Ultrastructural features of the starch sheath cells of the primary pulvinus after gravistimulation of the sensitive plant (Mimosa pudica L.)

Summary InMimosa pudica the primary and secondary motor organs (pulvini) of fully grown leaves are capable of graviresponse. These organs possess sedimentable amyloplasts in their starch sheath cells.In the primary pulvinus these cells are characterized by a structural polarity induced by the localization of nucleus at their (morphologically) apical part and the localization of amyloplasts at their (physically) basal part. These cells also display structural peculiarities including plasmodesmatal disposition, little development of the endoplasmic reticulum and an absence of vacuolar tannins; moreover, the sedimentation of the amyloplasts, induced by gravistimulation, is accompanied by the variation of localization of the cytoplasm, vacuole and mitochondria and by structural modifications of the nucleus and endoplasmic reticulum.     

Transport processes in stimulated and non-stimulated leaves of Mimosa pudica

Summary Using energy-dispersive X-ray microanalysis, the concentrations of ions, especially potassium and chlorine, were determined in different tissues of primary and tertiary pulvini of Mimosa pudica. It was shown that stimulating the leaf was followed by ion displacements which were most striking in the outer extensor cells, resulting in turgor loss. Since Ca concentration remains relatively constant in cell walls of collapsed cells, the changes of K concentration are best described by the K:Ca ratio. After stimulation the K:Ca ratio dropped in the outer extensor of the primary pulvinus from 775.3 to 2.37 in the cytoplasm, and from 542.2 to 9.25 in the cell wall. Changes in chlorine content were less striking in the primary pulvinus. The KCl ratios in some cases were lower than 1.0, which indicates that Cl content can increase, while K content is diminished. In the non-stimulated tertiary pulvini the outer extensor cells show high concentrations of Cl, but much lower Cl concentrations were found after stimulation. In contrast to the primary pulvinus the K content of the tertiary pulvini is very low. In the vascular tissues of both primary and tertiary pulvini stimulation is followed by a release of K and Cl out of the sieve element cytoplasm into the apoplast. K then appears accumulated in the cell walls of the collenchymatous tissue. These displacements lead to the assumption that the collenchymatous apoplast temporarily functions as a reservoir for K and to a lesser extent for Cl. With regard to the mechanism of leaf movement after stimulation, the accumulation of ions in the apoplast seems to be initiated by the decrease of water potential triggered by an apoplastic accumulation of unloaded sucrose (Fromm and Eschrich 1988a). The resulting turgor release in the outer extensor is accompanied by an efflux of ions.Supported by the Deutsche Forschungsgemeinschaft     

Indoleamines and calcium channels influence morphogenesis in in vitro cultures of Mimosa pudica L.

The present article reports the interplay of indoleamine neurohormones viz. serotonin, melatonin and calcium channels on shoot organogenesis in Mimosa pudica L. In vitro grown nodal segments were cultured on MS medium with B5 vitamins containing Serotonin (SER) and Melatonin (MEL) at 100 µM and indoleamine inhibitors viz. serotonin to melatonin conversion inhibitor p-chlorophenylalanine (p-CPA) at 40 µM, serotonin reuptake inhibitor (Prozac) 20 µM. In another set of experiment, calcium at 5 mM, calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) 100 µM, and calcium channel blocker varapamil hydrochloride (1 mM) a calcium chelator EGTA (100 µM) were administered to the culture medium. The percentage of shoot multiplication, endogenous MEL and SER were monitored during shoot organogenesis. At 100 µM SER and MEL treatment 60% and 70% explants responded for shoot multiplication respectively. Medium supplemented with either SER or MEL along with calcium (5 mM) 75%–80% explants responded for organogenesis. SER or MEL along with calcium ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187) at 100 µM 70% explants responded for shoot multiplication. p-CPA, prozac, verapamil and EGTA, shoot multiplication was reduced and endogenous pools of SER, MEL decreased by 40–70%. The results clearly demonstrated that indoleamines and calcium channels positively influenced shoot organogenesis in M. pudica L.     

