.45μg•ml-1, the correlation coefficients was 0.9996. The Fe3+-SCN-spectrophotometric method of monitoring SCN- in desulfurization waste solution was feasible.

Greenhouse temperature of the three different types was measured under sunny winter weather conditions. The variation regularity of greenhouses temperature with outdoor temperature was studied according to the monitoring data of experimental greenhouse, which was different roof and floor structure. The results showed that the greenhouse temperature of the herringbone roof construction higher than the greenhouse of the serration roof construction; the greenhouse temperature of the serration roof construction is higher than the greenhouse of arch roof construction. Through analysis of temperature variation on the different type greenhouse, it will provide theoretical basis to construct greenhouse in different areas.

The pebble thermoregulation system in greenhouse bottom corner is put forward according the characteristics of greenhouse structure and higher energy consumption. The indoor temperature of the pebble thermoregulation greenhouse is monitored in the course of experiment and the temperature change law of greenhouse bottom corner and greenhouse middle is analyzed and heat storage of pebble thermoregulation system is calculated. The results showed that heat storage of pebble thermoregulation system is higher, it is has important theoretical and realistic significance for greenhouse heating in winter.

According to the characteristics of non-uniform temperature distribution in the seeding greenhouse, a pebble thermoregulation system in the bottom corner of greenhouse was put forward. The changes regularity of indoor greenhouse temperature with outdoor weather conditions was studied according to monitoring the date of experimental greenhouse, which was a pebble thermoregulation greenhouse and contrast greenhouse with same structure, such as indoor temperature and out temperature, etc. The results showed that temperature distribution between greenhouse bottom corner and greenhouse middle is uniform and the temperature difference is less than 1°C.

Toosendanin (TSN) is a triterpenoid extracted from Melia toosendan Sieb et Zucc, which was used as a digestive tract-parasiticide and agricultural insecticide in ancient China. TSN was demonstrated to be a selective presynaptic blocker and an effective antibotulismic agent. By interfering with neurotransmitter release through an initial facilitation followed by a subsequent depression, TSN eventually blocks synaptic transmission at both the neuro-muscular junction and central synapses. Despite sharing some similar actions with botulinum neurotoxin (BoNT), TSN has a marked antibotulismic effect in vivo and in vitro. Studies suggest that the antibotulismic effect of TSN is achieved by preventing BoNT from approaching its enzymatic substrate, the SNARE protein. It is also found that TSN can induce differentiation and apoptosis in several cell lines, and suppress proliferation of various human cancer cells. TSN inhibits various K(+)-channels, selectively facilitates Ca(2+)-influx via L-type Ca(2+) channels and increases intracellular Ca(2+) concentration ([Ca(2+)](i)). The TSN-induced [Ca(2+)](i) increase and overload could be responsible for the TSN-induced biphasic effect on transmitter release, cell differentiation, apoptosis as well as the cytoxicity of TSN.

Aim: Botulinum neurotoxins (BoNT) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. The ability of botulinum toxin to produce its effect is dependent on the ability of the light chain to cleave the SNARE proteins associated with transmitter release. Translocation of the light chain protease through the heavy chain-formed channel is a pivotal step in the intoxication process. Toosendanin (TSN), a triterpenoid derivative extracted from a Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study was designed to explore the antibotulismic mechanisms of toosendanin. Methods: The inside-out singlechannel recording patch-clamp technique was used to record the BoNT/A-induced currents in the presence and absence of TSN. Results: Channel formation was delayed and the sizes of the channels were reduced in the TSN-treated PC12 cell membrane. Conclusion: The antibotulismic effect of TSN might occur via interference with toxin translocation.

