Peptides (v.21, #4)
Behavioral effect of mouse fibrinopeptide A on mouse forced swimming by Yutaka Masuda; Yoshihiko Kawarada; Toshihiro Sugiyama (459-461).
A specific dopamine 2 receptor antagonist, (−)sulpiride, induced an anti-depressive behavior, climbing, in mice forced to swim for 6 h after the injection. The effective fraction was divided from the mouse serum using an ion exchanger and an ultra filtration method. This fraction contained fibrinopeptide A. A peptide synthesized according to the primary 6-amino acid sequence (TDTEDK) of fibrinopeptide A also remarkably increased the behavior. The present findings clearly indicate that a peptide with TDTEDK showed anti-depressive activity.
Keywords: Mouse forced swimming; Behavioral depression model; Anti-depressive behavior; Dopamine 2 antagonism; Fibrinopeptide A; Amino acid sequence;
Induction of high level of specific antibody response to the neutralizing epitope ELDKWA on HIV-1 gp41 by peptide-vaccine☆ by Maofu Liao; Yun Lu; Yi Xiao; Manfred P. Dierich; Ying-Hua Chen (463-468).
The monoclonal antibody 2F5 recognizing the neutralizing epitope ELDKWA on the C-domain could neutralize 90% of the investigated HIV-1 isolates. Low levels of ELDKWA-epitope-specific antibodies were observed in HIV-1-infected individuals. To induce high levels of antibodies to ELDKW-epitope, C-domain peptide (P2) was conjugated with a carrier peptide (KGGG)7-K (K/G). P2-K/G-conjugate induced high level of antibodies in mice by titer 1:25 600 to ELDKWA-epitope. P2-K/G-BSA-conjugate induced antibody response to ELDKWA-epitope (1:320–6400) in mice. The ELDKWA-epitope-specific antibodies of 19.8 and 34.6 μg/per milliliter serum were isolated from two rabbit antiserums (1:25 600). The levels of ELDKWA-epitope-specific antibodies induced in rabbits were greater than 1 μg/ml, a level considered to confer long-term protection. These results demonstrate the potential role of the C-domain peptide of gp41 to develop an effective ELDKWA-based epitope/peptide-vaccine against HIV-1.
Keywords: Epitope-vaccine; Neutralizing epitope; HIV-1 gp41;
Purification and characterization of antimicrobial peptides from the skin of the North American green frog Rana clamitans☆ by Thomas Halverson; Yousef J. Basir; Floyd C. Knoop; J.Michael Conlon (469-476).
Ten peptides with differential growth-inhibitory activity against the Gram-positive bacterium, Staphylococcus aureus, the Gram-negative bacterium, Escherichia coli, and the yeast Candida albicans were isolated from an extract of the skin of a North American frog, the green frog Rana clamitans. Ranatuerin-1C (SMLSVLKNLGKVGLGLVACKINKQC), ranalexin-1Ca (FLGGLMKAFPALICAVTKKC), ranalexin-1Cb (FLGGLMKAFPAIICAVTKKC), ranatuerin-2Ca (GLFLDTLKGAAKDVAGKLLEGLKCKIAGC KP), and ranatuerin-2Cb (GLFLDTLKGLAGKLLQGLKCIKAGCKP), are members of three previously characterized families of antimicrobial peptides, first identified in the North American bullfrog Rana catesbeiana. In addition, five structurally related peptides (temporin-1Ca, -1Cb, -1Cc, -1Cd, and -1Ce), comprising 13 amino acid residues and containing a C-terminally α-amidated residue, belong to the temporin family first identified in the European common frog Rana temporaria. Peptides belonging to the brevinin-1, brevinin-2, esculentin-1, and esculentin-2 families, previously isolated from the skins of Asian and European Ranid frogs, were not identified in the extract. The data support the hypothesis that the distribution and amino acid sequences of the skin antimicrobial peptides are valuable tools in the identification and classification of Ranid frogs.
Keywords: Antimicrobial peptide; Ranatuerin; Ranalexin; Temporin; Amphibian;
Primary structure of CHH/MIH/GIH-like peptides in sinus gland extracts from Penaeus vannamei☆ by Yajun J Wang; Timothy K Hayes; G.Mark Holman; Antonio R Chavez; Larry L Keeley (477-484).
