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Biomedical subjects

A Pomés

Publications and source records attributed to A Pomés.

At least 19 recordsLinked to original sources
PubMed2006

Quantification of Ara h 1 in peanuts: why roasting makes a difference.

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.

Allergens↗
PubMed2004

Fel d 4, a cat lipocalin allergen.

BACKGROUND: Cat allergy is unique among allergy to mammals in that the major allergen Fel d 1 is a uteroglobin-like protein and not a lipocalin. The biochemical spectrum of the cat allergens is thus uncertain, particularly with regard to the role that a cat lipocalin protein may play in sensitization to cats in allergic individuals. OBJECTIVE: To analyse cDNA encoding a lipocalin allergen and the corresponding recombinant allergen at both the molecular and immunological levels. METHODS: A submandibular salivary gland cDNA expression library was constructed and screened for clones producing IgE-binding polypeptides. cDNA encoding a lipocalin allergen and its corresponding recombinant allergen were analysed. RESULTS: An IgE binding molecule with high sequence identity to the boar salivary lipocalin and the horse lipocalin Equ c 1 allergen was isolated and designated, Fel d 4. Serum from 62.96% of cat-allergic subjects examined had measurable IgE antibody to Fel d 4 but typically at low levels. Despite this in 47% of sera the anti-Fel d 4 IgE titres were higher than the anti-Fel d 1 titres. IgE binding to the lipocalin allergen could be blocked by an allergen extract from cow and to a lesser degree by extracts from horse and dog. CONCLUSION: Fel d 4 is a lipocalin allergen produced by the cat, which binds IgE at relatively high frequency in cat-sensitive individuals. The allergen provides not only a means for investigating differences in the immune response to lipocalin allergens from that found for other mammalian species but also an important reagent for the diagnosis of cat allergy.

Allergens↗
PubMed2004

Peanut allergen (Ara h 1) detection in foods containing chocolate.

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.

Allergens↗
PubMed2001

Cockroach allergens: environmental distribution and relationship to disease.

Cockroach allergy has been recognized as an important cause of asthma. Exposure to high levels of cockroach allergens in the home is a major risk factor for symptoms in sensitized individuals. Previously identified allergens from Blatella germanica and Periplaneta americana include Bla g 2 (inactive aspartic proteinase), Bla g 4 (calycin), Bla g 5 (glutathione-S-transferase), Bla g 6 (troponin), the Group 1 cross-reactive allergens Bla g 1 and Per a 1, Per a 3 (arylphorin), and Per a 7 (tropomyosin). The primary site of cockroach allergen accumulation is the kitchen. However, lower levels of allergen can be found in bedding, on the bedroom floor, and in sofa dust. Strategies for decreasing exposure to cockroach have been investigated. The results suggest that a sustained decrease in cockroach allergen levels is difficult to accomplish, even after successful extermination of cockroach populations. The use of recombinant cockroach allergens may lead to the development of new approaches to asthma treatment in the future.

Air Pollutants↗
PubMed2001

Molecular cloning, expression and modelling of cat allergen, cystatin (Fel d 3), a cysteine protease inhibitor.

BACKGROUND: Cats are an important source of indoor allergens. However, only two cat allergens, Fel d 1 and albumin, have been cloned and sequenced. IgE antibodies to Fel d 1 and albumin do not fully account for IgE responses to cat and there is good immunochemical evidence that cats produce other allergens. OBJECTIVE: To identify and define the molecular structure of the other potential cat allergens. METHODS: A cat skin cDNA library was screened using pooled serum obtained from five asthmatic patients which contained high levels of IgE antibody to cat dander. Selected cDNA clones were screened by plaque immunoassay and one cDNA clone, encoding cystatin, was expressed in E. coli. The three dimensional structure of cat cystatin was modelled using the SWISS-MODEL computer program. RESULTS: Three positive cDNA clones (A, B and C) were identified, two of which were fully sequenced. Clones A and C encoded the same 98 amino acid residue sequence which showed 79% and 75% homology with bovine and human cystatin A, respectively. The cat cystatin sequence contained the conserved cysteine protease inhibitor signature and two of three lipocalin motifs. By plaque immunoassay, 60-90% of cat allergic sera had IgE ab to the expressed cystatin clones. The cysteine protease inhibitor motif was also partially conserved in dog allergen sequences, Can f 1 and Can f 2, which are lipocalins. The recombinant protein was expressed in E. coli as an 11-kDa protein, corresponding to the predicted MW of cat cystatin. The three-dimensional structure of cat cystatin was modelled on human cystatin structures. CONCLUSION: A newly identified allergen, cystatin (Fel d 3), has been cloned from cat skin and is a member of the cysteine protease inhibitor family.

