Antiapoptotic role of major royal jelly protein 8 of honeybee (Apis mellifera) venom

The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H₂O₂ and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses.     

Early prostaglandin E synthesis is an obligatory event in the induction of cell proliferation in mouse epidermis in vivo by the phorbol ester TPA

Following the topical application of the phorbol ester TPA to mouse skin $\text{in vivo}$ a rapid increase of the prostaglandin E content after 10 and 60 minutes was observed. Pretreatment of mouse skin with indomethacin abolished the first PGE peak as well as the cellular proliferation induced by TPA. Both effects could not be prevented when indomethacin was applied 30 to 60 minutes after TPA treatment, suggesting that the early increase in epidermal PGE is an obligatory event in the course of the induction of epidermal cell proliferation by TPA. A small increase of epidermal PGE was also seen after treatment with the TPA-analogue “Ti₈”, whereas 4-O-methyl-TPA was inactive in this respect. “Ti₈”-induced epidermal cell proliferation could be partially inhibited by indomethacin, whereas 4-O-methyl-TPA-induced cell proliferation was insensitive to the drug.     

Single-molecule phospholipase A2 becomes processive on melittin-induced membrane deformations

While it is established that the topology of lipid membranes plays an important role in biochemical processes, few direct observations exist regarding how the membranes are actively restructured and its consequences on subsequent reactions. In this work, we investigated how the two major components of bee venom, melittin and phospholipase A₂ (PLA₂), achieve activation by such membrane remodeling. Their membrane-disrupting functions have been reported to increase when both are present, but the mechanism of this synergism had not been established. Using membrane reconstitution, we found that melittin can form large-scale membrane deformities upon which PLA₂ activity is 25-fold higher. Tracking of single-molecule PLA₂ revealed that its processive behavior on these deformities underlies the enhanced activity. These results show how melittin and PLA₂ work synergistically to enhance the lytic effects of the bee venom. More broadly, they also demonstrate how the membrane topology may be actively altered to modulate cellular membrane-bound reactions.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Design of a Modern Liposome and Bee Venom Formulation for the Traditional VIT-Venom Immunotherapy

Traditional venom immunotherapy uses injections of whole bee venom in buffer or adsorbed in Al (OH)₃ in an expensive, time-consuming way. New strategies to improve the safety and efficacy of this treatment with a reduction of injections would, therefore, be of general interest. It would improve patient compliance and provide socio-economic benefits. Liposomes have a long tradition in drug delivery because they increase the therapeutic index and avoid drug degradation and secondary effects. However, bee venom melittin (Mel) and phospholipase (PLA₂) destroy the phospholipid membranes. Our central idea was to inhibit the PLA₂ and Mel activities through histidine alkylation and or tryptophan oxidation (with pbb, para-bromo-phenacyl bromide, and/or NBS- N-bromosuccinimide, respectively) to make their encapsulations possible within stabilized liposomes. We strongly believe that this formulation will be nontoxic but immunogenic. In this paper, we present the whole bee venom conformation characterization during and after chemical modification and after interaction with liposome by ultraviolet, circular dichroism, and fluorescence spectroscopies. The PLA₂ and Mel activities were measured indirectly by changes in turbidity at 400_{n m}, rhodamine leak-out, and hemolysis. The native whole bee venom (BV) presented 78.06%?of α-helical content. The alkylation (A-BV) and succynilation (S-BV) of BV increased 0.44 and 0.20%?of its α-helical content. The double-modified venom (S-A-BV) had a 0.74%?increase of α-helical content. The BV chemical modification induced another change on protein conformations observed by Trp that became buried with respect to the native whole BV. It was demonstrated that the liposomal membranes must contain pbb (SPC:Cho:pbb, 26:7:1) as a component to protect them from aggregation and/or fusion. The membranes containing pbb maintained the same turbidity (100%) after incubation with modified venom, in contrast with pbb-free membranes that showed a 15%?size decrease. This size decrease was interpreted as membrane degradation and was corroborated by a 50%?rhodamine leak-out. Another fact that confirmed our interpretation was the observed 100%?inhibition of the hemolytic activity after venom modification with pbb and NBS (S-A-BV). When S-A-BV interacted with liposomes, other protein conformational changes were observed and characterized by the increase of 1.93%?on S-A-BV α-helical content and the presence of tryptophan residues in a more hydrophobic environment. In other words, the S-A-BV interacted with liposomal membranes, but this interaction was not effective to cause aggregation, leak-out, or fusion. A stable formulation composed by S-A-BV encapsulated within liposomes composed by SPC:Cho:pbb, at a ratio of 26:7:1, was devised. Large unilamellar vesicles of 202.5 nm with a negative surface charge (–24.29 mV) encapsulated 95%?of S-A-BV. This formulation can, now, be assayed on VIT.     

