RESUMEN
Aminocarboxylic acid (monoamine-based) chelating agents such as GLDA, MGDA, NTA, and EDG are widely used in a variety of products and processes. In the European Union, based on the Green Deal and the Chemicals Strategy for Sustainability (CSS), there is an increasing tendency to speed up chemical hazard evaluation and to regulate chemicals by grouping substances based on molecular structure similarity. Recently, it was proposed to group polycarboxylic acid monoamines, hydroxy derivatives and their salts with monovalent cations, and to consider all group members as potential carcinogens based on the official CLP classification of one group member, viz. NTA, which is classified as suspected carcinogen Cat. 2. In this review, we show that a grouping approach for harmonized classification and labeling based on molecular structure alone, disregarding existing animal test data as well as current scientific and regulatory knowledge, would result in incorrect classification. Using such a simplistic, although considered pragmatic approach, classification of all group members upfront would not improve protection of human health. Instead, it could not only lead to unnecessary additional vertebrate animal testing but also to onerous and disproportionate restrictions being placed on the use of these valuable substances; some of these even being considered as green chemicals.
Asunto(s)
Carcinógenos , Quelantes , Animales , Humanos , Aminas , Medición de RiesgoRESUMEN
Aminocarboxylic acid (ethylenediamine-based) chelating agents such as DTPA are widely used in a variety of products and processes. Recently, DTPA was classified in the European Union as a developmental toxicant CLP Category 1B. However, according to the CLP regulation (CLP, 2008) classification as a developmental toxicant requires a chemical to possess an intrinsic, specific property to do so. This paper provides overwhelming evidence that shows the developmental toxicity only seen at a sustained high dose of 1000 mg DTPA/kg bw/day in rats during pregnancy is mediated by zinc depletion which leads to non-specific secondary effects associated with zinc deficiency. Therefore, based on the CLP regulation itself, viz. the lack of a specific, intrinsic property, supported by significant differences in zinc kinetics and physiology between pregnant rats and pregnant women, DTPA should not be classified as a developmental toxicant. Moreover, classification for developmental toxicity resulting from zinc deficiency, and only observed at high doses, would not increase protection of human health; instead, it will only lead to onerous and disproportionate restrictions being placed on the use of this substance.
Asunto(s)
Quelantes , Zinc , Femenino , Ratas , Humanos , Embarazo , Animales , Quelantes/toxicidad , Zinc/toxicidad , Ácido Pentético/toxicidadRESUMEN
Over the last decade, research into methodologies to identify skin sensitization hazards has led to the adoption of several non-animal methods as OECD test guidelines. However, predictive accuracy beyond the chemical domains of the individual validation studies remains largely untested. In the present study, skin sensitization test results from in vitro and in chemico methods for 12 plant extracts and 15 polymeric materials are reported and compared to available in vivo skin sensitization data. Eight plant extracts were tested in the DPRA and h-CLAT, with the 2 out of 3 approach resulting in a balanced accuracy of 50%. The balanced accuracy for the 11 plant extracts assessed in the SENS-IS was 88%. Excluding 5 polymers inconclusive in vitro, the remainder, assessed using the 2 out of 3 approach, resulted in 63% balanced accuracy. The SENS-IS method, excluding one polymeric material due to technical inapplicability, showed 68% balanced accuracy. Although based on limited numbers, the results presented here indicate that some substance subgroups may not be in the applicability domains of the method used and careful analysis is required before positive or negative results can be accepted.
