Proline and functioning of the antioxidant system in Thellungiella salsuginea plants and cultured cells subjected to oxidative stress

The effects of proline on the functioning of antioxidant enzymes — superoxide dismutase (SOD) and ascorbate peroxidase (APO) — in Thellungiella salsuginea plants and cultured cells under normal conditions of culturing and under the influence of hydrogen peroxide (500 μM) were studied. Proline addition (0.2, 2, or 5 mM) to the medium for suspension culture or nutrient medium for plant growing resulted in the increase in the content of intracellular proline in both cultured cells and intact plant leaves and also in the activation of proline dehydrogenase, i.e., the enzyme degrading proline. Under normal conditions, treatment with proline exerted prooxidant action on both cellular and organismal levels. This was manifested in MDA accumulation and changes in APO and SOD activities. The amino acid alanine, used as a control, did not exert similar strong effect as proline. Application of 500 μM H₂O₂ on plant leaves resulted in the development of oxidative stress, whereas hydrogen peroxide addition into the culture medium — to the death of 50% of suspension cells. When plants and cultured cells were treated with 2 mM proline and than with H₂O₂, the number of dead cells in suspension was 35%, the content of MDA was decreased, APO was activated, and SOD activity was decreased in both cell culture and plant leaves. Thus, an increase in the intracellular proline concentration changed the redox balance and induced functioning of APO and SOD at both normal conditions of plant growing and cell culturing and under stress.     

An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.     

FoxO1在胰岛β细胞代谢灵活性受损及失代偿进程中的作用

葡萄糖及脂肪酸是胰岛β细胞的关键代谢底物,葡萄糖刺激胰岛β细胞分泌胰岛素是维持机体血糖稳态平衡的关键。胰岛素抵抗发生时,β细胞对能量代谢底物的选择失调,加速胰岛β细胞由代偿到胰岛β细胞失代偿的进程,是肥胖胰岛素抵抗最终发展为2型糖尿病的始动因素。核转录因子FoxO1属于Fox家族成员,在胰腺内广泛表达,在β细胞的代谢,发育,增殖过程中发挥着重要的调节作用。鉴于FoxO1在维持胰岛β细胞功能中的关键作用,现着重对FoxO1在胰岛β细胞代谢灵活性受损及失代偿过程发生中的作用调节进行阐述。为其作为调控胰岛β细胞功能的关键靶点提供参考。     

Effect of spermine treatment on the functioning of Thellungiella salsuginea antioxidant system

The effect of spermine (Spm) treatment on the content of polyamines (PAs) and activities of antioxidant enzymes in the roots and leaves of Thellungiella salsuginea (Pall.) O.E. Schulz plants grown under optimal conditions were studied. The genes encoding three forms of ascorbate peroxidase (APX; APX1, APX2, and APX4) and genes of key enzymes of proline metabolism (Pro, P5CS1, 1P5CD) were identified, and their expression intensity was measured. Six-day-old plants were treated with Spm (1 and 2 mM) and with the inhibitor of polyamine oxidase (PAO) activity, N,N-(2-hydroxyethyl)hydrazine (HEH, 1 and 2 mM) separately or in combination by adding these compounds to nutrient medium. Roots and leaves responded differently to Spm treatment. In the leaves, the content of PAs reduced due to a decreased in the spermidine (Spd) content, whereas in the roots the total pool of PAs increased due to putrescine (Put) and Spd accumulation. Treatment with Spm activated PAO in the roots but not in the leaves; HEH removed this increase, but the intercellular Spm concentration was not substantially changed. It was suggested that treatment with Spm suppressed the biosynthesis of intracellular Spm and, on the other hand, stimulated the reverse conversion of Spm into Spd and further into Put due to the activation of one of the PAO isoforms. Plant treatment with Spm was not accompanied by a noticeable activation enzymes degrading hydrogen peroxide in the roots: APX, (except of peroxidase II), and catalase. However, the activity of Cu/Zn-SOD doubled and the activity of Mn-SOD reduced. In the leaves, slight activation of peroxidases I and III, the inhibition of Cu/Zn- and Mn-SOD, differential changes in the time-coursed of gene expression of three APX isoforms, and activated gene expression of key enzymes of Pro metabolism were observed. At the same time, the level of MDA did not increase either in the leaves or in the roots. This indicates that treatment of Th. salsuginea plants with Spm under optimal growing conditions did not enhance ROS generation and did not manifest prooxidant properties.     

Unique interaction of scorpion toxins with the hERG channel

ERG potassium channels specify one component of the delayed rectifier in the heart and are likely to play an important functional role in other excitable cells. Compared to other K+ channels, the human ERG (hERG) channel possesses an unusually long S5-P linker that presumably forms an alpha-helix important for channel function. hERG-specific toxins bind to the outer mouth of the hERG channel. Channel residues in the middle of the S5-P linker and at the pore entrance are critical for toxin binding. One of these scorpion toxins is BeKm-1. Residues critical for BeKm-1 binding to the hERG channel are located in the alpha-helix and the following loop, whereas the "traditional" interaction surface of other short scorpion toxins is formed by residues on the beta-sheet. This unique localization of BeKm-1's interaction surface and its specific action on the hERG channel suggest a unique outer mouth structure of the hERG channel. We used the mutant cycle analysis approach to define contacts in the toxin-channel complex. This information provides critical constraints and is important for molecular modeling of the hERG pore structure.     

Modulation of P2X3 receptors by spider toxins

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.     

促性腺激素释放激素Buserelin 稳定性的研究

目的:研究Buserelin原料药的性质在温度、湿度、光线等条件的影响下随时间变化的规律,为该原料药的生产、包装、储存、运输及有效期的制定提供依据。方法:根据中国药典2005版二部附录XIX C药物稳定性试验指导原则及化学药物稳定性研究技术指导原则进行强光照射、高温(60℃、40℃)、高湿(RH92.5%±5%、RH75%±5%)影响因素试验,加速试验(40℃±2℃、RH75%±5%;25℃±2℃、RH60%±10%);按Buserelin原料药标准规定的质量指标及相关的检验方法对产品在试验条件下的主要质量指标进行检测。结果:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,故应密封、于干燥、阴凉处保存。在加速试验中,Buserelin原料药的各项质量指标发生了小的变化,但均在质量标准规定的范围内。结论:强光照射、高温、高湿等影响因素对Buserelin的稳定性有明显影响,应在阴凉干燥处避光密封保存和运输。加速试验结果证明:在此条件下,它的各项质量指标变化均在质量标准范围内,符合Buserelin原料药质量标准规定的要求;故将其保质期暂定为两年。     

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE*

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.     

The Role of the C-terminal Intracellular Domain in Acid-Sensing Ion Channel 3 Functioning

Journal of Evolutionary Biochemistry and Physiology - An increase in the concentration of protons in the synaptic cleft during neurotransmitters release is considered as one of the possible ways...