Mast Cell Degranulation Activity Research Articles

Characterization of the Molecular Diversity and Degranulation Activity of Mastoparan Family Peptides from Wasp Venoms.

Wasp stings have become an increasingly serious public health problem because of their high incidence and mortality rates in various countries and regions. Mastoparan family peptides are the most abundant natural peptides in hornet venoms and solitary wasp venom. However, there is a lack of systematic and comprehensive studies on mastoparan family peptides from wasp venoms. In our study, for the first time, we evaluated the molecular diversity of 55 wasp mastoparan family peptides from wasp venoms and divided them into four major subfamilies. Then, we established a wasp peptide library containing all 55 known mastoparan family peptides by chemical synthesis and C-terminal amidation modification, and we systematically evaluated their degranulation activities in two mast cell lines, namely the RBL-2H3 and P815 cell lines. The results showed that among the 55 mastoparans, 35 mastoparans could significantly induce mast cell degranulation, 7 mastoparans had modest mast cell degranulation activity, and 13 mastoparans had little mast cell degranulation activity, suggesting functional variation in mastoparan family peptides from wasp venoms. Structure-function relationship studies found that the composition of amino acids in the hydrophobic face and amidation in the C-terminal region are critical for the degranulation activity of mastoparan family peptides from wasp venoms. Our research will lay a theoretical foundation for studying the mechanism underlying the degranulation activity of wasp mastoparans and provide new evidence to support the molecular design and molecular optimization of natural mastoparan peptides from wasp venoms in the future.

Nasal immunization with mast cell activating small molecules protects mice against West Nile Virus infection

Abstract Objective: Mast cell activators are a novel class of mucosal vaccine adjuvants. The polymeric compound, Compound 48/80 (C48/80), and cationic peptide, Mastoparan 7 (M7) are mast cell activators that provide adjuvant activity when administered by the nasal route. However, mast cell activating small molecules may be an adjuvant alternative that is easily synthesized with high purity compared to M7 or C48/80. The study objective is to identify small molecule mast cell activators that provide adjuvant activity in nasal West Nile virus vaccines. Methods: High-throughput screening assessed over 55,000 small molecules for mast cell degranulation activity. Fifteen hit compounds were down-selected to three by in vitro evaluation of immune activation activities, including cytokine production and cytotoxicity, and selection for functional diversity. These three molecules, ST101036, ST048871, and R529877, were then examined for their ability to evoke protection against West Nile virus infections in BALB/c mice when administered nasally with West Nile virus envelope domain III (EDIII) protein. Results: All three adjuvants evoked high levels of EDIII-specific antibody and conferred similar levels of protection, ST101036 (67%), ST048871 (73%) and R529877 (75%), which was also comparable to the protection induced by M7 (67%) but markedly higher than the levels seen with mice immunized with EDIII alone (no adjuvant 33%). Conclusion: Novel small molecule mast cell activators are as efficacious as previously described mast cell activating peptides and serve as potential replacements in in vivo studies.

Deletion of ΔdblGata motif leads to increased predisposition and severity of IgE-mediated food-induced anaphylaxis response.

