RESUMEN
Magnetite mineralization in human tissue is associated with various pathological processes, especially neurodegenerative disorders. Ferritin's mineral core is believed to be a precursor of magnetite mineralization. Magnetoferritin (MF) was prepared with different iron loading factors (LFs) as a model system for pathological ferritin to analyze its MRI relaxivity properties compared to those of native ferritin (NF). The results revealed that MF differs statistically significantly from NF, with the same LF, for all studied relaxation parameters at 7 T: r1, r2, r2*, r2/r1, r2*/r1. Distinguishability of MF from NF may be useful in non-invasive MRI diagnosis of pathological processes associated with iron accumulation and magnetite mineralization (e.g., neurodegenerative disorders, cancer, and diseases of the heart, lung and liver). In addition, it was found that MF samples possess very strong correlation and MF's relaxivity is linearly dependent on the LF, and the transverse and longitudinal ratios r2/r1 and r2*/r1 possess complementary information. This is useful in eliminating false-positive hypointensive artefacts and diagnosis of the different stages of pathology. These findings could contribute to the exploitation of MRI techniques in the non-invasive diagnosis of iron-related pathological processes in human tissue.
Asunto(s)
Apoferritinas/análisis , Ferritinas/análisis , Hierro/análisis , Imagen por Resonancia Magnética/métodos , Óxidos/análisis , Animales , Caballos , Humanos , Hidrodinámica , Enfermedades Neurodegenerativas/diagnósticoRESUMEN
Brain iron accumulation has been found to accelerate disease progression in amyloid-ß(Aß) positive Alzheimer patients, though the mechanism is still unknown. Microglia have been identified as key players in the disease pathogenesis, and are highly reactive cells responding to aberrations such as increased iron levels. Therefore, using histological methods, multispectral immunofluorescence and an automated in-house developed microglia segmentation and analysis pipeline, we studied the occurrence of iron-accumulating microglia and the effect on its activation state in human Alzheimer brains. We identified a subset of microglia with increased expression of the iron storage protein ferritin light chain (FTL), together with increased Iba1 expression, decreased TMEM119 and P2RY12 expression. This activated microglia subset represented iron-accumulating microglia and appeared morphologically dystrophic. Multispectral immunofluorescence allowed for spatial analysis of FTL+Iba1+-microglia, which were found to be the predominant Aß-plaque infiltrating microglia. Finally, an increase of FTL+Iba1+-microglia was seen in patients with high Aß load and Tau load. These findings suggest iron to be taken up by microglia and to influence the functional phenotype of these cells, especially in conjunction with Aß.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/patología , Hierro/metabolismo , Microglía/metabolismo , Microglía/patología , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/patología , Adulto , Anciano , Anciano de 80 o más Años , Péptidos beta-Amiloides , Apoferritinas/análisis , Apoferritinas/metabolismo , Autopsia , Encéfalo/diagnóstico por imagen , Femenino , Humanos , Inmunohistoquímica , Hierro/análisis , Masculino , Fenotipo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Análisis EspacialRESUMEN
Protein-based nanoparticles have developed rapidly in areas such as drug delivery, biomedical imaging and biocatalysis. Ferritin possesses unique properties that make it attractive as a potential platform for a variety of nanobiotechnological applications. Here we synthesized magnetoferritin (P-MHFn) nanoparticles for the first time by using the human H chain of ferritin that was expressed by Pichia pastoris (P-HFn). Western blot results showed that recombinant P-HFn was successfully expressed after methanol induction. Transmission electron microscopy (TEM) showed the spherical cage-like shape and monodispersion of P-HFn. The synthesized magnetoferritin (P-MHFn) retained the properties of magnetoferritin nanoparticles synthesized using HFn expressed by E. coli (E-MHFn): superparamagnetism under ambient conditions and peroxidase-like activity. It is stable under a wider range of pH values (from 5.0 to 11.0), likely due to post-translational modifications such as N-glycosylation on P-HFn. In vivo near-infrared fluorescence imaging experiments revealed that P-MHFn nanoparticles can accumulate in tumors, which suggests that P-MHFn could be used in tumor imaging and therapy. An acute toxicity study of P-MHFn in Sprague Dawley rats showed no abnormalities at a dose up to 20 mg Fe Kg-1 body weight. Therefore, this study shed light on the development of magnetoferritin nanoparticles using therapeutic HFn expressed by Pichia pastoris for biomedical applications.
