Immunoglobulin E Epitope Mapping and Structure-Allergenicity Relationship Analysis of Crab Allergen Scy p 9.

Filamin C is an allergen of Scylla paramamosain (Scy p 9), and six IgE linear epitopes of the allergenic predominant region had previously been validated. However, the IgE epitope and structure-allergenicity relationship of Scy p 9 are unclear. In this study, a hydrophobic bond was found to be an important factor of conformation maintaining. The critical amino acids in the six predicted conformational epitopes were mutated, and the IgE-binding capacity and surface hydrophobicity of four mutants (E216A, T270A, Y699A, and V704A) were reduced compared to Scy p 9. Ten linear epitopes were verified with synthetic peptides, among which L-AA187-205 had the strongest IgE-binding capacity. In addition, IgE epitopes were mapped in the protruding surface of the tertiary structure, which were conducive to binding with IgE and exhibited high conservation among filamin genes. Overall, these data provided a basis for IgE epitope mapping and structure-allergenicity relationship of Scy p 9.

IgE Epitope Analysis and Hypo-Immunoreactivity Derivative of Arginine Kinase in Mantis Shrimp (Oratosquilla oratoria).

As the main allergenic food, shrimp can trigger allergic reactions in various degrees. In this study, arginine kinase (AK) was identified as an allergen in Oratosquilla oratoria by LC-MS/MS. The open reading frame of AK was obtained, which included 356 amino acids, and recombinant AK (rAK) was expressed in Escherichia coli. The results of immunological analysis and circular dichroism showed that rAK displayed similar IgG-/IgE-binding activity and structure as native AK. Besides, five IgE linear epitopes of AK were verified by serological analysis, on the basis of which an epitope-deleted derivative was obtained and named as mAK-L. It has been shown that mAK-L displayed hypo-immunoreactivity compared to rAK, and the contents of secondary structures were different. In conclusion, these discoveries enrich the overall understanding of crustacean allergens and epitopes and set the foundations for food allergy diagnosis and immunotherapy.

Identification of linear epitopes and their major role in the immunoglobulin E-binding capacity of tropomyosin from Alectryonella plicatula.

Tropomyosin (TM) is an important allergen in molluscans. However, there was a lack of information about TM as an allergen in oysters. TM was purified and identified from Alectryonella plicatula (ATM), and its primary sequence was cloned and encoded with 284 amino acids (AAs). Chemical denaturants were used to destroy the structure to confirm that linear epitopes played a major role in the immunoglobulin E-binding capacity of ATM. Subsequently, nine linear epitopes were identified using a serological test. The peptide with AA27-41 was regarded as the key epitope because it could be recognized strongly by most sera of oyster-sensitive individuals in comparison to other epitope peptides. Finally, the epitopes and the primary sequence of TM among shellfish were aligned to find the two conserved epitopes (AA117-132 and AA164-178) in oyster, octopus, abalone, scallop, clam, shrimp, and crab. Overall, these data provide a foundation for the allergenicity and cross-reactivity of TM.

Crystal Structure Analysis and IgE Epitope Mapping of Allergic Predominant Region in Scylla paramamosain Filamin C, Scy p 9.

Filamin C (FLN c) is a novel allergen in shellfish. In this study, FLN c from Scylla paramamosain was divided into three regions for recombinant expression based on the number of domains and amino acids. Using dot blot and basophil activation tests, the allergic predominant region of FLN c was determined to be 336-531 amino acid positions (named FLN c-M). It was confirmed that by X-ray diffraction, the crystal structure of FLN c-M with immunoglobulin-like folding at a resolution of 1.7 Å was obtained. The monomer was a barrel structure composed of 16 ß-strands and 2 α-helices. Three conformational epitopes were predicted, six linear epitopes were verified by serological test, and they were positioned on the crystal structure of FLN c-M. For the first time, the crystal structure of the allergic predominant region of FLN c was determined, and it provided an accurate template for the localization of IgE epitopes.

Two allergens from Scylla paramamosain share common epitopes showed different allergenic potential in Balb/c mice.