Phospholipases: melittin facilitation of bee venom phospholipase A2-catalyzed hydrolysis of unsonicated lecithin liposomes.

Melittin isolated from the venom of the common honey bee is a potent activator for bee venom phospholipase A₂-catalyzed hydrolysis of unsonicated liposomes of egg phosphatidyl choline. At 37 °C and pH 8, the rate of this enzymatic reaction is increased approximately 300-fold by the addition of 8 × 10^?5m melittin. The magnitude of facilitation of the phospholipase A₂ reaction is much greater than that previously reported by other workers for systems involving sonicated egg phosphatidyl choline liposomes or Escherichia coli membrane fragments as substrates. Melittin having lysines quantitatively modified through reaction with methyl acetimidate is as effective a potentiator of phospholipase A₂ activity as the unmodified material. The same result was obtained for melittin in which the single tryptophan residue was modified. Melittin modified by succinylation retained approximately 50% of its capacity to facilitate phospholipase A₂ activity. In contrast, a modified melittin in which the C-terminal four amino residues were removed, acetimidated des(23–26)melittin, is a very poor activator, as is a mixture of this peptide with the C-terminal tetrapeptide. In contrast to the results with egg lecithin liposomes, melittin has little influence on the susceptibility of monomolecular aqueous solutions of dihexanoylphosphatidyl choline to phospholipase A₂ attack.     

Effects of the bumblebee (Bombus ignitus) venom serine protease inhibitor on serine protease and phospholipase A2 of B. ignitus venom

Bumblebee venom contains serine proteases and serine protease inhibitors. In this study, we characterized whether the bumblebee (Bombus ignitus) venom serine protease inhibitor (Bi-KTI) inhibits B. ignitus venom serine protease (Bi-VSP) or phospholipase A₂ (Bi-PLA₂). Bi-KTI did not inhibit Bi-VSP activity at pH 5.4 or 7.4, whereas Bi-KTI slightly inhibited Bi-VSP activity at pH 7.4 after a 30 min preincubation. The Bi-VSP activity that converts prothrombin into thrombin and fibrin into fibrin degradation products was not significantly affected by Bi-KTI. Additionally, Bi-KTI or Bi-VSP did not inhibit Bi-PLA₂ activity. These findings indicate that each bee venom component appears to a play a toxic role via a unique function.     

In vivo regional levels of PGE and thromboxane in mouse brain: Effect of decapitation,focused microwave fixation,and indomethacin

A focused microwave fixation technique was tested for use in determining basal PGE and thromboxane B₂ levels of mouse brain. Focused microwave irradiation (3.5 Kw/0.4 sec) to the head of C3H mice produced basal values of PGE and TXB₂ which were five-fold less than those in animals killed by decapitation. Indomethacin (10 mg/kg) pretreatment blocked the decapitation rise in PGE and TXB₂ levels and gave values similar to focused microwave irradiation. Indomethacin pretreatment combined with microwave fixation did not reduce PG levels more tham microwave treatment alone. When microwave fixation was used, there was no difference in regional (cerebral cortex, whole cerebellum, midbrain, hypothalamus) levels of either PGE or TXB₂. However, PGE levels were significantly higher than TXB₂ in all regions. After decapitation there was a greater increase in TXB₂ than PGE. The cerebellum produced less PGE and TXB₂ after decapitation compared to the other regions. Our results confirm the usefulness of the focused microwave irradiation technique for examining $\text{in vivo}$ basal prostaglandin levels in mouse brain.     