Gossypol, a male antifertility compound isolated from cotton, has been proved to inhibit capacitation and the acrosome reaction in human and mammalian sperm. Here, by using whole-cell recording, we observed the effects of gossypol on Ca(2+) and Cl(-) currents in mouse spermatogenic cells obtained by mechanical dissociation. The results showed that gossypol concentration-dependently and irreversibly inhibited T-type Ca(2+) currents in the cells. When the concentration of gossypol was > or =5 microM, the currents were blocked completely. The time to current block was progressively shortened as the gossypol concentration was increased from 5 to 80 microM. Moreover, the drug increased the time constant of inactivation in a concentration-dependent manner, while it did not affect the activation of the current. The inhibitory effect on the T-type Ca(2+) current did not correlate with signaling mediated by G proteins and tyrosine phosphorylation. No obvious effect of gossypol on Cl(-) currents was observed. These data suggest that the gossypol-induced inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of the compound, indicating a possibility to use gossypol as a local contraceptive drug.

By perineurial recording, the effects of toosendanin (TSN), a presynaptic blocker, on nerve terminal calcium currents (I(Ca)) were observed in innervated triangularis sterni of the mouse and cutaneous pectoris of the frog. It was found that TSN blocked the slow component of I(Ca) insensitive to nifedipine and omega-conotoxin-GVIA, and increasing the extracellular Ca(2+) concentration partially antagonized the inhibitory effect in mouse motor nerve terminals. However, in the frog, TSN increased the slow component of I(Ca) and this effect disappeared in the presence of nifedipine in perfusion solution. Based on previous data showing that the slow component of I(Ca) were mediated by different subtypes of calcium channels in mouse and frog motor nerve terminals, we presume that TSN could exercise different effects on various subtypes of calcium channels.

Beta-agkistrodotoxin (beta-AgTx), a polypeptide purified from the venom of Agkistrodon blomhoffii brevicaudus, is a presynaptic blocker acting on neurotransmitter release. In this work, perineural recording technique was employed to study the effects of beta-AgTx on sodium, potassium and calcium currents of mouse motor nerve terminals. The results showed that beta-AgTx selectively inhibited Ca2+-dependent (I(K,Ca)) and fast (I(K,f) K+ currents, but did not affect slow K+ current (I(K,s)), sodium and calcium currents. However there are other components in A. blomhoffii brevicaudus venom which inhibit perineural sodium current. The present data have provided additional evidence that the site of action of beta-AgTx is different from that of beta-bungarotoxin.

The effect of prejunctional blocker Toosendanin (TSN) on acetylcholine (ACh) level in striatum and parietal cortex was investigated by means of method of brain dialysis coupled with microbore high-performance liquid chromatograph (HPLC)-electrochemical detection (ECD) with enzyme immobilized post-column at freely moving rats. The results are as follows: (1) TSN inhibits high K+ (100 mM)-induced ACh release from the mentioned two regions of brain, completely and irreversibly. (2) Before the inhibition, there appears a transient rise of ACh level. (3) This rise of ACh level can be blocked by TTX. (4) Calcium is necessary for the onset of this rise, but not for that of the sustained decrease of ACh level. These results indicate that the effect of TSN on central cholinergic synapses is similar to that of neuromuscular junction, namely, its blocking action is always preceded by a Ca(2+)-dependent facilitatory phase with result of a TSN-induced transient rise of ACh, but the sustained decline and final blockade of ACh release are Ca(2+)-independent.

Cinnamomin, a new type II ribosome-inactivating protein, purified from the seeds of Cinnamonum camphora is reconstituted into the membranes of planar lipid bilayer and giant liposome. The channel-forming activity of the cinnamomin is found and cation permeability of the channel is characterized by patch clamp. In an asymmetric solution system, bath 150/pipette 100 mM KCl, the unit conductance is 140+/-7 pS and the reversal potential is 10.4+/-0.6 mV, very close to the theoretical value of the K+ electrode. The results offer an interpretation for internalization of the RIP and the cytotoxicity difference between single and two chain RIP.

