RESUMO
Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.
Assuntos
Aminoácido Oxirredutases/fisiologia , Caderinas/genética , Metástase Neoplásica/genética , Fator 3 de Transcrição/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Células Cultivadas , Cães , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos NusRESUMO
Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative-type breast cancers (BTNBCs) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of lysyl oxidase (LOX), a metastasis-promoting, matrix-remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by small interfering RNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between LOX and GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBCs to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBCs.
Assuntos
Neoplasias da Mama/metabolismo , Fator de Transcrição GATA3/metabolismo , Metástase Neoplásica/prevenção & controle , Neoplasia de Células Basais/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasia de Células Basais/patologia , Neoplasias Hormônio-Dependentes/metabolismo , Prognóstico , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Pirimidinas , TiofenosRESUMO
Clinical evaluation of a young woman with subcutaneous fibrotic nodules, progressive distal joint contractures and marfanoid stature revealed a previously unrecognized fibrotic disorder characterized by several unique phenotypic features and some features overlapping with known disorders. Mutational analysis of the FBN1 and FBN2 genes excluded Marfan syndrome and congenital contractural arachnodactyly. MMP2 gene sequence analysis excluded multicentric osteolysis, nodulosis and arthropathy. The lack of mutations within the MAGP2 gene also excluded an MAGP2-associated disorder. In order to establish the mechanistic basis for the severe skin pathology noted in this patient, we performed transcriptional profiling of dermal fibroblasts, and candidate gene expression studies in conjunction with immunocytochemistry and cell-based and functional assays. Data from these experiments have further excluded any previously recognized fibrotic disorder and identified a unique pattern of gene expression in this patient consistent with progressive fibrosis. The pathogenic mechanisms included persistent proliferation of dermal fibroblasts in coexistence with a disarray of the microfibrillar network. Collagen accumulation, moreover, could be linked to extensive crosslinking resulting from increased activities of lysyl oxidases (LOX and LOXL), and lack of remodelling due to deficiencies in collagenolytic matrix metalloproteinases. The disorder may represent a novel syndrome in which transforming growth factor-ß1-independent dermal fibrosis, unlike known microfibrillar disorders caused by single gene deficiencies, associates with a disarray of the microfibrillar network.
Assuntos
Derme/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/diagnóstico , Fibrose/genética , Adulto , Biópsia , Proliferação de Células , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Citocinas/metabolismo , Análise Mutacional de DNA , Derme/ultraestrutura , Regulação para Baixo , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibrilina-1 , Fibrilina-2 , Fibrilinas , Fibrose/metabolismo , Perfilação da Expressão Gênica , Glicoproteínas/genética , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular , Metaloproteinase 2 da Matriz/genética , Proteínas dos Microfilamentos/genética , Reação em Cadeia da Polimerase/métodos , Fatores de Processamento de RNA , Análise de Sequência de DNA , Adulto JovemRESUMO
The extracellular matrix (ECM) plays a critical role during the development and invasion of primary brain tumours. However, the function of ECM components and signalling between a permissive ECM and invasive astrocytes is not fully understood. We have recently reported the ECM enzyme, lysyl oxidase (LOX), in the central nervous system and observed up-regulation of LOX in anaplastic astrocytoma cells. While the catalytic function of LOX is essential for cross-linking of ECM proteins, we also reported that LOX induced invasive and metastatic properties in breast tumour epithelial cells through hydrogen peroxide-mediated FAK/Src activation. In this study, we tested the hypothesis that active LOX is expressed in anaplastic astrocytes and promotes FAK activation and invasive/migratory behaviour. Results demonstrate that increased expression and activity of LOX positively correlated with invasive phenotype of malignant astrocytoma cell lines. Immunohistochemistry detected increased LOX within tumour cells and ECM in grade I-IV astrocytic neoplasm compared with normal brain and coincidence of increased LOX with the loss of glial fibrillary acidic protein in higher-grade tumours. Increased active LOX in invasive astrocytes was accompanied by phosphorylation of FAK[Tyr576] and paxillin[Tyr118]; furthermore, both FAK and paxillin tyrosine phosphorylation were diminished by beta-aminopropionitrile inhibition of LOX activity and depletion of H(2)O(2) via catalase treatment. Additionally, we provide evidence that in astrocytes, LOX is likely processed by bone morphogenic protein-1 and LOX activity might be further stimulated by the expression of fibronectin in these cells. These results demonstrate an important LOX-mediated mechanism that promotes migratory/invasive behaviour of malignant astrocytes.