Bee venom ameliorates cardiac dysfunction in diabetic hyperlipidemic rats

High levels of blood glucose and lipids are well-known risk factors for heart diseases. Bee venom is a natural product that has a potent hypoglycemic, hypolipidemic, anti-inflammatory, and antioxidant effects. The current study aimed to determine the bee venom effects on cardiac dysfunction compared to combined therapy of metformin and atorvastatin in diabetic hyperlipidemic rats. The median lethal dose of bee venom was estimated, and then 50 adult male albino rats were categorized into five groups. One group was fed a standard diet and served as a negative control, while the other groups were given nicotinamide and streptozotocin injections to induce type 2 diabetes. After confirming diabetes, the rats were fed a high-fat diet for four weeks. The four groups were divided as follows: one group served as a positive control, whereas the other three groups were treated with bee venom (0.5 mg/kg), bee venom (1.23 mg/kg), and combined therapy of metformin (60 mg/kg) and atorvastatin (10 mg/kg), respectively, for four weeks. Upon termination of the experiment, blood samples and heart tissue were obtained. Administration of bee venom using both doses (0.5 and 1.23 mg/kg) and combined therapy of metformin and atorvastatin revealed a significant decrease in the concentrations of glucose, total cholesterol, triacylglycerol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol, troponin I, creatine kinase, and lactate dehydrogenase activities. Moreover, a significant decrease had been detedcted in malondialdehyde, nuclear factor-kappa-β levels, and relative mRNA expression of vascular cell adhesion molecule-1 and galectin-3 in heart tissue compared to the positive control (P < 0.0001). Furthermore, there was a significant increase in bodyweight levels of insulin, high-density lipoprotein cholesterol, and total antioxidant capacity in heart tissue compared to the positive control (P < 0.0001). The results indicate that bee venom can ameliorate cardiac dysfunction through attenuating oxidative stress and downregulating the NF-κβ signaling pathway.     

Phospholipase A2 inhibitors from marine algae

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A₂ (PLA₂) (> 50%) in the M range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., B furcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA₂ inhibition in vitro by pure compounds isolated from marine algae.     

Bee Venom Phospholipase A2 Protects against Acetaminophen-Induced Acute Liver Injury by Modulating Regulatory T Cells and IL-10 in Mice

The aim of this study was to investigate the protective effects of phospholipase A2 (PLA2) from bee venom against acetaminophen-induced hepatotoxicity through CD4⁺CD25⁺Foxp3⁺ T cells (Treg) in mice. Acetaminophen (APAP) is a widely used antipyretic and analgesic, but an acute or cumulative overdose of acetaminophen can cause severe hepatic failure. Tregs have been reported to possess protective effects in various liver diseases and kidney toxicity. We previously found that bee venom strongly increased the Treg population in splenocytes and subsequently suppressed immune disorders. More recently, we found that the effective component of bee venom is PLA2. Thus, we hypothesized that PLA2 could protect against liver injury induced by acetaminophen. To evaluate the hepatoprotective effects of PLA2, C57BL/6 mice or interleukin-10-deficient (IL-10^−/−) mice were injected with PLA2 once a day for five days and sacrificed 24 h (h) after acetaminophen injection. The blood sera were collected 0, 6, and 24 h after acetaminophen injection for the analysis of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). PLA2-injected mice showed reduced levels of serum AST, ALT, proinflammatory cytokines, and nitric oxide (NO) compared with the PBS-injected control mice. However, IL-10 was significantly increased in the PLA2-injected mice. These hepatic protective effects were abolished in Treg-depleted mice by antibody treatment and in IL-10^−/− mice. Based on these findings, it can be concluded that the protective effects of PLA2 against acetaminophen-induced hepatotoxicity can be mediated by modulating the Treg and IL-10 production.     

