APITOXINA & ANTIBIOTICO

Items 1 - 48 of 48

 

1: Antimicrob Agents Chemother. 2005 Oct;49(10):4085-92.

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Atomic force microscopy study of the effect of antimicrobial peptides on the cell envelope of Escherichia coli.

Meincken M, Holroyd DL, Rautenbach M.

UNESCO Associated Centre for Macromolecules, Department of Chemistry, University of Stellenbosch, Matieland, South Africa.

The influences of the antibacterial magainin 2 and PGLa from the African clawed frog (Xenopus laevis) and the hemolytic bee venom melittin on Escherichia coli as the target cell were studied by atomic force microscopy (AFM). Nanometer-scale images of the effects of the peptides on this gram-negative bacterium's cell envelope were obtained in situ without the use of fixing agents. These high-resolution AFM images of the surviving and intact target cells before and after peptide treatment showed distinct changes in cell envelope morphology as a consequence of peptide action. Although all three peptides are lytic to E. coli, it is clear from this AFM study that each peptide causes distinct morphological changes in the outer membrane and in some cases the inner membrane, probably as a consequence of different mechanisms of action.

PMID: 16189084 [PubMed - indexed for MEDLINE]


2: FEBS Lett. 2005 Mar 14;579(7):1658-64.

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Cross-presentation of a CMV pp65 epitope by human dendritic cells using bee venom PLA2 as a membrane-binding vector.

Babon A, Almunia C, Boccaccio C, Beaumelle B, Gelb MH, Menez A, Maillere B, Abastado JP, Salcedo M, Gillet D.

Protein Engineering and Research Department (DIEP), bat 152, CEA-Saclay, 91191 Gif sur Yvette cedex, France.

We have used bee venom phospholipase A2 as a vector to load human dendritic cells ex vivo with a major histocompatibility complex (MHC) class I-restricted epitope fused to its C-terminus. The fusion protein bound to human monocyte-derived dendritic cells and was internalized into early endosomes. In vitro immunization experiments showed that these dendritic cells were able to generate specific CD8 T cell lines against the epitope carried by the fusion protein. Cross-presentation did not require proteasome, transporter associated with antigen processing, or endosome proteases, but required newly synthesized MHC molecules. Comparison of the antigen presentation pathway observed in this study to that followed by other toxins used as vectors is discussed.

PMID: 15757657 [PubMed - indexed for MEDLINE]


3: Peptides. 2005 Feb;26(2):217-25.

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Conformation and activity of delta-lysin and its analogs.

Dhople VM, Nagaraj R.

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500007, India.

Delta-Lysin is a 26-residue hemolytic peptide secreted by Staphylococcus aureus. Unlike the bee venom peptide melittin, delta-lysin does not exhibit antibacterial activity. We have synthesized delta-lysin and several analogs wherein the N-terminal residues of the toxin were sequentially deleted. The toxin has three aspartic acids, four lysines and no prolines. Analogs were also generated in which all the aspartic acids were replaced with lysines. A proline residue was introduced in the native sequences as well as in the analogs where aspartic acids were replaced with lysines. We observed that 20- and 22-residue peptides corresponding to residues 7-26 and 5-26 of delta-lysin, respectively, had greater hemolytic activity than the parent peptide. These shorter peptides, unlike delta-lysin, did not self-associate to adopt alpha-helical conformation in water, at lytic concentrations. Introduction of proline or substitution of aspartic acids by lysines resulted in loss in propensity to adopt helical conformation in water. When proline was introduced in the peptides corresponding to the native toxin sequence, loss of hemolytic activity was observed. Substitution of all the aspartic acids with lysines resulted in enhanced hemolytic activity in all the analogs. However, when both proline and aspartic acid to lysine changes were made, only antibacterial activity was observed in the shorter peptides. Our investigations on delta-lysin and its analogs provide insights into the positioning of anionic, cationic residues and proline in determining hemolytic and antibacterial activities.

PMID: 15629533 [PubMed - indexed for MEDLINE]


4: J Vet Sci. 2001 Aug;2(2):121-4.

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Therapeutic effect of bee venom in sows with hypogalactia syndrome postpartum.

Choi SH, Kang SS.

Department of Veterinary Surgery, College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Korea. shchoi@cbucc.chungbuk.ac.kr

The objective of this study was to determine the clincotherapeutic effect of whole bee venom in hypogalactic sows postpartum. Sows after parturition were assigned to treated and nontreated control groups. In the treated group, 22 sows were bee acupunctured once a day for 3 consecutive days. Honeybees (Apis mellifera L.) for bee acupuncture were about 15 days after metamorphosis. One live bee was used to sting the acupoints known as Yang-ming (ST-18, 1.5 cm lateral to the base of the last 2 pairs of teats) and Jiao-chao (GV-1, at the indentation between the base of tail and the anus). In the control group, 20 sows were intramuscularly injected with a standard dosage of penicillin G (400,000 IU/head) once a day for 3 consecutive days. At post-treatment, 85.0% of the drug-treated control and 90.9% of the bee venomtreated group recovered from hypogalactia syndrome. The advantages of apitherapy were that the patients did not have stress because they were not restrained for a long period. The result suggested that apitherapy using bee venom is an effective treatment for sows with hypogalactia syndrome postpartum.

Publication Types:

·       Clinical Trial

·       Randomized Controlled Trial


PMID: 14614282 [PubMed - indexed for MEDLINE]


5: Am J Chin Med. 2003;31(1):149-55.

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Effect of bee venom treatment in sows with oligogalactic syndrome postpartum.

Choi SH, Kang SS, Bae CS, Cho SK, Pak SC.

College of Veterinary Medicine and Research Institute of Veterinary Medicine Chungbuk National University, Cheongju 361-763, Korea. shchoi@cbucc.chungbuk.ac.kr

The objective of this study was to determine the clinico-therapeutic effect of worker honeybee venom in sows with oligogalactic syndrome postpartum. Comparison between bee venom- and drug-treated groups was our main concern in the present study. Sows after parturition were assigned to bee venom- and drug-treated groups, respectively. In the bee venom-treated group, 22 sows were bee-acupunctured once a day for 3 consecutive days. Honeybees (Apis mellifera L.) forbee acupuncture were about 15 days old after metamorphosis. Live bees were used to sting the acupoints known as yang-ming (ST-18, 1.5 cm lateral to the base of the last two pairs of teats) and jiao-chao (GV- , at the indentation between the base of tail and the anus). In the drug-treated group, 20 sows were intramuscularly injected with a standard dose of penicillin G (400,000 IU/head) once a day for 3 consecutive days. On post-treatment day 4, 85.0% of the drug-treated group and 90.9% of the bee venom-treated group recovered from oligogalactic syndrome postpartum. The result suggested that apitherapy using worker honeybee is an effective treatment for sows with oligogalactic syndrome postpartum.

Publication Types:

·       Clinical Trial


PMID: 12723765 [PubMed - indexed for MEDLINE]


6: Antimicrob Agents Chemother. 2003 Mar;47(3):965-71.

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Modulation of the activity of secretory phospholipase A2 by antimicrobial peptides.

Zhao H, Kinnunen PK.

Helsinki Biophysics & Biomembrane Group, Institute of Biomedicine, FIN-00014 University of Helsinki, Finland.

The antimicrobial peptides magainin 2, indolicidin, and temporins B and L were found to modulate the hydrolytic activity of secretory phospholipase A(2) (sPLA(2)) from bee venom and in human lacrimal fluid. More specifically, hydrolysis of phosphatidylcholine (PC) liposomes by bee venom sPLA(2) at 10 micro M Ca(2+) was attenuated by these peptides while augmented product formation was observed in the presence of 5 mM Ca(2+). The activity of sPLA(2) towards anionic liposomes was significantly enhanced by the antimicrobial peptides at low [Ca(2+)] and was further enhanced in the presence of 5 mM Ca(2+). Similarly, with 5 mM Ca(2+) the hydrolysis of anionic liposomes was enhanced significantly by human lacrimal fluid sPLA(2), while that of PC liposomes was attenuated. These results indicate that concerted action of antimicrobial peptides and sPLA(2) could improve the efficiency of the innate response to infections. Interestingly, inclusion of a cationic gemini surfactant in the vesicles showed an essentially similar pattern on sPLA(2) activity, suggesting that the modulation of the enzyme activity by the antimicrobial peptides may involve also charge properties of the substrate surface.

