RESUMO
BACKGROUND: Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. OBJECTIVE: This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population and to determine the structure for functional characterization. METHODS: IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7 and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analysed by X-ray crystallography and NMR. RESULTS: Despite a high prevalence of Der p 23, (75% vs. 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n = 47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, thirty fold less than Der p 1 and sevenfold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulphide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. CONCLUSIONS AND CLINICAL RELEVANCE: Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expression of other Dermatophagoides allergens.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Antígenos de Dermatophagoides/química , Antígenos de Dermatophagoides/imunologia , Hipersensibilidade/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Dermatophagoides/sangue , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Genômica , Humanos , Imunoglobulina E/sangue , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Conformação ProteicaRESUMO
BACKGROUND: Only limited evidence is available regarding the cytokine repertoire of effector T cells associated with peanut allergy, and how these responses relate to IgE antibodies to peanut components. OBJECTIVE: To interrogate T cell effector cytokine populations induced by Ara h 1 and Ara h 2 among peanut allergic (PA) children in the context of IgE and to evaluate their modulation during oral immunotherapy (OIT). METHODS: Peanut-reactive effector T cells were analysed in conjunction with specific IgE profiles in PA children using intracellular staining and multiplex assay. Cytokine-expressing T cell subpopulations were visualized using SPICE. RESULTS: Ara h 2 dominated the antibody response to peanut as judged by prevalence and quantity among a cohort of children with IgE to peanut. High IgE (> 15 kU(A)/L) was almost exclusively associated with dual sensitization to Ara h 1 and Ara h 2 and was age independent. Among PA children, IL-4-biased responses to both major allergens were induced, regardless of whether IgE antibodies to Ara h 1 were present. Among subjects receiving OIT in whom high IgE was maintained, Th2 reactivity to peanut components persisted despite clinical desensitization and modulation of allergen-specific immune parameters including augmented specific IgG4 antibodies, Th1 skewing and enhanced IL-10. The complexity of cytokine-positive subpopulations within peanut-reactive IL-4(+) and IFN-γ(+) T cells was similar to that observed in those who received no OIT, but was modified with extended therapy. Nonetheless, high Foxp3 expression was a distinguishing feature of peanut-reactive IL-4(+) T cells irrespective of OIT, and a correlate of their ability to secrete type 2 cytokines. CONCLUSION: Although total numbers of peanut-reactive IL-4(+) and IFN-γ(+) T cells are modulated by OIT in highly allergic children, complex T cell populations with pathogenic potential persist in the presence of recognized immune markers of successful immunotherapy.
Assuntos
Citocinas/biossíntese , Hipersensibilidade a Amendoim/imunologia , Hipersensibilidade a Amendoim/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th2/imunologia , Células Th2/metabolismo , Albuminas 2S de Plantas/imunologia , Administração Oral , Adolescente , Alérgenos/administração & dosagem , Alérgenos/imunologia , Antígenos de Plantas/administração & dosagem , Antígenos de Plantas/imunologia , Criança , Pré-Escolar , Dessensibilização Imunológica , Feminino , Glicoproteínas/imunologia , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Imunofenotipagem , Lactente , Interleucina-4/biossíntese , Masculino , Hipersensibilidade a Amendoim/terapiaRESUMO
The IUIS Allergen Nomenclature Sub-Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, www.allergen.org. In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cow's milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub-Committee encourages researchers to use these updated allergen names in future publications.
Assuntos
Alérgenos/classificação , Bases de Dados Factuais , Terminologia como Assunto , Alérgenos/química , Animais , HumanosRESUMO
BACKGROUND: Recent immunological data demonstrated that dendritic cells preferentially recognize advanced glycation end product (AGE)-modified proteins, upregulate expression of the receptor for AGE (RAGE), and consequently bias the immune response toward allergy. METHODS: Peanut extract was characterized by mass spectrometry (MS) to elucidate the specific residues and specific AGE modifications found in raw and roasted peanuts and on rAra h 1 that was artificially glycated by incubation with glucose or xylose. The binding of the RAGE-V1C1 domain to peanut allergens was assessed by PAGE and Western analysis with anti-Ara h 1, 2, and 3 antibodies. IgE binding to rAra h 1 was also assessed using the same methods. RESULTS: AGE modifications were found on Ara h 1 and Ara h 3 in both raw and roasted peanut extract. No AGE modifications were found on Ara h 2. Mass spectrometry and Western blot analysis demonstrated that RAGE binds selectively to Ara h 1 and Ara h 3 derived from peanut extract, whereas the analysis failed to demonstrate Ara h 2 binding to RAGE. rAra h 1 with no AGE modifications did not bind RAGE; however, after AGE modification with xylose, rAra h 1 bound to RAGE. CONCLUSIONS: AGE modifications to Ara h 1 and Ara h 3 can be found in both raw and roasted peanuts. Receptor for AGE was demonstrated to selectively interact with AGE-modified rAra h 1. If sensitization to peanut allergens occurs in dendritic cells via RAGE interactions, these cells are likely interacting with modified Ara h 1 and Ara h 3, but not Ara h 2.
