RESUMO
Environmental signals in the cellular milieu such as hypoxia, growth factors, extracellular matrix (ECM), or cell-surface molecules on adjacent cells can activate signaling pathways that communicate the state of the environment to the nucleus. Several groups have evaluated gene expression or signaling pathways in response to increasing cell density as an in vitro surrogate for in vivo cell-cell interactions. These studies have also perhaps assumed that cells grown at various densities in standard in vitro incubator conditions do not have different pericellular oxygen levels. However, pericellular hypoxia can be induced by increasing cell density, which can exert profound influences on the target cell lines and may explain a number of findings previously attributed to normoxic cell-cell interactions. Thus, we first sought to test the hypothesis that cell-cell interactions as evaluated by the surrogate approach of increasing in vitro cell density in routine normoxic culture conditions results in pericellular hypoxia in prostate cancer cells. Second, we sought to evaluate whether such interactions affect transcription mediated by the hypoxia response element (HRE). Thirdly, we sought to elucidate the signal transduction pathways mediating the induction of HRE in response to cell density induced pericellular hypoxia in routine normoxic culture conditions. Our results indicate that paracrine cell interactions can induce nuclear localization of HIF-1a protein and this translocation is associated with strong stimulation of the HRE-reporter activity. We also make the novel observation that cell density-induced activity of the HRE is dependent on nitric oxide production, which acts as a diffusible paracrine factor secreted by densely cultured cells. These results suggest that paracrine cell interactions associated with pericellular hypoxia lead to the physiological induction of HRE activity via the cooperative action of Ras, MEK1, HIF-1a via pericellular diffusion of nitric oxide. In addition, these results highlight the importance of examining pericellular hypoxia as a possible stimulus in experiments involving in vitro cell density manipulation even in routine normoxic culture conditions.
Assuntos
Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas ras/metabolismo , 2,2'-Dipiridil/farmacologia , Contagem de Células , Hipóxia Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Quelantes de Ferro/farmacologia , MAP Quinase Quinase 1 , Masculino , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II , Neoplasias da Próstata , Elementos de Resposta , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
BACKGROUND: Signals from a cell's environment are sensed by receptors, which activate pathways that, in turn, transmit the signals to the nucleus, informing decisions on growth, angiogenesis, and other cell functions. Transcription of vascular endothelial growth factor (VEGF), a potent angiogenic factor, can be induced by cell-cell contact. In the current work, we sought to determine if this induction is dependent on transformation of cells to a malignant phenotype and subsequently to determine which signaling molecules mediate activation of VEGF transcription. METHODS: Normal and transformed prostate epithelial cell lines were examined at various cell densities to simulate the effect of increased cell contact on expression of VEGF messenger RNA. Transformed cells were also cotransfected with a VEGF promoter-reporter construct and with constructs that express dominant negative or activated versions of signal transduction proteins hypothesized to be involved in the cell-cell contact process, and reporter activity was assessed at various cell densities. All P values are two-sided. RESULTS: Direct cell-cell contact, but not extracellular matrix components, resulted in transcriptional activation of a VEGF promoter-reporter construct in malignant (P<.0001) but not in nonmalignant (P =.37) prostate cells. This process was mediated via a mitogen-activated protein kinase (MAPK); it required the activity of focal adhesion kinase (FAK), Rap1, and Raf and was Ras independent. In addition, transcriptional activation of a Ras-sensitive Elk-1 chimeric reporter by cell-cell contact suggests that Rap1 is a key factor in regulating the specificity of convergent MAPK-signaling pathways arising from different upstream extracellular stimuli. CONCLUSIONS: Cell contact induction of VEGF transcription via FAK and Rap1 provides a novel Ras-independent, but transformation-dependent, mechanism for stimulus-specific regulation of tumor VEGF expression via MAPK.