PMID: 12604528 [PubMed - indexed for MEDLINE]


7: Chembiochem. 2002 Jul 2;3(7):664-71.

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Erratum in:

·       Chembiochem. 2002 Aug 29;3(8):687.


Molecular basis of phospholipase A2 inhibition by petrosaspongiolide M.

Dal Piaz F, Casapullo A, Randazzo A, Riccio R, Pucci P, Marino G, Gomez-Paloma L.

Dipartimento di Chimica Organica e Biochimica Universita di Napoli Federico II via Cinthia 6, 80126 Napoli, Italy.

Petrosaspongiolide M (PM) is an anti-inflammatory marine metabolite that displays a potent inhibitory activity toward group II and III secretory phospholipase A(2) (PLA(2)) enzymes. The details of the mechanism, which leads to a covalent adduct between PLA(2) and gamma-hydroxybutenolide-containing molecules such as PM, are still a matter of debate. In this paper the covalent binding of PM to bee venom PLA(2) has been investigated by mass spectrometry and molecular modeling. The mass increment observed for the PM-PLA(2) adduct is consistent with the formation of a Schiff base by reaction of a PLA(2) amino group with the hemiacetal function (masked aldehyde) at the C-25 atom of the PM gamma-hydroxybutenolide ring. Proteolysis of the modified PLA(2) by the endoprotease LysC followed by HPLC MS analysis allowed us to establish that the PLA(2) alpha-amino terminal group of the Ile-1 residue was the only covalent binding site for PM. The stoichiometry of the reaction between PM and PLA(2) was also monitored and results showed that even with excess inhibitor, the prevalent product is a 1:1 (inhibitor:enzyme) adduct, although a 2:1 adduct is present as a minor component. The 2:1 adduct was also characterized, which showed that the second site of reaction is located at the epsilon -amino group of the Lys-85 residue. Similar results in terms of the reaction profile, mass increments, and location of the PLA(2) binding site were obtained for manoalide, a paradigm for irreversible PLA(2) inhibitors, which suggests that the present results may be considered of more general interest within the field of anti-inflammatory sesterterpenes that contain the gamma-hydroxybutenolide pharmacophore. Finally, a 3D model, constrained by the above experimental results, was obtained by docking the inhibitor molecule into the PLA(2) binding site through AFFINITY calculations. The model provides an interesting insight into the PM-PLA(2) inhibition process and may prove useful in the design of new anti-inflammatory agents that target PLA(2) secretory enzymes.

PMID: 12325001 [PubMed - indexed for MEDLINE]


8: FEBS Lett. 1999 Apr 1;448(1):62-6.

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Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity.

Subbalakshmi C, Nagaraj R, Sitaram N.

Centre for Cellular and Molecular Biology, Hyderabad, India.

Melittin, the 26-residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C-terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5-7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been transpositioned to the N-terminal region from the C-terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.

PMID: 10217411 [PubMed - indexed for MEDLINE]


9: Yakugaku Zasshi. 1997 May;117(5):253-64.

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[Molecular action mechanisms and membrane recognition of membrane-acting antimicrobial peptides]

[Article in Japanese]

Matsuzaki K.

Faculty of Pharmaceutical Sciences, Kyoto University, Japan.

A number of antimicrobial peptides have been isolated in the animal kingdom, serving as defensive or offensive weapons. The mechanisms of their action are considered to be the permeability of bacterial membranes, although the details are not yet clarified. I have studied the interactions of several antibiotic peptides with both artificial lipid bilayers and biomembranes to elucidate the molecular mechanisms of the action and to find out the rationale for their membrane specificity. Magainin 2 from the Xenopus skin was found to form a peptide-lipid supramolecular complex pore in the membrane, followed by peptide internalization, simultaneously dissipating the transmembrane potential and the lipid asymmetry. This novel mechanism also works for a wasp bee venom, mastoparan X. Tachyplesin I from Tachypleus and a bee venom, melittin, also translocate across the membrane by forming a pore. The membrane selectivity of these peptides is closely related to their affinity for the lipids constituting the membrane surface. A strategy for developing a potent antibiotic was discussed based on these results.

Publication Types:

·       Review


PMID: 9194394 [PubMed - indexed for MEDLINE]


10: Biochemistry. 1997 Feb 18;36(7):1826-35.

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Selective lysis of bacteria but not mammalian cells by diastereomers of melittin: structure-function study.

Oren Z, Shai Y.

Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.

Studies on lipid-peptide interactions of cytolytic polypeptides tend to emphasize the importance of the amphipathic alpha-helical structure for their cytolytic activity. In this study, diasetereomers of the bee venom melittin (26 a.a.), a non-cell-selective cytolysin, were synthesized and investigated for their structure and cytolytic activity toward bacteria and mammalian cells. Similarly to the findings with the diastereomers of the less cytolytic peptide pardaxin (33 a.a.) (Shai & Oren. 1996), the melittin diastereomer, lest their alpha-helical structure, which abrogated their hemolytic activity toward human erythrocytes. However, they retained their antibacterial activity and completely lysed both Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy. To understand the molecular mechanism underlying this selectivity, binding experiments utilizing the intrinsic tryptophan of melittin, tryptophan quenching experiments using brominated phospholipids, and membrane destabilization studies were done. The data revealed that the melittin diastereomers bound to and destabilized only negatively-charged phospholipid vesicles, in contrast to native melittin, which binds strongly to both negatively-charged and zwitterionic phospholipids. However, the partition coefficient, the depth of penetration into the membrane, and the membrane-permeating activity of the diastereomers with negatively-charged phospholipids were similar to those obtained with melittin. The results obtained do not support the formation of transmembrane pores as the mode of action of the diastereomers, but rather suggest that these peptides bind to the surface of the bacterial membrane, cover it in a "carpet-like" manner, and dissolve it like a detergent. The results presented here together with those obtained with the cytolytic peptide pardaxin suggest that the combination of hydrophobicity and net positive charge may be sufficient in the design of potent diastereomers of antibacterial polypeptides for the treatment of infectious diseases.

PMID: 9048567 [PubMed - indexed for MEDLINE]


11: Res Microbiol. 1997 Feb;148(2):163-75.

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Effect of natural amphipathic peptides on viability, membrane potential, cell shape and motility of mollicutes.

Beven L, Wroblewski H.

Groupe Membranes et Osmoregulation, UPRES-A CNRS Q6026, Universite de Rennes 1, Rennes, France.

The antibiotic activity of ten amphipathic peptides was investigated in six species of mollicutes belonging to the genera Acholeplasma, Mycoplasma and Spiroplasma. A. laidlawii was the most sensitive and M. mycoides subsp. mycoides SC the most resistant. Animal defence peptides (cecropins A and P1, and magainin 2) proved to be less potent than bee-venom mellitin and most of the peptides produced by bacteria (globomycin, gramicidin S, surfactin and valinomycin) or fungi (alamethicin). Gramicidin S was by far the most active peptide, with minimal inhibitory concentrations ranging from 2 to 50 nM. Alamethicin, gramicidin S, mellitin and surfactin had a cidal effect, whilst cecropins, globomycin, magainin 2, polymyxin B and valinomycin proved to be static. The peptides altered the membrane potential of spiroplasma cells with a potency independent of their linear or cyclic structure. However, globomycin depolarized the plasma membrane only weakly, whilst polymyxin B, in order to be active, required prior hyperpolarization of the membrane. The peptides also induced the loss of cell motility and helicity in spiroplasmas, suggesting that motility and cell shape in these bacteria are coupled to the transmembrane electrochemical gradient. Globomycin, an inhibitor of signal-peptidase II, prevented the growth of spiroplasmas, M. gallisepticum, and M. genitalium, but not that of A. laidlawii and M. mycoides subsp. mycoides SC, although the latter also synthesized membrane lipoproteins. Inhibition of spiralin processing by globomycin was demonstrated in S. citri and S. melliferum, with a more pronounced effect in the second species.

PMID: 9765797 [PubMed - indexed for MEDLINE]


12: J Steroid Biochem Mol Biol. 1997 Jan;60(1-2):51-7.

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Functional dissociation between glucocorticoid-induced decrease in arachidonic acid release and inhibition of adrenocorticotropic hormone secretion in AtT-20 corticotrophs.

Pompeo A, Luini A, Buccione R.

