RESUMO
Cancer and cardiovascular diseases are the leading causes of death in high-income countries. Cardiovascular complications can be found in cancer patients, being the result of so-called 'cardio-toxicity'. Therefore, it becomes essential to thoroughly investigate the origin of cardiac damage and the strategy to prevent it or to reverse the negative remodelling associated with cardiotoxicity. In this review the beneficial effects of physical exercise in cancer patients were analysed, particularly to prevent cardio-toxicity before its clinical manifestation. According to the relevance of exercise, we suggest strategies for exercise prescription with a tailored approach in these patients. In conclusion, physical exercise seems to be a promising and effective treatment for cancer patients during and after therapy and seems to counteract the negative effects induced by drugs on the cardiovascular system. Exercise prescription should be tailored according to patient's individual characteristics, to the drugs administered, to the personal history, and to his/her response to exercise, taking into account that different types of training can be prescribed according also to the patient's choice. A cardiological evaluation including exercise testing is essential for an appropriate prescription of exercise in these patients.
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Breast cancer is the most common cancer in women worldwide, and the high incidence of this cancer coupled with improvements in initial treatments has led to an ever-increasing number of breast cancer survivors. Among the prospective epidemiological studies on diet and breast cancer incidence and recurrence, to date, there is no association that is strong, reproducible and statistically significant, with the exception of alcohol intake, overweight, and weight gain. Nevertheless, many beliefs about food and breast cancer persist in the absence of supporting scientific evidence. After a comprehensive review regarding the role of lifestyle on breast cancer outcomes and a thorough study of the dissemination field including mass media, clinical institutions, and academic figures, we briefly reported the most common presumptions and also facts from the literature regarding lifestyle, nutrition, and breast cancer. The randomised controlled trial is the best study-design that could provide direct evidence of a causal relationship; however, there are methodological difficulties in applying and maintaining a lifestyle intervention for a sufficient period; consequently, there is a lack of this type of study in the literature. Instead, it is possible to obtain indirect evidence from observational prospective studies. In this article, it becomes clear that for now the best advice for women's health is to follow the World Cancer Research Fund/American Institute of Cancer Research (WCRF/AICR) recommendations on diet, nutrition, physical activity, and weight management for cancer prevention, because they are associated with a lower risk of developing most types of cancer, including breast cancer. Despite current awareness of the role of nutrition in cancer outcomes, there is inadequate translation from research findings into clinical practice. We suggest the establishment of a multidisciplinary research consortium to demonstrate the real power of lifestyle interventions.
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Group B Streptococcus (GBS) has evolved several strategies to avoid host defences. We have shown that interaction of macrophages with GBS causes macrophage calpain activation, cytoskeletal disruption and apoptosis, consequences of intracellular calcium increase induced by membrane permeability alterations provoked by GBS-ß-haemolysin. Open question remains about what effect calcium influx has on other calcium-sensing proteins such as gelsolin, involved in cytoskeleton modulation and apoptosis. Therefore we analysed the effect of GBS-III-COH31:macrophage interaction on gelsolin expression. Here we demonstrate that an early macrophage response to GBS-III-COH31 is a very strong gelsolin increase, which occurs in a time- and infection-ratio-dependent manner. This is not due to transcriptional events, translation events, protein turnover alterations, or protein-kinase activation, but to calcium influx, calpain activation and caspase-3 degradation. In fact, EGTA and PD150606 (calpain inhibitor) prevented gelsolin increase while BAF (caspase inhibitor) enhanced it. Since gelsolin increase is induced by highly ß-haemolytic GBS-III-NEM316 and GBS-V-10/84, but not by weakly ß-haemolytic GBS, or GBS-III-COH31 in conditions suppressing ß-haemolysin expression/activity and the presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor), GBS-ß-haemolysin is solely responsible for gelsolin increase causing, through membrane permeability defects, calcium influx and calpain activation. Early gelsolin increase could represent a macrophage response to antagonize apoptosis since gelsolin knockdown increases macrophage susceptibility to GBS-induced apoptosis. This response seems to be GBS specific because macrophage apoptosis by Staurosporine or Cycloeximide does not induce gelsolin.