Asunto(s)
Apoferritinas/análisis , Colorantes Fluorescentes/análisis , Hierro/análisis , Nanopartículas/análisis , Imagen Óptica/métodos , Óxidos/análisis , Animales , Apoferritinas/genética , Apoferritinas/toxicidad , Apoferritinas/ultraestructura , Colorantes Fluorescentes/toxicidad , Expresión Génica , Humanos , Hierro/toxicidad , Nanopartículas/ultraestructura , Óxidos/toxicidad , Ratas Sprague-Dawley , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/toxicidad , Proteínas Recombinantes/ultraestructura , Saccharomycetales/genéticaRESUMEN
Background In hereditary hyperferritinaemia-cataract syndrome (HHCS), single nucleic acid alterations in the ferritin light chain (L-ferritin) iron response element (IRE) constitutively derepress ferritin synthesis, resulting in hyperferritinaemia, L-ferritin deposits in the lens of the eye and early bilateral cataract onset. Methods In this study, six German families with putative HHCS were analysed. Clinical diagnosis of HHCS was based on medical history, evaluation of ferritin serum levels, transferrin saturation and clinical ophthalmological examination. Diagnosis was confirmed by polymerase chain reaction (PCR)-based DNA sequencing of the L-ferritin IRE. Results Genetic analysis of the L-ferritin IRE revealed relevant single nucleic acid alterations in each of the affected families. Variants c.-168G > A, c.-168G > U and c.-167C > U were located in the C-bulge region; and variants c.-161C > U and c.-157G > A were located in the hexanucleotide loop of the L-ferritin IRE. Conclusions Family history of hyperferritinaemia and juvenile cataracts are strong indicators of HHCS. Genetic analysis of the L-ferritin IRE is a straightforward procedure to confirm the diagnosis. Accurate diagnosis of hyperferritinaemia can avoid unnecessary treatment by venesection, and focus attention on early cataract detection in offspring at risk.
Asunto(s)
Apoferritinas/genética , Catarata/congénito , Trastornos del Metabolismo del Hierro/congénito , Adulto , Anciano , Anciano de 80 o más Años , Apoferritinas/análisis , Apoferritinas/sangre , Secuencia de Bases/genética , Catarata/diagnóstico , Catarata/epidemiología , Familia , Femenino , Ferritinas/genética , Pruebas Genéticas/métodos , Alemania/epidemiología , Humanos , Hierro/metabolismo , Trastornos del Metabolismo del Hierro/diagnóstico , Trastornos del Metabolismo del Hierro/epidemiología , Masculino , Persona de Mediana Edad , Mutación/genética , LinajeRESUMEN
An iron-responsive optical probe has been developed for simultaneous sensing of both ferritin and apoferritin proteins at pH 7.4 in water. The compound showed an exclusive response (turn-off signal) towards ferritin among a wide range of proteins even at nanomolar concentration. In contrast, apoferritin dissociates the preformed iron complex and revives the green colored fluorescence of the native probe (turn-on signal). Subsequently, various parameters associated with the serum iron level are evaluated, which are beneficial for clinical diagnosis of many iron-related diseases, including anemia. Estimation of iron was achieved in a wide range of edible plant materials as well as pharmaceutical formulations. Subsequently, different kinds of natural water samples were screened for quantification of soluble iron contents. In addition to traditional spectroscopic tools, dye-coated paper strips were developed as an alternative strategy for onsite 'instrument-free' detection of iron. Highly specific bioimaging of Fe3+ was achieved in cervical cancer cells (HeLa).
Asunto(s)
Apoferritinas/análisis , Ferritinas/análisis , Colorantes Fluorescentes/química , Hierro/sangre , Agua Dulce/análisis , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Hierro/análisis , Microscopía Confocal , Teoría Cuántica , Espectrofotometría , Rayos UltravioletaRESUMEN
Inflammation and calcification concomitantly drive atherosclerotic plaque progression and rupture and are the compelling targets for identifying plaque vulnerability. However, current imaging modalities for vulnerable atherosclerotic plaques are often limited by inadequate specificity and sensitivity. Here, we show that natural H-ferritin nanocages radiolabeled with technetium-99m (99mTc-HFn) can identify and accurately localize macrophage-rich, atherosclerotic plaques in living mice using combined SPECT and CT. Focal 99mTc-HFn uptake was observed in the atherosclerotic plaques with multiple high-risk features of macrophage infiltration, active calcification, positive remodeling, and necrosis on histology and in early active ongoing lesions with intense macrophage infiltration. The uptake of 99mTc-HFn in plaques enabled quantitative measuring of the dynamic changes of inflammation during plaque progression and anti-inflammation treatment. This strategy lays the foundation of using bioengineered endogenous human ferritin nanocages for the identification of vulnerable and early active plaques as well as potential assessment of anti-inflammation therapy.
