RESUMEN
Gliomas are responsible for more than 60% of all primary brain tumors. Glioblastoma multiforme (GBM), a grade IV tumor (WHO), is one of the most frequent and malignant gliomas. Despite two decades of advances in the discovery of new markers for GBM, the chemotherapy of choice falls to temozolomide after surgery and radiotherapy, which are not enough to increase the survival of patients to more than 15 months. It is urgent to discover new anti-glioma compounds. Many compounds derived from natural products have been used in the development of anti-tumor drugs. In this work, we have screened six low molecular weight sesquiterpene lactones, isolated from Eremanthus spp., and studied their function as anti-proliferative agents against GBM strains. We demonstrated that two of them, goyazensolide and lychnofolide, were effective in reducing cell viability, preventing the formation of anchorage-dependent colony and were able to pass through a mimetic blood-brain barrier making them candidates for glioma therapy, being more potent than temozolomide, according to in vitro assays for the cell lines tested. Proteomic analysis revealed a number of altered proteins involved in glycolytic metabolism and cellular catabolism.
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Lactonas/farmacología , Vernonia/metabolismo , Antineoplásicos/farmacología , Asteraceae , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Brasil , Hidrocarburos Aromáticos con Puentes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Furanos/farmacología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Lactonas/metabolismo , Extractos Vegetales/farmacología , Sesquiterpenos/farmacología , Sesterterpenos/farmacología , Vernonia/fisiologíaRESUMEN
Candida albicans is a human pathogenic fungus mainly affecting immunocompromised patients. Resistance to the commonly used fungicides can lead to poor treatment of mucosal infections which, in turn, can result in life-threatening systemic candidiasis. In this scenario, antimicrobial photodynamic treatment (PDT) has emerged as an effective alternative to treat superficial and localized fungal infections. Microbial death in PDT is a consequence of the oxidation of many cellular biomolecules, including proteins. Here, we report a combination of two-dimensional electrophoresis and tandem mass spectrometry to study the protein damage resulting from treating C. albicans with PDT with new methylene blue N and red light. Two-dimensional gels of treated cells showed an increase in acidic spots in a fluence-dependent manner. Amino acid analysis revealed a decrease in the histidine content after PDT, which is one plausible explanation for the observed acidic shift. However, some protein spots remained unchanged. Protein identification by mass spectrometry revealed that both modified and unmodified proteins could be localized to the cytoplasm, ruling out subcellular location as the only explanation for damage selectivity. Therefore, we hypothesize that protein modification by PDT is a consequence of both photosensitizer binding affinity and the degree of exposure of the photooxidizable residues on the protein surface.
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Candidiasis/terapia , Azul de Metileno/análogos & derivados , Fotoquimioterapia , Proteoma/efectos de los fármacos , Proteoma/efectos de la radiación , Candida albicans/efectos de los fármacos , Candida albicans/efectos de la radiación , Humanos , Luz , Azul de Metileno/farmacología , Fármacos Fotosensibilizantes/farmacologíaRESUMEN
BACKGROUND: Gliomas account for more than 60 % of all primary central nervous system neoplasms. Low-grade gliomas display a tendency to progress to more malignant phenotypes and the most frequent and malignant gliomas are glioblastomas (GBM). Another type of glioma, oligodendroglioma originates from oligodendrocytes and glial precursor cells and represents 2-5 % of gliomas. The discrimination between these two types of glioma is actually controversial, thus, a molecular distinction is necessary for better diagnosis. METHODS: iTRAQ-based quantitative proteomic analysis was performed on non-neoplastic brain tissue, on astrocytoma grade II, glioblastoma with short and long survival and oligodendrogliomas. RESULTS: We found that expression of nucleophosmin (NPM1), glucose regulated protein 78 kDa (GRP78), nucleolin (NCL) and heat shock protein 90 kDa (HSP90B1) were increased, Raf kinase inhibitor protein (RKIP/PEBP1) was decreased in glioblastoma and they were associated with a network related to tumor progression. Expression level of heat shock protein 27 (HSPB1/HSP27) discriminated glioblastoma presenting short (6 ± 4 months, n = 4) and long survival (43 ± 15 months, n = 4) (p = 0.00045). Expression level of RNA binding protein nova 1 (NOVA1) differentiated low-grade oligodendroglioma and astrocytoma grade II (p = 0.0082). Validation were done by Western blot, qRT-PCR and immunohistochemistry in a larger casuistry. CONCLUSION: Taken together, our quantitative proteomic analysis detected the molecular triad, NPM1, GRP78 and RKIP participating together with NCL and HSP27/HSPB1 in a network related to tumor progression. Additionally, two new important targets were uncovered: NOVA1 useful for diagnostic refinement differentiating astrocytoma from oligodendroglioma, and HSPB1/HSP27, as a predictive factor of poor prognosis for GBM.