Istituto di Richerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Department of Cell Biology and Oncology, Chieti, Italy.

The biochemical basis of the short-term inhibitory effects of glucocorticoids on corticotropin release from pituitary corticotrophs is still obscure. A well-characterized effect of glucocorticoids in several cell types is the inhibition of arachidonic acid (AA) generation by phospholipase A2 (PLA2). Arachidonic acid and its metabolites have been implicated in the secretory process from a number of pituitary cells, such as the corticotrophs. We have thus examined the role of AA in the anti-secretagogue effects of glucocorticoids in a corticotropin-secreting clonal corticotroph line (AtT-20 D16/16). Glucocorticoids decreased AA release induced by melittin, a bee venom protein related to extracellular PLA2. When a possible role of AA in corticotropin release was studied, the following results were obtained: (a) all corticotropin secretagogues tested, including corticotropin-releasing factor (CRF), did not alter AA generation; (b) calcium and guanine nucleotides, which stimulate corticotropin release in permeabilized cells, inhibited the release of AA under the same conditions; (c) administration of melittin or of exogenous AA had no effect on basal and CRF-stimulated corticotropin release; (d) administration of large amounts of exogenous AA was unable to restore the ability to secrete corticotropin under suppression by glucocorticoids. Altogether, the data suggest that whereas glucocorticoids can inhibit both AA generation and corticotropin release, these two effects appear to be causally unrelated.

PMID: 9182858 [PubMed - indexed for MEDLINE]


13: Curr Microbiol. 1996 Nov;33(5):317-22.

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Inhibition of spiralin processing by the lipopeptide antibiotic globomycin.

Beven L, Le Henaff M, Fontenelle C, Wroblewski H.

Equipe "Membranes et Osmoregulation," CNRS URA No. 256, Universite de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France.

The cyclic lipopeptide globomycin, a specific inhibitor of signal-peptidase II (Lsp A), proved toxic for the mollicute Spiroplasma melliferum with a minimal inhibitory concentration (MIC) in the range 6.25-12.5 microM, about one order of magnitude higher (that is, less efficient) than bee-venom mellitin. SDS-PAGE analysis of cell proteins followed by immunolabeling ("Western blotting") and by crossed immunoelectrophoresis demonstrated that the cleavage of the prespiralin leader peptide was prevented by globomycin. Cell fractionation experiments showed that prespiralin was membrane bound and did not accumulate in the cytoplasm or in the culture medium. Furthermore, the use of the potential-sensitive fluorescent dye 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide (diS-C3-[5]) revealed that, in contrast to valinomycin and mellitin, globomycin up to 30 microM has no effect on the electrical transmembrane potential of S. melliferum. This indicates that the toxicity of globomycin for spiroplasma cells is mainly if not exclusively owing to the inhibition of spiralin processing. Added to previously published data, these results suggest that spiralin and probably other lipoproteins of mollicutes are acylated and membrane targeted by a mechanism involving notably the processing of the prelipoprotein precursor by a type II, globomycin-sensitive signal peptidase.

PMID: 8875913 [PubMed - indexed for MEDLINE]


14: Singapore Med J. 1996 Aug;37(4):389-91.

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Case reports and mini review of bee stings of the cornea.

Chuah G, Law E, Chan WK, Ang CL.

Singapore National Eye Centre, Singapore.

Bee stings of the eye are not uncommon. Quite a few clinical case reports have documented the various ocular reactions to the venom of the bee stings, which may range from mild conjunctivitis to sudden loss of vision. This report presents 2 patients who suffered bee stings to the cornea and their different outcomes. The properties of bee venom as well as the treatment of various possible complications are also discussed.

Publication Types:

·       Case Reports

·       Review


PMID: 8993139 [PubMed - indexed for MEDLINE]


15: Biochim Biophys Acta. 1994 Jul 21;1222(3):471-6.

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Melittin inhibits epidermal growth factor-induced protein tyrosine phosphorylation: comparison with phorbol myristate acetate and calcium ionophore A23187.

Errasfa M, Stern A.

Department of Pharmacology, New York University Medical Center, NY 10016.

In HER14 cells, epidermal growth factor (EGF) induces tyrosine phosphorylation of several proteins, including its own receptor. The bee venom peptide, melittin, impaired EGF-dependent protein tyrosine phosphorylation in a calcium-dependent manner. The melittin effect was similarly reproduced by calcium ionophore A23187. The effect of melittin and calcium ionophore A23187 on EGF-dependent protein tyrosine phosphorylation was abolished by treatment of cells with the calcium chelator EGTA. Phorbol-myristate acetate (PMA) inhibited EGF-dependent protein tyrosine phosphorylation, and when compared to melittin or calcium ionophore A23187, only PMA potentiated the EGF-induced tyrosine phosphorylation of two proteins immunologically related to mitogen activated protein (MAP) kinases of 40 kDa and 44 kDa molecular mass. Unlike PMA, the effect of melittin and calcium ionophore A23187 on inhibition of EGF-dependent protein tyrosine phosphorylation was lost neither in protein kinase C-depleted cells nor in cells treated with the protein tyrosine phosphatase inhibitors NaF and Na3VO4. Melittin inhibited high affinity binding of EGF to its receptor in intact cells, but this effect was not prevented by EGTA. It is concluded that melittin and calcium ionophore A23187 differ from PMA in their inhibition of EGF-dependent protein tyrosine phosphorylation in vivo, by acting via a Ca(2+)-dependent pathway, that is independent of protein kinase C, protein tyrosine phosphatase activity and high affinity binding of EGF to its receptor.

PMID: 8038217 [PubMed - indexed for MEDLINE]


16: J Med Chem. 1993 Jun 25;36(13):1866-79.

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Molecular model of the interaction of bee venom phospholipase A2 with manoalide.

Ortiz AR, Pisabarro MT, Gago F.

Departamento de Fisiologia y Farmacologia, Universidad de Alcala de Henares, Madrid, Spain.

A molecular model of the interaction between manoalide (MLD) and bee venom phospholipase A2 (bv-PLA2) has been derived making use of a combination of computational methods. MLD was built in its open form and simulated by using molecular dynamics techniques. It is shown that the polar part of the molecule, which is thought to be the reactive region, is endowed with considerable conformational flexibility whereas the apolar region is rather rigid. The proposed active conformation of MLD and the main putative binding site for MLD on this enzyme were identified by matching potential energy GRID maps for both ligand and receptor with the chemical structure of the respective counterpart. The binding site is found in the C-terminal region of bv-PLA2, forming part of the proposed interfacial surface for binding to aggregated substrates, and comprises two distinct regions: (i) a hydrophobic cavity delimited by the C-terminal beta-sheet and the antiparallel beta-sheet, which interacts with the apolar zone of MLD, and (ii) a cationic site made up of residues Arg-58 and Lys-94, which interacts with the polar zone. Molecular dynamics and molecular orbital calculations indicate that the most likely initial reaction between MLD and bv-PLA2 is formation of a Schiff base between Lys-94 and the aldehyde generated upon opening of MLD's gamma-lactone ring, supporting recent model reaction studies. The inhibition seems to be a consequence of the occupation by MLD of a site overlapping a phosphocholine binding site in bv-PLA2 presumably involved in the interface desolvation process. The present model represents a starting point for further structural studies on the mechanism of phospholipases A2 inactivation by MLD and MLD-like compounds.

PMID: 8515424 [PubMed - indexed for MEDLINE]


17: Oncogene. 1993 Apr;8(4):939-47.

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Melittin-induced hyperactivation of phospholipase A2 activity and calcium influx in ras-transformed cells.

Sharma SV.

Department of Microbiology and Immunology, University of Tennessee, Memphis 38163.

The activated ras oncogene is a key mediator of cellular transformation and is present in a wide variety of primary human neoplasms. The biochemical role of the ras oncogene in cellular transformation is at present unclear, and hence approaches to control its activities in transformed cells have met with limited success. Previous studies have demonstrated the ability of melittin, a 26 amino acid amphipathic peptide from bee venom, to specifically counterselect for cells in culture that express high levels of the ras oncogene product. The biochemical basis for this counterselection is currently unknown. This study demonstrates the ability of melittin to hyperactivate phospholipase A2 (PLA2) in ras-transformed cells by the mediation of enhanced influx of calcium ions (Ca2+). This hyperactivation of PLA2 and Ca2+ mobilization in ras-transformed cells by melittin is mimicked by the calcium ionophore, A23187. Both melittin- and A23187-mediated PLA2 hyperactivation require Ca2+. However, the action of melittin is strongly dependent on extracellular Ca2+, whereas that of A23187 is not. Melittin-induced Ca2+ influx and PLA2 hyperactivation is inhibited by manganese ions (Mn2+). These studies reveal a close correlation between the extent of PLA2 hyperactivation and Ca2+ mobilization, suggesting a causal relationship.