Assuntos
Gelsolina/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Streptococcus agalactiae/imunologia , Relação Dose-Resposta Imunológica , Proteínas Hemolisinas/metabolismo , Interações Hospedeiro-Patógeno , Macrófagos/metabolismoRESUMO
Hairy cell leukemia (HCL) shows unique clinicopathological and biological features. HCL responds well to purine analogs but relapses are frequent and novel therapies are required. BRAF-V600E is the key driver mutation in HCL and distinguishes it from other B-cell lymphomas, including HCL-like leukemias/lymphomas (HCL-variant and splenic marginal zone lymphoma). The kinase-activating BRAF-V600E mutation also represents an ideal therapeutic target in HCL. Here, we investigated the biological and therapeutic importance of the activated BRAF-mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) pathway in HCL by exposing in vitro primary leukemic cells purified from 26 patients to clinically available BRAF (vemurafenib; dabrafenib) or MEK (trametinib) inhibitors. Results were validated in vivo in samples from vemurafenib-treated HCL patients within a phase 2 clinical trial. BRAF and MEK inhibitors caused, specifically in HCL (but not HCL-like) cells, marked MEK/ERK dephosphorylation, silencing of the BRAF-MEK-ERK pathway transcriptional output, loss of the HCL-specific gene expression signature, downregulation of the HCL markers CD25, tartrate-resistant acid phosphatase, and cyclin D1, smoothening of leukemic cells' hairy surface, and, eventually, apoptosis. Apoptosis was partially blunted by coculture with bone marrow stromal cells antagonizing MEK-ERK dephosphorylation. This protective effect could be counteracted by combined BRAF and MEK inhibition. Our results strongly support and inform the clinical use of BRAF and MEK inhibitors in HCL.
Assuntos
Antineoplásicos , Imidazóis , Indóis , Leucemia de Células Pilosas/tratamento farmacológico , Leucemia de Células Pilosas/genética , Oximas , Piridonas , Pirimidinonas , Sulfonamidas , Transcriptoma/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Imidazóis/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Oximas/farmacologia , Oximas/uso terapêutico , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêutico , Células Tumorais Cultivadas , VemurafenibRESUMO
We proposed that group IIA secretory phospholipase A(2) (GIIA) participates in neuritogenesis based on our observations that the enzyme migrates to growth cones and neurite tips when PC12 cells are induced to differentiate by nerve growth factor (NGF) (Ferrini et al., Neurochem Res 35:2168-2174, 2010). The involvement of other secretory PLA(2) isoforms in neuronal development has been suggested by others but through different mechanisms. In the present study, we compared the subcellular distribution of GIIA and group X sPLA(2) (GX) after stimulation of PC12 cells with NGF. We found that GIIA, but not GX, localized at the neuritic tips after treatment with NGF, as demonstrated by immunofluorescence analysis. We also found that NGF stimulated the expression and the activity of GIIA. In addition, NGF induced the expressed myc-tagged GIIA protein to migrate to neurite tips in its active form. We propose that GIIA expression, activity, and subcellular localization is regulated by NGF and that the enzyme may participate in neuritogenesis through intracellular mechanisms, most likely by facilitating the remodelling of glycerophospholipid molecular species by deacylation-reacylation reactions necessary for the incorporation of polyunsaturated fatty acids.
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Fosfolipases A2 do Grupo II/metabolismo , Fator de Crescimento Neural/farmacologia , Neuritos/enzimologia , Neurogênese/efeitos dos fármacos , Animais , Fosfolipases A2 do Grupo II/genética , Neuritos/efeitos dos fármacos , Células PC12 , RatosRESUMO
The endosomal-lysosomal system plays important roles in cellular physiology. Beyond the well-known function as terminal degradative compartment, necessary to maintain the health of the cell, lysosomes are critical for many other cellular processes, such as termination of signaling mediated by cell surface receptors and processing of internalized peptides in antigen-presenting cells. Moreover, the intracellular membrane trafficking related to the endosomal-lysosomal system plays a pivotal role in diverse physiological and pathological processes, such as exocytosis, plasma membrane repair, and endocytosis. Increasing evidences suggest that several lysosomal glycohydrolases, together with nonlysosomal glycohydrolases, are associated with cell membranes in their active form, and they are localized into lipid microdomains. The role of these forms in physiological and pathological conditions, such as differentiation and aging, neurodegenerative diseases, and cancer spreading, is under investigation. Here we provide general methods to purify lipid microdomain proteins and to discriminate cell surface lipid microdomains-associated glycohydrolases from those not exposed on cell surface. The methods reported here have been developed to characterize the membrane-associated forms of the acidic glycohydrolases ß-hexosaminidase and ß-galactosidase, but they may be applied to any other protein of interest.
