RESUMEN

The interaction of Plasmodium falciparum-infected red blood cells (iRBCs) with the vascular endothelium plays a crucial role in malaria pathology and disease. KAHRP is an exported P. falciparum protein involved in iRBC remodelling, which is essential for the formation of protrusions or "knobs" on the iRBC surface. These knobs and the proteins that are concentrated within them allow the parasites to escape the immune response and host spleen clearance by mediating cytoadherence of the iRBC to the endothelial wall, but this also slows down blood circulation, leading in some cases to severe cerebral and placental complications. In this work, we have applied genetic and biochemical tools to identify proteins that interact with P. falciparum KAHRP using enhanced ascorbate peroxidase 2 (APEX2) proximity-dependent biotinylation and label-free shotgun proteomics. A total of 30 potential KAHRP-interacting candidates were identified, based on the assigned fragmented biotinylated ions. Several identified proteins have been previously reported to be part of the Maurer's clefts and knobs, where KAHRP resides. This study may contribute to a broader understanding of P. falciparum protein trafficking and knob architecture and shows for the first time the feasibility of using APEX2-proximity labelling in iRBCs.

Asunto(s)

Eritrocitos , Plasmodium falciparum , Proteómica , Proteínas Protozoarias , Eritrocitos/parasitología , Eritrocitos/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Humanos , Proteómica/métodos , Malaria Falciparum/parasitología , Malaria Falciparum/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Ascorbato Peroxidasas/metabolismo , Unión Proteica , Biotinilación , Endonucleasas , Péptidos , Proteínas , Enzimas Multifuncionales

RESUMEN

L-Lactate is an important bioanalyte in the food industry, biotechnology, and human healthcare. In this work, we report the development of a new L-lactate electrochemical biosensor based on the use of multiwalled carbon nanotubes non-covalently functionalized with avidin (MWCNT-Av) deposited at glassy carbon electrodes (GCEs) as anchoring sites for the bioaffinity-based immobilization of a new recombinant biotinylated lactate oxidase (bLOx) produced in Escherichia coli through in vivo biotinylation. The specific binding of MWCNT-Av to bLOx was characterized by amperometry, surface plasmon resonance (SPR), and electrochemical impedance spectroscopy (EIS). The amperometric detection of L-lactate was performed at -0.100 V, with a linear range between 100 and 700 µM, a detection limit of 33 µM, and a quantification limit of 100 µM. The proposed biosensor (GCE/MWCNT-Av/bLOx) showed a reproducibility of 6.0% and it was successfully used for determining L-lactate in food and enriched serum samples.

Asunto(s)

Avidina , Técnicas Biosensibles , Ácido Láctico , Oxigenasas de Función Mixta , Nanotubos de Carbono , Nanotubos de Carbono/química , Oxigenasas de Función Mixta/química , Avidina/química , Técnicas Electroquímicas , Resonancia por Plasmón de Superficie , Enzimas Inmovilizadas/química , Escherichia coli , Biotinilación , Electrodos , Espectroscopía Dieléctrica , Límite de Detección

RESUMEN

The characterization of protein-protein interactions (PPIs) is of high value for understanding protein function. Two strategies are popular for identification of PPIs direct from the cellular environment: affinity capture (pulldown) isolates the protein of interest with an immobilized matrix that specifically captures the target and potential partners, whereas in BioID, genetic fusion of biotin ligase facilitates proximity biotinylation, and labeled proteins are isolated with streptavidin. Whilst both methods provide valuable insights, they can reveal distinct PPIs, but the basis for these differences is less obvious. Here, we compare both methods using four different trypanosome proteins as baits: poly(A)-binding proteins PABP1 and PABP2, mRNA export receptor MEX67, and the nucleoporin NUP158. With BioID, we found that the population of candidate interacting proteins decreases with more confined bait protein localization, but the candidate population is less variable with affinity capture. BioID returned more likely false positives, in particular for proteins with less confined localization, and identified low molecular weight proteins less efficiently. Surprisingly, BioID for MEX67 identified exclusively proteins lining the inner channel of the nuclear pore complex (NPC), consistent with the function of MEX67, whereas the entire NPC was isolated by pulldown. Similarly, for NUP158, BioID returned surprisingly few PPIs within NPC outer rings that were by contrast detected with pulldown but instead returned a larger cohort of nuclear proteins. These rather significant differences highlight a clear issue with reliance on a single method to identify PPIs and suggest that BioID and affinity capture are complementary rather than alternative approaches.

