专一识别脱落酸甲酯的单克隆抗体的制备与应用

专一识别２－顺（Ｓ）ＡＢＡ甲酯的单克隆抗体来源于以ＡＢＡ分子中的１－ＣＯＯＨ为偶联位点合成的免疫原。它与游离态ＡＢＡ和结合态ＡＢＡ葡萄糖酯的交叉反应仅分别为１％与３．５％,而与ＡＢＡ类似物,如２－顺－黄质醛、紫黄质以及ＡＢＡ的２－反式异构体和（Ｒ）－对映体则无交叉反应。利用该抗体建立的高度灵敏和精确的ＡＢＡｍｅ酶联免疫测定法,其检测线性范围为０．０４８～１．５２ｐｍｏｌ。通过ＡＢＡｍｅＥＬＩＳＡ和ＧＡ１＋３ＥＬＩＳＡ分析可知羊蹄叶片衰老与内源ＧＡ１＋３／ＡＢＡ比值的下降有关。     

黄腐酸对冬小麦幼苗IAA、ABA水平的影响及作用机理的探讨

本实验用酶联免疫（ＥＬＩＳＡ） 法测定了黄腐酸（ＦＡ） 处理后冬小麦幼苗吲哚乙酸（ＩＡＡ） 、脱落酸（ＡＢＡ） 的水平。结果表明，ＦＡ 能使ＩＡＡ、ＡＢＡ 水平增加，这与ＦＡ 能刺激植物生长、抑制气孔开启是一致的，而且ＦＡ 与这两种激素都没有协同作用。说明ＦＡ 既能直接对植物起作用，也能通过改变内源激素水平间接起作用     

苹果犁花芽分化期内源激素含量的变化（简报）

苹果梨花芽分化期间,可测到Ｚ、ＺＲ、ＩＡＡ、ＡＢＡ、ＣＡ３。生理分化期（花芽诱导期）,ＧＡ３、ＡＢＡ含量降低,Ｚ、ＺＲ、ＩＡＡ增加;形态分化期,ＩＡＡ降低,ＧＡ３、Ｚ、ＺＲ增加,ＡＢＡ初期降低,后期增加。在分化期间,Ｚ／ＧＡ３、ＺＲ／ＧＡ３、Ｚ／ＡＢＡ、ＺＲ／ＡＢＡ、ＩＡＡ／ＡＢＡ比值均增高。     

苹果梨花芽分化期内源激素含量的变化(简报)

苹果梨花芽分化期间,可测到Ｚ、ＺＲ、ＩＡＡ、ＡＢＡ、ＧＡ３ 。生理分化期（ 花芽诱导期） ,ＧＡ３ 、ＡＢＡ含量降低,Ｚ、ＺＲ、ＩＡＡ 增加;形态分化期,ＩＡＡ 降低,ＧＡ３ 、Ｚ、ＺＲ增加,ＡＢＡ初期降低,后期增加。在分化期间,Ｚ／ＧＡ３ 、ＺＲ／ＧＡ３ 、Ｚ／ＡＢＡ、ＺＲ／ＡＢＡ、ＩＡＡ／ＡＢＡ比值均增高。     

植物激素脱落酸的信号转导

介绍了与ＡＢＡ信号转导直接相关的ＡＢＡ结合蛋白、第二信使和ＡＢＡ响应基因的诱导表达等方面的最新研究进展。     

苹果果实组织脱落酸结合位点的亚细胞定位

在苹果（ＭａｌｕｓｐｕｍｉｌａＭｉｌ）果实细胞的可溶性组分中存在ＡＢＡ特异结合位点,这些位点能与ＡＢＡ形成稳定的３ＨＡＢＡ蛋白结合复合物,而微粒体部分未表现出３ＨＡＢＡ结合活性。可溶性组分对３ＨＡＢＡ的结合效率与结合稳定性需要活体组织完整细胞的存在。ＡＢＡ共价交联物ＡＢＡＬＹＣＨ,具有与ＡＢＡ相同的抑制红苋菜（ＡｍａｒａｎｔｈｕｓｔｒｉｃｏｌｏｒＬ．）种子萌发的生物活性,能有效地竞争抑制果实组织圆片对３ＨＡＢＡ的结合。通过ＡＢＡＬＹＣＨ对苹果果实组织圆片及果实组织游离细胞的荧光染色表明,未成熟果实的细胞外周被特异性地染色。在ＢＳＡ存在的情况下,ＡＢＡＬＹＣＨ复合物被转运进细胞,形成密集的强烈荧光团。结果表明,ＡＢＡＬＹＣＨ与３ＨＡＢＡ的竞争性结合发生在细胞质膜水平。     