Asunto(s)
Apoferritinas/análisis , Aterosclerosis/diagnóstico por imagen , Nanopartículas/análisis , Animales , Aorta/diagnóstico por imagen , Apolipoproteínas E/deficiencia , Compuestos Azo/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Seasonal Weight Loss (SWL) is one of the most pressing issues in animal production in the tropics and Mediterranean. This work aims to characterize muscle proteome changes as a consequence of SWL in meat producing sheep, using a label-free proteomics approach. We compare three breeds: the Australian Merino (SWL susceptible), the Damara (SWL tolerant) and the Dorper (SWL intermediate tolerance). We identified 668 proteins of the sheep proteome, 95 with differential regulation. Also we observe that the more vulnerable to SWL a breed is, the more differential abundance proteins we find. Protein binding was the most frequently altered molecular function identified. We suggest 6 putative markers for restricted nutritional conditions independently of breed: ferritin heavy-chain; immunoglobulin V lambda chain; transgelin; fatty acid synthase; glutathione S-transferase A2; dihydrodiol dehydrogenase 3-like. Moreover, we suggest as related to SWL tolerance: S100-A10 Serpin A3-5-like and Catalase, subject however to necessary validation assays. The identification of SWL-tolerance related proteins using proteomics will lead to increased stock productivity of relevant interest to animal production, particularly if identified at the muscle level, the tissue of economic importance in meat production. BIOLOGICAL SIGNIFICANCE: Seasonal Weight Loss (SWL) is the most pressing issue in animal production in the tropics and the Mediterranean. To counter SWL, farmers often use animal breeds that have a natural ability to withstand pasture scarcity. Here we study the sheep muscle proteome at the muscle level, the tissue of economic importance in meat production. Furthermore, the identification of proteins that change their abundance in response to SWL using proteomics can contribute to increased stock productivity of relevant interest to animal production. We identified 668 proteins of the sheep proteome. We demonstrate that the following proteins are affected by restricted nutritional conditions: ferritin heavy chain; immunoglobulin V lambda chain; transgelin; fatty acid synthase; glutathione S-transferase A2; dihydrodiol dehydrogenase 3-like. Furthermore, S100-A10, Serpin A3-5-like and Catalase are proteins that changed their abundance in response to SWL. Nevertheless, it is important to highlight that Catalase values for the merino breed were close to significance and therefore catalase validation is of utmost importance.
Asunto(s)
Adaptación Fisiológica , Músculos/metabolismo , Proteoma/metabolismo , Ovinos/fisiología , Pérdida de Peso , Animales , Apoferritinas/análisis , Biomarcadores/análisis , Ácido Graso Sintasas/análisis , Perfilación de la Expresión Génica , Glutatión Transferasa/análisis , Región Variable de Inmunoglobulina/análisis , Isoenzimas/análisis , Proteínas de Microfilamentos/análisis , Proteínas Musculares/análisis , Proteoma/análisis , Proteómica/métodos , Estaciones del Año , Especificidad de la EspecieRESUMEN
PURPOSE: The development of glioma therapy in clinical practice (e.g., gene therapy) calls for efficiently visualizing and tracking glioma cells in vivo. Human ferritin heavy chain is a novel gene reporter in magnetic resonance imaging. This study proposes hFTH as a reporter gene for MR molecular imaging in glioma xenografts. METHODS: Rat C6 glioma cells were infected by packaged lentivirus carrying hFTH and EGFP genes and obtained by fluorescence-activated cell sorting. The iron-loaded ability was analyzed by the total iron reagent kit. Glioma nude mouse models were established subcutaneously and intracranially. Then, in vivo tumor bioluminescence was performed via the IVIS spectrum imaging system. The MR imaging analysis was analyzed on a 7T animal MRI scanner. Finally, the expression of hFTH was analyzed by western blotting and histological analysis. RESULTS: Stable glioma cells carrying hFTH and EGFP reporter genes were successfully obtained. The intracellular iron concentration was increased without impairing the cell proliferation rate. Glioma cells overexpressing hFTH showed significantly decreased signal intensity on T2-weighted MRI both in vitro and in vivo. EGFP fluorescent imaging could also be detected in the subcutaneous and intracranial glioma xenografts. Moreover, the expression of the transferritin receptor was significantly increased in glioma cells carrying the hFTH reporter gene. CONCLUSION: Our study illustrated that hFTH generated cellular MR imaging contrast efficiently in glioma via regulating the expression of transferritin receptor. This might be a useful reporter gene in cell tracking and MR molecular imaging for glioma diagnosis, gene therapy and tumor metastasis.