BackgroundPrevious studies have revealed an important role for the transcription factor GATA-1 in mast cell maturation and degranulation. However, there have been conflicting reports with respect to the requirement of GATA-1 function in mast cell dependent inflammatory processes. Herein, we examine the requirement of GATA-1 signaling in mast cell effector function and IgE-mast cell-dependent anaphylaxis.ObjectiveTo study the requirement of GATA-1 dependent signaling in the development and severity of IgE-mast cell-dependent anaphylaxis in mice.MethodsWild type (Balb/c) and mutant ΔdblGata (Balb/c) mice were employed to study the role of GATA-1 signaling in in vitro IgE-mediated activation of bone marrow derived mast cells (BMMCs). Murine models of passive IgE-mediated and oral antigen-induced IgE-mediated anaphylaxis were employed in mice. Frequency of steady state mast cells in various tissues (duodenum, ear, and tongue), peritoneal cavity, and clinical symptoms (diarrhea, shock, and mast cell activation) and intestinal Type 2 immune cell analysis including CD4+ Th2 cells, type 2 innate lymphoid cells (ILC2), and IL-9 secreting mucosal mast cells (MMC9) were assessedResultsIn vitro analysis revealed that ΔdblGata BMMCs exhibit a reduced maturation rate, decreased expression of FcεRIα, and degranulation capacity when compared to their wildtype (WT) counterparts. These in vitro differences did not impact tissue resident mast cell numbers, total IgE, and susceptibility to or severity of IgE-mediated passive anaphylaxis. Surprisingly, ΔdblGata mice were more susceptible to IgE-mast cell-mediated oral antigen induced anaphylaxis. The increased allergic response was associated with increased Type 2 immunity (antigen-specific IgE, and CD4+ TH2 cells), MMC9 cells and small intestine (SI) mast cell load.ConclusionDiminished GATA-1 activity results in reduced in vitro mast cell FcεRIα expression, proliferation, and degranulation activity. However, in vivo, diminished GATA-1 activity results in normal homeostatic tissue mast cell levels and increased antigen-induced CD4+ Th2 and iMMC9 cell levels and heightened IgE-mast cell mediated reactions.

1030 Role of mast cells in psoriasis associated-pruritus

Whilst a key role of mast cells in itch (pruritogenic pruritus) is well-established, the exact underlying mechanisms remain to be fully elucidated. Here, we have examined the role of mast cells in psoriasis-associated pruritus. By immunofluorescence microscopy we confirmed not only that lesional psoriasis skin showed increased mast cell numbers, degranulation activity and overall innervation, but also an increased density of PAR2-positive nerve fibres. Moreover, the proximity of visualized nerve fibres and mast cells was increased suggesting enhanced mast cell-nerve interaction. To characterized mast cell related-itch pathways, healthy human skin was exposed in vitro for 18 hours to known pruritogens which induce mast cell activation such as compound 48/80, substance P (SP), and the selective proteinase-activated receptor 2 (PAR2)-activating peptide, SLIGKV, to mimic conditions of itch and neurogenic skin inflammation and to compare the expression of itch-associated genes with unexposed skin. RNA-Seq with DESeq2 was used for analysis of transcript expression. In organ-cultured healthy human skin, TSLP transcripts were upregulated 1.65, 1.53, and 1.59 fold following stimulation with 100 μg/ml of compound 48/80, 2 μM of SP and 100 μM of SLIGKV, respectively. In addition, SP stimulation upregulated IL31RA and TLR7 gene expression 3.22 and 2.56 fold, respectively, whilst stimulation with SLIGKV also upregulated MRGPRX2, TRPA1, and IL2RA 1.56, 2.21, and 1.76 fold, respectively. The differences in itch-associated genes, other than TSLP, upregulated following stimulation of skin with compound 48/80, SP, and SLIGKV may suggest that SP and PAR-2 agonists could induce itch through multiple, distinct and synergistic signalling pathways. Furthermore, these findings indicate that SP stimulation increases the sensitivity of the skin to potent pruritogens, such as IL-31 or TLR7 ligands, by upregulating their receptors.

