Cnidarian Toxins Acting on Voltage-Gated Ion Channels

Voltage-gated ion channels generate electrical activity in excitable cells. As such, they are essential components of neuromuscular and neuronal systems, and are targeted by toxins from a wide variety of phyla, including the cnidarians. Here, we review cnidarian toxins known to target voltage-gated ion channels, the specific channel types targeted, and, where known, the sites of action of cnidarian toxins on different channels.     

Antimicrobial Activity of the Marine Alkaloids,Clathrodin and Oroidin,and Their Synthetic Analogues

Marine organisms produce secondary metabolites that may be valuable for the development of novel drug leads as such and can also provide structural scaffolds for the design and synthesis of novel bioactive compounds. The marine alkaloids, clathrodin and oroidin, which were originally isolated from sponges of the genus, Agelas, were prepared and evaluated for their antimicrobial activity against three bacterial strains (Enterococcus faecalis, Staphylococcus aureus and Escherichia coli) and one fungal strain (Candida albicans), and oroidin was found to possess promising Gram-positive antibacterial activity. Using oroidin as a scaffold, 34 new analogues were designed, prepared and screened for their antimicrobial properties. Of these compounds, 12 exhibited >80% inhibition of the growth of at least one microorganism at a concentration of 50 µM. The most active derivative was found to be 4-phenyl-2-aminoimidazole 6h, which exhibited MIC₉₀ (minimum inhibitory concentration) values of 12.5 µM against the Gram-positive bacteria and 50 µM against E. coli. The selectivity index between S. aureus and mammalian cells, which is important to consider in the evaluation of a compound’s potential as an antimicrobial lead, was found to be 2.9 for compound 6h.     

The Structural Basis and Functional Consequences of Interactions Between Tetrodotoxin and Voltage-Gated Sodium Channels

Tetrodotoxin (TTX) is a highly specific blocker of voltage-gated sodium channels. The dissociation constant of block varies with different channel isoforms. Until recently, channel resistance was thought to be primarily imparted by amino acid substitutions at a single position in domain I. Recent work reveals a novel site for tetrodotoxin resistance in the P-region of domain IV.     

Pathophysiological Responses to Conotoxin Modulation of Voltage-Gated Ion Currents

Voltage-gated ion channels are plasma membrane proteins that generate electrical signals following a change in the membrane voltage. Since they are involved in several physiological processes, their dysfunction may be responsible for a series of diseases and pain states particularly related to neuronal and muscular systems. It is well established for decades that bioactive peptides isolated from venoms of marine mollusks belonging to the Conus genus, collectively known as conotoxins, can target different types and isoforms of these channels exerting therapeutic effects and pain relief. For this reason, conotoxins are widely used for either therapeutic purposes or studies on ion channel mechanisms of action disclosure. In addition their positive property, however, conotoxins may generate pathological states through similar ion channel modulation. In this narrative review, we provide pieces of evidence on the pathophysiological impacts that different members of conotoxin families exert by targeting the three most important voltage-gated channels, such as sodium, calcium, and potassium, involved in cellular processes.     

Brevetoxin and Conotoxin Interactions with Single-Domain Voltage-Gated Sodium Channels from a Diatom and Coccolithophore

The recently characterized single-domain voltage-gated ion channels from eukaryotic protists (EukCats) provide an array of novel channel proteins upon which to test the pharmacology of both clinically and environmentally relevant marine toxins. Here, we examined the effects of the hydrophilic µ-CTx PIIIA and the lipophilic brevetoxins PbTx-2 and PbTx-3 on heterologously expressed EukCat ion channels from a marine diatom and coccolithophore. Surprisingly, none of the toxins inhibited the peak currents evoked by the two EukCats tested. The lack of homology in the outer pore elements of the channel may disrupt the binding of µ-CTx PIIIA, while major structural differences between mammalian sodium channels and the C-terminal domains of the EukCats may diminish interactions with the brevetoxins. However, all three toxins produced significant negative shifts in the voltage dependence of activation and steady state inactivation, suggesting alternative and state-dependent binding conformations that potentially lead to changes in the excitability of the phytoplankton themselves.     