Allergens↗
PubMed2001

Cockroach allergens and asthma.

Asthma and allergy are the most common diseases associated with cockroach infestation of houses in the United States and other parts of the world. Sensitization and exposure to cockroach allergens is associated with increased asthma morbidity in the United States, especially among lower socioeconomic groups, including African American and Hispanic populations. Exposure to cockroach allergens in the first 3 months of life has been associated with repeated wheezing and asthma. The principal domestic cockroach species are Blattella germanica and Periplaneta americana. Both species produce several potent allergens, including Bla g 2 (inactive aspartic proteinase), Bla g 4 (calycin), Bla g 5 (glutathione-S-transferase), the group 1 cross-reactive allergens Bla g 1 and Per a 1, and tropomyosin. Structural homology between tropomyosins from cockroaches, mites, and shrimp may explain clinical cases of the oral allergy syndrome. The 3-dimensional structures of several cockroach allergens are known, and biologically active recombinant allergens have been produced in high-level expression vectors. The use of recombinant cockroach allergens should allow mechanisms of cockroach-induced asthma to be investigated and may lead to the development of new approaches to asthma treatment. Environmental allergen measurements of Bla g 1 and Bla g 2 have allowed exposure levels that cause allergic sensitization to be established. Abatement studies have shown that a sustained decrease in cockroach allergen levels is difficult but can be accomplished by professional application of insecticides, together with rigorous household cleaning. Cockroach asthma is an important public health problem that affects patients who are the least likely to be compliant with treatment with asthma medications or environmental control. Patient education, improvements in the housing stock, and improvements in environmental and immunologic treatment strategies are likely to be the most successful approaches to reduce the prevalence of cockroach-induced asthma.

Allergens↗
PubMed2001

Can knowledge of the molecular structure of allergens improve immunotherapy?

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.

Allergens↗
PubMed2000

Adenosine: A partial agonist of the growth hormone secretagogue receptor.

The growth hormone secretagogue receptor (GHS-R) is involved in the regulation of pulsatile GH release. However, until recently, natural endogenous ligands for the receptor were unknown. We fractionated porcine hypothalamic extracts and assayed fractions for activity on HEK293 cells expressing GHS-R and aequorin. A partial agonist was isolated and identified using microspray tandem mass spectrometry as adenosine. GHS-R activation by adenosine and synthetic adenosine agonists is inhibited by the GHS-R selective antagonists L-765,867, D-Lys(3)-GHRP-6, and by theophylline and XAC. Cross desensitization of the GHS-R occurs with both MK-0677 and adenosine. Ligand binding and site directed mutagenesis studies show that adenosine binds to a binding site that is distinct from the previously characterized MK-0677 and GHRP-6 binding pocket. We propose, that adenosine is a physiologically important endogenous GHS-R ligand and speculate that GHS-R ligands modulate dopamine release from hypothalamic neurons.

Adenosine↗
PubMed2000

Recombinant allergens for diagnosis and therapy of allergic disease.