Peptides belonging to the CHH/MIH/GIH-family of crustacean hormones were isolated from acetic acid extracts of sinus glands isolated from eyestalks of the shrimp, Penaeus vannamei. The peptides were isolated by chromatography and molecular weights determined by MALDI mass spectrometry. Peptides in the range of 7–9 kDa and containing three disulfide bridges were selected for amino acid sequence analysis. Three peptides with the requisite properties were present in sufficient amounts for sequence analysis. Two peptides had unique sequences similar to CHH/MIH/GIH peptides from other crustaceans. A third peptide seemed to be a truncated form of one of the previous sequences.
Keywords: Crustacean neuropeptides; Peptide chemistry; Shrimp hormones; Amino acid sequence; CHH; MIH; GIH family;
A peptide derived from pICln induced a strong hypotonic resistance in Escherichia coli cells☆ by Guo-Zhong Tao; Yohtalou Tashima (485-490).
We demonstrated previously that expression of rat pICln in Escherichia coli conferred a strong resistance to hypotonic stress. To define the intramolecular functional domain responsible for the resistance, molecular dissection of pICln was performed and the obtained peptides were expressed in E. coli. The cells expressing the peptides were exposed to a hypotonic solution, and their ‘survival rates’ were observed. The cells expressing only the peptides including the second acidic domain of pICln exhibited significantly higher ‘survival rates’ after hypotonic stress. The functional domain against hypotonicity was finally narrowed down to a peptide consisting of a 46-amino acid residue, P107–152. We conclude that the expression of P107–152 in E. coli cells could enhance their resistance to a hypotonic environment.
Keywords: Anti-hypotonic peptide; Cell protection from a hypotonic environment; Survival rate assay after hypotonic stress;
Bioavailability of Ziconotide in brain: influx from blood, stability, and diffusion by Robert Newcomb; Thomas J. Abbruscato; Tej Singh; Laszlo Nadasdi; Thomas P. Davis; George Miljanich (491-501).
Ziconotide is a selective peptide antagonist of the N-type calcium channel currently in clinical trials for analgesia. Ziconotide reached a maximal brain concentration of between 0.003 and 0.006% of the injected material per gram of tissue at 3–20 min after i.v. injection, and this decayed to below 0.001%/g after 2 h. The structurally distinct conopeptide SNX-185 (synthetic TVIA) was considerably more persistent in brain after i.v. administration, with 0.0035% of the injected material present at 2–4 h after i.v. injection, and 0.0015% present at 24 h. Similar results (i.e. greater persistence of SNX-185) were obtained when the peptides were perfused through in vivo dialysis probes implanted into the hippocampus. Image analysis and serial sectioning showed that diffusion of Ziconotide in the extracellular fluid around the dialysis probe was minimal, with the peptide located within 1 mm of the probe after 2 h. In vitro diffusion through cultured bovine brain microvessel endothelial cells (BBMEC) verified that a close structural analog of Ziconotide (SNX-194) passed through this blood–brain barrier (BBB) model as expected for peptides of similar physical properties (permeability coefficient of 6.5 × 10−4 cm/g). Passage from blood to brain was also verified by in situ perfusion through the carotid artery. A statistically greater amount of radioactivity was found to cross the BBB after perfusion of radioiodinated Ziconotide compared to [14C]inulin. Capillary depletion experiments and HPLC analysis defined the brain location and stability.
Keywords: Ziconotide; SNX-111; Omega-conopeptide; MVII-A; Blood–brain barrier; In vivo dialysis;
Characterization of tynorphin, a potent endogenous inhibitor of dipeptidyl peptidaseIII by Yukio Yamamoto; Jun-ichi Hashimoto; Mariko Shimamura; Teruhide Yamaguchi; Tadahiko Hazato (503-508).
