RESUMEN
Intense electromagnetic fields (EMFs) induce DNA double stranded breaks (DSBs) in exposed lymphocytes.We study developing pre-B lymphocytes following V(D)J recombination at their Immunoglobulin light chain loci (IgL). Recombination physiologically induces DNA DSBs, and we tested if low doses of EMF irradiation affect this developmental stage. Recombining pre-B cells, were exposed for 48 h to low intensity EMFs (maximal radiative power density flux S of 9.5 µW/cm2 and electric field intensity 3 V/m) from waves of frequencies ranging from 720 to 1224 MHz. Irradiated pre-B cells show decreased levels of recombination, reduction which is dependent upon the power dose and most remarkably upon the frequency of the applied EMF. Although 50% recombination reduction cannot be obtained even for an S of 9.5 µW/cm2 in cells irradiated at 720 MHz, such an effect is reached in cells exposed to only 0.45 µW/cm2 power with 950 and 1000 MHz waves. A maximal four-fold recombination reduction was measured in cells exposed to 1000 MHz waves with S from 0.2 to 4.5 µW/cm2 displaying normal levels of γH2AX phosphorylated histone. Our findings show that developing B cells exposure to low intensity EMFs can affect the levels of production and diversity of their antibodies repertoire.
Asunto(s)
Campos Electromagnéticos , Células Precursoras de Linfocitos B/efectos de la radiación , Ondas de Radio , Animales , Anticuerpos/efectos de la radiación , Línea Celular , Roturas del ADN de Doble Cadena/efectos de la radiación , Ratones , Terapia por Radiofrecuencia/tendenciasRESUMEN
The effect of ionizing radiation (1 Gy) on the immunological characteristics of spleen in the mice that consumed essential oils of oregano, clove bud and the mixture of lemon oil with ginger extract at low doses with drinking water for 6 months was studied. It was found that the essential oils increased the content of antibody forming lymphocyte cells in the spleen. The maximal effect in comparison with control was found for essential oil of clove bud.
Asunto(s)
Anticuerpos/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Linfocitos/efectos de los fármacos , Bazo/efectos de los fármacos , Animales , Anticuerpos/efectos de la radiación , Zingiber officinale/química , Inmunidad Innata/efectos de la radiación , Linfocitos/efectos de la radiación , Ratones , Aceites Volátiles/administración & dosificación , Origanum/química , Aceites de Plantas/administración & dosificación , Radiación Ionizante , Bazo/efectos de la radiación , Syzygium/químicaRESUMEN
Photonic immobilization technique (PIT) has been used to develop an immunosensor for the detection of parathion. An antibody solution has been activated by breaking the disulfide bridge in the triad Trp/Cys-Cys through absorption of ultrashort UV laser pulses. The free thiol groups so produced interact with gold lamina making the antibody oriented upside, that is, with its variable parts exposed to the environment, thereby greatly increasing the detection efficiency. PIT has been applied to anchor polyclonal antiparathion antibodies to the gold electrode of a Quartz Crystal Microbalance (QCM) giving rise to very high detection sensitivity once the parathion is made heavier by complexion with BSA (bovine serum albumin), this latter step only required by the mass based transducer used in this case. The comparison of the sensor response with irradiated antibodies against different analytes shows that the high degree of antibody specificity is not affected by PIT nor is it by the complexion of parathion with BSA. These results pave the way to important applications in biosensing, since the widespread occurrence of the Trp/Cys-Cys residues triads in proteins make our procedure very general and effective to detect light analytes.