不同贮藏温度下马铃薯的萌芽和内源激素含量变化

马铃薯品种晋薯２号休眠期间,ＡＢＡ含量下降,ＺＲ,ＧＡ３,ＧＡ３／ＡＢＡ增加,高温下种薯的ＡＢＡ含量下降时间,ＧＡ３和ＺＲ含量以及ＧＡ３／ＡＢＡ比值增加时间均提前,休眠期缩短,低温下种薯的ＡＢＡ含量下降,ＧＡ３含量和ＧＡ３／ＡＢＡ比值增加均缓慢,ＺＲ含量有升有降,休眠期延长。     

牛奶子的组织培养及植株再生

1　植物名称　牛奶子 (Ｅｌａｅａｇｎｕｓｕｍｂｅｌｌａｔａ) ,又称甜枣、羊奶子。2　材料类别　幼苗的下胚轴。3　培养条件　 ( 1 )ＭＳ 6 ＢＡ 1ｍｇ·Ｌ- 1 (单位下同 ) 2 ,4 Ｄ 3 ＩＡＡ 0 .5 ＣＨ 30 0 ;( 2 )ＭＳ 6 ＢＡ2 2 ,4 Ｄ 2 ＮＡＡ 0 .5 ＣＨ 30 0 ;( 3)ＭＳ 6 ＢＡ3 2 .4 Ｄ 1 ＩＢＡ 0 .5 ＣＨ 30 0 ;( 4 )ＭＳ 6 ＢＡ1 .5 ＩＡＡ 0 .5;( 5)ＭＳ 6 ＢＡ 1 .5 ＮＡＡ 0 .5;( 6)ＭＳ 6 ＢＡ 1 .5 ＩＢＡ 0 .5;( 7)ＭＳ 6 ＢＡ 0 .2 ＩＡＡ 0 .5 ＧＡ 0 .5;( 8)ＭＳ 6 ＢＡ 0 .2 ＩＡＡ 0 .1 ＧＡ…     

识别未衍生化的13—羟化GAs及其葡萄糖苷的单克隆抗体

抗ＧＡ３ 及其葡萄糖苷的ＭＡＢ１０单克隆抗体源于以ＧＡ３ 中的３ 位羟基（３－ＯＨ）为偶联位点,人血清白蛋白（ＨＳＡ）为载体合成的ＧＡ３－３－ＨＳＡ 免疫原． 该抗体对１３－羟化ＧＡｓ（１３－ＯＨＧＡｓ、ＧＡ１、ＧＡ３、ＧＡ５ 等）和ＧＡ３ 葡萄糖苷具有高亲和力． ７ 位羧基的甲酯化可显著降低ＭＡＢ１０ 对１３－ＯＨ ＧＡｓ的亲和力,而３－ＯＨ 的糖苷化却未降低其亲和力． 用该抗体建立的两种分别用于ＧＡ３ 及其葡萄糖苷测定的酶联免疫吸附法（ＥＬＩＳＡ）,其检测线性范围均为０．２～２０ ｐｍ ｏｌ． 借助这两种ＥＬＩＳＡｓ,研究了羊蹄（Ｒｕｍ ｅｘ ｊａｐｏｎｉｃｕｓ）叶片中ＧＡ３ 及其类似ＧＡｓ和葡萄糖苷的动态变化．结果表明,叶片衰老与游离态ＧＡｓ的糖苷化有关;而６－ＢＡ 延缓衰老则可能与其减缓ＧＡｓ的糖苷化有关     