To find a more effective inhibitor than spinorphin (LVVYPWT), an endogenous factor derived from bovine spinal cord, we synthesized spinorphin analogues and assayed their inhibitory activity toward DPPIII among enkephalin-degrading enzymes. Tynorphin (VVYPW), an N-terminal and C-terminal truncated form of spinorphin, exhibited more potent inhibitory activity and an IC50 value of 0.086 ± 0.05 μg/ml (n = 4), whereas structures smaller than four amino acid residues exhibited almost no or less activity, suggesting that a five amino acid structure containing a Tyr-Pro residue is essential for the inhibition. The inhibition of DPPIII by tynorphin was predominantly competitive and the K i value was found to be 7.50 ± 1.19 × 10−8 M on Lineweaver–Burk plotting. The inhibitory activity of tynorphin toward other enkephalin-degrading enzymes such as neutral endopeptidase, aminopeptidase, and angiotensin-converting enzyme was not as high as that toward DPPIII, suggesting that tynorphin is a specific inhibitor of DPPIII. In HPLC analysis, human serum cleaved tynorphin rapidly (38% of control at 2 h and background level at 4 h), but in the presence of leuhisitin, an aminopeptidase inhibitor, tynorphin was maintained at the original level for 24 h. These results indicated that tynorphin had a more effective structure for expression of inhibitory activity toward DPPIII.
Keywords: Spinorphin; Tynorphin; DPPIII; Enkephalin-degrading enzymes;
Subcellular ontogeny of brain pyroglutamyl peptidase I☆ by Juan M De Gandarias; Jon Irazusta; Javier Gil; Adolba Varona; Fernando Ortega; Luis Casis (509-517).
The present work studies pyroglutamyl-peptidase I activity in several subcellular fractions of the developing brain. In the synaptosomal fraction, soluble pGlu-peptidase I activity is low until postnatal Day 9 (with a peak at postnatal Day 2) and the activity increases at postnatal Day 15. In later stages there are not significant changes of synaptosomal pGlu-peptidase I activity. However, in the cytosolic fraction the activity is high from ED22 until postnatal Day 9, and afterwards decreases. The changes in the particulate fractions are generally less drastic.
Keywords: TRH; Development; pGlu-peptidase I; Synaptosomes; Subcellular fractions;
Hypothalamic galanin is up-regulated during hyperphagia and increased body weight gain induced by disruption of signaling in the ventromedial nucleus☆ by Michael G. Dube; Satya P. Kalra; Pushpa S. Kalra (519-526).
Disruption of signaling in the ventromedial nucleus (VMN) by colchicine (COL) produces transient (4 days) hyperphagia and weight gain. Microinjection of galanin into various hypothalamic sites stimulates feeding, so we tested the hypothesis that galanin is up-regulated in COL-treated rats by analyzing galanin concentrations in micropunched hypothalamic sites. Galanin was increased in the paraventricular nucleus on Days 1 through 4 after COL-injection. Galanin was also elevated in three other hypothalamic sites, the dorsomedial nucleus, lateral hypothalamic area, and perifornical hypothalamus, on Days 2–4 and in the lateral preoptic area, on Day 1 only. In the median eminence-arcuate nucleus and amygdala an initial decrease on Day 1 was followed by a then progressive increase through Day 4. These increases occurred despite marked elevations in blood insulin and leptin, hormones known to suppress hypothalamic galanin. When COL- or saline-treated rats were injected intracerebroventricularly with galanin, it stimulated feeding further in the hyperphagic COL-treated rats, but the relative response over basal consumption was similar in both COL-treated and control rats. These results in VMN disrupted rats suggest that neurochemical rearrangements, including increased availability of galanin, may contribute to the hyperphagia and increased weight gain; additionally, it seems that neurons in the VMN normally exert a restraint on galanin signaling.
Keywords: Colchicine; Paraventricular nucleus; Dorsomedial nucleus; Lateral hypothalamic area; Perifornical hypothalamus; Amygdala; Insulin; Leptin;
Structure activity relationships for bradykinin antagonists on the inhibition of cytokine release and the release of histamine☆ 1 1 Abbreviations: Aca, adamantane carboxyl-; bApG, N,N-bis(3-aminopropyl)-glycine; BK, bradykinin; Chg, α-cyclohexylglycine; Cpg, α-cyclopentylglycine; DDD, dodecanedioyl; EGS, ethylene glycol bis-succinyl-; Dhq, 2-dehydroquinuclidine-3-carboxyl; f5f, pentafluorophenylalanine; Gun guanidyl-; Hyp, trans hydroxyproline; Igl, α-(2-indanyl)glycine; Mosi, methoxy-suberimido; MPIV, 2.4-methanoproline; Nchg, N-cyclohexylglycine; Nc7g, N-cycloheptylglycine; Nig, N-(2-indanyl)glycine; NMF, N-methylphenylalanine; Oic, octahydroindole-2-carboxylic acid; Sub, suberyl:-CO-(CH2)6-CO-; Suim, suberimidyl: -C(NH)-CH2)6-C(NH)-; Thi, β-(2-thienyl)-alanine; Tic, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. by Siegmund Reissmann; Felipe Pineda; Gabriele Vietinghoff; Heinz Werner; Lajos Gera; John M Stewart; Inge Paegelow (527-533).