Asunto(s)
Anticuerpos/efectos de la radiación , Paratión/análisis , Plaguicidas/análisis , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Rayos Ultravioleta , Animales , Bovinos , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/química , Albúmina Sérica Bovina/efectos de la radiaciónRESUMEN
Antibody microarrays are becoming frequently used tools for analytical purposes. A key factor for optimal performance is the stability of the immobilized (capturing) antibodies as well as those that have been fluorescently labeled to achieve the immunological test (tracers). This is especially critical for long-distance transport, field testing, or planetary exploration. A number of different environmental stresses may affect the antibody integrity, such as dryness, sudden temperature shift cycles, or, as in the case of space science, exposure to large quantities of the highly penetrating gamma radiation. Here, we report on the effect of certain stabilizing solutions for long-term storage of printed antibody microarrays under different conditions. We tested the effect of gamma radiation on printed and freeze- or vacuum-dried fluorescent antibodies at working concentrations (tracer antibodies), as well as the effect of multiple cycles of sudden and prolonged temperature shifts on the stability of fluorescently labeled tracer antibody cocktails. Our results show that (i) antibody microarrays are stable at room temperature when printed on stabilizing spotting solutions for at least 6 months, (ii) lyophilized and vacuum-dried fluorescently labeled tracer antibodies are stable for more than 9 months of sudden temperature shift cycles (-20°C to 25°C and 50°C), and (iii) both printed and freeze- or vacuum-dried fluorescent tracer antibodies are stable after several-fold excess of the dose of gamma radiation expected during a mission to Mars. Although different antibodies may exhibit different susceptibilities, we conclude that, in general, antibodies are suitable for use in planetary exploration purposes if they are properly treated and stored with the use of stabilizing substances.
Asunto(s)
Anticuerpos/análisis , Colorantes Fluorescentes , Rayos gamma , Análisis por Matrices de Proteínas , Vuelo Espacial , Temperatura , Anticuerpos/efectos de la radiación , Anticuerpos Inmovilizados/análisis , Anticuerpos Inmovilizados/efectos de la radiación , Liofilización , SolucionesRESUMEN
Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Biochips have been proposed as a way to detect organic matter on extraterrestrial objects. A biochip is a miniaturized device composed of biologically sensitive systems, such as antibodies, which are immobilized on a slide. In the case of in situ measurements, the main concern is to ensure the survival of the antibodies under space radiation. Our recent computing simulation of cosmic ray interactions with the martian environment shows that neutrons are one of the dominant species at soil level. Therefore, we have chosen, in a first approach, to study antibody resistance to neutrons by performing irradiation experiments at the Applications Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine (AIFIRA) platform, a French ion beam facility at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in Bordeaux. Antibodies and fluorescent dyes, freeze-dried and in buffer solution, were irradiated with 0.6 MeV and 6 MeV neutrons. Sample analyses demonstrated that, in the conditions tested, antibody recognition capability and fluorescence dye intensity are not affected by the neutrons.
Asunto(s)
Anticuerpos/efectos de la radiación , Colorantes/efectos de la radiación , Radiación Cósmica , Exobiología/métodos , Fluoresceína/efectos de la radiación , Neutrones , Sitios de Unión , Tampones (Química) , Simulación por Computador , Liofilización , Soluciones , Análisis Espectral , Volatilización/efectos de la radiaciónRESUMEN
In an effort to identify proteomic changes that may be useful for radiation biodosimetry, human cells of hematological origin were treated with ionizing radiation or mock-irradiated and then harvested at different times after treatment. Protein lysates were generated from these cells and evaluated by Western blotting using a panel of 301 commercially available antibodies targeting 161 unique proteins. From this screen, we identified 55 ionizing radiation-responsive proteins, including 14 proteins not previously reported to be radiation-responsive at the protein level. The data from this large-scale screen have been assembled into a public website ( http://labs.fhcrc.org/paulovich/biodose_index.html ) that may be of value to the radiation community both as a source of putative biomarkers for biodosimetry and also as a source of validation data on commercially available antibodies that detect radiation-responsive proteins. Using a panel of candidate radiation biomarkers in human cell lines, we demonstrate the feasibility of assembling a complementary panel of radiation-responsive proteins. Furthermore, we demonstrate the feasibility of using blood cell-based proteomic changes for biodosimetry by demonstrating detection of protein changes in circulating cells after total-body irradiation in a canine model.