Ion environment and ionic fluxes through membrane are thought to be important in the spermatozoa's maturation, capacitation, and the initiating process of gamete interaction. In this work, the membrane proteins isolated from human sperm plasma membrane were reconstituted into planar lipid bilayers via fusion, and the ion channels activities were observed under voltage clamp mode. In cis 200//trans 100 mM KCl solution, a TEA-sensitive cation-selective channel with a unit conductance of 40 pS was recorded. In a gradient of 200//100 mM NaCl solutions, a Na(+)-selective channel with a unit conductance of 26 pS was recorded. In both cases, reversal potential was about-18 mV, which is close to the predicated value of a perfect Nernst K+ or Na+ electrode. In 50//10 mM CaCl2 solution, a cation channel activity with a unit conductance of 40 pS and reversal potential of about -20 mV was usually observed. In 200//100 mM NMDG(N-methyl-D-glucamine)-Cl solution, where the cation ions were substituted with NMDG, a 30-pS anion-selective channel activity was also detected. The variety in the types of ion channels observed in human spermatozoa plasma membrane suggests that ion channels may play a range of different roles in sperm physiology and gamete interaction.

The paper puts forward a new classification method of association rules that is based on support-significant structure. Starting from the characteristics of customer segmentation, this method introduces a up-to-date rule evaluation index significance during the produce process of classification rules, therefore, the evaluation and selection of principle have serious statistical basis. After simple comparison of the real example, we prove the comparative superiority of introducing significance into the customer segmentation.

The fact that the fruit and bark of plant belonging to family Melia could be used as digestive tract-parasiticide and agricultural insecticide was recorded about two thousand years ago in ancient China. Toosendanin (TSN, C30H38O11, FW=574), a triterpenoid derivative, was extracted from the bark of Melia toosendan Sieb. et Zucc. by Chinese scientists in 1950os and used as an ascarifuge in China instead of imported sendanin. Studies have demonstrated that TSN possesses special biological actions as well as considerable various values in scientific research, clinic medicine and agriculture. The first is that by interfering with neurotransmitter release by causing an initial facilitation, TSN eventually blocks synaptic transmission at both the neuromuscular junction and central synapses. The action might result from TSN-induced Ca(2+)-sensitivity change and final elimination of transmitter release machinery. The second is that despite sharing many similar actions with botulinum neurotoxin (BoNT) on blocking neuromuscular transmission, TSN has a markedly antibotulismic action in vivo and in vitro: TSN-treatment saves the botulism mice or monkeys from death; TSN-incubation in vitro or TSN-injection in vivo endows neuromuscular junction with a high tolerance to BoNT. Studies suggest that the antibotulismic action is achieved by preventing BoNT from approaching its enzymatic substrate, SNARE protein. The third, in recent years, it is also observed that TSN can induce differentiation and apoptosis in several cell lines, and suppress proliferation of various human cancer cells. The TSN-induced differentiation is Ca(2+)-dependent and the mitochondria-dependent apoptosis pathway is involved in the TSN-induced apoptosis. The fourth is that TSN inhibits various K(+) channels and selectively facilitates Ca(2+) current through L-type Ca(2+) channels and hence elevates [Ca(2+)](i). The TSN-induced [Ca(2+)](i) increase and overload could be responsible for the TSN-induced biphasic effect on neurotransmitter release, cell differentiation, apoptosis as well as the cytotoxicity of TSN.

Botulinum neurotoxins (BoNT) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. The ability of botulinum toxin to produce its effect is dependent on the ability of the light chain to cleave the SNARE proteins associated with transmitter release. Translocation of the light chain protease through the heavy chain-formed channel is a pivotal step in the intoxication process. Toosendanin (TSN), a triterpenoid derivative extracted from a Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study was designed to explore the antibotulismic mechanisms of toosendanin.The inside-out single-channel recording patch-clamp technique was used to record the BoNT/A-induced currents in the presence and absence of TSN.Channel formation was delayed and the sizes of the channels were reduced in the TSN-treated PC12 cell membrane.The antibotulismic effect of TSN might occur via interference with toxin translocation.

