Analysis of particles containing alpha emitters in stagnant water in Fukushima Daiichi Nuclear Power Station's Unit 3 reactor building.

Particles containing alpha (α) nuclides were identified from sediment in stagnant water in the Unit 3 reactor building of the Fukushima Daiichi Nuclear Power Station (FDiNPS). We analyzed different concentrations of α-nuclide samples collected at two sampling sites, the torus room and the main steam isolation valve (MSIV) room. The solids in the stagnant water samples were classified, and the uranium (U) and total alpha concentrations of each fraction were measured by dissolution followed by inductively coupled plasma mass spectrometry and α-spectrometry. Most of the α-nuclides in the stagnant water samples from the torus and MSIV rooms were in particle fractions larger than 10 µm. We detected uranium-bearing particles ranging from sub-µm to 10 µm in size by scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) observations. The chemical forms of U particles were determined in U-Zr oxides, oxidized UO2, and U3O8 with micro-Raman spectroscopy. Other short-lived α-nuclides (plutonium [Pu], americium [Am], and curium [Cm]) were detected by alpha track detection, and the particles with α-nuclides was characterized by SEM-EDX analysis. α-nuclide-containing particles with several tens to several 100 µm in size mainly comprised iron (Fe) oxyhydroxides. In addition, we detected adsorbed U onto Fe oxyhydroxide particles in the MSIV room sample, which indicated nuclear fuel dissolution and secondary U accumulation. This study clarifies the major characteristics of U and other α-nuclides in sediment in stagnant water in the FDiNPS Unit 3 reactor building, which significantly contribute to the consideration of removal methods for particles containing α-nuclides in the stagnant water.

Spider and Wasp Acylpolyamines: Venom Components and Versatile Pharmacological Leads, Probes, and Insecticidal Agents.

Polyamines (PAs) are polycationic biogenic amines ubiquitously present in all life forms and are involved in molecular signaling and interaction, determining cell fate (e.g., cell proliferation, dif-ferentiation, and apoptosis). The intricate balance in the PAs' levels in the tissues will determine whether beneficial or detrimental effects will affect homeostasis. It's crucial to note that endoge-nous polyamines, like spermine and spermidine, play a pivotal role in our understanding of neu-rological disorders as they interact with membrane receptors and ion channels, modulating neuro-transmission. In spiders and wasps, monoamines (histamine, dopamine, serotonin, tryptamine) and polyamines (spermine, spermidine, acyl polyamines) comprise, with peptides and other sub-stances, the low molecular weight fraction of the venom. Acylpolyamines are venom components exclusively from spiders and a species of solitary wasp, which cause inhibition chiefly of iono-tropic glutamate receptors (AMPA, NMDA, and KA iGluRs) and nicotinic acetylcholine receptors (nAChRs). The first venom acylpolyamines ever discovered (argiopines, Joro and Nephila toxins, and philanthotoxins) have provided templates for the design and synthesis of numerous analogs. Thus far, analogs with high potency exert their effect at nanomolar concentrations, with high se-lectivity toward their ionotropic and ligand receptors. These potent and selective acylpolyamine analogs can serve biomedical purposes and pest control management. The structural modification of acylpolyamine with photolabile and fluorescent groups converted these venom toxins into use-ful molecular probes to discriminate iGluRs and nAchRs in cell populations. In various cases, the linear polyamines, like spermine and spermidine, constituting venom acyl polyamine backbones, have served as cargoes to deliver active molecules via a polyamine uptake system on diseased cells for targeted therapy. In this review, we examined examples of biogenic amines that play an essential role in neural homeostasis and cell signaling, contributing to human health and disease outcomes, which can be present in the venom of arachnids and hymenopterans. With an empha-sis on the spider and wasp venom acylpolyamines, we focused on the origin, structure, derivatiza-tion, and biomedical and biotechnological application of these pharmacologically attractive, chemically modular venom components.

Isolation of Paraphaeoketones: A Plausible Biosynthetic Explanation for Paraphaeolactones via a Benzilic Acid Rearrangement Rather than a Favorskii Rearrangement.