Asunto(s)

Proteínas , Proteómica , Biotinilación , Poro Nuclear , Proteínas/química , Proteómica/métodos , Estreptavidina/química

RESUMEN

Broadly neutralizing antibodies against HIV-1 are rare with the 2F5 antibody being one of the most protective. Insertion of an antibody epitope into a stable and small protein scaffold overcomes many of the obstacles found to produce antibodies. However, the design leads to grafting of epitopes that may cause protein aggregation. Here, I investigated the 2F5 epitope grafted into the Top7 as the scaffold in which the resulting immunoreactive protein precipitates along the storage time, as opposed to its completely soluble biotinylated version. Molecular dynamics showed that biotinylation eliminates the intermediate state of the scaffold-epitope Top7-2F5 by switching a noncooperative to a cooperative folding. The aggregation propensity of the Top7-designed proteins is examined in light of thermodynamic cooperativity and kinetic traps along the decreasing depth of the intermediate ensemble in the free energy landscape. This protocol may predict stable and soluble scaffold-epitopes with the purpose of composing novel therapeutic and diagnostic platforms.

Asunto(s)

VIH-1 , Biotinilación , Anticuerpos ampliamente neutralizantes , Epítopos , Anticuerpos Anti-VIH/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , VIH-1/metabolismo , Agregado de Proteínas , Proteínas/metabolismo

RESUMEN

Solid-phase synthesis represents the methodological showcase for technological advances such as split-and-pool combinatorial chemistry and the automated synthesis of peptides, nucleic acids and polysaccharides. These strategies involve iterative coupling cycles that do not generate functional diversity besides that incorporated by the amino acids, nucleosides and monosaccharide building blocks. In sharp contrast, multicomponent reactions (MCRs) are traditionally used to generate both skeletal and appendage diversity in short, batchwise procedures. On-resin MCRs have traditionally been employed for the construction of heterocycle and peptidomimetic libraries, but that scenario has changed recently, and today the focus is more on the solid-phase derivatization of peptides and oligonucleotides. This review presents relevant experimental details and addresses the synthetic scope of such on-resin multicomponent protocols employed to accomplish specific biopolymer covalent modifications that are practically inviable by traditional solution-phase methodologies. Recommendations are provided to facilitate the implementation of solid-supported protocols and avoid possible pitfalls associated with the selection of the polymeric resin, the solvent and the order and amount of the reagents employed. We describe procedures comprising the multicomponent lipidation, biotinylation and labeling of both termini and the side chains, as well as the use of MCRs in the traceless on-resin synthesis of ligated and cyclic peptides. Solid-phase protocols for the assembly of α-helical and parallel ß-sheet peptides as well as hybrid peptide-peptoid and peptide-peptide nucleic acid architectures are described. Finally, the solid-supported multicomponent derivatization of DNA oligonucleotides is illustrated as part of the DNA-encoded library technology relying on MCR-derived heterocyclic compounds.

Asunto(s)

Biopolímeros/química , Técnicas Químicas Combinatorias/métodos , Técnicas de Síntesis en Fase Sólida/métodos , Aminas , Aminoácidos , Biopolímeros/biosíntesis , Biotinilación , ADN , Compuestos Heterocíclicos , Oligonucleótidos , Péptidos/síntesis química , Péptidos Cíclicos , Resinas Sintéticas/química

