中华大蟾蜍蝌蚪变态过程中脊椎骨化次序

两栖动物在幼体变态即由水栖到陆栖的环境转变中,骨骼系统会发生重塑。本文采用茜素红和阿利新蓝的双染色技术对不同发育阶段中华大蟾蜍(Bufo gargarizans)蝌蚪变态过程中(Gosner 38~46)脊椎骨的发育进程进行了形态学研究。结果显示,在中华大蟾蜍蝌蚪Gosner 39期,椎板中线处发生融合;荐前椎Ⅱ-Ⅷ和荐椎的椎体、椎弓起始骨化发生在Gosner 42期;其次荐前椎Ⅱ-Ⅷ和荐椎的横突、底索和荐后椎Ⅰ开始骨化;荐后椎Ⅱ骨化最晚;在Gosner 46期,尾杆骨最终形成。荐后椎愈合形成尾杆骨反映无尾类幼体由水栖环境转变为陆生环境中骨骼系统的机能适应。     

高温刺激对蟾蜍免疫活性和能量消耗的影响

全球气候变化是导致全世界两栖动物种群衰退和灭绝的重要因素之一。为了解无尾两栖类在气候异常导致的极端高温下的生理反应特征和相关适应机制,以雄性中华大蟾蜍(Bufo bufo gargarizan)为对象,研究该物种在高温刺激(环境温度由22 ℃迅速升至32 ℃)0、6、12、24、48 h后的非特异性免疫活性和能量消耗的变化情况。研究结果:与对照组(22 ℃)比较,高温刺激可显著抑制外周血细胞吞噬活性;而脾脏巨噬细胞呼吸爆发在高温刺激下呈现出先增强后减弱的趋势。高温刺激导致血糖含量在第24和48 h显著低于对照组,而对肝糖原含量无显著影响。处理组精巢ATP含量逐渐升高,并于24和48 h显著高于对照组;处理组Na⁺-K⁺-ATPase活性随时间呈现出先下降后上升稳定的趋势,但与对照组相比均无显著性差异,;处理组Ca²⁺-Mg²⁺-ATPase活性在不同时期与对照组相比均无显著性差异。研究结果说明,高温刺激可显著抑制中华大蟾蜍的非特异性免疫功能,并导致血糖的大量消耗;当面对高温胁迫时,雄性大蟾蜍可能会投入更多的能量来维持性腺的正常发育。     

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

花背蟾蜍蝌蚪皮肤的结构与发育

为研究花背蟾蜍(Bufo raddei)蝌蚪在变态发育期皮肤的显微结构特点,选取G19、G22、G26、G36、G41、G43和G46共7个发育期蝌蚪的连续石蜡切片及成体的背部皮肤切片,采用H.E和AB-PAS染色方法,观察了皮肤各层结构的发育时序并进行了相应的测量.结果表明,在G19、G22和G26蝌蚪表皮均为1层细胞;G36蝌蚪皮肤细胞形态和层数在背腹部出现了显著的区别;在G41基本完成了表皮2层细胞的构建;G43期完成完整的真皮构建,其中分布有毛细血管和2种皮肤腺.G46皮肤在厚度、腺体和毛细血管分布等方面表现出了明显的区域性差异,并与成体皮肤结构有明显的差别,显示出蝌蚪在发育过程中皮肤结构的变化与其生存环境之间紧密的关联性.     

中国林蛙与中华蟾蜍蝌蚪颅骨形态的系统进化比较

对无尾两栖动物系统进化的研究,主要是基于经典的形态学、现代分子生物学等技术。无尾两栖动物蝌蚪的进化与成体的进化可能是两个独立的过程,所以无尾两栖类蝌蚪形态及发育特征也可以作为研究系统进化的重要指征。本文对中国林蛙(Rana chensinensis)及中华蟾蜍(Bufo gargarizans)变态前蝌蚪的软骨性颅骨及鳃部骨骼进行形态学描述,基于幼体形态特征,进行系统建树。建树结果支持Orton将无尾两栖类蝌蚪划分为4种类型,认为最原始的类群为Ⅲ型蝌蚪,与Tihen的观点一致。新蛙亚目的中国林蛙和中华蟾蜍蝌蚪属Ⅳ型,是最进化的类群。     

Five new C21 steroidal glycosides from the stems of Marsdenia tenacissima

Five new C₂₁ steroidal glycosides (1-5) were isolated from the stems of Marsdenia tenacissima. The chemical structures and relative configurations of the new compounds were elucidated by mass spectrometry and NMR spectroscopy. Cellular assay of these compounds showed that they are weak cytotoxic to various cell lines.     