Asunto(s)
Anticuerpos/efectos de la radiación , Proteoma/efectos de la radiación , Dosis de Radiación , Animales , Western Blotting , Línea Celular , Perros , Humanos , Fosforilación , Proyectos de InvestigaciónRESUMEN
Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/efectos de la radiación , Rayos Láser , Fotoblanqueo , Anticuerpos/química , Anticuerpos/efectos de la radiación , Reacciones Antígeno-Anticuerpo , FotonesRESUMEN
Here we report new photoactivable antibody binding proteins, which site-selectively capture antibodies and form covalent conjugates with captured antibodies upon irradiation. The proteins allow the site-selective tagging and/or immobilization of antibodies with a highly preferred orientation and omit the need for prior antibody modifications. The minimal Fc-binding domain of protein G, a widely used antibody binding protein, was genetically and chemically engineered to contain a site-specific photo cross-linker, benzophenone. In addition, the domain was further mutated to have an enhanced Fc-targeting ability. This small engineered protein was successfully cross-linked only to the Fc region of the antibody without any nonspecific reactivity. SPR analysis indicated that antibodies can be site-selectively biotinylated through the present photoactivable protein. Furthermore, the system enabled light-induced covalent immobilization of antibodies directly on various solid surfaces, such as those of glass slides, gold chips, and small particles. Antibody coupling via photoactivable antibody binding proteins overcomes several limitations of conventional approaches, such as random chemical reactions or reversible protein binding, and offers a versatile tool for the field of immunosensors.
Asunto(s)
Anticuerpos/química , Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Inmunoensayo/métodos , Fragmentos Fc de Inmunoglobulinas/química , Secuencia de Aminoácidos , Anticuerpos/efectos de la radiación , Proteínas Bacterianas/efectos de la radiación , Benzofenonas/síntesis química , Benzofenonas/química , Sitios de Unión , Biotina/metabolismo , Fragmentos Fc de Inmunoglobulinas/efectos de la radiación , Datos de Secuencia Molecular , Resonancia por Plasmón de SuperficieRESUMEN
Rapidly accumulating evidence indicates that inflammatory T cells sensitively respond to their redox environment by activating signal transduction pathways. The hypothesis that T-cell receptors have the potential to catalytically transform singlet oxygen into H(2)O(2) attracted our attention since the biophysical regulation of this process would provide a new tool for therapeutically directing T cells down a preferred signaling pathway. Light-dependent production of H(2)O(2) was first described in antibodies, and we reproduced these findings. Using a real-time H(2)O(2) sensor we extended them by showing that the reaction proceeds in a biphasic way with a short-lived phase that is fast compared to the slow second phase of the reaction. We then showed that Jurkat T cells biophotonically produce about 30nM H(2)O(2)/min/mg protein when pretreated with NaN(3). This activity was concentrated 4 to 5 times in T-cell membrane preparations. The implications of these observations for the development of new therapeutic tools for inflammatory diseases are discussed.
Asunto(s)
Anticuerpos/química , Membrana Celular/metabolismo , Membrana Celular/efectos de la radiación , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/metabolismo , Linfocitos T/metabolismo , Linfocitos T/efectos de la radiación , Anticuerpos/efectos de la radiación , Células Cultivadas , Relación Dosis-Respuesta en la Radiación , Humanos , Peróxido de Hidrógeno/efectos de la radiación , Células Jurkat , Luz , Dosis de RadiaciónAsunto(s)
Anticuerpos/efectos de la radiación , Especificidad de Anticuerpos/efectos de la radiación , Western Blotting/métodos , Microondas , Ácido Nitrilotriacético/análogos & derivados , Proteínas/efectos de la radiación , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Compuestos Férricos/administración & dosificación , Compuestos Férricos/farmacología , Inyecciones Intraperitoneales , Riñón/química , Riñón/efectos de los fármacos , Ácido Nitrilotriacético/administración & dosificación , Ácido Nitrilotriacético/farmacología , Proteínas/inmunología , Ratas , Ratas Wistar , Organismos Libres de Patógenos EspecíficosRESUMEN
A modified western blotting protocol was developed to increase the binding specificity of antigens and antibodies, using intermittent microwave irradiation (IMWI) with seven antibodies and two cell lines. The method was based on IMWI of the blotting membrane in the immunoblotting step using 5% skim milk as the diluting buffer. For some antibodies against p53, CDK4 and cyclinE, there were no distinct differences between the IMWI(+) and IMWI(-) counterparts; but improvement over the standard protocol was noted in both. For some antibodies, such as the polyclonal antibody against tubulin and the monoclonal antibodies against beta-tubulin, cyclinA and cyclinB1 (which were otherwise difficult to obtain good results with), IMWI was extremely effective, resulting in clear, specifically binding bands and a clean background. Moreover, the times were reduced from 8 to 3 h. Both the IMWI(+) and IMWI(-) protocols can be applied as simple, rapid and highly specific detection techniques for applications with various antigens, reducing background 'noise' to a minimum.