A Tale of Toxin Promiscuity: The Versatile Pharmacological Effects of Hcr 1b-2 Sea Anemone Peptide on Voltage-Gated Ion Channels

Sea anemones are a rich source of biologically active compounds. Among approximately 1100 species described so far, Heteractis crispa species, also known as sebae anemone, is native to the Indo-Pacific area. As part of its venom components, the Hcr 1b-2 peptide was first described as an ASIC1a and ASIC3 inhibitor. Using Xenopus laevis oocytes and the two-electrode voltage-clamp technique, in the present work we describe the remarkable lack of selectivity of this toxin. Besides the acid-sensing ion channels previously described, we identified 26 new targets of this peptide, comprising 14 voltage-gated potassium channels, 9 voltage-gated sodium channels, and 3 voltage-gated calcium channels. Among them, Hcr 1b-2 is the first sea anemone peptide described to interact with isoforms from the Kv7 family and T-type Cav channels. Taken together, the diversity of Hcr 1b-2 targets turns this toxin into an interesting tool to study different types of ion channels, as well as a prototype to develop new and more specific ion channel ligands.     

Selective Blocking Effects of 4,9-Anhydrotetrodotoxin,Purified from a Crude Mixture of Tetrodotoxin Analogues,on NaV1.6 Channels and Its Chemical Aspects

Tetrodotoxin (TTX) is a potent neurotoxin found in a number of marine creatures including the pufferfish, where it is synthesized by bacteria and accumulated through the food chain. It is a potent and selective blocker of some types of voltage-gated Na⁺ channel (Na_V channel). 4,9-Anhydrotetrodotoxin (4,9-anhydroTTX) was purified from a crude mixture of TTX analogues (such as TTX, 4-epiTTX, 6-epiTTX, 11-oxoTTX and 11-deoxyTTX) by the use of liquid chromatography-fluorescence detection (LC-FLD) techniques. Recently, it has been reported that 4,9-anhydroTTX selectively blocks the activity of Na_V1.6 channels with a blocking efficacy 40–160 times higher than that for other TTX-sensitive Na_V1.x channel isoforms. However, little attention has been paid to the molecular properties of the α-subunit in Na_V1.6 channels and the characteristics of binding of 4,9-anhydroTTX. From a functional point of view, it is important to determine the relative expression of Na_V1.6 channels in a wide variety of tissues. The aim of this review is to discuss briefly current knowledge about the pharmacology of 4,9-anhydroTTX, and provide an analysis of the molecular structure of native Na_V1.6 channels. In addition, chemical aspects of 4,9-anhydroTTX are briefly covered.     

花生生产潜力与高产途径

据估算，中熟大花生和早熟小花生最高单产可达到17.3t/hm^2和11.9t/hm^2，实际生产中，中熟大花生和早熟小花生最高单产已达到11.2t/hm^2和9.5t/hm^2。目前全球花生平均单产不到1.0t/hm^2，我国平均约3.7t/hm^2，花生生产仍有较大的增产余地。延长作物生长期、提高植株光合效率和增加光合产物向生殖体的分配比例，是花生品种改良的有效途径。适期播种、创造良好的土壤环境、选择适宜的种植方式和密度、合理调控叶面积系数、在增加总生物产量的基础上适当提高经济系数等，是目前花生高产栽培有效的措施途径。     

Computational Design of High-Affinity Blockers for Sodium Channel NaV1.2 from μ-Conotoxin KIIIA

The voltage-gated sodium channel subtype 1.2 (Na_V1.2) is instrumental in the initiation of action potentials in the nervous system, making it a natural drug target for neurological diseases. Therefore, there is much pharmacological interest in finding blockers of Na_V1.2 and improving their affinity and selectivity properties. An extensive family of peptide toxins from cone snails (conotoxins) block Na_V channels, thus they provide natural templates for the design of drugs targeting Na_V channels. Unfortunately, progress was hampered due to the absence of any Na_V structures. The recent determination of cryo-EM structures for Na_V channels has finally broken this impasse. Here, we use the Na_V1.2 structure in complex with μ-conotoxin KIIIA (KIIIA) in computational studies with the aim of improving KIIIA’s affinity and blocking capacity for Na_V1.2. Only three KIIIA amino acid residues are available for mutation (S5, S6, and S13). After performing molecular modeling and simulations on Na_V1.2–KIIIA complex, we have identified the S5R, S6D, and S13K mutations as the most promising for additional contacts. We estimate these contacts to boost the affinity of KIIIA for Na_V1.2 from nanomole to picomole domain. Moreover, the KIIIA[S5R, S6D, S13K] analogue makes contacts with all four channel domains, thus enabling the complete blocking of the channel (KIIIA partially blocks as it has contacts with three domains). The proposed KIIIA analogue, once confirmed experimentally, may lead to novel anti-epileptic drugs.     