Asunto(s)
Dermatitis Alérgica por Contacto , Animales , Alternativas a las Pruebas en Animales/métodos , Polímeros/toxicidad , PielRESUMEN
Aminocarboxylic acid (ethylenediamine-based) chelating agents, such as DTPA and EDTA, are widely used in a variety of products and processes. Recently the European RAC proposed to classify DTPA as a developmental toxicant Category 1B according to CLP. This paper provides unequivocal and significant evidence that developmental effects cannot be considered an intrinsic property of the chelating substances themselves since: (1) animals fed a zinc deficient diet during gestation exhibit developmental toxicity of a similar nature and severity to that observed in studies involving such chelates, (2) sufficient supplementation of zinc in the diet, or administration of zinc bound chelates, completely negates the developmental effects. Moreover, the bioavailability of DTPA is very low with >95% of oral doses excreted unchanged via the feces within 24â¯h. If DTPA would possess the intrinsic property to be developmentally toxic, simple zinc supplementation should not be sufficient to negate these effects. Furthermore, the relevance of classification is highly questionable since worker or consumer exposure could not lead to a scenario whereby sufficient zinc deficiency would manifest itself. Therefore classification of DTPA for such effects is not protective of human health; instead it leads to onerous and disproportionate restrictions being placed on this substance.
Asunto(s)
Quelantes/toxicidad , Ácido Pentético/toxicidad , Animales , Quelantes/administración & dosificación , Humanos , Ácido Pentético/administración & dosificación , Ácido Pentético/antagonistas & inhibidores , Zinc/farmacologíaRESUMEN
The IL-4-inducing principle from Schistosoma mansoni eggs (IPSE/α-1), the major secretory product of eggs from the parasitic worm S. mansoni, efficiently triggers basophils to release the immunomodulatory key cytokine interleukin-4. Activation by IPSE/α-1 requires the presence of IgE on the basophils, but the detailed molecular mechanism underlying activation is unknown. NMR and crystallographic analysis of IPSEΔNLS, a monomeric IPSE/α-1 mutant, revealed that IPSE/α-1 is a new member of the ßγ-crystallin superfamily. We demonstrate that this molecule is a general immunoglobulin-binding factor with highest affinity for IgE. NMR binding studies of IPSEΔNLS with the 180-kDa molecule IgE identified a large positively charged binding surface that includes a flexible loop, which is unique to the IPSE/α-1 crystallin fold. Mutational analysis of amino acids in the binding interface showed that residues contributing to IgE binding are important for IgE-dependent activation of basophils. As IPSE/α-1 is unable to cross-link IgE, we propose that this molecule, by taking advantage of its unique IgE-binding crystallin fold, activates basophils by a novel, cross-linking-independent mechanism.
Asunto(s)
Antígenos Helmínticos/metabolismo , Basófilos/metabolismo , Cristalinas/inmunología , Proteínas del Huevo/metabolismo , Proteínas del Helminto/metabolismo , Inmunoglobulina E/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Helmínticos/química , Antígenos Helmínticos/genética , Sitios de Unión/genética , Western Blotting , Cromatografía en Gel , Cristalinas/genética , Cristalinas/metabolismo , Cristalografía por Rayos X , Proteínas del Huevo/química , Proteínas del Huevo/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Inmunoglobulina E/química , Interleucina-4/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.
Asunto(s)
Anticuerpos/química , Reactivos de Enlaces Cruzados/química , Técnicas de Sonda Molecular , Ácido 2,4-Diclorofenoxiacético/inmunología , 2,4-Dinitrofenol/inmunología , Afinidad de Anticuerpos , Haptenos , Límite de DetecciónRESUMEN
Intestinal edema remains a serious clinical problem, and novel approaches to study its pathophysiology are needed. It was our aim to develop a long-term stable isolated perfused rat small bowel preparation permitting analysis of vascular, luminal, interstitial, and lymphatic compartments and to demonstrate the utility of this model by studying the effects of the proinflammatory mediator platelet-activating factor (PAF). A temperature-controlled chamber with an integrated balance was designed to perfuse isolated intestines through the mesenteric artery and the gut lumen. Steroids or oxygen carriers were not needed. Functional and morphological integrity of the tissue was preserved for several hours as confirmed by oxygen consumption, venous lactate-to-pyruvate ratio, arterial and venous pH, lactose digestion and galactose uptake, intravascular and luminal pressures, maintained fluid homeostasis, gut motility, and quantitative light microscopic analysis. Administration of PAF caused typical effects such as vasoconstriction, gut atony, and loss of galactose uptake. PAF also elicited a transient loss of 20% of the perfusate liquid from the mesenteric vascular bed, two-thirds of which were transferred to the lumen. All these responses were entirely reversible. This new model provides detailed insights into the physiology of the small intestine and will allow to study fundamental processes such as fluid homeostasis, barrier functions, transport mechanisms, and immune responses in this organ. Using this model, here we show a dramatic and yet reversible response of the rat small bowel to PAF, suggesting luminal water clearance as a novel safety factor in the intestine that may be of clinical relevance.