Bee Venom Induces the Interaction between Phosphorylated Histone Variant,H2AX,and the Intracellular Site of beta-Actin in Liver and Breast Cancer Cells

Bee venom is a natural mixture and candidate anti-cancer agent with selective cytotoxic effect on some cancer cells. However, the cellular mechanisms of how bee venom selectively targets cancer cells remain elusive. The aim of this study was to reveal the genotoxic effect of bee venom in concordance with the location of β-actin protein throughout the nucleus or/and cytoplasm. For this aim, the level of H2AX phosphorylation (γH2AX) and intracellular location of β-actin were assessed by immunofluorescence in liver (HEPG2) and metastatic breast (MDA-MB-231) cancer cell lines compared to normal fibroblasts (NIH3T3) after bee venom treatment. Colocalisation profiles of γH2AX and β-actin in each cell line were also analysed. The results showed that the levels of γH2AX staining decreased in normal cells but increased in cancer cells. The majority of β-actin was localised within the cytoplasm of normal cells after bee venom treatment, but it was mostly accumulated within the nucleus in cancer cells. Colocalisation of β-actin and γH2AX both in nucleus and cytoplasm was induced in each cancer cell by different patterns. The results showed that normal and cancerous cells had different responses against bee venom, and suggested that bee venom induced a cellular response by the interaction between γH2AX and β-actin.     

Effects of petrosaspongiolide R on the surface topology of bee venom PLA(2): a limited proteolysis and mass spectrometry analysis

Petrosaspongiolides are sponge metabolites belonging to the family of the γ-hydroxybutenolide marine terpenoids. They possess a remarkable in vitro and in vivo anti-inflammatory profile, due to the specific inhibition of group II and III secretory phospholipase A₂ enzymes, and for this reason can be considered as potential lead for the development of anti-inflammatory drugs. The molecular mechanism of bee venom phospholipase A₂ inactivation has been identified, and the ligand-enzyme complex formation is guided by either non-covalent and covalent interactions. In this work we have analyzed the conformational changes induced by petrosaspongiolide R on the bee venom phospholipase A₂ topology during the molecular recognition process, through the application of limited proteolysis and mass spectrometric methodologies. The results are indicative of structural changes at the N- and C-terminal domains producing a more compact conformational arrangement of the enzyme.     

Cytotoxicity of pilosulin 1, a peptide from the venom of the jumper ant Myrmecia pilosula

The synthetic peptide pilosulin 1, corresponding to the largest defined allergenic polypeptide found in the venom of the jumper ant Myrmecia pilosula, inhibited the incorporation of [methyl-³H]thymidine into proliferating Epstein–Barr transformed (EBV) B-cells. The LD₅₀ was four-fold lower in concentration than melittin, a cytotoxic peptide found in honey bee venom. Loss of cell viability was assessed by flow cytometry by measuring the proportion of cells that fluoresced in the presence of the fluorescent dye 7-aminoactinomycin D. Examination of proliferating EBV B-cells indicated that the cells lost viability within a few minutes exposure to pilosulin 1. Partial peptides of pilosulin 1 were less efficient in causing loss of cell viability and the results suggest that the 22 N-terminal residues are critical to the cytotoxic activity of pilosulin 1. Normal blood white cells were also labile to pilosulin 1. T- and B-lymphocytes, monocytes and natural killer cells, however, were more labile than granulocytes. Analysis of pilosulin 1 using circular dichroism indicated that, in common with melittin and other Hymenoptera venom toxins, it had the potential to adopt an α-helical secondary structure.     

Purification from bee venom of melittin devoid of phospholipase A2 contamination

Conditions for the inactivation of phospholipase A₂ which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A₂ activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A₂ still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.     