Assuntos
Astrócitos/enzimologia , Neoplasias Encefálicas/enzimologia , Ativação Enzimática/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Paxilina/metabolismo , Proteína-Lisina 6-Oxidase/metabolismo , Astrócitos/patologia , Astrocitoma/enzimologia , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Humanos , Imuno-Histoquímica , Invasividade Neoplásica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Members of the lysyl oxidase family (LOX) are copper and lysyl-tyrosine quinone cofactor-containing amine oxidases that are important for the assembly and maintenance of components of the extracellular matrix. Our previous results demonstrated that a novel member, LOXL4, is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. Results of the current study showed overexpression of the LOXL4 transcript in 74% (46 of 62) of invasive HNSCC tumours and 90% of both primary and metastatic HNSCC cell lines. Significant correlation was found between LOXL4 expression and local lymph node metastases versus primary tumour types (p<0.01) and higher tumour stages (p<0.01). Immunocytochemistry demonstrated cellular overexpression of the LOXL4 protein that correlated with the increased mRNA transcription in HNSCC cells. HNSCC cell lines displayed in significant subset of nuclei increased copies of the LOX4 gene locus on chromosome 10q24, demonstrated by fluorescence in situ hybridization (FISH). Extensive metaphase cytogenetic analysis was performed on UTSCC19A cells, identifying an isochromosome i(10)(q10). Taken together, these results highlight LOXL4 expression as a distinctive trait and suggest a functional role for LOXL4 in the molecular pathogenesis of invasive head and neck carcinomas.
Assuntos
Aminoácido Oxirredutases/metabolismo , Carcinoma de Células Escamosas/enzimologia , Neoplasias de Cabeça e Pescoço/enzimologia , Regulação para Cima , Adulto , Idoso , Aminoácido Oxirredutases/genética , Northern Blotting/métodos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/secundário , Estudos de Casos e Controles , Linhagem Celular Tumoral , Cromossomos Humanos Par 10 , Análise Citogenética , Ativação Enzimática , Matriz Extracelular/enzimologia , Feminino , Neoplasias de Cabeça e Pescoço/genética , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente , Masculino , Pessoa de Meia-Idade , Proteína-Lisina 6-OxidaseRESUMO
Various microsatellite and CGH studies in prostate cancer identify deletions on the short arm of chromosome 8 especially at band 8p21-22 searching for unknown putative tumor suppressor genes. By means of microsatellite markers several candidate genes were detected which may play different roles in early prostate cancer progression. We established a quantitative gene dosage PCR based on the real time PCR method serving the purpose of genomic fine mapping. Therefore we used 10 Assays-on Demand (ABI) for the detection of deletions located between and nearby the microsatellite markers D8S258 and NEFL spanning a genomic region of approximate 7 mbp. Comparative immunohistochemical analysis from tissue micro arrays (TMA) of 1122 independent cases followed. We were able to detect three clearly separated deletion intervals on 8p21-22. One on LZTS1, second on NEFL and third a deletion hot spot on LOXL2, which was affected in 72% of all investigated cases. Our comparative immunohistochemical TMA based studies demonstrate that LOXL2 is nearly lost in most prostate cancer tissues. LOXL2 catalyze the crosslinking of collagen and elastin in the extracellular matrix and it has been assumed that it is involved in tumor suppression and cell adhesion. LOXL2 is frequently expressed in proliferating tissues and shows a high expression in benign prostate tissue too. In prostate cancer the expression is positive correlated with the MIB1-score.
Assuntos
Cromossomos Humanos Par 8 , Dosagem de Genes , Deleção de Sequência , Mapeamento Cromossômico , Humanos , Masculino , Repetições de Microssatélites , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Neoplasias da Próstata/genéticaRESUMO
Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.
Assuntos
Aminoácido Oxirredutases/química , Proteína-Lisina 6-Oxidase/química , Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/fisiologia , Animais , Drosophila/enzimologia , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Miocárdio/enzimologia , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/fisiologiaRESUMO
We have identified a novel 14-exon human lysyl oxidase-like gene, LOXL4, on chromosome 10q24. The cDNA and derived amino acid sequence of LOXL4 demonstrates a conserved C-terminal region including the characteristic copper-binding site, lysyl and tyrosyl residues and a cytokine receptor-like domain. One of the four N-terminal SRCR domains contains a 13 amino acid insertion encoded by a short exon not present within the closely homologous LOXL2 and LOXL3 genes. The 3.5-kb LOXL4 mRNA is present in pancreas and testis and at lower levels in several other tissues. Fibroblasts, smooth muscle and osteosarcoma (HOS) cells express LOXL4. No expression was detected in HCT-116 and DLD-1 colon, MCF-7 breast and DU-145 prostate cancer cell lines.