Asunto(s)
Intestino Delgado/fisiología , Técnicas de Cultivo de Órganos/métodos , Equilibrio Hidroelectrolítico/fisiología , Animales , Femenino , Intestino Delgado/irrigación sanguínea , Músculo Liso/fisiología , Técnicas de Cultivo de Órganos/normas , Perfusión , Peristaltismo/fisiología , Factor de Activación Plaquetaria/farmacología , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiologíaRESUMEN
The gastrointestinal tract is a hostile biological environment, yet not all ingested materials are destroyed. The minute differences that determine whether a substance persists or is digested, liberated, adsorbed, excreted, or taken up are still poorly understood. Most attempts to investigate the events occurring during an orogastrointestinal passage rely on simplified in vitro systems where an analyte is exposed to artificial intestinal fluids. To closely mimic the events in the gastrointestinal tract, the exact intestinal fluid composition and the in vivo concentration of its constituents must be known. The widely used lavage procedures, however, dilute the intestinal fluids to an extent that precludes recalculation to the original concentrations. Thus, we developed procedures with which undiluted murine intestinal fluid can be harvested; determined the in vivo concentrations of the digestive enzymes trypsin, chymotrypsin, and elastase and the adsorbents mucin and immunoglobulin A in small intestinal fluid of fasted and unfasted female Balb/c mice; and identified chymotrypsin and immunoglobulin A as valid endogenous dilution markers for the recalculation of aqueous lavages. With these technologies and information at hand, more reliable investigations on the fate of allergens, pathogens, food, and anthropogenic xenobiotics in the gastrointestinal tract will be possible.
Asunto(s)
Bioquímica/métodos , Líquidos Corporales/metabolismo , Intestino Delgado/metabolismo , Proteínas/metabolismo , Animales , Digestión , Ayuno/metabolismo , Femenino , Inmunoglobulina A/metabolismo , Intestino Delgado/citología , Ratones , Ratones Endogámicos BALB C , Mucinas/metabolismo , Péptido Hidrolasas/metabolismo , Irrigación TerapéuticaRESUMEN
All solid-phase immunoassay techniques depend on so-called blocking reagents to suppress the background that is caused by unwanted adhesion of assay system components to the solid support. Commonly used blocking reagents based on biological materials bear severe inherent drawbacks such as heterogeneity and cross-reactivity, while synthetic alternatives often show insufficient background prevention. In this study, polyethylene glycol-conjugated alkylamines were synthesized via a versatile building block approach and were studied as novel blocking reagent candidates in immunoassays. The newly developed substances outperformed commonly used blocking reagents in two different ELISA setups, enabling both, excellent prevention of non-specific binding and particularly high assay sensitivity. This class of surfactants therefore may contribute significantly to the field of assay technology.
Asunto(s)
Aminas/química , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoensayo/métodos , Polietilenglicoles/química , Tensoactivos/química , HumanosRESUMEN
Many bioanalytic and diagnostic procedures rely on labels with which the molecule of interest can be tracked in or discriminated from accompanying like substances. Herein, we describe a new labeling and detection system based on derivatives of 2,4-dichlorophenoxyacetic acid (2,4-D) and anti-2,4-D antibodies. The 2,4-D system is highly sensitive with a K(D) of 7 x 10(-11) M for the hapten-antibody pair, can be used on a large variety of biomolecules such as proteins, peptides, carbohydrates, and nucleic acids, is not hampered by endogenous backgrounds because 2,4-D is a xenobiotic, and is robust because 2,4-D is a very stable compound that withstands the conditions of most reactions usually performed on biomolecules. With this unique blend of properties, the 2,4-D system compares favorably with its rivals digoxigenin (DIG)/anti-DIG and biotin/(strept)avidin and provides an interesting and powerful tool in biomolecular labeling.