Toosendanin, a triterpenoid derivative isolated from the barks of Melia toosendan Sieb et Zucc, has been used as an anthelmintic vermifuge against ascaris for more than fifty years in China. In the present study, we investigated the growth inhibition and apoptosis-induced effect of toosendanin on human cancer cells. The result showed that toosendanin significantly suppressed the proliferation of tested human cancer cell lines. The IC(50) values were less than 1.7 x 10(-7) M and U937 was the most sensitive cell line with a IC(50) of 5.4 x 10(-9) M. Flow cytometric analysis revealed that treatment of U937 cells with toosendanin resulted in a dose- and time-dependent accumulation of cells in the S phase with a concomitant decrease in cells processing to G(0)/G(1) phase. The growth inhibition of U937 cells after exposure to toosendanin was subsequently associated with the induction of apoptosis, as evidence by the typical condensed and fragmented nuclei, DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of plasma membrane. All these results indicated that toosendanin could serve as a potential candidate for anticancer drug.

Botulinum neurotoxins (BoNTs), a group of bacterial proteins that comprise a light chain disulfide linked a heavy chain, are the most lethal biotoxins known to mankind. By inhibiting neurotransmitter release, BoNTs cause severe neuroparalytic disease, botulism. A series of important findings in the past 10 years which displayed the molecular targets of BoNTs and hence proposed a four-step action mechanism to explain BoNT intoxication greatly advanced the study of antibotulismic drug. In this article, we reviewed these progresses and anti-botulismic compounds found in recent years. These compounds function due to their facilitation on neurotransmitter release or to their interference on the binding, internalization, translocation, and endopeptidase activity of the toxins. Toosendanin is a triterpenoid derivative extracted from a digestive tract-parasiticide in Chinese traditional medicine. Chinese scientists have found that the compound is a selective prejunctional blocker. In spite of sharing some similar action with BoNT, toosendanin can protect botulism animals that have been administrated with lethal doses of BoNT/A or BoNT/B for several hours from death and make them restore normal activity. The neuromuscular junction preparations isolated from the rats that have been injected with toosendanin tolerate BoNT/A challenge. Toosendanin seems to have no effect on endopeptidase activity of BoNT, but blocks the toxin approach to its enzymatic substrate.

To examine if toosendanin (TSN) affects intracellular free-Ca(2+) concentration ([Ca(2+)](i)) in neuroblastoma pluglioma hybrid cells (NG108-15 cells).The [Ca(2+)](i) was determined by laser-scanning confocal microscopic imaging technique in which Fluo-3 was used as Ca(2+) indicator.TSN induced an increase in resting [Ca(2+)](i) and in high K(+)-evoked Ca(2+) transient in differentiated NG108-15 cells. The TSN-induced increase in [Ca(2+)](i) was dose-dependent and disappeared in CdCl(2-), nifedipine-containing or Ca(2+)-free solution, and appeared after washing out the Ca(2+) channel blockers or adding Ca(2+).TSN increased [Ca(2+)](i) in differentiated NG108-15 cells. The [Ca(2+)](i) enhancement was due to the influx of extracellular Ca(2+) and related to L-type Ca(2+) channels.

Toosendanin, a triterpenoid derivative extracted from Melia toosendan Sieb et Zucc, was demonstrated to be potentially useful in medical and scientific researches. Here, we investigated the effects of toosendanin on L-type voltage-dependent Ca 2+ channels in cultured neonatal rat ventricular cells, using whole-cell patch-clamp method. Toosendanin irreversibly increased L-type Ca 2+ current ( I Ca(L) ) in a concentration-dependent manner and shifted the maximum of the current/voltage relationship from 8.3±3.7 to 1.7±3.7 mV, without modifying the threshold potential of the current. Toosendanin shifted the steady-state activation and inactivation curves to the left. The deactivation kinetics of the I Ca(L) was significantly slowed by toosendanin while the activation kinetics was not affected. The cells pretreated with 100 nM 1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester ( S (−)-BayK8644) still respond to further addition of 87 μM toosendanin, and vice versa. These results prove toosendanin to be a novel L-type Ca 2+ channel agonist, which possesses a distinct binding site from BayK8644.