Redistribution of potassium,chloride and calcium during the gravitropically induced movement of Mimosa pudica pulvinus

When the leaves of Mimosa pudica are changed from their normal position in the gravitational field, they perform reversible compensatory movements by means of pulvini. These movements are not the result of growth processes but involve reversible turgor variations. These variation are concomitant with ion migrations within pulvini: during the gravitropic movement, K⁺ and Cl^- shift towards the adaxial half of the motor organ whereas Ca²⁺ shifts towards the abaxial half. Compounds known to affect K⁺ transport, tetraethylammonium chloride and valinomycin, do not hinder the gravitropic movement but inhibit strongly the seismonastic reaction. The same general result is obtained with compounds affecting anion transport, disulfonic stilbenes and 9-anthracene carboxylic acid. Calcium chelators inhibit the gravitropic movement more efficiently than the seismonastic reaction and the calcium ionophore A 23 187 increases both movements. The data obtained with these various compounds indicate that ions do not have the same functional importance in the regulation of the two different pulvinar movements.Abbreviations abx abaxial half of the pulvinus - adx adaxial half of the pulvinus - 9-AC 9-anthracene carboxylic acid - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid - TEA tetraethylammonium chloride     

Absence of plasma membrane H+-ATPase in plasmodesmata located in pit-fields of the young reactive pulvinus ofMimosa pudica L.

Summary Immunocytochemical techniques were employed to study the spatial distribution of the plasma membrane H⁺-ATPase within various cell types of the young reactive primary pulvinus ofMimosa pudica L. These cells were interconnected by large numbers of plasmodesmata, being concentrated within pit-fields. Although we could routinely detect evidence of the H⁺-ATPase along the plasma membrane, immunolabelling was rarely, if ever, observed along the plasma membranes of the plasmodesmata. This finding is discussed with respect to the likely specialized supramolecular structure of the plasmodesma.Abbreviations SEL size exclusion limit of plasmodesmata     

银杏苦内酯B对缺血豚鼠心室肌动作电位、L-型钙电流和延迟整流钾电流的作用

目的:研究银杏苦内酯B对正常和缺血心室肌细胞动作电位(action potential,AP),L-型钙电流(L-type calcium current,ICa-L)、延迟整流钾电流(Delayed Rectifier Currennt,IK)的影响.方法:用常规细胞内微电极方法记录豚鼠心室肌细胞动作电位,用全细胞膜片钳技术记录游离心室肌细胞离子流.结果:①在生理条件下,银杏苦内酯B可缩短心室肌细胞动作电位时程 (action potential duration,APD),但对AP其他参数无影响,银杏苦内酯B可增大IK,呈浓度依赖性,但对ICa-L无显著作用;②在缺血条件下,APD50、APD90明显缩短,RP、APA减小,Vmax减慢,而银杏苦内酯B则可延缓和减轻缺血所引起上述参数的变化;3.在缺血条件下,IK和ICa-L均受到抑制,但加入银杏苦内酯B后可逆转缺血所造成这两种离子流的减小.结论:银杏苦内酯B可对抗心肌缺血所引起的心肌电生理的变化,提示银杏苦内酯B可预防心律失常的发生.     

蛋白激酶A和蛋白激酶C对豚鼠心肌细胞延迟整流钾电流的作用

目的 :研究蛋白激酶A和蛋白激酶C对豚鼠心室肌细胞延迟整流钾电流 (Ik)的影响。方法 :采用电极内液浓度差扩散法进行细胞内给药 ,利用全细胞膜片箝技术测定单细胞Ik。结果 :cAMP15 0 μmol/L使Ik及Ik ,tail(pA/pF)从 13.7± 2 .1和 6 .1± 0 .3增至 18.5± 3.3和 6 .4± 2 .1(P <0 .0 1,n =6 ) ;8 CPT cAMP15 0 μmol/L使电流 (pA/pF)从 11.4± 1.8及 5 .3± 0 .6增至 17.9± 4 .0和 6 .2± 1.3,PKA的选择性抑制剂 6 2 2 1.0 μmol/L的可逆转二者的作用。cAMP使Ik的激活曲线左移 ,半激活电压 (V1/ 2 )从 2 3.3mV移至 18.7mV ,激活曲线斜率 (k)在用药前后变化较小。 10 μmol/LPMA可以分别使Ik和Ik ,tial(pA/pF)从 12 .9± 1.8和 5 .0± 1.7升至 2 3.7± 2 .8和 7.5±1.1。PMA使I V曲线幅值增加 ,并随去极化电压的升高其作用加强 ,同时PMA使通道的激活曲线k从 15 .3mV升到 2 5 .6mV ,但对V1/ 2 基本无影响。结论 :蛋白激酶A和蛋白激酶C均可增加豚鼠心肌细胞Ik,但二者作用特点有所不同     

A dual action of taurine on the delayed rectifier K+ current in embryonic chick cardiomyocytes