Asunto(s)
Ácido 2,4-Diclorofenoxiacético/análogos & derivados , Ácido 2,4-Diclorofenoxiacético/metabolismo , Inmunoensayo/métodos , Coloración y Etiquetado/métodos , Ácido 2,4-Diclorofenoxiacético/análisis , Ácido 2,4-Diclorofenoxiacético/inmunología , Aminas/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , ADN/metabolismo , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Ambiente , Límite de Detección , Ratones , Polietilenglicoles/metabolismo , Proteínas/química , Proteínas/metabolismoRESUMEN
The notorious degradation susceptibility of peptides is a major obstacle to their use as medicinal drugs. Assays with which the stability of peptides in complex proteolytic environments can be determined are thus indispensable for peptide drug development. Herein, we describe a new peptide proteolysis assay that meets that demand. It unites the high-throughput capacity of heterogeneous with the well-defined kinetic characteristics of homogeneous assay formats and operates on the cleavage-caused loss of a detection handle. We have confirmed the assay's accuracy with well-defined model interactions and proved its high versatility and robustness with a representative application where we determined the half-lives of 375 different peptides in a crude intestinal protease preparation. With this reliable, reproducible, and efficient assay the enzyme kinetics of any peptide-protease interaction is accessible even for complex protease solutions.
Asunto(s)
Péptidos/química , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Ensayo de Inmunoadsorción Enzimática , Semivida , Intestino Delgado/enzimología , Cinética , Ratones , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Biblioteca de Péptidos , Péptidos/análisis , Poliestirenos/química , Estabilidad Proteica , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
Detection of cancer at an early stage is pivotal for successful treatment and long term survival, yet early diagnosis requires sensitive and specific markers that can be easily detected by screening procedures. Differences in the surface structure of tumor and healthy cells, if sufficiently pronounced and discernible, may serve that purpose. We analyzed the luminal surface of healthy and neoplastic human colorectal tissues for the presence and architecture of the glycocalyx-a dense network of highly glycosylated proteins-using transmission electron microscopy. The ultrastructural analyses showed that 93% of healthy mucosae were covered by an intact glycocalyx. Contrarily, on over 90% of the surface of neoplastic cells the glycocalyx was absent. The sensitivity and specificity of our marker "absence of a glycocalyx" are excellent, being 91% (83-96%) and 96% (89-99%) for adenocarcinomas and 94% (73-100%) and 92% (85-97%) for precancerous polyps (means and 95% confidence intervals). Using a cell culture model we could demonstrate that a particulate probe targeting a cell surface receptor usually concealed beneath the glycocalyx can bind selectively to glycocalyx-free areas of a tumor cell layer. We propose that the absence of a glycocalyx may serve as novel type of tumor marker. If the absence of the glycocalyx can be detected e.g. via binding of imaging probes to non-shielded surface receptors of anomalously differentiated cells, this tumor marker could be used to enable early diagnosis of colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/metabolismo , Detección Precoz del Cáncer , Glicocálix/metabolismo , Mucosa Intestinal/metabolismo , Anciano , Anciano de 80 o más Años , Células CACO-2 , Neoplasias Colorrectales/patología , Femenino , Humanos , Mucosa Intestinal/patología , Masculino , Persona de Mediana Edad , Receptores de Superficie Celular/metabolismoRESUMEN
Tetanus neurotoxin binds via its carboxyl-terminal H(C)-fragment selectively to neurons mediated by complex gangliosides. We investigated the lactose and sialic acid binding pockets of four recently discovered potential binding sites employing site-directed mutagenesis. Substitution of residues in the lactose binding pocket drastically decreased the binding of the H(C)-fragment to immobilized gangliosides and to rat brain synaptosomes as well as the inhibitory action of recombinant full length tetanus neurotoxin on exocytosis at peripheral nerves. The conserved motif of S(1287)XWY(1290) em leader G(1300) assisted by N1219, D1222, and H1271 within the lactose binding site comprises a typical sugar binding pocket, as also present, for example, in cholera toxin. Replacement of the main residue of the sialic acid binding site, R1226, again caused a dramatic decline in binding affinity and neurotoxicity. Since the structural integrity of the H(C)-fragment mutants was verified by circular dichroism and fluorescence spectroscopy, these data provide the first biochemical evidence that two carbohydrate interaction sites participate in the binding and uptake process of tetanus neurotoxin. The simultaneous binding of one ganglioside molecule to each of the two binding sites was demonstrated by mass spectroscopy studies, whereas ganglioside-mediated linkage of native tetanus neurotoxin molecules was ruled out by size exclusion chromatography. Hence, a subsequent displacement of one ganglioside by a glycoprotein receptor is discussed.