Isolation of an acidic phospholipase A2 from the venom of the snake Bothrops asper of Costa Rica: Biochemical and toxicological characterization

Phospholipases A₂ (PLA₂) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA₂s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA₂-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA₂-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA₂-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA₂-II also differed from other acidic PLA₂s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA₂-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA₂-II revealed that acidic and basic PLA₂s form two different antigenic groups in B. asper venom.     

花背蟾蜍消化道5-羟色胺细胞的分布及形态学特征

采用卵白素-生物素-过氧化物酶复合物（avidin-biotin-peroxidase complex technique,ABC）免疫组织化学方法对花背蟾蜍（Bufo raddei）消化道5-羟色胺（5-HT）细胞的分布密度及形态学特征进行了观察。结果显示,5-HT细胞在花背蟾蜍整个消化道中均有分布,食管、贲门、胃体和幽门的分布密度都显著高于肠道各段,胃幽门部密度最高,胃体部其次,直肠部最低。消化道各个部位5-HT开放型和闭合型细胞的比值变化范围为2.48~4.71。消化道各段均以开放型细胞为主,大多呈锥体形、梭形或不规则形,少数为闭合型细胞,呈圆形或椭圆形。花背蟾蜍5-HT细胞的形态学特征与其他两栖动物相似,但分布密度有自身特征,可能与其食性和善于摄取活动性小的食物的生活习性有关。     

Identification, cloning and expression of a second gene (vpr1) from the venom of the endoparasitic wasp, Pimpla hypochondriaca that displays immunosuppressive activity

Previously, we biochemically isolated an immunosuppressive protein (VPr3) from the venom of Pimpla hypochondriaca and cloned and expressed the gene in bacteria. The deduced amino acid sequence for VPr3 shares 63% identity with a second P. hypochondriaca protein, venom protein one (VPr1). We have now cloned and expressed the gene for vpr1. The expression of His-tagged recombinant VPr1 (rVPr1) in E. coli BL21 Star™ (DE3) cells was induced by the addition of 0.5 mM IPTG. Cultures were grown at 24 and 37 °C, and VPr1 more readily partitioned into the soluble fraction at 24 °C. Soluble rVPr1 was purified using the MagneHis purification system and a modified elution buffer to allow the protein to be directly tested for activity against haemocytes. It was observed that rVPr1 prevented the ability of haemocytes to spread and form aggregates in vitro in a dose-dependent manner. Furthermore, comparable levels of activity were observed when similar concentrations of rVPr1 and rVPr3 were tested. In addition, the encapsulation of Sephadex beads in vivo was reduced by the presence of rVPr1 and beads were unencapsulated (negative) or only weakly encapsulated. The functional and physio-chemical properties of rVPr1 and rVPr3 are compared and discussed.     

New 4-methylated and 19-oxygenated steroids from the Formosan soft coral Nephthea erecta

Two new 4-methylated steroids, erectasteroids A and B (1 and 2), six new 19-oxygenated steroids, erectasteroids C-H (3-8) and two known 19-oxygenated steroids (9 and 10) were isolated from the acetone solubles of the Formosan soft coral Nephthea erecta. The structures were elucidated by extensive NMR spectroscopic analysis and their cytotoxicity against selected cancer cells was measured in vitro.     

