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1.
Cancers (Basel) ; 13(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207464

RESUMO

Targeting multiple cellular populations is of high therapeutic relevance for the tackling of solid tumors heterogeneity. Herein, the ability of pegylated and pH-sensitive liposomes, functionalized with the nucleolin-binding F3 peptide and containing doxorubicin (DXR)/C6-ceramide synergistic combination, to target, in vitro, ovarian cancer, including ovarian cancer stem cells (CSC), was assessed. The underlying molecular mechanism of action of the nucleolin-mediated intracellular delivery of C6-ceramide to cancer cells was also explored. The assessment of overexpression of surface nucleolin expression by flow cytometry was critical to dissipate differences identified by Western blot in membrane/cytoplasm of SKOV-3, OVCAR-3 and TOV-112D ovarian cancer cell lines. The former was in line with the significant extent of uptake into (bulk) ovarian cancer cells, relative to non-targeted and non-specific counterparts. This pattern of uptake was recapitulated with putative CSC-enriched ovarian SKOV-3 and OVCAR-3 sub-population (EpCAMhigh/CD44high). Co-encapsulation of DXR:C6-ceramide into F3 peptide-targeted liposomes improved cytotoxic activity relative to liposomes containing DXR alone, in an extent that depended on the intrinsic resistance to DXR and on the incubation time. The enhanced cytotoxicity of the targeted combination was mechanistically supported by the downregulation of PI3K/Akt pathway by C6-ceramide, only among the nucleolin-overexpressing cancer cells presenting a basal p-Akt/total Akt ratio lower than 1.

2.
Front Plant Sci ; 12: 631239, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33912202

RESUMO

Somatic embryogenesis is the process by which bipolar structures with no vascular connection with the surrounding tissue are formed from a single or a group of vegetative cells, and in conifers it can be divided into five different steps: initiation, proliferation, maturation, germination and acclimatization. Somatic embryogenesis has long been used as a model to study the mechanisms regulating stress response in plants, and recent research carried out in our laboratory has demonstrated that high temperatures during initial stages of conifer somatic embryogenesis modify subsequent phases of the process, as well as the behavior of the resulting plants ex vitro. The development of high-throughput techniques has facilitated the study of the molecular response of plants to numerous stress factors. Proteomics offers a reliable image of the cell status and is known to be extremely susceptible to environmental changes. In this study, the proteome of radiata pine somatic embryos was analyzed by LC-MS after the application of high temperatures during initiation of embryonal masses [(23°C, control; 40°C (4 h); 60°C (5 min)]. At the same time, the content of specific soluble sugars and sugar alcohols was analyzed by HPLC. Results confirmed a significant decrease in the initiation rate of embryonal masses under 40°C treatments (from 44 to 30.5%) and an increasing tendency in the production of somatic embryos (from 121.87 to 170.83 somatic embryos per gram of embryogenic tissue). Besides, heat provoked a long-term readjustment of the protein synthesis machinery: a great number of structural constituents of ribosomes were increased under high temperatures, together with the down-regulation of the enzyme methionine-tRNA ligase. Heat led to higher contents of heat shock proteins and chaperones, transmembrane transport proteins, proteins related with post-transcriptional regulation (ARGONAUTE 1D) and enzymes involved in the synthesis of fatty acids, specific compatible sugars (myo-inositol) and cell-wall carbohydrates. On the other hand, the protein adenosylhomocysteinase and enzymes linked with the glycolytic pathway, nitrogen assimilation and oxidative stress response were found at lower levels.

3.
Mar Drugs ; 19(1)2021 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-33445445

RESUMO

As Yondelis joins the ranks of approved anti-cancer drugs, the benefit from exploring the oceans' biodiversity becomes clear. From marine toxins, relevant bioproducts can be obtained due to their potential to interfere with specific pathways. We explored the cytotoxicity of toxin-bearing secretions of the polychaete Eulalia onto a battery of normal and cancer human cell lines and discovered that the cocktail of proteins is more toxic towards an ovarian cancer cell line (A2780). The secretions' main proteins were identified by proteomics and transcriptomics: 14-3-3 protein, Hsp70, Rab3, Arylsulfatase B and serine protease, the latter two being known toxins. This mixture of toxins induces cell-cycle arrest at G2/M phase after 3h exposure in A2780 cells and extrinsic programmed cell death. These findings indicate that partial re-activation of the G2/M checkpoint, which is inactivated in many cancer cells, can be partly reversed by the toxic mixture. Protein-protein interaction networks partake in two cytotoxic effects: cell-cycle arrest with a link to RAB3C and RAF1; and lytic activity of arylsulfatases. The discovery of both mechanisms indicates that venomous mixtures may affect proliferating cells in a specific manner, highlighting the cocktails' potential in the fine-tuning of anti-cancer therapeutics targeting cell cycle and protein homeostasis.