Asunto(s)
Gangliósidos/metabolismo , Toxina Tetánica/metabolismo , Animales , Sitios de Unión , Encéfalo/metabolismo , Dicroismo Circular , Modelos Moleculares , Conformación Proteica , Ratas , Ratas Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidad , Espectrofotometría Ultravioleta , Sinaptosomas/metabolismo , Toxina Tetánica/química , Toxina Tetánica/toxicidadAsunto(s)
Síndrome de Creutzfeldt-Jakob/prevención & control , Encefalopatía Espongiforme Bovina/prevención & control , Enfermedades por Prión/prevención & control , Vacunas , Animales , Bovinos , Síndrome de Creutzfeldt-Jakob/transmisión , Infección Hospitalaria/prevención & control , Encefalopatía Espongiforme Bovina/fisiopatología , Encefalopatía Espongiforme Bovina/transmisión , Humanos , Enfermedades por Prión/transmisión , Enfermedades por Prión/veterinaria , Priones/metabolismo , Factores de RiesgoRESUMEN
Endowing mucosal vaccines with ligands that target antigen to mucosal lymphoid tissues may improve immunization efficacy provided that the ligands withstand the proteolytic environment of the gastro-intestinal tract until they reach their destination. Our aim was to investigate whether and how three renowned ligands - Ulex europaeus agglutinin I and the B subunits of cholera toxin and E. coli heat-labile enterotoxin - master this challenge. We assessed the digestive power of natural murine intestinal fluid (natIF) using assays for trypsin, chymotrypsin and pancreatic elastase along with a test for nonspecific proteolysis. The natIF was compared with simulated murine intestinal fluid (simIF) that resembled the trypsin, chymotrypsin and elastase activities of its natural counterpart but lacked or contained albumins as additional protease substrates. The ligands were exposed to the digestive fluids and degradation was determined. The studies revealed that (i) the three pancreatic endoproteases constitute only one third of the total protease activity of natIF and (ii) the ligands resist proteolysis in natIF and protein-enriched simIF over 3 h but (iii) are partially destroyed in simIF that lacks additional protease substrate. We assume that the proteins of natIF are preferred substrates for the intestinal proteases and thus can protect vaccine-targeting ligands from destruction.
Asunto(s)
Toxinas Bacterianas/metabolismo , Toxina del Cólera/metabolismo , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Intestino Delgado/enzimología , Péptido Hidrolasas/metabolismo , Lectinas de Plantas/metabolismo , Ulex/metabolismo , Vibrio cholerae/metabolismo , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxina del Cólera/genética , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Femenino , Contenido Digestivo/enzimología , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Lectinas de Plantas/genética , Inhibidores de Proteasas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Transmissible spongiform encephalopathies are fatal neurodegenerative disorders that affect humans and certain animals and are caused by prions. In most cases, infection occurs by ingestion of prions. Their long-time persistence in the environment creates a reservoir of potentially infectious matter that renders the eradication of the disease problematic. Unfortunately, no cure is available to date. Yet, for both the treatment of infected and the protection of uninfected individuals, active and passive immunizations have been shown to have a beneficial effect on the course of the disease. The current review provides an overview of such antibody-based approaches and assesses their feasibility and potential in prophylaxis and therapy of transmissible spongiform encephalopathies.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Inmunización Pasiva , Proteínas PrPC/inmunología , Proteínas PrPSc/inmunología , Enfermedades por Prión/terapia , Vacunación , Animales , Diseño de Fármacos , Humanos , Inmunización Pasiva/tendencias , Enfermedades por Prión/inmunología , Enfermedades por Prión/prevención & control , Enfermedades por Prión/transmisión , Enfermedades por Prión/veterinaria , Vacunación/tendencias , ZoonosisRESUMEN
A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was prepared by a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligand Escherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When an epitope mapping of several ganglioside binding proteins and protein fragments was performed by screening a cellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displaying ganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile tool for manifold biochemical investigations.