Asunto(s)
Anticuerpos/efectos de la radiación , Especificidad de Anticuerpos/efectos de la radiación , Western Blotting/métodos , Microondas , Proteínas/efectos de la radiación , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Tampones (Química) , Bovinos , Humanos , Leche/química , Proteínas/inmunología , Células Tumorales CultivadasRESUMEN
To study the effect of microwaves on immunolabeling, ultrathin cryosections or diluted antibodies were irradiated prior to antibody application, and gold labeling was quantified. In addition, affinity purified, polyclonal antibodies and protein A-gold were applied to ultrathin cryosections of aldehyde-fixed material in the presence and absence of microwaves. Amylase, a soluble protein secreted by pancreatic acinar cells, MHC class II, an integral membrane protein, and 3-(2,4-dinitroanilino)-3-amino-N-methyldipropylamine (DAMP), an exogenously added antigen, were localized with monospecific antibodies. Each was chosen for their contrasting subcellular characteristics. Results demonstrated that for some antigens, antibody labeling efficiency was quantitatively improved by microwave irradiation of sections prior to antibody application. Irradiation of diluted antibodies prior to their application also resulted in improved labeling. In contrast, the results obtained using rapid immunolabeling protocols in the presence of microwaves resulted in labeling levels similar to those obtained in the absence of microwaves. We conclude that microwave irradiation can improve the labeling efficiency of some antibodies. However, improvements in labeling density are dependent on the antigen under study and on variable irradiation times, unique to each antibody. This suggests that the routine use of microwaves to reduce incubation times may not be a viable alternative to bench protocols.
Asunto(s)
Aumento de la Imagen , Inmunohistoquímica/métodos , Amilasas/análisis , Animales , Anticuerpos/efectos de la radiación , Línea Celular , Crioultramicrotomía/métodos , Dinitrobencenos/análisis , Humanos , Complejo Mayor de Histocompatibilidad/inmunología , Masculino , Proteínas de la Membrana/análisis , Ratones , Microondas , Páncreas/química , Conejos , Ratas , Ratas Sprague-DawleyRESUMEN
El objetivo del presente trabajò fuè evaluar la presencia de anticuerpos antiplaquetarios de tipo IgG e IgM en pacientes con enfermedades trombocitopenicas. Fueron estudiados 31 pacientes con el diagnòstico clìnico de purpura trombocitopenica, 23 niños y 8 adultos, provenientes del hospital de clìnicas. Se determinò la presencia de anticuerpos antiplaquetarios de tipo IgG e IGM por el mètodo de Inmunofluorescencia indirecta. Del total de pacientes estudiados, 87 por ciento de los pacientes presentaron anticuerpos antiplaquetarios. De los cuales 36 por ciento fueron anticuerpos de tipo IgG positivo, 1,6 por ciento anticuerpos de tipo IgM positivo y 35 por ciento presentaron ambos anticuerpos, IgG e IgM. este estudio demuestra que el test de Inmunofluorescencia es un mètodo ùtil para la detecciòn de anticuerpos antiplaquetarios que se deberìa utilizar de rutina para el diagnòstico de las trombocitopenias.