Assuntos
Anelídeos , Antineoplásicos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Toxinas Marinhas/uso terapêutico , Neoplasias Ovarianas/patologia , Animais , Antineoplásicos/isolamento & purificação , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Células K562 , Células MCF-7 , Toxinas Marinhas/isolamento & purificação , Toxinas Marinhas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo
4.
J Pharm Biomed Anal ; 187: 113323, 2020 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-32470692

RESUMO

Since dopamine (DA) was discovered as an essential neurotransmitter, with a profound impact on motor control, memory, and behavioral impulses, the pathogenesis of several neurodegenerative and neuropsychiatric disorders have been associated with the dysfunction of the dopaminergic system. Regarding this, the most common drugs used to treat these pathologies act on the dopaminergic neurons. Therefore, the measurement of DA and its precursors and metabolites levels can be a useful tool to help the diagnosis and development of targeted therapeutic approaches to neurological disorders. Furthermore, monitoring and detecting DA metabolism (DA, precursors, and metabolites) in biological samples, like plasma, urine, and cerebrospinal fluid, constitute an interesting subject from a clinical perspective. However, the development of suitable and efficient methods to determine these compounds in biological samples remains a challenge. Thus, this review provides an overview of the recent advances and available methodologies to quantify DA and its precursors and metabolites in plasma samples focusing on previous reports which used less than two milliliters. Also, it deals with the actual extraction and separation techniques, as well as detection modes; and it gives a perspective, on the present-day, about the use of analytical methods as a helpful tool to improve diagnosis.


Assuntos
Dopamina/metabolismo , Terapia de Alvo Molecular , Doenças do Sistema Nervoso/diagnóstico , Animais , Dopamina/análise , Dopamina/sangue , Humanos , Doenças do Sistema Nervoso/fisiopatologia , Doenças do Sistema Nervoso/terapia
5.
J Am Med Dir Assoc ; 21(10): 1513.e1-1513.e17, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32001171

RESUMO

OBJECTIVES: Nutritional insufficiencies have been associated with cognitive impairment. Understanding whether nutritional biomarker levels are associated with clinical progression could help to design dietary intervention trials. This longitudinal study examined a panel of nutritional biomarkers in relation to clinical progression in patients with subjective cognitive decline (SCD) or mild cognitive impairment (MCI). DESIGN, SETTING AND PARTICIPANTS: We included 299 patients without dementia (n = 149 SCD; age 61 ± 10 years, female 44%, n = 150 MCI; age 66 ± 8 years, female 38%). Median (interquartile range) follow-up was 3 (2-5) years. METHODS: We measured 28 nutritional biomarkers in blood and 5 in cerebrospinal fluid (CSF), associated with 3 Alzheimer's disease pathologic processes: vascular change (lipids), synaptic dysfunction (homocysteine-related metabolites), and oxidative stress (minerals and vitamins). Nutritional biomarker associations with clinical progression to MCI/dementia and cognitive decline based on the Mini-Mental State Examination score were evaluated using Cox proportional hazard models and linear mixed models. We used partial least squares Cox models (PLS-Cox) to examine nutritional biomarker profiles associated with clinical progression. RESULTS: In the total group, high high-density lipoprotein (HDL) levels were associated with clinical progression and cognitive decline. In SCD, high folate and low bilirubin levels were associated with cognitive decline. In MCI, low CSF S-adenosylmethionine (SAM) and high theobromine were associated with clinical progression to dementia and high HDL, cholesterol, iron, and 1,25(OH)2 vitamin D were associated with cognitive decline. PLS-Cox showed 1 profile for SCD, characterized by high betaine and folate and low zinc associated with clinical progression. In MCI, a profile with high theobromine and HDL and low triglycerides and a second profile with high plasma SAM and low cholesterol were associated with risk of dementia. CONCLUSION AND IMPLICATIONS: High HDL was most consistently associated with clinical progression. Moreover, different nutritional biomarker profiles for SCD and MCI showed promising associations with clinical progression. Future dietary (intervention) studies could use nutritional biomarker profiles to select patients, taking into account the disease stage.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Idoso , Biomarcadores , Disfunção Cognitiva/diagnóstico , Progressão da Doença , Feminino , Humanos , Estudos Longitudinais , Pessoa de Meia-Idade , Testes Neuropsicológicos
6.
Int J Mol Sci ; 21(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861944

