Assessment of the In Vivo and In Vitro Release of Chemical Compounds from Vespa velutina

Vespa velutina has been rapidly expanding throughout Galicia since 2012. It is causing human health risks and well-known losses in the beekeeping sector. Control methods are scarce, unspecific, and ineffective. Semiochemicals are insect-derived chemicals that play a role in communication and they could be used an integrated pest management tool alternative to conventional pesticides. A previous determination of the organic chemical profile should be the first step in the study of these semiochemicals. HS-SPME in living individuals and the sting apparatus extraction followed by GC-MS spectrometry were combined to extract a possible profile of these compounds in 43 hornets from Galicia. The identified compounds were hydrocarbons, ketones, terpenes, and fatty acid, and fatty acid esters. Nonanal aldehyde appeared in important concentrations in living individuals. While pentadecane, 8-hexyl- and ethyl oleate were mainly extracted from the venom apparatus. Ketones 2-nonanone, 2-undecanone and 7-nonen-2-one, 4,8-dimethyl- were identified by both procedures, as was 1,7-Nonadiene, 4,8-dimethyl-. Some compounds were detected for the first time in V. velutina such as naphthalene, 1,6-dimethyl-4-(1-methylethyl). The chemical profile by caste was also characterized.     

Apis mellifera syriaca Venom: Evaluation of Its Anticoagulant Effect,Proteolytic Activity,and Cytotoxicity along with Its Two Main Compounds—MEL and PLA2—On HeLa Cancer Cells

Bee venom (BV) is one of the most remarkable natural products that has been a subject of studies since ancient times. Recent studies have shown that Apis mellifera syriaca venom possesses antibacterial as well as cytotoxic effects on cancer cell lines. The venom contains a variety of bioactive molecules—mainly melittin (MEL) and phospholipase A2 (PLA2), as well as other compounds that are not well characterized. In this work, we continue the biological characterization of A. mellifera syriaca venom by testing its anticoagulant effect on human plasma using the prothrombin time (PT) test, as well as assessing its proteolytic activity. In addition, the cytotoxicity of the crude venom—and of its two main components, MEL and PLA2—was tested on HeLa cancer cell lines for the first time. The results obtained showed the capacity of A. mellifera syriaca venom to increase clotting time, thereby proving its anticoagulant effect. Moreover, the venom did not demonstrate a significant proteolytic activity unless administrated at concentrations ≥ 5 mg/mL. Finally, we showed that crude A. mellifera syriaca venom, along with MEL, exhibit a strong in vitro cytotoxic effect on HeLa cancer cell lines, even at low concentrations. In summary, our findings could serve as a basis for the development of new natural-based drug candidates in the therapeutic field.     

Thermal characterization of social vespid silk

In this paper we try to establish a link between the microclimate in the wasp nest and the structure and thermal stability of vespid silk. We suggest that there are at least two types of water that is absorbed by the silk of Oriental hornets, namely, surface water and intrinsic structural water. The release of both types of water was found to be reversible. The enthalpy values of the endothermic peaks associated with the release of water from different silk samples do not differ substantially and are in the range of 106 to 130 J g^-1 for the Vespa orientalis male larvae silk (sample #1), Paravespula germanica (yellowjacket) worker larvae silk (#3) and Vespa orientalis nest envelope(#4). For the Vespa orientalis worker larvae silk (sample #2), however, it is twice as large (228 J g^-1). This is in agreement with the increased total amount of absorbed water. The silk studied has a fibrilar structure with interconnecting surfaces overlying entire regions. It is assumed that the initial water loss stems from water evaporation from the coat of the fibers - a daily occurrence in the hornets' nest. Heating to above 70°C may result in structural changes in the silk core. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low-molecular weight hyaluronidase from the venom of Bungarus caeruleus (Indian common krait) snake: Isolation and partial characterization