Asunto(s)
Anticuerpos/efectos de la radiación , Anticuerpos/inmunología , Púrpura Trombocitopénica/diagnóstico , Púrpura Trombocitopénica/enfermería , Púrpura Trombocitopénica/inmunología , Pruebas Inmunológicas , Técnica del Anticuerpo Fluorescente Directa/enfermeríaRESUMEN
El objetivo del presente trabajò fuè evaluar la presencia de anticuerpos antiplaquetarios de tipo IgG e IgM en pacientes con enfermedades trombocitopenicas. Fueron estudiados 31 pacientes con el diagnòstico clìnico de purpura trombocitopenica, 23 niños y 8 adultos, provenientes del hospital de clìnicas. Se determinò la presencia de anticuerpos antiplaquetarios de tipo IgG e IGM por el mètodo de Inmunofluorescencia indirecta. Del total de pacientes estudiados, 87 por ciento de los pacientes presentaron anticuerpos antiplaquetarios. De los cuales 36 por ciento fueron anticuerpos de tipo IgG positivo, 1,6 por ciento anticuerpos de tipo IgM positivo y 35 por ciento presentaron ambos anticuerpos, IgG e IgM. este estudio demuestra que el test de Inmunofluorescencia es un mètodo ùtil para la detecciòn de anticuerpos antiplaquetarios que se deberìa utilizar de rutina para el diagnòstico de las trombocitopenias
Asunto(s)
Anticuerpos/inmunología , Anticuerpos/efectos de la radiación , Púrpura Trombocitopénica/diagnóstico , Púrpura Trombocitopénica/enfermería , Púrpura Trombocitopénica/inmunología , Técnica del Anticuerpo Fluorescente Directa/enfermería , Pruebas InmunológicasRESUMEN
The ability to activate biological macromolecules remotely, at specific locations and times, will allow the manipulation of a wide range of cellular activities and give rise to many practical applications. Interest has been shown in the theoretical possibility of accomplishing this by means of photochemical approaches. Photochemical changes of the guest-binding cavity of cyclodextrins has been suggested; however, these changes require organic solvent. What is needed is a widely and readily applicable method allowing activation under physiological conditions. We have developed such a method. This is based on our demonstration that relatively large amounts of the a-methyl substituted 2-nitrobenzyl alcohol, namely, 1-(2-nitrophenyl)ethanol (NPE) can be coupled to proteins using diphosgene. Previous work involved "caging" of small molecules such as ATP (ref. 5-9) and blocking amino acids in peptide synthesis with 2-nitrobenzyl compounds. For large molecules, site-specific reversible inactivation of T4-lysozyme has been reported following introduction of an aspartyl beta-nibenzyl ester into its active site by mutagenesis. In contrast, the present simple procedure allows an existing protein to be deactivated and then, when and where required, reactivated by exposure to ultraviolet-A (UV-A) light. We have employed antibodies as models for both receptors and ligands and have successfully modulated: antibody binding sites for antigen; antigen binding sites for antibody, and antibody Fc binding sites for Protein A.
Asunto(s)
Anticuerpos/efectos de la radiación , Luz , Anticuerpos/inmunología , Afinidad de Anticuerpos , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Proteína Estafilocócica A/inmunologíaRESUMEN
Coupling a fluorochrome (e.g. fluorescein-isothiocyanate, FITC) to a molecule can enhance specific laser light absorption, thus leading to alteration or even destruction of the molecule itself. Therefore antibodies were labelled with FITC (absorption maximum 480 nm) and irradiated with laser light (488 nm) under various conditions. Inactivation of antibodies could only be achieved at the absorption maximum of FITC (as measured by direct and indirect immunofluorescence). Positive linear correlation exists between the amount of destruction and both exposure time and energy. Similar destructive effects were obtained when FITC-labelled peroxidase was irradiated. In these cases, enzyme activities measured by absorption photometry also showed a positive correlation to the total amount of energy transferred. Non-labelled proteins were not affected by irradiation. So we conclude that labelling of proteins with fluorochromes provides a highly specific means of selection of target molecules to be destroyed or inactivated. The method is based upon the laws of linear optics and is different from photodynamic or photochemical actions. The destructions observed are most likely caused by thermally induced changes of the molecules' tertiary structure.
Asunto(s)
Fluoresceína-5-Isotiocianato , Rayos Láser , Proteínas/efectos de la radiación , Anticuerpos/efectos de la radiación , Peroxidasa de Rábano Silvestre/efectos de la radiación , HumanosRESUMEN
UV irradiation of donor rhesus-positive blood in apparatus, applied in Soviet hospitals for autotransfusion of UV-irradiated blood produces a 2-fold increase of the blood capacity to bind antirhesus antibodies in blood or serum from sensibilized women. The above data can be used for increase in therapeutic effect of blood exchange transfusion in children with rhesus-conflict hemolytic disease.