Many of the problems associated with using natural allergenic products for allergy diagnosis and treatment can be overcome with use of genetically engineered recombinant allergens. Over the past 10 years, the most important allergens from mites, pollens, animal dander, insects, and foods have been cloned, sequenced, and expressed. In many cases the three-dimensional allergen structure has been determined and B-cell and T-cell epitopes have been mapped. These studies show that allergens have diverse biologic functions (they may be enzymes, enzyme inhibitors, lipocalins, or structural proteins) and that as a rule the allergen function is unrelated to its ability to cause IgE antibody responses. High-level expression systems have been developed to produce recombinant allergens in bacteria, yeast, or insect cells. Recombinant allergens show comparable IgE antibody binding to their natural counterparts (where available) and show excellent reactivity on skin testing and in in vitro diagnostic tests. Cocktails of recombinant allergens can be formulated with predetermined and uniform allergen levels, which could replace natural allergens and result in the development of innovative, patient-based tests for allergy diagnosis. Recombinant allergens also offer the exciting possibility of developing new forms of allergen immunotherapy, including the use of hypoallergens, allergens coupled to IgE suppressive adjuvants, and peptide-based therapies. The production of recombinant allergens as defined molecular entities makes it feasible to consider the possibility of developing prophylactic allergen vaccines. The introduction of recombinant allergens in research and in clinical trials should lead to significant improvements in allergy diagnosis and treatment.

Allergens↗
PubMed1999

Molecular cloning of Per a 1 and definition of the cross-reactive Group 1 cockroach allergens.

BACKGROUND: Sensitization to allergens produced by German and American cockroaches is strongly associated with the cause of asthma. Most of the cockroach allergens identified to date have been species specific. OBJECTIVE: The aim of this study was to identify and sequence cross-reactive cockroach allergens. METHODS: A Periplaneta americana cDNA library was screened with IgE antibody from patients in the United States who were allergic to cockroach and who were sensitized to Blattella germanica. RESULTS: A cDNA clone was isolated that contained an 870-bp sequence with a 695-bp open reading frame, encoding a 231 amino acid protein, molecular weight 26.2 kd. Plaque immunoassays using anti-Bla g 1 and anti-Per a 1 mAbs and a panel of human IgE antibodies showed that the protein expressed by these clones was Per a 1. Sequence homology searches showed that Per a 1 was homologous to 5 previously reported, but unidentified, sequences from B germanica and P americana. These sequences encoded proteins with multiple molecular sizes containing approximately 100 amino acid repeats. The Per a 1 sequence also showed 31% identity to a mosquito precursor protein, ANG12, which may be involved in digestion. The Per a 1 cDNA was expressed in Pichia pastoris to produce purified recombinant allergen (yield, 14 mg/L). CONCLUSION: The results define the molecular structure and antigenic relationships between a new family of cross-reactive "Group 1" allergens produced by both P americana and B germanica. These recombinant allergens and specific mAbs will provide tools to improve the diagnosis and treatment of allergic diseases caused by cockroaches.

Allergens↗
PubMed1998

Novel allergen structures with tandem amino acid repeats derived from German and American cockroach.

Cockroaches produce potent allergens that are an important cause of asthma. The two principal domiciliary cockroach species, Blattella germanica and Periplaneta americana, secrete major allergens, Bla g 1 and Per a 1. Here, we report the molecular cloning of three Bla g 1 cDNA clones, which showed 70% amino acid sequence identity with Per a 1. Plaque immunoassays with human IgE antibodies or murine monoclonal antibodies showed that these allergens were antigenically cross-reactive. The Bla g 1 sequences also showed homology to five previously undefined cockroach allergen sequences. An unusual feature of all these sequences was that they contained multiple tandem amino acid repeats of approximately 100 amino acid residues. Between one and seven repeat units were identified by dot-plot matrix analysis. The sequences also showed homology to a mosquito protein involved in digestion (ANG12 precursor) and to mitochondrial energy transfer proteins. High levels of Bla g 1 were found in cockroach hindgut and proventriculus. Amino acid sequencing of natural Bla g 1 and Per a 1 suggested that these allergens are cleaved by trypsin-like enzymes following secretion into the digestive tract. The repeat sequences appear to have evolved by duplication of an ancestral amino acid domain, which may have arisen from the mitochondrial energy transfer proteins.

Allergens↗
PubMed1997

Allosteric interactions between gamma-aminobutyric acid, benzodiazepine and picrotoxinin binding sites in primary cultures of cerebellar granule cells. Differential effects induced by gamma- and delta-hexachlorocyclohexane.

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.

Animals↗
PubMed1997

The mechanism for hexachlorocyclohexane-induced cytotoxicity and changes in intracellular Ca2+ homeostasis in cultured cerebellar granule neurons is different for the gamma- and delta-isomers.