Synthesis, toxicity study and anti-inflammatory effect of MHTP, a new tetrahydroisoquinoline alkaloid

The alkaloid 2-methoxy-4-(7-methoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)phenol (MHTP) was synthesized to prospect new compounds with therapeutic properties. Thus, the goal of this study was to evaluate the MHTP anti-inflammatory effect by in vivo and in vitro assays. The MHTP toxicity was analyzed. We found that MHTP pre-treatment (2.5–10 mg/kg) showed antiedematogenic effect (p < 0.05) in carrageenan-induced paw edema by inhibiting the PGE2 action independently of mast cell degranulation or histamine activity. MHTP also diminished (p < 0.01) total leukocyte migration in 41.5% into peritoneal cavity during carrageenan-induced peritonitis, reducing polymorphonuclear cells (PMNs) (59.6%) and proteins levels (29.4%). MHTP in an experimental model of acute lung injury inhibited (p < 0.001) total inflammatory cell migration into the lungs and PMNs in 58% and 67.5%, respectively. Additionally, MHTP did not present cytotoxicity at concentrations of 10, 25 or 50 μM but decreased (p < 0.001) the NO production in 24%, 47% and 39%, respectively. The alkaloid also reduced (p < 0.001, in lipopolysaccharide (LPS)-stimulated macrophages (1 μg/mL), IL-1β, IL-6 and IL-10 levels in 35.7%, 31.0% and 33.4%, respectively. The results obtained in this study allow us to conclude that the inedited synthetic alkaloid, MHTP has anti-inflammatory effect by inhibiting PGE2 function as well as inhibiting inflammatory cell migration to the inflamed site and attenuated the acute lung injury disease by inhibiting the migration of neutrophil to the lung. However, further studies will be carried out to demonstrate the mechanisms of action of the molecule and explore its potential as a future drug to treat inflammatory processes.

Role of peptide-peptide interactions in aggregation: Protonectins observed in equilibrium and replica exchange molecular dynamics simulations.

Protonectin (ILGTILGLLKGL-NH2), a peptide extracted from the venom of the wasp Agelaia pallipes pallipes, promotes mast cell degranulation activity, antibiosis against Gram-positive and -negative bacteria, and chemotaxis in polymorphonucleated leukocytes. Another peptide from the same venom, Protonectin (1-6), corresponding to the first six residues of Protonectin, exhibits only chemotaxis. A 1:1 mixture of these two peptides showed positive synergistic antimicrobial effects, attributed to the formation of a heterodimer.16 The antimicrobial activity is probably related to the peptides' interaction with membrane phospholipids. Equilibrium and replica exchange molecular dynamics simulations were used to investigate two systems: the interaction of Protonectins (two molecules) and that of a mixture Protonectin and Protonectin (1-6) in the environment of sodium dodecyl sulfate (SDS) micelles, which mimic bacterial membranes and are also highly anionic. We found that in both systems the peptides tend to aggregate in the aqueous environment and are held together by hydrophobic interactions and hydrogen bonds. In the equilibrium simulations, aggregated Protonectin/Protonectin (1-6) dissociates after penetrating the SDS micelle, whereas the two Protonectins remain associated throughout the simulation time. Also, in the replica exchange simulations, the Protonectins remain closer, associating through a greater number of hydrogen bonds, and were found at only one free energy minimum, whereas the peptides in the mixture display other probable distances from each other, which are significantly longer than those observed with two Protonectin molecules. Coulomb contributions and the free energy of the systems containing micelles were calculated and show that the interactions of the mixed peptides are favored, whereas the interactions between pure Protonectins are more probable. As a consequence of the preferential interaction with the micelle, the Protonectin molecule of the mixed system presents a higher helical structure content. The enhancement of the amphipathic features caused by Protonectin (1-6) can be related to the increase in the antimicrobial activity experimentally observed.

Molecular Cloning and Multifunctional Characterization of Host Defense Peptides from the Bullfrog Harderian Gland with Special Reference to Catesbeianalectin