RESUMO

Casuarina glauca displays high levels of salt tolerance, but very little is known about how this tree adapts to saline conditions. To understand the molecular basis of C. glauca response to salt stress, we have analyzed the proteome from branchlets of plants nodulated by nitrogen-fixing Frankia Thr bacteria (NOD+) and non-nodulated plants supplied with KNO3 (KNO3+), exposed to 0, 200, 400, and 600 mM NaCl. Proteins were identified by Short Gel, Long Gradient Liquid Chromatography coupled to Tandem Mass Spectrometry and quantified by Sequential Window Acquisition of All Theoretical Mass Spectra -Mass Spectrometry. 600 proteins were identified and 357 quantified. Differentially Expressed Proteins (DEPs) were multifunctional and mainly involved in Carbohydrate Metabolism, Cellular Processes, and Environmental Information Processing. The number of DEPs increased gradually with stress severity: (i) from 7 (200 mM NaCl) to 40 (600 mM NaCl) in KNO3+; and (ii) from 6 (200 mM NaCl) to 23 (600 mM NaCl) in NOD+. Protein-protein interaction analysis identified different interacting proteins involved in general metabolic pathways as well as in the biosynthesis of secondary metabolites with different response networks related to salt stress. Salt tolerance in C. glauca is related to a moderate impact on the photosynthetic machinery (one of the first and most important stress targets) as well as to an enhancement of the antioxidant status that maintains cellular homeostasis.


Assuntos
Frankia/fisiologia , Magnoliopsida/fisiologia , Proteínas de Plantas/metabolismo , Nódulos Radiculares de Plantas/fisiologia , Tolerância ao Sal , Magnoliopsida/microbiologia , Espectrometria de Massas/métodos , Proteoma/metabolismo , Proteômica/métodos , Nódulos Radiculares de Plantas/microbiologia , Salinidade , Simbiose
7.
Molecules ; 24(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394755

RESUMO

Caffeine is one of the most widely consumed psycho-stimulants. The study of the beneficial effects of caffeine consumption to decrease the risk of developing several neuropsychiatric pathologies is receiving increasing attention. Thus, accurate and sensitive methods have been developed, mainly by LC-MS/MS, in order to quantify caffeine and its metabolites. These quantifications of caffeine and its metabolites by LC-MS/MS require a considerable effort to select or find a surrogate matrix, without the compounds of interest, to be used in the calibration curves. Thus, we evaluated the possibility of using calibration curves prepared in solvent instead of calibration curves prepared in human plasma. Results show that the calibration curves prepared in solvent and in human plasma were similar by comparing their slopes and interceptions, and the accuracy and precision were within the limits of acceptance for both calibration curves. This work demonstrates that, by using internal standards, it is possible to use a calibration curve in solvent instead of a calibration curve in plasma to perform an accurate and precise quantification of caffeine and theobromine.


Assuntos
Cafeína/análise , Cromatografia Líquida , Espectrometria de Massas em Tandem , Teobromina/análise , Cafeína/sangue , Cafeína/química , Cromatografia Líquida/métodos , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Teobromina/sangue , Teobromina/química , Teofilina/análise , Teofilina/sangue , Teofilina/química
8.
Methods Mol Biol ; 2044: 321-336, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432422

RESUMO

Cerebrospinal fluid (CSF) has been considered the key source for the search of biomarkers, in particular for neurological diseases, such as Alzheimer's and Parkinson's disease, since it reflects the state of the central nervous system (CNS). Finding biomarkers in the earliest stages of neurodegenerative diseases has become imperative, since, at the moment, there are no drugs that can reverse these pathological processes. Untargeted metabolomics analysis by liquid chromatography combined with SWATH-MS relative quantification is an emerging approach to search for potential biomarkers. In this chapter, we describe a method for untargeted metabolomics analysis of CSF samples that can also be used in parallel to a proteomics approach. The analysis is focused on the SWATH acquisition mode, where beyond precursor's relative quantification, the information of the MS/MS relative quantification is also used to help in the search of new potential biomarkers.


