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1.
Allergy ; 69(7): 888-97, 2014 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-24773443

RESUMEN

BACKGROUND: Food allergens have been evidenced in breast milk under physiological conditions, but the kinetic and the role of this passage in food allergies are still unclear. We then aimed to analyze the passage of peanut allergens in human breast milk and their allergenicity/immunomodulatory properties. METHODS: Human breast milk was collected from two non-atopic peanut-tolerant mothers before and at different time points after ingestion of 30 g of commercial roasted peanut. Ara h 6, Ara h 6 immune complexes, and the IgE binding capacity of breast milk samples were measured using specific immunoassays. Their allergenic functionality was then assessed using cell-based assay. Finally, human breast milk obtained before or after peanut ingestion was administered intragastrically to BALB/c mice at different ages, and mice were further experimentally sensitized to peanut using cholera toxin. RESULTS: Ara h 6 is detected as soon as 10 min after peanut ingestion, with peak values observed within the first hour after ingestion. The transfer is long-lasting, small quantities of peanut allergens being detected over a 24-h period. IgG-Ara h 6 and IgA-Ara h 6 immune complexes are evidenced, following a different kinetic of excretion than free allergens. Peanut allergens transferred in milk are IgE reactive and can induce an allergic reaction in vitro. However, administration of human breast milk to young mice, notably before weaning, does not lead to sensitization, but instead to partial oral tolerance. CONCLUSION: The low quantities of immunologically active allergens transferred through breast milk may prevent instead of priming allergic sensitization to peanut.


Asunto(s)
Albuminas 2S de Plantas/inmunología , Antígenos de Plantas/inmunología , Tolerancia Inmunológica/inmunología , Leche Humana/química , Hipersensibilidad al Cacahuete/inmunología , Animales , Lactancia Materna , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Leche Humana/inmunología , Hipersensibilidad al Cacahuete/prevención & control
2.
J Neurosci ; 21(4): 1104-9, 2001 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-11160380

RESUMEN

Recently, the cannabinoid (CB) receptor agonist anandamide (AEA) has been shown to excite perivascular terminals of primary sensory neurons via activation of the vanilloid receptor-1 (VR-1). To determine whether AEA stimulates central terminals of these neurons, via VR-1 activation, we studied the release of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities (LI) from slices of rat dorsal spinal cord. Mobilization of Ca(2+) in rat dorsal root ganglion (DRG) neurons in culture was also studied. AEA (0.1-10 micrometer) increased the outflow of CGRP-LI and SP-LI from slices of the rat dorsal spinal cord in a Ca(2+)-dependent manner and increased [Ca(2+)](i) in capsaicin-sensitive cultured DRG neurons. Both effects of AEA were abolished by capsaicin pretreatment and by the VR-1 antagonist capsazepine but not affected by the CB receptor antagonists AM281 or AM630. Both neuropeptide release and Ca(2+) mobilization induced by electrical field stimulation (EFS) were inhibited by a low concentration of AEA (10 nm). Inhibition by AEA of EFS-induced responses was reversed by AM281 and AM630, but was not affected by capsazepine. Results indicate that stimulation of VR-1 with high concentrations of AEA excites central terminals of capsaicin-sensitive DRG neurons, thus causing neuropeptide release in the dorsal spinal cord. This novel activity opposes the CB receptor-mediated inhibitory action of low concentrations AEA. However, only if large amounts of endogenous AEA could be produced at the level of the dorsal spinal cord, they may not inhibit, but rather activate, nociceptive sensory neurons.


Asunto(s)
Ácidos Araquidónicos/farmacología , Capsaicina/análogos & derivados , Ganglios Espinales/metabolismo , Neuronas Aferentes/metabolismo , Terminales Presinápticos/efectos de los fármacos , Receptores de Droga/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Calcio/metabolismo , Capsaicina/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Endocannabinoides , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Técnicas In Vitro , Masculino , Neuronas Aferentes/citología , Neuronas Aferentes/efectos de los fármacos , Alcamidas Poliinsaturadas , Ratas , Receptores de Cannabinoides , Receptores de Droga/agonistas , Receptores de Droga/antagonistas & inhibidores , Médula Espinal/citología , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Sustancia P/metabolismo , Canales Catiónicos TRPV
3.
Biochim Biophys Acta ; 1254(3): 333-40, 1995 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-7857974

RESUMEN

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.