Filamin C (FLN c) and triosephosphate isomerase (TIM) are novel allergens of crab (Scylla paramamosain) which are sharing common epitopes. This work aimed to assess their contributions to the induction and elicitation of allergenic responses. Balb/c mice were sensitized by intraperitoneal injections and challenged by intragastric gavage with purified proteins. Upon oral challenge, FLN c triggered more severe anaphylactic symptoms, higher levels of specific antibodies and histamine in serum than TIM, while TIM was a more active promotor of early specific antibody production and stimulated stronger Th2-biased responses. Combined with the results of in vitro assays, the data demonstrated that though with common epitopes, the two allergens showed a different allergenicity, TIM favored Th2 polarization in sensitization stage, while FLN c had a better ability to stimulate B cells and is highly immunogenic in oral challenge stage. The findings can help with the better understanding of allergenicity of crab allergens.

Analysis of Immunoreactivity of α/α2-Tropomyosin from Haliotis discus hannai, Based on IgE Epitopes and Structural Characteristics.

Tropomyosin (TM) was reported to be a supercoil allergen of shellfish. However, little information is available about its link between structure and allergenicity. In this study, the subunit of TM (α-TM) and supercoil of TM (α2-TM) were identified from Haliotis discus hannai. α2-TM showed higher immunoreactivity than α-TM. Meanwhile, seven linear epitopes in α-TM and α2-TM were verified, and two conformational epitopes in α2-TM were predicted. The physicochemical properties and chemical bond assays confirmed the existence of the disulfide bond in α2-TM. According to spectroscopy and hydrophobicity analysis, α-TM showed higher α-helix features and blueshift of the fluorescence intensity peak compared with those of α2-TM. The structure analysis revealed the possibility of conformational epitopes in α2-TM, which could explain the immunoreactivity differences between α-TM and α2-TM further. These results improved the understanding of Haliotis discus hannai TM, which lay the foundation for the food processing of abalone.

MicroRNA­181a promotes epithelial­mesenchymal transition in esophageal squamous cell carcinoma via the TGF­ß/Smad pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most debilitating and invasive tumors. Although previous reports have demonstrated the critical role microRNA­181a (miR­181a) serves in the progression of ESCC, how miR­181a induces epithelial­mesenchymal transition (EMT) remains to be elucidated. In the present study, the expression profiles of TGF­ß1 and Smad4 proteins in 88 patients with ESCC and 21 adjacent non­cancerous tissues were analyzed using immunohistochemistry. The expression of miR­181a in ESCC cells (ECA109 and TE­1) and HEEC was analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The role of miR­181a in ESCC was analyzed using miR­181a mimics and inhibitor in the same system. Migration, proliferation and apoptosis of cells were assessed using wound­healing assays and cell proliferation assays and flow cytometry, respectively. The expression levels of TGF­ß1 and Smad4 in ESCC cell lines transfected with miR­181a mimics and inhibitor were measured using RT­qPCR and western blotting. The expression of E­cadherin and vimentin was also assessed following transfection. The findings demonstrated that expression of TGF­ß1 was upregulated, in contrast to Smad4 expression which was downregulated. Expression levels of Smad4 affected the prognosis of patients with ESCC. Higher expression of miR­181a promoted migration and proliferation but inhibited apoptosis of ESCC cells. miR­181a promoted EMT by modulating Smad4 expression in ESCC cells. Overall, these findings revealed that miR­181a induced EMT in ESCC via the TGF­ß/Smad pathway in ESCC. Consequently, miR­181a is a potential novel target against ESCC.

Cloning, expression and comparison of the properties of Scy p 9, a Scylla paramamosain allergen.

This study investigated the properties of Scy p 9 in mud crab (Scylla paramamosain). The gene sequence of filamin C obtained from crabs, which was denoted as Scy p 9, contains a 2544 bp open reading frame and encodes 848 amino acid residues. Recombinant Scy p 9 (rScy p 9) is expressed in Escherichia coli, which exhibits tertiary structure changes, and the IgE binding activity of rScy p 9 is higher than that of native Scy p 9 (nScy p 9). Moreover, this study explored the possibility of the presence and cross-reactivity of filamin C in 8 shellfish. IgE-specific binding to nScy p 9 and rScy p 9 in patients allergic to shellfish revealed that rScy p 9 was more sensitive than nScy p 9. The gene sequence of filamin C fills in the blank in shellfish. This study contributes to the understanding of the properties of Scy p 9, and the results indicate that rScy p 9 can be used as a candidate for component-resolved diagnosis in shellfish.