Molecular characterization of a venom acid phosphatase Acph-1-like protein from the Asiatic honeybee Apis cerana

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45 °C and pH 4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.     

15-Keto-13,14-dihydroprostaglandin E2- and F2 alpha-metabolite levels in blood from men and women given prostaglandin E2 orally

Prostaglandin E2 (PGE2) was administered orally in a dose of 1 mg to healthy males (n = 20) and females (n = 10). Blood levels of 15-keto-13,14-dihydroprostaglandin F2 alpha (PGF2 alpha-M) and 15-keto-13,14-dihydroprostaglandin E2 (PGE2-M), determined as the rearrangement product 11-deoxy-15-keto-13,14-dihydro-11 beta, 16-cycloprostaglandin E2 (PGE2-cyclo-M), were measured. The levels of the two PG metabolites increased already 10 minutes after ingestion of the tablet and the mean peak value for PGE2-cyclo-M in the men was 4.64 nmol/l which was reached 50 minutes after PGE2 administration. The mean peak value in women was 4.99 nmol/l which was obtained after 30 minutes. The increase in PGE2-cyclo-M concentration was significantly faster (p less than 0.05) in women than in the men. The mean plasma concentration of PGF2 alpha in males were 0.20 nmol/l prior to treatment and rose after PGE2 ingestion to mean peak level of 0.84 nmol/l after 70 minutes. The corresponding values for the females were 0.18 nmol/l and 0.88 nmol/l 50 minutes into treatment. When the data from both sexes were amalgamated PGE2-cyclo-M peak levels were reached significantly (p = 0.004) sooner than the PGF2 alpha-M peak. The two PG metabolites returned to baseline levels in 70% of the individuals after 240 minutes. The increase in PGF2 alpha-M concentration following oral administration of PGE2 indicates that part of the PGE2 was reduced to PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Dose-dependence of protection from systemic reactions to venom immunotherapy by omalizumab

Background

Systemic reactions (SR) to venom immunotherapy (VIT) are rare but may occur, with a rate significantly higher for honeybee than for vespid VIT. In patients with repeated SRs to VIT it is difficult to reach the maintenance dose of venom and pre-treatment with omalizumab is indicated, as shown by some studies reporting its preventative capacity, when antihistamines and corticosteroids are ineffective.

Case presentation

We present the case of a 47 years old woman allergic to bee venom who experienced two severe SRs after bee stings and several SRs to VIT with bee venom. Pre-treatment with antihistamines and corticosteroids as well as omalizumab at doses up to 300 mg was unsuccessful, while an omalizumab dose of 450 mg finally achieved in our patient the protection from SRs to VIT with 200 mcg of bee venom.

Conclusions

The search of the dose of omalizumab able to protect a patient with repeated SRs to VIT may be demanding, but this search is warranted by the need to provide to this kind of patient, by an adequate VIT, the protection from potentially life-threatening reactions.

     

Crude honey bee venom and Bacillus thuringiensis L. Cry1c toxin in controlling wax moth Achroia grisella F. (Lepidoptera: Pyralidae)

Achroia grisella, is a noxious pest of honey bee hives. In this study the toxicity of honey bee venom and bacterial Cry1C toxin of Bacillus thuringiensis on A. grisella were assessed. In addition, the synergistic interaction between BT Cry1c and crude honey bee venom was determined. The combination of HBV and Cry1C increased the activity for both toxins. Therefore, further analysis was carried out to assess the synergistic interaction of each compound assay separately and in mixture. The results showed LC₅₀ value of 1.105 μg/μl for HBV and LC₅₀ value of 0.201 μg/μl for Cry1C. The combination bioassay result showed an LC₅₀ value of 0.549 μg/μl. The combined calculated LC₅₀ is containing lower concentration of the component toxins, this result proved that the toxicity was enhanced due to the combination. A chi square value of 39.93and relative synergistic factor ratio of 1.14 confirmed that a synergic interaction was achieved in combination.