Asunto(s)
Biomarcadores de Tumor/análisis , Glioblastoma/metabolismo , Proteínas de Choque Térmico HSP27/análisis , Oligodendroglioma/metabolismo , Proteoma/análisis , Proteínas de Unión al ARN/análisis , Adulto , Anciano , Biomarcadores de Tumor/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Chaperón BiP del Retículo Endoplásmico , Glioblastoma/mortalidad , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas de Choque Térmico , Humanos , Marcaje Isotópico , Persona de Mediana Edad , Chaperonas Moleculares , Antígeno Ventral Neuro-Oncológico , Nucleofosmina , Oligodendroglioma/mortalidad , Valor Predictivo de las Pruebas , Proteoma/metabolismo , Proteómica , Proteínas de Unión al ARN/metabolismo , Análisis de Supervivencia , Adulto JovenRESUMEN
Malnutrition programs the neuroendocrine axis by disruption of food-intake control, leading to obesity. Taurine (Tau) is neuroprotective and improves anorexigenic actions in the hypothalamus. We evaluated the hypothalamic gene-expression profile and food-intake control in protein-restricted mice submitted to a high-fat diet (HFD) and Tau supplementation. Mice were fed on a control (14 % protein-C) or a protein-restricted diet (6 % protein-R) for 6 weeks. Thereafter, mice received, or not, HFD for 8 weeks (CH and RH) with or without 5 % Tau supplementation (CHT and RHT). Protein restriction led to higher food intake, but calories were matched to controls. Excessive calorie intake occurred in HFD mice and this was prevented by Tau supplementation only in the CH group. Additionally, RH and CH mice developed hypothalamic leptin resistance, which was prevented by Tau. Global alterations in the expressions of genes involved in hypothalamic metabolism, cellular defense, apoptosis and endoplasmic reticulum stress pathways were induced by dietary manipulations and Tau treatment. The orexigenic peptides NPY and AgRP were increased by protein restriction and lowered by the HFD. The anorexigenic peptide Pomc was increased by HFD, and this was prevented by Tau only in CH mice. Thus, food intake was disrupted by dietary protein restriction and obesity. HFD-induced alterations were not enhanced by previous protein deficiency, but the some beneficial effects of Tau supplementation upon food intake were blunted by protein restriction. Tau effects upon feeding behavior control are complex and involve interactions with a vast gene network, preventing hypothalamic leptin resistance.
Asunto(s)
Grasas de la Dieta/farmacología , Suplementos Dietéticos , Hipotálamo/metabolismo , Leptina/metabolismo , Deficiencia de Proteína/mortalidad , Taurina/farmacología , Animales , Apoptosis/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Conducta Alimentaria/efectos de los fármacos , Hipotálamo/patología , Masculino , Ratones , Deficiencia de Proteína/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Lipid rafts are highly ordered membrane domains rich in cholesterol and sphingolipids that provide a scaffold for signal transduction proteins; altered raft structure has also been implicated in cancer progression. We have shown that 25 µm 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate (ODPC), an alkylphospholipid, targets high cholesterol domains in model membranes and induces apoptosis in leukemia cells but spares normal hematopoietic and epithelial cells under the same conditions. We performed a quantitative (SILAC) proteomic screening of ODPC targets in a lipid-raft-enriched fraction of leukemic cells to identify early events prior to the initiation of apoptosis. Six proteins, three with demonstrated palmitoylation sites, were reduced in abundance. One, the linker for activation of T-cell family member 2 (LAT2), is an adaptor protein associated with lipid rafts in its palmitoylated form and is specifically expressed in B lymphocytes and myeloid cells. Interestingly, LAT2 is not expressed in K562, a cell line more resistant to ODPC-induced apoptosis. There was an early loss of LAT2 in the lipid-raft-enriched fraction of NB4 cells within 3 h following treatment with 25 µm ODPC. Subsequent degradation of LAT2 by proteasomes was observed. Twenty-five µm ODPC inhibited AKT activation via myeloid growth factors, and LAT2 knockdown in NB4 cells by shRNA reproduced this effect. LAT2 knockdown in NB4 cells also decreased cell proliferation and increased cell sensitivity to ODPC (7.5×), perifosine (3×), and arsenic trioxide (8.5×). Taken together, these data indicate that LAT2 is an early mediator of the anti-leukemic activity of alkylphospholipids and arsenic trioxide. Thus, LAT2 may be used as a target for the design of drugs for cancer therapy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Apoptosis/efectos de los fármacos , Fosfolípidos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Trióxido de Arsénico , Arsenicales/farmacología , Caspasa 3/metabolismo , Línea Celular , Proliferación Celular , Colesterol/metabolismo , Activación Enzimática , Humanos , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Microdominios de Membrana , Óxidos/farmacología , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfolípidos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Estructura Terciaria de Proteína , Proteoma/análisis , Interferencia de ARN , ARN Interferente PequeñoRESUMEN