To examine the effects of opioid receptor antagonists and norepinephrine on intracellular free Ca2+ concentration ([Ca2+]i) in mesenteric arterial (MA) smooth muscle cells (SMC) isolated from normal and hemorrhagic shocked rats in the vascular hyporesponse stage.The rat model of hemorrhagic shock was made by withdrawing blood to decrease the artery mean blood pressure to 3.73-4.26 kPa and keeping at the level for 3 h. [Ca2+]i of vascular smooth muscle cells (VSMC) were detected by the laser scan confocal microscopy.In the hyporesponse VMSC of rats in hemorrhagic shock, selective delta-, kappa-, and mu-opioid receptor antagonists (naltrindole, nor-binaltorphimine, and beta-funaltrexamine, 100 nmol/L) as well as norepinephrine 5 micromol/L significantly increased [Ca2+]i by 47 %+/-13 %, 37 %+/-14 %, 33 %+/-10 %, and 54 %+/-17 %, respectively, although their effects were lower than those in the normal rat cells (the increased values were 148 %+/-54 %, 130 %+/-44 %, 63 %+/-17 % and 110 %+/-38 %, respectively); and the norepinephrine-induced increase in [Ca2+]i was further augmented by three delta-, kappa-, and mu-opioid receptor antagonists (50 nmol/L, respectively) application (from 52 %+/-16 % to 99 %+/-29 %, 146 %+/-54 % and 137 %+/-47 %, respectively).The disorder of [Ca2+]i regulation induced by hemorrhagic shock was mediated by opioid receptor and alpha-adrenoceptor, which may be partly responsible for the vascular hyporesponse, and the opioid receptor antagonists improved the response of resistance arteries to vascular stimulants in decompensatory stage of hemorrhagic shock.

Our previous study showed that toosendanin, a triterpenoid derivative isolated from a Chinese traditional medicine, could induce apoptosis in PC12 cells. In this study we confirmed the apoptosis-inducing effect of toosendanin in PC12 cells with new evidences in morphology and biochemistry: the shrinkage of cytosol, the condensation and fragmentation of nuclei and the formation of DNA ladder. It was also demonstrated that toosendanin decreased the PC12 cell viability in a time- and concentration-dependent manner. To elucidate the pathway linked with the toosendanin-induced apoptosis, the cytochrome c in the cytosol and the cleavage of poly(ADP-ribose) polymerase (PARP) were examined. The obtained results showed that toosendanin caused the release of cytochrome c from mitochondria into the cytosol and then led to the activation of caspase, indicating that the cytochrome c release and caspase activation were involved in the toosendanin-induced apoptosis process. These results suggested the possibility that toosendanin could serve as a candidate for anti-cancer drug.

Toosendanin, a triterpenoid derivative extracted from Melia toosendan Sieb et Zucc, was demonstrated to be a selective presynaptic blocker and an effective antibotulismic agent in previous studies. Here, we observed its effects on Ca(2+) channels in NG108-15 cells by whole-cell patch-clamp recording. Obtained data showed that toosendanin concentration dependently increased the high-voltage-activated (HVA) Ca(2+) current with an EC(50) of 5.13 microM in differentiated NG108-15 cells. The enhancement effect was still observed when the cells were pretreated with 5 microM omega-conotoxin MVIIC. However, when the cells were preincubated with 5 microM nifedipine or 10 microM verapamil-containing solution, the effect was absent. In undifferentiated NG108-15 cells, which only express T-type Ca(2+) channels, toosendanin did not affect Ca(2+) currents. These results show that toosendanin increases Ca(2+) influx in NG108-15 cells via L-type Ca(2+) channels.

In the present study, the effects of toosendanin on cell differentiation and apoptosis were investigated in PC12 cells. The results showed that after 24-48 h of culture in a medium containing toosendanin (approximately 1-10x10(-7) M), cell differentiation and outgrowth of neuronal processes were promoted. Combined treatment with toosendanin and a calcium channel blocker, nifedipine or omega-conotoxin GVIA, resulted in a significant inhibition of the toosendanin-induced effects. Pretreatment of PC12 cells with BAPTA-AM also inhibited the toosendanin-induced effects; however, these effects were not inhibited by pertussis toxin and H-7 in the medium. Toosendanin also induced cell apoptosis. Based on the DNA content determined by flow cytometric analysis, the number of apoptotic cells significantly increased when the incubation time in the toosendanin-containing medium was lasted up to 72 h. Toosendanin at a higher concentration (> or =1 x 10(-6) M) caused cell death while it had no effect on cell division at concentrations lower than 1 x 10(-7) M.