RESUMEN

The direct rapid immunohistochemical test (dRIT) has been recommended for laboratorial diagnosis of rabies, especially in developing countries. The absence of commercial primary antibodies, however, still represents a major limitation to its wider use in testing. We describe here the development of a biotinylated polyclonal antibody against Rabies lyssavirus (RABV) ribonucleoprotein (RNP) and its use as a primary reagent in dRIT. Anti-RNP polyclonal horse IgG was purified by ionic exchange chromatography followed by immunoaffinity column chromatography, and its affinity, diagnostic sensitivity, and specificity were evaluated. CNS samples (120) of suspected rabies cases in different animal species were tested by dRIT, with the positive (n = 14) and negative (n = 106) results confirmed by direct fluorescence antibody testing (dFAT). Comparing the results of dRIT and dFAT, we found that the biotinylated anti-RNP IgG delivered 100% diagnostic specificity and sensibility for rabies diagnosis. Our findings show that the biotinylated anti-RNP polyclonal IgG can be produced with the quality required for application in dRIT. This work represents an important step in efforts to diagnose rabies in developing countries.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Antígenos Virales/inmunología , Inmunoglobulina G/inmunología , Virus de la Rabia/inmunología , Rabia/inmunología , Ribonucleoproteínas/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Biotinilación , Encéfalo/inmunología , Encéfalo/virología , Gatos , Bovinos , Quirópteros , Perros , Técnica del Anticuerpo Fluorescente Directa/métodos , Caballos , Inmunoglobulina G/metabolismo , Inmunohistoquímica/métodos , Primates , Rabia/diagnóstico , Rabia/virología , Sensibilidad y Especificidad , Especificidad de la Especie , Porcinos

RESUMEN

BACKGROUND: The diagnosis of active Toxocara canis infections in humans is challenging. Larval stages of T. canis do not replicate in human tissues and disease may result from infection with a single T. canis larva. Recently, we developed a nanobody-based electrochemical magnetosensor assay with superior sensitivity to detect T. canis excretory-secretory (TES) antigens. Here, we evaluate the performance of the assay in children from an Ecuadorian birth cohort that followed children to five years of age. METHODS: Samples were selected based on the presence of peripheral blood eosinophilia and relative eosinophil counts. The samples were analyzed by the nanobody-based electrochemical magnetosensor assay, which utilizes a bivalent biotinylated nanobody as capturing agent on the surface of streptavidin pre-coated paramagnetic beads. Detection was performed by a different nanobody chemically labelled with horseradish peroxidase. RESULTS: Of 87 samples tested, 33 (38%) scored positive for TES antigen recognition by the electrochemical magnetosensor assay. The average concentration of TES antigen in serum was 2.1 ng/ml (SD = 1.1). The positive result in the electrochemical assay was associated with eosinophilia > 19% (P = 0.001). Parasitological data were available for 57 samples. There was no significant association between positivity by the electrochemical assay and the presence of other soil-transmitted helminth infections. CONCLUSIONS: Our nanobody-based electrochemical assay provides highly sensitive quantification of TES antigens in serum and has potential as a valuable tool for the diagnosis of active human toxocariasis.

Asunto(s)

Antígenos Helmínticos/sangre , Técnicas Electroquímicas/métodos , Eosinofilia/parasitología , Proteínas del Helminto/sangre , Anticuerpos de Dominio Único/inmunología , Toxocariasis/diagnóstico , Animales , Biotinilación , Camelidae , Preescolar , Ecuador/epidemiología , Eosinofilia/epidemiología , Humanos , Separación Inmunomagnética , Lactante , Población Rural , Toxocara canis , Toxocariasis/epidemiología

RESUMEN

Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi, which is transmitted by insects of the family Reduviidae. Since conventional treatments with nitroheterocyclic drugs show serious adverse reactions and have questionable efficiency, different research groups have investigated polypeptide-based approaches to interfere with the parasite cell cycle in other Trypanosomatids. These strategies are supported by the fact that surface players are candidates to develop surface ligands that impair function since they may act as virulence factors. In this study, we used a phage display approach to identify peptides from one library-LX8CX8 (17 aa) (where X corresponds to any amino acid). After testing different biopanning conditions using live or fixed epimastigotes, 10 clones were sequenced that encoded the same peptide, named here as EPI18. The bacteriophage expressing EPI18 binds to epimastigotes from distinct strains of T. cruzi. To confirm these results, this peptide was synthetized, biotinylated, and assayed using flow cytometry and confocal microscopy analyses. These assays confirmed the specificity of the binding capacity of EPI18 toward epimastigote surfaces. Our findings suggest that EPI18 may have potential biotechnological applications that include peptide-based strategies to control parasite transmission.