The Harderian gland (HG) is an orbital gland found in many terrestrial vertebrates that possess a nictitating membrane. Using reverse-transcription polymerase chain reaction (RT-PCR), we cloned five cDNAs encoding antimicrobial peptide (AMP)-homologs, catesbeianalectin, ranacyclin-CBa, ranatuerin-1CBa, ranatuerin-2CBa, and ranatuerin-2CBb, from the bullfrog HG total RNA. Of these, catesbeianalectin has not been thoroughly studied in terms of its biological activities. We examined antimicrobial activities of the synthetic replicate of catesbeianalectin and its putative unprocessed precursor, catesbeianalectin-GK. Both peptides showed slight but significant growth inhibitory activity against the Gram-negative bacterium Escherichia coli. Subsequently, we tested catesbeianalectin and catesbeianalectin-GK for mast cell degranulation activity as a criterion of the release of N-acetyl-β-D-glucosaminidase from the mouse-derived mastocytoma cell line P-815, followed by the standard MTT assay to assess cell survival and recovery after peptide treatment. We found that catesbeianalectin and catesbeianalectin-GK invariably exhibited mast cell degranulation activity without cytotoxic effects. Hemagglutination assay revealed the presence of lectin-like activity in both catesbeianalectin and catesbeianalectin-GK. Our findings strongly suggest that these multifunctional host defense peptides in the amphibian HG are involved in innate immunodefense of the eye of the host against pathogenic environmental microorganisms.

Model membrane interaction and DNA-binding of antimicrobial peptide Lasioglossin II derived from bee venom

Lasioglossins, a new family of antimicrobial peptide, have been shown to have strong antimicrobial activity with low haemo-lytic and mast cell degranulation activity, and exhibit cytotoxic activity against various cancer cells in vitro. In order to understand the active conformation of these pentadecapeptides in membranes, we have studied the interaction of Lasioglossin II (LL-II), one of the members of Lasioglossins family with membrane mimetic micelle Dodecylphosphocholine (DPC) by fluorescence, Circular Dichroism (CD) and two dimensional (2D) 1H NMR spectroscopy. Fluorescence experiments provide evidence of interaction of the N-terminal tryptophan residue of LL-II with the hydrophobic core of DPC micelle. CD results show an extended chain conformation of LL-II in water which is converted to a partial helical conformation in the presence of DPC micelle. Moreover we have determined the first three-dimensional NMR structure of LL-II bound to DPC micelle with rmsd of 0.36Å. The solution structure of LL-II shows hydrophobic and hydrophilic core formation in peptide pointing towards different direction in the presence of DPC. This amphipathic structure may allow this peptide to penetrate deeply into the interfacial region of negatively charged membranes and leading to local membrane destabilization. Further we have elucidated the DNA binding ability of LL-II by agarose gel retardation and fluorescence quenching experiments.

Structural and biological characterization of mastoparans in the venom of Vespa species in Taiwan

Mastoparans, a family of small peptides, are isolated from the wasp venom. In this study, six mastoparans were identified in the venom of six Vespa species in Taiwan. The precursors of these mastoparans are composed of N-terminal signal sequence, prosequence, mature mastoparan, and appendix glycine at C-terminus. These mature mastoparans all have characteristic features of linear cationic peptides rich in hydrophobic and basic amino acids without disulfide bond. Therefore, these peptides could be predicted to adopt an amphipathic α-helical secondary structure. In fact, the CD (circular dichroism) spectra of these peptides show a high content α-helical conformation in the presence of 8mM SDS or 40% 2,2,2-trifluoroethanol (TFE). All mastoparans exhibit mast cell degranulation activity, antimicrobial activity against both Gram-positive and -negative bacteria tested, various degree of hemolytic activity on chicken, human, and sheep erythrocytes as well as membrane permeabilization on Escherichia coli BL21. Our results also show that the hemolytic activity of mastoparans is correlated to mean hydrophobicity and mean hydrophobic moment.