Summary Effects of taurine on the delayed rectifier K⁺ channel in isolated 10-day-old embryonic chick ventricular cardiomyocytes were examined at different intracellular Ca²⁺ concentrations ([Ca]_i), using whole-cell voltage and current clamp techniques. Experiments were performed at room temperature (22°C). Test pulses were applied between -20 to +90m V from a holding potential of -40mV. When [Ca]_i was pCa 7, addition of 10 and 20 mM taurine to the bath solution reduced the delayed rectifier K⁺ current (I_K) at +90mV by 17.4 ± 2.8% (n = 5, P < 0.01) and 25.5 ± 2.6% (n = 5, P < 0.001), respectively. In contrast, when [Ca]_i was pCa 10, I_K at +90 mV was enhanced by 19.1 ± 3.1% (n = 7, P < 0.01) at 10mM taurine, and by 29.3 ± 2.4% (n = 7, P < 0.001) at 20mM taurine. The voltage of half-maximum activation (V_1/2) was shifted in a hyperpolarizing direction; at pCa 7, the value was +0.2 ± 2.2mV (n = 5) in control and -10.6 ± 1.8mV (n = 5) in 20mM taurine. At pCa 10, the V_1/2 value was +18.5 ± 4.6mV (n = 5) in control and +6.6 ± 5.2mV (n = 5) in taurine (20mM). Taurine decreased the action potential duration (APD) at pCa 10, but at pCa 7 did not affect it. In addition, taurine enhanced the transient outward current in a concentration-dependent manner. These results indicate that taurine modulates the delayed rectifier K⁺ channel, an effect dependent on [Ca]_i and capable of regulating APD.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

Effects of calcium,magnesium, potassium and boron on sperm cells isolated from pollen of Zea mays L.

Our previous studies showed that Brewbaker and Kwack salts, which have been widely used in pollen germination and sperm isolation, are not appropriate for the maintenance of isolated maize (Zea mays L.) sperm cells. In the present study, we have characterized the effects of each BKS component salt on the integrity of isolated sperm cells using hemacytometry. At 0.01 and 0.1 mM, there were no differences in cell number between control and any salt-treated cells except a 22% decrease with 0.1 mM MgSO₄ at 48 h. At the 1 mM level, cell number decreased with time in the presence of Ca(NO₃)₂ and MgSO₄, with loss of integrity of most cells at 48 h, while KNO₃ and H₃BO₃ had little or no effect. Further characterization of calcium-induced reduction in cell integrity using flow cytometry showed that depletion of possible residual free calcium by addition of EGTA to the suspension medium improved cell longevity and viability. Exposure of isolated sperm cells to 1 mM calcium had no effect on cell integrity and viability in 5 h; however, only 12% of cells remained intact at 24 h. The reduction in cell integrity was hastened when cells were pretreated with the calcium ionophore A23187 prior to exposure to 1 mM calcium, with a 54% reduction in cell number at 1 h and complete cell lysis at 24 h. However, depletion of cytosolic free calcium by pretreatment of cells with the calcium ionophore followed by resuspension in the presence of EGTA resulted in rapid reduction of cell integrity as well. These results collectively suggest that maize sperm cells are sensitive to exogenous free calcium; however, a certain level of cytosolic free calcium is necessary for maintenance of integrity. Mechanisms of calcium-induced reduction in cell integrity are discussed along with possible roles of the sensitivity of sperm cells to calcium in fertilization.     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

Modulation of A-type potassium current in smooth-muscle cells of the rat Vas Deferens by Ca2+/calmodulin-dependent protein kinase II

Using a standard patch-clamp technique in the perforated patch configuration, we studied the effect of a highly specific membrane-permeable inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII), KN-93, on fast outward A-type potassium current in isolated smooth-muscle cells (SMCs) of an epididymal region of the rat vas deferens. This inhibitor significantly changed the dynamics of the studied current; in particular, it increased the rate of inactivation and considerably slowed down the recovery after inactivation. In the presence of 5 μM KN-93, we observed a moderate (nearly by 20%) decrease in the peak amplitude of fast A-type current. Based on the data obtained, we conclude that voltage-sensitive fast A-type potassium current in SMCs of the epididymal part of the rat vas deferens can be significantly regulated by the activity of CaM-KII. Therefore, by influencing the kinetic characteristics of the above current, this enzyme can be indirectly involved in the control of electrical activity in SMCs. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 419–422, July–October, 2007.     

Relationship between plant phosphorus status and the kinetics of arsenate influx in clones ofdeschampsia cespitosa (L.) beauv. that differ in their tolerance to arsenate

Uptake kinetics of arsenate were determined in arsenate tolerant and non-tolerant clones of the grassDeschampsia cespitosa under differing root phosphorus status to investigate the mechanism controlling the suppression of arsenate influx observed in tolerant clones. Influx was always lower in tolerants compared to non-tolerants. Short term influx of arsenate by the high affinity uptake system in both tolerant clones was relatively insensitive to root phosphorus status. This was in contrast to the literature where the regulation of the phosphate (arsenate) uptake system is normally much more responsive to plant phosphorus status. The low affinity uptake system in both tolerant and non-tolerant clones, unlike the high affinity uptake system, was more closely regulated by root phosphate status and was repressed to a much greater degree under increasing root phosphorus levels than the high affinity system.     