Asunto(s)

Enfermedad de Chagas/tratamiento farmacológico , Péptidos/farmacología , Trypanosoma cruzi/efectos de los fármacos , Secuencia de Aminoácidos , Bacteriófagos/aislamiento & purificación , Bioprospección , Biotinilación , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/prevención & control , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Microscopía Confocal , Microscopía Fluorescente , Biblioteca de Péptidos , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Temperatura , Trypanosoma cruzi/genética

RESUMEN

We are reporting an original supramolecular architecture based on a rationally designed new nanohybrid with enhanced peroxidase-like activity and site-specific biorecognition properties using avidin-functionalized multi-walled carbon nanotubes (MWCNTs-Av) and Ru nanoparticles (RuNPs). The nanohybrid-electrochemical interface was obtained by drop-coating of MWCNTs-Av dispersion at glassy carbon electrodes (GCE) followed by solvent evaporation and further electrodeposition of RuNPs (50 ppm RuCl2 for 15 s at -0.600 V). The simultaneous presence of MWCNTs and RuNPs produces a synergic effect on the non-enzymatic catatalytic reduction of H2O2 and allows the quantification of H2O2 in a wide linear range (from 5.0 × 10-7 M to 1.75 × 10-3 M) with a low limit of detection (65 nM). The avidin residues present in MWCNTs-Av/RuNPs hybrid nanomaterial allowed the anchoring by bioaffinity of biotinylated glucose oxidase (biot-GOx) as proof-of-concept of the analytical application of MWCNTs-Av platform for biosensors development. The resulting nanoarchitecture behaves as a bienzymatic-like glucose biosensor with a competitive analytical performance: linear range between 2.0 × 10-5 M and 1.23 × 10-3 M, sensitivity of (0.343 ±â€¯0.002) µA mM-1 or (2.60 ±â€¯0.02) µA mM-1 cm-2, detection limit of 3.3 µM, and reproducibility of 5.2% obtained with five different GCE/MWCNTs-Av/RuNPs/biot-GOx bioplatforms prepared the same day using the same MWCNTs-Av dispersion, and 9.1% obtained with nine biosensors prepared in different days with nine different MWCNTs-Av dispersions. The average concentrations of glucose in Gatorade®, Red bull® and Pepsi® with the biosensor demonstrated excellent agreement with those reported in the commercial beverages.

Asunto(s)

Avidina/química , Técnicas Biosensibles/métodos , Nanopartículas/química , Nanotubos de Carbono/química , Rutenio/química , Aspergillus niger/enzimología , Bebidas/análisis , Materiales Biomiméticos/química , Biotinilación , Catálisis , Técnicas Electroquímicas/métodos , Glucosa/análisis , Glucosa Oxidasa/química , Peróxido de Hidrógeno/análisis , Límite de Detección , Nanopartículas/ultraestructura , Nanotubos de Carbono/ultraestructura , Peroxidasa/química

RESUMEN

Antibodies against the HIV-1 2F5 epitope are known as one of the most powerful and broadly protective anti-HIV antibodies. Therefore, vaccine strategies that include the 2F5 epitope in their formulation require a robust method to detect specific anti-2F5 antibody production by B cells. Towards this goal, we have biotinylated a previously reported computer-designed protein carrying the HIV-1 2F5 epitope aiming the further development of a platform to detect human B-cells expressing anti-2F5 antibodies through flow cytometry. Biophysical and immunological properties of our devised protein were characterized by computer simulation and experimental methods. Biotinylation did not affect folding and improved protein stability and solubility. The biotinylated protein exhibited similar binding affinity trends compared to its unbiotinylated counterpart and was recognized by anti-HIV-1 2F5 antibodies expressed on the surface of patient-derived peripheral blood mononuclear cells. Moreover, we present a high affinity marker for the identification of epitope-specific B cells that can be used to measure the efficacy of vaccine strategies based on the HIV-1 envelope protein.