Asunto(s)
Prostaglandina-Endoperóxido Sintasas/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Plaquetas/enzimología , Humanos , Sueros Inmunes/inmunología , Técnicas para Inmunoenzimas , Masculino , Datos de Secuencia Molecular , Péptidos/inmunología , Prostaglandina-Endoperóxido Sintasas/análisis , Prostaglandina-Endoperóxido Sintasas/inmunología , Vesículas Seminales/enzimología , Ovinos
4.
Biochim Biophys Acta ; 1254(3): 341-8, 1995 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-7857975

RESUMEN

We have produced and characterized monoclonal antibodies (mAbs) directed against a specific carboxyterminal sequence of human cyclooxygenase-2 (residues 580-598). A rabbit polyclonal antiserum was also raised against another sequence of 10 amino acids (residues 570-581) not present in human constitutive cyclooxygenase-1. Affinity-purified polyclonal antibodies, coated on microtiter plates, were used as capture antibodies in a two-site immunometric assay, with an mAb-acetylcholinesterase conjugate used as tracer. The detection limit was 500 fmol/ml of peptide C3-COX2 (residues 570-595). The assay was specific for the cyclooxygenase-2 (COX-2) isoform, since no immunoreactivity could be detected in platelet extracts known to be rich in cyclooxygenase-1 (COX-1). In contrast, extracts from cultured human umbilical vein endothelial cells challenged with 20 nM phorbol myristate acetate (PMA) showed an increase in COX-2 immunoreactivity related both to the increase in enzyme activity and the variations observed by Western blot analysis. Under these conditions, analysis of the same cell lysates with another immunometric assay specific for COX-1 revealed insignificant variation of this enzyme. The specificity of detection was further assessed by measuring the immunoreactivity of the fractions obtained after molecular sieve chromatography of control and stimulated cell extracts, and corroborated the marked enhancement of COX-2 by comparison with COX-1. Treatment of PMA-activated cells with H-7 or actinomycin D totally abolished the COX-2 signal and had little effect on COX-1. No significant variation in COX-2 immunoreactivity was observed using the inactive isomer 4 alpha-PMA, even at 100 nM. These assays constitute the first quantitative analysis of constitutive COX-1 and of inducible COX-2 in nucleated cells at the protein level.


Asunto(s)
Endotelio Vascular/enzimología , Isoenzimas/análisis , Prostaglandina-Endoperóxido Sintasas/análisis , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Inducción Enzimática , Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Isoenzimas/biosíntesis , Isoenzimas/inmunología , Datos de Secuencia Molecular , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/inmunología
5.
Biochim Biophys Acta ; 1526(1): 13-6, 2001 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-11287117

RESUMEN

Unstimulated RAW 264.7 macrophages express negligible heme oxygenase-1 (HO-1) protein but incubation with the nitric oxide (NO) donor spermine nonoate (SPNO) induced HO-1 and weakly cyclo-oxygenase-2 (COX-2) protein. This effect was potentiated by coincubation with the COX-2 selective inhibitor, SC58125. Cells incubated with SPNO showed a strong increase in HO-1 mRNA levels after 4 h with a significant potentiation in the presence of SC58125, which did not modify HO-1 mRNA stability. The induction of HO-1 by NO and its potentiation by anti-inflammatory agents may play a role in inflammatory and immune responses.


Asunto(s)
Hemo Oxigenasa (Desciclizante)/biosíntesis , Macrófagos/efectos de los fármacos , Óxido Nítrico/farmacología , Animales , Línea Celular , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Sinergismo Farmacológico , Hemo-Oxigenasa 1 , Isoenzimas/antagonistas & inhibidores , Isoenzimas/biosíntesis , Macrófagos/enzimología , Proteínas de la Membrana , Ratones , Óxidos de Nitrógeno , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Pirazoles/farmacología , ARN Mensajero/biosíntesis , Espermina/análogos & derivados , Regulación hacia Arriba
6.
Biochim Biophys Acta ; 1339(2): 253-67, 1997 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-9187246

