Title: Peptide Leucine Arginine, a Potent Immunomodulatory Peptide Isolated and Structurally Characterized from the Skin of the Northern Leopard Frog, Rana pipiens
Abstract: On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys5 and Cys15 are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes,i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin. On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys5 and Cys15 are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes,i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin. peptide leucine arginine high performance liquid chromatography 9 fluorenylmethoxycarbonyl lactate dehydrogenase 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol colony forming units-granulocyte macro- phage formation concentration required to elicit 50% release of calcein or produced 50% hemolysis concentration required to elicit release of 25% of the total cellular histamine content concentration of POPG or POPC in the large unilamellar vesicles Studies on the array of biologically active substances present in amphibian skin have been reported in the scientific literature for almost 40 years (1Erspamer V. Comp. Biochem. Physiol. 1984; 79C: 1-7Google Scholar). In particular, skin is a rich source of bioactive peptides, many of which are present in copious amounts (mg/g wet weight skin) (2Erspamer V. Erspamer G.F. Cei J.M. Comp. Biochem. Physiol. 1986; 85C: 125-137Google Scholar). Many peptides occur at concentrations that are several orders of magnitude higher than the circulating levels in the frog, indicating that a major source of biosynthesis is in the skin (3Nakajima T. Trends Pharmacol. Sci. 1981; 2: 202-204Abstract Full Text PDF Scopus (46) Google Scholar). These peptides are putative components in the defense of the frog against predation or invading microorganisms (1Erspamer V. Comp. Biochem. Physiol. 1984; 79C: 1-7Google Scholar). Several peptides have been isolated from the skins of ranid frog species, which have structural analogs in mammalian neuroendocrine systems. Bradykinin and related family members have been identified in several Rana species (4Anastasi A. Erspamer V. 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Protein Research Foundation, Osaka, Japan1986: 363-368Google Scholar) and the family of temporin peptides (15Simmaco M. Mignogna G. Canofeni S. Miele R. Mangoni M.L. Barra D. Euro. J. Biochem. 1996; 242: 788-792Crossref PubMed Scopus (296) Google Scholar) demonstrate structural similarity to the mast cell-activating and phospholipase A2-facilitating peptide, crabrolin, from the venom of the wasp, Vespa crabro (16Argiolas A. Pisano J.J. J. Biol. Chem. 1984; 259: 10106-10111Abstract Full Text PDF PubMed Google Scholar). Many peptides activate rat peritoneal mast cells; thus, histamine release can be utilized as a screen for putative bioactive peptides. There are several frog skin peptides that have been identified in this manner, namely, peptide XO-4 from Kassina maculata (17Yasuhara T. Nakajima T. Erspamer V. Tsukamoto Y. Mori M. Peptide Chemistry 14th Symposium 1986. Protein Research Foundation, Osaka, Japan1987: 69-74Google Scholar), granuliberin R from Rana rugosa (18Yasuhara T. Ishikawa O. Nakajima T. Chem. Pharm. Bull. ( Tokyo ). 1979; 27: 492-498Crossref PubMed Scopus (15) Google Scholar), and the pipinins fromRana pipiens (19Horikawa R. Parker D.S. Herring P.L. Pisano J.J Fed. Proc. 1985; 44: 695Google Scholar). A number of peptides have also been isolated from the venoms of hornets, wasps, and bees on the basis of histamine release (Refs. 16Argiolas A. Pisano J.J. J. Biol. Chem. 1984; 259: 10106-10111Abstract Full Text PDF PubMed Google Scholar and 20Yasuhara T. Itokawa H. Suzuki N. Nakamura H. Nakajima T. Peptide Chemistry 1984. Protein Research Foundation, Osaka, Japan1985Google Scholar, 21Yasuhara T. Nakajima T. Fukuda K. Tsukamoto Y. Mori M. Kitada C. Fujino M. Peptide Chemistry 1983. Protein Research Foundation, Osaka, Japan1984Google Scholar, 22Yasuhara T. Nakajima T. Erspamer V. Peptide Chemistry 1982. Protein Research Foundation, Osaka, Japan1983Google Scholar, 23Miroshnikov A.I. Snezhkova L.G. Nazimov I.V. Reshetova O.I. Rozynov B.V. Gushchin I.S. Bioorg. Khim. 1981; 7: 1467-1477Google Scholar, 24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar, 25Argiolas A. Herring P. Pisano J.J. Peptides. 