沙蜇(Stomolophus meleagris)刺丝囊毒素生物活性的初步分析

从沙蜇触手提取刺丝囊细胞毒素,并对该毒素进行溶血活性、致死活性、SOD活性和抗肿瘤活性的研究。结果显示,沙蜇毒素具有明显的溶血活性,其半溶血率(HU50)约为10.5μg/ml;该毒素还对草鱼显示出较强的致死活性,半致死量(LD50)为50μg毒素/g鱼;同时该毒素具有明显的SOD活性和抗肿瘤活性,当毒素浓度为18μg/ml时其总SOD活性为161 U/mg,而毒素浓度为1 mg/ml时,该毒素对肝癌细胞Bel-7402表现出显著的抑制效果,其抑制率达到54.9%。因此,有必要对沙蜇毒素内的生物活性组分进行深入研究,为沙蜇毒素的开发利用提供依据。     

Toxinological and immunological studies of capillary electrophoresis fractionated Chrysaora quinquecirrha (Desor) fishing tentacle and Chironex fleckeri Southcott nematocyst venoms

Repeated runs of capillary electrophoresis (CE) were used to study partially-purified jellyfish nematocyst venom protein in concentrations sufficient to perform toxinological assays. Nematocyst venoms from Chironex fleckeri (Cf) and Chysaora quinquecirrha were processed. The CE eluate was divided into quadrants by scanning protein content. The fourth fraction of both jellyfish venoms, contained proteins with the smallest molecular weight components, which were responsible for the highest hemolysins and the humoral and cell-mediated immunological activity. Cytotoxic Cf lethal factor activity against human liver cells was widely dispersed throughout both venoms but more prominent in fraction 4. A V(beta) receptor human T-cell repertoire was not species-specific for either crude or fractionated jellyfish nematocyst venom.     

Antiproliferative and antioxidant properties of nematocysts crude venom from jellyfish Acromitus flagellatus against human cancer cell lines

This study aimed to investigate the antiproliferative and antioxidant properties of crude venom from the nematocyst of Jellyfish Acromitus flagellates on human lung cancer (A549) and liver cancer (HepG2) cell lines. The prepared crude venom was subjected to analyses of the biochemical constituents, protein profiles, antioxidant and anticancer activities by standard methods. The extracted venom was pale-yellow in color and viscous/sticky. The biochemical composition such as, protein (1.547 mg/ml), lipid (0.039 mg/ml) and carbohydrate (0.028 mg/ml) was estimated. Protein profiles were determined by SDS PAGE, the result revealed that the molecular weight range from 205 ? 3.5 kDa. The free radical scavenging activity was analyzed by the reducing potential (56.36%), DPPH (72.47%), hydroxyl (68.50%), superoxide anion (65.75%), and nitric oxide (33.04%). The cell viability was observed by using different concentrations (20 to 100 µg/ml) of crude venom on A549 and HepG2 cancer cell lines and the IC₅₀ values were recorded in (60 μg/ml and 40 μg/ml) respectively, while it had none cytotoxic effects on Vero cell line up to the concentration of 90 μg/ml. These results suggest that crude venom from nematocyst of A. flagellatus possesses anti-cancer activity and able to develop novel drugs on marine-derived compounds.     

Evaluation of the effects of various chemicals on discharge of and pain caused by jellyfish nematocysts

Jellyfish tentacles in contact with human skin can produce pain swelling and redness. The pain is due to discharge of jellyfish nematocysts and associated toxins and discharge can be caused by a variety of mechanical and chemical stimuli. A series of tests were carried out with chemicals traditionally used to treat jellyfish stings e.g. acetic acid ammonia meat tenderizer baking soda and urea to determine if these chemicals stimulated or inhibited nematocyst discharge and if they brought relief to testers who were exposed to jellyfish tentacles. Chrysaora quinquecirrha (sea nettle) Chiropsalmus quadrumanus (sea wasp) and Physalia physalis (Portuguese man-of-war) were used in the study. It was found that many of the chemicals traditionally used to treat jellyfish stings stimulated nematocyst discharge and did not relieve the pain. However there was immediate relief when a common anesthetic lidocaine was sprayed on the skin of testers in contact with jellyfish tentacles. Initial exposure of tentacle suspensions to lidocaine prevented the nematocyst discharge by subsequent exposure to acetic acid ethanol ammonia or bromelain. Thus lidocaine in addition to acting as an anesthetic on skin in contact with jellyfish tentacles inhibited nematocyst discharge possibly by blocking sodium and/or calcium channels of the nematocytes.     