Paraphaeoketones A-C (1-3) were isolated from the culture broth of Paraphaeosphaeria sp. KT4192. Their structures and relative configurations were determined using spectroscopic analysis and verified through density functional theory (DFT)-based chemical shift calculations. The absolute configurations of these compounds were determined by comparing the experimental electronic circular dichroism (ECD) spectra with those based on DFT calculations. We also propose a plausible biosynthetic route to 1-3. While our prior studies on the isolation and structural elucidation of paraphaeolactones (e.g., 4) led us to suggest a Favorskii rearrangement for their biosynthesis, the isolation of 2 prompted the proposal of an alternative biosynthesis for 4, featuring a benzilic acid rearrangement of 2. Moreover, an in vitro conversion of 2 into 4 was achieved successfully, suggesting that a biosynthetic pathway for paraphaeolactones involving a benzilic acid rearrangement is more plausible than the previously presumed Favorskii rearrangement pathway. Arguments based on DFT calculations for these pathways are also described.

Comprehensive analysis and biological characterization of venom components from solitary scoliid wasp Campsomeriella annulata annulata

Venoms of solitary wasps are utilized for prey capture (insects and spiders), paralyzing them with a stinger injection to be offered as food for their larvae. Thus, the identification and characterization of the components of solitary wasp venoms can have biotechnological application. In the present study, the venom components profile of a solitary scoliid wasp, Campsomeriella annulata annulata, was investigated through a comprehensive analysis using LC-MS and -MS/MS. Online mass fingerprinting revealed that the venom extract contains 138 components, and MS/MS analysis identified 44 complete sequences of the peptide components. The peptides are broadly divided into two classes: bradykinin-related peptides, and linear α-helical peptides. Among the components of the first class, the two main peptides, α-campsomerin (PRLRRLTGLSPLR) and β-campsomerin (PRLRRLTGLSPLRAP), had their biological activities evaluated. Both peptides had no effects on metallopeptidases [human neprilysin (NEP) and angiotensin-converting enzyme (ACE)] and acetylcholinesterase (AChE), and had no cytotoxic effects. Studies with PC12 neuronal cells showed that only α-campsomerin was able to enhance cell viability, while β-campsomerin had no effect. It is noteworthy that the only difference between the primary structures from these peptides is the presence of the AP extension at the C-terminus of β-campsomerin, compared to α-campsomerin. Among the linear α-helical peptides, annulatin (ISEALKSIIVG-NH2) was evaluated for its biological activities. Annulatin showed histamine releasing activity from mast cells and low hemolytic activity, but no antimicrobial activities against all microbes tested were observed. Thus, in addition to providing unprecedented information on the whole components, the three peptides selected for the study suggest that molecules present in solitary scoliid wasp venoms may have interesting biological activities.

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.

Novel neuroprotective peptides in the venom of the solitary scoliid wasp Scolia decorata ventralis

Background Solitary wasp venoms may be a rich source of neuroactive substances, since their venoms are used for paralyzing preys. We have been exploring bioactive constituents of solitary wasp venoms and, in this study, the component profile of the venom from a solitary scoliid wasp, Scolia decorata ventralis, was investigated through a comprehensive analysis using LC-MS. Two peptides were synthesized, and their neuroprotective properties were evaluated. Methods A reverse-phase HPLC connected to ESI-MS was used for LC-MS analyses. Online mass fingerprinting was performed from TIC, and data-dependent tandem mass spectrometry gave the MS/MS spectra. The sequences of two major peptide components were determined by MALDI-TOF/TOF MS analysis, confirmed by solid phase synthesis. Using the synthetic peptides, biological activities were assessed. Cell integrity tests and neuroprotection analyzes using H2O2 as an oxidative stress inducer were performed for both peptides. Results Online mass fingerprinting revealed that the venom contains 123 components, and the MS/MS analysis resulted in 33 full sequences of peptide components. The two main peptides, α-scoliidine (DYVTVKGFSPLR) and β-scoliidine (DYVTVKGFSPLRKA), present homology with the bradykinin C-terminal. Despite this, both peptides did not behave as substrates or inhibitors of ACE, indicating that they do not interact with this metallopeptidase. In further studies, β-scoliidine, but not α -scoliidine, showed protective effects against oxidative stress-induced neurotoxicity in PC12 cells through integrity and metabolism cell assays. Interestingly, β-scoliidine has the extension of the KA dipeptide at the C-terminal in comparison with α-scoliidine. Conclusion Comprehensive LC-MS and MS/MS analyses from the Scolia decorata ventralis venom displayed the component profile of this venom. β-scoliidine showed an effective cytoprotective effect, probably due to the observed increase in the number of cells. This is the first report of solitary wasp venom peptides showing neuroprotective activity.(AU)