1985; 3 (Suppl. 6): 431-436Crossref Scopus (13) Google Scholar, 26Gmachl M. Kreil G. J. Biol. Chem. 1995; 270: 12704-12708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar and TableI). Many mammalian peptides can activate mast cells, including substance P, neurokinin A, calcitonin gene-related peptide, neuropeptide Y, and neurotensin (27Heaney L.G. Cross L.J.M. Stanford C.F. Ennis M. Clin. Exp. Allergy. 1995; 25: 179-186Crossref PubMed Scopus (80) Google Scholar, 28Cross L.J. M Heaney L.G. Ennis M. Inflamm. Res. 1997; 46: 306-309Crossref PubMed Scopus (25) Google Scholar, 29Forsythe P. Ennis M. Clin. Exp. Allergy. 1998; 28: 1171-1173Crossref PubMed Scopus (3) Google Scholar, 30Grundemar L. Hakanson R. Brit. J. 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Commun. 1987; 148: 1440-1445Crossref PubMed Scopus (140) Google Scholar).Table IMast cell activating peptides identified on the basis of histamine releasing abilityPeptide1-aReferences are given in parentheses.Primary sequenceXO-4(17Yasuhara T. Nakajima T. Erspamer V. Tsukamoto Y. Mori M. Peptide Chemistry 14th Symposium 1986. Protein Research Foundation, Osaka, Japan1987: 69-74Google Scholar)FIKQLLPHLPGWIDAVSNAFS-NH2Granuliberin R (18Yasuhara T. Ishikawa O. Nakajima T. Chem. Pharm. Bull. ( Tokyo ). 1979; 27: 492-498Crossref PubMed Scopus (15) Google Scholar)FGFLPIYRRPAS-NH2Pipinin I (19Horikawa R. Parker D.S. Herring P.L. Pisano J.J Fed. Proc. 1985; 44: 695Google Scholar)FLPIIAGVAAKVFPKIFCAISKKCPipinin II (19Horikawa R. Parker D.S. Herring P.L. Pisano J.J Fed. Proc. 1985; 44: 695Google Scholar)FLPIIAGIAAKVFPKIFCAISKKCPipinin III (19Horikawa R. Parker D.S. Herring P.L. Pisano J.J Fed. Proc. 1985; 44: 695Google Scholar)FLPIIASVAAKVFSKIFCAISKKCMastoparan C (16Argiolas A. Pisano J.J. J. Biol. Chem. 1984; 259: 10106-10111Abstract Full Text PDF PubMed Google Scholar,23Miroshnikov A.I. Snezhkova L.G. Nazimov I.V. Reshetova O.I. Rozynov B.V. Gushchin I.S. Bioorg. Khim. 1981; 7: 1467-1477Google Scholar)INLKAIAALVKKVL-NH2Crabrolin (16Argiolas A. Pisano J.J. J. Biol. Chem. 1984; 259: 10106-10111Abstract Full Text PDF PubMed Google Scholar)FLPLILRKIVTAL-NH2Vespid chemotactic peptide L (20Yasuhara T. Itokawa H. Suzuki N. Nakamura H. Nakajima T. Peptide Chemistry 1984. Protein Research Foundation, Osaka, Japan1985Google Scholar)FLPIIAKLVSGLL-NH2Vespid chemotactic peptide X (20Yasuhara T. Itokawa H. Suzuki N. Nakamura H. Nakajima T. Peptide Chemistry 1984. Protein Research Foundation, Osaka, Japan1985Google Scholar)FLPIIAKLLGGLL-NH2Ropalidian chemotactic peptide (20Yasuhara T. Itokawa H. Suzuki N. Nakamura H. Nakajima T. Peptide Chemistry 1984. Protein Research Foundation, Osaka, Japan1985Google Scholar)IVPFLGPLLGLLT-NH2Vespid chemotactic peptide A (21Yasuhara T. Nakajima T. Fukuda K. Tsukamoto Y. Mori M. Kitada C. Fujino M. Peptide Chemistry 1983. Protein Research Foundation, Osaka, Japan1984Google Scholar)FLPMIAKLLGGLL-NH2Vespid chemotactic peptide M (21Yasuhara T. Nakajima T. Fukuda K. Tsukamoto Y. Mori M. Kitada C. Fujino M. Peptide Chemistry 1983. Protein Research Foundation, Osaka, Japan1984Google Scholar)FLPIIGKLLSGLL-NH2Vespid chemotactic peptide T (22Yasuhara T. Nakajima T. Erspamer V. Peptide Chemistry 1982. Protein Research Foundation, Osaka, Japan1983Google Scholar)FLPILGKILGGLL-NH2Histamine releasing peptide II (23Miroshnikov A.I. Snezhkova L.G. Nazimov I.V. Reshetova O.I. Rozynov B.V. Gushchin I.S. Bioorg. Khim. 1981; 7: 1467-1477Google Scholar)FLPLILGKLKGLL-NH2Bombolitin I (24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar)IKITTMLAKLGKVLAHVBombolitin II (24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar)SKITDILAKLGKVLAHVBombolitin III (24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar)IKIMDILAKLGKVLAHVBombolitin IV (24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar)INIKDILAKLVKVLGHVBombolitin V (24Argiolas A. Pisano J.J. J. Biol. Chem. 1985; 260: 1437-1444Abstract Full Text PDF PubMed Google Scholar)INVLGILGLLGKALSHLMast cell degranulating peptide (25Argiolas A. Herring P. Pisano J.J. Peptides. 