发育和早萌中花生胚胚轴一子叶内BAPAase活性的差异

花生胚发育过程中,子叶和胚轴中都出现ＢＡＰＡａｓｅ活性,花生种子明发时,子叶和胚轴中的ＢＡＰＡａｓｅ活性迅速上升,子叶中无新的ＢＡＰＡａｓｅ合成,胚轴中能重新合成ＢＡＰＡａｓｅ。ＡＢＡ抑制子叶和胚轴中ＢＡＰＡａｓｅ的活性,抑制胚轴中ＢＡＰＡａｓｅ活性所需的外源ＡＢＡ浓度更高,Ａｃｔ＝Ｄ和ＣＨＭ不能逆ＡＢＡ对ＢＡＰＡａｓｅ活性的抑制作用。     

锌离子对急性分离的大鼠骶髓后连合核神经元GABAA受体介导电流的抑制作用

采用制霉菌素穿孔膜片箝技术,研究了锌离子(Zn2+)对急性分离的大鼠骶髓后连合核神经元GABAA受体介导电流的作用.结果表明:(1)在箝制电压为-40mV时,GABA可通过GABAA受体介导产生内向电流;(2)此电流可被Zn2+呈非电压依赖性可逆地阻断;(3)在Zn2+存在的情况下,GABA的浓度-效应曲线平行右移.上述结果提示,Zn2+可能通过变构调控机制对GABAA介导的反应产生抑制作用.     

PSD-95 eliminates Src-induced potentiation of NR1/NR2A-subtype NMDA receptor channels and reduces high-affinity zinc inhibition

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high-affinity, voltage-independent Zn(2+) inhibition. However, it has been reported that Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition. The post-synaptic density protein PSD-95 interacts with the C-terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD-95 eliminates the Src-induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn(2+). Our results reveal that the absence of Src-induced potentiation of PSD-95-coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn(2+). These results indicate that PSD-95 functionally modulates NR1/NR2A channels and explain why Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition.     

Promiscuity of GABAA-receptor beta 3 subunits as demonstrated by their presence in alpha 1, alpha 2 and alpha 3 subunit-containing receptor subpopulations.

Polyclonal antibodies were raised in rabbits against the GABAA-receptor beta 3 subunit peptide sequence, KQSMPREGHGRHMDR-NH2 coupled to keyhole limpet haemocyanin. These anti-beta 3 379-393 antibodies immunoprecipitated in a dose-dependent manner specific benzodiazepine agonist binding sites from Na+ deoxycholate extracts of bovine cerebral cortex. In immunoblots, anti-beta 3 379-393 antibodies recognised two species with Mr 59,900 and Mr 57,200 in all preparations tested, which included crude detergent-solubilised, benzodiazepine affinity chromatography-purified receptor, anti-alpha 1 324-341 antibody, anti-Cys alpha 2 414-424 antibody and anti-Cys alpha 3 454-467 antibody immunoaffinity-purified GABAA-receptor subpopulations. These results provide evidence for the ubiquity and promiscuity of the GABAA-receptor beta 3 subunit.     

Hyperekplexia phenotype of glycine receptor alpha1 subunit mutant mice identifies Zn(2+) as an essential endogenous modulator of glycinergic neurotransmission

Zn(2+) is thought to modulate neurotransmission by affecting currents mediated by ligand-gated ion channels and transmitter reuptake by Na(+)-dependent transporter systems. Here, we examined the in vivo relevance of Zn(2+) neuromodulation by producing knockin mice carrying the mutation D80A in the glycine receptor (GlyR) alpha1 subunit gene (Glra1). This substitution selectively eliminates the potentiating effect of Zn(2+) on GlyR currents. Mice homozygous for Glra1(D80A) develop a severe neuromotor phenotype postnatally that resembles forms of human hyperekplexia (startle disease) caused by mutations in GlyR genes. In spinal neurons and brainstem slices from Glra1(D80A) mice, GlyR expression, synaptic localization, and basal glycinergic transmission were normal; however, potentiation of spontaneous glycinergic currents by Zn(2+) was significantly impaired. Thus, the hyperekplexia phenotype of Glra1(D80A) mice is due to the loss of Zn(2+) potentiation of alpha1 subunit containing GlyRs, indicating that synaptic Zn(2+) is essential for proper in vivo functioning of glycinergic neurotransmission.     