The cytotoxic action of the delta- and gamma-isomers of hexachlorocyclohexane (HCH) as well as their ability to induce changes in intracellular Ca2+ homeostasis was studied in cultured rat cerebellar granule neurons. Changes in the free intracellular Ca2+ concentration ([Ca2+]i) related to Ca2+ influx and release from intracellular stores were investigated using, in addition to the physiological incubation medium, media without added Ca2+ (nominally Ca2+ free) or containing verapamil or dantrolene, drugs which block influx through voltage-gated Ca2+ channels and release from intracellular stores, respectively. Cytotoxicity was monitored using leakage of lactate dehydrogenase (LDH) and staining of damaged cells with either trypan blue or propidium iodide. In the latter case, when fluorescence microscopy was employed, undamaged cells were visualized using the vital stain calcein acetoxymethyl ester (calcein-AM). The delta-isomer of HCH was found to be more potent and active as a cytotoxic agent than the gamma-isomer (lindane) and it was demonstrated that the moderate cytotoxic action of lindane could be ameliorated by dantrolene which on the other hand had no effect on cytotoxicity induced by delta-HCH. It was noticed that pronounced (50% increase in fluorescence) staining with propidium iodide was associated with only a marginal (20%) LDH leakage and staining of only a few (<25%) cells with trypan blue. Both delta- and gamma-HCH induced an increase in [Ca2+]i which was most pronounced in case of delta-HCH. Analysis of the different mechanisms governing the increase in [Ca2+li using dantrolene, verapamil, and Ca2+-free medium revealed distinct differences between the two isomers with regard to the Ca2+ pools affected. Both isomers stimulated Ca2+ influx through voltage-gated Ca2+ channels but only the gamma-isomer affected a Ca2+-dependent, dantrolene-sensitive pool. On the other hand, delta-HCH affected mainly a Ca2+-independent dantrolene-insensitive pool. The finding that delta-HCH was more toxic than lindane may be correlated to the differences between the isomers with regard to the action on the different Ca2+ pools. Thus, delta-HCH affected primarily the dantrolene-insensitive Ca2+ pools which partly reflect influx. On the contrary, lindane had little effect on these Ca2+ pools but affected primarily dantrolene-sensitive intracellular Ca2+ stores. This may suggest that delta-HCH may exert its cytotoxic action by stimulating a large influx of Ca2+ possibly leading to release of Ca2+ from dantrolene-insensitive stores. In contrast, the toxic action of lindane may be primarily related to release of Ca2+ from the dantrolene-sensitive stores.

Animals↗
PubMed1996

Solubilization and characterization of a growth hormone secretagogue receptor from porcine anterior pituitary membranes.

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.

Animals↗
PubMed1994

Lindane cytotoxicity in cultured neocortical neurons is ameliorated by GABA and flunitrazepam.

The effect of lindane (gamma-hexachlorocyclohexane) on [35S]t-butylbicyclophosphorothionate ([35S]TBPS) binding and GABA-stimulated 36Cl- influx was investigated in cultured cerebral cortical neurons. In addition, the cytotoxic action of lindane as well as a protection by GABA and flunitrazepam were studied together with the ability of lindane to increase the intracellular concentration of free Ca2+. Lindane was found to be toxic to the neurons, an effect that could be completely prevented by the simultaneous presence of GABA (0.1 microM) and flunitrazepam (100 microM) and reduced by GABA alone. An interaction with the GABA receptor-gated chloride channel was demonstrated by an inhibitory action of lindane on [35S]TBPS binding (IC50 188 +/- 51 nM) and on GABA-stimulated 36Cl- influx in the neurons. Lindane only marginally increased the intracellular Ca2+ concentration in the neurons. It is concluded that the cytotoxic action of lindane is mediated through interaction with GABA receptors in a manner essentially independent of changes in intracellular Ca2+ homeostasis.

Animals↗
PubMed1994

Effects of organochlorine pesticides on 36Cl- flux in primary neuronal cultures.

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.

Animals↗
PubMed1994

Disruption of GABA-dependent chloride flux by cyclodienes and hexachlorocyclohexanes in primary cultures of cortical neurons.

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.

Animals↗