The antibodies against pre-synaptic membrane receptor (PsmR) and acetylcholine receptor (AChR) in serum samples of myasthenia gravis (MG) patients and healthy donors were tested by enzyme-linked immunosorbent assays (ELISA). The serum samples of eight MG patients with different autoantibodies and those of six healthy donors without these two kinds of autoantibodies were collected to investigate their effects on the peri-neurially recorded membrane currents at mouse motor nerve terminals. After inhibition of both fast and Ca(2+)-dependent K+ currents by tetra-ethylammonium (TEA), a positive wave was revealed, which was a balance of the slow K+(Ik,s) and Ca2+ currents (ICa). Application of anti-PsmR antibody negative MG sera and healthy donor sera, whether anti-AChR antibody positive or negative, did not affect the positive wave. However, the positive wave shifted to prolonged Ca(2+)-plateau when adding two of four anti-PsmR antibody positive serum samples from MG patients, indicating an inhibition of Ik,s by anti-PsmR antibody positive sera. Meanwhile, all serum samples derived from either patients or healthy donors did not affect INa.

Previous study has indicated a significant enhancement of activity of large-conductance Ca 2+ -activated K + channel (BK Ca ) in mesenteric arterial vascular smooth muscle cells isolated from rats in vascular hyporesponsive stage of hemorrhagic shock. In the present study, the effect of opioid receptor antagonism on BK Ca activity in the vascular smooth muscle cells of rats in the hyporesponse stage of hemorrhagic shock was investigated by using inside-out configuration of the patch-clamp technique. The results showed that naloxone (10 μM) down-regulated the activity of BK Ca by reducing open probability (P o ) and open frequency of the channels. The reduction of P o resulted from a decrease of mean open time and an increase of the slow closed time constant. Naltrindole and nor-binaltorphimine (100 nM) had the similar effects to that of naloxone, but no significant effect of β-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that δ- and K-opioid receptors, but not μ-receptors, may be involved in the regulation of BK Ca in vascular hyporesponse stage, and that inhibition of BK Ca may be one of the mechanisms of the opioid receptor antagonists improving the response of resistance arteries to vasoactive stimulants during the decompensatory stage of hemorrhagic shock.

Ion channel plays a key role in maturation, capacitation and acrosome reaction of sperm. However, as it is difficult to record channel currents from a mature mammal sperm directly by patch-clamp technique, there were no basic data on the types and properties of the channels in human sperm until the method reconstituting the channels into bilayer was used. By reconstituting the channel proteins isolated from sperm membrane into phospholipid-forming bilayer, we have characterized several kinds of Ca2+-, Na+-, K+-, and Cl--permeable channels with different conductance and properties in human sperm membrane. To study the channels in spermatogenic cells is another approach to understand the ion-channels in mature sperm. The cell is used as a model to analyze the effects of male antifertility agents on Ca2+-channel. To date, several male contraceptives derived from Chinese traditional medicine have been attached worldwide interest, a lot of compounds have been purified from them, and the antifertility effects of some compounds were demonstrated. We studied the effects of gossypol and several compounds isolated from Tripterygium wilffordii on Ca2+ channel in mouse spermatogenic cells and found that each of them inhibited the channel and sperm acrosome reaction at a proximate concentration, suggesting that the inhibition of Ca2+ channels may be one mechanism of the antifertility effects of these contraceptives.