Assuntos
Endossomos/química , Lisossomos/química , Microdomínios da Membrana/química , beta-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Biotinilação , Cromatografia de Afinidade , Cromatografia DEAE-Celulose , Endossomos/metabolismo , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Células Jurkat , Lisossomos/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Fluorescência , Transporte Proteico , beta-Galactosidase/química , beta-Galactosidase/isolamento & purificação , beta-N-Acetil-Hexosaminidases/química , beta-N-Acetil-Hexosaminidases/isolamento & purificaçãoRESUMO
OBJECTIVES: It has been recently observed that a T-cell subset, lacking of both CD4 and CD8 molecules and defined as double negative (DN), is expanded in the blood of patients with systemic lupus erythematosus, produces IL-17 and accumulates in the kidney during nephritis. Since IL-17 production is enhanced in salivary gland infiltrates of primary Sjögren's syndrome (SS) patients, we investigated whether DN T cells may be involved in the pathogenesis of salivary gland damage. METHODS: Phenotypic characterisation of peripheral blood mononuclear cells from SS patients and controls was performed by flow cytometry in freshly isolated and anti-CD3-stimulated cells. SS minor salivary glands were processed for immunofluorescence staining. RESULTS: CD3(+)CD4(-)CD8(-) DN T cells were major producers of IL-17 in SS and expressed ROR-γt. They were expanded in the peripheral blood, spontaneously produced IL-17 and infiltrated salivary glands. In addition, the expansion of αß-TCR(+) DN T cells was associated with disease activity. Notably, IL-17-producing DN T cells from SS patients, but not from healthy controls, were strongly resistant to the in vitro effect of dexamethasone. CONCLUSIONS: These findings appear to be of great interest since the identification of a peculiar T-cell subset with pro-inflammatory activity, but resistant to corticosteroids, in an autoimmune disorder such as SS may help to design new specific treatments for the disease.
Assuntos
Interleucina-17/biossíntese , Glândulas Salivares/imunologia , Síndrome de Sjogren/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th17/imunologia , Corticosteroides/farmacologia , Dexametasona/farmacologia , Feminino , Citometria de Fluxo , Humanos , Interleucina-17/imunologia , Pessoa de Meia-Idade , Glândulas Salivares/patologia , Síndrome de Sjogren/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
BACKGROUND: Hairy-cell leukemia (HCL) is a well-defined clinicopathological entity whose underlying genetic lesion is still obscure. METHODS: We searched for HCL-associated mutations by performing massively parallel sequencing of the whole exome of leukemic and matched normal cells purified from the peripheral blood of an index patient with HCL. Findings were validated by Sanger sequencing in 47 additional patients with HCL. RESULTS: Whole-exome sequencing identified five missense somatic clonal mutations that were confirmed on Sanger sequencing, including a heterozygous mutation in BRAF that results in the BRAF V600E variant protein. Since BRAF V600E is oncogenic in other tumors, further analyses were focused on this genetic lesion. The same BRAF mutation was noted in all the other 47 patients with HCL who were evaluated by means of Sanger sequencing. None of the 195 patients with other peripheral B-cell lymphomas or leukemias who were evaluated carried the BRAF V600E variant, including 38 patients with splenic marginal-zone lymphomas or unclassifiable splenic lymphomas or leukemias. In immunohistologic and Western blot studies, HCL cells expressed phosphorylated MEK and ERK (the downstream targets of the BRAF kinase), indicating a constitutive activation of the RAF-MEK-ERK mitogen-activated protein kinase pathway in HCL. In vitro incubation of BRAF-mutated primary leukemic hairy cells from 5 patients with PLX-4720, a specific inhibitor of active BRAF, led to a marked decrease in phosphorylated ERK and MEK. CONCLUSIONS; The BRAF V600E mutation was present in all patients with HCL who were evaluated. This finding may have implications for the pathogenesis, diagnosis, and targeted therapy of HCL. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).