Asunto(s)

Vacunas contra el SIDA/inmunología , Linfocitos B/metabolismo , Biotinilación , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Leucocitos Mononucleares/metabolismo , Simulación de Dinámica Molecular , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Simulación por Computador , Epítopos/inmunología , Humanos

RESUMEN

Understanding the kinetics of protein interactions plays a key role in biology with significant implications for the design of analytical methods for disease monitoring and diagnosis in medical care, research and industrial applications. Herein, we introduce a novel plasmonic approach to study the binding kinetics of protein-ligand interactions following the formation of silver nanoparticles (Ag NPs) dimers by UV-Vis spectroscopy that can be used as probes for antigen detection and quantification. To illustrate and test the method, the kinetics of the prototype biotin-streptavidin (Biot-STV) pair interaction was studied. Controlled aggregates (dimers) of STV functionalized Ag NPs were produced by adding stoichiometric quantities of gliadin-specific biotinylated antibodies (IgG-Biot). The dimerization kinetics was studied in a systematic way as a function of Ag NPs size and at different concentrations of IgG-Biot. The kinetics data have shown to be consistent with a complex reaction mechanism in which only the Ag NPs attached to the IgG-Biot located in a specific STV site are able to form dimers. These results help in elucidating a complex reaction mechanism involved in the dimerization kinetics of functionalized Ag NPs, which can serve as probes in surface plasmon resonance-based bioassays for the detection and quantification of different biomarkers or analytes of interest.

Asunto(s)

Dimerización , Nanopartículas del Metal/química , Proteínas/análisis , Proteínas/metabolismo , Plata/química , Resonancia por Plasmón de Superficie/métodos , Biotina/química , Biotina/metabolismo , Biotinilación , Humanos , Ligandos , Dominios y Motivos de Interacción de Proteínas , Proteínas/química , Estreptavidina/química , Estreptavidina/metabolismo , Propiedades de Superficie

RESUMEN

The high incidence of cancer, necessity of treatment, and prognosis times are urgent issues that need to be addressed. In this work, we present DPPC liposomes coated with F127 triblock copolymers as a promising alternative in drug delivery systems for cancer therapy. The proposed mixed liposomes exhibit adequate size, high stability, and passive targeting that result from the EPR effect. An interesting strategy to obtain both passive and active targeting is the vectorization with a covalent bond between F127 and Biotin (a vitamin). Cancer cells can overexpress Biotin receptors, such as Avidin. Here, we evaluate the cytotoxic effects of the erythrosine-decyl ester (ERYDEC). This is a photosensitizer that can be utilized in photodynamic therapy (PDT) and incorporated in DPPC liposomes coated with F127 (F127/DPPC) and the biotinylated-F127 (F127-B/DPPC). The results showed that DPPC liposomes were efficiently mixed with common F127 or F127B, exhibiting adequate physical properties with simple and low-cost preparation. An HABA/Avidin assay showed the amount of Biotin available at the liposome surface. In addition, ERYDEC interaction with lipid vesicles showed high encapsulating efficiency and slow release kinetics. The ERYDEC monomeric species are represented by high light absorption and high singlet oxygen generation (1O2), which confirm the presence of the drug in its monomeric state, as required for PDT. The ERYDEC/liposome system showed high stability and absence of significant cytotoxic effects (absence of light) in fibroblasts of the Mus musculus cell line. In addition, phototoxicity studies showed that ERYDEC/liposomes were able to inhibit cancer cells. However, in the biotinylated system, the effect was much greater than the common F127 coating. This dramatically decreased the inhibitory concentration of CC50 and CC90. In addition, cellular uptake studies based on fluorescence properties of ERYDEC showed that a two-hour incubation period was enough for the uptake by the cell. Therefore, the new vectorized-coated liposome is a potential system for use in cancer treatments, considering that it is a theranostic platform.