PMID: 8455945 [PubMed - indexed for MEDLINE]


18: Agents Actions. 1991 Sep;34(1-2):70-2.

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AGN 190383, a novel phospholipase inhibitor with topical anti-inflammatory activity.

De Vries GW, Lee G, Amdahl L, Wenzel M, Garst M, Wheeler LA.

Department of Biological Sciences, Allergan, Inc., Irvine, CA 92715.

AGN 190383 is a 5-hydroxy-2(5H)-furanone ring analog of the marine natural product manoalide. When applied topically, AGN 190383 inhibits phorbol ester induced mouse ear edema. It is a potent inhibitor of bee venom phospholipase A2 and blocks the release of arachidonic acid from calcium ionophore A23187 stimulated human neutrophils. AGN 190383 also inhibits both hormone-operated and depolarization-dependent calcium mobilization in GH3 cells, as well as fMLP stimulated increases in free cytosolic calcium in human PMNs. Furthermore, it is also able to block the release of the neutral protease elastase from stimulated neutrophils. The effects of AGN 190383 on arachidonic acid metabolism and leukocyte function may account, in part, for its anti-inflammatory activity in vivo.

PMID: 1793055 [PubMed - indexed for MEDLINE]


19: Chem Phys Lipids. 1991 Mar;57(2-3):327-40.

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Polymorphic phospholipid phase transitions as tools to understand peptide-lipid interactions.

Tournois H, de Kruijff B.

aATO Agrotechnology, Wageningen, The Netherlands.

The effect of peptides on bilayer----non-bilayer phase transitions can be used as a tool to investigate the molecular aspects of peptide-lipid interactions. In this contribution the action on membranes of the peptide antibiotic gramicidin A and the bee venom component melittin are compared. Although the known structures and locations of these peptides upon membrane binding are very different, their actions on membranes show striking parallels. A general model is proposed that explains the seemingly complex peptide-lipid interactions by making use of simple concepts.

Publication Types:

·       Review


PMID: 1711420 [PubMed - indexed for MEDLINE]


20: J Biol Chem. 1991 Feb 15;266(5):2753-8.

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Membrane interactions of amphiphilic polypeptides mastoparan, melittin, polymyxin B, and cardiotoxin. Differential inhibition of protein kinase C, Ca2+/calmodulin-dependent protein kinase II and synaptosomal membrane Na,K-ATPase, and Na+ pump and differentiation of HL60 cells.

Raynor RL, Zheng B, Kuo JF.

Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322.

Interactions of certain naturally occurring, amphiphilic polypeptides with membranes were investigated. Mastoparan (wasp venom toxin), melittin (bee venom toxin), cardiotoxin (cobra venom toxin), and polymyxin B (antibacterial antibiotic) inhibited protein kinase C stimulated by phosphatidylserine bilayer or arachidonate monomer and blocked binding of [3H] phorbol 12,13-dibutyrate to protein kinase C in the presence of phosphatidylserine bilayer, with IC50 values (concentrations causing 50% inhibition) of 1-8 microM. Mastoparan and polymyxin B were much less inhibitory (IC50, 10-20 microM), whereas melittin and cardiotoxin were similarly inhibitory (IC50, 1-4 microM), when protein kinase C was activated instead by synaptosomal membrane. Kinetic analysis indicate that mastoparan inhibited protein kinase C, assayed using phosphatidylserine or synaptosomal membrane as the phospholipid cofactor, competitively with the phospholipid cofactor, in a mixed manner with CaCl2 or diacylglycerol, noncompetitively with histone, and uncompetitively with ATP, with apparent Ki values of 1.6-18.7 microM. Inhibition of Na,K-ATPase in the membrane by these polypeptides had relative potencies different from those for their inhibition of protein kinase C activated by the same membrane preparation; mastoparan and melittin inhibited the two activities with comparable potencies, but polymyxin B and cardiotoxin were far less effective in inhibiting Na,K-ATPase. The same relative inhibitory potencies of the polypeptides (melittin greater than mastoparan greater than polymyxin B) for inhibition of Na,K-ATPase were also noted for their inhibition of Ca2+/calmodulin-dependent protein kinase II, 86Rb uptake (Na+ pump) by HL60 cells and the phorbol ester-induced differentiation of the leukemia cells. These findings were consistent with discrete interactions of the polypeptides with functionally distinct sites on the membrane, leading to differential inhibition of biological activities associated with the membrane. Actions of certain polypeptides appeared to be more specific compared to those of lipid second messengers such as lyso-phosphatidylcholine and sphingosine, and the antineoplastic ether lipid analogs such as 1-O-octadecyl-2-methyl-rac-glycero-3-ophosphocholine.

PMID: 1847132 [PubMed - indexed for MEDLINE]


21: Infect Immun. 1990 Aug;58(8):2678-82.

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Effect of staphylococcal delta-toxin and bee venom peptide melittin on leukotriene induction and metabolism of human polymorphonuclear granulocytes.

Raulf M, Alouf JE, Konig W.

Lehrstuhl fur Medizinische Mikrobiologie und Immunologie, Ruhr-Universitat Bochum, Federal Republic of Germany.

The abilities of delta-toxin from Staphylococcus aureus and melittin to induce and modulate the generation of leukotriene from human polymorphonuclear granulocytes (PMNs) were studied. Stimulation of PMNs with melittin (10 micrograms) induced leukotriene formation, whereas stimulation with delta-toxin did not. Preincubation of the PMNs with delta-toxin modulated the subsequent generation of leukotriene from PMNs induced by Ca ionophore A23187 or opsonized zymosan. The generation of leukotriene B4 (LTB4), induced by the Ca ionophore A23187, was increased when the PMNs were preincubated with delta-toxin for 5 min. When opsonized zymosan was used as a secondary stimulus to activate the delta-toxin-pretreated PMNs, LTB4 generation decreased. In contrast, melittin showed no significant modulatory effect on the generation of leukotriene from PMNs. In addition, preincubation of PMNs with delta-toxin inhibited the conversion of LTB4 to omega-oxidation products. Our data suggest that peptides with similar structures, e.g., delta-toxin and melittin, induce and modify leukotriene generation in different manners.

PMID: 2164512 [PubMed - indexed for MEDLINE]


22: FEBS Lett. 1989 Dec 18;259(1):103-6.

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Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids.

Boman HG, Wade D, Boman IA, Wahlin B, Merrifield RB.

Department of Microbiology, University of Stockholm, Sweden.

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1-13)-melittin(1-13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.

PMID: 2689223 [PubMed - indexed for MEDLINE]


23: Zentralbl Bakteriol. 1989 Oct;271(4):521-31.

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Inhibition of in vitro and in vivo mast cell degranulation by Taenia crassiceps metacestodes in vitro incubation products.

Seifert B, Geyer E.

Fachbereich Biologie, Philipps-Universitat, Marburg.

In vitro released products of T. crassiceps metacestodes (TcIP) harvested from the peritoneal cavity of NMRI mice were tested for inhibitory effects on the in vitro degranulation of peritoneal mast cells (MCs) of normal mice (NMRI) and rats (Wistar) and on the in vivo degranulation of rat (Wistar) skin MCs (PCA-assay). In vitro degranulation was elicited chemically (compound 48/80, polymyxin B or the bee venom peptide, mellitin). In vivo degranulation was triggered immunologically (anaphylactic systems ovalbumin/anti-ovalbumin or Fasciola hepatica crude fluke extract antigen/serum of fluke-infected rats (Wistar]. In vitro degranulation of murine peritoneal MCs or the in vitro histamine release of rat peritoneal MCs normally induced chemically was significantly inhibited when the MCs were preincubated with the TcIP or with serum of T. crassiceps-infected NMRI mice from day 35 post infection and thereafter. In vitro degranulation of peritoneal MCs of infected mice was strongly inhibited beginning on day 10 after infection. Also in vivo degranulation of the IgE-sensitized rat skin MCs was significantly reduced by intradermal injection of the TcIP before (6, 3 and 1 h) antigen challenge and by preinjection (1 h) of serum from infected mice (day 80 p.i.)-The inhibitory effect was also demonstrated after immunoadsorption of mouse serum proteins naturally contaminating the TcIP. Heating (100 degrees C/15 min), even in the presence of 0.25 M HCl, did not suppress the inhibitory activity.