Highly potent bradykinin antagonists were found to inhibit bradykinin-induced release of cytokines but to stimulate histamine release. Both actions show structural requirements completely different from those for bradykinin B1 and B2 receptors, indicating that the release of some cytokines from spleen mononuclear cells and of histamine from rat mast cells is not mediated by these receptors. Most potent bradykinin antagonists release histamine at lower concentrations than does bradykinin itself. Dimers of bradykinin antagonists are the most potent compounds for histamine release. In contrast to enhanced histamine release, potent inhibition of cytokine release enhances the applicability of these compounds as anti-inflammatory drugs. Many of the peptides designed for high B2-receptor antagonism were found to be compared by their concentrations far more potent for inhibition of cytokine release than for smooth muscle contraction. Thus, for some antagonists inhibition of cytokine release was detected at concentrations as low as 10−15 M. The rational design of peptide and nonpeptide bradykinin antagonists for therapeutic use requires not only knowledge about the potency but also knowledge about the structure–activity relationships of such important side effects as cytokine and histamine release.
Keywords: Bradykinin receptor antagonists; Inhibition of cytokine release; Histamine release; Mast cells; Structure activity relationships; Spleen mononuclear cells; Tanned erythrocyte electrophoretic mobility (TEEM) test; QSAR;
Enhanced ganglionic responses to substance P in spontaneously hypertensive rats☆ by John C Hancock; Gregory W Lindsay (535-541).
Intravenous injection of substance P (SP) increases blood pressure in normotensive rats by stimulating sympathetic ganglia. This study compared the effects of SP to increase renal nerve firing and blood pressure in normotensive and hypertensive rats treated with chlorisondamine. The increase in renal nerve firing was greatest in spontaneously hypertensive rats (SHR), intermediate in Wistar rats, and least in Wistar-Kyoto (WKY) rats. Blood pressure was increased more in SHR than in Wistar rats. Blood pressure was not increased in WKY rats. Responses to the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium were the same in the three strains. These results suggest that there is a selective increase in the action of SP on sympathetic ganglia of SHR and that ganglion responsiveness to SP is correlated with its effect on blood pressure.
Keywords: Hypertension; Substance P; Sympathetic nervous system; Ganglion stimulants; Rats; Inbred SHR;
Characterization of functional endothelin receptors in the porcine myometrium by Mitsuhiro Isaka; Kazuhiro Takaoka; Yuko Yamada; Yoshihiro Abe; Takio Kitazawa; Tetsuro Taneike (543-551).
To characterize the endothelin (ET) receptor that mediates the contraction induced by ET-1 in the porcine myometrium, we carried out a contraction study, radioligand binding study and molecular study (reverse transcription polymerase chain reaction) for detection of ET receptor-coding genes (mRNA). ET-1 (1 nM–1 μM) caused a tetrodotoxin-insensitive contraction in both longitudinal and circular muscles, but the longitudinal muscle was more sensitive to ET-1 than was the circular muscle. On the other hand, ET-3 and sarafotoxin S6c were less effective to cause a contractile response. The contraction induced by ET-1 was markedly inhibited by BQ123 and FR139317, but BQ788 only slightly inhibited the response induced by ET-1. The radioligand binding study indicated the presence of a single class of 125I-ET-1 binding sites with the same K d value in both muscle layers. However, Bmax in the longitudinal muscle (3252 fmol/mg protein) was significantly higher than that in the circular muscle (1883 fmol/mg protein). ET-1 and FR139317 inhibited the specific 125I-ET-1 binding completely, but ET-3, sarafotoxin S6c and BQ3020 only slightly inhibited the specific binding (inhibition, 10–20%), suggesting that ETA is the dominant ET receptor subtype in the porcine myometrium. The results of the molecular study indicated the expression of both ETA and ETB receptor-coding genes in the porcine myometrium. In conclusion, ET-1 causes contraction of the porcine myometrium through activation of the ETA receptor present on smooth muscle cells. There is a marked muscle layer-related difference (longitudinal muscle > circular muscle) in the ET-1-induced contraction and the ETA receptor concentration.