Assuntos
Alérgenos/química , Arachis/química , Produtos Finais de Glicação Avançada/metabolismo , Reação de Maillard , Alérgenos/imunologia , Sequência de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/imunologia , Antígenos de Plantas/metabolismo , Arachis/imunologia , Produtos Finais de Glicação Avançada/química , Produtos Finais de Glicação Avançada/imunologia , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Glicosilação , Humanos , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Proteínas de Membrana , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Peanut allergy affects 1% of the population and causes the most fatal food-related anaphylactic reactions. The protein Ara h 2 is the most potent peanut allergen recognized by 80-90% of peanut allergic patients. METHODS: The crystal structure of the major peanut allergen Ara h 2 was determined for the first time at 2.7 Å resolution using a customized maltose-binding protein (MBP)-fusion system. IgE antibody binding to the MBP fusion construct vs the natural allergen was compared by ELISA using sera from peanut allergic patients. RESULTS: The structure of Ara h 2 is a five-helix bundle held together by four disulfide bonds and related to the prolamin protein superfamily. The fold is most similar to other amylase and trypsin inhibitors. The MBP--Ara h 2 fusion construct was positively recognized by IgE from 76% of allergic patients (25/33). Two populations of patients could be identified. Subpopulation 1 (n = 14) showed an excellent correlation of IgE antibody binding to natural vs recombinant Ara h 2. Subpopulation 2 (n = 15) showed significantly reduced IgE binding to the MBP fusion protein. Interestingly, about 20% of the IgE binding in subpopulation 2 could be recovered by increasing the distance between MBP and Ara h 2 in a second construct. DISCUSSION: The reduced IgE binding to the MBP--Ara h 2 of subpopulation 2 indicates that the MBP molecule protects an immunodominant epitope region near the first helix of Ara h 2. Residues involved in the epitope(s) are suggested by the crystal structure. The MBP--Ara h 2 fusion constructs will be useful to further elucidate the relevance of certain epitopes to peanut allergy.
Assuntos
Albuminas 2S de Plantas/química , Albuminas 2S de Plantas/metabolismo , Antígenos de Plantas/química , Antígenos de Plantas/metabolismo , Arachis/imunologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Epitopos Imunodominantes/química , Imunoglobulina E/metabolismo , Hipersensibilidade a Amendoim/classificação , Albuminas 2S de Plantas/genética , Albuminas 2S de Plantas/imunologia , Antígenos de Plantas/genética , Antígenos de Plantas/imunologia , Arachis/genética , Arachis/metabolismo , Cristalização , Cristalografia por Raios X , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Epitopos Imunodominantes/genética , Epitopos Imunodominantes/imunologia , Imunoglobulina E/imunologia , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Hipersensibilidade a Amendoim/diagnóstico , Hipersensibilidade a Amendoim/imunologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.
Assuntos
Alérgenos/química , Alérgenos/imunologia , Baratas/imunologia , Alérgenos/metabolismo , Animais , Baratas/química , Baratas/enzimologia , Humanos , Hipersensibilidade/enzimologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologiaRESUMO
A high-affinity avermectin binding site from Drosophila melanogaster head membranes was subjected to target size analysis by radiation inactivation in order to determine the functional unit size. Using the [3H]ivermectin binding assay to assess ligand binding activity, the target size was determined to be 44.3 +/- 3.9 kDa. This result suggests that the size of the functional unit required for high-affinity ligand binding is a monomer. The membrane-associated acetylcholinesterase present in the Drosophila head membranes was also analyzed by radiation inactivation and served as a control. By monitoring enzymatic activity, the functional unit size of the Drosophila acetylcholinesterase was determined to be 70 +/- 9.7 kDa. This corresponds to the molecular weight of a dimer composed of a 55 kDa subunit and a 16-18 kDa subunit.