Assuntos
Aminoácido Oxirredutases/genética , Cromossomos Humanos Par 10 , Cisteína/metabolismo , Proteínas de Membrana , Receptores de Lipoproteínas , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Aminoácido Oxirredutases/classificação , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Cisteína/genética , DNA Complementar , Éxons , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Estrutura Terciária de Proteína , Proteína-Lisina 6-Oxidase , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Distribuição TecidualRESUMO
It has been documented that alpha-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Inibidores de Caspase , Fármacos Neuroprotetores/uso terapêutico , Óxidos de Nitrogênio/uso terapêutico , Animais , Isquemia Encefálica/patologia , Caspase 3 , Óxidos N-Cíclicos , Modelos Animais de Doenças , Masculino , Ratos , Ratos Wistar , Detecção de Spin , Taxa de SobrevidaRESUMO
BACKGROUND: There is an urgent need to identify genes involved in familial ALS (FALS), as mutations in the CuZn superoxide dismutase (SOD1) gene can account for 20% of FALS cases. The mechanisms by which the many mutations in the SOD1 gene lead to motoneuron degeneration are unknown, although current experimental evidence supports a toxic gain of function, possibly through copper-induced cytotoxicity. Copper is an integral component of a number of enzymes as well as SOD1. Since abnormalities in connective tissue cross-linking have been reported in ALS patients, an enzyme of possible relevance is lysyl oxidase (LOX), a copper-containing enzyme which catalyses the crosslinking of collagens and elastin. The aim of this study was to investigate the hypothesis that allelic variants or mutants of LOX gene result in altered function of LOX in ALS patients. METHODS: The coding regions of the LOX gene were screened for polymorphism and mutations in a cohort of sporadic and familial ALS patients. RESULTS: A novel polymorphism, Pro159Gln, was identified in eight individuals with sporadic ALS (5.0%) and five controls (3.6%). The previously identified Arg158Gln polymorphism was also detected in ALS patients and controls. These polymorphisms were genotyped in 192 ALS patients, including 31 unrelated familial cases and 138 controls, and no association was found between any of these polymorphisms and amyotrophic lateral sclerosis or its phenotype. CONCLUSION: Mutations in the LOX gene are unlikely to be directly causative of ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Polimorfismo Genético , Proteína-Lisina 6-Oxidase/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Lateral Amiotrófica/enzimologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Cobre/metabolismo , Humanos , Pessoa de Meia-Idade , Mutação , RatosRESUMO
Lysyl oxidase (LOX), a copper-containing amine oxidase, belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. LOX has been traditionally known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. More recently, diverse roles have been attributed to lysyl oxidase and these novel activities cover a spectrum of diverse biological functions such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and our recent discovery of three novel members of this amine oxidase family, LOX-like (LOXL), LOXL2, and LOXL3, indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine-oxidase family. Structural similarities of the highly conserved copper-binding and lysyl-tyrosylquinone cofactor sites among the LOX and LOX-like proteins may result in similar amine oxidase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, will be determined by other, conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SRCR) domains present in LOXL2 and LOXL3. Furthermore, these functions may be carried out in a temporally and spatially regulated fashion.
Assuntos
Proteína-Lisina 6-Oxidase/metabolismo , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Humanos , Conformação Proteica , Proteína-Lisina 6-Oxidase/química , Proteína-Lisina 6-Oxidase/genética , Homologia de Sequência de AminoácidosRESUMO
A recent study reported that hyperglycemia of a brief duration worsens, and of long duration reduces, ischemic brain damage. To test whether this is a valid conception, we induced 10 min of transient forebrain ischemia, recorded postischemic seizures, and evaluated brain morphology. The results showed that administration of glucose 2 h before ischemia aggravated brain damage, induced seizures, and caused animal death in the same manner as was previously observed when glucose was given 30 min before ischemia. Thus, the conclusion that the influence of glucose on an ischemic transient is dependent upon the duration of hyperglycemia is unsubstantiated.