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Leucemia de Células Pilosas/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adulto , Idoso , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Humanos , Leucemia de Células Pilosas/metabolismo , Leucemia de Células Pilosas/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
Group B Streptococcus (GBS) has evolved several strategies to avoid host defences where macrophages are one of main targets. Since pathogens frequently target the cytoskeleton to evade immune defences, we investigated if GBS manipulates macrophage cytoskeleton. GBS-III-COH31 in a time- and infection ratio-dependent manner induces great macrophage cytoskeleton alterations, causing degradation of several structural and regulatory cytoskeletal proteins. GBS ß-haemolysin is involved in cytoskeleton alterations causing plasma membrane permeability defects which allow calcium influx and calpain activation. In fact, cytoskeleton alterations are not induced by GBS-III-COH31 in conditions that suppress ß-haemolysin expression/activity and in presence of dipalmitoylphosphatidylcholine (ß-haemolysin inhibitor). Calpains, particularly m-calpain, are responsible for GBS-III-COH31-induced cytoskeleton disruption. In fact, the calpain inhibitor PD150606, m-calpain small-interfering-RNA and EGTA which inhibit calpain activation prevented cytoskeleton degradation whereas µ-calpain and other protease inhibitors did not. Finally, calpain inhibition strongly increased the number of viable intracellular GBS-III-COH31, showing that cytoskeleton alterations reduced macrophage phagocytosis. Marked macrophage cytoskeleton alterations are also induced by GBS-III-NEM316 and GBS-V-10/84 through ß-haemolysin-mediated plasma membrane permeability defects which allow calpain activation. This study suggests a new GBS strategy to evade macrophage antimicrobial responses based on cytoskeleton disruption by an unusual mechanism mediated by calcium influx and calpain activation.
Assuntos
Actinas/antagonistas & inibidores , Actinas/metabolismo , Calpaína/metabolismo , Citoesqueleto/metabolismo , Macrófagos Peritoneais/microbiologia , Microtúbulos/metabolismo , Streptococcus agalactiae/patogenicidade , Animais , Proteínas de Bactérias/toxicidade , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hemolisinas/toxicidade , CamundongosRESUMO
The mitochondrial electron transport chain is a source of oxygen superoxide anion (O(2)(-)) that is dismutated to H(2)O(2). Although low levels of ROS are physiologically synthesized during respiration, their increase contributes to cell injury. Therefore, an efficient machinery for H(2)O(2) disposal is essential in mitochondria. In this study, the ability of brain mitochondria to acquire cardiolipin (CL), phosphatidylglycerol (PG), and phosphatidylserine (PS) in vitro through a fusion process was exploited to investigate lipid effects on ROS. MTT assay, oxygen consumption, and respiratory ratio indicated that the acquired phospholipids did not alter mitochondrial respiration and O(2)(-) production from succinate. However, in CL-enriched mitochondria, H(2)O(2) levels where 27% and 47% of control in the absence and in the presence of antimycin A, respectively, suggesting an increase in H(2)O(2) elimination. Concomitantly, cytochrome c (cyt c) was released outside mitochondria. Since free oxidized cyt c acquired peroxidase activity towards H(2)O(2) upon interaction with CL in vitro, a contribution of cyt c to H(2)O(2) disposal in mitochondria through CL conferred peroxidase activity is plausible. In this model, the accompanying CL peroxidation should weaken cyt c-CL interactions, favouring the detachment and release of the protein. Neither cyt c peroxidase activity was elicited by PS in vitro, nor cyt c release was observed in PS-enriched mitochondria, although H(2)O(2) levels were significantly decreased, suggesting a cyt c-independent role of PS in ROS metabolism in mitochondria.
