RESUMEN
Crustacean shellfish are major allergens in East Asia. In the present study, a major allergic protein in crustaceans, tropomyosin, was detected accurately using multiple reaction monitoring mode-based mass spectrometry, with shared signature peptides identified through proteomic analysis. The peptides were deliberately screened through thermal stability and enzymatic digestion efficiency to improve the suitability and accuracy of the developed method. Finally, the proposed method demonstrated a linear range of 0.15 to 30 mgTM/kgfood (R2 > 0.99), with a limit of detection of 0.15 mgTM/kg food and a limit of quantification of 0.5mgTM/kgfood and successfully applied to commercially processed foods, such as potato chips, biscuits, surimi, and hot pot seasonings, which evidenced the applicability of proteomics-based methodology for food allergen analysis.
Asunto(s)
Alérgenos , Crustáceos , Espectrometría de Masas , Péptidos , Proteómica , Mariscos , Tropomiosina , Tropomiosina/química , Tropomiosina/inmunología , Tropomiosina/análisis , Animales , Proteómica/métodos , Alérgenos/química , Alérgenos/análisis , Péptidos/química , Mariscos/análisis , Espectrometría de Masas/métodos , Crustáceos/química , Proteínas de Artrópodos/química , Proteínas de Artrópodos/inmunología , Hipersensibilidad a los Mariscos/inmunología , Hipersensibilidad a los Alimentos/inmunología , Alimentos ProcesadosRESUMEN
Regulations of the EU obliges the indication of the presence of allergens on food labels. This work reports the development of an electrochemical immunosensor to determine tropomyosin (TPM) - a major shellfish allergen - prevailing in the muscles of crustacean species. Two linear ranges between the signal and TPM concentration were obtained: between 2.5 and 20 ng mL-1 and between 30 and 200 ng mL-1, with a lowest limit of detection of 0.47 ng mL-1. The selectivity of the optimized immunoassay, tested with other food allergens (e.g., Cyp c 1, a fish allergen), assures the effective detection of TPM, enabling successful control of foodstuff labelling. Several (12) foods, containing high and low TPM concentrations and TPM-free samples, were analysed using the sensor. A conventional ELISA kit and recovery assays were used to evaluate the accuracy of the results.
Asunto(s)
Técnicas Biosensibles , Hipersensibilidad a los Alimentos , Alérgenos/análisis , Animales , Técnicas Biosensibles/métodos , Análisis de los Alimentos/métodos , Inmunoensayo/métodos , Tropomiosina/análisisRESUMEN
This paper presents the development and the application of a multisyringe flow injection analysis system for the fluorimetric determination of the major heat-stable known allergen in shrimp, rPen a 1 (tropomyosin). This muscle protein, made up of 284 amino acids, is the main allergen in crustaceans and can be hydrolyzed by microwave in hydrochloric acid medium to produce glutamic acid, the major amino acid in the protein. Glutamic acid can then be quantified specifically by thermal conversion into pyroglutamic acid followed by chemical derivatization of the pyroglutamic acid formed by an analytical protocol based on an OPA-NAC reagent. Pyroglutamic acid can thus be quantified between 1 and 100 µM in less than 15 min with a detection limit of 1.3 µM. The method has been validated by measurements on real samples demonstrating that the response increases with the increase in the tropomyosin content or with the increase in the mass of the shrimp sample.