Asunto(s)

Biotina/química , Liberación de Fármacos , Fármacos Fotosensibilizantes/farmacología , Animales , Biotinilación , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Eritrosina/farmacología , Humanos , Hidrodinámica , Liposomas , Tamaño de la Partícula , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Poloxámero/química , Espectrofotometría Ultravioleta

RESUMEN

Transient receptor potential melastatin 4 (TRPM4) is a Ca2+-activated nonselective cationic channel involved in a wide variety of physiologic and pathophysiological processes. Bioinformatics analyses of the primary sequence of TRPM4 allowed us to identify a putative motif for interaction with end-binding (EB) proteins, which are microtubule plus-end tracking proteins. Here, we provide novel data suggesting that TRPM4 interacts with EB proteins. We show that mutations of the putative EB binding motif abolish the TRPM4-EB interaction, leading to a reduced expression of the mature population of the plasma membrane channel and instead display an endoplasmic reticulum-associated distribution. Furthermore, we demonstrate that EB1 and EB2 proteins are required for TRPM4 trafficking and functional activity. Finally, we demonstrated that the expression of a soluble fragment containing the EB binding motif of TRPM4 reduces the plasma membrane expression of the channel and affects TRPM4-dependent focal adhesion disassembly and cell invasion processes.-Blanco, C., Morales, D., Mogollones, I., Vergara-Jaque, A., Vargas, C., Álvarez, A., Riquelme, D., Leiva-Salcedo, E., González, W., Morales, D., Maureira, D., Aldunate, I., Cáceres, M., Varela, D., Cerda, O. EB1- and EB2-dependent anterograde trafficking of TRPM4 regulates focal adhesion turnover and cell invasion.

Asunto(s)

Adhesiones Focales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Biotinilación/fisiología , Células COS , Adhesión Celular/genética , Adhesión Celular/fisiología , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Chlorocebus aethiops , Electrofisiología , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Proteínas Asociadas a Microtúbulos/genética , Simulación de Dinámica Molecular , Mutación/genética , Plásmidos/genética , Canales Catiónicos TRPM/genética

RESUMEN

The preparation of subproteome fractions prior to proteome analysis provides both the enrichment of proteins sub-represented in global proteome analysis and information on the cellular localization of the identified proteins. Here we describe protocols for the preparation of Trypanosoma cruzi surface and extracellular and nuclear fractions for further subproteome analysis.

Asunto(s)

Fraccionamiento Celular/métodos , Proteoma/análisis , Proteómica/métodos , Proteínas Protozoarias/análisis , Espectrometría de Masas en Tándem/métodos , Trypanosoma cruzi/química , Biotinilación , Membrana Celular/química , Núcleo Celular/química , Precipitación Química , Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Humanos , Ultracentrifugación/métodos

RESUMEN

One of the most used formats in inmuno-polymerase chain reaction (IPCR) is known as "Universal" IPCR (signal-generating complexes is based on conjugates of biotinylated DNA, biotinylated IgG and avidin). In the present study, we evaluated the utility of using mono- and bi-biotinylated DNA probes, pre-self-assembled DNA-neutravidin complex, blocking step and glutaraldehyde pretreatment of standard PCR tubes to improve the analytical performance of the hTSH-IPCR assay. The use of pre-self-assembled mono-biotinylated DNA-neutravidin complex enhances both the sensitivity and the reproducibility of the hTSH-IPCR assay, even without blocking step: hTSH-IPCR assay showed an improved limit of detection (LOD: 0.01 µIU/ml), calibration sensitivity (SEN: 2.4) and analytic sensitivity (γ: 9 µIU/ml-1) in comparison with both a self-made ELISA and a commercial one.