Assuntos
Lesões Encefálicas/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Infarto Cerebral/tratamento farmacológico , Glucose/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Lesões Encefálicas/patologia , Lesões Encefálicas/fisiopatologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Infarto Cerebral/patologia , Infarto Cerebral/fisiopatologia , Esquema de Medicação , Masculino , Degeneração Neural/tratamento farmacológico , Degeneração Neural/patologia , Degeneração Neural/fisiopatologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/patologia , Traumatismo por Reperfusão/fisiopatologia , Taxa de SobrevidaRESUMO
A BLASTN search using the mouse lor-2 cDNA identified three overlapping ESTs (AI752772, AA852888, and R55706) in the GenBank database. These expressed sequence tags were assembled into a contig of 3121 nucleotides with an open reading frame of 2262 bp. The encoded putative polypeptide of 754 amino acids presented all structural characteristics of the lysyl oxidase (LOX) enzyme family, a copper-binding site with four histidyl residues, the lysyl and tyrosyl residues known to be involved in LOX enzyme in the formation of the quinone cofactor and surrounding sequences, and the cytokine receptor-like domain. In addition, four scavenger receptor cysteine-rich (SRCR) domains were found in the N-terminal region of the protein. The gene encoding this new cDNA, which we have referred to as human lysyl oxidase-like 3 (humanLOXL3), has been mapped to chromosome 2p13.3, overlapping at its 3' end the HtrA2 serine protease gene. The structure of the humanLOXL3 gene was deduced from the BAC clone bac91a19 sequence and contained 14 exons. The expression pattern of this new member of the LOX gene family appears to be different from that of the LOX and LOX-like genes, as the central nervous system, neurons, and also leukocytes expressed humanLOXL3. A BLASTN search of the human EST database indicated the presence of ESTs, corresponding to alternative splice variants of LOXL3, that lacked exon 5 and exon 8. The putative resulting protein retained the region encoding the structural and functional elements of the amine oxidase but the second and fourth SRCR domains were truncated and the potential BMP-1 cleavage site was not present. The presence of domains unrelated to the traditional amine oxidase activity is a strong indication that humanLOXL3 might fulfill other functions in addition to intrinsic enzyme activity.
Assuntos
Aminoácido Oxirredutases/biossíntese , Aminoácido Oxirredutases/genética , Sistema Nervoso Central/metabolismo , Leucócitos/metabolismo , Miocárdio/metabolismo , Proteína-Lisina 6-Oxidase/biossíntese , Proteína-Lisina 6-Oxidase/genética , Útero/metabolismo , Processamento Alternativo , Aminoácido Oxirredutases/química , Sequência de Aminoácidos , Sítios de Ligação , Northern Blotting , Mapeamento Cromossômico , DNA Complementar/metabolismo , Bases de Dados Factuais , Éxons , Etiquetas de Sequências Expressas , Feminino , Humanos , Íntrons , Modelos Genéticos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Distribuição TecidualRESUMO
A novel human gene, SARM, encodes the orthologue of a Drosophila protein (CG7915) and contains a unique combination of the sterile alpha (SAM) and the HEAT/Armadillo motifs. The SARM gene was identified on chromosome 17q11, between markers D17S783 and D17S841 on BAC clone AC002094, which also included a HERV repeat and keratin-18-like, MAC30, TNFAIP1, HSPC017, and vitronectin genes in addition to three unknown genes. The mouse SARM gene was located on a mouse chromosome 11 BAC clone (AC002324). The SARM gene is 1.8 kb centromeric to the vitronectin gene, and the two genes share a promoter region that directs a high level of liver-specific expression of both the SARM and the vitronectin genes. In addition to the liver, the SARM gene was highly expressed in the kidney. A 0.4-kb antisense transcript was coordinately expressed with the SARM gene in the kidney and liver, while in the brain and malignant cell lines, it appeared independent of SARM gene transcription. The SARM gene encodes a protein of 690 amino acids. Based on amino acid sequence homology, we have identified a SAM motif within this derived protein. Structure modeling and protein folding recognition studies confirmed the presence of alpha-alpha right-handed superhelix-like folds consistent with the structure of the Armadillo and HEAT repeats of the beta-catenin and importin protein families. Both motifs are known to be involved in protein-protein interactions promoting the formation of diverse protein complexes. We have identified the same conserved SAM/Armadillo motif combination in the mouse, Drosophila, and Caenorhabditis elegans SARM proteins.
Assuntos
Cromossomos Humanos Par 17 , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila , Proteínas de Insetos/genética , Transativadores , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Proteínas do Domínio Armadillo , Northern Blotting , Encéfalo/metabolismo , Caenorhabditis elegans , Mapeamento Cromossômico , Sequência Conservada , Proteínas do Citoesqueleto/biossíntese , Drosophila , Evolução Molecular , Éxons , Etiquetas de Sequências Expressas , Marcadores Genéticos , Humanos , Recém-Nascido , Íntrons , Camundongos , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Distribuição Tecidual , Fatores de Transcrição , Transcrição Gênica , Células Tumorais Cultivadas , beta CateninaRESUMO
Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.