RESUMEN

We analyzed 45 batches of venom from 20 different species belonging to 11 genera from the 3 main families of venomous snakes (Elapidae, Viperidae and Crotalidae). We found high acetylcholinesterase (AChE) activity in all venoms from Elapidae, except in those from the Dendroaspis genus. AChE was particularly abundant in Bungarus venoms which contain up to 8 mg of enzyme per gram of dried venom. We could not detect acetylcholinesterase activity in any batch of venom from Viperidae or Crotalidae. Titration of active sites with an organophosphorous agent (MPT) revealed that the AChE of all venoms have similar turnovers (6000 to 8000 s(-1)) which are clearly higher than those of Torpedo and mammalian enzymes but lower than that of Electrophorus. AChEs from the venom of elapid snakes of the Bungarus, Naja, Ophiophagus and Haemacatus genera were purified by affinity chromatography. SDS-PAGE analysis and sucrose gradient centrifugation demonstrated that AChE is exclusively present as a nonamphiphilic monomer. These enzymes are true AChEs, hydrolyzing acetylthiocholine faster than propionylthiocholine and butyrylthiocholine and exhibiting excess substrate inhibition. Twenty-seven different monoclonal antibodies directed against AChE from Bungarus fasciatus venom were raised in mice. Half of them recognized exclusively the Bungarus enzyme while the others cross-reacted with AChEs from other venoms. Polyspecific mAbs were used to demonstrate that venoms from Dendroaspis, which contain the AChE inhibitor fasciculin but lack AChE activity, were also devoid of immunoreactive AChE protein. AChE inhibitors acting at the active site (edrophonium, tacrine) and at the peripheral site (propidium, fasciculin), as well as bis-quaternary ligands (BW284C51, decamethonium), were tested against the venom AChEs from 11 different species. All enzymes had a very similar pattern of reactivity with regard to the different inhibitors, with the exception of fasciculin. AChEs from Naja and Haemacatus venoms were relatively insensitive to fasciculin inhibition (IC50 >> 10(-6) M), while Bungarus (IC50 approximately 10(-8) M) and especially Ophiophagus (IC50 < 10(-10) M) AChEs were inhibited very efficiently. Ophiophagus and Bungarus AChEs were also efficiently inhibited by a monoclonal antibody (Elec-410) previously described as a specific ligand for the Electrophorus electricus peripheral site. Taken together, these results show that the venoms of most Elapidae snakes contain large amounts of a highly active non-amphiphilic monomeric AChE. All snake venom AChEs show strong immunological similarities and possess very similar enzymatic properties. However, they present quite different sensitivity to peripheral site inhibitors, fasciculin and the monoclonal antibody Elec-410.


Asunto(s)
Acetilcolinesterasa/metabolismo , Venenos Elapídicos/enzimología , Acetilcolinesterasa/inmunología , Acetilcolinesterasa/aislamiento & purificación , Anticuerpos Monoclonales/inmunología , Catálisis , Reacciones Cruzadas , Venenos Elapídicos/metabolismo , Inhibidores Enzimáticos/farmacología , Conformación Proteica
7.
Biochim Biophys Acta ; 1541(3): 150-60, 2001 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-11755209

RESUMEN

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.


Asunto(s)
Ciclodextrinas/farmacología , Sistemas de Liberación de Medicamentos , Receptores de Neuroquinina-1/efectos de los fármacos , Sustancia P/análogos & derivados , beta-Ciclodextrinas , gamma-Ciclodextrinas , Animales , Anticuerpos/inmunología , Autorradiografía , Unión Competitiva , Células CHO , Cricetinae , Ciclodextrinas/química , Ciclodextrinas/inmunología , Diseño de Fármacos , Estructura Molecular , Receptores de Neuroquinina-1/biosíntesis , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes/biosíntesis , Sustancia P/química , Sustancia P/inmunología
8.
Cell Signal ; 11(10): 743-51, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10574329

RESUMEN

The vasopressin V1a receptor undergoes homologous and heterologous desensitizations which can be mimicked by activation of protein kinase C. This suggests that phosphorylation of the V1a receptor may be involved in the desensitization mechanisms. Such a phosphorylation was presently investigated in HEK 293 cells stably transfected with rat vasopressin V1a receptor. Metabolic labelling and immunoprecipitation of epitope-tagged V1a receptor evidenced a 52-kDa band and a 92-kDa band. Glycosidase treatments and immunoblotting experiments suggest that the 52-kDa band corresponds to an immature unprocessed receptor protein, whereas the 92-kDa band would correspond to a highly glycosylated form of the mature V1a receptor. Exposure of the cells to vasopressin induced a selective 32P phosphate incorporation in the 92-kDa form of the receptor. This homologous ligand-induced phosphorylation was dose dependent with maximal phosphate incorporation corresponding to four times the basal level. Stimulation of the endogenous phospholipase C-coupled m3 muscarinic receptor by carbachol-induced heterologous phosphorylation of the V1a receptor whose amplitude was half that of the homologous phosphorylation. This heterologous phosphorylation was associated with a reduced vasopressin-dependent increase in intracellular calcium.