Isolation and characterization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata

The box jellyfish (sea wasp) Carybdea alata Reynaud, 1830 (Cubozoa) is distributed widely in the tropics. The sting of C. alata causes severe pain and cutaneous inflammation in humans. We successfully isolated C. alata toxin-A (CaTX-A, 43 kDa) and -B (CaTX-B, 45 kDa) for the first time from the tentacle of C. alata collected at a site along the Hawaiian shore. The experimental results showed that CaTX-A, but not CaTX-B, is present in the nematocyst, the organ responsible for stinging. Both CaTX-A and -B showed potent hemolytic activity, with CaTX-A being lethally toxic to crayfish when administered via intraperitoneal injection. Furthermore, we sequenced the cDNA encoding CaTX-A. The deduced amino acid sequence of CaTX-A (463 amino acids) showed 43.7% homology to Carybdea rastoni toxins (CrTXs) but not with any other known proteins. Therefore, these jellyfish toxins potentially represent a novel class of bioactive proteins. Secondary structure analysis of CaTX-A and CrTXs suggested the presence of amphiphilic alpha-helices, which are also seen in several known hemolytic or cytolytic protein toxins, including peptide toxins.     

霞水母刺丝囊细胞热激蛋白60(Hsp60)的分离纯化与鉴定

热激蛋白60作为分子伴侣家族中的重要成员,在蛋白质的运输、组装以及折叠等方面起到重要的作用。利用离子交换层析和凝胶过滤层析两步纯化方法,从霞水母刺丝囊细胞中分离到热激蛋白60。SDS-PAGE结果显示,在分子量为60kDa处显示为单一清晰的蛋白条带,并且通过N末端测序进行鉴定,其序列为APKEIKFGADAKSLM与热激蛋白60相吻合;此外,还利用ELISA法对其进一步确定,同时对分离过程的热激蛋白60的回收率进行了测定。该方法为进一步研究霞水母热激蛋白60的功能及其应用奠定了基础。     

Length Is Associated with Pain: Jellyfish with Painful Sting Have Longer Nematocyst Tubules than Harmless Jellyfish

A large number of humans are stung by jellyfish all over the world. The stings cause acute pain followed by persistent pain and local inflammation. Harmful jellyfish species typically cause strong pain, whereas harmless jellyfish cause subtle or no pain. Jellyfish sting humans by injecting a tubule, contained in the nematocyst, the stinging organ of jellyfish. The tubule penetrates into the skin leading to venom injection. The detailed morphology of the nematocyst tubule and molecular structure of the venom in the nematocyst has been reported; however, the mechanism responsible for the difference in pain that is caused by harmful and harmless jellyfish sting has not yet been explored or explained. Therefore, we hypothesized that differences in the length of the nematocyst tubule leads to different degrees of epithelial damage. The initial acute pain might be generated by penetration of the tubule, which stimulates pain receptor neurons, whilst persistent pain might be caused by injection of venom into the epithelium. To test this hypothesis we compared the lengths of discharged nematocyst tubules from harmful and harmless jellyfish species and evaluated their ability to penetrate human skin. The results showed that the harmful jellyfish species, Chrysaora pacifica, Carybdea brevipedalia, and Chironex yamaguchii, causing moderate to severe pain, have nematocyst tubules longer than 200 μm, compared with a jellyfish species that cause little or no pain, Aurelia aurita. The majority of the tubules of harmful jellyfishes, C. yamaguchii and C. brevipedalia, were sufficiently long to penetrate the human epidermis and physically stimulate the free nerve endings of Aδ pain receptor fibers around plexuses to cause acute pain and inject the venom into the human skin epithelium to cause persistent pain and inflammation.     

Novel proteinaceous toxins from the box jellyfish (sea wasp) Carybdea rastoni

During summer and autumn, the box jellyfish (sea wasp) Carybdea rastoni is one of the most bothersome stinging pests to swimmers and bathers on the Japanese coast. Two labile but potent hemolytic toxins from the tentacles of Carybdea rastoni were isolated in their active forms using newly developed purification methods. The molecular masses of the isolated C. rastoni toxin-A and toxin-B (CrTX-A and CrTX-B) are 43 and 46 kDa, respectively, as calculated from SDS-PAGE. In the present study, we sequenced the full-length cDNA (1600 bp), which encodes both CrTX-A and CrTX-B. The deduced 450 amino acid sequence of the CrTXs, showed no significant homology with any known protein. This report presents the first complete sequence of a proteinaceous jellyfish toxin. Furthermore, it was revealed that CrTX-A was primarily localized in the nematocyst, whereas CrTX-B was detected only in the tentacle. Because the nematocyst is the organ responsible for the cnidarian sting, the remainder of the study focused on the toxicity of CrTX-A. We found that CrTX-A was fatally toxic to mice at 20 microg/kg (i.v.) and crayfish at 5 microg/kg (i.p.). Subcutaneously injected CrTX-A (0.1 microg) caused inflammation of mouse skin. These results showed that CrTX-A is responsible for the cutaneous inflammation observed in humans stung by C. rastoni.     