Cu2+ suppresses GABA(A) receptor-mediated responses in rat sacral dorsal commissural neurons

The effect of copper ions (Cu(2+)) on gamma-aminobutyric acid (GABA)-induced responses in acutely dissociated neurons from the rat sacral dorsal commissural nucleus (SDCN) was investigated using a nystatin-perforated patch recording configuration under voltage clamp conditions. The application of Cu(2+) to SDCN neurons reversibly suppressed the GABA (10 microM)-activated Cl(-) current (I(GABA)) in a concentration-dependent manner (1-1000 microM; IC(50) = 24.5 microM). In the presence of Cu(2+) (30 microM), the concentration-response curve of GABA was shifted rightward without reducing I(GABA) recorded under the maximally effective concentration of GABA, thus indicating a dependence of Cu(2+) action on GABA concentration. Inhibition of GABA (10 microM) responses by 30 microM Cu(2+) was essentially voltage independent and was not accompanied by a shift in the reversal potential of the currents. Cu(2+) antagonized the suppressive effect of Zn(2+) in a concentration-dependent manner, suggesting competition between Cu(2+) and Zn(2+) for similar binding sites. These data demonstrate that Cu(2+) is a potent inhibitor of GABA(A) receptor-mediated responses, implying a possible modulatory effect of Cu(2+) on GABAergic synaptic transmission in the mammalian SDCN.     

Zinc has opposite effects on NMDA and non-NMDA receptors expressed in Xenopus oocytes

Pharmacological characterization of Zn2+ effects on glutamate ionotropic receptors was investigated in Xenopus oocytes injected with rat brain mRNA, using a double microelectrode, voltage-clamp technique. At low concentration, Zn2+ inhibited NMDA currents (IC50 = 42.9 +/- 1.3 microM) and potentiated both AMPA (EC50 = 30.0 +/- 1.2 microM) and desensitized kainate responses (EC50 = 13.0 +/- 0.1 microM). At higher concentrations, Zn2+ inhibited non-NMDA responses with IC50 values of 1.3 +/- 0.1 mM and 1.2 +/- 0.3 mM for AMPA and kainate, respectively. The potentiation of AMPA or quisqualate currents by Zn2+ was more than 2-fold, whereas that of the kainate current was only close to 30%. This potentiating effect of Zn2+ on AMPA current modified neither the affinity of the agonist for its site nor the current-voltage relationship. In addition, 500 microM Zn2+ differentially affected NMDA and non-NMDA components of the glutamate-induced response. The possible physiological relevance of Zn2+ modulation is discussed.     

Neuron–astrocyte interactions in the medial nucleus of the trapezoid body

The calyx of Held (CoH) synapse serves as a model system to analyze basic mechanisms of synaptic transmission. Astrocyte processes are part of the synaptic structure and contact both pre- and postsynaptic membranes. In the medial nucleus of the trapezoid body (MNTB), midline stimulation evoked a current response that was not mediated by glutamate receptors or glutamate uptake, despite the fact that astrocytes express functional receptors and transporters. However, astrocytes showed spontaneous Ca²⁺ responses and neuronal slow inward currents (nSICs) were recorded in the postsynaptic principal neurons (PPNs) of the MNTB. These currents were correlated with astrocytic Ca²⁺ activity because dialysis of astrocytes with BAPTA abolished nSICs. Moreover, the frequency of these currents was increased when Ca²⁺ responses in astrocytes were elicited. NMDA antagonists selectively blocked nSICs while D-serine degradation significantly reduced NMDA-mediated currents. In contrast to previous studies in the hippocampus, these NMDA-mediated currents were rarely synchronized.     

Increased expression of NR2A subunit does not alter NMDA-evoked responses in cultured fetal trisomy 16 mouse hippocampal neurons

The trisomy 16 (Ts16) mouse is an animal model for human trisomy 21 (Down's syndrome). The gene encoding the NR2A subunit of the NMDA receptor has been localized to mouse chromosome 16. In the present study, western blot analysis revealed a 2.5-fold increase of NR2A expression in cultured Ts16 embryonic hippocampal neurons. However, this increase did not affect the properties of NMDA-evoked currents in response to various modulators. The sensitivity of NMDA receptors to transient applications of NMDA, spermine, and Zn(2+) was investigated in murine Ts16 and control diploid cultured embryonic hippocampal neurons. Peak and steady-state currents evoked by NMDA were potentiated by spermine at concentrations < 1 mM, and inhibited by Zn(2+) in a dose-dependent and voltage-independent manner. No marked difference was observed between Ts16 and control diploid neurons for any of these modulators with regard to IC(50) and EC(50) values or voltage dependency. Additionally, inhibition by the NR2B selective inhibitor, ifenprodil, was similar. These results demonstrate that NMDA-evoked currents are not altered in cultured embryonic Ts16 neurons and suggest that Ts16 neurons contain similar functional properties of NMDA receptors as diploid control neurons despite an increased level of NR2A expression.     