BACKGROUND: The epithelial-mesenchymal transition (EMT) promotes cell signaling and morphology alterations, contributing to cancer progression. Exosomes, extracellular vesicles containing proteins involved in cell-cell communication, have emerged as a potential source of biomarkers for several diseases. METHODS: Our aim was to assess the proteome content of exosomes secreted after EMT-induction to identify potential biomarkers for ovarian cancer classification. EMT was induced in the ovarian cancer cell line CAOV3 by treating it with EGF (10 ng/mL) for 96 h following 24 h of serum deprivation. Subsequently, exosomes were isolated from the supernatant using selective centrifugation after decellularization, and their characteristics were determined. The proteins present in the exosomes were extracted, identified, and quantified using Label-Free-Quantification (LFQ) via Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS). To identify potential biomarkers, the obtained proteomic data was integrated with the TGGA database for mRNA expression using principal component analysis and a conditional inference tree. RESULTS: The exosomes derived from CAOV3 cells exhibited similar diameter and morphology, measuring approximately 150 nm, regardless of whether they were subjected to EMT stimulation or not. The proteomic analysis of proteins from CAOV3-derived exosomes revealed significant differential regulation of 157 proteins, with 100 showing upregulation and 57 downregulation upon EMT induction. Further comparison of the upregulated proteins with the TCGA transcriptomic data identified PLAU, LAMB1, COL6A1, and TGFB1 as potential biomarkers of the mesenchymal HGSOC subtype. CONCLUSIONS: The induction of EMT, the isolation of exosomes, and the subsequent proteomic analysis highlight potential biomarkers for an aggressive ovarian cancer subtype. Further investigation into the role of these proteins is warranted to enhance our understanding of ovarian cancer outcomes.
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Exosomas , Neoplasias Ováricas , Femenino , Humanos , Exosomas/metabolismo , Transición Epitelial-Mesenquimal/genética , Proteómica , Cromatografía Liquida , Espectrometría de Masas en Tándem , Biomarcadores/metabolismo , Neoplasias Ováricas/metabolismo , Línea Celular TumoralRESUMEN
Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.
Asunto(s)
Glioblastoma/metabolismo , Proteínas Nucleares/metabolismo , Proteoma/metabolismo , ARN Interferente Pequeño/genética , Adulto , Antineoplásicos Alquilantes/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Chaperón BiP del Retículo Endoplásmico , Femenino , Regulación Neoplásica de la Expresión Génica , Glioblastoma/diagnóstico , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Humanos , Masculino , Persona de Mediana Edad , Proteínas Nucleares/genética , Nucleofosmina , Pronóstico , Proteoma/genética , Proteómica , Interferencia de ARN , Temozolomida , TransfecciónRESUMEN
BACKGROUND: The most frequent and malignant brain cancer is glioblastoma multiforme (GBM). In gliomas, tumor progression and poor prognosis are associated with the tumorigenic ability of the cells. U87MG cells (wild-type p53) are known to be tumorigenic in nude mice, but T98G cells (mutant p53) are not tumorigenic. We investigated the proteomic profiling of these two cell lines in order to gain new insights into the mechanisms that may be involved in tumorigenesis. RESULTS: We found 24 differentially expressed proteins between T98G and U87MG cells. Gene Ontology supports the notion that over-representation of differentially expressed proteins is involved in glycolysis, cell migration and stress oxidative response. Among those associated with the glycolysis pathway, TPIS and LDHB are up-regulated in U87MG cells. Measurement of glucose consumption and lactate production suggests that glycolysis is more effective in U87MG cells. On the other hand, G6PD expression was 3-fold higher in T98G cells and this may indicate a shift to the pentose-phosphate pathway. Moreover, GRP78 expression was also three-fold higher in T98G than in U87MG cells. Under thapsigargin treatment both cell lines showed increased GRP78 expression and the effect of this agent was inversely correlated to cell migration. Quantitative RT-PCR and immunohistochemistry of GRP78 in patient samples indicated a higher level of expression of GRP78 in grade IV tumors compared to grade I and non-neoplastic tissues, respectively. CONCLUSIONS: Taken together, these results suggest an important role of proteins involved in key functions such as glycolysis and cell migration that may explain the difference in tumorigenic ability between these two glioma cell lines and that may be extrapolated to the differential aggressiveness of glioma tumors.