Asunto(s)
Alérgenos/análisis , Análisis de Inyección de Flujo/métodos , Alimentos Marinos/análisis , Tropomiosina/análisis , Animales , MariscosRESUMEN
Cachexia, a condition prevalent in many chronically ill patients, is characterized by weight loss, fatigue, and decreases in muscle mass and function. Cachexia is associated with tumor burden and disease-related malnutrition, but other studies implicate chemotherapy as being causative. We investigated the effects of a chemotherapy drug cocktail on myofibrillar protein abundance and synthesis, anabolic signaling mechanisms, and substrate availability. On day 4 of differentiation, L6 myotubes were treated with vehicle (1.4 µl/ml DMSO) or a chemotherapy drug cocktail (a mixture of cisplatin [20 µg/ml], leucovorin [10 µg/ml], and 5-fluorouracil [5-FLU; 50 µg/ml]) for 24-72 h. Compared to myotubes treated with vehicle, those treated with the drug cocktail showed 50%-80% reductions in the abundance of myofibrillar proteins, including myosin heavy chain-1, troponin, and tropomyosin (p < 0.05). Cells treated with only a mixture of cisplatin and 5-FLU had identical reductions in myofibrillar protein abundance. Myotubes treated with the drug cocktail also showed >50% reductions in the phosphorylation of AKTSer473 and of mTORC1 substrates ribosomal protein S6Ser235/236 , its kinase S6K1Thr389 and eukaryotic translation initiation factor 4E-binding protein 1 (all p < 0.05). Drug treatment impaired peptide chain initiation in myofibrillar protein fractions and insulin-stimulated glucose uptake (p = 0.06) but increased the expression of autophagy markers beclin-1 and microtubule-associated proteins 1A/1B light chain 3B (p < 0.05), and of apoptotic marker, cleaved caspase 3 (p < 0.05). Drug treatment reduced the expression of mitochondrial markers cytochrome oxidase and succinate dehydrogenase (p < 0.05). The observed profound negative effects of this chemotherapy drug cocktail on myotubes underlie a need for approaches that can reduce the negative effects of these drugs on muscle metabolism.
Asunto(s)
Fibras Musculares Esqueléticas/efectos de los fármacos , Proteínas Musculares/efectos de los fármacos , Animales , Western Blotting , Caquexia/inducido químicamente , Células Cultivadas , Cisplatino/administración & dosificación , Cisplatino/farmacología , Quimioterapia Combinada , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacología , Leucovorina/administración & dosificación , Leucovorina/farmacología , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/análisis , Proteínas Musculares/fisiología , Cadenas Pesadas de Miosina/análisis , Ratas , Tropomiosina/análisis , Troponina/análisisRESUMEN
Hepatocellular carcinoma (HCC) is known as the fifth most common cancer in the world for its poor prognosis. New diagnostic markers and treatments are urgent to discover. To evaluate the protein expression of Tropomyosin4 (TPM4) and investigate its prognostic value in HCC, we collected 110 patients with different degrees of HCC and 10 patients with normal hepatic tissues and performed immunohistochemistry. Western bot was used to evaluate the expression of TPM4 in three HCC cell lines (HepG2, Huh7, SMMC-7721) and normal liver cell line LO2, as well as 7 HCC tissues and 7 normal hepatic tissues. The results of TPM4 staining revealed that TPM4 expression in HCC was higher than that in normal hepatic tissues, which was positive in 51.8% (n=57) and negative in 48.2% (n=53) while in normal hepatic tissues positive staining was in 10% (n=1) and negative staining was in 90% (n=9) (P=0.011). And the expression of TPM4 was related to pT status, grade and stage (P<0.001, P=0.015 and P<0.001, respectively). Western blot results indicated that TPM4 was high expressed in HCC cell line and HCC tissues. In conclusion, we believe that TPM4 can be applied as a diagnostic and prognostic marker to assist the management of HCC.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/diagnóstico , Neoplasias Hepáticas/diagnóstico , Hígado/patología , Tropomiosina/metabolismo , Biomarcadores de Tumor/análisis , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Neoplasias Hepáticas/patología , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Estadificación de Neoplasias , Pronóstico , Tropomiosina/análisisRESUMEN
This study focuses on developing a highly sensitive immunosensor by immobilizing oxidized carbon nanohorns/Nafion/Fe3O4@Pd nanocomposite on carbon screen-printed electrodes (SPEs) for the detection of tropomyosin (Tro-Ag). The performance of the fabricated immunosensor was investigated via electrochemiluminescence (ECL) method, resulting from the chemical reaction between tris(bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) and tripropylamine (TPrA), and the peak intensity is recorded at 1.0 V. The nanocomposite is able to enhance the ECL intensity of [Ru(bpy)3]2 +/TPrA system and achieves high sensitivity of 28.16 fg/mL with a dynamic working range of 28.16 fg/mL to 100 ng/ml.. Furthermore, the immunosensor demonstrated a decent stability and good repeatability for Tro-Ag detection in food products. Graphical abstract The schematic representation of the modified carbon SPE with CNHs-OH/Nafion/Fe3O4@Pd and the signal produced in the (a) absence and (b) presence of Tro-Ag.