Expression and epitope identification of myosin light chain isoform 1, an allergen in Procambarus clarkii.

Myosin light chain isoform 1 (MLC1) is reported to be a novel allergen in crayfish (Procambarus clarkii). However, little information is available about its allergic epitopes. In this study, recombinant crayfish MLC1 (rMLC1) was expressed and confirmed by mass spectrometry. Circular dichroic analysis and serological test were performed for the measuring of structural and immunological properties of rMLC1. Specific-protein-A-enriched IgG raised in rabbits against purified rMLC1 was used to screen a phage display random peptide library. Nine MLC1 mimotope clones were identified among 16 random clones after biopanning. Five conformational epitopes were identified with the program LocaPep, and mapped into 3 epitope regions at the antibody-binding interface of MLC1. MLC1 of crayfish showed high primary and secondary structure identity to MLC of other allergenic species, its epitopes were located in the structure conserved regions, and its cross-reactivity among related species was indicated by immunological assays.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab.

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.

[Effect of Draconis Sanguis-containing serum on NGF, BDNF, CNTF, LNGFR, TrkA, GDNF, GAP-43 and NF-H expressions in Schwann cells].

OBJECTIVE: To observe the effect of Draconis Sanguis-containing serum on the expressions of NGF, BDNF, CNTF, LNG-FR, TrkA, GDNF, GAP-43 and NF-H in Schwann cells, and investigate the possible mechanism of Draconis Sanguis to promote peripheral nerve regeneration. METHOD: SD rats were randomly divided into 2 groups: the Draconis Sanguis group (orally administered with Draconis Sanguis-containing balm solution) and the blank group (equivoluminal balm) to prepare Draconis Sanguis-containing serum and blank control serum. Schwann cells were extracted from double sciatic nerves of three-day-old SD rats, divided into 2 groups: the Draconis Sanguis group and the blank control group, and respectively cultured with 10% Draconis Sanguis-containing serum or blank control serum. The mRNA expressions of NGF, BDNF, CNTF and other genes in Schwann cells were measured by RT-PCR analysis 48 hours later. RESULT: Most of the Schwann cells were bipolar spindle and arranged shoulder to shoulder or end to end under the microscope and identified to be positive with the immunocytochemical method. To compare with the blank group, mRNA expressions of NGF, LNGFR, GDNF and GAP-43 significantly increased (P < 0.01). Whereas that of BDNF decreased significantly (P < 0.05), and so did that of TrkA, CNTF (P < 0.01), with no remarkable difference in NF-H-mRNA. CONCLUSION: Traditional Chinese medicine Draconis Sanguis may show effect in nerve regeneration by up-regulating mRNA expressions of NGF, LNGFR, GDNF and GAP-43 and down-regulating mRNA expressions of TrkA, BDNF and CNTF.

The protective effect of ginko bilboa leaves injection on the brain dopamine in the rat model of cerebral ischemia/reperfusion injury.

BACKGROUND: Ginkgo Bilboa injection has had been clinically applied to restore the damaged cells and tissues due to the ischemia through improving the cerebral blood supply and decreasing the oxygen consumption. OBJECTIVE/AIM: To evaluate the Ginkgo Bilboa injection's therapeutic role towards ischemia/ reperfusion (I/R) injury through determination of monoamine neurotransmitter dopamine (DA) in corpus striatum. METHODS: After the incomplete global cerebral ischemia and reperfusion models were prepared, rats were randomly assigned into four groups: sham-operated group, ischemia-reperfusion group, nimodipine injection group, and Ginkgo Biloba injection group. The cerebrospinal fluid in the rat brain striatum at different time points was collected with microdialysis, and the level of monoamine neurotransmitters dopamineDA was determined by high performance liquid chromatography (HPLC) with electrochemical detector (ECD). RESULTS: The dopamineDA content in cerebral ischemia model group was significantly higher than that in the sham-operated group (P < 0.05) at the 30 min. However, the DA level in nimodipine injection group and Ginkgo Biloba injection group were lower than the model group (P < 0.05). The dopamineDA level in Ginkgo Biloba injection group gradually decreased, and was significantly different from the model group (P < 0.05). CONCLUSION: Ginkgo Biloba injection can could significantly inhibit brain I/R injury, as demonstrated by prevention of excessive release of dopamineDA in striatum.