PMID: 2479392 [PubMed - indexed for MEDLINE]


24: J Pharm Pharmacol. 1989 Jul;41(7):450-8.

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Inhibition of Na+,K+-ATPase activity by phospholipase A2 and several lysophospholipids: possible role of phospholipase A2 in noradrenaline release from cerebral cortical synaptosomes.

Nishikawa T, Tomori Y, Yamashita S, Shimizu S.

Department of Pharmacology, Kagoshima University Dental School, Japan.

p-Bromophenacyl bromide (PBPB), quinacrine and indomethacin, which inhibit phospholipase A2 (PLA2; EC 3.1.1.4) activity in several tissues, caused a dose-dependent inhibition of prelabelled [3H]noradrenaline ([3H]NA) release evoked by high concentrations of K+ from rat cerebral cortical synaptosomes. Release of prelabelled [3H]NA was caused by natural lysophosphatidic acid (LPA; 10(-6)-10(-5) g mL-1) and lysophosphatidylcholine (LPC; 10(-6)-10(-5) g mL-1) and synthetic LPA (6 x 10(-6), 2 x 10(-5) M) and LPC (6 x 10(-6), 2 x 10(-5) M), but not by natural lysophosphatidylserine (LPS; 10(-5) g mL-1), lysophosphatidylethanolamine (LPE; 10(-5) g mL-1) and lysophosphatidylinositol (LPI; 10(-5) g mL-1). The release evoked by natural LPA and LPC could be inhibited only marginally by PBPB and quinacrine. Phosphatidic acid (PA)-specific and phosphatidylcholine (PC)-specific PLA2 activities from rat cerebral cortical synaptosomes were stimulated in incubation medium containing high concentrations of K+ or calcium ionophore A23187. Low concentrations of PLA2 (10(-6)-10(-8) g mL-1, from bee venom) inhibited the synaptic membrane Na+,K+-ATPase activity in incubation media with intracellular levels of free Ca2+. Several lysophospholipids (LPLs), metabolites of the PLA2 type, also inhibited the synaptic membrane Na+,K+-ATPase activity in a dose-dependent manner. The minimum effective concentrations of natural LPA, LPC, LPS, LPI and LPE were 10(-6), 4.7 x 10(-6), 10(-5), 4.7 x 10(-5) and 4.7 x 10(-5) g mL-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 2570849 [PubMed - indexed for MEDLINE]


25: Biochem Pharmacol. 1988 Oct 1;37(19):3639-46.

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Inactivation of phospholipase A2 by manoalide. Localization of the manoalide binding site on bee venom phospholipase A2.

Glaser KB, Vedvick TS, Jacobs RS.

Department of Biological Sciences, University of California, Santa Barbara 93106.

The marine natural product manoalide (MLD), a potent inhibitor of phospholipases, completely inactivates bee venom phospholipase A2 (PLA2) by an irreversible mechanism. It has been proposed [K. B. Glaser and R. S. Jacobs, Biochem. Pharmac. 36, 2079 (1987)] that the reaction of MLD with PLA2 may involve the selective reactivity of MLD to a peptide sequence, possibly a Lys-X-X-Lys peptide. Localization of the MLD binding site on bee venom PLA2 demonstrated that upon MLD modification of bee venom PLA2 the only change in amino acid content was an apparent loss of Lys, corresponding to approximately three of the eleven Lys residues present. Selective chemical modification of Lys residues with [14C]maleic anhydride demonstrated that all eleven Lys residues on bee venom PLA2 were accessible to this reagent (11.6 mol maleyl group incorporated/mol of PLA2). Pretreatment of PLA2 with MLD (less than 0.7% residual activity) resulted in a molar ratio of 8.7, also consistent with the loss of three Lys residues upon modification by MLD. Reverse phase high performance liquid chromatography (RP-HPLC) of the cyanogen bromide (CNBr) digestion product of MLD-treated PLA2 produced three peaks (A280). The second peak showed the most intense absorbance at 434 nm. This material corresponded to residues 81-128, as determined by gas-phase microsequence analysis. Sequencing failure was observed at Lys-88 in the MLD-treated fragment. The control carboxymethylated-PLA2 fragment corresponding to residues 81-128 sequenced beyond Lys-88 without significant change in the expected yield. These data suggest that Lys-88 may correspond to one of the three MLD-modified Lys residues. The minor absorbance at 434 nm of the CNBr fragments containing residues 42-80 and 1-36 as compared to the fragment of residues 81-128 suggests that the major MLD binding fragment residues in residues 81-128.

PMID: 3178877 [PubMed - indexed for MEDLINE]


26: Biochem Pharmacol. 1987 Jul 1;36(13):2079-86.

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Inactivation of bee venom phospholipase A2 by manoalide. A model based on the reactivity of manoalide with amino acids and peptide sequences.

Glaser KB, Jacobs RS.

The marine natural product manoalide (MLD), a potent irreversible inhibitor of bee venom phospholipase A2 (PLA2), was shown to produce a chromophore (lambda max = 437 nm) during incubation with the enzyme. MLD also developed an identical chromophore when incubated with free lysine (Lys), cysteine (Cys) or tryptophan (Trp) but not with their N-alpha-amino-blocked analogs. These results suggest that the chromophore product was dependent on the presence of two nucleophilic groups which react by an ordered mechanism rather than by simple random collision. Lys polymers prevented MLD from inhibiting PLA2, whereas monomeric Lys did not. The optimal active polymer of Lys appeared to be a tetralysine (L4) peptide, and a degree of selectivity was obtained when the Lys residues were in a 1,4-Lys arrangement. The rate of chromophore development with PLA2 and the rate of inactivation of PLA2 by MLD appear to be independent processes. Based on these data, it is possible that the irreversible inactivation of PLA2 may involve an ordered reaction with a peptide sequence in PLA2 containing a 1,4-Lys arrangement.

PMID: 3111475 [PubMed - indexed for MEDLINE]


27: Biochem Pharmacol. 1987 Mar 1;36(5):733-40.

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Differential effects of manoalide on secreted and intracellular phospholipases.

Bennett CF, Mong S, Clarke MA, Kruse LI, Crooke ST.

Manoalide, a novel nonsteroidal sesterterpenoid, is a potent inhibitor of phospholipase A2 isolated from bee and cobra venoms. This report compares the inhibition by manoalide of phospholipase A2 in crude cytosol fractions from four mammalian tissues with that of four purified extracellular phospholipase A2's. Phospholipase A2 isolated from bee venom (Apis mellifera) was the most sensitive to inactivation by manoalide (IC50 approximately equal to 0.12 microM). Extracellular phospholipase A2 from rattlesnake and cobra venom was intermediate in sensitivity to manoalide (IC50 values of 0.7 and 1.9 microM respectively). Porcine pancreatic phospholipase A2 was relatively resistant to inactivation by manoalide (IC50 approximately equal to 30 microM). The phospholipase A2 assayed in crude cytosol fractions from four mammalian tissues exhibited IC50 values of 30 microM or greater. Cytosolic proteins as well as bovine serum albumin and poly-L-lysine (Mr = 57,000) protected purified bee venom phospholipase A2 from inactivation by manoalide. In contrast, amino acids such as lysine and alanine failed to protect the purified enzyme from inactivation. Proteins and certain amino acids, such as lysine, formed a chromogenic product when incubated with manoalide. These data suggest that lysine is capable of reacting with manoalide, but only when it is present in macromolecules is it capable of protecting phospholipase A2 from inactivation by manoalide. Because cellular proteins protect PLA2 from inactivation by manoalide, high concentrations of manoalide must be applied topically to produce statistically significant inactivation of intracellular phospholipase A2. Finally, a chemical model is presented which explains the formation of a chromogenic product when manoalide is incubated with proteins and amino acids.

PMID: 3103628 [PubMed - indexed for MEDLINE]


28: Arch Biochem Biophys. 1987 Feb 1;252(2):478-86.