1985; 3 (Suppl. 6): 431-436Crossref Scopus (13) Google Scholar,26Gmachl M. Kreil G. J. Biol. Chem. 1995; 270: 12704-12708Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar)IKCNCKRHVIKPHICRKICGKN-NH2Magainin-2-amide (36Cross L.J.M. Ennis M. Krause E. Dathe M. Lorenz D. Krause G. Beyermann M. Bienert M. Eur. J. Pharmacol. 1995; 291: 291-300Crossref PubMed Scopus (20) Google Scholar)GIGKFLHSAKKFGKAFVGGIMNS-NH2Melittin (45Vlasak R. Unger-Ullmann C. Kreil G. Frischauf A.-M. Eur. J. Biochem. 1983; 135: 123-126Crossref PubMed Scopus (59) Google Scholar)GIGAVLKVLTTGLPALISWIKRKRQQ-NH2Peptide leucine arginine (pLR)LVRGCWTKSYPPKPCFVRComparison of the primary sequence of peptide leucine arginine (pLR) with other known peptides that elicit histamine release from rat peritoneal mast cells. Single letter notations for amino acid residues have been used.1-a References are given in parentheses. Open table in a new tab Comparison of the primary sequence of peptide leucine arginine (pLR) with other known peptides that elicit histamine release from rat peritoneal mast cells. Single letter notations for amino acid residues have been used. The present study describes the isolation and structural characterization of peptide leucine arginine (pLR)1 from the skin of the frog R. pipiens. We have shown that pLR is one of the most active, noncytolytic histamine-liberating peptides, which also inhibits granulocyte macrophage colony formation without induction of neutrophil apoptosis. This novel amphibian peptide may have an unrecognized counterpart with a regulatory function in the mammalian immune system. The frogs were lightly anesthetized and killed by pithing. Dorsal skin (6.2 g wet weight) was removed from six adult specimens of R. pipiens, chopped into small pieces, and incubated in acidified ethanol (ethanol, 0.7 m HCl (3:1 v/v) and 8 ml/g of tissue) for 12 h at 4 °C. Tissue debris was removed by centrifugation (3000 × g, 4 °C, 30 min), and the ethanol was removed from the decanted supernatant prior to lyophilization. The extract was reconstituted with acetic acid (3 ml, 2 m) and applied to a precalibrated 90 × 1.6 cm chromatographic column packed with Sephadex G50 (fine) gel, equilibrated with acetic acid (2 m). The column was eluted at a flow rate of 10 ml/h, and 2.5-ml fractions were collected. Size exclusion chromatographic fractions 55–64 (500 μl of each fraction) were pooled and subjected to reversed-phase HPLC using a 30 × 1-cm Vydac 218TP1010 C18 column and a Waters HPLC system: mobile phase A, trifluoroacetic acid and water (0.1:99.9; v/v); mobile phase B, trifluoroacetic acid, acetonitrile, and water (0.1:49.9:50.0 v/v/v); and linear gradient 0–35% B in 70 min (gradient rate of 0.25% acetonitrile/min). Aliquots (200 μl) of each fraction were removed, lyophilized, and assayed for mast cell histamine-releasing activity. The fraction demonstrating maximum histamine release was purified in two consecutive runs using a 0.46 × 15 cm Hypersil PEP 300 C3 column: mobile phase A and B, as above; linear gradient, 0–25% B in 50 min (gradient rate of 0.25% acetonitrile/min). Aliquots (100 μl) of each fraction were removed, lyophilized, and assessed for mast cell histamine-releasing activity. The molecular mass of the purified histamine-releasing peptide was determined by 252Cf plasma desorption mass spectroscopy using a BioIon 20 K time-of-flight instrument. Spectra were recorded at 16 kV for 106 primary fission events, and internal mass calibration of the instrument with known standards established the accuracy of mass determination as ± 0.1%. Automated Edman degradation was performed using an Applied Biosystems 470A gas phase sequencer. The limit for detection of phenylthiohydantoin-amino acids was 0.5 pmol. Subsequent to the initial sequencing run, a further sample of the peptide was pyridylethylated, using 4-vinylpyridine under reducing conditions. This was achieved by incubation in 100 μl of