Asunto(s)

Reacción en Cadena de la Polimerasa/métodos , Tirotropina/análisis , Biotinilación , Sondas de ADN/química , Sondas de ADN/metabolismo , Humanos , Inmunoensayo , Límite de Detección , Reacción en Cadena de la Polimerasa/instrumentación , Reproducibilidad de los Resultados , Tirotropina/genética

RESUMEN

Vitamin C (in the reduced form ascorbate or in the oxidized form dehydroascorbate) is implicated in signaling events throughout the central nervous system (CNS). In the retina, a high-affinity transport system for ascorbate has been described and glutamatergic signaling has been reported to control ascorbate release. Here, we investigated the modulatory role played by vitamin C upon glutamate uptake and N-methyl-d-aspartate (NMDA) receptor activation in cultured retinal cells or in intact retinal tissue using biochemical and imaging techniques. We show that both forms of vitamin C, ascorbate or dehydroascorbate, promote an accumulation of extracellular glutamate by a mechanism involving the inhibition of glutamate uptake. This inhibition correlates with the finding that ascorbate promotes a decrease in cell surface levels of the neuronal glutamate transporter excitatory amino acid transporter 3 in retinal neuronal cultures. Interestingly, vitamin C is prone to increase the activity of NMDA receptors but also promotes a decrease in glutamate-stimulated [3 H] MK801 binding and decreases cell membrane content of NMDA receptor glutamate ionotropic receptor subunit 1 (GluN1) subunits. Both compounds were also able to increase cAMP response element-binding protein phosphorylation in neuronal nuclei in a glutamate receptor and calcium/calmodulin kinase-dependent manner. Moreover, the effect of ascorbate is not blocked by sulfinpyrazone and then does not depend on its uptake by retinal cells. Overall, these data indicate a novel molecular and functional target for vitamin C impacting on glutamate signaling in retinal neurons.

Asunto(s)

Ácido Ascórbico/farmacología , Glutamatos/metabolismo , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Retina/efectos de los fármacos , Retina/metabolismo , Vitaminas/farmacología , Animales , Biotinilación , Células Cultivadas , Embrión de Pollo , Pollos , Transportador 3 de Aminoácidos Excitadores/metabolismo , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

OBJECTIVE: To determine the pathogenic mechanisms of autoantibodies to the cell adhesion molecule Caspr2 in acquired neuromyotonia and autoimmune encephalitis. METHODS: Caspr2-positive samples were confirmed using a cell-based assay, and their IgG subtypes were determined by enzyme-linked immunosorbent assay and cell-based assay. A solid phase binding assay quantified the binding of Caspr2 to contactin-2 in the presence of Caspr2 autoantibodies. Living cultures of primary rat hippocampal neurons were incubated with Caspr2-positive or control sera, and the distribution of Caspr2-positive immunofluorescent puncta and total surface Caspr2 was quantified. HEK cells transfected to express Caspr2 were incubated with Caspr2-positive or control samples, and cell-surface biotinylation and Western blot were used to assess total, internalized, and surface levels of Caspr2. RESULTS: We confirmed 6 samples with strong Caspr2 reactivity. IgG4 Caspr2 antibodies were present in all 6 cases. Caspr2 interacted with another cell adhesion molecule, contactin-2, with nanomolar affinity in the solid phase assay, and Caspr2 autoantibodies inhibited this interaction. Caspr2 autoantibodies did not affect the surface expression of Caspr2 in rat primary hippocampal neurons or transfected HEK cells. INTERPRETATION: Caspr2 autoantibodies inhibit the interaction of Caspr2 with contactin-2 but do not cause internalization of Caspr2. Functional blocking of cell adhesion molecule interactions represents a potential mechanism with therapeutic implications for IgG4 autoantibodies to cell adhesion molecules in neurological diseases. Ann Neurol 2018;83:40-51.

