RESUMO
Black-pearl (Blp) is a highly conserved, essential inner-mitochondrial membrane protein. The yeast Blp homologue, Magmas/Pam16, is required for mitochondrial protein transport, growth, and survival. Our purpose was to determine the role of Drosophila Blp in mitochondrial function, cell survival, and proliferation. To this end, we performed mitotic recombination in Drosophila melanogaster, RNAi-mediated knockdown, MitoTracker staining, measurement of reactive oxygen species (ROS), flow cytometry, electron transport chain complex assays, and hemocyte isolation from Drosophila larvae. Proliferation-defective, Blp-deficient Drosophila Schneider cells exhibited mitochondrial membrane depolarization, a 60% decrease in ATP levels, increased amounts of ROS (3.5-fold), cell cycle arrest, and activation of autophagy that were associated with a selective 65% reduction of cytochrome c oxidase activity. N-acetyl cysteine (NAC) rescued Blp-RNAi-treated cells from cell cycle arrest, indicating that increased production of ROS is the primary cause of the proliferation and survival defects in Blp-depleted cells. blp hypomorph larvae had a 35% decreased number of plasmatocytes with a 45% reduced active mitochondrial staining and their viability was increased 2-fold by administration of NAC, which blocked melanotic lesions. Loss of Blp decreases cytochrome c oxidase activity and uncouples oxidative phosphorylation, causing ROS production, which selectively affects mitochondria-rich plasmatocyte survival and function, leading to melanotic lesions in Blp-deficient flies.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Mitocôndrias/fisiologia , Acetilcisteína/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Autofagia/fisiologia , Sequência de Bases , Western Blotting , Primers do DNA , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Citometria de Fluxo , Fase G1 , Microscopia Eletrônica de Varredura , Mitocôndrias/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Fase SRESUMO
Magmas (mitochondria associated, granulocyte-macrophage colony stimulating factor signaling molecule), is a highly conserved and essential gene, expressed in all cell types. We designed and synthesized several small molecule Magmas inhibitors (SMMI) and assayed their effects on proliferation in yeast. We found that the most active compound 9 inhibited growth at the 4 µM scale. This compound was shown by fluorometric titration to bind to Magmas with a K(d)=33 µM. Target specificity of the lead compound was established by demonstrating direct binding of the compound to Magmas and by genetic studies. Molecular modeling suggested that the inhibitor bound at the predicted site in Magmas.
Assuntos
Desenho de Fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Proteínas Mitocondriais/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/síntese química , Proliferação de Células/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Leveduras/efeitos dos fármacosRESUMO
A goal of behavior-analytic interventions is to produce behavior that is maintained under naturalistic conditions. In this experiment, we studied the effects of a speaker immersion protocol (SIP) on the number of speaker responses (tacts and mands) emitted by 3 preschool students under naturalistic, not directly targeted, conditions. During the SIP, the researchers provided 100 daily opportunities for the participants to emit mands using the target mand form by contriving establishing operations (EOs) throughout the school day. The effects of the intervention were evaluated using a multiple-probe design by measuring target mands during EO probe sessions and the number of mands and tacts emitted during noninstructional-setting probe sessions. The researchers found that the SIP produced increases in both targeted and generalized verbal behavior.
RESUMO
A female infant who presented with autoimmune hemolytic anemia and thrombocytopenia subsequently developed hepatic, dermatologic, renal, pulmonary, gastrointestinal, endocrine, and nervous system involvement. Prolonged and intensive treatment with prednisone, IVIG, mycophenolate mofetil, and anti-CD20 and anti-CD52 antibodies was necessary to control the symptoms. Laboratory evaluation showed normal lymphocyte subsets and function. There was normal Foxp3 and CD25 expression, no increased CD4(-)CD8(-) T-cell population, and the AIRE and Fas genes were without mutations. These features place the patient at the most severe portion of the Evans syndrome spectrum, and suggest that this case may represent a rare, new immunodeficiency disorder.