Assuntos
Drosophila melanogaster/química , Inseticidas/química , Ivermectina/análogos & derivados , Acetilcolinesterase/química , Animais , Sítios de Ligação , Canais de Cloreto/metabolismo , Drosophila melanogaster/metabolismo , Ivermectina/química , Ivermectina/metabolismoRESUMO
Conventional immunotherapy may be associated with the development of adverse reactions, including anaphylaxis, due to the use of increasing doses of allergen. Standardization of extracts is necessary in order to assess the correct amount of allergen administered. In recent years, increased knowledge on the molecular structure of allergens has allowed the development of novel alternatives for immunotherapy. Initially, allergens were cloned and expressed as recombinant proteins in eukaryotic and prokaryotic systems. Crystallization of the purified proteins led to the elucidation of the tertiary structure of the allergen. Molecular biology techniques were used to construct modified allergens whose new IgE binding properties were studied. IgE antibody mapping combined with molecular modeling has allowed the recognition of IgE binding sites on the surface of the molecule. This information has been applied to the engineering of new modified allergens, with and without adjuvants, that retain immunogenicity but with reduced allergenicity. The use of these molecules for immunotherapy should allow the administration of greater doses of allergen, without the undesired side effects characteristic of conventional immunotherapy.
Assuntos
Alérgenos , Hipersensibilidade/terapia , Imunoterapia/métodos , Alérgenos/química , Alérgenos/genética , Alérgenos/imunologia , Humanos , Biologia Molecular/métodosRESUMO
Allosterism between gamma-aminobutyric acid (GABA), benzodiazepine and picrotoxinin recognition sites on the GABAA receptor was studied in primary cultures of cerebellar granule cells. The increase in [3H]flunitrazepam binding induced by GABA was inhibited by bicuculline and picrotoxinin and the decrease in [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding mediated by GABA was reverted by bicuculline. The effects of hexachlorocyclohexanes (the convulsant gamma- and the depressant delta-isomers, both acting at the picrotoxinin recognition site) on GABA and benzodiazepine sites were studied. delta-Hexachlorocyclohexane, but not the gamma-isomer (lindane), increased [3H]flunitrazepam binding in a concentration-dependent manner (EC50: 8.3 microM). This increase in [3H]flunitrazepam binding was reduced by bicuculline and picrotoxinin. The gamma-isomer reduced the increase in [3H]flunitrazepam binding induced by GABA or delta-hexachlorocyclohexane. Neither delta- nor gamma-hexachlorocyclohexane inhibited [3H]GABA binding. Moreover, the inhibition of [35S]TBPS binding induced by delta-hexachlorocyclohexane was not reverted by bicuculline. The results obtained in this study in vitro agree with the pharmacological properties and the effects of gamma- and delta-hexachlorocyclohexane in vivo. It is concluded that delta-hexachlorocyclohexane acts as a positive allosteric modulator and gamma-hexachlorocyclohexane acts as a non-competitive antagonist of the GABAA receptor.
Assuntos
Cerebelo/metabolismo , Hexaclorocicloexano/farmacologia , Receptores de GABA-A/metabolismo , Animais , Bicuculina/farmacologia , Células Cultivadas , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Flunitrazepam/metabolismo , Antagonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/metabolismo , Hexaclorocicloexano/química , Isomerismo , Cinética , Ratos , Receptores de GABA-A/química , Ácido gama-Aminobutírico/metabolismoRESUMO
The characteristics of the picrotoxinin binding site present on the gamma-aminobutyric acidA (GABAA) receptor were studied in neurons using primary cultures of cerebellar granule cells. The binding properties of these sites in intact cultured cells were compared with those measured in cultured cell membrane preparations. t-[35S]Butylbicyclophosphorothionate (TBPS) binding was performed in cultured rat cerebellar neurons grown for 13 days. Binding parameters (Kd and Bmax) were similar to those reported in the literature determined using brain membranes. However, equilibrium was reached faster when using intact cultured neurons. Convulsant compounds like picrotoxinin (PTX) and pentylenetetrazol (PTZ) competitively inhibited the binding of TBPS in this in vitro system. Convulsant organochlorine pesticides (gamma-hexachlorocyclohexane gamma-HCH or lindane and the cyclodienes aldrin, endrin, dieldrin and alpha-endosulfan) competitively inhibited [35S]TBPS binding in cerebellar neuronal cultures. Inhibitory affinity constant (Ki) values were in the nanomolar range, alpha-endosulfan and endrin being the most potent inhibitors corresponding to their high toxicity in mammals. Stereospecificity was also shown for HCH isomers, the non-convulsant isomers (alpha- and delta-HCH) being 15-30 times less potent in inhibiting [35S]TBPS binding than the convulsant gamma-HCH, while the beta-isomer was inactive.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes , Compostos Bicíclicos com Pontes/metabolismo , Cerebelo/metabolismo , Convulsivantes/metabolismo , Neurônios/metabolismo , Animais , Ligação Competitiva , Células Cultivadas , Cerebelo/citologia , Radioisótopos de EnxofreRESUMO
The case of a 68-year-old woman who had relatively acute, unilateral ophthalmoplegia is reported. Radiological studies indicated a mass lesion involving the pituitary gland and left cavernous sinus. Pathological tissue obtained by the transsphenoidal approach revealed the presence of a Coccidioides granuloma. This pathological entity should be considered when evaluating patients with a pituitary mass and ophthalmoplegia.