Assuntos
Encéfalo/metabolismo , Cardiolipinas/metabolismo , Citocromo-c Peroxidase/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Consumo de Oxigênio/fisiologia , Fosfatidilgliceróis/metabolismo , Fosfatidilserinas/metabolismo , Ratos , Superóxidos/metabolismoRESUMO
Plastids are considered promising bioreactors for the production of recombinant proteins, but the knowledge of the mechanisms regulating foreign protein folding, targeting, and accumulation in these organelles is still incomplete. Here we demonstrate that a plant secretory signal peptide is able to target a plastome-encoded recombinant protein to the thylakoid membrane. The fusion protein zeolin with its native signal peptide expressed by tobacco (Nicotiana tabacum) transplastomic plants was directed into the chloroplast thylakoid membranes, whereas the zeolin mutant devoid of the signal peptide, Δzeolin, is instead accumulated in the stroma. We also show that zeolin folds in the thylakoid membrane where it accumulates as trimers able to form disulphide bonds. Disulphide bonds contribute to protein accumulation since zeolin shows a higher accumulation level with respect to stromal Δzeolin, whose folding is hampered as the protein accumulates at low amounts in a monomeric form and it is not oxidized. Thus, post-transcriptional processes seem to regulate the stability and accumulation of plastid-synthesized zeolin. The most plausible zeolin targeting mechanism to thylakoid is discussed herein.
Assuntos
Proteínas de Plantas/metabolismo , Sinais Direcionadores de Proteínas , Tilacoides/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Frações Subcelulares/metabolismo , Nicotiana/metabolismoRESUMO
Shaker-like (KV1.1) channels contain a highly conserved Pro-Val-Pro (PVP) motif at the base of S6 that produces a kink in the S6 helices and provides a flexible element thought to be essential for channel gating. The role of proline-induced kinks in transmembrane helices is well known, but the contribution of the small hydrophobic valine between these two prolines is not known, and interestingly, Shab-like (KV2.1) channels possess an isoleucine at this position (PIP). Here we show that the exact nature of this central hydrophobic residue within the PXP motif confers unique functional properties to KV1 channels, including changes in activation and deactivation kinetics, voltage-dependent properties and open probabilities, but single-channel conductance and cell expression levels are not affected. In support of these functional changes, molecular dynamic simulations demonstrate that valine and isoleucine contribute differently to S6 flexibility within this motif. These results therefore indicate that the nature of the central hydrophobic residue in the PXP motif is an important functional determinant of KV channel gating by contributing, at least in part, to the relative flexibility of this motif.
Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.1/metabolismo , Potássio/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Simulação por Computador , Humanos , Interações Hidrofóbicas e Hidrofílicas , Isoleucina , Cinética , Canal de Potássio Kv1.1/química , Canal de Potássio Kv1.1/genética , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Oócitos , Conformação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade , Valina , Xenopus laevisRESUMO
Creation of a nuclear export signal (NES) motif and loss of tryptophans (W) 288 and 290 (or 290 only) at the COOH terminus of nucleophosmin (NPM) are both crucial for NPM aberrant cytoplasmic accumulation in acute myelogenous leukemia (AML) carrying NPM1 mutations. Hereby, we clarify how these COOH-terminal alterations functionally cooperate to delocalize NPM to the cytoplasm. Using a Rev(1.4)-based shuttling assay, we measured the nuclear export efficiency of six different COOH-terminal NES motifs identified in NPM mutants and found significant strength variability, the strongest NES motifs being associated with NPM mutants retaining W288. When artificially coupled with a weak NES, W288-retaining NPM mutants are not exported efficiently into cytoplasm because the force (W288) driving the mutants toward the nucleolus overwhelms the force (NES) exporting the mutants into cytoplasm. We then used this functional assay to study the physiologic NH(2)-terminal NES motifs of wild-type NPM and found that they are weak, which explains the prominent nucleolar localization of wild-type NPM. Thus, the opposing balance of forces (tryptophans and NES) seems to determine the subcellular localization of NPM. The fact that W288-retaining mutants always combine with the strongest NES reveals mutational selective pressure toward efficient export into cytoplasm, pointing to this event as critical for leukemogenesis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/química , Proteínas Nucleares/genética , Motivos de Aminoácidos , Animais , Transformação Celular Neoplásica , Citoplasma/metabolismo , Análise Mutacional de DNA , Fibroblastos/metabolismo , Leucemia Mieloide Aguda/metabolismo , Camundongos , Microscopia de Fluorescência , Mutação , Células NIH 3T3 , Nucleofosmina , Plasmídeos/metabolismo , Estrutura Terciária de ProteínaRESUMO