Asunto(s)
Apoferritinas/análisis , Apoferritinas/genética , Neoplasias Encefálicas/diagnóstico , Genes Reporteros , Glioma/diagnóstico , Imagen Molecular/métodos , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Glioma/genética , Glioma/patología , Células HEK293 , Xenoinjertos , Humanos , Imagen por Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , RatasRESUMEN
Highly blue luminescent nitrogen-doped carbon dots (N-CDs) with a fluorescence quantum yield of 42.3% were prepared by an efficient one-step pyrolytic route from ethylenediaminetetraacetic acid and urea. The as-synthesized N-CDs were demonstrated as an effective fluorescent probe for label-free, selective and sensitive recognition of Fe(3+) with a linear range of 0.5µM to 2mM and a detection limit of 13.6nM due to Fe(3+)-quenched fluorescence (turn-off). The quenched fluorescence could be turned on after the addition of apoferritin owing to the removal of ferric species from the surface of N-CDs by apoferritin, making complex N-CDs/Fe(3+) a selective apoferritin probe with a linear range of 0.1-25µM and a detection limit as low as 2.6nM. In addition, the application of this novel N-CDs-based probe for imaging Fe(3+) ions and apoferritin in living cells suggest that this sensing system has great potential applications in biosensing, bioimaging, and many other fields.
Asunto(s)
Apoferritinas/análisis , Carbono/química , Colorantes Fluorescentes/química , Hierro/análisis , Imagen Óptica/métodos , Puntos Cuánticos/química , Cationes/análisis , Línea Celular , Humanos , Límite de Detección , Espectrometría de Fluorescencia/métodosRESUMEN
The heterogeneity of atherosclerotic tissue has limited comprehension in proteomic and metabolomic analyses. To elucidate the functional implications, and differences between genders, of atherosclerotic lesion formation we investigated protein profiles from different regions of human carotid atherosclerotic arteries; internal control, fatty streak, plaque shoulder, plaque centre, and fibrous cap. Proteomic analysis was performed using 2-DE with MALDI-TOF, with validation using nLC-MS/MS. Protein mapping of 2-DE identified 52 unique proteins, including 15 previously unmapped proteins, of which 41 proteins were confirmed by nLC-MS/MS analysis. Expression levels of 18 proteins were significantly altered in plaque regions compared to the internal control region. Nine proteins showed site-specific alterations, irrespective of gender, with clear associations to extracellular matrix remodelling. Five proteins display gender-specific alterations with 2-DE, with two alterations validated by nLC-MS/MS. Gender differences in ferritin light chain and transthyretin were validated using both techniques. Validation of immunohistochemistry confirmed significantly higher levels of ferritin in plaques from male patients. Proteomic analysis of different plaque regions has reduced the effects of plaque heterogeneity, and significant differences in protein expression are determined in specific regions and between genders. These proteomes have functional implications in plaque progression and are of importance in understanding gender differences in atherosclerosis.