Assuntos
Adenoma/diagnóstico , Coccidioidomicose/diagnóstico , Neoplasias Hipofisárias/diagnóstico , Idoso , Encefalopatias/diagnóstico , Encefalopatias/microbiologia , Diagnóstico Diferencial , Feminino , Humanos , Sela Túrcica/microbiologia , Tomografia Computadorizada por Raios XRESUMO
Primary cultures from cortical neuronal cells were used to study the effect of organochlorine pesticides on GABA-dependent Cl- fluxes. In this system cyclodienes inhibited GABA-induced 36Cl- uptake with similar potencies to those described using brain synaptoneurosomes whereas lindane showed greater potency than that described using other in vitro systems. Basal Cl- uptake was not affected by these compounds although alpha-endosulfan exhibited a small inhibition. DDT, which mechanism of neurotoxic action has been postulated through activation of Na+ channels, did not inhibit GABA-induced 36Cl- uptake. These results clearly demonstrate that the stimulant neurotoxic effects of cyclodienes and lindane are produced by their interaction with the Cl- channel coupled to the GABAA receptor. Furthermore, these results show the usefulness of cortical neuronal cultures to study the effects of toxic agents on GABAA receptor.
Assuntos
Cloretos/metabolismo , Inseticidas/toxicidade , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , DDT/toxicidade , Feminino , Hexaclorocicloexano/toxicidade , Camundongos , Neurônios/metabolismo , Gravidez , Receptores de GABA-A/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Inadvertent exposure to peanut in foods poses health risks for peanut-allergic individuals that can be reduced by improving detection systems for allergen contaminants in food products and manufacturing processes. Detection of peanut in chocolate has been especially difficult. We report the optimization of conditions for measuring a major peanut allergen, Ara h 1, in chocolate with the use of a two-site monoclonal antibody sandwich enzyme-linked immunosorbent assay. Ara h 1 was extracted from peanut in the presence or absence of chocolate with phosphate buffer, salt, and three dried milks (goat, soy, or nonfat) (0 to 25% wt/vol) for 15 min at 60 degrees C or for 2.5 h at room temperature. The best conditions for Ara h 1 extraction in the presence of chocolate were 5% nonfat dry milk for 2.5 h at room temperature. Spiking experiments of chocolate with peanut confirmed improvement of the extraction: Ara h 1 was detected in extractions of 0.16 to 0.33% peanut in chocolate. Interestingly, the best conditions for Ara h 1 extraction were different for peanut alone than with chocolate, regarding time, temperature, and percentage of nonfat dry milk in the extraction buffer. In chocolate with peanut foods, the total Ara h 1 values were 10-fold higher than when products were extracted with phosphate buffer alone and could be up to 400-fold higher for individual foods. The dramatic improvement of Ara h 1 extraction should allow specific allergen monitoring in chocolate-containing food products and assessment of Ara h 1 exposure.