Snake venom hyaluronidases known as “spreading factor” are not extensively studied. Recently, it is argued that beyond its role as a spreading factor, venom hyaluronidase (HYL) deserves to be explored as a possible therapeutic target for inhibiting the systemic distribution of venom/toxins and also for minimizing local tissue destruction. In this context, in the present study, a low-molecular weight HYL has been isolated from Bungarus caeruleus (Indian krait) venom by single step chromatography on HPLC system. The apparent molecular weight determined by SDS-PAGE is 14 ± 2 kDa, as confirmed by zymogen study and LC–MS as well. The enzyme had optimal pH 6 and temperature 37°C. The Michaelis–Menten constant (K_m) was found to be 8.48 µg/mL at 37°C. The activity of purified enzyme was completely inhibited by Ba²⁺ metal ion and N-acetyl imidazole group-specific agents. This work yielded a highly active HYL from B. caeruleus the first one to be isolated. Further studies on its pharmacological actions will be interesting to develop lead molecules for better management of snakebite.     

Inhibition of Snake Venom Metalloproteinase by β-Lactoglobulin Peptide from Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Colostrum

Bioactive peptide research has experienced considerable therapeutic interest owing to varied physiological functions, efficacy in excretion, and tolerability of peptides. Colostrum is a rich natural source of bioactive peptides with many properties elucidated such as anti-thrombotic, anti-hypertensive, opioid, immunomodulatory, etc. In this study, a variant peptide derived from β-lactoglobulin from buffalo colostrum was evaluated for the anti-ophidian property by targeting snake venom metalloproteinases. These are responsible for rapid local tissue damages that develop after snakebite such as edema, hemorrhage, myonecrosis, and extracellular matrix degradation. The peptide identified by LC-MS/MS effectively neutralized hemorrhagic activity of the Echis carinatus venom in a dose-dependent manner. Histological examinations revealed that the peptide mitigated basement membrane degradation and accumulation of inflammatory leucocytes at the venom-injected site. Inhibition of proteolytic activity was evidenced in both casein and gelatin zymograms. Also, inhibition of fibrinolytic and fibrinogenolytic activities was seen. The UV-visible spectral study implicated Zn²⁺ chelation, which was further confirmed by molecular docking and dynamic studies by assessing molecular interactions, thus implicating the probable mechanism for inhibition of venom-induced proteolytic and hemorrhagic activities. The present investigation establishes newer vista for the BLG-col peptide with anti-ophidian efficacy as a promising candidate for therapeutic interventions.     

Excretion kinetics of 13C-urea breath test: influences of endogenous CO2 production and dose recovery on the diagnostic accuracy of Helicobacter pylori infection

We report for the first time the excretion kinetics of the percentage dose of ¹³C recovered/h (¹³C-PDR %/h) and cumulative PDR, i.e. c-PDR (%) to accomplish the highest diagnostic accuracy of the ¹³C-urea breath test (¹³C-UBT) for the detection of Helicobacter pylori infection without any risk of diagnostic errors using an optical cavity-enhanced integrated cavity output spectroscopy (ICOS) method. An optimal diagnostic cut-off point for the presence of H. pylori infection was determined to be c-PDR (%)?=?1.47 % at 60 min, using the receiver operating characteristic curve (ROC) analysis to overcome the “grey zone” containing false-positive and false-negative results of the ¹³C-UBT. The present ¹³C-UBT exhibited 100 % diagnostic sensitivity (true-positive rate) and 100 % specificity (true-negative rate) with an accuracy of 100 % compared with invasive endoscopy and biopsy tests. Our c-PDR (%) methodology also manifested both diagnostic positive and negative predictive values of 100 %, demonstrating excellent diagnostic accuracy. We also observed that the effect of endogenous CO₂ production related to basal metabolic rates in individuals was statistically insignificant (p?=?0.78) on the diagnostic accuracy. However, the presence of H. pylori infection was indicated by the profound effect of urea hydrolysis rate (UHR). Our findings suggest that the current c-PDR (%) is a valid and sufficiently robust novel approach for an accurate, specific, fast and noninvasive diagnosis of H. pylori infection, which could routinely be used for large-scale screening purposes and diagnostic assessment, i.e. for early detection and follow-up of patients. Figure

The excretion kinetics of the ¹³C-urea breath test with an ICOS system is demonstrated for accurate, specific, fast and noninvasive diagnosis of H. pylori infection in the human stomach     

A new peptide from venom of the East-European hornet Vespa orientalis. Mass spectrometric de novo sequence

The de novo sequence of a new peptide from venom of the East-European hornet Vespa orientalis L. was determined by mass spectrometry. It was used as an example to show that time-of-flight mass spectrometry with an electrospray ion source can be used effectively to determine the amino-acid sequence of microquantities of peptides. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 49–51, January–February, 2008.     