Comprehensive analysis of peptides and low molecular weight components of the giant ant Dinoponera quadriceps venom

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that include one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by on-line peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g., glutamic acid and proline), free thymidine and cytosine. To the best of our knowledge most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptides variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and defending D. quadriceps against aggressors, predators and potential microbial infection.

Sa12b peptide from solitary wasp inhibits ASIC currents in rat dorsal root ganglion neurons

In this work, we evaluate the effect of two peptides Sa12b (EDVDHVFLRF) and Sh5b (DVDHVFLRF-NH2) on Acid-Sensing Ion Channels (ASIC). These peptides were purified from the venom of solitary wasps Sphex argentatus argentatus and Isodontia harmandi, respectively. Voltage clamp recordings of ASIC currents were performed in whole cell configuration in primary culture of dorsal root ganglion (DRG) neurons from (P7-P10) CII Long-Evans rats. The peptides were applied by preincubation for 25 s (20 s in pH 7.4 solution and 5 s in pH 6.1 solution) or by co-application (5 s in pH 6.1 solution). Sa12b inhibits ASIC current with an IC50 of 81 nM, in a concentration-dependent manner when preincubation application was used. While Sh5b did not show consistent results having both excitatory and inhibitory effects on the maximum ASIC currents, its complex effect suggests that it presents a selective action on some ASIC subunits. Despite the similarity in their sequences, the action of these peptides differs significantly. Sa12b is the first discovered wasp peptide with a significant ASIC inhibitory effect.

Chemical and biological characteristics of antimicrobial alpha-helical peptides found in solitary wasp venoms and their interactions with model membranes

Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an a-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial a-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.

New mastoparan peptides in the venom of the solitary eumenine wasp eumenes micado

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic a-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly a-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes

Comprehensive analysis of peptides and low molecular weight components of the giant ant Dinoponera quadriceps venom

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that include one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by on-line peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g., glutamic acid and proline), free thymidine and cytosine. To the best of our knowledge most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptides variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and defending D. quadriceps against aggressors, predators and potential microbial infection.

Sa12b peptide from solitary wasp inhibits ASIC currents in rat dorsal root ganglion neurons

In this work, we evaluate the effect of two peptides Sa12b (EDVDHVFLRF) and Sh5b (DVDHVFLRF-NH2) on Acid-Sensing Ion Channels (ASIC). These peptides were purified from the venom of solitary wasps Sphex argentatus argentatus and Isodontia harmandi, respectively. Voltage clamp recordings of ASIC currents were performed in whole cell configuration in primary culture of dorsal root ganglion (DRG) neurons from (P7-P10) CII Long-Evans rats. The peptides were applied by preincubation for 25 s (20 s in pH 7.4 solution and 5 s in pH 6.1 solution) or by co-application (5 s in pH 6.1 solution). Sa12b inhibits ASIC current with an IC50 of 81 nM, in a concentration-dependent manner when preincubation application was used. While Sh5b did not show consistent results having both excitatory and inhibitory effects on the maximum ASIC currents, its complex effect suggests that it presents a selective action on some ASIC subunits. Despite the similarity in their sequences, the action of these peptides differs significantly. Sa12b is the first discovered wasp peptide with a significant ASIC inhibitory effect.