Immunoglobulin E oligomers identified in blood components activate mast cells: relevance to anaphylactic transfusion reaction

In most cases of anaphylactic transfusion reaction, the mechanisms underlying its development are unclear. We found a donor whose transfused blood components were implicated in two cases of anaphylactic transfusion reaction, and we found that the donor plasma showed mast cell degranulation activity. The donor plasma was examined to identify the mast cell-activating factors in it. Cultured mast cells prepared from cord blood were used for in vitro degranulation assay. Serum prepared from the donor plasma was fractionated by three-step chromatography using mast cell degranulation activity as a marker. The fractions selected from the third step of chromatography were analyzed by mass spectrometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The characteristics of the identified proteins and other plasma samples that had been donated by the donor over several years were examined. Two plasma proteins of high molecular weight were detected in the anion-exchange fractions and identified as human immunoglobulin (Ig)Es of 500 kDa and higher. The mast cell degranulation activity of the IgEs decreased in the presence of monomeric human IgE as well as an anti-human IgE antibody. Mast cell degranulation activity was detected in the donor plasma since January 4, 2002, when the first case was reported. We identified high-molecular-weight IgEs as the mast cell-activating factors in the donor plasma. Results of analysis suggest that these IgEs were dimeric and trimeric and that they directly activated the transfusion recipient's mast cells by triggering the crosslinking of Fcε receptor I, thereby inducing an anaphylactic transfusion reaction.

Lasioglossins: Three Novel Antimicrobial Peptides from the Venom of the Eusocial Bee Lasioglossum laticeps (Hymenoptera: Halictidae)

Three novel structurally related pentadecapeptides, named lasioglossins, were isolated from the venom of the eusocial bee Lasioglossum laticeps. Their primary sequences were established as H-Val-Asn-Trp-Lys-Lys-Val-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-I), H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Ala-Lys-NH(2) (LL-II) and H-Val-Asn-Trp-Lys-Lys-Ile-Leu-Gly-Lys-Ile-Ile-Lys-Val-Val-Lys-NH(2) (LL-III). These lasioglossins exhibited potent antimicrobial activity against both Gram-positive and Gram-negative bacteria, low haemolytic and mast cell degranulation activity, and a potency to kill various cancer cells in vitro. The lasioglossin CD spectra were measured in the presence of trifluoroethanol and sodium dodecyl sulfate solution and indicated a high degree of alpha-helical conformation. NMR spectroscopy, which was carried out in trifluoroethanol/water confirmed a curved alpha-helical conformation with a concave hydrophobic and convex hydrophilic side. To understand the role of this bend on biological activity, we studied lasioglossin analogues in which the Gly in the centre of the molecule was replaced by other amino acid residues (Ala, Lys, Pro). The importance of the N-terminal part of the molecule to the antimicrobial activity was revealed through truncation of five residues from both the N and C termini of the LL-III peptide. C-terminal deamidation of LL-III resulted in a drop in antimicrobial activity, but esterification of the C terminus had no effect. Molecular modelling of LL-III and the observed NOE contacts indicated the possible formation of a bifurcated H-bond between hydrogen from the Lys15 CONH peptide bond and one H of the C-terminal CONH(2) to the Ile11 oxygen atom. Such interactions cannot form with C-terminal esterification.

Antidiarrhoeal activity of the standardised extract of Cinnamomum tamala in experimental rats

The present study was designed to investigate the antidiarrhoeal potential of 50% ethanolic extract of Cinnamomum tamala on experimentally induced castor oil diarrhoea, gastric emptying of phenol red meal, gastrointestinal transit of charcoal meal and in vitro mast cell degranulation activity. C. tamala extract (25, 50 and 100 mg/kg, orally) produced a dose-dependent reduction in the total amount of faecal matter in castor oil-induced diarrhoea. The mean distance travelled by charcoal meal at 50 and 100 mg/kg of extract showed a significant reduction in the secretion of gastrointestinal fluid accumulation by 32.5-65.0%. The Na(+) and K(+) concentrations on castor oil-induced fluid accumulation showed a greater inhibitory effect on Na(+) levels than on K(+) concentrations. C. tamala significantly reduced the lipid peroxidation (P < 0.001) and increased the catalase (P < 0.01) activity in comparison to the castor oil-induced groups. C. tamala leaf extract did not show any significant effect at a higher dose (15 mg/ml) on mast cell degranulation. However, the extract in the dose of 5 and 10 mg/ml conferred significant mast cell protective action (P < 0.001). The percentage of eugenol in extract is 3.8% w/w, and total tannin is 247.5 mg/g. The result indicates the Indian spice C. tamala is useful for diarrhoea.