Asunto(s)
Receptores de Vasopresinas/metabolismo , Animales , Calcio/metabolismo , Carbacol/farmacología , Línea Celular Transformada , Agonistas Colinérgicos/farmacología , AMP Cíclico/metabolismo , Humanos , Fosforilación , Ratas , Receptor Muscarínico M1 , Receptor Muscarínico M3 , Receptor Muscarínico M5 , Receptores Muscarínicos/genética , Receptores de Vasopresinas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Vasopresinas/metabolismo , Vasopresinas/farmacología
9.
J Leukoc Biol ; 67(4): 545-52, 2000 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10770288

RESUMEN

The central role of alveolar macrophages in the establishment of lipopolysaccharide (LPS)-induced lung inflammation is well demonstrated. They produce and release numerous proinflammatory molecules, among which is tumor necrosis factor alpha (TNF-alpha), a cytokine responsible in part for the neutrophilic alveolitis. Interleukin-10 (IL-10) produced by LPS-activated mononuclear phagocytes is a major anti-inflammatory cytokine that down-regulates TNF-alpha synthesis. We studied the ability of murine alveolar macrophages to produce IL-10 in vivo and in vitro, in response to LPS. Unexpectedly, the IL-10 protein was not detected in the whole lung and airspaces after LPS intranasal instillation. In addition, no IL-10 protein was found in supernatants of isolated and LPS-stimulated alveolar macrophages. The lack of IL-10 synthesis was confirmed by the absence of specific RNA transcripts. By contrast and as expected, autologous peritoneal macrophages produced IL-10 upon LPS challenge. Drugs that usually modify the TNF-alpha/IL-10 balance in favor of IL-10 were used without success. Thus, maneuvers allowing an increase in intracellular cAMP concentrations did not reverse this unexpected phenotype. Moreover, direct activation of protein kinase C with PMA was unable to trigger IL-10 formation by alveolar, by contrast to peritoneal, macrophages. The current findings describe a specific phenotype for murine alveolar macrophages during LPS-induced inflammation.


Asunto(s)
Interleucina-10/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos Alveolares/metabolismo , Macrófagos Peritoneales/metabolismo , Animales , Ratones , Especificidad de Órganos , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/biosíntesis
10.
Mol Immunol ; 34(12-13): 829-38, 1997.
Artículo en Inglés | MEDLINE | ID: mdl-9464518

RESUMEN

In a previous study (Boquet et al., 1995, Molec. Immunol. 32, 303-308) we observed remarkable inversions of hydropathic profiles between complementarity determining regions (CDRs) of an anti-substance P monoclonal antibody (SP31) and the corresponding 5 amino acid C-terminal peptide epitope. Here we demonstrate the importance this hydropathic complementarity by measuring the immunoreactivity of SP-related peptides which have been modified in their C-terminal parts so that hydropathic profile has been conserved (by substituting amino acids in the epitope) or modified (by mixing the sequence of amino acids in the epitope). Experiments performed in equilibrium conditions using a competitive enzyme immunoassay showed that most of the peptides conserving the hydropathic profile of SP epitope were recognized by mAb SP31 (even if marked variations in affinity were observed), while those for which the hydropathic profile was modified exhibited very low or undetectable affinity. The kinetic parameters (ka and kd) of peptide-antibody interactions were determined using Surface Plasmon Resonance technology (BIACORE 2000). These measurements showed that all peptides recognized by mAb SP31 had similar association rate constants (close to 2 x 10[5] M[-1] s[-1]), differences in binding affinities essentially resulting from differences in dissociation rate constants (ranging from 1.61 x 10[-4] to 1.15 x 10[-1] s[-1]). From these results, it was concluded that hydropathic complementarity between the epitope and the paratope could be a necessary but not sufficient condition for establishing high-affinity binding. We hypothesize that hydropathic interactions may play a critical role during the first contacts between antibody CDRs and the peptide, possibly by favouring reorganization of water molecules at the antibody-peptide interface.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Sustancia P/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Técnicas Biosensibles , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Cinética , Fragmentos de Péptidos/inmunología , Relación Estructura-Actividad , Sustancia P/química
11.
Mol Immunol ; 37(3-4): 161-7, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-10865115