Chemical and biological characteristics of antimicrobial alpha-helical peptides found in solitary wasp venoms and their interactions with model membranes

Solitary wasps use their stinging venoms for paralyzing insect or spider prey and feeding them to their larvae. We have surveyed bioactive substances in solitary wasp venoms, and found antimicrobial peptides together with some other bioactive peptides. Eumenine mastoparan-AF (EMP-AF) was the first to be found from the venom of the solitary eumenine wasp Anterhynchium flavomarginatum micado, showing antimicrobial, histamine-releasing, and hemolytic activities, and adopting an a-helical secondary structure under appropriate conditions. Further survey of solitary wasp venom components revealed that eumenine wasp venoms contained such antimicrobial a-helical peptides as the major peptide component. This review summarizes the results obtained from the studies of these peptides in solitary wasp venoms and some analogs from the viewpoint of (1) chemical and biological characterization; (2) physicochemical properties and secondary structure; and (3) channel-like pore-forming properties.

New mastoparan peptides in the venom of the solitary eumenine wasp eumenes micado

Comprehensive LC-MS and MS/MS analysis of the crude venom extract from the solitary eumenine wasp Eumenes micado revealed the component profile of this venom mostly consisted of small peptides. The major peptide components, eumenine mastoparan-EM1 (EMP-EM1: LKLMGIVKKVLGAL-NH2) and eumenine mastoparan-EM2 (EMP-EM2: LKLLGIVKKVLGAI-NH2), were purified and characterized by the conventional method. The sequences of these new peptides are homologous to mastoparans, the mast cell degranulating peptides from social wasp venoms; they are 14 amino acid residues in length, rich in hydrophobic and basic amino acids, and C-terminal amidated. Accordingly, these new peptides can belong to mastoparan peptides (in other words, linear cationic a-helical peptides). Indeed, the CD spectra of these new peptides showed predominantly a-helix conformation in TFE and SDS. In biological evaluation, both peptides exhibited potent antibacterial activity, moderate degranulation activity from rat peritoneal mast cells, and significant leishmanicidal activity, while they showed virtually no hemolytic activity on human or mouse erythrocytes. These results indicated that EMP-EM peptides rather strongly associated with bacterial cell membranes rather than mammalian cell membranes

Peripheral 5-HT3 Receptors Are Involved in the Antinociceptive Effect of Bunodosine 391

Bunodosine 391 (BDS 391), a low molecular weight compound isolated from the sea anemone Bunodosoma cangicum, increases the nociceptive threshold and inhibits inflammatory hyperalgesia. Serotonin receptors are involved in those effects. In this study, we have expanded the characterization of the antinociceptive effect of BDS 391 demonstrating that, in rats: (a) the compound inhibits (1.2-12 ng/paw) overt pain, in the formalin test, and mechanical hyperalgesia (0.6-6.0 ng/paw) detected in a model of neuropathic pain; (b) intraplantar administration of ondansetron, a selective 5-HT3 receptor antagonist, blocks the effect of BDS 391, whereas ketanserin, a 5-HT2 receptor antagonist, partially reversed this effect, indicating the involvement of peripheral 5-HT2 and 5-HT3 receptors in BDS 391 antinociception; and (c) in binding assay studies, BDS 391 was not able to displace the selective 5-HT receptor antagonists, suggesting that this compound does not directly bind to these receptors. The effect of biguanide, a selective 5-HT3 receptor agonist, was also evaluated. The agonist inhibited the formalin's nociceptive response, supporting an antinociceptive role for 5-HT3 receptors. Our study is the first one to show that a non-peptidic low molecular weight compound obtained from a sea anemone is able to induce antinociception and that activation of peripheral 5-HT3 receptors contributes to this effect.

Peripheral 5-HT3 Receptors Are Involved in the Antinociceptive Effect of Bunodosine 391

Bunodosine 391 (BDS 391), a low molecular weight compound isolated from the sea anemone Bunodosoma cangicum, increases the nociceptive threshold and inhibits inflammatory hyperalgesia. Serotonin receptors are involved in those effects. In this study, we have expanded the characterization of the antinociceptive effect of BDS 391 demonstrating that, in rats: (a) the compound inhibits (1.2-12 ng/paw) overt pain, in the formalin test, and mechanical hyperalgesia (0.6-6.0 ng/paw) detected in a model of neuropathic pain; (b) intraplantar administration of ondansetron, a selective 5-HT3 receptor antagonist, blocks the effect of BDS 391, whereas ketanserin, a 5-HT2 receptor antagonist, partially reversed this effect, indicating the involvement of peripheral 5-HT2 and 5-HT3 receptors in BDS 391 antinociception; and (c) in binding assay studies, BDS 391 was not able to displace the selective 5-HT receptor antagonists, suggesting that this compound does not directly bind to these receptors. The effect of biguanide, a selective 5-HT3 receptor agonist, was also evaluated. The agonist inhibited the formalin's nociceptive response, supporting an antinociceptive role for 5-HT3 receptors. Our study is the first one to show that a non-peptidic low molecular weight compound obtained from a sea anemone is able to induce antinociception and that activation of peripheral 5-HT3 receptors contributes to this effect.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