Asunto(s)
Técnicas Electroquímicas/métodos , Inmunoensayo/métodos , Mediciones Luminiscentes/métodos , Nanocompuestos/química , Tropomiosina/análisis , Animales , Anticuerpos Inmovilizados/inmunología , Carbono/química , Complejos de Coordinación/química , Técnicas Electroquímicas/instrumentación , Electrodos , Óxido Ferrosoférrico/química , Polímeros de Fluorocarbono/química , Compuestos Organometálicos/química , Paladio/química , Penaeidae/química , Propilaminas/química , Conejos , Tropomiosina/inmunologíaRESUMEN
The development of a sensitive and rapid detection approach for allergens in various food matrices is essential to assist patients in managing their allergies. The most common methods used for allergen detection are based on immunoassays, PCR and mass spectrometry. However, all of them are very complex and time-consuming. Herein, an aptamer biosensor for the detection of the major shrimp allergen tropomyosin (TM) was developed. Graphene oxide (GO) was used as a platform for screening of the minimal-length aptamer sequence required for high-affinity target binding. A fluorescein dye labeled GO quenches the truncated aptamer by π-stacking interactions. After the addition of TM, the fluorescence was restored due to the competitive binding of the aptamer to GO. One of the truncated aptamers was found to bind to TM with four-fold higher affinity (30 nM) compared to the full-length aptamer (124 nM), with a limit of detection (LOD) of 2 nM. The aptamer-based sensor demonstrates the sensitive, selective, and specific detection of TM in 30 min. The performance of the sensor was confirmed using TM spiked chicken soup, resulting in a high percentage recovery (~97 ± 10%). The association of GO and labelled aptamer sensor platform has shown the rapid detection of TM in food, which is compared to other methods very sensitive, specific and performs in high throughput application.
Asunto(s)
Técnicas Biosensibles/métodos , Crustáceos/química , Tropomiosina/análisis , Alérgenos/análisis , Alérgenos/genética , Animales , Aptámeros de Nucleótidos/genética , Grafito/química , Límite de Detección , Tropomiosina/genéticaRESUMEN
A colorimetric assay is described for simultaneous detection of multiple analytes related to food safety. It is based on the use of a sandwich aptasensor and terminal deoxynucleotidyl transferase (TdT) which produces a primer for subsequent rolling circle amplification (RCA). Two split aptamer fragments (Apt1 and Apt2) are firstly immobilized, Apt1 on gold nanoparticles (AuNPs), and Apt2 on magnetic beads (MBs). They are then used in a sandwich aptasensor. In the presence of analyte, two probes could specifically recognize target and form a ternary assembly, and the magnetic beads also act to separate rapidly and enrich the target. Then, the extension of template-free DNA is triggered by TdT at the exposed 3'-hydroxy terminals of Apt1. This produces polyA sequences that serve as primers for subsequent RCA. The product of RCA is hybridized with a complementary horse radish peroxidase (HRP) DNA probe. HRP catalyzes the H2O2-mediated oxidation of tetramethylbenzidine (TMB) and forms a blue chromogenic product. After magnetic separation, the absorption values of the blue product in the supernatant are measured at a wavelength of 600 nm. Based on this dual amplification mechanism, the assay was applied to multiplexed determination of enrofloxacin (ENR), lead(II), Escherichia coli O157:H7 and tropomyosin. Exemplarily, ENR is detectable at concentrations down to 2.5 pg mL-1 with a linear range that extends from 1 pg mL-1 to 1 µg·mL-1. The assay was validated by analysis of spiked fish samples. Recoveries range between 87.5 and 92.1%. Graphical abstractSchematic representation of a TdT-RCA based aptasensor for multiple analytes related to food safety. It makes use of sandwich aptasensors and TdT-produced universal primer-triggered RCA reaction. dATP: deoxyadenosine triphosphate, TdT: Terminal Deoxynucleotidyl Transferase, RCA: rolling circle amplification, TMB: 3,3',5,5'-Tetramethylbenzidine.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Colorimetría/métodos , ADN Nucleotidilexotransferasa/química , Contaminación de Alimentos/análisis , Animales , Aptámeros de Nucleótidos/genética , Armoracia/enzimología , Bencidinas/química , Colorantes/química , ADN/química , ADN/genética , Enrofloxacina/análisis , Escherichia coli O157/aislamiento & purificación , Oro/química , Peroxidasa de Rábano Silvestre/química , Peróxido de Hidrógeno/química , Plomo/análisis , Límite de Detección , Nanopartículas del Metal/química , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , Salmón , Alimentos Marinos/análisis , Tropomiosina/análisisRESUMEN