The effect of borneol on the concentration of meropenem in rat brain and blood.

Meropenem is a carbapenem antibiotic with a wide spectrum of activity against both Gram-positive and Gram-negative bacteria. Because of its clinical efficacy, meropenem is an excellent choice for the treatment of serious infections in both adults and children. The knowledge of tissue concentrations of antibiotic in an infection site is valuable for the prediction of treatment outcome. The aim of the present study is to investigate the effect of borneol on the concentration of meropenem in rat brain and blood and to find the potential relationships of the combined use of medicine and traditional Chinese medicine. Analysis of meropenem in the dialysates was achieved using the microdialysis technique and HPLC. At 40 min after the administration of an intraperitoneal injection of meropenem, the concentration of meropenem in brain in borneol+meropenem group was 2.25 (0.35) µg ml(-1), which was significantly higher than that in meropenem group [1.20 (0.12) µg ml(-1); P < 0.01]. Within 80 min of drug administration, the AUCbrain/AUCblood (area under the curve, AUC) in the borneol+meropenem group was 1.2 times that of the meropenem group. Borneol can increase the concentration of meropenem in the cerebrospinal fluid, but has no influence on its blood concentration. This study represents a successful application of the microdialysis technique, which is an effective method for the study of pharmacokinetics of meropenem.

Involvement of the cerebral monoamine neurotransmitters system in antidepressant-like effects of a chinese herbal decoction, baihe dihuang tang, in mice model.

Baihe Dihuang Tang (BDT) is a renowned Chinese herbal formula which is commonly used for treating patients with mental instability, absentmindedness, insomnia, deficient dysphoria, and other psychological diseases. These major symptoms closely associated with the depressive disorders. BDT was widely popular use for treating emotion-thought disorders for many years in China. In the present study, the antidepressant-like effect of BDT in mice was investigated by using the forced swim test (FST) and the tail suspension test (TST). The underlying mechanism was explored by determining the effect of BDT on the level of cerebral monoamine neurotransmitters. BDT (9 and 18 g/kg, p.o. for 14 days) administration significantly reduced the immobility time in both the FST and the TST without changing locomotion in the open field-test (OFT). Moreover, BDT treatment at the dose of 18 g/kg inhibited reserpine-induced ptosis. Meanwhile, BDT enhanced 5-HT and NA levels in mouse cerebrum as well as decreased the ratio of 5-HT compared to its metabolite, 5-HIAA, (turnover, 5-HIAA/5-HT) after TST. The results demonstrated that the antidepressant-like effect of BDT is mediated, at least partially, via the central monoaminergic neurotransmitter system.

[Effect of storax on concentration of sulpiride in brain and blood by MD-HPLC].

OBJECTIVE: To observe the effect of traditional Chinese medicine storax on the concentration of combined western medicine sulpiride in brain and blood, discuss the effect of storax in inducing resuscitation and increasing the permeability of the gastrointestinal barrier (GB) and the blood-brain-barrier (BBB), and explore the interaction between storax and sulpiride. METHOD: Rats were orally administered with the drugs for one week, probes were implanted in their brains and necks by surgery. After balance for 60 min, brain microdialysis and blood microdialysis were adopted for collect dislysates from blood in right atrum and cerebral hippocampus at time periods of 30, 60, 90, 120, 150, 180 min. The concentration of sulpirde in the samples was detected by RP-HPLC. Statistical approaches were adopted to compare the contents of sulpirde in brain and blood of the two groups. RESULT: The sulpiride combined with storax group showed a significant higher concentration of sulpiride than the pure sulpiride group. The pure sulpiride group showed a concentration ratio between sulpiride in brain and blood of 1:0.2; while the sulpiride combined with storax group increased the concentration ratio between sulpiride in brain and blood to 1:0.3. Compared with the pure sulpiride group, the sulpiride combined with storax group showed an increase of concentration by 39% in brain and 69% in blood. CONCLUSION: Storax can notably increase the concentration of sulpiride in rat brain and blood, indicating that it can increase the permeation of sulpiride through gastrointestinal barrier and BBB. This study reveals the mechanism of storax in inducing resuscitation by promoting the permeation through gastrointestinal barrier and BBB.