Essential sulfhydryl groups in the catalytic center of the tonoplast H+-ATPase from coleoptiles ofZea mays L. as demonstrated by the biotin-streptavidin-peroxidase system

Functional properties and the localization of essential SH-groups of the tonoplast H⁺-ATPase fromZea mays L. were studied. In contrast to the pyrophosphate-dependent H⁺-translocation activity of the tonoplast, the H⁺-ATPase activity was inhibited by SH-blocking agents, such as N-ethylmaleimide and iodoacetic acid. In the case ofp-hydroxymercuribenzoate, HgCl₂ and oxidized glutathione, the inhibition could be reversed by adding reduced glutathione or dithiothreitol. Incubation of tonoplast vesicles with oxidized glutathione or N-ethylmaleimide in the presence of Mg·ADP—a competitive inhibitor of the ATP-dependent H⁺ pump—avoided the inhibition of the H⁺-pumping activity. This effect is an indication for the occurrence of essential SH-groups at the catalytic site of the H⁺-ATPase. In order to characterize the active center these thiols were specifically labeled with maleimidobutyrylbiocytin. Subsequently, the membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to an immobilizing membrane. The maleimidobutyrylbiocytin-labeled active-center protein was detected by a biotin-streptavidin-peroxidase staining system and was shown to be a 70-kDa subunit of the tonoplast H⁺-ATPase. It is suggested that the oxidation state of the critical sulfhydryl groups within the active center of the enzyme and their reversible blocking by endogenous compounds might be of great importance for the regulation of the enzyme activity in vivo.     

Down-regulation of prostate specific antigen in LNCaP cells by flavonoids from the pollen of Brassica napus L.

The pollen of Brassica napus L. has been used in China to treat benign prostatic hyperplasia (BPH) for over decades. In this study, the pollen of Brassica napus L. was extracted successively with chloroform, ethyl acetate and ethanol. The ethyl acetate extract showed strong activity in decreasing the secretion of prostate specific antigen (PSA) in LNCaP cells as compared to two other extracts, measured by ELISA with finasteride as positive control in the assay. Five flavonoids were subsequently isolated from the active extract using bioassay-guided fractionation. They were Naringenin (1); Luteolin (2); Kaempferol (3); Kaempferol 3-(3-E-p-coumaroyl-alpha-L-rhamnopyranoside) (4); and Kaempferol 3-(2,3-di-E-p-coumaroyl-alpha-L-rhamnopyranoside) (5). All these compounds inhibited PSA secretion significantly, with IC50 values in the range of 5-50 microM. Compounds 2, 4 and 5 showed moderate cytotoxicity to LNCaP cells within the active concentration range, while compounds 1 and 3 showed no cytotoxicity. Further studies on the mechanism action of these compounds were performed by evaluating their activation of estrogen receptor (ER) and antagonistic activities on androgen receptor (AR) in cell-based reporter gene assays. All compounds described here were first isolated from the pollen of Brassica napus L.     

Molecular and biological studies on male-sterile cytoplasm in the Cruciferae. I. The origin and distribution of Ogura male-sterile cytoplasm in Japanese wild radishes (Raphanus sativus L.) revealed by PCR-aided assay of their mitochondrial DNAs

Ogura male-sterile cytoplasm was surveyed in common Japanese radish cultivars and in wild radishes growing in various localities in Japan. Mitochondrial (mt) DNA rearrangement involving the atp6 gene was used as a molecular marker. To detect the mtDNA rearrangement, polymerase chain reactions (PCR) were designed to amplify the upstream region of the atp6 gene. The oligonucleotides homologous to the following three regions were synthesized: (1) trnfM, (2) ORF105 and (3) atp6. PCRs were conducted with a pair of the first and the third primers to detect normal mtDNA, and with the second and the third primers for Ogura-type mtDNA. All 15 Japanese cultivars yielded an amplification product which was the same as that of normal mtDNA, whereas some wild radishes gave the product specific to Ogura mtDNA. Twenty-four populations of wild radish were classified into three groups according to the frequency of Ogura-type mtDNA: (1) in ten populations, all four plants analyzed per population had normal type mtDNA, (2) in five populations, only plants with Ogura-type mtDNA were found, and (3) nine populations included both normal and Oguratype mtDNAs. There were no geographical restrictions and no cline in the distribution of the plants with Ogura-type mtDNA. These results suggested that the Ogura-type male-sterile cytoplasm originated in wild radishes.