guanidine hydrochloride (6 m) containing dl-dithiothreitol (10 mm) and 4-vinylpyridine (1 μl) for 1 h at room temperature in the dark. The reaction mixture was purified by reversed-phase HPLC, and the pyridylethylated peptide was subjected to automated Edman degradation on an Applied Biosystems 491 Procise sequencer with phenylthiohydantoin-pyridylethyl-cysteine incorporated into the phenylthiohydantoin-amino acid standards. The primary structure was compared with those deposited in the SWISSPROTTM data base. The initial quantity of peptide was estimated using automated sequence analysis data (∼20–30% accuracy). The peptide pLR was synthesized automatically using the solid phase method (Wang-resin, 0.6 mmol/g, Calbiochem-Novabiochem AG, Läufelfingen) and standard 9 Fmoc chemistry (double couplings with 8 equivalent of Fmoc-amino acid derivatives). Couplings were performed by use of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (Calbiochem-Novabiochem AG, Läufelfingen). The final cleavage/deblocking was performed using trifluoroacetic acid, phenol, water, and triisopropylsian (88:5:5:2 v/v/v/v) for 3 h. The crude peptide (213 mg, 0.01 mmol) was then dissolved in 100 ml of dimethyl sulfoxide with water (2:8 v/v, pH 8). The final purification was performed by preparative HPLC (Nucleosil C-18, 250 × 20 mm): mobile phase A, trifluoroacetic acid and water (0.1:99.9 v/v); mobile phase B, trifluoroacetic acid, acetonitrile, and water (0.1:80.0:19.9 v/v/v); linear gradient 10–70% B in 70 min (gradient rate 0.68% acetonitrile/min). The mass of the purified peptide was verified by electrospray mass spectroscopy. Stock solutions of pLR were prepared by dissolving the samples in 10 mm Tris buffer (pH 7.4 and 150 mm NaCl). For CD measurements, aliquots of the solution were diluted with buffer or mixed with 2,2,2-trifluoroethanol (50%) to give a final concentration of 50 μm and the desired solvent composition. Measurements were carried out on a J 720 spectrometer (Jasco, Japan). Protein data base screening for similar sequences was performed using BLAST and FASTA programs available in the bioinformatic program GCG Wisconsin package. The Sybyl program package version 6.4 was used to construct the cyclic peptide. The initial conformation of pLR was predicted using fragments of consensus conformations found within the three-dimensional protein data bank. The conformation was then minimized by AMBER 4.1 force field and was subjected to an annealing simulation protocol, where, in a cyclic procedure (50 cycles) the peptide was heated to 1000 K during 2000 fs and cooled down during 5000 fs. The phi/psi torsion angles of the polyproline motif PPKP were weakly constrained (force constant 2.0 kcal mol−1 rad−2) during the cooling down phase. Male Hooded Lister rats (150–250 g body weight) were lightly anesthetized with CO2 and then killed by cervical dislocation and exsanguination. Mixed peritoneal cells were obtained as previously described (36Cross L.J.M. Ennis M. Krause E. Dathe M. Lorenz D. Krause G. Beyermann M. Bienert M. Eur. J. Pharmacol. 1995; 291: 291-300Crossref PubMed Scopus (20) Google Scholar). These cells were washed twice in Tyrode's buffer (137 mm NaCl, 5.6 mm glucose, 10 mm HEPES, 2.7 mm KCl, 1 mmMgCl2·6H2O, 1 mmCaCl2·2H2O, and 0.4 mmNaH2PO4·2H2O, pH 7.4) and recovered by centrifugation (100 × g, 4 °C, 2 min). Isolated peritoneal cells (100 μl) were aliquoted into conical polystyrene test tubes and prewarmed to 37 °C for 5 min. Lyophilized aliquots of chromatographic fractions were reconstituted in Tyrode's buffer (100 μl) and added to the cell suspensions. Following incubation (10 min, 37 °C), the reaction was quenched by addition of ice-cold Tyrode's (2.8 ml). The cell suspensions were centrifuged as above, and the supernatants removed for histamine assay. The remaining cell pellets were resuspended in buffer (3 ml) and then placed in a boiling water bath (10 min) to release the residual histamine. The histamine content was determined in both the supernatants and the cell pellets using the fluorimetric method based on the method described previously (37Shore