In Silico and In Vitro Structure–Activity Relationship of Mastoparan and Its Analogs

Antimicrobial peptides are an important class of therapeutic agent used against a wide range of pathogens such as Gram-negative and Gram-positive bacteria, fungi, and viruses. Mastoparan (MpVT) is an α-helix and amphipathic tetradecapeptide obtained from Vespa tropica venom. This peptide exhibits antibacterial activity. In this work, we investigate the effect of amino acid substitutions and deletion of the first three C-terminal residues on the structure–activity relationship. In this in silico study, the predicted structure of MpVT and its analog have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bonds. The secondary structure and the biological activity of six designed analogs are studied. The biological activity assays show that the substitution of phenylalanine (MpVT1) results in a higher antibacterial activity than that of MpVT without increasing toxicity. The analogs with the first three deleted C-terminal residues showed decreased antibacterial and hemolytic activity. The CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 40% 2,2,2-trifluoroethanol (TFE). In conclusion, the first three C-terminal deletions reduced the length of the α-helix, explaining the decreased biological activity. MpVTs show that the hemolytic activity of mastoparan is correlated to mean hydrophobicity and mean hydrophobic moment. The position and spatial arrangement of specific hydrophobic residues on the non-polar face of α-helical AMPs may be crucial for the interaction of AMPs with cell membranes.     

Cytotoxicity of Silica Nanoparticles with Transcaucasian Nose-Horned Viper, <Emphasis Type="Italic">Vipera ammodytes transcaucasiana</Emphasis>, Venom on U87MG and SHSY5Y Neuronal Cancer Cells

Highly bioactive compounds of the snake venom make them particular sources for anticancer agent development. They contain very rich peptide-protein structures. Therefore, they are very susceptible to environmental conditions such as temperature, pH, and light. In this study, Vipera ammodytes transcaucasiana venom was encapsulated in PAMAM-G4 dendrimer by sol-gel method in order to prevent degradation of venom contents from the environmental conditions. For this purpose, nanoparticles were prepared by sol-gel methodology and SEM analyses were performed. U87MG and SHSY5Y neuronal cancer cell lines were treated with different concentrations of venom-containing nanoparticles and cytotoxicity was determined by MTT assay. IC₅₀ values of nanoparticles with snake venom were calculated as 37.24 and 44.64 μg/ml for U87MG and SHSY5Y cells, respectively. The IC₅₀ values of nanoparticles with snake venom were calculated as 10.07 and 7.9 μg/ml for U87MG and SHSY5Y cells, respectively. As a result, nanoparticles with V. a. transcaucasiana venom showed remarkably high cytotoxicity. Encapsulation efficiency of nanoparticles with 1 mg/ml snake venom was determined as %67 via BCA? protein analysis. In conclusion, this method is found to be convenient and useful for encapsulating snake venom as well as being suitable for drug delivery systems.     

Nomad Jellyfish Rhopilema nomadica Venom Induces Apoptotic Cell Death and Cell Cycle Arrest in Human Hepatocellular Carcinoma HepG2 Cells