Assuntos
Anemia Hemolítica Autoimune/imunologia , Anemia Hemolítica Autoimune/patologia , Síndromes de Imunodeficiência/classificação , Púrpura Trombocitopênica Idiopática/imunologia , Púrpura Trombocitopênica Idiopática/patologia , Anemia Hemolítica Autoimune/sangue , Anemia Hemolítica Autoimune/complicações , Feminino , Humanos , Síndromes de Imunodeficiência/sangue , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/imunologia , Lactente , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/complicaçõesRESUMO
The primary focus in the pathogenesis and treatment of human malignancies has been the tumor cell. However, the biologic properties of a malignancy are not all intrinsically determined. Interactions between heterogeneous cell populations influence the growth and survival of both normal and malignant cells. Studies defining the origin of endothelial cells involved in tumor angiogenesis first demonstrated the contributions of normal cellular environment. Recently, the mononuclear phagocyte lineage has been found to have biologically and clinically significant tumor enhancing and tumor suppressive effects. This article reviews the multiple roles of mononuclear phagocytes in cancer biology. A companion manuscript (J Pediatr Hematol Oncol. 2008, in press) describes the targeting of these cells for therapeutic benefit. Incorporating these strategies into future childhood cancer protocols could be an innovative approach for improving patient outcome.
Assuntos
Sistema Fagocitário Mononuclear/fisiologia , Neoplasias/imunologia , Fagócitos/fisiologia , Diferenciação Celular/fisiologia , Feminino , Humanos , Fagócitos/citologiaRESUMO
Magmas, is a 13-kDa mitochondrial protein which is ubiquitously expressed in eukaryotic cells. It was identified as a granulocyte-macrophage-colony stimulating factor (GM-CSF) inducible gene in hematopoietic cells and has a key role in the transport of mitochondrial proteins in yeast. Because GM-CSF receptor levels are elevated in prostate cancer, Magmas expression was examined in normal and neoplastic tissue. Magmas protein levels were barely detectable in non-neoplastic prostate glands. Increased amounts were observed in some samples of intraepithelial neoplasia. Approximately one half of the adenocarcinoma samples examined had weak Magmas expression, while the remainder had intermediate to high levels. The increased Magmas observed in malignant tissue was a result of higher protein expression and not from changes in mitochondrial content. Interestingly, in some patients, the normal prostate tissue had more Magmas message than the malignant portion. The results indicated that Magmas expression in prostate cancer is heterogeneous and independent of clinical stage and Gleason score. Further studies are needed to determine if Magmas expression has prognostic significance in prostate cancer.
Assuntos
Proteínas Mitocondriais/metabolismo , Neoplasias da Próstata/metabolismo , Humanos , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/análise , Proteínas Mitocondriais/genética , Estadiamento de Neoplasias , Prognóstico , Próstata/química , Próstata/metabolismo , Neoplasias da Próstata/química , Neoplasias da Próstata/diagnóstico , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
Magmas is a protein that is involved in GM-CSF signaling in a myeloid cell line. Its precise role in the signal transduction process is unclear. To accurately characterize Magmas expression in a variety of cells, mouse embryos and adult murine tissues were analyzed for both mRNA and protein content. Magmas expression was detected as early as the day 6.5 embryo. The level of expression was developmentally regulated. During embryogenesis, elevated Magmas was observed in several structures, including heart, liver, notochord, choroid plexus, cervical ganglion, and nasal mucosa. Muscle, pancreas, intestinal mucosa, and testes were among the adult tissues with high Magmas expression. Most cell types, including hepatocytes and skeletal, smooth, and cardiac myocytes, also expressed the GM-CSF receptor (GMR) but the relative tissue levels of GMR were not always proportional to Magmas. The expression patterns suggest that Magmas has a role in both developing and mature tissues.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/biossíntese , Animais , Northern Blotting , Embrião de Mamíferos/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Imuno-Histoquímica , Camundongos , Microscopia Eletrônica , Especificidade de Órgãos , Subunidades Proteicas , RNA Mensageiro/biossíntese , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genéticaRESUMO
Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.