The effects of hemorrhagic shock on Ba 2+ currents (I Ba ) via Ca 2+ channels and the regulation of the channels in the vascular hyporesponse stage of hemorrhagic shock by opioid receptor antagonists were examined by using the whole-cell recording of patch-clamp technique in mesenteric arterial smooth muscle cells of rats. The results showed that hemorrhagic shock induced an inhibition of Ca2 + channels in the cells; 10 μM of naloxone and 100 nM of naltrindole, nor-binaltorphimine, and (3-funaltrexamine increased the I Ba in the cells of rats in shock. After inhibition of protein kinase C by using 1-(5-isoquindinesulfonyl)-2-methylpiperazine via electrodes, the enhancement of I Ba by the antagonists was not observed. These results suggested that the inhibition of Ca 2+ channel induced by hemorrhagic shock was mediated by 8-, K-, and μ-opioid receptors in the cells and may be partly responsible for vascular hyporesponse. The enhancement of I Ba was mediated by activation of protein kinase C and may be responsible for the antagonist-caused improvement in the response of resistance arteries to vasoactive stimulants at the decompensatory stage of hemorrhagic shock.

The male antifertility effect of a water-chloroform extract (GTW) from the root xylem of Tripterygium wilfordii has attracted worldwide interest. In the present study, by using whole-cell recording, the effects of GTW and two isolated monomers from GTW, demethylzeylasteral and L-epicatechin, on the T-type Ca(2+) channels in mouse spermatogenic cells were investigated. The results showed that each of them concentration-dependently and partially reversibly inhibited T-type Ca(2+) current in the cells. The IC(50) of GTW and demethylzeylasteral were approximate, while L-epicatechin inhibited the channels at a much higher concentration. The voltage dependence of the inhibitory effect and the changes in activation and inactivation time constants after application of these compounds were also examined. These data suggest that the inhibition of T-type Ca(2+) currents could be responsible for the antifertility activity of these compounds.

Effect of beta-agkistrodotoxin (beta-AgTx), a presynaptic neurotoxin purified from snake venom, on large-conductance calcium-activated potassium channels (BK(Ca)) was studied in rat hippocampal CA1 pyramidal neurons using inside-out configuration of patch-clamp technique. The results showed that in equimolar K+ (150 mM) and 1 microM intracellular Ca2+ conditions, internal application of beta-AgTx inhibited the activity of BK(Ca) by reducing open probability (P(o)) of the channels in a concentration-dependent manner. High concentration (74 nM) of beta-AgTx completely eliminated opening of the channels. However, 37 nM beta-AgTx (at -40 mV) decreased P(o) from 0.49+/-0.07 to 0.03+/-0.03, switched two open time constants (0.51+/-0.32 and 8.77+/-1.63 ms) to be a single time constant of 0.46+/-0.40 ms. The results indicate that inhibition of BK(Ca) by beta-AgTx may account for the facilitatory phase of the toxin on acetylcholine release from nerve terminals.

The effect of toosendanin, a presynaptic blocker, on the inward rectifier potassium channel (K(Kir)) of hippocampal CA1 pyramidal neurons of rats was studied by the single-channel patch-clamp technique. The results showed that toosendanin had an inhibitory effect on K(Kir) in an excised inside-out patch of the neuron under a symmetrical 150 mM K(+) condition. By decreasing the slower open time constant and increasing the slower close time constant, toosendanin (1x10(-6)-1x10(-4) g/ml) significantly reduced the open probability of the channel in a concentration-dependent manner. Meanwhile, a dose-dependent reduction in unitary conductance of the channel was also detected after toosendanin application. These data offer an explanation for toosendanin-induced facilitation of neurotransmitter release and antibotulismic effect of the drug.

The effect of a delta-selective opioid agonist, DPDPE([D-Pen(2, 5)]-enkephalin), on the inward rectifier potassium current (I(KIR)) of NG108-15 cell was studied by whole cell voltage-clamp technique. It was found that microM DPDPE increased the amplitude and delayed the activation and inactivation of I(KIR) rapidly and reversibly. These effects could be reversed by naloxone, but were still obtained in pertussis toxin (PTX) preincubated cells or when using GDP-betas (guanosine 5'-o-[2-thio] diphoaphate) containing electrodes to block the G-protein coupled events. The above results suggest that DPDPE-induced change of I(KIR) is mediated by delta-opioid receptor but does not involve G-proteins.

