Molecular characterization on the genome structure of hemolysin toxin isoforms isolated from sea anemone Actineria villosa and Phyllodiscus semoni

We recently identified the existence of new isoforms of Avt-I (from sea anemone Actineria villosa) and Pstx20 (from sea anemone Phyllodiscus semoni) hemolytic toxins, and named them Avt-II and Pst-I. Avt-II and Pst-I differ in length by 14 and 7 bp, respectively, as compared to their corresponding isoform genes. Both newly found isoform genes have the coding regions with the identical length of 1033 bp. The restriction fragment length polymorphism analysis with endonuclease HphI was able to clearly distinguish between the two Avt isoforms, but not Pstx isoforms, and based on the densitometric analysis of DNA bands, it indicated that relative expression levels of Avt-I and Avt-II genes were 18.3% and 81.7%, respectively. PCR amplification of the two Avt isoform genes using the genomic DNA as template indicated the existence of two introns within each toxin isoform gene. The first intron with the identical 242 bp in length for both Avt isoform was found within the 5′-untranslated region, and the second intron with lengths of 654 bp and 661 bp in Avt-I and Avt-II isoforms, respectively, was found within the signal sequence coding region. This is for the first time to identify the existence of introns within hemolysin genes of sea anemone. Having several unique characteristics that have identified only for a new member of actinoporin family of A. villosa and P. semoni, e.g., strong toxicity and genes with introns, it is plausible to speculate that these toxins have a unique genetic evolutionary linage differed from that for other sea anemone hemolytic toxins.     

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A₂ activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.     

Amino acid sequence of RTX-A's isoform actinoporin from the sea anemone, Radianthus macrodactylus.

The amino acid sequence of actinoporin RTX-A (175 aa) from the sea anemone Radianthus macrodactylus was determined by sequencing of clones obtained via amplification of cDNA. It was established that RTX-A possessed high homology with HmgIII from Heteractis magnifica (87%) and StI, StII from Stichodactyla helianthus (84 and 87%, respectively). The analysis of structural and functional relationships within RTX-A was carried out. The some disagreement concerning to significant role of several amino acid residues for actinoporins exhibition of hemolytic activity was found.     

The crude extract from the sea anemone, Bunodosoma caissarum elicits convulsions in mice: possible involvement of the glutamatergic system

The crude extract from the sea anemone, Bunodosoma caissarum caused dose-dependent convulsions by i.c.v. route in mice. The involvement of the glutamatergic system in the convulsions was investigated. MK-801 and ketamine, non-competitive NMDA receptor antagonists, prolonged the latencies for convulsion onset. AP-5, a competitive NMDA receptor antagonist, reduced the number of animals convulsing and also increased the latency for convulsion onset. 7-Chlorokynurenic acid, an antagonist of the glycine site on the NMDA receptor, reduced the incidence of convulsions. GMP, a nucleotide known to antagonize some NMDA actions, reduced the incidence and the severity of convulsions and prolonged the latency for their onset. Riluzole, a neuroprotective and anticonvulsant agent, blocked the appearance of convulsions. In vitro, the crude extract inhibited [³H]glutamate binding to cerebral cortical membranes and enhanced [³H]glutamate release from cortical synaptosomes. Heating the crude extract to 100 °C for 30 min or preincubating it with sphingomyelin, abolished its effect on glutamate release, but did not alter its ability to induce convulsions and to inhibit glutamate binding. However, the convulsant action was inhibited when the crude extract was submitted to trypsin treatment. Our data suggest that the convulsions elicited by the crude extract are not due to the presence of cytolysin and are not related to an increase in glutamate release, but seem to be dependent on the interaction between a peptide component of the extract and NMDA receptors.     

曲茎石斛及其近似种鉴别的形态和DNA分子证据

目的　探讨曲茎石斛(南阳居群)与同属近似种:细茎石斛(南阳居群)、霍山石斛(霍山居群)、铁皮石斛(天台居群)药材鉴别的形态及DNA分子证据。方法　对曲茎石斛(南阳居群)及其近似种的药材进行了外部形态和rDNA ITS序列比较。结果　曲茎石斛及其近似种药材的外部形态虽无明显区别,但在rDNA ITS序列上却存在着显著而稳定的差异。曲茎石斛(南阳居群)与铁皮石斛(天台居群)的rDNA ITS序列的差异最小,绝对遗传距离为7;但与细茎石斛(南阳居群)及霍山石斛(霍山居群)rDNA ITS区的差异较大,绝对遗传距离分别为40和42。在rDNA ITS区域中,作者共挑选了7个碱基位点用作鉴别曲茎石斛(南阳居群)及其近似种的DNA证据。结论　根据药材的形态特征及rDNA ITS区碱基序列差异,可准确鉴别曲茎石斛及其近似种药材。     