Background: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic alpha-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic alpha-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic alpha-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

Background: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic α-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic α-helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic α-helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.(AU)

Peptidomic analysis of the venom of the solitary bee Xylocopa appendiculata circumvolans

Abstract Background: Among the hymenopteran insect venoms, those from social wasps and bees - such as honeybee, hornets and paper wasps - have been well documented. Their venoms are composed of a number of peptides and proteins and used for defending their nests and themselves from predators. In contrast, the venoms of solitary wasps and bees have not been the object of further research. In case of solitary bees, only major peptide components in a few venoms have been addressed. Therefore, the aim of the present study was to explore the peptide component profile of the venom from the solitary bee Xylocopa appendiculata circumvolans by peptidomic analysis with using LC-MS. Methods: A reverse-phase HPLC connected to ESI-OrbiTrap MS was used for LC-MS. On-line mass fingerprinting was made from TIC, and data-dependent tandem mass spectrometry gave MSMS spectra. A major peptide component was isolated by reverse-phase HPLC by conventional way, and its sequence was determined by Edman degradation, which was finally corroborated by solid phase synthesis. Using the synthetic specimen, biological activities (antimicrobial activity, mast cell devaluation, hemolysis, leishmanicidal activity) and pore formation in artificial lipid bilayer were evaluated. Results: On-line mass fingerprinting revealed that the crude venom contained 124 components. MS/MS analysis gave 75 full sequences of the peptide components. Most of these are related to the major and novel peptide, xylopin. Its sequence, GFVALLKKLPLILKHLH-NH2, has characteristic features of linear cationic -helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic -helix secondary structure. In biological evaluation, xylopin exhibited broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. Additionally, the peptide was able to incorporate pores in artificial lipid bilayers of azolectin, confirming the mechanism of the cytolytic activity by pore formation in biological membranes. Conclusions: LC-ESI-MS and MS/MS analysis of the crude venom extract from a solitary bee Xylocopa appendiculata circumvolans revealed that the component profile of this venom mostly consisted of small peptides. The major peptide components, xylopin and xylopinin, were purified and characterized in a conventional manner. Their chemical and biological characteristics, belonging to linear cationic -helical peptides, are similar to the known solitary bee venom peptides, melectin and osmin. Pore formation in artificial lipid bilayers was demonstrated for the first time with a solitary bee peptide.

Chemical and biological characterization of four new linear cationic á-helical peptides from the venoms of two solitary eumenine wasps

Four novel peptides were isolated from the venoms of the solitary eumeninewasps Eumenes rubrofemoratus and Eumenes fraterculus. Their sequences were determined by MALDI-TOF/ TOF (matrix assisted laser desorption/ionization time-of-flight mass spectrometry)analysis, Edman degradation and solid-phase synthesis. Two of them, eumenitin-R (LNLKGLIKKVASLLN) and eumenitin-F (LNLKGLFKKVASLLT), are highly homologous to eumenitin, an antimicrobial peptide from a solitary eumeninewasp, whereas the other two, EMP-ER (FDIMGLIKKVAGAL-NH2) and EMP-EF (FDVMGIIKKIAGAL-NH2), are similar to eumenine mastoparan-AF (EMP-AF), a mast cell degranulating peptide from a solitary eumeninewasp. These sequences have the characteristic features of linear cationic cytolyticpeptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, they can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD (circular dichroism) spectra of these peptides showed significant a-helical conformation content in the presence of TFE (trifluoroethanol), SDS (sodium dodecylsulfate) and asolectin vesicles. In the biological evaluation, all the peptides exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity.