RESUMO
In recent years, human dihydroorotate dehydrogenase inhibitors have been associated with acute myelogenous leukemia as well as studied as potent host targeting antivirals. Starting from MEDS433 (IC50 1.2 nM), we kept improving the structure-activity relationship of this class of compounds characterized by 2-hydroxypyrazolo[1,5-a]pyridine scaffold. Using an in silico/crystallography supported design, we identified compound 4 (IC50 7.2 nM), characterized by the presence of a decorated aryloxyaryl moiety that replaced the biphenyl scaffold, with potent inhibition and pro-differentiating abilities on AML THP1 cells (EC50 74 nM), superior to those of brequinar (EC50 249 nM) and boosted when in combination with dipyridamole. Finally, compound 4 has an extremely low cytotoxicity on non-AML cells as well as MEDS433; it has shown a significant antileukemic activity in vivo in a xenograft mouse model of AML.
Assuntos
Leucemia Mieloide Aguda , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Animais , Humanos , Camundongos , Antivirais/farmacologia , Di-Hidro-Orotato Desidrogenase , Dipiridamol/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Piridinas/farmacologia , Piridinas/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Lapatinib is a highly selective reversible inhibitor of the tyrosine kinase domains of HER2 and EGFR, approved for the treatment of advanced stage HER2-overexpressing breast cancers. Although targeted therapy with lapatinib provides initial clinical advantage, cancer cells' adaptive responses can overcome the inhibitory effects of lapatinib. HER3 upregulation and autocrine induction of HER3 ligand neuregulin-1 (NRG), have been implicated in the restoration of AKT and ERK1/2 activity and rescue of cell proliferation. In this study we evaluated the effects of lapatinib alone and in combination with AMPK activator GSK-621 in HER2-overexpressing breast cancer cell lines SKBR3 and BT474. Our results show that in cells exposed to lapatinib and GSK-621 in combination, lapatinib-mediated HER3 upregulation was reduced and reactivation of AKT and ERK1/2 kinases was prevented. The two drugs in combination decreased cell viability in a synergistic manner and greatly reduced the ability of NRG to rescue cell proliferation. Finally, we provide evidence that in cells exposed to lapatinib and GSK-621 in combination the establishment of a transcriptionally permissive chromatin structure at the HER3 promoter is hampered. The results of this study highlight a potential role for AMPK activation in counteracting lapatinib-induced adaptive responses of HER2-overexpressing breast cancer cells.
Assuntos
Antineoplásicos , Neoplasias da Mama , Proteínas Quinases Ativadas por AMP/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Cromatina , Feminino , Humanos , Lapatinib/farmacologia , Ligantes , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/farmacologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
The connection with acute myelogenous leukemia (AML) of dihydroorotate dehydrogenase (hDHODH), a key enzyme in pyrimidine biosynthesis, has attracted significant interest from pharma as a possible AML therapeutic target. We recently discovered compound 1, a potent hDHODH inhibitor (IC50 = 1.2 nM), able to induce myeloid differentiation in AML cell lines (THP1) in the low nM range (EC50 = 32.8 nM) superior to brequinar's phase I/II clinical trial (EC50 = 265 nM). Herein, we investigate the 1 drug-like properties observing good metabolic stability and no toxic profile when administered at doses of 10 and 25 mg/kg every 3 days for 5 weeks (Balb/c mice). Moreover, in order to identify a backup compound, we investigate the SAR of this class of compounds. Inside the series, 17 is characterized by higher potency in inducing myeloid differentiation (EC50 = 17.3 nM), strong proapoptotic properties (EC50 = 20.2 nM), and low cytotoxicity toward non-AML cells (EC30(Jurkat) > 100 µM).
Assuntos
Compostos de Bifenilo/química , Inibidores Enzimáticos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirazóis/química , Piridinas/química , Animais , Apoptose/efeitos dos fármacos , Sítios de Ligação , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Meia-Vida , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Pirazóis/metabolismo , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
NF-κB-inducing kinase (NIK), an oncogenic drug target that is associated with various cancers, is a central signalling component of the non-canonical pathway. A blind screening process, which established that amino pyrazole related scaffolds have an effect on IKKbeta, led to a hit-to-lead optimization process that identified the aminopyrazole 3a as a low µM selective NIK inhibitor. Compound 3a effectively inhibited the NIK-dependent activation of the NF-κB pathway in tumour cells, confirming its selective inhibitory profile.
RESUMO
Human dihydroorotate dehydrogenase ( hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5- a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This Article reports the design, synthesis, SAR, X-ray crystallography, biological assays, and physicochemical characterization of the new class of hDHODH inhibitors.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Desenho de Fármacos , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirazóis/química , Pirazóis/farmacologia , Sítios de Ligação , Di-Hidro-Orotato Desidrogenase , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Células Jurkat , Modelos Moleculares , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
A new generation of potent hDHODH inhibitors designed by a scaffold-hopping replacement of the quinolinecarboxylate moiety of brequinar, one of the most potent known hDHODH inhibitors, is presented here. Their general structure is characterized by a biphenyl moiety joined through an amide bridge with an acidic hydroxyazole scaffold (hydroxylated thiadiazole, pyrazole and triazole). Molecular modelling suggested that these structures should adopt a brequinar-like binding mode involving interactions with subsites 1, 2 and 4 of the hDHODH binding site. Initially, the inhibitory activity of the compounds was studied on recombinant hDHODH. The most potent compound of the series in the enzymatic assays was the thiadiazole analogue 4 (IC50 16 nM). The activity was found to be dependent on the fluoro substitution pattern at the biphenyl moiety as well as on the choice/substitution of the heterocyclic ring. Structure determination of hDHODH co-crystallized with one representative compound from each series (4, 5 and 6) confirmed the brequinar-like binding mode as suggested by modelling. The specificity of the observed effects of the compound series was tested in cell-based assays for antiproliferation activity using Jurkat cells and PHA-stimulated PBMC. These tests were also verified by addition of exogenous uridine to the culture medium. In particular, the triazole analogue 6 (IC50 against hDHODH: 45 nM) exerted potent in vitro antiproliferative and immunosuppressive activity without affecting cell survival.
Assuntos
Azóis/química , Inibidores Enzimáticos/química , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Sítios de Ligação , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Di-Hidro-Orotato Desidrogenase , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxilação , Terapia de Imunossupressão , Células Jurkat , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Difração de Raios XRESUMO
The NF-κB signaling pathway is a validated oncological target. Here, we applied scaffold hopping to IMD-0354, a presumed IKKß inhibitor, and identified 4-hydroxy-N-[3,5-bis(trifluoromethyl)phenyl]-1,2,5-thiadiazole-3-carboxamide (4) as a nM-inhibitor of the NF-κB pathway. However, both 4 and IMD-0354, being potent inhibitors of the canonical NF-κB pathway, were found to be inactive in human IKKß enzyme assays.
RESUMO
Long term durability of osseointegrated implants depends on bone adaptation to stress and strain occurring in proximity of the prosthesis. Mechanical overloading, as well as disuse, may reduce the stability of implants by provoking bone resorption. However, an appropriate mechanical environment can improve integration. Several studies have focused on the definition of numerical methods to predict bone peri-implant adaptation to the mechanical environment. Existing adaptation models differ notably in the type of mechanical variable adopted as stimulus but also in the bounds and shape of the adaptation rate equation. However, a general comparison of the different approaches on a common benchmark case is still missing and general guidelines to determine physically sound parameters still need to be developed. This current work addresses these themes in two steps. Firstly, the histograms of effective stress, strain and strain energy density are compared for rat tibiae in physiological (homeostatic) conditions. According to the Mechanostat, the ideal stimulus should present a clearly defined, position and tissue invariant lazy zone in homeostatic conditions. Our results highlight that only the octahedral shear strain presents this characteristic and can thus be considered the optimal choice for implementation of a continuum level bone adaptation model. Secondly, critical modeling parameters such as lazy zone bounds, type of rate equation and bone overloading response are classified depending on their influence on the numerical predictions of bone adaptation. Guidelines are proposed to establish the dominant model parameters based on experimental and simulated data.
Assuntos
Adaptação Fisiológica , Análise de Elementos Finitos , Próteses e Implantes , Tíbia/fisiologia , Animais , Feminino , Osseointegração , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Suporte de CargaRESUMO
OBJECTIVES: (i) To assess the effects of mechanical overloading on implant integration in rat tibiae, and (ii) to numerically predict peri-implant bone adaptation. MATERIALS AND METHODS: Transcutaneous titanium implants were simultaneously placed into both tibiae of rats (n = 40). After 2 weeks of integration, the implants of the right tibiae were stimulated daily for 4 weeks with loads up to 5N (corresponding to peak equivalent strains of 3300 ± 500 µÎµ). The effects of stimulation were assessed by ex vivo mechanical tests and quantification of bone mineral density (BMD) in selected regions of interests (ROIs). Specimen-specific finite element models were generated and processed through an iterative algorithm to mimic bone adaptation. RESULTS: Bilateral implantation provoked an unstable integration that worsened when mild (2-4N) external loads were applied. In contrast, a stimulation at 5N tended to "counterbalance" the harmful effects of daily activity and, if applied to well-integrated specimens, significantly augmented the implants' resistance to failure (force: +73% P < 0.01, displacement: +50% P < 0.01 and energy: +153% P < 0.01). Specimen-specific numerical predictions were in close agreement with the experimental findings. Both local and overall BMD variations, as well as the implants' lateral stability, were predicted with small errors (0.14 gHA/cm3 and 0.64%, respectively). CONCLUSIONS: The rats' daily activity detrimentally affects implant integration. Conversely, external stimulations of large magnitudes counterbalance this effect and definitively improve integration. These changes can be predicted using the proposed numerical approach.
Assuntos
Implantação Dentária Endóssea/métodos , Implantes Dentários , Osseointegração/fisiologia , Tíbia/cirurgia , Animais , Fenômenos Biomecânicos , Densidade Óssea , Feminino , Análise de Elementos Finitos , Implantes Experimentais , Ratos , Ratos Sprague-Dawley , Estresse Mecânico , Tíbia/diagnóstico por imagem , Titânio , Tomografia Computadorizada por Raios XRESUMO
Hydrogen peroxide synthesis from hydrogen and oxygen in the gas phase is postulated to be a key reaction step in the gas phase epoxidation of propene using gold-titanium silicate catalysts. During this process H2O2 is consumed in a secondary step to oxidise an organic molecule so is typically not observed as a reaction product. We demonstrate that using AuPd nanoparticles, which are known to have high H2O2 synthesis rates in the liquid phase, it is possible to not only oxidise organic molecules in the gas phase but to detect H2O2 for the first time as a reaction product in both a fixed bed reactor and a pulsed Temporal Analysis of Products (TAP) reactor without stabilisers present in the gas feed. This observation opens up possibility of synthesising H2O2 directly using a gas phase reaction.
RESUMO
Implanted rat bones play a key role in studies involving fracture healing, bone diseases or drugs delivery among other themes. In most of these studies the implants integration also depends on the animal daily activity and musculoskeletal loads, which affect the implants mechanical environment. However, the tissue adaption to the physiological loads is often filtered through control groups or not inspected. This work aims to investigate experimentally and numerically the effects of the daily activity on the integration of implants inserted in the rat tibia, and to establish a physiological loading condition to analyse the peri-implant bone stresses during gait. Two titanium implants, single and double cortex crossing, are inserted in the rat tibia. The animals are caged under standard conditions and divided in three groups undergoing progressive integration periods. The results highlight a time-dependent increase of bone samples with significant cortical bone loss. The phenomenon is analysed through specimen-specific Finite Element models involving purpose-built musculoskeletal loads. Different boundary conditions replicating the post-surgery bone-implant interaction are adopted. The effects of the gait loads on the implants integration are quantified and agree with the results of the experiments. The observed cortical bone loss can be considered as a transient state of integration due to bone disuse atrophy, initially triggered by a loss of bone-implant adhesion and subsequently by a cyclic opening of the interface.
Assuntos
Análise de Elementos Finitos , Marcha , Teste de Materiais , Próteses e Implantes , Tíbia/fisiologia , Suporte de Carga , Animais , Ratos , TitânioRESUMO
Imatinib mesylate is a tyrosine kinase inhibitor with selectivity for abelson tyrosine-protein kinase 1 (c-Abl), breakpoint cluster region (Bcr)-Abl fusion protein (Bcr-Abl), mast/stem cell growth factor receptor Kit (c-Kit), and platelet-derived growth factor receptor (PDGFR). Previous studies demonstrated that imatinib in the low micromolar range exerted antiproliferative effects on neuroblastoma cell lines. However, although neuroblastoma cells express c-Kit and PDGFR, the imatinib concentrations required to achieve significant growth inhibitory effects (≥ 10 µM) are substantially higher than those required for inhibition of ligand-induced phosphorylation of wild type c-Kit and PDGFR (≤ 1 µM), suggesting that additional mechanisms are responsible for the antitumor activity of imatinib on these cells. In this study, we show that treatment of neuroblastoma cell lines with 1-15 µM imatinib resulted in a dose dependent inhibition of 5-bromo-2'-deoxyuridine (BrdU) incorporation into newly synthesized DNA. The antiproliferative effect of imatinib was dependent on the upregulation of the cyclin-dependent kinase (CDK) inhibitor p27(KIP1) in the nuclear compartment as a result of increased p27(KIP1) protein stability. We demonstrate that the mechanism of p27(KIP1) stabilization relied on inhibition of p27(KIP1) phosphorylation on tyrosine residues by c-Abl. We provide evidence that in neuroblastoma cell lines a significant fraction of cellular c-Abl is phosphorylated on Tyr-245, consistent with an open and active conformation. Notably, exposure to imatinib did not affect Tyr-245 phosphorylation. Given the low affinity of active c-Abl for imatinib, these data provide a molecular explanation for the relatively high imatinib concentrations required to inhibit neuroblastoma cell proliferation.
Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Genes abl/fisiologia , Neuroblastoma/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Relação Dose-Resposta a Droga , Genes abl/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
The direct synthesis of hydrogen peroxide offers a potentially green route to the production of this important commodity chemical. Early studies showed that Pd is a suitable catalyst, but recent work indicated that the addition of Au enhances the activity and selectivity significantly. The addition of a third metal using impregnation as a facile preparation method was thus investigated. The addition of a small amount of Pt to a CeO2-supported AuPd (weight ratio of 1:1) catalyst significantly enhanced the activity in the direct synthesis of H2O2 and decreased the non-desired over-hydrogenation and decomposition reactions. The addition of Pt to the AuPd nanoparticles influenced the surface composition, thus leading to the marked effects that were observed on the catalytic formation of hydrogen peroxide. In addition, an experimental approach that can help to identify the optimal nominal ternary alloy compositions for this reaction is demonstrated.
RESUMO
Accumulation of transactive response DNA binding protein (TDP-43) fragments in motor neurons is a post mortem hallmark of different neurodegenerative diseases. TDP-43 fragments are the products of the apoptotic caspases-3 and -7. Either excessive or insufficient cellular Ca(2+) availability is associated with activation of apoptotic caspases. However, as far as we know, it is not described whether activation of caspases, due to restricted intracellular Ca(2+), affects TDP-43 cleavage. Here we show that in various cell lineages with restricted Ca(2+) availability, TDP-43 is initially cleaved by caspases-3 and -7 and then, also by caspases-6 and -8 once activated by caspase-3. Furthermore, we disclose the existence of a TDP-43 caspase-mediated fragment of 15kDa, in addition to the well-known fragments of 35 and 25kDa. Interestingly, with respect to the other two fragments this novel fragment is the major product of caspase activity on murine TDP-43 whereas in human cell lines the opposite occurs. This outcome should be considered when murine models are used to investigate TDP-43 proteinopathies.
Assuntos
Apoptose/genética , Cálcio/metabolismo , Caspases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , CamundongosRESUMO
The authors propose a protocol to derive finite element (FE) models from micro computer tomography scans of implanted rat bone. A semi-automatic procedure allows segmenting the images using specimen-specific bone mineral density (BMD) thresholds. An open-source FE model generator processes the segmented images to a quality tetrahedral mesh. The material properties assigned to each element are integrated from the BMD field. Piecewise, threshold-dependent density-elasticity relationships are implemented to limit the effects of metal artefacts. A detailed sensitivity study highlights the coherence of the generated models and quantifies the influence of the modelling parameters on the results. Two applications of the protocol are proposed. The stiffness of bare and implanted rat tibiae specimens is predicted by simulating three-point bending and inter-implant displacement, respectively. Results are compared with experimental tests. The mean value and the variability between the specimens are well captured in both tests.