忍冬属植物中咖啡酰奎宁酸类成分及其吸收与代谢研究进展

迄今为止从忍冬属植物中分离出24种咖啡酰奎宁酸类化合物,此类化合物显示有多种药理活性,具备广阔的应用前景,吸收与代谢研究发现其主要吸收部位为小肠,以被动扩散为主,代谢部位主要在大肠,代谢途径有甲基化、葡萄糖醛酸结合、水解、异构化和还原反应等。本文对忍冬属植物中咖啡酰奎宁酸类成分及其吸收与代谢研究进行总结,以期为咖啡酰基奎宁酸类药物研究开发提供参考。     

A simple biochemical method in the search for bioactive polypeptides in a sea anemone (Anemonia sulcata)

The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of ¹²⁵I-dendrotoxin to rat brain membranes and block the voltage-sensitive K⁺ channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.     

1H-吡咯-2(5H)-酮类化合物的合成及生物活性

目的研究5位连有取代苯基的1H-吡咯-2(5H)-酮类化合物生物活性。方法在吡咯酮的5位引入各种取代苯基,合成出目标化合物,所有化合物1H NMR和MS等谱确证;通过荧光偏振法测定化合物对蛋白的抑制常数Ki。结果所有13个目标化合物都能抑制蛋白的结合,其中化合物4e的Ki达到阳性对照药的5倍。结论 5位苯环的取代基团对活性影响很大,吸电子基团取代活性高于亲电子基团,其中硝基取代活性最强。     

梅花鹿鹿茸和马鹿鹿茸中5种核苷类成分的含量比较

目的 建立HPLC测定梅花鹿鹿茸和马鹿鹿茸药材中尿嘧啶、次黄嘌呤、尿苷、鸟苷和肌苷5种核苷成分含量的方法,并比较不同批次的梅花鹿鹿茸和马鹿鹿茸药材中5种核苷类成分含量的差异。方法 采用HPLC对15批梅花鹿鹿茸药材和15批马鹿鹿茸药材进行测定,并对测定结果进行聚类分析。结果 测定了梅花鹿鹿茸和马鹿鹿茸药材中5种核苷类成分的含量,二者中核苷类成分含量差异显著;不同产地不同养殖户的鹿茸药材中,5种核苷成分含量也存在明显差异。采用聚类分析能将不同产地的15批梅花鹿鹿茸和15批马鹿鹿茸药材分为2类。结论 不同产地梅花鹿鹿茸和马鹿鹿茸药材核苷成分的组成存在显著性差异,鹿茸药材的质量受鹿的品种、自然环境、养殖技术等因素的影响。该方法适用于梅花鹿鹿茸和马鹿鹿茸中核苷类成分的含量测定,为二者质量控制提供保证。     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

黄柏与关黄柏的化学成分及生物活性研究进展

黄皮树Phellodendron chinense、黄檗P.amurense的干燥树皮分别作为黄柏、关黄柏入药,二者在2005、2010、2015年版《中国药典》被分别收录。从这两种药材中分离出来的化学成分主要包括生物碱类、柠檬苦素类、酚酸类、萜类、苯丙素类、挥发性类等,具有抗菌、抗癌、抗糖尿病、保护心脑血管等多种生物活性。综述近年来对黄柏、关黄柏化学成分、生物活性的研究进展,为进一步研究两者药效物质基础、药理作用及临床合理用药提供参考依据。     

Effects of guggulsterone isolated from Commiphora mukul in high fat diet induced diabetic rats

Sedentary lifestyle, consumption of energy-rich diet, obesity and longer lifespan are some of the major reasons for the rise of metabolic disorders like type II diabetes, obesity, hypertension and dyslipidemia among people of various age groups. High fat diet induced diabetic rodent models resembling type II diabetic condition in human population were used to assess the anti-diabetic and hypolipidemic activity of guggulsterone (isolated from Commiphora mukul resin). Four groups of rats were fed high fat diet, for 16 weeks. On feeding the normal rats with fat rich diet they showed increased serum glucose, cholesterol and triglyceride levels along with increase in insulin resistance significantly (p < 0.05) in comparison to control animals. Different biochemical parameters like GTT, glycogen content, glucose homeostatic enzymes (like glucose-6-phosphatase, hexokinase), insulin release in vivo and expression profiles of various genes involved in carbohydrate and lipid metabolism clearly demonstrated the hypoglycemic effect of this extract. Guggulsterone demonstrated a differential effect with a significantly improved PPARγ expression and activity in in vivo and in vitro conditions, respectively. However, it inhibited 3T3-L1 preadipocytes differentiation in vitro. The results presented here suggest that the guggulsterone has both hypoglycemic and hypolipidemic effect which can help to cure type II diabetes.     