Keywords: Porcine myometrium; Endothelin; ETA receptor; 125I-Endothelin-1 binding; RT-PCR;
Lovastatin is a potent inhibitor of cholecystokinin secretion in endocrine tumor cells in culture☆ by Daesety Vishnuvardhan; Margery C Beinfeld (553-557).
Lovastatin prevents isoprene synthesis thereby affecting the structural organization of proteins involved in protein transport and secretion. Lovastatin at 1 μM decreases CCK 8 secretion by over 50% in WE cells and in CCK 8 expressing AtT20 cells. At 10 μM CCK 8 secretion was inhibited by two thirds and at 100 μM, cytotoxic effects were observed in both cell types. Addition of mevalonate does not restore CCK secretion and stimulation of secretion by forskolin is also partially inhibited. Cellular content of CCK 8 and pro-CCK were not altered in either of these cell lines except at 100 μM lovastatin. Our results clearly demonstrate that lovastatin at 1 μM strongly inhibits CCK 8 secretion at multiple levels while having little or no effect on its synthesis. This effect on secretion may be partly responsible for the adverse gastrointestinal side effects of lovastatin in patients.
Keywords: Lovastatin; Cholecystokinin; Prenylation;
Effects of CGRP on human osteoclast-like cell formation: a possible connection with the bone loss in neurological disorders?☆ by Aram Akopian; Anne Demulder; Frank Ouriaghli; Francis Corazza; Pierre Fondu; Pierre Bergmann (559-564).
Osteoclast-like cell (OCL-like) differentiation is increased in long term cultures of bone marrow taken from paralyzed areas of paraplegic patients. Among the neuropeptides recently described in bone, calcitonin gene-related peptide (CGRP) has been shown in animal studies to inhibit bone resorption in vivo and OCL-like differentiation in vitro: its deficiency could thus be a link between the neural lesion and increased OCL-like production in paraplegia and some other neurologic disorders. We therefore investigated in this study the effects of CGRP on human OCL-like formation and found that it indeed has an inhibitory effect mediated at least in part via cAMP.
Keywords: CGRP; Osteoclast-like cell; Disuse osteoporosis; Neurological disorders;
Orexin-A in the human brain and tumor tissues of ganglioneuroblastoma and neuroblastoma by Zenei Arihara; Kazuhiro Takahashi; Osamu Murakami; Kazuhito Totsune; Masahiko Sone; Fumitoshi Satoh; Sadayoshi Ito; Yutaka Hayashi; Hironobu Sasano; Toraichi Mouri (565-570).
Regional distribution of orexin-A-like immunoreactivity in the human brain and pituitary, and the presence of orexin-A-like immunoreactivity in the tumor tissues of pheochromocytomas, ganglioneuroblastomas and neuroblastomas were studied by radioimmunoassay. Expression of orexin mRNA was studied by reverse transcriptase polymerase chain reaction (PCR) method. Orexin-A-like immunoreactivity was detected in every region of human brain, but not in the pituitary. The highest concentration of orexin-A-like immunoreactivity in the human brain was found in hypothalamus (17.8 ± 4.3 pmol/g wet weight, mean ± SEM, n = 7), followed by thalamus, medulla oblongata, and pons. Orexin-A-like immunoreactivity was detected in the tumor tissues of ganglioneuroblastoma and neuroblastoma, but not in the tumor tissues of pheochromocytoma. Reverse phase high performance liquid chromatographic analyses of the orexin-A-like immunoreactivity in the human brain extracts and neuroblastoma extracts showed a single immunoreactive peak, which was eluted in an identical position to synthetic human orexin-A. Orexin mRNA was expressed in the hypothalamus and in the tumor tissues of ganglioneuroblastoma and neuroblastoma. These findings suggest that orexin-A is produced in the hypothalamus and transported to various brain regions via axons. In addition, this study has shown for the first time the production of orexin-A by ganglioneuroblastomas and neuroblastomas.