RESUMEN

Twenty patients allergic to cow's milk proteins and with high levels of specific IgE directed against bovine whole casein were selected to evaluate reactivity of their IgE antibodies with human beta-casein. Highly purified human and bovine beta-caseins were prepared by selective precipitations and FPLC separation. Their identity and purity were assessed by HPLC, analysis of amino acid composition, sequencing of the five N-terminal amino acid residues and immunochemical tests. Direct and indirect ELISAs were performed using human and bovine beta-casein coated into microtiter plates and monoclonal anti-human IgE antibody AChE labelled for revelation. Seven sera contained specific IgE directed against human beta-casein. Inhibition studies using native human and bovine beta-caseins as well as bovine beta-casein-derived peptides demonstrated that, depending on the sera, one or several common epitopes located in different parts of the molecule were shared by the two homologous proteins.


Asunto(s)
Alérgenos/inmunología , Especificidad de Anticuerpos , Caseínas/inmunología , Inmunoglobulina E/inmunología , Hipersensibilidad a la Leche/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bovinos , Niño , Preescolar , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Homología de Secuencia de Aminoácido , Especificidad de la Especie
12.
Mol Immunol ; 37(8): 423-33, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11090877

RESUMEN

Monoclonal antibody (mAb) PS12, obtained using the complementary peptide methodology, mimics the neuropeptide substance P (SP) in recognizing the SP-binding domain of the neurokinin-1 receptor (NK1R) and eliciting production of polyclonal antibodies cross-reacting with SP with a high affinity (Déry et al., 1997. J. Neuroimmunol. 76, 1-9). The aim of the present study was to investigate which structural features of mAb PS12 might account for this molecular mimicry. Cloning and sequencing of variable regions of both light (VL) and heavy (VH) chains of this 'SP-like' antibody did not indicate any primary sequence homology between SP and any antibody region. Instead, they revealed a striking similarity between the hydropathic profile of SP and that of an 11-amino-acid region in the light chain encompassing the second complementarity determining region (CDR2). When applied to CHO cells expressing the human NK1R, a synthetic extended 17-amino-acid peptide (denoted CDR2L) corresponding to this VL region inhibited the high-affinity binding of radiolabeled SP and antagonized the SP-induced inositol phosphate production. Moreover, a re-examination of the sequences of several antibodies that previously served in the design of CDR-derived bioactive peptides indicated that these antibodies also carried the hydropathic image of the respective ligands that they mimic. In agreement with previous observations on artificial synthetic peptides, our data thus suggest that the molecular mimicry between natural proteins (i.e. antibody and hormone, for example) could be understood on a structural level directly related, at least in part, to hydropathic homology. These results could then guide the search for bioactive paratope-derived peptides of potential pharmacological interest. We also observed inverse hydropathy between multiple CDRs of mAb PS12 (including CDR3H and CDR3L) and the peptide epitope, confirming the importance of hydropathic complementarity in antigen-antibody interactions.


Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Imitación Molecular , Receptores de Neuroquinina-1/inmunología , Sustancia P/química , Secuencia de Aminoácidos , Animales , Antígenos/inmunología , Antígenos/metabolismo , Células CHO , Cricetinae , Humanos , Hibridomas , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/inmunología , Fosfatos de Inositol/metabolismo , Ligandos , Datos de Secuencia Molecular , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-1/metabolismo , Alineación de Secuencia , Transducción de Señal/efectos de los fármacos , Sustancia P/metabolismo , Sustancia P/farmacología
13.
Endocrinology ; 138(5): 2163-71, 1997 May.
Artículo en Inglés | MEDLINE | ID: mdl-9112416

RESUMEN

In this study we investigated the expression of the two cyclooxygenases, cox-1 and -2, in sheep uterine tissues during the estrous cycle and early pregnancy. We identified the cox-2 isoform in the ovine uterus by Western blot and demonstrated that the two cyclooxygenases exhibited different patterns of expression. Cox-1 was expressed at steady state levels in the endometrium during the estrous cycle and comparable stages of pregnancy. In contrast, cox-2 was highly and transiently expressed from days 12-15 of the estrous cycle and declined thereafter to undetectable levels. Endometrium from early pregnant ewes showed a similar pattern of cox-2 expression, although there was a slower decrease beyond day 15. Immunohistochemical studies demonstrated that cox-1 was localized in both epithelial and stromal cells, whereas cox-2 was localized solely in the luminal epithelium and to a lesser extent in the superficial glands. Treatment of ovariectomized ewes with steroids indicated that expression of cox-1 remained at constant levels whatever the treatment. In contrast, endometrial cox-2 was highly induced by a 10-day progesterone treatment. Estradiol slightly increased cox-2 expression but only after progesterone priming. Collectively these results suggest that the developing ability of the uterus to synthesize PGs is due to the induction of cox-2.