A shrimp tropomyosin (TPM) immunosensor has been developed and optimized to detect trace amounts of shrimp (in the ppm range), based on a combination of an amperometric transduction, magnetic particles and disposable screen-printed electrodes. The approach is based on the implementation of a sandwich immunoassay format on the surface of magnetic beads and their coupling onto disposable screen-printed electrodes to finally register the amperometric response at -200 mV vs. Ag pseudo-reference electrode, using H2O2 as enzymatic substrate and hydroquinone as redox mediator. The use of carboxyl-functionalized magnetic microbeads (MBs) and in-house made magnetic nanoparticles (MNPs) as solid supports have been evaluated and compared. Our experimental results confirm that the use of MBs, in addition to simplifying the test protocol, improves the resulting sensitivity, so they were selected for the implementation of the immunosensor. In the optimized experimental conditions, the developed immunosensor offered a LOD of 47â¯pgâ¯mL-1 for amperometric determination of shrimp TPM standards and great selectivity against TPM from other sources, thus allowing differentiation between crustaceans (shrimp) and mollusks (squid). Applicability studies demonstrated successful determination both in crude and cooked samples using very simple protocols. Additionally, processed foods based on fish and mollusks that could potentially include crustaceans in their composition have been analyzed using the sensor and compared to the declared ingredients. The sensitivity and specificity showed by the sensor in the analysis of heterogeneous food samples without a previous purification or enrichment stage, also outperforms existing solutions in terms of time and cost effectiveness and permits its direct and smooth implementation in the food industry for routine allergen analyses.
Asunto(s)
Alérgenos/análisis , Contaminación de Alimentos/análisis , Tropomiosina/análisis , Alérgenos/inmunología , Animales , Anticuerpos/inmunología , Armoracia/enzimología , Técnicas Electroquímicas/métodos , Análisis de los Alimentos/métodos , Peroxidasa de Rábano Silvestre/química , Peróxido de Hidrógeno/química , Hidroquinonas/química , Inmunoensayo/métodos , Límite de Detección , Penaeidae/química , Tropomiosina/inmunologíaRESUMEN
In Taiwan, crab is one of the main causes for food allergy. Several proteins are recognized as crustacean allergens, and tropomyosin is known to be the major one. However, sensitization patterns of Taiwanese patients to crustacean allergens remain unclear. Therefore, we analyzed the specific-IgE binding ability of crucifix crab (Charybdis feriatus) allergens by western blot using patients' sera. In particular, we found a 56â¯kDa protein in crucifix crab reacted with specific-IgEs in patients' sera, and we further identified the protein as a novel crab allergen pyruvate kinase 2. Additionally, little is known about tropomyosin contents in crabs consumed in Taiwan. Thus, we also quantified the levels of tropomyosin by using enzyme-linked immunosorbent assay (ELISA) among raw and cooked crab species. Our results showed tropomyosin levels varied depending on crab species. In summary, these findings improve the understanding of crustacean allergens and contribute to the clinical diagnosis of crustacean allergies.
Asunto(s)
Alérgenos/análisis , Braquiuros/inmunología , Piruvato Quinasa/análisis , Piruvato Quinasa/inmunología , Mariscos/análisis , Adulto , Animales , Western Blotting , Braquiuros/química , Ensayo de Inmunoadsorción Enzimática , Hipersensibilidad a los Alimentos/sangre , Hipersensibilidad a los Alimentos/inmunología , Humanos , Inmunoglobulina E/sangre , Alimentos Marinos/análisis , Taiwán , Tropomiosina/análisisRESUMEN
Asymmetrical flow field flow fractionation (AF4) combined with ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to screen allergen protein epitopes. The selected allergen protein (tropomyosin, TM) was enzymatically digested into peptide segments and analyzed via UPLC-QTOF-MS to establish a protein-specific peptide database. The peptide segments were incubated with immunoglobulin E (IgE) for 30 min. During the incubation procedure, the specific peptide segments (with the antigen epitope) combine with IgE while the other peptide segments remain in solution. After incubation, the solution was injected into the AF4 device. The combined peptide segments flowed out of the outlet along with IgE, and the other peptide segments flowed into the waste liquid. The components of outlet were then collected, analyzed by UPLC-QTOF-MS, and the results matched with the spectra of the protein peptides. Eventually the specific peptide segments were identified to detect the antigen epitopes. This study extends the application of AF4 with a preliminary exploration of the detection of an allergen protein epitope, providing a novel research strategy for the screening of allergen epitopes.