[Effects of human ERMAP-siRNA on erythroid differentiation of K562 cells induced by Ara-C].

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% (p < 0.05), but still lower than that in group K562 + Ara-C. The percentage of CD36(-)/CD235a(+) increased from 8.83% to 11.28%, CD36(+)/CD235a(+) increased from 1.23% to 2.64%, and CD36(+)/CD235a(-) increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 + Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52 x 10(-3) to 4.53 x 10(-3), which was also significantly lower than that of group K562 + Ara-C. It is concluded that the ERMAP-shRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.

[Expression of human ermap gene in umbilical cord blood mononuclear cells during differentiation and development towards erythroid lineage].

The aim of study was to explore the potential of human erythroid membrane associated protein (ERMAP) gene in erythroid cell differentiation and development, mononuclear cells (MNCs) were isolated from umbilical cord blood and induced to erythroid cell differentiation by SCF, IL-3 and EPO. The cell morphology was observed by using optical microscopy, the positive rate of cells was counted by biphenylamine staining and the ratios of CD36+/CD235a-, CD36+/CD235a+, CD36-/CD235a+ cells were detected by flow cytometry, the change of human ermap gene expression level was analyzed by using fluorescent quantitative PCR (FQ-PCR). The results showed that the ermap gene expression level increased while MNCs were induced to erythroid lineage after treatment with SCF, IL-3 and EPO. It is concluded that the human ermap gene plays an important role in differentiation and development of erythroid cells.

[Research for dependablity of administration of platycodi radix in Tianwang Buxinwan decoction with change of brain inhibitive neurotransmitte in rats by microdialysis].

OBJECTIVE: To study the effects of Tianwang Buxinwan decoction on the contents of amino acids neurotransmitters in corpus striatum of rats to implicate the mechanism of Tianwang Buxinwan promoting and Improving sleeping. METHOD: Contents of two amino acids neurotransmitters in corpus striatum of rats were prepared by microdialysis technology and were determined by HPLC which involved pre-column derivation with orthophthaladehyde, recersed-phase gradient elution and fluorescence detection. RESULT: In the experimental separation condition, Tianwang Buxinwan seemed do not influence three kinds of contents of amino acids neurotransmiters (glutamic acid, glycin, aspartic acid), but TBW seemed increase the content of gamma-GABA in corpus striatum of rats. CONCLUSION: The effects of Tianwang Buxinwan to relieve uneasiness may relate with the inhibitory amino acids gamma-GABA. Tianwang Buxinwan may promote increasing the content of gamma-GABA. This discovery may be helpful for the deep study of related mechanism of Tianwang Buxinwan.

[Expression of human ERMAP gene in fetal tissues].

Human ERMAP (hERMAP) is a novel gene coding for erythroid membrane-associated protein, which may play a role in erythropoiesis. To explore the role of hERMAP in fetal hematopoiesis, 29 fetuses of inevitable abortion with the fetal ages of 9 - 36 weeks were collected, and the total RNAs were isolated from the liver, spleen, kidney, heart, lung, thymus, brain, bone marrow and skeletal muscle, the RNAs from which at 25(+5) week fetal age were used to detect hERMAP expressions in different fetal tissues with Northern blot, and the hERMAP in various fetal tissues were quantified by FQ-PCR. As a result, Northern blot showed that hERMAP was expressed in liver and bone marrow at 25(+5) fetal age, but not in the other organ tissues. FQ-PCR results indicated that the hERMAP had been still expressed in 9 - 36th week fetal liver, increased starting from 12th-week, reaching a peak between 18 - 20th week and declining slowly starting from 21st-week. In the fetal bone marrow, expression of the hERMAP began from 15th week, reached a highest level between 27 - 32nd week and fell rapidly from 33rd week, the expression level of which was lower than that in liver. The low level expression of this gene was observed in some specimens of kidney, heart, lung, thymus, brain and skeletal muscle. It is concluded that the expression of hERMAP in fetal liver approximately consistent with haematopoiesis of fetal liver, and the expression of this gene in bone marrow aged 15 - 32nd fetal weeks coincides with haematopoiesis of fetal bone marrow. It suggests that the function of the hERMAP is possibly related with the migration of erythroid cells to liver and bone marrow during the fetal development process.