RESUMEN
Proteomic approaches have been useful for the identification of aberrantly expressed proteins in complex diseases such as cancer. These proteins are not only potential disease biomarkers, but also targets for therapy. The aim of this study was to identify differentially expressed proteins in diffuse astrocytoma grade II, anaplastic astrocytoma grade III and glioblastoma multiforme grade IV in human tumor samples and in non-neoplastic brain tissue as control using 2-DE and MS. Tumor and control brain tissue dissection was guided by histological hematoxylin/eosin tissue sections to provide more than 90% of tumor cells and astrocytes. Six proteins were detected as up-regulated in higher grade astrocytomas and the most important finding was nucleophosmin (NPM) (p<0.05), whereas four proteins were down-regulated, among them raf kinase inhibitor protein (RKIP) (p<0.05). We report here for the first time the alteration of NPM and RKIP expression in brain cancer. Our focus on these proteins was due to the fact that they are involved in the PI3K/AKT/mTOR and RAS/RAF/MAPK pathways, known for their contribution to the development and progression of gliomas. The proteomic data for NPM and RKIP were confirmed by Western blot, quantitative real-time PCR and immunohistochemistry. Due to the participation of NPM and RKIP in uncontrolled proliferation and evasion of apoptosis, these proteins are likely targets for drug development.
Asunto(s)
Astrocitoma/genética , Neoplasias Encefálicas/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Nucleares/genética , Proteínas de Unión a Fosfatidiletanolamina/genética , Proteómica , Adulto , Secuencia de Aminoácidos , Astrocitoma/patología , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/patología , Electroforesis en Gel Bidimensional , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteínas Nucleares/aislamiento & purificación , Nucleofosmina , Proteínas de Unión a Fosfatidiletanolamina/aislamiento & purificación , Proteínas/genética , Proteínas/aislamiento & purificaciónRESUMEN
Micronutrients and their metabolites are cofactors in proteins involved in lipid metabolism. The present study was a subproject of the Harmonized Micronutrient Project (ClinTrials.gov # NCT01823744). Twenty participants were randomly selected from 136 children and adolescents that consumed a daily dose of 12 vitamins and 5 minerals supplementation for 6 weeks. The 20 individuals were divided into two pools of 10 individuals, according to their lipid profile at baseline (Pool 1 with lower triglycerides, LDL, and VLDL). The individuals were analyzed at baseline, after 6 weeks of daily supplementation, and after 6 weeks of a washout period in relation to anthropometric, body composition, food intake, lipid profile, micronutrient levels, and iTRAQ proteomic data. Genetic ancestry and its association with vitamin serum levels were also determined. After supplementation, LDL levels decreased while alpha-tocopherol and pantothenic acid levels increased in pool 2; lipid profiles in pool 1 did not change but had higher plasma levels of pantothenic acid, pyridoxal, and pyridoxic acid. In pool 2, expression of some proteins increased, and expression of other ones decreased after intervention, while in pool 1, the same proteins responded inversely or did not change their levels. Plasma alpha-tocopherol and Native American genetic ancestry explained a significant fraction of LDL plasma levels at baseline and in response to the intervention. After intervention, changes in expression of alpha-1 antitrypsin, haptoglobin, Ig alpha-1 chain C region, plasma protease C1 inhibitor, alpha-1-acid glycoprotein 1, fibrinogen alpha, beta, and gamma-chain in individuals in pool 2 may be associated with levels of LDL and vitamin E. Vitamin E and Native American genetic ancestry may also be implicated in changes of vitamin E and LDL levels. The results of this pilot study must be validated in future studies with larger sample size or in in vitro studies.