Cotiarinase is a novel prothrombin activator from the venom of Bothrops cotiara

Snake venom serine proteinases (SVSPs) may affect hemostatic pathways by specifically activating components involved in coagulation, fibrinolysis and platelet aggregation or by unspecific proteolytic degradation. In this study, we purified and characterized an SVSP from Bothrops cotiara venom, named cotiarinase, which generated thrombin upon incubation with prothrombin. Cotiarinase was isolated by a two-step procedure including gel-filtration and cation-exchange chromatographies and showed a single protein band with a molecular mass of 29 kDa by SDS-polyacrylamide gel electrophoresis under reducing conditions. Identification of cotiarinase by mass spectrometric analysis revealed peptides that matched sequences of viperid SVSPs. Cotiarinase did not show fibrinogen-clotting, platelet-aggregating, fibrinogenolytic and factor X activating activities. Upon incubation with prothrombin the generation of thrombin was detected using the peptide substrate d-Phe-Pip-Arg-pNA. Moreover, mass spectrometric identification of prothrombin fragments generated by cotiarinase in the absence of co-factors (phospholipids, factor Va, factor Xa and Ca²⁺ ions), indicated the limited proteolysis of this protein to release prothrombin 1, fragment 1 and thrombin. Cotiarinase is a novel SVSP that acts on prothrombin to release active thrombin that does not match any group of the current classification of snake venom prothrombin activators.     

Structural and functional properties of BaTX,a new Lys49 phospholipase A2 homologue isolated from the venom of the snake Bothrops alternatus

BaTX PLA₂, a K49 phospholipase A₂ homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA₂. The complete amino acid sequence of BaTX PLA₂ contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA₂s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA₂s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD₅₀ of BaTX was 7 μg/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 μM: 40 ± 0.4 min and 0.07 μM: 35 ± 0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA₂ family, which presents the typical bioactivities described for such proteins.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Identification and characterization of a phospholipase A2 from the venom of the Saw-scaled viper: Novel bactericidal and membrane damaging activities

Phospholipase A₂ (PLA₂), a common toxic component of snake venom, has been implicated in various pharmacological effects. In this study, a basic myotoxic PLA₂, named EcTx-I was isolated from Echis carinatus snake venom by using gel filtration on Superdex G-75, and reverse phase HPLC on C18 and C8 Sepharose columns. PLA₂, EcTx-I was 13,861.72 molecular weight as estimated by MALDI-TOF (15 kD by SDS-PAGE), and consisted of 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with basic myotoxic PLA₂s from other snake venoms. The purified PLA₂ EcTx-I was evaluated (250 μg/ml) for bactericidal activity of a wide variety of human pathogens against Burkholderia pseudomallei (KHW&TES), Enterobacter aerogenes, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Pseudomonas aeruginosa and Staphylococcus aureus. EcTx-I showed strong antibacterial activity against B. pseudomallei (KHW) and E. aerogenes among the tested bacteria. Other Gram-negative and Gram-positive bacteria showed only a moderate effect. However, the Gram-positive bacterium E. aerogenes failed to show any effect on EcTx-I protein at tested doses. The most significant bacteriostatic and bactericidal effect of EcTx-I was observed at MICs of >15 μg/ml against (B. pseudomallei, KHW) and MICs >30 μg/ml against E. aerogenes. Mechanisms of bactericidal and membrane damaging effects were proved by ultra-structural analysis. EcTx-I was able to induce cytotoxicity on THP-1 cells in vitro as well as lethality in BALB/c mice. EcTx-I also induced mild myotoxic effects on mouse skin, but was devoid of hemolytic effects on human erythrocytes up to 500 μg/ml. It is shown that the toxic effect induced by E. carinatus venom is due to the presence of myotoxic PLA₂ (EcTx-I). The result also corroborates the hypothesis of an association between toxic and enzymatic domains. In conclusion, EcTx-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.     

Alkaloids in the venom of Messor ants

Tobacco alkaloids of the anabasine type have been found or confirmed in the venom of five species of arid-dwelling Messor ants. They are frequently accompanied by alkylpyrazines. Messor mediorubra contains four alkaloids, with anabasine the major component and also minor pyrazines. Anabasine was found alone in the venom of Messor semirufus and confirmed in Messor ebeninus. Messor rugosus from Tel Aviv contained a mixture of alkaloids and pyrazines, but those from Ein Yahav contained 3-ethyl-2,5-dimethylpyrazine, some 2-phenylethylamine, and N-ethylidene 2-phenylethylamine. Messor arenarius is confirmed as having a complex but variable mixture of alkaloids and pyrazines.     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

C14-Oxylipin glucosides isolated from Lemna paucicostata

Oxylipin glucosides (2-4) were isolated from Lemna paucicostata with their structures and absolute configurations elucidated by spectroscopic and chemical methods. Compounds 2-4 were glucosides of C₁₄ oxylipin which were synthesized from α-linolenic acid via the 9-lipoxygenase pathway.     