Asunto(s)
Alérgenos/análisis , Epítopos/análisis , Fraccionamiento de Campo-Flujo , Proteínas/análisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masas en Tándem , Tropomiosina/análisisRESUMEN
A biosensor based on a chiral assembly of polymer of gold nanoparticle (AuNP) trimers was developed for the detection and quantification of the major shellfish allergen tropomyosin (TROP). TROP and anti-TROP monoclonal antibodies (mAb) were immobilized on 20â¯nm and 30â¯nm 16-mercaptohexadecanoic acid (16-MHDA) functionalized AuNPs to assemble a trimer, which has a Circular dichroism (CD) signal. The free TROP from samples was quantified as an inhibitor for the formation of the AuNP trimer. The AuNP trimer-based biosensor allowed for the selective determination of TROP in the range of 0.1-15â¯ngâ¯mL-1 with the limit of detection (LOD) of 21â¯pgâ¯mL-1 (S/Nâ¯=â¯3) and the limit of quantitation (LOQ) of 70â¯pgâ¯mL-1 (S/Nâ¯=â¯10). The use of a AuNP trimer-based biosensor with simple sample preparation functions with specificity and accuracy; this highlights its applicability for the detection of allergens in shellfish products, related products and their production lines. Furthermore, based on the less conserved sequences of TROP in phylogenetically different species, this biosensor is currently being used to identify the adulteration of shellfish products using TROP as biomarker.
Asunto(s)
Alérgenos/análisis , Técnicas Biosensibles/métodos , Oro/química , Nanopartículas del Metal/química , Proteínas de Mariscos/análisis , Mariscos/análisis , Tropomiosina/análisis , Anticuerpos Inmovilizados/química , Dimerización , Análisis de los Alimentos/métodos , Inmunoensayo/métodos , Límite de DetecciónRESUMEN
A label-free proteomics method was used to explore the effects of differentially expressed proteins on the tenderness of yak rumen smooth muscle during postmortem storage (0, 3 and 7â¯days) at 3⯱â¯1⯰C. The tenderness improved significantly during storage. A total of 212 differentially expressed proteins were identified by the following comparisons: Day 3 vs.0, day 7 vs.0, and day 7 vs.3. Twenty-eight proteins were correlated with the WBSF of yak rumen smooth muscle. Calpastatin, ADP/ATP translocase 1, zyxin, LMOD1 protein, tropomyosin α-3 chain, thrombospondin-4 and UQCRC1 protein are highly related to smooth muscle tenderness, and thus, they are candidates indicators of yak rumen smooth muscle tenderness during storage. Furthermore, bioinformatics analyses revealed that the identified proteins were related to focal adhesion, vascular smooth muscle contraction, cardiac muscle contraction and necroptosis. The present results could provide proteomic insights into changes in yak rumen smooth muscle tenderness during storage and may be a valuable resource for future investigations.
Asunto(s)
Espectrometría de Masas/métodos , Músculo Liso/química , Proteoma/análisis , Rumen/química , Animales , Autopsia , Bovinos , Biología Computacional , Proteínas del Citoesqueleto/análisis , Complejo III de Transporte de Electrones , Necroptosis , Trombospondinas/análisis , Factores de Tiempo , Tropomiosina/análisis , Zixina/análisisRESUMEN
Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico-chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high-performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Carne/análisis , Músculos/metabolismo , Tropomiosina/química , Tropomiosina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Fenómenos Químicos , Pollos , Calidad de los Alimentos , Miofibrillas/metabolismo , Cambios Post Mortem , Factores de Tiempo , Tropomiosina/análisis , Tropomiosina/metabolismoRESUMEN
No disponible
Asunto(s)
Humanos , Masculino , Niño , Tropomiosina/análisis , Alérgenos/análisis , Hipersensibilidad a los Alimentos/diagnóstico , Anafilaxia/diagnóstico , Productos Pesqueros/efectos adversos , Factores de RiesgoRESUMEN