Assuntos
Arachis/imunologia , Análise de Alimentos/métodos , Proteínas de Plantas/análise , Proteínas de Plantas/isolamento & purificação , Alérgenos , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/imunologia , Antígenos de Plantas , Cacau/química , Ensaio de Imunoadsorção Enzimática/métodos , Glicoproteínas , Humanos , Proteínas de Membrana , Sensibilidade e Especificidade , Temperatura , Fatores de TempoRESUMO
The study population was composed of 43 patients affected by idiopathic facial paralysis (20 males and 23 females), aged between 11 and 67. The study was carried out in a double-blind, randomized, placebo controlled manner. Acetyl-L-carnitine was given in an oral dose of 3 x 1 g daily for 1 month, along with a daily oral administration of 50 mg of methylprednisolone for 14 days. The evaluation was made by means of electromyograms (EMG) of the orbicularis oculi and oris muscles, by the Schirmes lacrimation test, by stapedial reflex test and a score scale for clinical assessment of paralysis. Results so far obtained have shown an earlier functional recovery of the nerve in those patients treated with acetyl-L-carnitine. Comparison between the affected and unaffected sides of the face revealed a statistical significance in the treated group (p < 0.05) as well as the amplitudes of the muscle action potentials (MAP) between the affected sides (p < 0.01).
Assuntos
Acetilcarnitina/uso terapêutico , Paralisia Facial/tratamento farmacológico , Acetilcarnitina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Adolescente , Adulto , Idoso , Criança , Método Duplo-Cego , Paralisia Facial/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We investigated 19 patients affected by chronic peripheral neurological disorders treated with therapeutic plasma exchange (TPE) to verify the efficacy of the therapeutic protocol used in these diseases. Every patient was clinically considered after 5 TPE. Those who showed an improvement started chemotherapy and continued TPE at the rate of 2 procedures/week for 2 weeks, then 1 procedure/week for 1 month and finally 1 procedure every 2 weeks for 2 months. Intravenous immunoglobulins (IVIg) were infused at the end of apheretic treatment in one of the patients affected by neurological disorders due to monoclonal gammopathy undetermined significance. HCV-positive patients with cryoglobulins were treated with alpha-interferon (alpha-IFN) for 6 months before TPE. Eleven patients (58%) had a symptomatic improvement, 2 (1.5%) stopped TPE treatment owing to side effects and 6 (31.5%) did not respond to apheretic therapy. In order to improve the advantages of TPE we suggest using IVIg at the end of apheretic therapy, while in HCV-positive patients, at least one year of alpha-IFN therapy is required before initiating TPE.
Assuntos
Doenças Desmielinizantes/terapia , Paraproteinemias/complicações , Doenças do Sistema Nervoso Periférico/terapia , Troca Plasmática , Adulto , Idoso , Doença Crônica , Feminino , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Masculino , Pessoa de Meia-Idade , Paraproteinemias/terapia , Troca Plasmática/efeitos adversosAssuntos
Poluição do Ar em Ambientes Fechados , Alérgenos/genética , Proteínas/genética , Alérgenos/biossíntese , Alérgenos/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/genética , Endopeptidases/biossíntese , Endopeptidases/genética , Técnicas Genéticas , Humanos , Imunoterapia , Dados de Sequência Molecular , Biossíntese de Proteínas , Proteínas/química , Proteínas Recombinantes/biossíntese , Homologia de Sequência de Aminoácidos , Testes CutâneosRESUMO
BACKGROUND: Increased allergenicity of roasted vs. raw peanut has been reported by showing higher IgE binding to roasted peanut extracts. OBJECTIVE: To study the effect of roasting on Ara h 1 quantification in peanut using a specific monoclonal antibody-based ELISA, and to compare the Ara h 1 content from different kernel size peanuts from four runner cultivars. METHODS: Raw or oven-roasted (177 degrees C for 5-30 min) runner peanuts were crushed and extracted at 60 degrees C. Inhibition ELISA was used to study binding of Ara h 1 purified from raw or roasted peanut. Runner peanuts of four different cultivars were collected, shelled, sized and roasted for 15 min at 177 degrees C. Ara h 1 in the extracts was compared by ELISA. RESULTS: Ara h 1 levels were up to 22-fold higher in roasted than in raw peanuts (820 vs. 37 microg/mL, in a representative experiment) with an Ara h 1 peak at 10-15 min of roasting. Inhibition ELISA indicated that this increase was not due to conformational changes in the Ara h 1 monoclonal antibody epitopes. Ara h 1 was found at lower levels in number 1 than in jumbo- and medium-sized peanuts, and no differences were found among cultivars. CONCLUSION: These results suggest that roasting increases the efficiency of Ara h 1 extraction, and/or that the monoclonal antibody binding epitopes were more accessible in roasted peanut. Expression of Ara h 1 is associated with peanut maturity.