Asunto(s)
Toxinas Bacterianas/química , Enterotoxinas/química , Proteínas de Escherichia coli/química , Gangliósido G(M1)/síntesis química , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Secuencias de Aminoácidos , Sitios de Unión , Escherichia coli/química , Escherichia coli/metabolismo , Fluorescencia , Gangliósido G(M1)/química , Gangliósido G(M1)/metabolismo , Ligandos , Fragmentos de Péptidos/químicaRESUMEN
To test whether prion protein (PrP) specific secretory immunoglobulin A (sIgA) can be induced and protect against oral transmission of spongiform encephalopathy (SE) we immunized Balb/c mice either intragastrically or intranasally (i.n.) with a recombinant PrP-fragment (PrP90-231) and cholera toxin (CT) adjuvant. Since PrP90-231 was rapidly digested in intestinal lavage, aprotinin was added to some vaccine formulations. While an anti-CT response was elicited via both routes, solely i.n. immunization without aprotinin induced PrP-specific sIgA. They recognize predominantly PrP-oligomers as the antigen was aggregated in the vaccine formulations. Challenge experiments showed that the immune response induced by our protocol could not prevent disease, but increases the median survival of the animals. We conclude that PrP-specific sIgA reduce the infectivity of the inoculum and that complete protection against transmission of SE should be achievable by optimized immunization regimens.
Asunto(s)
Fragmentos de Péptidos/administración & dosificación , Enfermedades por Prión/prevención & control , Priones/administración & dosificación , Vacunas Sintéticas/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Administración Intranasal , Animales , Aprotinina/administración & dosificación , Aprotinina/farmacología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/farmacología , Modelos Animales de Enfermedad , Heces , Femenino , Inmunidad Mucosa , Inmunoglobulina A Secretora/sangre , Inmunoglobulina A Secretora/inmunología , Inmunoglobulina G/análisis , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Enfermedades por Prión/inmunología , Enfermedades por Prión/transmisión , Priones/inmunología , Inhibidores de Serina Proteinasa/administración & dosificación , Inhibidores de Serina Proteinasa/farmacología , Orina , Vacunas Sintéticas/inmunologíaRESUMEN
The biological function of prion protein (PrP) and the physiological relevance of its truncated subtypes and glycoforms is still enigmatic. In this paper, we adduce evidence that recombinant murine PrP fragment 90-231 (mPrP90-231) contains a biotin-mimicking sequence motif that causes binding of the bacterial protein streptavidin to mPrP90-231. As indicated by epitope mapping and proven by analysis of a deletion mutant (mPrP101-231), streptavidin binding is primarily mediated by the amino-terminus of mPrP90-231 with the core-binding sequence represented by residues 94-100. Competition with biotin significantly reduces the interaction pointing to an involvement of streptavidin's biotin-binding site (BBS). Since the BBS of streptavidin shares similarities with the active sites of proteins involved in biotin metabolism we speculate that biotin mimicry by truncated PrP-species may have an impact in vivo.
Asunto(s)
Priones/química , Precursores de Proteínas/química , Estreptavidina/química , Secuencias de Aminoácidos , Animales , Sitios de Unión , Biotina/química , Epítopos/química , Eliminación de Gen , Indicadores y Reactivos/química , Ratones , Mutación , Proteínas Priónicas , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/químicaRESUMEN
The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).