Structural and functional characterization of two novel peptide toxins isolated from the venom of the social wasp Polybia paulista

Two novel inflammatory peptides were isolated from the venom of the social wasp Polybia paulista. They had their molecular masses determined by ESI-MS and their primary sequences were elucidated by Edman degradation chemistry as: • Polybia-MPI: I D W K K L L D A A K Q I L-NH 2 (1654.09 Da), • Polybia-CP: I L G T I L G L L K S L-NH 2 (1239.73 Da). Both peptides were functionally characterized by using Wistar rat cells. Polybia-MPI is a mast cell lytic peptide, which causes no hemolysis to rat erythrocytes and presents chemotaxis for polymorphonucleated leukocytes (PMNL) and with potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Polybia-CP was characterized as a chemotactic peptide for PMNL cells, presenting antimicrobial action against Gram-positive bacteria, but causing no hemolysis to rat erythrocytes and no mast cell degranulation activity at physiological concentrations.

Structural and biological characterization of two novel peptides from the venom of the neotropical social wasp Agelaia pallipes pallipes

The venom of the neotropical social wasp Agelaia pallipes pallipes was fractionated by RP-HPLC resulting in the elution of seven fractions; the last two were re-fractionated under RP-HPLC by using isocratic elution conditions and the purity of the fractions were confirmed by using ESI-MS analysis. Both fractions are constituted of peptide components, which were sequenced by Edman degradation chemistry, resulting in the following sequences: Protonectin I-L-G-T-I-L-G-L-L-K-G-L-NH 2 Agelaia-MP I-N-W-L-K-L-G-K-A-I-I-D-A-L-NH 2 Both peptides are manually synthesized on solid-phase and functionally characterized by using Wistar rats cells. Protonectin is a non-hemolytic chemotactic peptide for polymorphonucleated leukocytes (PMNL), presenting some mast cell degranulating activity and potent antimicrobial action both against Gram-positive and Gram-negative bacteria. Agelaia-MP was characterized as a hemolytic mast cell degranulator toxin, presenting a poor antimicrobial action and no chemotaxis for PMNL.

Jelleines: a family of antimicrobial peptides from the Royal Jelly of honeybees ( Apis mellifera)

Four antimicrobial peptides were purified from Royal Jelly of honeybees, by using reverse phase-HPLC and sequenced by using Q-Tof-MS/MS: PFKLSLHL-NH 2 (Jelleine-I), TPFKLSLHL-NH 2 (Jelleine-II), EPFKLSLHL-NH 2 (Jelleine-III), and TPFKLSLH-NH 2 (Jelleine-IV). The peptides were synthesized on-solid phase, purified and submitted to different biological assays: antimicrobial activity, mast cell degranulating activity and hemolysis. The Jelleines-I–III presented exclusively antimicrobial activities against yeast, Gram+ and Gram− bacteria; meanwhile, Jelleine-IV was not active in none of the assays performed. These peptides do not present any similarity with the other antimicrobial peptides from the honeybees; they are produced constitutively by the workers and secreted into Royal Jelly.

Glycoprotein derived from the hot water extract of mint plant, Perilla frutescens britton.