RESUMEN
BACKGROUND: Childhood-onset systemic lupus erythematosus (c-SLE) is a chronic autoimmune disease which increases cardiovascular risk factors (CRF) such as elevated homocysteine, TNF-α, and hs-C reactive protein. METHODS: We evaluated BMI, waist circumference (WC), 24-h recalls, SLEDAI-2 K, SLICC/ACR-DI, serum levels of homocysteine, folate, TNF-α, hs-C reactive protein, lipid profile, proteomic data, and duration of corticosteroid therapy in 19 c-SLE and 38 healthy volunteers. Physiological and anthropometric variables of c-SLE and healthy controls were compared by ANCOVA. k-cluster was used to separate c-SLE into two different groups with the best and the worst metabolic profile according to previous analysis showing some metabolites that were statistically different from controls, such as homocysteine, TNF-α, hs-CRP and folate levels. These two clusters were again compared with the control group regarding nutritional parameters, lipid profile and also proteomic data. RESULTS: Individuals with c-SLE presented higher BMI, WC, homocysteine, triglycerides, TNF-α, hs-CRP and lower folate levels when compared to controls. We found 10 proteins whose relative abundances were statistically different between control group and lupus clusters with the best (LCBMP) and the worst metabolic profile (LCWMP). A significant positive correlation was found between TNF-α and triglycerides and between hs-CRP and duration of corticosteroid therapy. CONCLUSION: Cardiovascular disease (CVD) risk parameters were worse in c-SLE. A less protective CVD proteomic profile was found in LCWMP.
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Proteína C-Reactiva/metabolismo , Enfermedades Cardiovasculares/etiología , Ácido Fólico/sangre , Homocisteína/sangre , Lupus Eritematoso Sistémico/sangre , Factor de Necrosis Tumoral alfa/sangre , Adolescente , Antropometría , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Estudios Transversales , Femenino , Glucocorticoides/administración & dosificación , Humanos , Lípidos/sangre , Lupus Eritematoso Sistémico/complicaciones , Estado Nutricional , Proteómica/métodos , Factores de RiesgoRESUMEN
Nine chromatographic components containing trypsin inhibitor activity were isolated from Sechium edule seeds by acetone fractionation, gel filtration, affinity chromatography and RP-HPLC in an overall yield of 46% of activity and 0.05% of protein. The components obtained with highest yield of total activity and highest specific activity were sequenced by Edman degradation and their molecular masses determined by mass spectrometry. The inhibitors contained 31, 32 and 27 residues per molecule and their sequences were: SETI-IIa, EDRKCPKILMRCKRDSDCLAKCTCQESGYCG; SETI-IIb, EEDRKCPKILMRCKRDSDCLAKCTCQESGYCG and SETI-V, CPRILMKCKLDTDCFPTCTCRPSGFCG. SETI-IIa and SETI-IIb, which differed by an amino-terminal E in the IIb form, were not separable under the conditions employed. The sequences are consistent with consensus sequences obtained from 37 other inhibitors: CPriI1meCk_DSDCla_C_C_G_CG, where capital letters are invariant amino acid residues and lower case letters are the most preserved in this position. SETI-II and SETI-V form complexes with trypsin with a 1:1 stoichiometry and have dissociation constants of 5.4x10(-11)M and 1.1x10(-9)M, respectively.
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Cucurbita/química , Proteínas de Plantas/química , Semillas/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Peso Molecular , Proteínas de Plantas/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 1 , Tripsina/química , Tripsina/metabolismo , Inhibidores de Tripsina/aislamiento & purificaciónRESUMEN
The properties of an Fe(3+)-peptide complex containing 5.6% Fe, obtained by the reaction of ferric chloride with an enzymatic hydrolysate of casein, are described. The major site of iron binding corresponds primarily to the carboxylate groups and to a lesser extent to the peptide bonds. The Fe(3+)-peptide complex is insoluble at acid pH and completely soluble at neutral to alkaline pH. When soluble, the Fe(3+) is tightly bound to the complex peptide mixture but can be displaced and complexed by a low molecular weight ligand such as cysteine. Its efficacy in relation to iron sulfate was compared in rats. Both iron sources were administrated in Milli-Q water by gastric gavage to male Wistar rats (180-200 g) after an 18 h fast with water ad libitum. Fe(3+) from the Fe(3+)-peptide complex was transferred to the blood in a dose-dependent manner (1-8 mg of Fe/kg), and the serum iron levels were significantly higher (p < 0.001) than in a similar group of rats treated with iron sulfate. In the comparative kinetics experiments, the rats received 4 mg of Fe/kg. Both iron sources presented maximum absorption, as indicated by the elevation of serum iron levels, 30 min after administration, and the AUC(0)(-->2h) of the Fe(3+)-peptide complex was significantly higher (p < 0.05) than that observed with iron sulfate. The simultaneous administration of free peptides (0-192 mg) with the Fe(3+)-peptide complex or iron sulfate did not modify the extent of absorption of iron from both sources, suggesting that the absorption is due to the complex formed and probably not to exchange reactions in the gastrointestinal tract. In the hemoglobin repletion experiments carried out on newly weaned rats with anemia induced by a low-iron diet, supplementation of the diet with the the Fe(3+)-peptide complex was as efficient as supplementation with iron sulfate in the conversion from diet to hemoglobin iron. These results, taken together, suggest that the Fe(3+)-peptide complex is a potential compound for use as an iron source in biological situations.