The present study aimed to investigate protein expression levels of intra and extracranial atherosclerosis in rabbits following administration of a highfat diet. Rabbits were randomly divided into control (group A; n=9) and highfat diet (group B; n=9) groups. At week 12, tissues were sectioned from the common carotid artery (CCA) and middle cerebral artery (MCA). Pathological analysis was performed. Differential protein expression levels were examined by 2D gel electrophoresis (2DE) and mass spectrometry (MS) analysis and validated by western blotting. Serum lipid levels, the intimamedia thickness (IMT) and degree of atherosclerosis of the CCA and MCA were increased at week 12 in the highfat diet group compared with rabbits that received a normal diet. 2DE and MS analysis of the protein extracted from CCA and MCA detected >439 different proteins; the expression of 25 proteins was altered, and 8 proteins [albumin A chain, tropomyosin α1 chain (TPM1), heat shock protein 70 (HSP70), αsmooth muscle actin, ßgalactose binding agglutinin, TPM4 isoform 2, cell keratin 9, single octylic acid glyceride ß2) demonstrated significant alterations in expression levels. Due to limited antibody sources, only three differentially expressed proteins (TPM1, HSP70 and αsmooth muscle actin) were examined by western blotting. The results of our previous study demonstrated that hyperlipidemia affected the IMT of intracranial and extracranial cerebral arteries. In the present study, protein expression levels of TPM1 and αsmooth muscle actin from extracranial cerebral arteries were significantly increased compared with intracranial cerebral arteries; however, protein expression levels of HSP70 from intracranial cerebral arteries was increased compared with extracranial cerebral arteries. The differences may be closely associated with cell proliferation and metastasis, and oxidoreduction, in intra and extracranial cerebral atherosclerosis. HSP70 may have protective properties against atherosclerosis via underlying antiinflammatory mechanisms, furthermore, differential protein expression levels (TPM1, HSP70 and αsmooth muscle actin) between intra and extracranial cerebral arteries may facilitate the identification of novel biological markers for the diagnosis and treatment of cerebral arteriosclerosis.
Asunto(s)
Arteriosclerosis/complicaciones , Arteria Carótida Común/patología , Arterias Cerebrales/patología , Hiperlipidemias/complicaciones , Arteriosclerosis Intracraneal/complicaciones , Proteoma/análisis , Actinas/análisis , Animales , Arteriosclerosis/sangre , Arteriosclerosis/patología , Grosor Intima-Media Carotídeo , Dieta Alta en Grasa/efectos adversos , Proteínas HSP70 de Choque Térmico/análisis , Hiperlipidemias/sangre , Hiperlipidemias/patología , Arteriosclerosis Intracraneal/sangre , Arteriosclerosis Intracraneal/patología , Lípidos/sangre , Masculino , Proteómica , Conejos , Tropomiosina/análisisRESUMEN
The purpose of this study was to find discrimination markers for four major meat species such as beef, pork, chicken and duck. Myofibrillar and sarcoplasmic proteins isolated from each meat type were analyzed by one-dimensional gel electrophoresis and some proteins were identified through LC-MS/MS analysis. We confirmed that troponin I (TnI), enolase 3, l-lactate dehydrogenase (LDH) and triose-phosphate isomerase (TPI) could be useful markers for discrimination of mammals from poultry due to their different electrophoretic mobility. Tropomyosin 1 and carbonic anhydrase 3 were observed as muscle fiber type-related proteins and these could also be markers to distinguish mammals from poultry. Species-specific peptides identified by LC-MS/MS spectra allow the identification of each species regardless of the same protein. Therefore, it is easy to discriminate between mammals and poultry by comparing the electrophoretic mobility of TnI, enolase 3, LDH, TPI and CA3, and each species could be identified through LC-MS/MS analysis.