Jellyfish venom is a rich source of bioactive proteins and peptides with various biological activities including antioxidant, antimicrobial and antitumor effects. However, the anti-proliferative activity of the crude extract of Rhopilema nomadica jellyfish venom has not been examined yet. The present study aimed at the investigation of the in vitro effect of R. nomadica venom on liver cancer cells (HepG2), breast cancer cells (MDA-MB231), human normal fibroblast (HFB4), and human normal lung cells (WI-38) proliferation by using MTT assay. The apoptotic cell death in HepG2 cells was investigated using Annexin V-FITC/PI double staining-based flow cytometry analysis, western blot analysis, and DNA fragmentation assays. R. nomadica venom displayed significant dose-dependent cytotoxicity on HepG2 cells after 48 h of treatment with IC₅₀ value of 50 μg/mL and higher toxicity (3:5-fold change) against MDA-MB231, HFB4, and WI-38 cells. R. nomadica venom showed a prominent increase of apoptosis as revealed by cell cycle arrest at G2/M phase, upregulation of p53, BAX, and caspase-3 proteins, and the down-regulation of anti-apoptotic Bcl-2 protein and DNA fragmentation. These findings suggest that R. nomadica venom induces apoptosis in hepatocellular carcinoma cells. To the best of the authors’ knowledge, this is the first scientific evidence demonstrating the induction of apoptosis and cell cycle arrest of R. nomadica jellyfish venom.     

Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na⁺/K⁺‐ATPase pump by inhibiting the cardiotonic steroid dependent‐site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt‐sensitive hypertensive states such as essential hypertension, congestive heart‐failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin‐layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid‐phase extraction coupled with fractionation on high‐performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid‐phase extraction combined with high‐performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time‐consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.     

Determination of Defibrase in Pharmaceutical Formulation by LC with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate Derivatization and Fluorescence Detection

A sensitive LC method with pre-column fluorescence derivatization using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate has been developed for the determination of defibrase from the venom of Agkistrodon acutus (also named defibrase in the Chinese Pharmacopoeia) in pharmaceutical formulation. By a denaturing procedure prior to derivatization, full and homogeneously derivatization of defibrase was obtained and demonstrated using MALDI-TOF-MS. The specificity of the method was validated by assessing interference from the placebo and by forced degradation. The method has demonstrated good linearity (r = 0.9998), accuracy (mean recovery 98.53%) and precision (RSD < 2.5%). Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL^?1) and limit of quantitation (10.16 ng mL^?1). The method was successfully applied to the quantitative determination of defibrase in pharmaceutical formulation.     

Synthesis and Insecticidal Activity of Fire Ant Venom Alkaloid-Based 2-Methyl-6-alkyl-Δ1,6-piperideines

2,6-dialkylpiperideines found in the venom of Solenopsis (Hymenoptera, Formicidae) fire ants are a range of compounds possessing various biological activities. A series of racemic 2-methyl-6-alkyl-Δ^1,6-piperideines were synthesized for chemical confirmation of the natural products found in fire ant venom, and the evaluation of their biological activity. Synthetic Δ^1,6-piperideines and the natural compounds in the cis-alkaloid fraction of Solenopsis invicta had identical mass spectra and retention times. Their insecticidal activities against the third-instar larvae of cotton bollworm (Helicoverpa armigera) were evaluated by using injection and topical application methods. All three compounds exhibited no lethal effect at concentrations of 0.05–0.4 mol/L by topical treatment, but moderate lethal effect at 0.4 mol/L through injection treatment. Compound 6a showed significantly higher activity than the natural insecticide nicotine. The differences in activity among compounds 6b, 6c and nicotine were not significant. The elongation of the carbon chain at the 6-position of the piperideine ring appears to decrease insecticidal activity.     

Selective detection of phosphopeptides in complex mixtures by electrospray liquid chromatography/mass spectrometry

A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (≤320-μm i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phospho|peptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO₂ ^?; and PO₃ ^?, respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.     

Determination of the inclusion complex constant between oleuropein and cyclodextrins by complexation theory

Oleuropein (OLE) is a major phenolic compound of olive leaf (Olea europaea) and has many therapeutic properties associated with olive leaf extracts. This work concerns the determination of the inclusion complex constant between OLE and cyclodextrins (CDs), based on the competition of two guests for the CD cavity, one being a dye and the other OLE. The dye used was methylorange (MO) and pH 3 was selected, since MO molar absorptivity at 500 nm is at maximum in this condition. A solution of MO, OLE, and α-CD or β-CD, with citrate buffer was used for determining the absorbance values. From these data and by appropriate mathematical modeling, the equilibrium constant for the formation of OLE:CD complexes were obtained: for OLE:α-CD K = 1,352.4 L mol^?1 (R ² = 0.9975) and for OLE:β-CD K = 1,827.9 L mol^?1 (R ² = 0.9991). The results show that OLE has a greater affinity for β-CD than for α-CD and given the relatively high constants, OLE:CD complexes have potential for giving longer shelf lives for OLE medicinal and food additive preparations.     