BACKGROUND INFORMATION: AQP4 (aquaporin 4) internalization and a concomitant decrease in the osmotic water permeability coefficient (Pf) after histamine exposure has been reported in AQP4-transfected gastric HGT1 cells. RESULTS: In the present study we report that AQP4 internalization is followed by an increase in AQP4 phosphorylation. Histamine treatment for 30 min resulted in an approx. 10-fold increase in AQP4 phosphorylation that was inhibited by 1 microM H89, a specific PKA (protein kinase A) inhibitor, but not by PKC (protein kinase C) and CK2 inhibitors. Moreover, measurement of PKA activity after 30 min of histamine treatment showed that PKA activity was approx. 3-fold higher compared with basal conditions. AQP4 phosphorylation was prevented in cells treated with histamine for 30 min after pre-incubation with PAO (phenylarsine oxide), an inhibitor of protein endocytosis. Using an endo-exocytosis assay we showed that, after histamine washed out, internalized AQP4 recycled back to the cell surface, even in cells in which de novo protein synthesis was inhibited by cycloheximide. CONCLUSIONS: Phosphorylation experiments, combined with immunolocalization studies, indicated that AQP4 phosphorylation is mediated by PKA and occurs subsequently to its internalization in late endosomes. We suggest that phosphorylation might be a mechanism involved in retaining AQP4 in a vesicle-recycling compartment.
Assuntos
Aquaporina 4/metabolismo , Histamina/farmacologia , Células Parietais Gástricas/citologia , Células Parietais Gástricas/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocitose/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Transfecção , Vírus da Estomatite Vesicular IndianaRESUMO
Because of a lack of specific clonality markers, information on lineage involvement and cell of origin of acute myeloid leukemia with normal karyotype (AML-NK), is missing. Because Nucleophosmin (NPM) gene is frequently mutated in AML-NK and causes aberrant NPM cytoplasmic localization (NPMc+), it was used as an AML lineage clonality marker. Clonal NPM exon 12 mutations were detected in myeloid, monocytic, erythroid, and megakaryocytic cells but not in fibroblasts or endothelia that were laser-microdissected from 3 patients with NPMc+ AML. Aberrant cytoplasmic expression of mutated NPM proteins was identified with anti-NPM antibodies in 2 or more myeloid hemopoietic cell lineages in 99 (61.5%) of 161 of NPMc+ AML paraffin-embedded bone marrow biopsies; lymphoid involvement was excluded in 3 investigated cases. These findings suggest that NPMc+ AML derives from either a common myeloid or earlier progenitor. Immunohistochemical studies show that varying combinations and ratios of NPMc+ leukemic cells from distinct lineages are responsible for heterogeneity within each French-American-British (FAB) classification type and for NPMc+ AML falling into different FAB categories. These findings question the value of FAB criteria in subdividing the WHO category of "AML not otherwise characterized" and suggest that, for clinical use, NPMc+ AML be provisionally regarded as a separate AML with prognostic significance.
Assuntos
Biomarcadores Tumorais/genética , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Medula Óssea/metabolismo , Medula Óssea/patologia , Humanos , Cariotipagem/métodos , Leucemia Mieloide Aguda/classificação , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Nucleofosmina , Transporte Proteico/genética , Organização Mundial da SaúdeRESUMO
Group B Streptococcus (GBS) has developed several strategies to evade immune defenses. We show that GBS induces macrophage (Mphi) membrane permeability defects and apoptosis, prevented by inhibition of calcium influx but not caspases. We analyze the molecular mechanisms of GBS-induced murine Mphi apoptosis. GBS causes a massive intracellular calcium increase, strictly correlated to membrane permeability defects and apoptosis onset. Calcium increase was associated with activation of calcium-dependent protease calpain, demonstrated by casein zymography, alpha-spectrin cleavage to a calpain-specific fragment, fluorogenic calpain-substrate cleavage, and inhibition of these proteolyses by calpain inhibitors targeting the calcium-binding, 3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid, or active site (four different inhibitors), by calpain small-interfering-RNA (siRNA) and EGTA. GBS-induced Mphi apoptosis was inhibited by all micro- and m-calpain inhibitors used and m-calpain siRNA, but not 3-(5-Fluoro-3-indolyl)-2-mercapto-(Z)-2-propenoic acid (micro-calpain inhibitor) and micro-calpain siRNA indicating that m-calpain plays a central role in apoptosis. Calpain activation is followed by Bax and Bid cleavage, cytochrome c, apoptosis-inducing factor, and endonuclease G release from mitochondria. In GBS-induced apoptosis, cytochrome c did not induce caspase-3 and -7 activation because they and APAF-1 were degraded by calpains. Therefore, apoptosis-inducing factor and endonuclease G seem the main mediators of the calpain-dependent but caspase-independent pathway of GBS-induced apoptosis. Proapoptotic mediator degradations do not occur with nonhemolytic GBS, not inducing Mphi apoptosis. Apoptosis was reduced by Bax siRNA and Bid siRNA suggesting Bax and Bid degradation is apoptosis correlated. This signaling pathway, different from that of most pathogens, could represent a GBS strategy to evade immune defenses.
Assuntos
Apoptose/imunologia , Calpaína/metabolismo , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/microbiologia , Streptococcus agalactiae/imunologia , Acrilatos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Fator Apoptótico 1 Ativador de Proteases , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Calpaína/antagonistas & inibidores , Caspase 3 , Caspase 7 , Caspases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Ativação Enzimática/imunologia , Feminino , Hidrólise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Proteínas Mitocondriais/metabolismo , Proteínas/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Nucleophosmin (NPM) exon-12 mutations occur in 50% to 60% of adult acute myeloid leukemia (AML) with normal karyotype and are predictors of favorable prognosis. We evaluated bone marrow or peripheral blood samples from 450 adult patients with AML of the GIMEMA (Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto)/AML12 EORTC (European Organization for Research and Treatment of Cancer) trial to (1) search for new exon-12 NPM mutations; (2) determine whether NPM immunostaining on paraffin-embedded biopsies predicts NPM mutations; and (3) investigate altered nucleocytoplasmic NPM traffic in primary AML cells. Fourteen NPM mutations, including 8 new variants, were identified. All 200 AML cases expressing cytoplasmic NPM (NPMc(+) AML) carried NPM mutations. None of the 250 cases with nucleus-restricted NPM (NPMc(-) AML) was mutated. At the C-terminus, NPM leukemic mutants carried mutations of only tryptophan 290 or of both tryptophans 288 and 290 and a new nuclear export signal (NES) motif, which appear to underlie their nuclear export. The specific Crm1/exportin-1 inhibitor leptomycin-B relocated NPM mutants from cytoplasm to nucleus of primary NPMc(+) AML cells, demonstrating that nuclear export is NES dependent. NPM mutants bound and recruited wild-type NPM into leukemic cell cytoplasm. Because alterations at C-terminus of leukemic NPM mutants are similar, immunohistochemistry detects all exon-12 NPM mutations and is a valuable, inexpensive tool in the diagnostic-prognostic work-up of patients with AML with normal karyotype.
Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Adolescente , Adulto , Sequência de Aminoácidos , Sequência de Bases , Citoplasma/metabolismo , DNA de Neoplasias/genética , Éxons , Humanos , Imuno-Histoquímica , Pessoa de Meia-Idade , Sinais de Exportação Nuclear/genética , Proteínas Nucleares/química , Nucleofosmina , Triptofano/genéticaRESUMO
We recently identified aberrant cytoplasmic expression of nucleophosmin (NPM) as the immunohistochemical marker of a large subgroup of acute myeloid leukemia (AML) (about one-third of adult AML) that is characterized by normal karyotype and mutations occurring at the exon-12 of the NPM gene. In this paper, we have elucidated the molecular mechanism underlying the abnormal cytoplasmic localization of NPM. All 29 AML-associated mutated NPM alleles so far identified encode abnormal proteins which have acquired at the C-terminus a nuclear export signal (NES) motif and lost both tryptophan residues 288 and 290 (or only the residue 290) which determine nucleolar localization. We show for the first time that both alterations are crucial for NPM mutant export from nucleus to cytoplasm. In fact, the cytoplasmic accumulation of NPM is blocked by leptomycin-B and ratjadones, specific exportin-1/Crm1-inhibitors, and by reinsertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. NPM leukemic mutants in turn recruit the wild-type NPM from nucleoli to nucleoplasm and cytoplasm. These findings indicate that potential therapeutic strategies aimed to retarget NPM to its physiological sites will have to overcome 2 obstacles, the new NES motif and the mutated tryptophan(s) at the NPM mutant C-terminus.