Assuntos
Biomarcadores/líquido cefalorraquidiano , Metabolômica/métodos , Biomarcadores/química , Proteínas do Líquido Cefalorraquidiano/química , Proteínas do Líquido Cefalorraquidiano/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Humanos , Software , Extração em Fase Sólida , Espectrometria de Massas em Tandem , Fluxo de Trabalho
9.
Artigo em Inglês | MEDLINE | ID: mdl-27107853

RESUMO

A liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-(2)H2]glucose and [U-(13)C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-(13)C6]glucose and an oral glucose load enriched with [6,6-(2)H2]glucose. The sensitivity is sufficient for analysis of the equivalent to <5µL of blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments.


Assuntos
Glicemia/análise , Cromatografia Líquida/métodos , Teste em Amostras de Sangue Seco/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Isótopos de Carbono/administração & dosagem , Isótopos de Carbono/farmacocinética , Glucose/administração & dosagem , Glucose/farmacocinética , Limite de Detecção , Modelos Lineares , Ratos , Reprodutibilidade dos Testes
10.
Biopreserv Biobank ; 14(4): 289-97, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26937781

RESUMO

Biobank saliva sample quality depends on specific criteria applied to collection, processing, and storage. In spite of the growing interest in saliva as a diagnostic fluid, few biobanks currently store large collections of such samples. The development of a standard operating procedure (SOP) for saliva collection and quality control is fundamental for the establishment of a new saliva biobank, which stores samples to be made available to the saliva research community. Different collection methods were tested regarding total volume of protein obtained, protein content, and protein profiles, and the results were used to choose the best method for protein studies. Furthermore, the impact of the circadian variability and inter- and intraindividual differences, as well as the saliva sample stability at room temperature, were also evaluated. Considering our results, a sublingual cotton roll method for saliva collection proved to produce saliva with the best characteristics and should be applied in the morning, whenever possible. In addition, there is more variability in salivary proteins between individuals than in the same individual for a 5-month period. According to the electrophoretic protein profile, protein stability is guaranteed for 24 hours at room temperature and the protein degradation profile and protein identification were characterized. All this information was used to establish an SOP for saliva collection, processing, and storage in a biobank. We conclude that it is possible to collect saliva using an easy and inexpensive protocol, resulting in saliva samples for protein analysis with sufficient quality for biobanking purposes.


Assuntos
Saliva/metabolismo , Proteínas e Peptídeos Salivares/metabolismo , Manejo de Espécimes/normas , Adulto , Bancos de Espécimes Biológicos , Ritmo Circadiano , Feminino , Humanos , Masculino , Estabilidade Proteica , Controle de Qualidade , Proteínas e Peptídeos Salivares/química , Manejo de Espécimes/métodos , Temperatura , Adulto Jovem
11.
J Alzheimers Dis ; 47(4): 1069-78, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26401784

RESUMO

Caffeine may be protective against Alzheimer's disease (AD) by modulating amyloid-ß (Aß) metabolic pathways. The present work aimed to study a possible association of caffeine consumption with the cerebrospinal fluid (CSF) biomarkers, particularly Aß. The study included 88 patients with AD or mild cognitive impairment. The consumption of caffeine and theobromine was evaluated using a validated food questionnaire. Quantification of caffeine and main active metabolites was performed with liquid chromatography coupled to tandem mass spectrometry. The levels of A(1-42), total tau, and phosphorylated tau in the CSF were determined using sandwich ELISA methods and other Aß species, Aß(X-38), Aß(X-40), and Aß(X-42), with the MSD Aß Triplex assay. The concentration of caffeine was 0.79±1.15 µg/mL in the CSF and 1.20±1.88 µg/mL in the plasma. No correlation was found between caffeine consumption and Aß42 in the CSF. However, a significant positive correlation was found between the concentrations of theobromine, both in the CSF and in the plasma, with Aß42 in the CSF. Theobromine in the CSF was positively correlated with the levels of other xanthines in the CSF, but not in the plasma, suggesting that it may be formed by central metabolic pathways. In conclusion, caffeine consumption does not modify the levels of CSF biomarkers, and does not require to be controlled for when measuring CSF biomarkers in a clinical setting. Since theobromine is associated with a favorable Aß profile in the CSF, the possibility that it might have a protective role in AD should be further investigated.


Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Cafeína/líquido cefalorraquidiano , Dieta , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/sangue , Biomarcadores/líquido cefalorraquidiano , Cafeína/administração & dosagem , Cafeína/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Teobromina/sangue , Teobromina/líquido cefalorraquidiano , Proteínas tau/sangue , Proteínas tau/líquido cefalorraquidiano
12.
J Diabetes Res ; 2015: 542029, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26236747

RESUMO

Mice deficient in adipose triglyceride lipase (ATGL(-/-)) present elevated ectopic lipid levels but are paradoxically glucose-tolerant. Measurement of endogenous glucose production (EGP) and Cori cycle activity provide insights into the maintenance of glycemic control in these animals. These parameters were determined in 7 wild-type (ATGL(+/-)) and 6 ATGL(-/-) mice by a primed-infusion of [U-(13)C6]glucose followed by LC-MS/MS targeted mass-isotopomer analysis of blood glucose. EGP was quantified by isotope dilution of [U-(13)C6]glucose while Cori cycling was estimated by analysis of glucose triose (13)C-isotopomers. Fasting plasma free fatty-acids were significantly lower in ATGL(-/-) versus control mice (0.43 ± 0.05 mM versus 0.73 ± 0.11 mM, P < 0.05). Six-hour fasting EGP rates were identical for both ATGL(-/-) and control mice (79 ± 11 versus 71 ± 7 µmol/kg/min, resp.). Peripheral glucose metabolism was dominated by Cori cycling (80 ± 2% and 82 ± 7% of glucose disposal for ATGL(-/-) and control mice, resp.) indicating that peripheral glucose oxidation was not significantly upregulated in ATGL(-/-) mice under these conditions. The glucose (13)C-isotopomer distributions in both ATGL(-/-) and control mice were consistent with extensive hepatic pyruvate recycling. This suggests that gluconeogenic outflow from the Krebs cycle was also well compensated in ATGL(-/-) mice.


Assuntos
Glicemia/metabolismo , Ciclo do Ácido Cítrico , Ácidos Graxos não Esterificados/metabolismo , Lipase/genética , Fígado/metabolismo , Animais , Isótopos de Carbono , Cromatografia Líquida , Camundongos , Camundongos Knockout , Oxirredução , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas em Tandem
13.
Biochem J ; 459(3): 441-53, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24564673

RESUMO

PRRXL1 [paired related homeobox-like 1; also known as DRG11 (dorsal root ganglia 11)] is a paired-like homeodomain transcription factor expressed in DRG and dSC (dorsal spinal cord) nociceptive neurons. PRRXL1 is crucial for the establishment and maintenance of nociceptive circuitry, as Prrxl1(-/-) mice present neuronal loss, reduced pain sensitivity and failure to thrive. In the present study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, whereas the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified, Ser¹¹9 and Ser²³8. Phosphorylation at Ser¹¹9 is shown to be determinant for PRRXL1 conformation and transcriptional activity. Ser¹¹9 phosphorylation is thus proposed as a mechanism for regulating PRRXL1 function and conformation during nociceptive system development.


Assuntos
Gânglios Espinais/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Nociceptores/metabolismo , Processamento de Proteína Pós-Traducional , Serina/metabolismo , Medula Espinal/metabolismo , Fatores de Transcrição/metabolismo , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Desenvolvimento Embrionário , Feminino , Proteínas Ligadas por GPI , Gânglios Espinais/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Camundongos Endogâmicos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Medula Espinal/embriologia , Fatores de Transcrição/química , Fatores de Transcrição/genética
14.
Artigo em Inglês | MEDLINE | ID: mdl-21777686