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Caseínas/metabolismo , Compuestos Férricos/metabolismo , Compuestos Férricos/uso terapéutico , Deficiencias de Hierro , Péptidos/metabolismo , Anemia Ferropénica/terapia , Animales , Cloruros , Dieta , Compuestos Férricos/administración & dosificación , Compuestos Férricos/farmacocinética , Hemoglobinas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Absorción Intestinal , Hierro/sangre , Hierro de la Dieta/administración & dosificación , Hierro de la Dieta/uso terapéutico , Cinética , Masculino , Ratas , Ratas Wistar , Cloruro de Sodio/farmacología , Solubilidad , Espectrofotometría Infrarroja , Estómago/efectos de los fármacosRESUMEN
Shotgun proteomics (liquid chromatography-electrospray ionization-mass spectrometry, LC-ESI-MS/MS) has dominated the strategies for global protein expression in subcells, cells, tissues, and whole organisms with several types of approaches, as isobaric tags for relative and absolute quantification (iTRAQ), isotope-coded affinity tags (ICAT), or stable isotope labeling using amino acids in cell culture (SILAC) and non-labeling (label free) methods. Shotgun proteomics practically replaced the classical 2D gel electrophoresis. Selected reaction monitoring (SRM), also denominated multiple reaction monitoring (MRM), is a targeted quantitative technology that uses a complex mixture of tryptic peptides that can be selectively detected by liquid chromatography coupled to electrospray triple-quadrupole mass spectrometer; this system can select precursor ions in combination with their correspondent product ions during collision-induced dissociation to produce specific detection related to a particular protein. Here we describe protocols that are efficient to produce a complete enzymatic trypsin digestion from complex biological matrices and concomitant material to be used for LC-SRM-MS and LC-ESI-MS/MS (labeled or label free).
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Biomarcadores , Proteómica , Marcadores de Afinidad , Cromatografía Liquida , Marcaje Isotópico , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
We describe the preparation of pancreatic enzymes hydrolysate of milk whey proteins containing low levels of aromatic amino acids. Pancreatin and trypsin/chymotrypsin (6.3 percent w/w protein) when used to hydrolyze whey proteins for 27 h at 37±2 °C, released 74 percent of the Phe, 100 percent of the Tyr and 100 percent of the Trp as free amino acids. Most of the free aromatic amino acids present in 2 kg hydrolysate were separated from the remaining peptides and other amino acids by gel filtration on a 15 liter Sephadex G-25 column eluted with 5 percent acetic acid at 60 liters 'h POT. -1' 25°C. The product, recovered in 37 percent yield, contained 0.70 mmol Phe, 0.41 mmol Tyr, and <0.01mmol Trp/100mmol recovered amino acids. The hydrolysate had a general amino acid composition similar to the whey proteins from which it was prepared and could be use as a nitrogen source for patients with phenylketonuria or tyrosinemia after the addition of appropriate aromatic amino acids...
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Errores Innatos del Metabolismo , Fenilalanina Hidroxilasa , Fenilcetonurias , Tirosinemias , Clarificación Química , Cromatografía en Gel , Filtración/métodosRESUMEN
In the present study, the effect of dietary fish oil, a highly unsaturated fat source, on the intestinal mucosa before and after proximal small bowel resection in rats was studied. Forty Wistar rats were fed defined diets containing fish oil (experimental group) or corn oil (control group). After 2 weeks, animals underwent a 50 por cento proximal small bowel resection. Mucosal disaccharidases, alkaline phosphatase, aminopeptidase, protein, DNA, and TBARs levels were assessed in samples immediately before and 6 and 12 days after surgery. Disaccharidase activities were reduced in the ileal mucosa from rats in the fish oil-fed group compared to the control group before and 12 days after surgery...