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Mitogen-stimulated release of inositol phosphates in human fibroblasts.

Jamieson GA Jr, Villereal ML.

Mitogenic stimulation of quiescent human fibroblasts (HSWP) with a growth factor mixture (consisting of epidermal growth factor (EGF), insulin, bradykinin, and vasopressin) rapidly induces an increase in Na influx via a Ca-mediated activation of an amiloride-sensitive Na/H exchanger. Inositol phosphates (specifically inositol-1',4',5'-phosphate) have been implicated in mediating the mobilization of intracellular Ca stores in other cell types and we have now completed a detailed analysis of the mitogen-induced release of inositol phosphates in HSWP cells. Stimulation of inositol trisphosphate release is rapid (within 5 s) and reaches a maximum level (416-485% basal) within 10-15 s after the addition of growth factor mixture. Inositol bisphosphate and inositol monophosphate reach maximum levels by 30 s (1257% basal) and 60 s (291% basal), respectively. Levels of all three compounds then decay toward basal levels but remain elevated (150-350% of basal levels) after 10 min of incubation with mitogens. The effects of different combinations of these growth factors and of the bee venom peptide, melittin, have also been determined. We have also found that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate, which prevents the mitogen-induced rise in intracellular calcium activity and activation of Na influx, does not alter the mitogen-stimulated accumulation of inositol trisphosphate. In addition, the calcium ionophore A23187, which increases cytosolic Ca activity and induces a Na influx, does not stimulate the release of inositol trisphosphate. Assays performed in the presence of lithium, which inhibits inositol phosphate monophosphatase, promotes the prolonged and enhanced accumulation of inositol monophosphate. Treatment with the phospholipase inhibitor mepacrine or pretreatment with dexamethasone reduces the amount of inositol phosphates released upon mitogenic stimulation. Hence mitogenic stimulation of HSWP cells leads to the rapid stimulation of inositol phosphate release via a calcium-independent mechanism and suggests inositol trisphosphate as a candidate to mediate the release of intracellular calcium stores which is involved in the processes responsible for the activation of the Na/H exchanger.

PMID: 3028268 [PubMed - indexed for MEDLINE]


29: Biochem Pharmacol. 1986 Feb 1;35(3):449-53.

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Molecular pharmacology of manoalide. Inactivation of bee venom phospholipase A2.

Glaser KB, Jacobs RS.

The marine natural product manoalide (MLD) was shown to directly inactivate bee venom phospholipase A2 (PLA2). Inactivation was pH dependent (maximum inactivation occurred at pH 8.0), time dependent and concentration dependent. The IC50 was estimated at 0.05 microM and virtually complete inactivation of the enzyme occurred at 4.0 microM. The time-dependent loss of PLA2 activity suggested that inactivation does not follow typical Michaelis-Menten kinetics. Reversibility was studied directly by dilution and dialysis; both methods were ineffective in dissociating the MLD-PLA2 complex. A kinetic plot of initial velocity (v) versus [PLA2] supported our hypothesis that MLD apparently inactivates bee venom PLA2 by an irreversible mechanism.

PMID: 3947381 [PubMed - indexed for MEDLINE]


30: Biochem Soc Symp. 1985;50:247-64.

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Cell damage by viruses, toxins and complement: common features of pore-formation and its inhibition by Ca2+.

Pasternak CA, Alder GM, Bashford CL, Buckley CD, Micklem KJ, Patel K.

Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs.
The mechanisms of these effects are discussed.

Publication Types:

·       Review


PMID: 3939403 [PubMed - indexed for MEDLINE]


31: Comp Biochem Physiol C. 1985;82(2):291-6.

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Melittin-induced changes in Na and Cl movements across the skin and cornea (in vitro) of the toad Bufo marinus.

McGahan MC, Hazard RL, Bentley PJ.

Melittin, from bee venom, increases short-circuit current (Isc) across the skin and cornea of toads. In skin this reflects a rise in the influx of Na and is inhibited by meclofenamic acid (inhibits prostaglandin synthetase). In corneas with melittin on the inside the rise in Isc is inhibited by bumetanide (inhibits Cl transport) and meclofenamic acid. Melittin on the tear side of the cornea causes a biphasic change in Isc, and a rise in all undirectional fluxes of Na and Cl. This effect was not changed by bumetanide or meclofenamic acid. Melittin apparently has two types of effects, one mediated by prostaglandins while the other is more direct.

PMID: 2866902 [PubMed - indexed for MEDLINE]


32: Br J Pharmacol. 1984 Apr;81(4):693-701.

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A comparison of histamine secretion from peritoneal mast cells of the rat and hamster.

Leung KB, Pearce FL.

Functional mast cells have been obtained by peritoneal lavage of the rat and hamster. Both cell types released histamine on stimulation with appropriate dilutions of anti-rat IgE and anti-hamster serum. The maximum response evoked by each reagent was significantly greater for the hamster cells. The release was non-cytotoxic and was in each case blocked by the corresponding soluble antigen. The rat and hamster cells responded to concanavalin A and the lectin from lentil. Phosphatidylserine (PS) potentiated the release only from the rat cells. In the absence of the lipid, the hamster cells were more reactive. The lectin from wheat germ, in the presence of PS, evoked histamine secretion only from the rat cells. Both populations were refractory to the lectin from soybean and to protein A. Rat peritoneal cells were more responsive to the basic secretagogues compound 48/80 and peptide 401 (the MCD-peptide from bee venom). These differences were less marked in the case of polylysine and polyarginine. The two cell populations responded to the calcium ionophores A23187, ionomycin and chlortetracycline. The hamster cells were significantly more sensitive to the former two liberators but markedly less reactive to chlortetracycline. Adenosine 5'-triphosphate (ATP) and dextran were potent histamine liberators from the rat cells but were totally ineffective against the hamster. Acetylcholine and carbamylcholine had no effect on either cell type. These results are discussed in terms of the functional heterogeneity of mast cells from different sources.

PMID: 6202354 [PubMed - indexed for MEDLINE]


33: Biochim Biophys Acta. 1983 Dec 7;736(1):57-66.

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Changes in membrane phospholipid distribution during platelet activation.

Bevers EM, Comfurius P, Zwaal RF.

Exposure of phospholipids at the outer surface of activated and control platelets was studied by incubation with a mixture of phospholipase A2 from Naja naja and bee venom, solely or in combination with sphingomyelinase from Staphylococcus aureus, using conditions under which cell lysis remained below 10%. Incubation with phospholipase A2 alone revealed a markedly increased susceptibility of the phospholipids in platelets activated by a mixture of collagen plus thrombin, by the SH-oxidizing compound diamide, or by calcium ionophore A23187, as compared to control platelets or platelets activated separately by collagen or thrombin. Collagen plus thrombin, diamide, and ionophore treated platelets revealed an increased exposure of phosphatidylserine at the outer surface accompanied by a decreased exposure of sphingomyelin, as could be concluded from incubations with a combination of phospholipase A2 and sphingomyelinase. These alterations were much less apparent in platelets activated either by thrombin or by collagen alone. The increased exposure of phosphatidylserine in activated platelets is accompanied by an increased ability of the platelets to enhance the conversion of prothrombin to thrombin by coagulation factor Xa, in the presence of factor Va and calcium. It is concluded that the altered orientation of the phospholipids in the plasma membrane of platelets activated by collagen plus thrombin, by diamide, or by calcium ionophore, is the result of a transbilayer movement. Moreover, the increased exposure of phosphatidylserine in platelets stimulated by the combined action of collagen and thrombin might be of considerable importance for the hemostatic process.

PMID: 6418205 [PubMed - indexed for MEDLINE]


34: FEBS Lett. 1983 Sep 5;161(1):41-4.

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Resistance to apamin of the Ca2+-activated K+ permeability in pancreatic B-cells.

Lebrun P, Atwater I, Claret M, Malaisse WJ, Herchuelz A.

The bee venom neurotoxin apamin failed to affect 86Rb outflow and insulin release from rat pancreatic islets stimulated by D-glucose or the Ca2+-ionophore A23187. Apamin, in contrast to quinine or A23187, also failed to affect bioelectrical activity in mouse islet cells. These findings suggest that, like in erythrocytes, and at variance with the situation found in smooth muscle, liver or neuroblastoma cells, the Ca2+-activated K+ permeability in the pancreatic B-cell is resistant to apamin.