Stimulation of Neurite Outgrowth in Cerebrocortical Neurons by Sodium Channel Activator Brevetoxin-2 Requires Both N-Methyl-D-aspartate Receptor 2B (GluN2B) and p21 Protein (Cdc42/Rac)-Activated Kinase 1 (PAK1)

N-methyl-D-aspartate (NMDA) receptors play a critical role in activity-dependent dendritic arborization, spinogenesis, and synapse formation by stimulating calcium-dependent signaling pathways. Previously, we have shown that brevetoxin 2 (PbTx-2), a voltage-gated sodium channel (VGSC) activator, produces a concentration-dependent increase in intracellular sodium [Na⁺]_I and increases NMDA receptor (NMDAR) open probabilities and NMDA-induced calcium (Ca²⁺) influxes. The objective of this study is to elucidate the downstream signaling mechanisms by which the sodium channel activator PbTx-2 influences neuronal morphology in murine cerebrocortical neurons. PbTx-2 and NMDA triggered distinct Ca²⁺-influx pathways, both of which involved the NMDA receptor 2B (GluN2B). PbTx-2-induced neurite outgrowth in day in vitro 1 (DIV-1) neurons required the small Rho GTPase Rac1 and was inhibited by both a PAK1 inhibitor and a PAK1 siRNA. PbTx-2 exposure increased the phosphorylation of PAK1 at Thr-212. At DIV-5, PbTx-2 induced increases in dendritic protrusion density, p-cofilin levels, and F-actin throughout the dendritic arbor and soma. Moreover, PbTx-2 increased miniature excitatory post-synaptic currents (mEPSCs). These data suggest that the stimulation of neurite outgrowth, spinogenesis, and synapse formation produced by PbTx-2 are mediated by GluN2B and PAK1 signaling.     

Obtaining Spheroplasts of Armored Dinoflagellates and First Single-Channel Recordings of Their Ion Channels Using Patch-Clamping

Ion channels are tightly involved in various aspects of cell physiology, including cell signaling, proliferation, motility, endo- and exo-cytosis. They may be involved in toxin production and release by marine dinoflagellates, as well as harmful algal bloom proliferation. So far, the patch-clamp technique, which is the most powerful method to study the activity of ion channels, has not been applied to dinoflagellate cells, due to their complex cellulose-containing cell coverings. In this paper, we describe a new approach to overcome this problem, based on the preparation of spheroplasts from armored bloom-forming dinoflagellate Prorocentrum minimum. We treated the cells of P. minimum with a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), and found out that it could also induce ecdysis and arrest cell shape maintenance in these microalgae. Treatment with 100–250 µM DCB led to an acceptable 10% yield of P. minimum spheroplasts and was independent of the incubation time in the range of 1–5 days. We show that such spheroplasts are suitable for patch-clamping in the cell-attached mode and can form 1–10 GOhm patch contact with a glass micropipette, allowing recording of ion channel activity. The first single-channel recordings of dinoflagellate ion channels are presented.     

茶叶叶绿素锌钠盐的制备及其稳定性研究

利用新鲜茶叶制备叶绿素锌钠盐,研究其最佳锌代工艺条件,并对其稳定性进行研究。结果表明,锌代反应最佳工艺条件为:茶叶粉(g):ZnSO4.7H2O(g)=5:7.1;温度为60℃;时间为2h,收率为2.1%。叶绿素锌钠盐水溶性、耐热性、耐氧化还原性、pH为2.8~12.78时的耐光性均较好,常见的食品添加剂和金属离子对叶绿素锌钠盐的稳定性无影响。     