Intracellular blockade of GABAA receptors in the rat hippocampal neurons

The intracellular blockade of GABA_A-receptor-mediated currents is a useful approach to suppress the GABAergic conductance in a single cell and to isolate the glutamatergic component of network-driven activities. Previously an approach has been described allowing intracellular blockade of GABA_A receptors by means of intracellular dialysis of a neuron with the pipette-filling solution, in which fluoride ions that hardly pass through the GABA_A receptor channels substitute for Cl^? and in which Mg²⁺ and ATP are omitted to induce rundown of the GABA_A receptors during whole-cell patch-clamp recordings. However, the kinetics of suppression of GABAergic conductance and the effect on the currents mediated by glutamate receptors remain unknown. Here, using whole-cell recordings with fluoride-based, Mg²⁺- and ATP-free solution on CA3 hippocampal neurons of neonatal rats, we show that after 1 h of such dialysis, both spontaneous and evoked GABA_A-receptor-mediated synaptic currents and responses induced by the GABA_A receptor agonist isoguvacine were completely suppressed. Inward GABAergic postsynaptic currents were suppressed prior to outward currents. Synaptic responses mediated by AMPA receptors were not affected by the dialysis, whereas the NMDA-receptor-mediated postsynaptic currents were reduced by approximately 20%. Dialysis with fluoride-based Mg²⁺, ATP-free solution either fully blocked giant depolarizing potentials (GDPs) in CA3 pyramidal cells (n = 2) or reduced the charge crossing the membrane during GDPs and shifted the GDP reversal potential to more positive values (n = 5). The dialysis-resistant component of GDPs was mediated by glutamate receptors, since: (i) it reversed around 0 mV; (ii) it demonstrated a negative slope conductance at negative membrane voltages, which is characteristic of NMDA receptor-mediated responses; (iii) kinetics of the individual events composing the dialysis-resistant component of GDPs at negative voltages were very similar to those of AMPA receptor-mediated synaptic currents. Thus, this procedure can be useful to isolate the glutamate receptor-mediated component of neuronal network-driven activities.     

Extracellular Zn2+ activates epithelial Na+ channels by eliminating Na+ self-inhibition

Inhibition of epithelial Na(+) channel (ENaC) activity by high concentrations of extracellular Na(+) is referred to as Na(+) self-inhibition. We investigated the effects of external Zn(2+) on whole cell Na(+) currents and on the Na(+) self-inhibition response in Xenopus oocytes expressing mouse alphabetagamma ENaC. Na(+) self-inhibition was examined by analyzing inward current decay from a peak current to a steady-state current following a fast switching of a low Na(+) (1 mm) bath solution to a high Na(+) (110 mm) solution. Our results indicate that external Zn(2+) rapidly and reversibly activates ENaC in a dose-dependent manner with an estimated EC(50) of 2 microm. External Zn(2+) in the high Na(+) bath also prevents or reverses Na(+) self-inhibition with similar affinity. Zn(2+) activation is dependent on extracellular Na(+) concentration and is absent in ENaCs containing gammaH239 mutations that eliminate Na(+) self-inhibition and in alphaS580Cbetagamma following covalent modification by a sulfhydryl-reactive reagent that locks the channels in a fully open state. In contrast, external Ni(2+) inhibition of ENaC currents appears to be additive to Na(+) self-inhibition when Ni(2+) is present in the high Na(+) bath. Pretreatment of oocytes with Ni(2+) in a low Na(+) bath also prevents the current decay following a switch to a high Na(+) bath but rendered the currents below the control steady-state level measured in the absence of Ni(2+) pretreatment. Our results suggest that external Zn(2+) activates ENaC by relieving the channel from Na(+) self-inhibition, and that external Ni(2+) mimics or masks Na(+) self-inhibition.