Isolation and characterization of the thrombin-like enzyme from Cryptelytrops purpureomaculatus venom

A thrombin-like enzyme, purpurase, was purified from the Cryptelytrops purpureomaculatus (mangrove pit viper) venom using high performance ion-exchange and gel filtration chromatography. The purified sample (termed purpurase) yielded a homogeneous band in SDS-polyacrylamide gel electrophoresis with a molecular weight of 35,000. The N-terminal sequence of purpurase was determined to be VVGGDECNINDHRSLVRIF and is homologous to many other venom thrombin-like enzymes. Purpurase exhibits both arginine ester hydrolase and amidase activities. Kinetic studies using tripeptide chromogenic anilide substrates showed that purpurase is not fastidious towards its substrate. The clotting times of fibrinogen by purpurase were concentration dependent, with optimum clotting activity at 3 mg fibronogen/mL. The clotting activity by purpurase was in the following decreasing order: cat fibrinogen > human fibrinogen > dog fibrinogen > goat fibrinogen >> rabbit fibrinogen. Reversed-phase HPLC analysis of the products of action of purpurase on bovine fibrinogen showed that only fibrinopeptide A was released. Indirect ELISA studies showed that anti-purpurase cross-reacted strongly with venoms of most crotalid venoms, indicating the snake venom thrombin-like enzymes generally possess similar epitopes. In the more specific double-sandwich ELISA, however, anti-purpurase cross-reacted only with venoms of certain species of the Trimeresurus complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex.     

Cloning,expression and characterization of a phospholipase D from Loxosceles gaucho venom gland

Loxosceles venom comprises a mixture of diverse toxins that induces intense local inflammatory reaction, dermonecrotic injury, platelet aggregation, hemolytic anemia and acute renal failure. Among several toxins in the venom, phospholipases D (PLDs), also called dermonecrotic toxins, are the most important and best studied, since they account for the main effects observed in loxoscelism. Despite their importance, biological analysis of PLDs is hampered by the minute amounts normally purified from the venom, and therefore many efforts have been made to clone those toxins. However, to date, no PLD from Loxosceles gaucho has been obtained in a heterologous system. Thus, in this work we show the cloning of a PLD from L. gaucho venom gland, named LgRec1, which was successfully expressed in a bacterial system. LgRec1 evoked local reaction (edema, erythema, ecchymosis, and paleness), dermonecrosis and hemolysis. It was also able to hydrolyze sphingomyelin and promote platelet aggregation. ELISA and Western blot analysis showed that LgRec1 was recognized by an anti-L. gaucho venom serum, a commercial arachnidic antivenom as well as a monoclonal antibody raised against the dermonecrotic fraction of L. gaucho venom. In addition, LgRec1 demonstrated to be highly immunogenic and antibodies raised against this recombinant toxin inhibited local reaction (∼65%) and dermonecrosis (∼100%) elicited by L. gaucho whole venom. Since PLDs are considered the major components accounting for the local and systemic envenomation effects caused by spiders from genus Loxosceles, the information provided here may help to understand the mechanisms behind clinical symptomatology.     

A novel phospholipase A2 from Agkistrodon blomhoffii ussurensis venom: Purification,proteomic, functional and structural characterizations

A phospholipase A₂ was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA₂. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA₂ was a new snake venom acidic PLA₂. Seven peptides were sequenced from Akbu-PLA₂ by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA₂ shared homolog peptides of phospholipases A₂ from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA₂ has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA₂ showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA₂ was shown to contain one Ca²⁺ per monomer by ICP-AES measurement. The Ca²⁺ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA₂. Ca²⁺ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA₂. The hydrolytic activity of Akbu-PLA₂ was accelerated due to the addition of Ca²⁺ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA₂ was found to be difficult to eliminate for the purification of Akbu-PLA₂. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA₂, which indicated that it should be a homodimer of Akbu-PLA₂. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA₂ monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA₂ monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca²⁺ ion was found to be able to promote the dimer formation of Akbu-PLA₂. We conclude that a novel PLA₂ was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA₂ reveal new threads and provide valuable inputs for the study of snake venom phospholipases A₂.