P.A. Burkhalter A. Cohn V.H. J. Pharmacol. Exp. Ther. 1959; 127: 182-186PubMed Google Scholar). Histamine release was expressed as a percentage of total content, and values were corrected for spontaneous release in the absence of added peptides (not exceeding 5%). Lactate dehydrogenase (LDH) release assay was performed utilizing a commercially available colorimetric assay kit (Sigma). LDH release was measured in both the supernatants and cell pellets and is expressed as a percentage of total LDH. Values were corrected for the spontaneous release in the absence of added stimuli. Stock solution (200 μm) of synthetic pLR was stored at −20 °C, and dilutions were prepared that gave final concentrations of between 0.001 and 50 μm. Assays were performed in duplicate, and the individual experiments were repeated four to six times. The potency of the synthetic peptide was compared with serial dilutions of the purified peptide from the skin. The time course of pLR-induced histamine release was investigated by incubating pLR (final concentration, 500 nm) with mast cells for time periods ranging from 1 s to 30 min. The temperature dependence of histamine release was investigated by incubating pLR (final concentration, 500 nm) at a range of temperatures between 4 and 45 °C. To determine the role of extracellular calcium in pLR-induced histamine release, cells were preincubated with Tyrode's buffer containing a range of different calcium concentrations (0–5 mm) prior to stimulation with pLR (final concentration, 500 nm) as above. To assess whether pLR-induced histamine release was via an energy-dependent mechanism, cells were preincubated for 20 min in the absence of glucose but in the presence of antimycin A (Sigma, 1 μm) and 2-deoxyglucose (Sigma, 5 mm). The cells were then stimulated with pLR (final concentration, 500 nm) as described above. To investigate whether pLR induced mast cell activation was via a cytolytic mechanism, LDH release was measured. In a further series of experiments, the histamine release induced by a range of pLR concentrations and various other known histamine-releasing peptides were compared. Membrane perturbation was studied by synthetic peptide induced dye release from negatively charged 1-palmitoyl-2-oleoylphosphatidyl-dl-glycerol (POPG) and neutral 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) large unilamellar vesicles. Large unilamellar vesicles (diameter, 0.1 μm) containing calcein at self-quenching concentrations were prepared by extrusion as described (38Dathe M. Schümann M. Wieprecht T. Winkler A. Beyermann M. Krause E. Matsuzaki K. Bienert M. Biochemistry. 1996; 35: 12612-12622Crossref PubMed Scopus (349) Google Scholar). Calcein release was monitored fluorimetrically by measuring the decrease of self-quenching (excitation, 490 nm; emission, 520 nm) on a PerkinElmer LS 50B spectrofluorimeter after mixing peptide solution and vesicle suspension. pLR was tested over the concentration range of 1–40 μm for POPG and 1–110 μm for POPC vesicles; magainin-2 amide was used at concentrations between 0.1 and 0.5 μm for POPG and 1 and 50 μm for POPC vesicles, and melittin was tested at concentrations ranging from 0.01 to 1 μm for POPG and 0.01 to 0.1 μm for POPC vesicles. The fluorescence intensity corresponding to 100% dye release was determined by addition of Triton X-100. The percentage of leakage after 1 min was used to create dose response curves, and the concentration required to evoke 50% dye release (EC50) for each peptide was determined from duplicate experiments. Hydrophobicity of pLR, magainin-2 amide and melittin were determined from methods using the Consensus Scale of Hydrophobicity (39Eisenberg D. Annu. Rev. Biochem. 