Assuntos
Acetiltransferases/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Esfingolipídeos/metabolismo , Acetiltransferases/deficiência , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Fermentação , Deleção de Genes , Perfilação da Expressão Gênica , Glucose/metabolismo , Humanos , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
We describe three siblings with holoprosencephaly, recurrent infections, and increased peripheral blood monocytes. These children were born to apparently healthy parents in a family with one unaffected child. Affected individuals had microcephaly, severe developmental delay, failure to thrive, and brachydactyly. The clinical courses were complicated by endocrine dysfunction, multiple respiratory, and skin infections. Laboratory studies showed normal karyotypes, normal lymphocyte function, and a peripheral blood monocytosis with markedly abnormal morphology. Mutation analysis of the seven genes (SHH, ZIC2, SIX3, TGI, FTDGF1, GLI2, and PTCH) known to be involved in holoprosencephaly was normal. This is the first report demonstrating an association between abnormal mononuclear phagocytes and holoprosencephaly.
Assuntos
Holoprosencefalia/complicações , Infecções/complicações , Leucocitose/complicações , Monócitos , Anormalidades Múltiplas/genética , Pré-Escolar , Feminino , Holoprosencefalia/genética , Humanos , Lactente , Infecções/genética , Cariotipagem , Leucocitose/genética , Masculino , Microcefalia/complicações , Microcefalia/genética , Mutação , Linhagem , Fenótipo , Recidiva , SíndromeRESUMO
Magmas is a nuclear encoded protein found in the mitochondria of mammalian cells. It participates in granulocyte-macrophage-colony stimulating factor (GM-CSF) signaling in hematopoietic cells and has an essential role in invertebrate development. In order to characterize the protein structural features and gene evolution of Magmas, a dataset containing 61 Magmas homologs from 52 species distributed among animals, plants and fungi was analyzed. All Magmas members were found to possess three novel sequence motifs in addition to a conserved leader peptide. Phylogenetic tree and dN/dS rate ratios showed that Magmas was evolutionarily conserved. Analysis of Magmas gene organization demonstrated incremental intron acquisition in plants and vertebrates. Significant genetic diversity in Magmas was observed from kingdom specific amino acid signatures, the presence of predicted signal peptides that target the protein to other intracellular locations besides the mitochondria, and the detection of multiple isoforms in higher animals. These studies demonstrate that Magmas members constitute an important family of conserved proteins having multifunctional activities, and provide a basis for future experiments.
Assuntos
Evolução Molecular , Proteínas Mitocondriais/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Duplicação Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Íntrons , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Proteínas Mitocondriais/fisiologia , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Transdução de Sinais/fisiologia , Especificidade da EspécieRESUMO
Antibody 26-10, obtained in a secondary immune response, binds digoxin with high affinity (K(a) = 1.3 x 10(10) M(-1)) because of extensive shape complementarity. We demonstrated previously that mutations of the hapten contact residue HTrp-100 to Arg (where H refers to the heavy chain) resulted in increased specificity for digoxin analogs substituted at the cardenolide 16 position. However, mutagenesis of H:CDR1 did not result in such a specificity change despite the proximity of the H:CDR1 hapten contact residue Asn-35 to the cardenolide 16 position. Here we constructed a bacteriophage-displayed library containing randomized mutations at H chain residues 30-35 in a 26-10 mutant containing Arg-100 (26-10-RRALD). Phage were selected by panning against digoxin, gitoxin (16-OH), and 16-acetylgitoxin coupled to bovine serum albumin. Clones that retained wild-type Asn at position 35 showed preferred binding to gitoxin, like the 26-10-RRALD parent. In contrast, clones containing Val-35 selected mainly on digoxin-bovine serum albumin demonstrated a shift back to wild-type specificity. Several clones containing Val-35 bound digoxin with increased affinity, approaching that of the wild type in a few instances, in contrast to the mutation Val-35 in the wild-type 26-10 background, which reduces affinity for digoxin 90-fold. It has therefore proven possible to reorder the 26-10 binding site by mutations including two major contact residues on opposite sides of the site and yet to retain high affinity for binding for digoxin. Thus, even among antibodies that have undergone affinity maturation in vivo, different structural solutions to high affinity binding may be revealed.