Assuntos
Fatores de Crescimento Endotelial/genética , Linfocinas/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transcrição Gênica , Proteínas rap1 de Ligação ao GTP/metabolismo , Northern Blotting , Células Cultivadas , Epitélio/metabolismo , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Genes Reporter/genética , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/genética , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Próstata/citologia , Neoplasias da Próstata/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA Mensageiro/genética , Transfecção , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteínas rap1 de Ligação ao GTP/genéticaRESUMO
Occupational exposure to lead or mercury was found to make protein better antigens. The production of autoantibodies to nervous system protein is one example of such effect. The present study aims to detect the possibility of induction of antisperm autoantibodies due to occupational exposure to lead or mercury. Male workers exposed to lead (n = 50) or to mercury (n = 39) were selected for this purpose and compared to a matched control group (n = 39). A negative control consisting of 17 females was also included. All subjects had two or more children. Blood samples were collected and the ELISA technique was applied to detect antisperm antibodies. Also, the levels of lead in blood and mercury in urine were determined as biological indices of exposure. Antisperm antibodies were detected in 90% of workers exposed to lead with the predominance of the IgG type and 84.6% of workers exposed to mercury with the predominance of the IgM type. Although the results did not correlate with the biological indices of exposure, it seems advisable to use the detection of sperm antibodies of sera of workers exposed to metals as a biological monitoring tool.
Assuntos
Autoanticorpos/sangue , Chumbo/urina , Mercúrio/sangue , Exposição Ocupacional/efeitos adversos , Espermatozoides/imunologia , Adulto , Poluentes Ambientais/toxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Chumbo/toxicidade , Masculino , Mercúrio/toxicidadeRESUMO
The nitric oxide synthases (NOS-I, neuronal, NOS-II, inducible, and NOS-III, endothelial) are the most recent additions to the large number of heme proteins that contain cysteine thiolate-liganded protoporphyrin IX heme prosthetic groups. This group of oxygenating enzymes also includes one of the largest gene families, that of the cytochromes P450, which have been demonstrated to be involved in the hydroxylation of a variety of substrates, including endogenous compounds (steroids, fatty acids, and prostaglandins) and exogenous compounds (therapeutic drugs, environmental toxicants, and carcinogens). The substrates for cytochromes P450 are universally hydrophobic while the physiological substrate for the nitric oxide synthases is the amino acid L-arginine, a hydrophilic compound. This review will discuss the approaches being used to study the structure and mechanism of neuronal nitric oxide synthase in the context of its known prosthetic groups and regulation by Ca(2+)-calmodulin and/or tetrahydrobiopterin (BH4).
Assuntos
Arginina/metabolismo , Isoenzimas/química , Neurônios/enzimologia , Óxido Nítrico Sintase/química , Óxido Nítrico/metabolismo , Animais , Cisteína/química , Radicais Livres , Hemeproteínas/química , Humanos , Hidroxilação , Isoenzimas/metabolismo , Óxido Nítrico Sintase/metabolismoRESUMO
The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain of E. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N omega-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.