PMID: 6411494 [PubMed - indexed for MEDLINE]


35: Biochim Biophys Acta. 1983 Jan 5;727(1):108-14.

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Melittin and a chemically modified trichotoxin form alamethicin-type multi-state pores.

Hanke W, Methfessel C, Wilmsen HU, Katz E, Jung G, Boheim G.

The bee venom constituent, melittin, is structurally and functionally related to alamethicin. By forming solvent-free planar bilayers of small area (approx. 100 microns 2) on the tip of fire-polished glass pipettes we could observe single melittin pores in these membranes. An increase in the applied voltage induced further non-integral conductance levels. This indicates that melittin forms multi-level pores similar to those formed by alamethicin. Trichotoxin A40, an antibiotic analogue of alamethicin, also induces a voltage-dependent bilayer conductivity, but no stable pore states are resolved. However, chemical modification of the C-terminal molecule part by introduction of a dansyl group leads to a steeper voltage-dependence and pore state stabilization. Comparing structure and activity of several natural and synthetic amphiphilic polypeptides, we conclude that a lipophilic, N-terminal alpha-helical part of adequate length (dipole moment) and a large enough hydrophilic, C-terminal region are sufficient prerequisites for voltage-dependent formation of multi-state pores.

PMID: 6824646 [PubMed - indexed for MEDLINE]


36: Adv Exp Med Biol. 1983;156:553-67.

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Influence of aprotinin and gabexate mesilate on arachidonic acid release by the Ca-ionophore A 23187 in the lung.

Seeger W, Wolf H, Graubert E, Moser U, Neuhof H, Roka L.

In a model of isolated, ventilated rabbit lungs, perfused with Krebs-Henseleit albumin buffer in a recirculating system, increased availability of free AA (arachidonic acid) results in an increase in pulmonary vascular resistance and permeability. The former can be ascribed to cyclooxygenase products of AA, among which thromboxane A2 is mainly responsible. The increase of vascular permeability is, at least partly, due to lipoxygenase products of AA. Availability of free AA for the different oxygenation pathways can be achieved either by direct application of free AA to the perfusion fluid or by stimulation of AA release from the membrane phospholipid pool by the Ca-ionophore A 23187. The serine proteinase inhibitor gebaxate mesilate in a concentration range between 1 microM and 10 microM and aprotinin in a concentration range between 8 and 200 KIE/ml dose-dependently reduce the increase in vascular resistance after stimulation with A 23187. Correspondingly the increase in vascular permeability due to A 23187 is significantly reduced by gabexate mesilate (5 microM) to 52% and by aprotinin (200 KIE/ml) to 73%. On the contrary the increase in pulmonary vascular resistance and permeability after direct application of free AA to the perfusion fluid is not affected by gabexate mesilate and aprotinin. AA metabolism by cyclooxygenase from ram vesicular gland microsomes is inhibited in vitro by gabexate mesilate and by aprotinin only in very high concentrations (greater than 1mM respectively greater than 2130 KIE/ml). Measurements with porcine pancreas and bee venom phospholipase A2 reveal no influence of aprotinin on these enzymes. Gabexate mesilate inhibits pancreas phospholipase A2 in concentrations more than 10-fold higher than those necessary in the isolated lungs (IC50 = 430 microM), bee venom phospholipase A2 not being affected at all. It is thus apparent that the release of AA from the membrane phospholipid pool rather than any particular step in its oxygenation metabolism is the site of action of these proteinase inhibitors in the pulmonary vascular bed. The possible involvement of an intracellular proteinase is discussed.

PMID: 6190378 [PubMed - indexed for MEDLINE]


37: J Immunol. 1982 Jun;128(6):2481-6.

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Mucosal mast cells. II. Effects of anti-allergic compounds on histamine secretion by isolated intestinal mast cells.

Pearce FL, Befus AD, Gauldie J, Bienenstock J.

Functional mast cells have been isolated from the lamina propria of the small intestine of rats infected with the nematode Nippostrongylus brasiliensis. The cells released histamine on challenge with specific antigen, anti-rat IgE, concanavalin A, and calcium ionophores but were less responsive than peritoneal mast cells (MMC) from the same animals. Intestinal mucosa mast cells (PMC) were refractory to the action of the basic secretagogues peptide 401 from bee venom and compound 48/80. The anti-allergic compounds disodium cromoglycate (less than or equal to 10(-3) M), AH 9679 (less than or equal to 10(-4) M), and theophylline (less than or equal to 10(-2)) did not inhibit antigen-induced histamine secretion by MMC, although these compounds were effective against PMC. In contrast, doxantrazole (10(-5) to 10(-3) M) inhibited the secretion of histamine from both MMC and PMC in a comparable dose-dependent fashion. Thus, we have established that mast cells from different sites are functionally heterogeneous not only in their response to various stimuli for histamine secretion, but also in their responses to different pharmacologic modulators of secretion. It cannot be assumed that anti-allergic compounds effective against mast cells in one tissue site or organ will be equally efficacious against mast cells in other sites. The extent of this functional heterogeneity must be established, and its investigation may provide new insights into the biochemical events involved in mast cell secretion.

PMID: 6176639 [PubMed - indexed for MEDLINE]


38: J Physiol. 1981 Aug;317:67-90.

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Effects of quinine and apamin on the calcium-dependent potassium permeability of mammalian hepatocytes and red cells.

Burgess GM, Claret M, Jenkinson DH.

1. K-sensitive electrodes placed in the extracellular fluid have been used to show that ATP and noradrenaline cause a rapid loss of up to 10% of the K content of isolated guinea-pig hepatocytes. 2. The hypothesis tha this response is a consequence of a rise in the K permeability of the hepatocyte membrane triggered by an increase in cytosolic Ca is supported by the finding that the divalent cation ionophore A23187 also initiated K loss, in this instance of up to 20-25% of the amount in the cells. 3. Under similar conditions A23187 caused a transient increase, followed by a larger decrease, in the 45Ca content of guinea-pig hepatocytes equilibrated with this isotope. The decrease alone was seen with ATP and noradrenaline. 4. Quinine (1 mM) and the bee venom neurotoxin apamin (10 nM) greatly reduced the effect of ATP, noradrenaline and A23187 on K content without affecting the changes in 45Ca movement. 5. Apamin (10 nM) also abolished the increase in 42K efflux which follows the application of the alpha-adrenoceptor agonist amidephrine to rabbit liver slices; the concurrent rises in 45Ca efflux and glucose release were unaffected. 6. It was concluded that quinine and apamin are able to block either the Ca-dependent K channels present in guinea-pig and rabbit liver cell membranes or the mechanism that controls them. 7. Surprisingly, rat hepatocytes took up rather than lost K when treated with the concentrations of ATP, noradrenaline or A23187 that initiated K loss from guinea-pig cells. This response was greatly reduced by ouabain. 8. Application of large concentrations of A23187 to rat hepatocytes caused K loss associated with cell death. 9. The influence of apamin (10-1000 nM) and quinine (200-1000 micro M) on the Ca-dependent K permeability of red blood cells and ghosts was also studied. Apamin was without effect even when applied to both sides of the ghost membrane, whereas quinine caused inhibition, as reported by others. 10. The results suggest that Ca-dependent K channels or carriers are present in the membranes of liver cells of the guinea-pig and rabbit, but are either lacking or inactive in rat liver. The finding that apamin blocks this mechanism in hepatocytes but not in erythrocytes may mean that the channels differ in these cells.

PMID: 6273550 [PubMed - indexed for MEDLINE]


39: Nature. 1981 Jul 16;292(5820):246-8.

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Sequence and specificity of two antibacterial proteins involved in insect immunity.

Steiner H, Hultmark D, Engstrom A, Bennich H, Boman HG.

Immune responses have been described for many different insect species. However, it is generally acknowledged that immune systems must therefore differ from those of vertebrates. An effective humoral immune response has been found in pupae of the cecropia moth, Hyalophora cecropia. The expression of this multicomponent system requires de novo synthesis of RNA and proteins and its broad antibacterial activity is due to at least three independent mechanisms, the most well known of which is the insect lysozyme. However, this enzyme is bactericidal for only a limited number of Gram-positive bacteria. WE recently purified and characterized P9A and P9B, which are two small, basic proteins with potent antibacterial activity against Escherichia coli and several other Gram-negative bacteria. We believe that P9A and P9B plays an important part in the humoral immune responses described previously and that the P9 proteins represent a new class of antibacterial agents for which we propose the name cecropins. We describe here the primary structures of cecropins A and B. We also show that cecropin A is specific for bacteria in contrast to melittin, the main lytic component in bee venom which lyses both bacteria and eukaryotic cells.