Asunto(s)
Proteínas en la Dieta/análisis , Aves de Corral , Carne Roja/análisis , Animales , Biomarcadores/análisis , Bovinos , Pollos , Cromatografía Liquida , Patos , L-Lactato Deshidrogenasa/análisis , Proteínas Musculares/análisis , Fosfopiruvato Hidratasa/análisis , Porcinos , Espectrometría de Masas en Tándem , Triosa-Fosfato Isomerasa/análisis , Tropomiosina/análisis , Troponina I/análisisRESUMEN
Sparganosis is one of the top three tissue-dwelling heterologous helminthic diseases, along with cysticercosis and paragonimiasis, in Korea. Due to a lack of effective early diagnosis and treatment methods, this parasitic disease is regarded as a public health threat. This study evaluated reactivity, against sparganum extracts, of sera from inhabitants of Cheorwon-gun, Goseong-gun and Ongjin-gun in Korea. The sera from 836 subjects were subjected to enzyme-linked immunosorbent assay and immunoblot analysis. The sera from 18 (5.8%) and 15 (5.1%) inhabitants in Cheorwon-gun (n = 312) and Goseong-gun (n = 294), respectively, exhibited highly positive reactions to the sparganum antigen, whereas only two (0.9%) inhabitants in Ongjin-gun (n = 230) showed positivity. We sought antigenic proteins for serodiagnosis of positive sera by immunoproteomic approaches. Total sparganum lysates were separated by two-dimensional electrophoresis and then subjected to immunoblot analysis with mixed sparganosis-positive sera. We found seven antigenic spots and identified paramyosin as an antigenic protein by liquid chromatography-mass spectrometry. By two-dimensional (2D)-based mass analysis and immunoblotting against sparganosis-positive sera, paramyosin was identified as a candidate antigen for serodiagnosis of sparganosis.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/inmunología , Pruebas Serológicas/métodos , Esparganosis/diagnóstico , Plerocercoide/inmunología , Tropomiosina/inmunología , Animales , Antígenos Helmínticos/análisis , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Immunoblotting , Espectrometría de Masas , Proteoma/análisis , República de Corea , Plerocercoide/química , Tropomiosina/análisisRESUMEN
Purpose: Tropomyosin-related kinase (Trk) receptors play critical roles in tumor development and are considered attractive targets for cancer therapy. We investigated correlations of the expression of TrkA, TrkB, and TrkC with clinicopathological features and outcomes in gastric cancer. Methods: Tumor samples were obtained from 221 patients with gastric cancer who underwent gastrectomy between 2003 and 2007. The expression of TrkA, TrkB, and TrkC was analyzed using immunohistochemical staining. The relationship of their expression to clinicopathological factors and outcomes was assessed. Results: High expression of TrkA, TrkB, or TrkC was significantly associated with histopathology (p = 0.022, p < 0.001, and p < 0.001). High expression of TrkA was significantly correlated with variables related to tumor progression, including lymph node metastasis (p = 0.024) and distant metastasis or recurrence (p < 0.001). Distant metastasis or recurrence was found in a significantly higher proportion of patients with high expression of TrkC than in those with low expression (p = 0.036). High expression of TrkA was significantly associated with poorer relapse-free survival (RFS) in univariate analysis (p = 0.001). High expression of TrkA or TrkC was significantly associated with poorer disease-specific survival (DSS) in univariate analysis (p < 0.001 and p = 0.008). In multivariate analysis, TrkA was an independent predictor of RFS [hazard ratio (HR), 2.294; 95 % confidence interval (CI), 1.309-4.032; p = 0.004] and DSS (HR, 2.146; 95 % CI, 1.195-3.861; p = 0.011). Expression of TrkB was not associated with RFS or DSS in univariate analysis. Conclusions: Our results demonstrated that TrkA expression was associated with tumor progression and poor survival, and was an independent predictor of poor outcomes in gastric cancer patients
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Asunto(s)
Humanos , Masculino , Femenino , Persona de Mediana Edad , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/cirugía , Pronóstico , Tropomiosina/análisis , Receptor trkA/análisis , Receptor trkB/análisis , Receptor trkC/análisis , Reacción en Cadena de la Polimerasa/métodos , Neoplasias Gástricas/patología , ARN/análisis , Reacción en Cadena de la Polimerasa/normas , Reacción en Cadena de la Polimerasa , 28599 , Análisis Multivariante , Modelos LogísticosRESUMEN
We herein analyzed the relationships between tropomyosin protein expression levels and clinicopathological factors in order to determine the significance of tropomyosins in lung cancers. Although neoplastic cells expressed different isoforms of tropomyosin, overall expression levels were lower than those in bronchial and alveolar epithelial cells. In adenocarcinomas, tropomyosin levels were markedly reduced in poorly differentiated or solid subtype carcinomas, suggesting that a loss in the expression of tropomyosins is involved in the progression of lung adenocarcinomas. The potential utility of the immunohistochemical expression of tropomyosins for a histopathological diagnosis was also investigated. The sensitivity and specificity of a loss in the expression of tropomyosins were 100% and 50%, respectively, which were superior to those for the strong expression of p53 (sensitivity 100% and specificity 44%), a conventional biomarker. An immunohistochemical examination of tropomyosins may assist in the histopathological detection of lung cancer cells in small biopsy specimens.