Assuntos
Aminoácido Oxirredutases/genética , Escherichia coli/genética , Neurônios/enzimologia , Animais , Arginina/análogos & derivados , Arginina/metabolismo , Sequência de Bases , Linhagem Celular , Chaperonina 10/genética , Chaperonina 60/genética , Clonagem Molecular , Sistema Enzimático do Citocromo P-450/genética , Primers do DNA , DNA Complementar , Hidrólise , Masculino , Dados de Sequência Molecular , Óxido Nítrico Sintase , Nitroarginina , Plasmídeos , Regiões Promotoras Genéticas , Ligação Proteica , Pterinas/metabolismo , Ratos , Proteínas Recombinantes/genética , Análise EspectralRESUMO
Nitric oxide synthase (NOS) catalyzes the NADPH-dependent, Ca2+/calmodulin-dependent formation of NO and citrulline from L-arginine and molecular oxygen. The localization of the heme-binding consensus sequence in the NH2-terminal half of NOS and of the binding sequences for nucleotides (FMN and FAD) in the COOH-terminal half suggests a bidomain structure. In addition, the presence of a putative calmodulin-binding sequence between the heme- and flavin-binding domains of the enzyme suggests a role for calmodulin in modulating a spatial orientation of these domains that is required for catalytic activity. First, to determine the effects of calmodulin and the functionality of the separated domains, Ca2+/calmodulin binding-induced conformational changes in NOS were measured by fluorescence quenching, from which a binding constant of approximately 1 nM for calmodulin was calculated. Second, electron transport to various artificial acceptors was measured. The addition of Ca2+/calmodulin increased cytochrome c reduction from 10-15-fold while stimulating the rate of 2,6-dichlorophenolindophenol and ferricyanide reduction only slightly, if at all. Calmodulin stimulation of NOS results in NADPH-mediated cytochrome c reduction, which is sensitive to superoxide dismutase, and the reduction of acetylated cytochrome c, which is only weakly reducible by unstimulated NOS. Thus, this stimulated activity is presumably superoxide anion-mediated. Third, limited proteolysis of NOS in the absence of calmodulin resulted in a time-dependent increase in cytochrome c reductase activity, which was not inhibitable by superoxide dismutase, and a decrease in catalysis of NO formation. SDS-polyacrylamide gel electrophoresis analysis of the tryptic digest demonstrated the formation of approximately 89- and approximately 79-kDa fragments. Sequence analysis of the peptides confirmed that trypsin cleaves the enzyme in the putative calmodulin-binding region beginning with Ala728. This region was protected from proteolysis by the addition of Ca2+/calmodulin. The separated NH2-terminal domain exhibited the characteristic spectrum of bound heme, while the COOH-terminal domain showed the characteristic spectrum of bound flavins. Other cleavage patterns were obtained in the presence of calmodulin. The data demonstrate that the heme- and flavin-binding domains of NOS can be isolated in functionally intact forms.
Assuntos
Aminoácido Oxirredutases/química , Encéfalo/enzimologia , Aminoácido Oxirredutases/metabolismo , Sequência de Aminoácidos , Calmodulina/metabolismo , Células Cultivadas , Heme/metabolismo , Humanos , Hidrólise , Dados de Sequência Molecular , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Óxido Nítrico Sintase , Superóxido Dismutase/metabolismoRESUMO
A six year old buffalo was admitted with a chronic indurated swelling at the left ramus of the mandible since 3 months. The radiographic picture showed a large osteolytic area and soft tissue swelling at the cheek teeth of the mandible. His topopathological examination disclosed neoplastic epithelial cells arranged in islands and cords. The cells were arranged in palisade form resembling the ameloblasts. The clinical findings, the radiographic features and the criteria of the neoplastic cells confirmed an ameloblastoma in a buffalo.
Assuntos
Ameloblastoma/veterinária , Búfalos , Neoplasias Mandibulares/veterinária , Ameloblastoma/patologia , Animais , Neoplasias Mandibulares/patologiaRESUMO
Chloroform anesthesia induced some histochemical changes in hepatic lobules in the form of diminution in succinic dehydrogenase and acid phosphatase activities accompanied by an increase in alpha esterase activity. In this respect more manifest changes were observed as a result of the delayed effect of chloroform as well as after administration of the inducer.
Assuntos
Clorofórmio/toxicidade , Fígado/efeitos dos fármacos , Fosfatase Ácida/metabolismo , Animais , Clorofórmio/metabolismo , Cães , Indução Enzimática , Esterases/metabolismo , Feminino , Histocitoquímica , Fígado/enzimologia , Masculino , Fenobarbital/farmacologia , Succinato Desidrogenase/metabolismoRESUMO
The effect of stress of anaesthesia and surgery was studied in non-bilharzial and bilharzial individuals. Estimation of ACTH and cortisol was carried out by the radio-immunoassay (RIA) technique. The level of ACTH and cortisol was found to be lower in unanaesthetized bilharzial subjects than in normal individuals. Under the effect of anaesthesia and surgery the elevation in the level of both hormones was also lower in bilharzial than in non-bilharzial patients. The response to the stress of anaesthesia and surgery was more marked during halothane than during nitrous oxide anaesthesia.