[Effects of sodium nitroprusside on growth and physiological characteristics of tomato seedlings under iron deficiency and NO3- stress]. / ç¡æ™®é’ å¯¹ç¼ºé“å’Œç¡é ¸ç›èƒè¿«ä¸‹ç•ªèŒ„å¹¼è‹—ç”Ÿé•¿åŠç”Ÿç†ç‰¹æ€§çš„å½±å“.

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 NO3- stress. The results indicated that after 7 d of treatment, iron deficiency inhibited the tomato seedling growth, decreased chlorophyll (a and b) and carotenoid contents, and led to obvious chlorosis; iron deficiency also reduced the activity of SOD, POD and CAT, resulting in significant accumulation of MDA contents and higher electrolytic leakage; proline and soluble sugar contents were not significantly changed; contents of N, P, K, Ca, Mg and Fe in both leaves and roots of tomato seedlings were decreased under iron deficiency in varying degrees. The combined stress of NO3- stress and iron deficiency promoted the inhibition of plant growth, decreased chlorophyll (a and b), carotenoid contents and the activities of SOD, POD and CAT; electrolytic leakage MDA, proline and soluble sugar contents of N, P, Mg, Fe in both leaves and roots of tomato seedlings were much lower, but the contents of K and Ca were significantly increased. Compared to the treatment without SNP, adding 0.1 mmol·L-1 SNP could alleviate the growth inhibition of stressed tomato seedlings. Adding 0.1 mmol·L-1 SF (sodium ferrocyanide) also showed alleviating or promoting effect on some above indexes including the activity of SOD, POD and CAT to some degree, but the other physiological indexes were not significantly changed because iron was contained in SF.

Biological effects of toosendanin, a triterpenoid extracted from Chinese traditional medicine.

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.

[Biological effects of toosendanin, an active ingredient of herbal vermifuge in Chinese traditional medicine].

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.

Toosendanin interferes with pore formation of botulinum toxin type A in PC12 cell membrane.

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 single-channel 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.

Growth inhibition and apoptosis-induced effect on human cancer cells of toosendanin, a triterpenoid derivative from chinese traditional medicine.

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.

The long-term effect of toosendanin on current through nifedipine-sensitive Ca2+ channels in NG108-15 cells.

Toosendanin is a triterpenoid derivative extracted from Melia toosendan Sieb et Zucc. Previous studies demonstrated that toosendanin could block neurotransmission and stimulate PC12 cell into differentiation and apoptosis. These actions of toosendanin were suggested to result from a continuous increase in Ca2+ influx, which led to intracellular Ca2+ overload. Here, we observed the long-term effect of toosendanin on Ca2+ channels in NG108-15 cells by whole-cell patch-clamp recording. Obtained data showed that a prolonged exposure to toosendanin induced a continuous increase in the Ca2+ influx in a concentration and time-dependent manner while a brief treatment induced an irreversible increase in Ca2+ influx in differentiated NG108-15 cells. The nifedipine-sensitive L-type currents were significantly increased after exposure to TSN while the nifedipine-resistant or omega-conotoxin MVIIC-sensitive currents were not affected.

Toosendanin, a triterpenoid derivative, acts as a novel agonist of L-type Ca2+ channels in neonatal rat ventricular cells.

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 microM 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.

Involvement of cytochrome c release and caspase activation in toosendanin-induced PC12 cell apoptosis.

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.

Cure of experimental botulism and antibotulismic effect of toosendanin.

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.

Toosendanin, a triterpenoid derivative, increases Ca2+ current in NG108-15 cells via L-type channels.

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.

Toosendanin increases free-Ca(2+) concentration in NG108-15 cells via L-type Ca(2+) channels.

AIM: To examine if toosendanin (TSN) affects intracellular free-Ca(2+) concentration ([Ca(2+)](i)) in neuroblastoma pluglioma hybrid cells (NG108-15 cells). METHODS: The [Ca(2+)](i) was determined by laser-scanning confocal microscopic imaging technique in which Fluo-3 was used as Ca(2+) indicator. RESULTS: 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+). CONCLUSION: 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.

Opioid receptor antagonists increase [Ca2+]i in rat arterial smooth muscle cells in hemorrhagic shock.

AIM: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Antagonism of botulinum toxin type A-induced cleavage of SNAP-25 in rat cerebral synaptosome by toosendanin.

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.

Effects of demethylzeylasteral and celastrol on spermatogenic cell Ca2+ channels and progesterone-induced sperm acrosome reaction.

The male antifertility effect of a water-chloroform extract of Tripterygium wilfordii Hook. f. (GTW) and several monomers isolated from GTW has attracted worldwide interest. In the present study, the effects of two isolated monomers from GTW, demethylzeylasteral and celastrol, on the Ca(2+) channels in mouse spermatogenic cells and on the sperm acrosome reaction were investigated by whole-cell patch-clamp recording and chlortetracycline staining methods, respectively. The results showed that demethylzeylasteral concentration-dependently and in a partially reversible manner inhibited the Ca(2+) current in spermatogenic cells with an IC(50) of 8.8 microg/ml. Celastrol decreased the Ca(2+) current in the cells time-dependently and irreversibly. The changes in the activation and inactivation time constants of Ca(2+) currents after application of these two compounds were also examined. Demethylzeylasteral increased both activation and inactivation time constants of Ca(2+) currents, and celastrol had no significant effect on them. The two compounds also inhibited significantly the sperm acrosome reaction initiated by progesterone. These data suggest that inhibition of Ca(2+) currents could be responsible for the antifertility activity of these compounds.

Toosendanin induces outgrowth of neuronal processes and apoptosis in PC12 cells.

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.

Effects of myasthenia gravis patients' sera with different autoantibodies on slow K+ current at mouse motor nerve terminals.

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.

Opioid receptor antagonists modulate Ca2+-activated K+ channels in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

Previous study has indicated a significant enhancement of activity of large-conductance Ca2+-activated K+ channel (BKCa) 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 BKCa 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 microM) down-regulated the activity of BKCa by reducing open probability (Po) and open frequency of the channels. The reduction of Po 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 beta-funaltrexamine (100 nM) on the activity of the channels could be found. These results suggest that delta- and kappa-opioid receptors, but not mu-receptors, may be involved in the regulation of BKCa in vascular hyporesponse stage, and that inhibition of BKCa 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-channels in human sperm membrane and contraceptive mechanisms of male antifertility compounds derived from Chinese traditional medicine.

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.

Modulation of Ca2+ channels by opioid receptor antagonists in mesenteric arterial smooth muscle cells of rats in hemorrhagic shock.

The effects of hemorrhagic shock on Ba currents ( ) via Ca 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 Ca channels in the cells; 10 micro M of naloxone and 100 n of naltrindole, nor-binaltorphimine, and beta-funaltrexamine increased the 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 by the antagonists was not observed. These results suggested that the inhibition of Ca channel induced by hemorrhagic shock was mediated by delta-, kappa-, and mu -opioid receptors in the cells and may be partly responsible for vascular hyporesponse. The enhancement of 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.

Inhibition of Ca(2+) channels in mouse spermatogenic cells by male antifertility compounds from Tripterygium wilfordii Hook. f.

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.