Asunto(s)

Autoanticuerpos/inmunología , Encefalitis/inmunología , Enfermedad de Hashimoto/inmunología , Síndrome de Isaacs/inmunología , Proteínas de la Membrana/inmunología , Proteínas del Tejido Nervioso/inmunología , Animales , Biotinilación , Contactina 2/inmunología , Contactina 2/metabolismo , Femenino , Humanos , Inmunoglobulina G/análisis , Masculino , Neuronas/inmunología , Neuronas/metabolismo , Ratas

RESUMEN

In the present study, we developed both a conventional enzyme-linked immunosorbent assay (ELISA) and a highly sensitive immuno-polymerase chain reaction (IPCR) assay specific for detection of human thyroid stimulating hormone (hTSH). Several anti-hTSH monoclonal antibodies (MAbs) were generated using hybridoma technology. Two pairs of MAbs (B-4 and B-9) were rationally selected and the optimal assay conditions of sandwich ELISAs were established. The ELISA prototypes were evaluated with standards calibrated with WHO 2nd International Reference Preparation for hTSH and in comparison with a commercial ELISA Kit. Although the limit of detection (LOD) was 0.1 µIU/ml in all cases, B-9-ELISA showed an analytical performance similar to commercial ELISA Kit. Therefore, we selected the B-9 ELISA to develop a hTSH-IPCR assay applying an "Universal-IPCR" format in standard PCR tubes without pretreatment. The signal amplification was achieved through the interaction between the biotinylated detection MAb and mono-biotinylated DNA probe pre-self-assembled with neutravidin. The hTSH-IPCR assay showed a significant increase in terms of the slope definition of sensitivity in low levels range. Our results support the potential of IPCR technique for being applied in clinical diagnosis of thyroid states.

Asunto(s)

Ensayo de Inmunoadsorción Enzimática/métodos , Reacción en Cadena de la Polimerasa , Tirotropina/sangre , Anticuerpos Monoclonales/inmunología , Avidina/química , Avidina/metabolismo , Biotina/química , Biotinilación , Sondas de ADN/química , Sondas de ADN/metabolismo , Humanos , Límite de Detección , Tirotropina/inmunología

RESUMEN

Although frank symptomatic biotin deficiency is rare, some evidence suggests that marginal biotin deficiency occurs spontaneously in a substantial proportion of women during normal human pregnancy and might confer an increased risk of birth defects. Herein I review 1) advances in assessing biotin status, including the relation between acylcarnitine excretion and biotin status; 2) recent studies of biotin status in pregnancy; 3) advances in understanding the role of biotin in gene expression and the potential roles of biotinylated proteins that are neither histones nor carboxylases; and 4) novel large-dose biotin supplementation as therapy for multiple sclerosis. The review concludes with a summary of recent studies that have reported potentially dangerous erroneous results in individuals consuming large amounts of biotin for measurements of various plasma hormones for common clinical assays that use streptavidin-biotin technology.

Asunto(s)

Biotina , Expresión Génica/efectos de los fármacos , Esclerosis Múltiple/tratamiento farmacológico , Estado Nutricional , Complicaciones del Embarazo/prevención & control , Complejo Vitamínico B , Animales , Biotina/sangre , Biotina/deficiencia , Biotina/farmacología , Biotina/uso terapéutico , Biotinilación , Carnitina/análogos & derivados , Carnitina/metabolismo , Femenino , Hormonas , Humanos , Esclerosis Múltiple/sangre , Embarazo , Complicaciones del Embarazo/sangre , Estreptavidina , Complejo Vitamínico B/sangre , Complejo Vitamínico B/farmacología , Complejo Vitamínico B/uso terapéutico

RESUMEN

This chapter describes a protocol for measuring prolyl oligopeptidase (POP) activity using a biotinylated peptide substrate coupled to magnetic microspheres. The complex is incubated in the presence of a pituitary extract and activity can be detected by loss of the biotin label. The assay can be multiplexed for measuring multiple proprotein-cleaving enzymes simultaneously and can be used in analyses of neuropsychiatric diseases in which proteolytic cleavage of biologically active peptides is known to play a role.

Asunto(s)

Separación Inmunomagnética/métodos , Proteínas del Tejido Nervioso/análisis , Hipófisis/enzimología , Serina Endopeptidasas/análisis , Biotinilación , Humanos , Microesferas , Fragmentos de Péptidos/química , Prolil Oligopeptidasas , Esquizofrenia/metabolismo , Estreptavidina