Effects of benzo[a]pyrene on mitochondrial and nuclear DNA damage in Atlantic killifish (Fundulus heteroclitus) from a creosote-contaminated and reference site

Benzo[a]pyrene (BaP) is a known genotoxicant that affects both mitochondrial and nuclear DNA (mtDNA, nDNA). Here, we examined mtDNA and nDNA damage in the Atlantic killifish (Fundulus heteroclitus) from a highly contaminated Superfund site (Elizabeth River, VA, USA) and from a reference site (King's Creek, VA, USA) that were dosed with 10 mg/kg BaP. Using the long amplicon quantitative PCR technique, we observed similar increases in mitochondrial and nuclear DNA damage in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish compared to King's Creek fish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Measurements of oxidative DNA damage (8-oxo-deoxyguanine by LC–MS/MS) showed no differences among treatment groups, suggesting that the majority of DNA damage is from covalent binding of BaP metabolites to DNA. This study shows for the first time that mitochondria can be an important target of BaP toxicity in fish, indicating that BaP exposures could have important energetic consequences. Results also suggest that multi-generational exposures in the wild may lead to adaptations that dampen DNA damage arising from BaP exposure.     

基于代谢组学和网络药理学分析福建金线莲叶和台湾银线兰叶治疗肝纤维化作用差异

目的 初步分析福建金线莲(Anoectochilus roxburghii)和台湾银线兰(Anoectochilus formosanus)的叶片在治疗肝纤维化疾病的作用机制差异，为分析两者的临床疗效差异及其资源的精准利用提供参考信息。方法 利用代谢组学技术分析福建金线莲叶(the leaves of A.roxburghii，Arl)和台湾银线兰叶(the leaves of A.formosanus，Afl)的差异代谢物；并结合网络药理学方法分析其差异代谢物治疗肝纤维化疾病的作用差异。结果 利用代谢组学的方法分析初步得到Arl和Afl的46个差异代谢物，主要为黄酮类、糖苷类和甘油磷脂类等，其中有23种差异化合物具有潜在药用活性。基于网络药理学的方法，预测了23种差异化合物的759个相关靶点，与肝纤维化疾病的相关靶点进行映射，得到交集靶点249个，其中AKT1、STAT3、VEGFA等48个关键靶点被预测为治疗肝纤维化的主要差异靶点；作用靶点较多的关键差异化合物为Arl相比Afl含量较高的化合物，预示Arl中富含更多可以用于治疗肝纤维化的有效化合物。GO富集功能和KEGG富集分析表明，福建金线莲与台湾银线兰的差异成分参与治疗肝纤维化疾病的信号通路有所不同，VEGF信号通路为Arl相比Afl含量较高的差异化合物的主要差异信号通路，磷脂酰肌酶3-激酶-蛋白激酶B(PI3K-Akt)信号通路为Arl相比Afl含量较低的差异化合物治疗肝纤维化疾病的主要差异信号通路。分子对接结果表明各差异成分与其对应的关键靶点均有较好的结合活性。结论 本研究表明福建金线莲与台湾银线兰叶片中存在一些差异代谢物，预测了其治疗肝纤维化疾病作用机制差异，并初步验证了其作用靶点，为金线莲资源的精准利用具有指导性意义。     

Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus.

A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD(50) of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel beta-structure, which is in good agreement with experimental data obtained from other sea anemones-actinoporins.     

Purification and characterization of the biological effects of phospholipase A2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA₂ isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA₂ proteins (named BcPLA₂1, BcPLA₂2 and BcPLA₂3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA₂1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA₂1 showed high amino acid sequence identity with PLA₂ group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA₂ and 1POC_A). In addition, BcPLA₂1 also showed significant overall homology to bee PLA₂. The enzymatic activity induced by native BcPLA₂1 (20 μg/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA₂1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA₂ from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA₂1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA₂, a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.     

Purification, cloning and characterization of fragaceatoxin C, a novel actinoporin from the sea anemone Actinia fragacea

Actinia fragacea is commonly called the “strawberry” anemone because of the distinctive yellow or green spots displayed on its red column. Its venom contains several haemolytic proteins with a molecular mass of 20 kDa that can be separated by ion-exchange column chromatography. One of them was purified to homogeneity and was named fragaceatoxin C (FraC). Its 15 N-terminal residues were identified by Edman degradation and served to obtain its complete DNA coding sequence by RT-PCR. The coding region of FraC was amplified and cloned in the expression vector pBAT-4. Purified recombinant FraC consists of 179 amino acids and multiple sequence alignment with other actinoporins clearly indicates that FraC belongs to this protein family. The secondary structure, thermal stability and lytic activity of native and recombinant FraC were practically identical and exhibit the same basic features already described for equinatoxin-II and sticholysin-II.     

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.