PMID: 7019715 [PubMed - indexed for MEDLINE]


40: Biokhimiia. 1980 Oct;45(10):1840-9.

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[Effects of melittin and its tetraacetyl derivative on rat liver mitochondria]

[Article in Russian]

Shol'ts KF, Reznik GI, Solov'eva NA, Shezhkova LG, Miroshnikov AI.

The uncoupling effect of melittin, the principal peptide of bee venom was studied. It was found that the activation of mitochondrial respiration by melittin and its derivative is due to the formation of channels equally penetrable for lithium, sodium, potassium and rubidium ions. The penetrability of the inner mitochondrial membrane appears at melittin or tetraacetyl melittin concentrations about 0.90 or 0.15 nmol/mg of protein, respectively. The appreciable increase of the uncoupling activity of melittin under blocking of its amino groups suggests that these groups are not very essential for the channel formation. In contrast to gramicidin S and similar to gramicidin A, the effect of melittin develops in time. The lag in the effects of melittin and its derivative indicates that the slow rearrangement stage precedes the formation of channels in the membrane. It is suggested that the effects of melittin are due to the formation of multimolecular complexes of the peptide with phosphatides of the inner mitochondrial membrane.

PMID: 6165401 [PubMed - indexed for MEDLINE]


41: Biochim Biophys Acta. 1979 Sep 12;570(1):198-209.

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Effect of alamethicin, gramicidin S and melittin upon the particulate guanylate cyclase from rat lung.

Lad PJ, White AA.

The channel-forming antibiotic alamethicin activated rat lung particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2), and the activated enzyme was further stimulated by sodium nitroprusside when a thiol such as 2-mercaptoethanol was present. Similar effects were seen with the antibiotic gramicidin S and with melittin, a polypeptide purified from bee venom. All of these agents are amphiphilic polypeptides. Nitroprusside was not able to stimulate both particulate and soluble enzyme treated with the nonionic amphiphile, Lubrol PX, suggesting that the membrane-active polypeptides had a different mechanism of action. These polypeptides are known to alter the membrane matrix by binding to phospholipid, and we suggest that this alteration allowed greater access of substrate and of nitroprusside to the enzyme. Lubrol PX, however, may interact preferentially with the enzyme, and thus block nitroprusside activation. The most potent of these agents was melittin, which stimulated nitroprusside activation at a concentration which had little effect by itself (7 microns), and at which others have demonstrated lytic effects on cells.

PMID: 90524 [PubMed - indexed for MEDLINE]


42: Med Biol. 1976 Feb;54(1):39-49.

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Cytotoxic effects on the plasma membrane of human diploid fibroblasts--a comparative study of leakage tests.

Thelestam M, Mollby R.

Confluent monolayers of human diploid lung fibroblasts were treated with cytolytic agents. The induced membrane damage was investigated by different test systems. Changes of membrane permeability were compared with morphological alterations. Four tests employed leakage of cytoplasmic markers of different sizes as criteria of membrane damage. Radioactive markers of the following decreasing size order were used: [3H]RNA (MW greater than 200,000) greater than 51Cr greater than [3H]nucleotides greater than [1-14C]alpha-amino-isobutyric acid (AIB, MW 103). Uptake of trypan blue was employed as a fifth criterior of changed membrane permeability. A comparison between the tests indicated the following order of sensitivity for detection of membrane damage: leakage of AIB-label greater than leakage of nucleotide label greater than leakage of 51Cr-label=uptake of trypan blue= morphological changes greater than leakage of RNA-label. Two distinct types of leakage patterns were evident: 1. Upon incubation with Triton X-100 all four release curves coincided. This was considered as representing large functional "holes". 2. With the polyene amphotericin B the smallest marker (AIB) was released to a strikingly greater extent than other markers. This was regarded as representing very small functional "holes". Melittin from bee venom and theta-toxin from Clostridium perfringens induced leakage patterns of two intermediate types. The results indicate that a combination of several different size markers may be useful for characterizing induced membrane lesions.

PMID: 1263610 [PubMed - indexed for MEDLINE]


43: Infect Immun. 1975 Aug;12(2):225-32.

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Sensitive assay for detection of toxin-induced damage to the cytoplasmic membrane of human diploid fibroblasts.

Thelestam M, Mollby R.

A sensitive assay was developed for detection and quantitation of subtle permeability changes in the cytoplasmic membrane of human diploid fibroblasts. Release of the non-metabolizable amino acid [1-14C]alpha-aminoisobutyric acid (AIB; molecular weight (103) from the cytoplasm of prelabeled cells was used as an indicator of toxin-induced membrane damage. An optimal procedure for labeling these cells was designed after varying the conditions with regard to pH, temperature, concentration of AIB, composition of medium, and incubation time. Toxin-induced release of AIB was compared with release of a previously described nucleotide label, [3H]uridine. Melittin from bee venom and the polyene antibiotics filipin and amphotericin B in low concentrations induced a strikingly greater release of AIB than of nucleotide label. The sensitivity of this assay was furthermore demonstrated by treatment with the following bacterial cytolysins: phospholipase C and theta-toxin from Clostridium perfringens, alpha-, beta-, delta-, and gamma-toxins from Staphylococcus aureus, and streptolysin S from Streptococcus pyogenes. In spite of their different modes of action, all these membrane-active toxins at low concentrations induced a significant release of AIB label. For an equal release of nucleotide label, several times higher concentrations were required.

PMID: 169201 [PubMed - indexed for MEDLINE]


44: Infect Immun. 1975 Apr;11(4):640-8.

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Determination of toxin-induced leakage of different-size nucleotides through the plasma membrane of human diploid fibroblasts.

Thelestam M, Mollby R.

Human diploid lung fibroblasts were treated with cytolytic bacterial toxins and the nature of the membrane damage was investigated. [3H] uridine was used for differential labeling of cytoplasmic components of small or large molecular size. Two principal size categories were achieved by labeling the fibroblasts in either early growth phase or stationary phase, a high-molecular weight ribonucleic acid label and a low-molecular-weight nucleotide label. The size of the labeled molecules was determined by perchloric acid precipitation and gel chromatography. Leakage of labeled molecules of different size indicated the size of the "functional pores" in the plasma membrane caused by the test substance. The nonionic detergent Triton X-100 produced large functional pores in the fibroblast membrane as evidenced by rapid leakage of both large and small labeled molecules. Theta-toxin from Clostridium perfringens and the polyene antibiotic filipin both gave rise to considerably small functional pores in the plasma membrane. Although small molecules easily passed the treated membrane, large molecules could not escape from the cells even after prolonged treatment with these substances or by increasing their concentration. By the contrast, the leakage profiles obtained with melittin from bee venom or with delta-toxin from Staphylococcus aureus in each case suggested the formation initially of pores of intermediate size that increased upon prolonged incubation or when higher concentrations were used.

PMID: 164404 [PubMed - indexed for MEDLINE]


45: FEBS Lett. 1974 Sep 15;46(1):141-4.

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Enhancement of bee venom phospholipase A2 activity by melittin, direct lytic factor from cobra venom and polymyxin B.

Mollay C, Kreil G.

PMID: 4371280 [PubMed - indexed for MEDLINE]


46: Z Naturforsch B. 1969 Feb;24(2):263.

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[On the biological activity of the bee venom melittin]

[Article in German]

Jentsch J.

PMID: 4388835 [PubMed - indexed for MEDLINE]


47: Proc Soc Exp Biol Med. 1968 Mar;127(3):707-10.

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Antibacterial action of melittin, a polypeptide from bee venom.

Fennell JF, Shipman WH, Cole LJ.

PMID: 4870538 [PubMed - indexed for MEDLINE]


48: Res Dev Tech Rep. 1967 Dec 5;:1-13.

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Antibacterial action of a bee venom fraction (melittin) against a penicillin-resistant staphylococcus and other microorganisms. USNRDL-TR-67-101.

Fennell JF, Shipman WH, Cole LJ.

PMID: 5300771 [PubMed - indexed for MEDLINE]