Assuntos
Alérgenos/análise , Arachis , Glicoproteínas/análise , Temperatura Alta , Hipersensibilidade a Amendoim/imunologia , Proteínas de Plantas/análise , Alérgenos/metabolismo , Animais , Especificidade de Anticorpos , Antígenos de Plantas , Ensaio de Imunoadsorção Enzimática/métodos , Manipulação de Alimentos , Glicoproteínas/metabolismo , Humanos , Imunodifusão , Imunoglobulina E/metabolismo , Proteínas de Membrana , Proteínas de Plantas/metabolismoRESUMO
A patient in whom transient global amnesia (TGA) led to the diagnosis of an ethmoidal meningioma is described. One year after neurosurgery, the patient showed an impairment of long-term memory, without any clinical or neuroradiological sign of relapse. We suggest that TGA may express a preexisting subclinical impairment of memory neuronal systems.
Assuntos
Amnésia/etiologia , Neoplasias Meníngeas/diagnóstico , Meningioma/diagnóstico , Feminino , Humanos , Neoplasias Meníngeas/complicações , Neoplasias Meníngeas/cirurgia , Meningioma/complicações , Meningioma/cirurgia , Pessoa de Meia-IdadeRESUMO
The effect of convulsant and nonconvulsant hexachlorocyclohexane (HCH) isomers and cyclodienes on GABA-induced Cl- flux was studied in primary cultures of neocortical neurons by measuring the GABA-stimulated 36Cl- uptake. GABA induced a dose-dependent chloride uptake. The convulsant agents gamma-HCH and cyclodienes alpha-endosulfan, dieldrin and aldrin blocked this 36Cl- uptake. A total or partial inhibition of GABA-induced 36Cl- uptake was produced by the noncompetitive GABAA antagonists picrotoxinin (PTX) and pentylenetetrazol, respectively. The inhibitory potencies of 36Cl- uptake by the organochlorine compounds (alpha-endosulfan > dieldrin > gamma-HCH > aldrin) were well correlated with their inhibitory potencies of [35S]TBPS binding. Positive modulators of GABAergic function (flunitrazepam and phenobarbital) prevented the blocking of GABA-induced chloride uptake by PTX but not that induced by alpha-endosulfan. The depressant beta- and delta-HCH isomers produced a biphasic response, increasing or decreasing the GABA-stimulated chloride uptake, depending on the HCH isomer and GABA concentrations used. The present results support the idea of cyclodienes and gamma-HCH action at the GABAA receptor by interacting with the TBPS binding site. A different interaction of PTX and alpha-endosulfan in the same recognition site is also suggested. An increase of GABA-induced 36Cl- flux by beta- and delta-HCH can account for the depressant activity of these compounds. This work also demonstrates the usefulness of primary neuronal cultures to perform functional studies of the GABAA receptor, taking into account allosteric interactions between the different recognition sites of the GABAA receptor.
Assuntos
Córtex Cerebral/metabolismo , Cloretos/metabolismo , Inseticidas/farmacologia , Ácido gama-Aminobutírico/farmacologia , Animais , Cálcio/metabolismo , Células Cultivadas , Endossulfano/farmacologia , Feminino , Camundongos , Pentilenotetrazol/farmacologia , Picrotoxina/análogos & derivados , Picrotoxina/farmacologia , Gravidez , Receptores de GABA-A/efeitos dos fármacos , SesterterpenosRESUMO
The discovery of a potential new GH therapy by small molecules that induce GH secretion (GHRP-6, L-692,429, MK-0677), has increased the interest in these GH secretagogues and their receptor and mechanism of action, which is different from the one of GHRH. We report the solubilization of the GH-secretagogue-receptor-ligand-G-protein complex (apparent molecular mass of approximately 255 kDa) from porcine anterior pituitary membranes using digitonin, after labelling the receptor with [35S]MK-0677. The solubilized receptor showed high affinity (KD = 122.2 +/- 14.4 pM) and low capacity (Bmax = 3.8 +/- 0.9 fmol/mg protein). These values and the inhibition constants (Ki) for a series of GH secretagogues were similar to the values determined in membranes isolated from porcine anterior pituitary gland. The solubilization of the GH secretagogue receptor opens up the possibility for further molecular characterization and sequencing of the receptor protein, necessary step prior to the identification of the natural ligand that would act as a GHRH amplifying hormone, and that the GH secretagogues would mimic.