Keywords: Orexin; Hypocretin; Radioimmunoassay; PCR; Human brain; Ganglioneuroblastoma; Neuroblastoma;
Neurotensin inhibits neuronal Na+ , K+-ATPase activity through high affinity peptide receptor by Marı́a Graciela López Ordieres; Georgina Rodrı́guez de Lores Arnaiz (571-576).
Neurotensin is a peptide present in mammalian CNS and peripheral tissues, which may play a major role in neurotransmission or neuromodulation, subserving diverse physiological functions. We studied the effect of added neurotensin on ATPase activities in synaptosomal membranes isolated from rat cerebral cortex. Neurotensin at 3 × 10−8–3 × 10−6 M concentration decreased 20–44% Na+, K+-ATPase activity but failed to modify Mg2+-ATPase activity; lower neurotensin concentrations (3 × 10−14–3 × 10−10 M) had no effect on enzyme activities. This inhibitory effect was abolished by neurotensin heating, by enzyme preincubation with neurotensin during periods exceeding 10 min, or by adding 1 × 10−6 M SR 48692, a high affinity neurotensin receptor antagonist. Levocabastine, which blocks low affinity neurotensin receptor, failed to alter enzyme inhibition by the peptide. It is suggested that the sodium pump may be a target for neurotensin effects at neuronal level involving the participation of high affinity neurotensin receptor.
Keywords: Neurotensin effect; Synaptosomal membrane enzymes; ATPases; Na+; K+-ATPase; Neurotensin receptor;
Binding of a pure 125I-monoiodoleptin analog to mouse tissues: a developmental study by Claude Dal Farra; Nicole Zsürger; Jean–Pierre Vincent; Anny Cupo (577-587).
The preparation of a pure 125I-labeled monoiododerivative of mouse leptin is described. This radiolabeled analog has been used to characterize and localize central and peripheral leptin binding sites (Ob-R) of the mouse at different stages of its development. The affinity values found in membrane homogenates of various mouse tissues are similar and range between 0.1 and 0.3 nM, indicating that all the Ob-R isoforms have a similar affinity. Leptin binding sites are highly expressed at the membrane level in lung, intestine, kidney, liver, and skin and to a lesser degree in stomach, heart, and spleen. Brain, thymus, and pancreas homogenates are devoid of any specific binding. The distribution of mouse Ob-R has also been explored by autoradiography and dipping techniques on whole mouse sections. In lung, leptin binding sites are located at the pulmonary parenchyma and at the bronchiolar epithelial level. Binding sites are expressed all along the digestive tract from the tongue to the rectum (esophagus, stomach, intestine, colon, and rectum). In muscular visceral structures (stomach, intestine, and bladder) the binding is mainly present in the lamina propria. During development, leptin receptors are early expressed in the liver, kidney, and bone. In the lung, the Ob-R level increased gradually from birth to adulthood where the expression is maximal. By contrast, leptin receptors located in the medulla of the kidney remain remarkably constant all along the development. A broad signal is present in cartilage and bone particularly in vertebrae, limb, and ribs. Interestingly, leptin receptors are barely detectable in the mouse brain except in the choroid plexus and leptomeninges, whereas in the rat brain leptin binding sites are located in the thalamus, the piriform cortex, the cerebellum (at the granular and molecular cell layer), and the pineal gland.
Keywords: Leptin; Iodination; Purification; Receptor; Mouse; Rat; Ontogeny; Brain; Peripheral tissues; Autoradiography; Dipping;
Fungal allergens and peptide epitopes☆ by Viswanath P. Kurup; Banani Banerjee (589-599).
Fungal allergens represent a major cause of atopic disorders. Immunochemical and molecular characterization of fungal allergens has been hampered by the lack of pure proteins and to inherent variation among fungal proteins and in their poor yields. With the advent of molecular biology techniques, a number of allergens have been cloned, sequenced, and expressed from a variety of fungal species. The knowledge of the primary, secondary, and tertiary structures of these allergens, the immunodominant regions of these proteins, and their interaction with T and B-cell epitopes, results in better understanding of the molecular mechanisms of allergy and may provide avenues of immunologic intervention to treat patients. The present review deals with the current understanding of fungal allergen epitopes.
Keywords: Fungal allergens; Recombinant allergens; T-cell epitopes; B-cell epitopes; IgE antibody; Epitope analysis; Epitopes in immunotherapy;