Asunto(s)
Estenosis Carotídea/patología , Proteoma/análisis , Anciano , Anciano de 80 o más Años , Apoferritinas/análisis , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Prealbúmina/análisis , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
In this paper, we aimed to evaluate the levels of ferritin enriched in H subunits (H-ferritin) and ferritin enriched in L subunits (L-ferritin) and the cells expressing these 2 molecules, in the bone marrow (BM) and liver biopsies obtained from adult onset Still's disease (AOSD) patients who developed macrophage activation syndrome (MAS), and correlating these data with the severity of the disease. Twenty-one patients with MAS-associated AOSD underwent BM biopsy and among them, 9 patients with hepatomegaly and elevated liver enzymes underwent liver biopsy. All the samples were stained by both immunohistochemistry and immunofluorescence. A statistical analysis was performed to estimate the possible correlation among both H-ferritin and L-ferritin tissue expression and the clinical picture of the disease. Furthermore, the same analysis was performed to evaluate the possible correlation among the number of CD68/H-ferritin or CD68/L-ferritin positive cells and the clinical picture. Both immunohistochemical and immunofluorescence analysis demonstrated an increased tissue H-ferritin expression, in the BM and liver samples of our patients. This increased expression correlated with the severity of the disease. An inflammatory infiltrate, enriched in CD68 macrophages, expressing H-ferritin was observed in both the BM and the liver samples of our patients. Furthermore, we observed, that this increased number of CD68/H-ferritin positive cells significantly correlated with the severity of clinical picture and this specific BM infiltrate correlated with the mortality rate, reported in our cohort. Our data showed an imbalance between the levels of H- and L-ferritin in different organs of patients with MAS-associated AOSD and the evidence of a strong infiltrate of CD68/H-ferritin positive cells in the same organs. Furthermore, a strong correlation among both the tissue H-ferritin and the CD68/H-ferritin positive cells and the clinical picture was observed.
Asunto(s)
Apoferritinas/análisis , Médula Ósea/química , Hígado/química , Síndrome de Activación Macrofágica/metabolismo , Enfermedad de Still del Adulto/metabolismo , Adulto , Edad de Inicio , Animales , Apoferritinas/metabolismo , HumanosRESUMEN
Estimating the amount of iron-replete ferritin versus iron-deficient apoferritin proteins is important in biomedical and nanotechnology applications. This work introduces a simple and novel approach to quantify ferritin by using magnetic force microscopy (MFM). We demonstrate how high magnetic moment probes enhance the magnitude of MFM signal, thus enabling accurate quantitative estimation of ferritin content in ferritin/apoferritin mixtures in vitro. We envisage MFM could be adapted to accurately determine ferritin content in protein mixtures or in small aliquots of clinical samples.
Asunto(s)
Apoferritinas/análisis , Ferritinas/análisis , Microscopía de Fuerza Atómica/métodos , Apoferritinas/ultraestructura , Ferritinas/ultraestructura , Humanos , Fenómenos Magnéticos , Microscopía Electrónica de TransmisiónRESUMEN
Novel prognostic biomarkers are imperatively needed to help direct treatment decisions by typing subgroups of node-negative breast cancer patients. Large screening of different biological compartments, such as the proteome, by means of high throughput techniques may greatly help scientists to find such markers. The present retrospective multicentric study included 268 node-negative breast cancer patients. We used a proteomic approach of SELDI-TOF-MS screening to identify differentially expressed cytosolic proteins with prognostic impact. The screening cohort was composed of 198 patients. Seventy supplementary patients were included for validation. Immunohistochemistry (IHC) and immunoassay (IA) were run to confirm the prognostic role of the marker identified by SELDI-TOF-MS screening. IHC was also used to explore links between selected marker and epithelial-mesenchymal transition (EMT)-like, proliferation and macrophage markers. Ferritin light chain (FTL) was identified as an independent prognostic marker (HR = 1.30-95% CI: 1.10-1.50, p = 0.001). Validation step by means of IHC and IA confirmed the prognostic value of FTL level. CD68 IHC showed that FTL was stored in tumor-associated macrophages (TAM), which exhibit an M2-like phenotype. We report here, first, the validation of FTL as a breast tumor prognostic biomarker in node-negative patients, and second, the fact that FTL is stored in TAM.