Assuntos
Transporte Ativo do Núcleo Celular/genética , Leucemia/genética , Mutação , Sinais de Exportação Nuclear/genética , Proteínas Nucleares/genética , Triptofano/genética , Motivos de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Nucléolo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Leucemia/metabolismo , Proteínas Nucleares/metabolismo , Nucleofosmina , TransfecçãoRESUMO
Plant pollens are an important source of environmental antigens that stimulate allergic responses. In addition to acting as vehicles for foreign protein antigens, they contain lipids that incorporate saturated and unsaturated fatty acids, which are necessary in the reproduction of higher plants. The CD1 family of nonpolymorphic major histocompatibility complex-related molecules is highly conserved in mammals, and has been shown to present microbial and self lipids to T cells. Here, we provide evidence that pollen lipids may be recognized as antigens by human T cells through a CD1-dependent pathway. Among phospholipids extracted from cypress grains, phosphatidyl-choline and phosphatidyl-ethanolamine were able to stimulate the proliferation of T cells from cypress-sensitive subjects. Recognition of phospholipids involved multiple cell types, mostly CD4(+) T cell receptor for antigen (TCR)alphabeta(+), some CD4(-)CD8(-) TCRgammadelta(+), but rarely Valpha24i(+) natural killer-T cells, and required CD1a(+) and CD1d(+) antigen presenting cell. The responding T cells secreted both interleukin (IL)-4 and interferon-gamma, in some cases IL-10 and transforming growth factor-beta, and could provide help for immunoglobulin E (IgE) production. Responses to pollen phospholipids were maximally evident in blood samples obtained from allergic subjects during pollinating season, uniformly absent in Mycobacterium tuberculosis-exposed health care workers, but occasionally seen in nonallergic subjects. Finally, allergic, but not normal subjects, displayed circulating specific IgE and cutaneous weal and flare reactions to phospholipids.
Assuntos
Alérgenos/imunologia , Antígenos CD1/imunologia , Cupressus/imunologia , Hipersensibilidade/imunologia , Fosfolipídeos/imunologia , Pólen/imunologia , Linfócitos T/imunologia , Adulto , Formação de Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Células Cultivadas , Cupressus/química , Citocinas/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Masculino , Pessoa de Meia-Idade , Mycobacterium tuberculosis/imunologia , Fosfolipídeos/química , Pólen/química , Receptores de Antígenos de Linfócitos T gama-delta/imunologiaRESUMO
Zeins, the main storage proteins of maize that accumulate in the endoplasmic reticulum of the endosperm cells, are particularly interesting because they are rich in the essential sulphur amino acids. Overexpression of certain zein genes in plants such as alfalfa would be expected to improve the nutritional characteristics of this crop. Recently, significant accumulation values have been reached, but still far from those considered useful for nutritional purposes. This study investigates whether targeting to compartments other than the endoplasmic reticulum (cytosol and chloroplasts) could result in increasing beta-zein accumulation in transgenic plants. To address beta-zein to the cytosol, the fragment which codes for the signal peptide has been removed. beta-zein has also been targeted to alfalfa and tobacco chloroplasts by a transit peptide signal. Both tobacco, as a model plant species, and alfalfa have been transformed with the assembled constructs. An alternative route to accumulate beta-zein in the chloroplasts is to synthesize beta-zein directly in the plastid lumen. Thus, the beta-zein gene has also been inserted into tobacco plastid DNA. The beta-zein gene in each different type of transformed plant was properly transcribed, as determined by northern blot analysis, but no accumulation of beta-zein was detected, either in the cytoplasm or in the chloroplasts of alfalfa and tobacco transformed plants. Therefore, it is concluded that chloroplasts and the cytosol are not favourable subcellular locations for zein protein accumulation.