Asunto(s)
Endometrio/enzimología , Estro/fisiología , Isoenzimas/metabolismo , Preñez/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Animales , Western Blotting , Ciclooxigenasa 1 , Ciclooxigenasa 2 , Dinoprost/metabolismo , Estradiol/farmacología , Femenino , Inmunohistoquímica , Isoenzimas/análisis , Ovariectomía , Embarazo , Progesterona/farmacología , Prostaglandina-Endoperóxido Sintasas/análisis , Ovinos
14.
J Clin Endocrinol Metab ; 88(1): 327-36, 2003 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-12519873

RESUMEN

Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350-400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.


Asunto(s)
Enfermedades de las Glándulas Suprarrenales/sangre , Líquidos Corporales/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Técnicas para Inmunoenzimas/métodos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Enfermedades del Sistema Nervioso/sangre , Adolescente , Adulto , Anciano , Proteínas de Unión al Calcio/sangre , Proteínas de Unión al Calcio/aislamiento & purificación , Factor de Crecimiento del Tejido Conjuntivo , Femenino , Humanos , Proteínas Inmediatas-Precoces/sangre , Proteínas Inmediatas-Precoces/aislamiento & purificación , Péptidos y Proteínas de Señalización Intercelular/sangre , Péptidos y Proteínas de Señalización Intercelular/aislamiento & purificación , Masculino , Persona de Mediana Edad , Proteína Hiperexpresada del Nefroblastoma , Sensibilidad y Especificidad , Células Tumorales Cultivadas
15.
FEBS Lett ; 289(2): 171-5, 1991 Sep 09.
Artículo en Inglés | MEDLINE | ID: mdl-1915845

RESUMEN

A competitive enzyme immunoassay using acetylcholinesterase as tracer for thymosin beta 4, has been developed. Using this assay and a previously described EIA for AcSDKP, a negative regulator of pluripotent haematopoietic stem cell proliferation, the levels of these two peptides were determined in mouse tissue extracts. The combination of EIAs with different HPLC procedures validated these methods and clearly demonstrated the ubiquity of these peptides in mouse tissues. Similar results are reported for rabbit thymus which suggest different hypotheses for AcSDKP biosynthesis.


Asunto(s)
Oligopéptidos/análisis , Timosina/análogos & derivados , Secuencia de Aminoácidos , Animales , División Celular , Reacciones Cruzadas , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Sueros Inmunes , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Oligopéptidos/aislamiento & purificación , Especificidad de Órganos , Ratas , Homología de Secuencia de Ácido Nucleico , Timosina/análisis , Timosina/aislamiento & purificación
16.
FEBS Lett ; 467(2-3): 239-44, 2000 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-10675546

RESUMEN

IgE response specific to those molecular regions of casein that contain a major phosphorylation site was analyzed using native and modified caseins and derived peptides. This study included (i) the naturally occurring common variants A1 and A from beta- and alphas2-caseins, respectively, which were purified in the native form and then dephosphorylated, (ii) a purified rare variant D of alphas2-casein which lacks one major phosphorylation site, and (iii) the native and dephosphorylated tryptic fragment f(1-25) from beta-casein. Direct and indirect ELISA using sera from patients allergic to milk showed that the IgE response to caseins is affected by modifying or eliminating the major phosphorylation site.