An immunochemical strategy based on peptidoglycan synthetic peptide epitopes to diagnose Staphylococcus aureus infections

The characteristic pentaglycyl cross-bridge of the Staphylococcus aureus peptidoglycan (PG) cell wall component is an attractive epitope to raise specific antibodies against this microorganism. Based on this approach, we report here for the first time a competitive ELISA able to detect S. aureus down to 10⁴ CFU mL⁻¹, without pre-enrichment on cell culture. The antibodies were raised against peptide-protein bioconjugates prepared by covalently coupling peptide haptens (PSau6 and PSau8) designed and synthesized taking into consideration the complex tridimensional structure in the PG polymer. Deglycosylation of the PG under acidic conditions has found to increase assay detectability. Assay performance has been evaluated in clinical samples such as bronchoalveolar lavage (BAL) and bronchoalveolar endotracheal aspirates (BAS) showing promising results for further implementation of this immunoassay as a daily routine diagnostic tool. Cross-reactivity studies have demonstrated that the immunoassay is specific for S. aureus.     

Resistance of vasoactive peptides from venom of Vespa orientalis to action of proteolytic enzymes

The kinetics of the hydrolysis by proteolytic enzymes of vasoactive peptides isolated from the venom of the hornetVespa orientalis has been investigated. It has been found that the half-period of decomposition of the peptides exceeds that of bradykinin. These facts are in harmony with in vivo results on the prolonged hypotensive action of the peptides.A. S. Sadykov Institute of Bioorganic Chemistry, Uzbek SSR Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 658–661, September–October, 1988.     

Analysis of the Composition of Deinagkistrodon acutus Snake Venom Based on Proteomics,and Its Antithrombotic Activity and Toxicity Studies

There is a strong correlation between the composition of Deinagkistrodon acutus venom proteins and their potential pharmacological effects. The proteomic analysis revealed 103 proteins identified through label-free proteomics from 30 different snake venom families. Phospholipase A2 (30.0%), snaclec (21.0%), antithrombin (17.8%), thrombin (8.1%) and metalloproteinases (4.2%) were the most abundant proteins. The main toxicity of Deinagkistrodon acutus venom is hematotoxicity and neurotoxicity, and it acts on the lung. Deinagkistrodon acutus venom may have anticoagulant and antithrombotic effects. In summary, the protein profile and related toxicity and pharmacological activity of Deinagkistrodon acutus venom from southwest China were put forward for the first time. In addition, we revealed the relationship between the main toxicity, pharmacological effects, and the protein components of snake venom.     

Isolation and characterization of a novel lepidopteran-selective toxin from the venom of South Indian red scorpion, Mesobuthus tamulus

Background

Scorpion venom contains insect and mammal selective toxins. We investigated the venom of the South Indian red scorpion, Mesobuthus tamulus for the purpose of identifying potent insecticidal peptide toxins.

Results

A lepidopteran-selective toxin (Buthus tamulus insect toxin; ButaIT) has been isolated from this venom. The primary structure analysis reveals that it is a single polypeptide composed of 37 amino acids cross-linked by four disulfide bridges with high sequence homology to other short toxins such as Peptide I, neurotoxin P2, Lqh-8/6, chlorotoxin, insectotoxin I5A, insect toxin 15 and insectotoxin I1. Three dimensional modeling using Swiss automated protein modeling server reveals that this toxin contains a short α-helix and three antiparallel β-strands, similar to other short scorpion toxins. This toxin is selectively active on Heliothis virescens causing flaccid paralysis but was non-toxic to blowfly larvae and mice.

Conclusion

This is the first report of a Heliothine selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.