1984; 53: 595-623Crossref PubMed Scopus (734) Google Scholar) The hemolytic activity of the synthetic peptide was determined using human red blood cells (40Dathe M. Wieprecht T. Nikolenko H. Handel L. Maloy W.L. MacDonald D.L. Beyermann M. Bienert M. FEBS Lett. 1997; 403: 208-212Crossref PubMed Scopus (344) Google Scholar). Suspensions containing 1.8 × 108 cells/ml where incubated (30 min, 37 °C) with varying concentrations of peptides. Concentration ranges used were 1–40 μm, 1 μm to 1 mm, and 0.1–10 μm for pLR, magainin-2 amide, and melittin, respectively. After cooling in ice water (5 min) followed by centrifugation (2000 × g, 4 °C, 5 min), the supernatant (200 μl) was diluted with NH4OH (1800 μl, 0.5%), and the optical density was measured at 540 nm. Peptide concentrations that produced 50% hemolysis (EC50) were determined from dose response curves. Semi-solid agar cultures were performed utilizing normal human bone marrow obtained from thoracotomy rib specimens as previously described (41Irvine A.E. French M.A. Bridges J.M. Crockard A.D. Desai Z.R. Morris T.C.M. Exp. Haematol. 1991; 19: 106-109PubMed Google Scholar). In brief, bone marrow cells (1 × 105) were cultured in the upper layer of triplicate cultures (1 ml), with human umbilical cord conditioned medium in the lower layer as the source of colony stimulating activity. Synthetic pLR was incorporated into both layers of the cultures in the range 0–4.7 μm. CFU-GM colonies (>20 cells) were scored using an inverted microscope after incubation (37 °C, 7 days, 5% CO2/air). Colony inhibition in the presence of pLR was expressed as percentage inhibition of granulopoiesis compared with that observed in the absence of any stimuli. These experiments were carried out in triplicate. Neutrophils were prepared by layering human peripheral blood on an equal volume of Histopaque 1119 and Histopaque 1077 (Sigma) (42English D Anderson B.R. J. Immunol. Methods. 1974; 5: 249-252Crossref PubMed Scopus (577) Google Scholar). Cells were washed in phosphate-buffered saline (0.01 m, pH 7.4, Sigma), resuspended in RPMI 1640 (Life Technologies, Inc.) containing 10% (v/v) fetal calf serum (Life Technologies, Inc.), and counted by hemocytometer. A suspension of neutrophils (1 × 106/ml) was cultured in the presence of synthetic pLR (0–4.7 μm) in 5% CO2/95% air at 37 °C in 6-well multi-dishes (Nunc). After 24 h the neutrophils were removed, and the cell viability was estimated by trypan blue exclusion. Three cytocentrifuge slide preparations were made per peptide concentration. One slide was stained with Giemsa and examined for morphological signs of apoptosis. At least 500 cells were counted per slide, and the percentage of apoptotic cells was calculated. Two slides were labeled using the terminal deoxyuridine triphosphate nick end labeling technique (43Irvine A.E. Magill M.K. Somerville L.E. McMullin M.F. Exp. Haematol. 1998; 26: 435-439PubMed Google Scholar) for further evaluation of apoptotic status. All experiments were repeated three times. Unless otherwise stated all values are given as the means ± S.E. Statistical analyses were performed using Student's t test. For multiple comparisons, an analysis of variance was first performed followed by the protected t test. The maximal histamine release was detected in the pool of fractions 33–36 from the reversed-phase HPLC (Fig.1A). Other histamine-releasing fractions were detected that were identified as related to the previously documented pipinins (44Morikawa N. Hagiwara K. Nakajima T. Biochem. Biophys. Res. Commun. 1992; 189: 184-190Crossref PubMed Scopus (242) Google Scholar). The individual fraction 35 possessed all of the activity (data not shown). Fraction 35 was further resolved into two major peptides (Fig. 1B). The second, more hydrophobic peptide evoked all histamine-releasing activity, and 2.23 nmol of peptide demonstrated a highly effective histamine-releasing ability of 80.6%. There was negligible LDH release evoked with this peptide (data not shown; LDH release 0.2% ± 0.01%). Furthermore, significant histamine release could still be detected at a 1:100 dilution (223 pmol) and had a comparable dose response curve to the synthetic pLR (data not shown). Therefore, synthetic pLR was used for all subsequent functional studies. 252Cf plasma desorption mass spectroscopy of the isolated peptide indicated a molecular mass of 2137 Da [MH]+ or 2136 Da in nonprotonated native form. Automated Edman degradation established the identity of residues through 18 cycles with two