Glycoprotein showing inhibitory activity against mast cell degranulation and hyaluronidase activity was purified from the hot water extract of mint plant (Perilla frutescens Britton). The purified inhibitor gave a single band detected with Coomassie brilliant blue staining and periodic acid-Schiff staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The molecular mass was estimated to be 6.0 kDa on SDS-PAGE. The inhibitor did not become inactivated when boiled for 30 min or digested with trypsin, V8 protease, or proteinase K but was inactivated by NaIO(4) oxidation. The inhibitor prevented mast cell degranulation and hyaluronidase activity (IC(50) = 0.42 mg/mL) in a dose-dependent manner. The inhibitor also inhibited the protein kinase C activity. It is possible to purify and characterize a glycoprotein with putative pharmacological properties from mint plants.

Anti‐inflammatory activity of bee venom peptide 401 (mast cell degranulating peptide) and compound 48/80 results from mast cell degranulation in vivo

1. The relationship between the anti-inflammatory activity of the bee venom peptide 401 in the carrageenin-induced oedema of the rat hind paw and its mast cell degranulating activity has been reinvestigated. 2. Mast cell degranulation caused by compound 48/80 (10 mg kg-1) or by allergen challenge in rats sensitized to Nippostrongylus brasiliensis also suppressed rat hind paw oedema in the same test. 3. The anti-inflammatory activities of peptide 401 and compound 48/80 were partially suppressed by pretreatment of rats with mepyramine and methysergide, at doses (2.5 mg kg-1) that completely suppressed skin reactions to these mast cell-derived amines. Pretreatment of rats with compound 48/80 also suppressed the apparent anti-inflammatory actions of peptide 401 and of compound 48/80. 4. Injection of peptide 401 together with carrageenin increased the inflammatory response in the rat hind paw. 5. The anti-inflammatory activity of peptide 401 and of compound 48/80 in the carrageenin-induced swelling of the rat hind paw arises from mast cell degranulation in vivo.

Long-term potentiation of synaptic transmission by synthetic mast cell degranulating(MCD) peptide.

A bee venom, mast cell degranulating peptide (MCD), was synthesized by stepwise formation of the two distinct disulfide bridges. This synthetic peptides with mast cell degrahulating activity MCD induced long-term potentiation (LTP) in the CAl region of hippocampus, while other peptides with mast cell degranulating activity apamin and mastoparan did not. This and other results using derivatives of the synthetic MCD suggest that a rigid structure is required for the LTP induction by MCD. Two antagonists of the N-methyl-D-aspartate (NMDA) receptor, AP5 and MK801, did not block the MCD induced-LTP. Moreover, the efficacy of the synaptic transmission in the slice which had been maximally activated by tetanic stimulation was further potentiated by MCD treatment. These results indicate that two quite distinct pathways for LTP formation exist in the CAl region of the hippocampus.

Fluorescence Assay for Membrane Lytic Peptides

Abstract Lytic peptides such as melittin and mastoparan are usually assayed by measuring the leakage of cell contents; e.g., hemolysis. When such peptides lyse liposomes containing concentration-quenched 6-carboxyfluorescein (6CF), the resulting fluorescence increase is proportional to the amount of lytic peptide added. Using this 6CF-liposome system, one can assay nanogram quantities of melittin. A protocol was developed to survey peptides for lytic activity and at the same time, to test for mast cell degranulating activity. Peptides possessed either, both, or neither of these activities. The dye-liposome system was used to assay HPLC fractions of bee venom. This fluorescence assay for lytic activity is more sensitive and convenient than the hemolysis method, does not require removal of unlysed structures, and does not require animal cells.

Studies on Wasp Venom, II A New Mast Cell Degranulating Peptide from the Wasp Paravespula vulgaris

Abstract From the extract of 3991 venom glands of the most common European wasp species Paravespula vulgaris a novel basic pentadecapeptide was isolated and investigated by amino acid analysis, micro-Edman-degradation and fast atom bombardment mass spectrometry. The peptide has the sequence H-Ile-Asn-Trp-Lys-Lys-Ile-Lys-Ser-Ile -lle -Lys-Ala-Ala-Met-Asn and exhibits strong mast cell degranulating activity similarly to the mastoparans isolated from various wasp species.