Asunto(s)
Apoferritinas/análisis , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Neoplasias de la Mama/diagnóstico , Macrófagos/química , Adulto , Anciano , Antígenos CD/análisis , Antígenos de Diferenciación Mielomonocítica/análisis , Neoplasias de la Mama/patología , Proliferación Celular , Estudios de Cohortes , Citosol , Transición Epitelial-Mesenquimal/fisiología , Femenino , Humanos , Persona de Mediana Edad , Pronóstico , Proteómica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Cellular MRI with a reporter gene offers the opportunity to track small numbers of tumor cells and to study metastatic processes in their earliest developmental stages in the target organs of interest. This study demonstrates the feasibility of using the MR reporter ferritin for the noninvasive imaging and quantification of metastatic melanoma cells in the lymph nodes (LNs) of living mice. A B16F10 murine melanoma cell line expressing human ferritin heavy chain (hFTH) and green fluorescent protein (GFP) was constructed to allow the detection of cells by MRI and fluorescence imaging. Stable overexpression of hFTH and GFP in B16F10 murine melanoma cells was feasible and showed no cellular toxicity. In addition, hFTH cells were detectable by 9.4-T MRI in vitro and in vivo, yielding significant changes in T(2)* relative to control cells. In BALB/c nude mice, the presence of hFTH- and GFP-expressing metastatic melanoma cells in deep-seated axillary LNs was demonstrated as areas of low T(2)* on MRI, but the same LNs were not visible by fluorescence imaging because the light was unable to penetrate the tissue. Furthermore, the metastatic volume of each LN, which was assessed by cumulative histogram analysis of the T(2)* MRI data, correlated well with tumor burden, which was determined by histology (r = -0.8773, p = 0.0001). This study is the first to use MRI and an MR reporter gene for both the visualization and quantification of metastatic cancer cells in LNs.
Asunto(s)
Apoferritinas/análisis , Ganglios Linfáticos/patología , Imagen por Resonancia Magnética/métodos , Melanoma Experimental/patología , Animales , Apoferritinas/biosíntesis , Apoferritinas/genética , Línea Celular Tumoral , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Ganglios Linfáticos/metabolismo , Metástasis Linfática , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía Fluorescente/métodos , TransfecciónRESUMEN
A chip-type design asymmetrical flow field-flow fractionation (AF4) channel has been developed for high-speed separation of proteins and top-down proteomic analysis using online coupled electrospray ionization mass spectrometry (ESI-MS). The new miniaturized AF4 channel was assembled by stacking multilayer thin stainless steel (SS, 1.5 mm each) plates embedded with an SS frit in such a way that the total thickness of the channel assembly was about 6 mm. The efficiency of the miniaturized AF4 channel at different channel lengths was examined with the separation of protein standards by adjusting flow rates in which an identical effective channel flow rate or an identical void time can be maintained at different channels. Detection limit, overloading effect, reproducibility, and influence of channel membrane materials on separation efficiency were investigated. Desalting and purification of proteins achieved during the AF4 operation by the action of an exiting crossflow and the use of aqueous mass-spectrometry-compatible (MS-compatible) buffer were advantageous for online coupling of the chip-type AF4 with ESI-MS. The direct coupling of AF4 and ESI-MS capabilities was demonstrated for the high-speed separation and identification of carbonic anhydrase (29 kDa) and transferrin (78 kDa) by full scan MS and for the first top-down identification of proteins with AF4-ESI-MS-MS using collision-induced fragmentation (CID). The presence of intact dimers (156 kDa) of transferrin was confirmed by AF4-ESI-MS via size separation of the dimers from monomers, followed by multiply charged ion spectral analysis of the dimers and molecular mass determinations. It was also found from these experiments that AF4-ESI-MS analysis of transferrin exhibited an increased signal-to-noise ratio compared to that of direct ESI-MS analysis due to online purification of the protein sample and size separation of dimers with AF4.
Asunto(s)
Apoferritinas/análisis , Anhidrasas Carbónicas/análisis , Fraccionamiento de Campo-Flujo , Transferrina/análisis , Animales , Anhidrasas Carbónicas/metabolismo , Bovinos , Fraccionamiento de Campo-Flujo/instrumentación , Caballos , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
In several brain regions, a subpopulation of neurons exists being characterized by the expression of a peculiar form of extracellular matrix, a so-called perineuronal net (PNN). We have previously shown that the PNN can bind large amounts of iron due to its polyanionic charge. Because free iron can generate reactive oxygen species thus being potentially toxic, the PNN may have a protective function by "scavenging" this free iron. Because of this ability, we have hypothesized that PNN-related neurons have an altered iron-specific metabolism. Thus, to compare the intracellular concentrations of iron containing proteins, specifically, the iron storage protein ferritin H between neurons with and without a PNN, we have used slide-based cytometry with image-based threshold-boundary cell detection on brain sections. In tissue sections, the integrity of the extracellular matrix, especially the characteristic PNNs, is preserved, which is necessary for the identification of the two neuronal subpopulations. A multilabeling approach was chosen to select neurons (neuronal marker NeuN), to classify the neurons according to their subtype (matrix marker Wisteria floribunda agglutinin), and to quantify the protein concentration (protein marker). Using this novel method, we were able to detect a relative difference in protein concentration as low as 12% between the two subpopulations of neurons in the neuronal population of the rat parietal cortex.