Polymer‐mediated flocculation of transient CHO cultures as a simple,high throughput method to facilitate antibody discovery

Most biopharmaceutical drugs, especially monoclonal antibodies (mAbs), bispecific antibodies (BsAbs) and Fc‐fusion proteins, are expressed using Chinese Hamster Ovary (CHO) cell lines. CHO cells typically yield high product titers and high product quality. Unfortunately, CHO cell lines also generate high molecular weight (HMW) aggregates of the desired product during cell culture along with CHO host cell protein (HCP) and CHO DNA. These immunogenic species, co‐purified during Protein A purification, must be removed in a multi‐step purification process. Our colleagues have reported the use of a novel polymer‐mediated flocculation step to simultaneously reduce HMW, HCP and DNA from stable CHO cell cultures prior to Protein A purification. The objective of this study was to evaluate this novel “smart polymer” (SmP) in a high throughput antibody discovery workflow using transiently transfected CHO cultures. SmP treatment of 19 different molecules from four distinct molecular categories (human mAbs, murine mAbs, BsAbs and Fabs) with 0.1% SmP and 25 mM stimulus resulted in minimal loss of monomeric protein. Treatment with SmP also demonstrated a variable, concentration‐dependent removal of HMW aggregates after Protein A purification. SmP treatment also effectively reduced HCP levels at each step of mAb purification with final HCP levels being several fold lower than the untreated control. Interestingly, SmP treatment was able to significantly reduce high concentrations of artificially spiked levels of endotoxin in the cultures. In summary, adding a simple flocculation step to our existing transient CHO process reduced the downstream purification burden to remove impurities and improved final product quality. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1393–1400, 2017     

Identification of jellyfish from Continuous Plankton Recorder samples

Previous analysis of the Continuous Plankton Recorder (CPR) long-term data set for the presence of ‘coelenterate’ tissue revealed changes in the frequency of jellyfish occurrence in the North Atlantic Ocean and North Sea; however, the identities of the jellyfish were unknown, causing uncertainty in interpreting these findings. To improve the utility of the ‘coelenterate’ data from the CPR, 62 CPR samples were selected for re-analysis from an area where the widespread occurrence of the holoplanktonic scyphomedusan, Pelagia noctiluca (Forsskål, 1775), was previously documented from net tows to test if the CPR sampled P. noctiluca. Examination of the samples revealed smears (<1 mm) of golden gelatinous tissue on the sampling mesh. Subsequent microscopic examination and measurement of nematocysts identified most of the smears as P. noctiluca. Indeed, P. noctiluca was identified on 53% of the CPR samples from the area that best coincided with the documented bloom area. Although hydrozoans were also identified in the samples, the characteristic golden colour of P. noctiluca and its distinct suite of nematocysts allowed identification to species level. The identification of other jellyfish may be more complicated due to the labour-intensive examination of samples and unclear delineation of nematocyst types when multiple similar species are present. Nevertheless, re-analysis of CPR samples holds great promise for identifying the underlying reason for increases in jellyfish occurrence in the CPR records.     

水母雪莲Myb转录调控因子SmP基因的克隆及序列分析

采用TDPCR(Touch down PCR)法从水母雪莲(Saussurea medusa Maxim)红色系愈伤组织cDNA文库中筛选并克隆了雪莲Myb转录调控因子SmP (S.medusa Maxim Mybrelated P gene)基因。序列分析表明该基因全长969bp,包括一个771bp的完整开放阅读框架（ORF）,编码一个256氨基酸残基的蛋白质。氨基酸序列的同源性分析表明在N-端具有两个典型的R2R3-Myb-DNA结合结构域。C-端富含亲水的丝氨酸S(18.38%),且以寡聚体的形式存在,具有转录调控因子激活结构域常见的特征。     

High zinc sensitivity and pore formation in an invertebrate P2X receptor

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn²⁺ ions (IC₅₀=0.4 μM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.     