石灰、硅酸钠和羟基磷灰石对烟草吸收镉的影响

为了探讨施用不同钝化剂对烟草吸收富集镉的效果,采用盆栽试验研究了石灰、硅酸钠和羟基磷灰石对Pb-Cd复合污染土壤上的烟草镉吸收富集的影响。结果表明,土壤pH值均随3种钝化剂用量的增大而升高,而土壤有效镉(DTPA-提取)含量则随之降低。土壤有效镉的最大降幅分别为:32 g/kg羟基磷灰石处理(52.4%)16 g/kg石灰处理(37.0%)12.5 g/kg硅酸钠处理(14.1%)。3种钝化剂都降低了烟株各部位镉的含量,其对烟叶镉含量的平均最大降幅分别为87%(16 g/kg石灰处理),74.7%(12.5 g/kg硅酸钠处理)和82.2%(32 g/kg羟基磷灰石处理)。石灰和羟基磷灰石对镉在根-茎之间的转移无显著影响,而硅酸钠则显著降低了镉在根-茎之间的转移。综上,镉污染的植烟土壤可以施用这些钝化剂以降低烟叶中镉的含量。     

脱氢醋酸钠的用量对椰花汁采后品质变化的影响

研究了不同浓度的脱氢醋酸钠(sodium dehydroacetate,SDA)对椰子花序汁液(coconut inflorescence sap,CIS)采后品质变化的影响.结果表明:CIS采集后,空白组中的折光糖度、还原糖、乙醇、总酸和挥发酸的含量均有显著变化.而添加SDA则可显著抑制这些指标含量的变化.在自然发酵第7 d.空白组和SDA浓度分别为0.02%,0.03%和0.04%的试验组中折光糖度分别为6.00%,11.60%,12.2%和12.4%,总酸度分别为3.91,1.51,1.05和0.95g/100 mL;在同一天内SDA浓度为0.02%的试验组乙醇含量显著高于另外2个试验组;挥发酸除第7 d中SDA浓度为0.02%的试验组比另2个试验组的显著高.其他3个试验组所测数据差异均不显著;3个试验组中的还原糖含量变化规律不明显,表明0.02%的SDA对CIS已有显著的保鲜效果.     

机插密度对盐碱地超级稻产量及钠钾离子吸收的影响

为筛选出江苏滨海盐碱地超级稻品种‘湘两优900’的适宜机插密度,研究了不同机插密度（高密度,T₁：25.0×10⁴穴/hm²;中密度,T₂：19.9×10⁴穴/hm²;低密度,T₃：16.6×10⁴穴/hm²）对水稻各指标的影响,并动态监测了土壤及田间水盐分的变化。结果表明：水稻种植能够显著降低土壤可溶性总盐含量,降幅可达28.68%;随机插密度变小,水稻单穴的分蘖数、干物质重能够显著提高,而群体的分蘖数和干物质重会相应的显著降低;水稻SPAD值在生殖生长期时,随密度下降呈增加的趋势,而叶面积指数在所有生育期均呈降低的趋势;机插密度下降能够显著增加单穴钠钾离子积累量和显著降低群体的钠钾离子积累。相关分析表明,不同机插密度水稻实际产量与单株钠离子积累量呈显著负相关,与群体有效穗数、群体干物质积累量呈显著正相关,与每穗总粒数、群体钾离子积累量呈极显著正相关。本试验条件下,江苏滨海盐碱地‘湘两优900’的适宜机插密度为25.0×10⁴穴/hm²。     

复方刺五加注射液对培养大鼠心肌细胞动作电位的影响

培养Wistar大鼠乳鼠心室肌细胞,向培养基中加入复方刺五加注射液200μg/mL,可使心肌细胞动作电位的波幅、波宽、阈电位、最大舒张电位、最大除极速度及复极(10%、50%、90%)水平的动作电位波宽一致减小,其作用与钙通道阻滞剂尼莫地平0.4μg/mL相似;Ca2 80μg/mL能使上述作用反转,提示复方刺五加注射液具有钙通道阻滞作用。     