The purified CF 0 -CF 1 complex of spinach was incorporated into planar lipid bilayer membranes (LBMs) formed with soybean phospholipid, and the transmembrane ion-transmission properties were studied under voltage-clamp mode. The results showed that the presence of both ADP and Pi decreased the membrane current while Dithiothreitol could evoke a stronger conductive change of CF 0 -CF 1 containing LBMs when Ca 2+ or Mg 2+ exists. Mg 2+ can dramatically increase the CF 0 -CF 1 conductance in various conditions. These results indicated that the H + -transitive function of CF 0 -CF 1 reconstituted in bilayer is sensitive to those factors which can affect its ATP synthase activity in vivo.

The effect of Toosendanin (TSN), a presynaptic transmission blocker, on the outward delayed rectifier potassium current (IKD) of NG108-15 cells was studied by using the whole-cell voltage-clamp technique. It was observed that externally applying TSN not only reduced IKD amplitude in a dose-dependent and partial reversible manner but also accelerated its inactivation. The effect of internally applying TSN was also examined by including TSN in the electrode, and it was the same as that of externally applying TSN. Further, comparison observations with TEA, 4-AP, verapamil, nifedipine, and (±)-Bay K 8644 were also made, and the results were as follows. The time courses of TSN's inhibition effect as well as its recovery after washing were much slower than those of TEA and 4-AP. Externally applying TEA or 4-AP reduced IKD amplitude but did not accelerate its inactivation. Externally applying verapamil, nifedipine, or (±)-Bay K 8644, however, similarly to the effect of TSN, not only reduced IKD amplitude but also accelerated its inactivation. Thus, from the obtained results it is suggested that TSN might diffuse into the cell interior and act intracellularly, and the underlying mechanism might be different from that of TEA and 4-AP but similar to that of verapamil, nifedipine, and (±)-Bay K 8644 to some extent. © 1997 Elsevier Science B.V. All rights reserved.

The effect of plasma from a myasthenia gravis (MG) patient, containing anti-presynaptic membrane receptor (PsmR) antibody on the membrane currents of motor nerve ending was investigated in mouse intercostal nerve triangularis sterni preparations by perineurial recording. After inhibition of both the fast K + current and Ca 2+ -dependent K + current by 30 mM Tetraethyl-ammonium (TEA) unmasked the voltage dependent fast Ca 2+ current and the “Ca plateau”, which was contributed by the voltage-dependent slow Ca 2+ current and slow K + current. Application of the MG plasma caused further prolongation and increase of the Ca plateau, due to blockage of the slow K + current. This effect was observed immediately after the application and could be partially reversed by washing, whereas no change was found by addition of the plasma from healthy persons. When K + current was completely blocked by 30 mM TEA and 300 μM 3,4-diaminopyridine (3,4-DAP), the fast Ca 2+ current and the slow Ca 2+ current were revealed. Neither the fast nor the slow Ca 2+ current could be affected by the MG plasma; It was also shown that the MG plasma was devoid of noticeable effect on the voltage dependent Na + current, fast K + current as well as the Ca 2+ -dependent K + current. So the effect of the MG plasma with antibody to PsmR was concluded to inhibit the slow K + current selectively. As we knew, the β-bungarotoxin binding protein was a kind of K + channel, these results further confirmed that the β-bungarotoxin binding protein should be the target of the antibody to PsmR found in the plasma of some patients suffering from MG.

Toosendanin (TSN), a triterpenoid derivative extracted from Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study is designed to explore its antibotulismic mechanism by Western blotting. The results showed that TSN incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes, but made the synaptosomes completely resistant to botulinum neurotoxin A (BoNT/A)-mediated cleavage of SNAP-25. After binding of BoNT/A to synaptosomes, TSN still partially antagonized the toxin-mediated cleavage of SNAP-25. However, TSN-incubated synaptosomal membrane fraction did not resist the cleavage of SNAP-25 by the light chain of BoNT/A. It is suggested that the antibotulismic effect of TSN results from blocking the toxin’s approach to its enzymatic substrate.