Predation by the phyllosoma larva of Ibacus novemdentatus on various kinds of venomous jellyfish

The phyllosoma, a larva of spiny and slipper lobsters, has an exceptionally flat body and long appendages. It is known to associate with several species of cnidarian jellyfish, a behavior that is not rare in crustaceans. Indeed, phyllosomas clinging onto jellyfish have been observed both in the laboratory and in the natural environment. Wild phyllosomas have been found to contain jellyfish tissues in their hepatopancreas and feces, suggesting that the larvae utilize jellyfish as a food source; however, how they capture jellyfish and what species of jellyfish they prefer have rarely been investigated. The few previous studies conducted have suggested that phyllosomas have a high specificity for jellyfish (preying on only a few species); in contrast, the results of our study indicate that specificity is low. We show that phyllosomas prey on a variety of jellyfish species including deadly stinging types, on a variety of jellyfish developmental stages, and on various parts of the jellyfish body. When making contact with a jellyfish, phyllosomas first cling onto its exumbrella, feed on its tentacles or oral arms, and then consume the exumbrella. Phyllosomas may be capable of defending themselves against any types of nematocyst sting, and it is likely that they have evolved to utilize venomous jellyfish as a food in the open sea, where food may be scarce.     

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.     

High zinc sensitivity and pore formation in an invertebrate P2X receptor

To investigate fast purinergic signaling in invertebrates, we examined the functional properties of a P2X receptor subunit cloned from the parasitic platyhelminth Schistosoma mansoni. This purinoceptor (SmP2X) displays unambiguous homology of primary sequence with vertebrate P2X subunits. SmP2X subunits assemble into homomeric ATP-gated channels that exhibit slow activation kinetics and are blocked by suramin and PPADS but not TNP-ATP. SmP2X mediates the uptake of the dye YO-PRO-1 through the formation of large pores and can be blocked by submicromolar concentrations of extracellular Zn2+ ions (IC50 = 0.4 microM). The unique receptor phenotype defined by SmP2X suggests that slow kinetics, modulation by zinc and the ability to form large pores are ancestral properties of P2X receptors. The high sensitivity of SmP2X to zinc further reveals a zinc regulation requirement for the parasite's physiology that could potentially be exploited for therapeutic purposes.     

Chemical modification of ascorbic acid and evaluation of its lipophilic derivatives as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

This investigation aims to evaluate the antitumor and antioxidant potential of Chrysaora quinquecirrha (sea nettle) nematocyst venom on Ehrlich ascites carcinoma (EAC) tumor model. Tumor was induced in mice by intraperitoneal injection of EAC cells. The antitumor effect of sea nettle nematocyst venom (SNV) peptide was evaluated by assessing in vitro cytotoxicity, survival time, hematological, and antioxidant parameters. Intraperitoneal injection of SNV peptide increased the survival time of the EAC-bearing mice. The SNV peptide brought back the altered levels of the hematological and antioxidant parameters in a dose dependent manner in EAC-bearing mice. The results were comparable to that of the result obtained from the animals treated with the standard drug 5-fluorouracil (20 mg/kg bw). Thus, present study revealed that SNV peptide possessed significant antitumor and antioxidant activity.     

Sequence-structure and structure-function analysis in cysteine-rich domains forming the ultrastable nematocyst wall

The nematocyst wall of cnidarians is a unique biomaterial that withstands extreme osmotic pressures, allowing an ultrafast discharge of the nematocyst capsules. Assembly of the highly robust nematocyst wall is achieved by covalent linkage of cysteine-rich domains (CRDs) from two main protein components, minicollagens and nematocyst outer wall antigen (NOWA). The bipolar minicollagens have different disulfide patterns and topologies in their N and C-terminal CRDs. The functional significance of this polarity has been elusive. Here, we show by NMR structural analysis that all representative cysteine-rich domains of NOWA are structurally related to N-terminal minicollagen domains. Natural sequence insertions in NOWA CRDs have very little effect on the tightly knit domain structures, nor do they preclude the efficient folding to a single native conformation. The different folds in NOWA CRDs and the atypical C-terminal minicollagen domain on the other hand can be directly related to different conformational preferences in the reduced states. Ultrastructural analysis in conjunction with aggregation studies argues for an association between the similar NOWA and N-terminal minicollagen domains in early stages of the nematocyst wall assembly, which is followed by the controlled association between the unusual structures of C-terminal minicollagen domains.     

Evolution of complex structures: minicollagens shape the cnidarian nematocyst

The generation of biological complexity by the acquisition of novel modular units is an emerging concept in evolutionary dynamics. Here, we review the coordinate evolution of cnidarian nematocysts, secretory organelles used for capture of prey, and of minicollagens, proteins constituting the nematocyst capsule. Within the Cnidaria there is an increase in nematocyst complexity from Anthozoa to Medusozoa and a parallel increase in the number and complexity of minicollagen proteins. This complexity is primarily manifest in a diversification of N- and C-terminal cysteine-rich domains (CRDs) involved in minicollagen polymerization. We hypothesize that novel CRD motifs alter minicollagen networks, leading to novel capsule structures and nematocyst types.