Assuntos
Osso e Ossos/fisiologia , Análise de Elementos Finitos , Animais , Densidade Óssea , Osso e Ossos/diagnóstico por imagem , Simulação por Computador , Elasticidade , Modelos Biológicos , Próteses e Implantes , Ratos , Ratos Sprague-Dawley , Tomografia Computadorizada por Raios XRESUMO
Engagement of the TCR and CD28 coreceptor by their respective ligands activates signal transduction cascades that ultimately lead to the activation of the transcription factors NFAT, AP-1, and NF-κB, which are required for the expression of cytokines and T cell clonal expansion. Previous studies have demonstrated that in mature T cells, activation of AP-1 and NF-κB is dependent on protein kinase C θ, suggesting the existence of a common signaling pathway. In this study, we show that in human primary CD4(+) T cells, exposure to the cell-permeable IKKß inhibitor PS-1145 or genetic ablation of IKKß abrogates cell proliferation and impairs the activation of NF-κB and AP-1 transcription factors in response to engagement of CD3 and CD28 coreceptor. In addition, we show that stimulation of T cells in the absence of IKKß activity promotes the time-dependent and cyclosporine-sensitive expression of negative regulators of T cell signaling leading to a hyporesponsive state of T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Quinase I-kappa B/imunologia , Ativação Linfocitária/imunologia , NF-kappa B/imunologia , Fator de Transcrição AP-1/imunologia , Western Blotting , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linhagem Celular , Separação Celular , Ensaio de Desvio de Mobilidade Eletroforética , Citometria de Fluxo , Humanos , Quinase I-kappa B/metabolismo , NF-kappa B/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/imunologia , Fator de Transcrição AP-1/metabolismoRESUMO
A recently described series of nitrooxyacyl derivatives of salicylic acid, displaying aspirin-like anti-inflammatory and platelet anti-aggregatory properties, were evaluated for their abilities to inhibit cyclooxygenase (COX). A number of these compounds irreversibly inhibited both COX-1 and COX-2 isoforms when tested in isolated human platelets and monocytes. Further studies using COX-1 expressed in human HEK293T cells showed that this inhibition mechanism is similar to that of aspirin; namely, the products are able to covalently bind to the Ser 530 residue present in the active cleft of the enzyme. Molecular modeling enabled us to rationalize this behavior. Because these products were previously found to display NO-dependent properties in rat animal models, particularly as they decreased inâ vivo gastrotoxicity and induced inâ vitro vasodilation, they represent a new and interesting class of potential aspirin-like antithrombotic agents worthy of further study.
Assuntos
Anti-Inflamatórios/química , Ciclo-Oxigenase 1/química , Inibidores da Agregação Plaquetária/química , Ácido Salicílico/química , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Aspirina/química , Domínio Catalítico , Linhagem Celular , Simulação por Computador , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Inibidores de Ciclo-Oxigenase/síntese química , Inibidores de Ciclo-Oxigenase/química , Inibidores de Ciclo-Oxigenase/farmacologia , Humanos , Inibidores da Agregação Plaquetária/síntese química , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Salicílico/síntese química , Ácido Salicílico/farmacologiaRESUMO
TDP-43, encoded by TARDBP, is a ubiquitously expressed, primarily nuclear protein. In recent years, TDP-43 has been identified as the major pathological protein in ALS due to its mislocalisation in the cytoplasm of motor neurons of patients with and without TARDBP mutations and expression in forms that do not match its predicted molecular weight. In this study, the TDP-43 profile was investigated using western immunoblot analysis in whole lysates, nuclei and cytoplasm of circulating lymphomonocytes from 16 ALS patients, 4 with (ALS/TDP+) and 12 without (ALS/TDP-) TARDBP mutations in the protein C-terminal domain, and thirteen age-matched, healthy donors (controls). Three disease-unaffected first-degree relatives of an ALS/TDP+ patient were also included: one carried the parent mutation (Rel/TDP+) whereas the other two did not (Rel/TDP-). In all ALS patients, relatives and controls, TDP-43 retained the predicted molecular weight in whole cell lysates and nuclei, but in the cytoplasm its molecular weight was slightly smaller than expected. In quantitative terms, TDP-43 was expressed at approximately the same levels in whole cell lysates of ALS patients, relatives and controls. In contrast, TDP-43 accumulated in the cytoplasm with concomitant nuclear depletion in all ALS/TDP+ patients, in about 50% of ALS/TDP- patients and in the Rel/TDP+ subject compared to the controls. In the remaining ALS/TDP- patients and in the two Rel/TDP- subjects, TDP-43 matched the control levels in both subcellular compartments. Were these findings further confirmed, circulating lymphomonocytes could be informative of TDP-43 mislocalisation in nervous tissue and used as a biomarker for future disease risk.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/patologia , Monócitos/metabolismo , Monócitos/patologia , Adulto , Idoso , Esclerose Lateral Amiotrófica/genética , Citoplasma/patologia , Proteínas de Ligação a DNA/química , Feminino , Humanos , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genéticaRESUMO
The direct synthesis of hydrogen peroxide from H(2) and O(2) has been studied using a high activity AuPd/TiO(2) catalyst. In particular, the effect of variation in the reaction conditions on the productivity of hydrogen peroxide formation is investigated in detail. The effect of H(2)/O(2) molar ratio, temperature, total pressure and solvent composition has been studied and optimised conditions identified. In addition, the effect of carrying out the synthesis reaction in the presence of hydrogen peroxide is investigated and the competing reactions of hydrogen peroxide formation, decomposition and hydrogenation are discussed and optimal operating conditions are identified.