Asunto(s)
Caseínas/química , Inmunoglobulina E/química , Procesamiento Proteico-Postraduccional , Sitios de Unión , Caseínas/genética , Caseínas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Humanos , Inmunoglobulina E/sangre , Hipersensibilidad a la Leche/sangre , Fosforilación , Isoformas de Proteínas/química , Tripsina
17.
FEBS Lett ; 234(1): 149-52, 1988 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-2899032

RESUMEN

Three putative processing enzymes, each with defined action in a prohormone system, a 'pro-ocytocin-neurophysin convertase' from bovine neurohypophysis secretory granules, a 'Leu-enkephalin Arg6 generating enzyme' from human CSF and the endoprotease from the 'S-28 convertase' complex of rat brain cortex, were tested for their ability to hydrolyze peptides deriving from pro-ocytocin, pro-enkephalin B and pro-somatostatin, respectively at pairs of basic amino acids. The observations suggest that structural parameters specified by the peptide region around the dibasic moieties govern recognition by the enzyme and define which peptide bond is hydrolyzed.


Asunto(s)
Endopeptidasas/metabolismo , Encefalinas/metabolismo , Oxitocina/análogos & derivados , Precursores de Proteínas/metabolismo , Serina Endopeptidasas/metabolismo , Somatostatina/metabolismo , Animales , Encéfalo/enzimología , Bovinos , Cromatografía Líquida de Alta Presión , Gránulos Citoplasmáticos/enzimología , Humanos , Oxitocina/metabolismo , Neurohipófisis/enzimología , Ratas , Especificidad por Sustrato
18.
FEBS Lett ; 460(2): 303-8, 1999 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-10544254

RESUMEN

The structural requirements for internalization and signalling of the vasopressin V1a receptor were investigated in stably transfected HEK-293 cells. Removal of the 51 C-terminal amino acids did not affect vasopressin binding, calcium signalling, heterologous desensitization or internalization of the receptor. Deletion of 14 additional amino acids reduced vasopressin-dependent calcium increase and impaired receptor internalization. Substitution of cysteines 371-372 did not affect intracellular signalling, but decreased endocytosis by 26%. Substitution of the 361-362 leucine by alanine residues reduced by 56% V1a receptor sequestration without affecting calcium signalling. These results indicate that di-cysteine and mostly di-leucine motifs present in the C-terminal region of the V1a receptor are involved in its internalization.


Asunto(s)
Cisteína/fisiología , Leucina/fisiología , Receptores de Vasopresinas/metabolismo , Calcio/metabolismo , Línea Celular , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Humanos , Cinética , Modelos Biológicos , Mutagénesis , Unión Proteica , Receptores de Vasopresinas/química , Transducción de Señal , Factores de Tiempo , Transfección , Vasopresinas/farmacología
19.
FEBS Lett ; 447(2-3): 155-9, 1999 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-10214937

RESUMEN

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS-7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193-197) region), selected because of its hydropathic complementarity with the common C-terminal extremity of tachykinins, abolish both the high-affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6-Pro9] SP 6-11). These results suggest that the (193-197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high-affinity binding domain for both NKA and septide, distinct from the SP binding site.


Asunto(s)
Neuroquinina A/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Neuroquinina-1/química , Receptores de Neuroquinina-1/metabolismo , Sustancia P/análogos & derivados , Sustancia P/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Células COS , Chlorocebus aethiops , ADN Complementario/genética , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Mutación Puntual , Ácido Pirrolidona Carboxílico/análogos & derivados , Receptores de Neuroquinina-1/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Taquicininas/agonistas
20.
FEBS Lett ; 536(1-3): 61-5, 2003 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-12586339

RESUMEN

Doppel protein has been discovered in prnp knock-out mouse lines, with overproduction of this protein in the brain causing ataxia and neurodegeneration. We investigated whether Doppel expression (i) affected or was affected by the course of prion propagation in neuroblastoma cells, or (ii) modulated Creutzfeldt-Jakob disease pathogenesis. No change in Doppel production was detected in N2a cells, before or after infection. Transient murine Doppel gene expression had no effect on N2a viability or PrP(Sc) production. A sensitive immunometric assay revealed low levels of Doppel in human brain, reflecting weak transcription of the corresponding gene. No difference in brain Doppel levels was observed between Creutzfeldt-Jakob disease patients and controls, adding further evidence that Doppel is unlikely to be involved in prion disease pathogenesis.


Asunto(s)
Encéfalo/metabolismo , Síndrome de Creutzfeldt-Jakob/metabolismo , Priones/metabolismo , Animales , Síndrome de Creutzfeldt-Jakob/genética , Femenino , Proteínas Ligadas a GPI , Humanos , Masculino , Ratones , Neuronas/metabolismo , Priones/genética , ARN Mensajero/biosíntesis , Scrapie/metabolismo , Transcripción Genética , Células Tumorales Cultivadas
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