Asunto(s)
Apoferritinas/análisis , Corteza Cerebral/química , Citoplasma/química , Citometría de Barrido por Láser/métodos , Neuronas/química , Coloración y Etiquetado/métodos , Animales , Apoferritinas/biosíntesis , Automatización de Laboratorios/métodos , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Citoplasma/metabolismo , Matriz Extracelular/metabolismo , Fluorescencia , Inmunohistoquímica , Hierro/metabolismo , Masculino , Microtomía , Neuronas/citología , Neuronas/metabolismo , Lectinas de Plantas/metabolismo , Ratas , Ratas Wistar , Receptores N-Acetilglucosamina/metabolismoRESUMEN
Swine hepatitis E virus (sHEV) has been discovered to be almost ubiquitous in pigs, and is antigenically and genetically related to human HEV. Proteomic analysis was used to identify altered protein expression in swine liver, using two-dimensional electrophoresis and peptide mass fingerprinting. A total of 10 protein spots exhibited significant alterations in the sHEV-infected organ. The upregulation of apolipoprotein E (Apo E) and downregulation of ferritin heavy chain were confirmed by Western analysis and by semi-quantitative reverse transcription-PCR. The elevated expression of Apo E may provide a novel insight into molecular responses to HEV infection in swine.
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Apoferritinas/análisis , Apolipoproteínas/análisis , Hepatitis E/veterinaria , Hígado/patología , Proteoma/análisis , Enfermedades de los Porcinos/patología , Animales , Western Blotting , Regulación hacia Abajo , Hepatitis E/patología , Hepatitis E/virología , Virus de la Hepatitis E/patogenicidad , Humanos , Hígado/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Enfermedades de los Porcinos/virología , Regulación hacia ArribaRESUMEN
Multinucleation is indispensable to the bone-resorbing activity of mature osteoclasts. Nevertheless, little is known about the regulatory networks among multi-nuclei in a single mature osteoclast. For this reason, we purified osteoclastic factors from the nuclear envelope by two-dimensional gel electrophoresis. Two annexin family proteins and ferritin light chain 1 protein were identified as osteoclastic candidates.
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Anexinas/análisis , Apoferritinas/análisis , Resorción Ósea/etiología , Proteínas Portadoras/análisis , Membrana Nuclear/química , Osteoclastos/química , Animales , Línea Celular , Células Cultivadas , Electroforesis en Gel Bidimensional , Redes Reguladoras de Genes , Macrófagos , RatonesRESUMEN
In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.
Asunto(s)
Apoferritinas/análisis , Gránulos Citoplasmáticos/química , Melaninas/análisis , Sustancia Negra/química , Western Blotting , Cromatografía Líquida de Alta Presión , Gránulos Citoplasmáticos/ultraestructura , Electroforesis en Gel de Poliacrilamida , Humanos , Microscopía Inmunoelectrónica , Cambios Post Mortem , Proteómica/métodos , Espectrometría de Masa por Ionización de Electrospray , Sustancia Negra/ultraestructuraRESUMEN
The appearance of small, dense, low-density lipoprotein in serum has been demonstrated to be associated with increased risk of coronary artery disease. The molecular diameter of low-density lipoprotein is usually measured on the basis of mobility differences on polyacrylamide gel electrophoresis. However, since mobility assessed by this method is seriously affected by the increased levels of serum free fatty acids associated with hypertriglyceridemia, we used polyacrylamide gradient gel electrophoresis to eliminate the interference by fatty acids and devised a simple, precise method of polyacrylamide gradient gel electrophoresis to measure the diameter of small, dense, low-density lipoproteins in serum. We used apoferritin and thyroglobulin, which have a molecular diameter of 12.2 nm and 17.0 nm, respectively, and standard low-density lipoprotein particles having a diameter of 25.7 and 27.0 nm as calibrators, estimated by measurement of negative staining of electron microscopy. We also included apoferritin as an internal standard for polyacrylamide gradient gel electrophoresis. The only stain used was Coomassie brilliant blue, and it was used for lipoprotein staining. When we used low-density lipoprotein of 25.73 nm in diameter as a quality control specimen, the coefficient of variation of the size measurements obtained by our method was less than 1.2%. The new method markedly improved the laboratory procedure for measuring the diameter of low-density lipoproteins.