采前喷施复硝酚钠对杧果果实品质和采后发病率的影响

探明采前喷施复硝酚钠对‘贵妃’杧果果实品质和采后发病率的影响,以期为复硝酚钠在杧果生产中的使用提供科学合理的理论依据。选用‘贵妃’杧果为试材,于盛花期后第15天开始喷施质量浓度为500 mg/L的复硝酚钠,每隔9~12 d喷施1次,共喷施6次。测定果实发育期间的纵横径、成熟期的单果质量、成熟过程中的果皮颜色参数（L、a、b值）、果肉硬度、可溶性固形物含量、总酸含量和果实病害等指标。试验结果表明,采前喷施复硝酚钠能够促进果实膨大,显著增加‘贵妃’杧果成熟期的单果质量,促进果实果皮转红和成熟期果皮转黄,延缓果肉硬度下降和可溶性固形物含量的上升,总酸含量基本保持不变,极大地降低了采后病害发病率和发病指数。     

Soil Physico-Chemical Properties and Microflora as Influenced by Bispyribac Sodium 10% SC in Transplanted Kharif Rice

The effects of bispyribac sodium 10% SC and butachlor 50% SC on soil physico-chemical properties and microflora in transplanted kharif rice were investigated over two seasons (2010 and 2011).Effects of the herbicide on bulk density,water holding capacity,moisture content,soil pH,organic matter content,electrical conductivity,as well as total nitrogen,available phosphorus and available potassium contents were analyzed along with microflora population (total bacteria,actinomycetes and fungi).No significant changes in soil physico-chemical properties were observed.Herbicide treatments resulted in decreases in microbial counts initially.With the degradation of applied herbicides within a considerable time,the microflora populations even exceeded the initial count at 60 d after application of the herbicide.     

硝酸钠对甜菜产质量和钾钠吸收的影响

硝酸钠在我国北方的黑土、白浆土等贫钾偏酸性土壤上施用,对甜菜有显著增产效果,与尿素比增产11.2％,在强石灰性土壤上施用也有一定的增产效果。故在钾素缺乏的土壤上施钠肥,可起到以钠代钾的作用。但施钠肥可使根中钠的含量增高,影响出糖率,也会增加土壤中钠的残留,因此应在低钠潮湿的土壤上合理使用。     

Enhancing Stability and Mucoadhesive Properties of Chitosan Nanoparticles by Surface Modification with Sodium Alginate and Polyethylene Glycol for Potential Oral Mucosa Vaccine Delivery

Background: The present study aimed to fabricate surface-modified chitosan nanoparticles with two mucoadhesive polymers (sodium alginate and polyethylene glycol) to optimize their protein encapsulation efficiency, improve their mucoadhesion properties, and increase their stability in biological fluids. Method: Ionotropic gelation was employed to formulate chitosan nanoparticles and surface modification was performed at five different concentrations (0.05, 0.1, 0.2, 0.3, 0.4% w/v) of sodium alginate (ALG) and polyethylene glycol (PEG), with ovalbumin (OVA) used as a model protein antigen. The functional characteristics were examined by dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and scanning electron microscopy (SEM)/scanning transmission electron microscopy (STEM). Stability was examined in the presence of simulated gastric and intestinal fluids, while mucoadhesive properties were evaluated by in vitro mucin binding and ex vivo adhesion on pig oral mucosa tissue. The impact of the formulation and dissolution process on the OVA structure was investigated by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and circular dichroism (CD). Results: The nanoparticles showed a uniform spherical morphology with a maximum protein encapsulation efficiency of 81%, size after OVA loading of between 200 and 400 nm and zeta potential from 10 to 29 mV. An in vitro drug release study suggested successful nanoparticle surface modification by ALG and PEG, showing gastric fluid stability (4 h) and a 96 h sustained OVA release in intestinal fluid, with the nanoparticles maintaining their conformational stability (SDS-PAGE and CD analyses) after release in the intestinal fluid. An in vitro mucin binding study indicated a significant increase in mucin binding from 41 to 63% in ALG-modified nanoparticles and a 27–49% increase in PEG-modified nanoparticles. The ex vivo mucoadhesion showed that the powdered particles adhered to the pig oral mucosa. Conclusion: The ALG and PEG surface modification of chitosan nanoparticles improved the particle stability in both simulated gastric and intestinal fluids and improved the mucoadhesive properties, therefore constituting a potential nanocarrier platform for mucosal protein vaccine delivery.