Assuntos
Fragmentação do DNA , Hiperglicemia/patologia , Ataque Isquêmico Transitório/patologia , Animais , Apoptose , Caspase 3 , Caspases/metabolismo , Corpo Estriado/patologia , Grupo dos Citocromos c/metabolismo , Giro Denteado/patologia , Ativação Enzimática , Hipocampo/patologia , Hiperglicemia/fisiopatologia , Marcação In Situ das Extremidades Cortadas , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Microscopia Eletrônica , Neocórtex/patologia , Neurônios/patologia , Ratos , Ratos Wistar , Tálamo/patologiaRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood-brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.
Assuntos
Esclerose Lateral Amiotrófica , Ciclosporina/farmacologia , Imunossupressores/farmacologia , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/imunologia , Esclerose Lateral Amiotrófica/mortalidade , Animais , Modelos Animais de Doenças , Humanos , Injeções Espinhais , Camundongos , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Mitocôndrias/enzimologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/mortalidade , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Taxa de SobrevidaRESUMO
The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.
Assuntos
Âmnio/enzimologia , Córion/enzimologia , Decídua/enzimologia , Proteína-Lisina 6-Oxidase/metabolismo , Aborto Legal , Adulto , Northern Blotting , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Gravidez , Proteína-Lisina 6-Oxidase/genética , RNA Mensageiro/metabolismoRESUMO
Several cytogenetic alterations affect the distal part of the long arm of human chromosome 15, including recurrent rearrangements between 12p13 and 15q25, which cause congenital fibrosarcoma (CFS). We present here the construction of a BAC/PAC contig map that spans 2 Mb from the neurotrophin-3 receptor (NTRK3) gene region on 15q25.3 to the proximal end of the Bloom's syndrome region on 15q26.1, and the identification of a set of new chromosome 15 duplicons. The contig reveals the existence of several regions of sequence similarity with other chromosomes (6q, 7p, and 12p) and with other 15q cytogenetic bands (15q11-q13 and 15q24). One region of similarity maps on 15q11-q13, close to the Prader-Willi/Angelman syndromes (PWS/AS) imprinting center. The 12p similar sequence maps on 12p13, at a distance to the ets variant 6 (ETV6) gene that is equivalent on 15q26.1 to the distance to the NTRK3 gene. These two genes are the targets of the CFS recurrent translocations, suggesting that misalignments between these two chromosomes regions could facilitate recombination. The most striking similarity identified is based on a low copy repeat sequence, mainly present on human chromosome 15 (LCR15), which could be considered a newly recognized duplicon. At least 10 copies of this duplicon are present on chromosome 15, mainly on 15q24 and 15q26. One copy is located close to a HERC2 sequence on the distal end of the PWS/AS region, three around the lysyl oxidase-like (LOXL1) gene on 15q24, and three on 15q26, one of which close to the IQ motif containing GTPase-activating protein 1 (IQGAP1) gene on 15q26.1. These LCR15 span between 13 and 22 kb and contain high identities with the golgin-like protein (GLP) and the SH3 domain-containing protein (SH3P18) gene sequences and have the characteristics of duplicons. Because duplicons flank chromosome regions that are rearranged in human genomic disorders, the LCR15 described here could represent new elements of rearrangements affecting different regions of human chromosome 15q.
Assuntos
Cromossomos Humanos Par 15/genética , Genes Duplicados , Sequência de Bases , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 7/genética , Mapeamento de Sequências Contíguas , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido NucleicoRESUMO
A 46,823 bp region of human chromosome 5q23.1 encompassing the seven-exon lysyl oxidase gene was characterized at the primary sequence level. Approximately 17.4% of this region is comprised of repetitive elements. The gene colocalizes with microsatellite marker D5S467. It is flanked by two candidate nuclear matrix association regions (MARs). The 5' MAR centered at position 12,500 is of the AT-rich and curved DNA class. This is followed by a large CpG island containing fifty-seven putative regulatory elements which extend from just upstream of exon 1 to intron 2. The larger 3' MAR, spans position 35,050-39,750 and is characterized by a TG-rich kinked structure that also contains a topoisomerase II binding site. Based on these results model of the transcriptional regulation of the lysy/oxidase gene is presented.
Assuntos
Proteína-Lisina 6-Oxidase/genética , Sequência de Bases , Biologia Computacional , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Three new glycosides were isolated from the fruits of noni (Morinda citrifolia). Their structures were determined to be 6-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose (1), 6-O-(beta-D-glucopyranosyl)-1-O-hexanoyl-beta-D-glucopyranose (2), and 3-methylbut-3-enyl 6-O-beta-D-glucopyranosyl-beta-D-glucopyranoside (3) using MS and NMR methods.