RESUMO

Glucose metabolism in free-swimming fasted and fed seabass was studied using deuterated water ((2)H(2)O). After transfer to seawater enriched with 4.9% (2)H(2)O for 6-h or for 72-h, positional and mole percent enrichment (MPE) of plasma glucose and water were quantified by (2)H NMR and ESI-MS/MS. Plasma water (2)H-enrichment reached that of seawater within 6h. In both fasted and fed fish, plasma glucose MPE increased asymptotically attaining ~55% of plasma water enrichment by 72 h. The distribution of (2)H-enrichment between the different glucose positions was relatively uniform. The gluconeogenic contribution to glucose that was synthesized during (2)H(2)O administration was estimated from the ratio of position 5 and 2 glucose enrichments. For both fed and fasted fish, gluconeogenesis accounted for 98±1% of the glucose that was produced during the 72-h (2)H(2)O administration period. For fasted fish, gluconeogenic contributions measured after 6h were identical to 72-h values (94±3%). For fed fish, the apparent gluconeogenic contribution at 6-h was significantly lower compared to 72-h (79±5% versus 98±1%, p<0.05). This may reflect a brief augmentation of gluconeogenic flux by glycogenolysis after feeding and/or selective enrichment of plasma glucose position 2 via futile glucose-glucose-6-phosphate cycling.


Assuntos
Bass/metabolismo , Glicemia/metabolismo , Animais , Glicemia/química , Óxido de Deutério/química , Europa (Continente) , Pesqueiros , Glicogenólise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas em Tandem
15.
Expert Rev Proteomics ; 7(5): 655-63, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20973639

RESUMO

Peptide fractionation is extremely important in proteomics approaches. Full proteome characterization is desired from complex organisms, and with growing interest in post-translational modifications an extended protein sequence coverage is required. Peptide fractionation techniques have the great challenge of feeding current mass spectrometers in a way in which these issues are met. Peptide fractionation can be divided into three simple components: the column characteristics; the mobile phase; and peptide properties (charge, polarity, hydrophobicity and size). The current challenges are in the combination of these three components to allow comprehensive proteomics studies to be improved.


Assuntos
Peptídeos/química , Proteômica/métodos , Cromatografia por Troca Iônica , Cromatografia Líquida , Interações Hidrofóbicas e Hidrofílicas , Focalização Isoelétrica/métodos
16.
Ann N Y Acad Sci ; 1139: 212-21, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18991867

RESUMO

The glutamate-glutamine cycle between neurons and glia is tightly related to excitatory glutamatergic and inhibitory GABAergic regulation in brain. The role of this neuron-astrocyte cross-talk on the neurotoxicity induced by amphetamines is not understood. Also, the impact of neurotoxic doses of amphetamines on the balance between glutamatergic and GABAergic circuits is largely unknown. The aim of this work was to assess the acute effect of a neurotoxic regimen of amphetamine (AMPH) on glutamine (GLN, an astrocytic marker) levels and on glutamine/glutamate (an index for glutamate-glutamine cycle) and GABA/glutamate ratios in rat brain. Sprague-Dawley rats were sacrificed 4 and 24 h after a single-dose regimen of AMPH (30 mg/kg, i.p.), and the caudate-putamen (CPu), frontal cortex (FC), and hippocampus (Hp) were dissected for analysis of glutamate (GLU), gamma-aminobutyric acid (GABA), and GLN. The total content of these amino acids was measured using a microbore HPLC electrochemical detector. Although AMPH did not change GLU levels, it increased both GLN content and GLN/GLU ratio (160-469%) at 4 h, but not at 24 h, in all regions after injection. Striatal GABA levels and GABA/GLU ratio were increased (46 and 100%, respectively) at 24 h. In hippocampus the GABA/GLU increase (60%) occurred as early as 4 h after treatment. To the contrary, AMPH exerted no effect in GABA/GLU balance in frontal cortex. These data strongly suggest that this neurotoxic AMPH regimen provoked an early increase in the glutamate-glutamine cycle between neurons and glia. This increase may ultimately lead to an upregulation of the inhibitory system as a compensatory response.


Assuntos
Anfetamina/farmacologia , Encéfalo , Estimulantes do Sistema Nervoso Central/farmacologia , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Animais , Astrócitos/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Núcleo Caudado/metabolismo , Lobo Frontal/metabolismo , Hipocampo/metabolismo , Masculino , Neurônios/metabolismo , Putamen/metabolismo , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismo
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