Assuntos
Ascite , Trato Gastrointestinal , Humanos , Ascite/diagnóstico por imagem , Ascite/etiologiaRESUMO
Variations in otolith patterns, sizes and body morphometrics of jack mackerel Trachurus japonicus juveniles were investigated. Under transmitted light, translucent (W(t)) and opaque otoliths (W(o)) were detected in juveniles collected from Wakasa Bay between July 2005 and April 2006, whereas only opaque otoliths (G(o)) were detected in Goto-nada Sea individuals between May and June 2006. Three groups of juveniles were distinguished based on differences in hatch season, otolith size and growth history, and body morphometrics. As T. japonicus has different spawning seasons according to spawning grounds, each group was estimated to hatch in different waters. Juveniles with W(t) otoliths were considered to have stayed in coastal habitat longer, as the hatch area was estimated to be near Wakasa Bay. Juveniles with W(o) and G(o) otoliths appear to recruit to coastal waters at larger size, since their hatch areas were estimated to be far from each collection area. Larger otoliths of W(t) were attributed to otolith accretion after the second growth flexion, which was observed only for W(t) . Standard length of W(t) fish at the second otolith growth flexion was estimated to correspond to recruitment size to coastal rocky reefs in Wakasa Bay. Body morphometrics were correlated with otolith size after removing body size effect, suggesting that morphological variations of T. japonicus juveniles were also associated with the timing of recruitment to coastal habitat.
Assuntos
Membrana dos Otólitos/crescimento & desenvolvimento , Perciformes/crescimento & desenvolvimento , Animais , Tamanho Corporal , Ecossistema , Japão , Membrana dos Otólitos/anatomia & histologia , Perciformes/anatomia & histologia , Estações do AnoRESUMO
The mutagens produced through chemical reaction between chlorine and the insecticide fenitrothion were studied by using a quadrupole GC-MS. The mutagenicity and the mutagen formation potential (MFP) of the identified by-products were evaluated by the Ames assay (preincubation method) using Salmonella typhimurium TA100 without exogenous activation by S9 mix (TA100-S9). Before conducting GC/MS analyses, six compounds were presumed to be produced in chlorinated fenitrothion. These compounds were confirmed to be produced by the GC/MS analyses, but none of them were mutagenic. One of the chlorination by-products, 3-methyl-4-nitrophenol, has 19 times greater MFP than that of fenitrothion. This result suggests that a major mutagen in chlorinated fenitrothion will be produced via a chemical reaction between chlorine and 3-methyl-4-nitrophenol.
Assuntos
Fenitrotion/química , Halogenação , Inseticidas/química , Mutagênicos/química , Cromatografia Gasosa-Espectrometria de Massas , Testes de MutagenicidadeRESUMO
The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Fosfoproteínas/fisiologia , Proteínas/fisiologia , Proteínas Repressoras , Transativadores , Proteínas Adaptadoras de Transdução de Sinal , Proteína Axina , Linhagem Celular , Cromatografia em Gel , Proteínas Desgrenhadas , Relação Dose-Resposta a Droga , Escherichia coli/metabolismo , Imunofluorescência , Microinjeções , Microscopia Confocal , Modelos Genéticos , Fosforilação , Plasmídeos , Proteínas Proto-Oncogênicas c-myc/fisiologia , Saccharomyces cerevisiae/genética , Fatores de Tempo , Transfecção , beta CateninaRESUMO
We have established a fabrication process for conductive carbon nanotube (CNT) tips for multiprobe scanning tunneling microscope (STM) with high yield. This was achieved, first, by attaching a CNT at the apex of a supporting W tip by a dielectrophoresis method, second, by reinforcing the adhesion between the CNT and the W tip by electron beam deposition of hydrocarbon and subsequent heating, and finally by wholly coating it with a thin metal layer by pulsed laser deposition. More than 90% of the CNT tips survived after long-distance transportation in air, indicating the practical durability of the CNT tips. The shape of the CNT tip did not change even after making contact with another metal tip more than 100 times repeatedly, which evidenced its mechanical robustness. We exploited the CNT tips for the electronic transport measurement by a four-terminal method in a multiprobe STM, in which the PtIr-coated CNT portion of the tip exhibited diffusive transport with a low resistivity of 1.8 kOmega/microm. The contact resistance at the junction between the CNT and the supporting W tip was estimated to be less than 0.7 kOmega. We confirmed that the PtIr thin layer remained at the CNT-W junction portion after excess current passed through, although the PtIr layer was peeled off on the CNT to aggregate into particles, which was likely due to electromigration or a thermally activated diffusion process. These results indicate that the CNT tips fabricated by our recipe possess high reliability and reproducibility sufficient for multiprobe STM measurements.
Assuntos
Condutividade Elétrica , Microscopia de Tunelamento , Nanotubos de Carbono , Microscopia de Tunelamento/instrumentação , Microscopia de Tunelamento/métodos , Nanotubos de Carbono/ultraestruturaRESUMO
To investigate the effect of estrogenic compounds on the marine mussel Mytilus edulis, an assay was developed to measure the expression of two vertebrate estrogen responsive genes-estrogen receptor (ER) and vitellogenin (VTG) genes. Expression was measured in M. edulis gonads following a 10-day exposure to 200 ng/l 17beta-estradiol (estradiol). The concentrations of esterified estradiol in mussel tissue increased 15-fold in a time-dependent manner-confirming uptake of the compound by the mussels, however there was no significant increase of free estradiol in mussel tissues during the exposure period. The ER and VTG mRNA levels in the gonads of both sexes were measured at days 1-3, 5, and 10 in control and exposed mussels. However, no significant change in the expression of either the ER or VTG genes was recorded at any of the sampled time points. The results suggest that either a regulatory mechanism exists in a mussel that is able to maintain constant levels of free estradiol by converting the excess estradiol into esterified products which may have reduced affinity for the estrogen receptor, or alternatively, that the ER and VTG genes are unresponsive to estrogens in these organisms. The significance of these findings in terms of the utility of ER and VTG as biomarkers of endocrine disruption in bivalve species is discussed.
Assuntos
Estradiol/toxicidade , Expressão Gênica/efeitos dos fármacos , Mytilus edulis/efeitos dos fármacos , Receptores de Estrogênio/genética , Vitelogeninas/genética , Poluentes Químicos da Água/toxicidade , Sequência de Aminoácidos , Animais , Primers do DNA/química , Estradiol/análise , Feminino , Gônadas/química , Gônadas/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Mytilus edulis/química , RNA Mensageiro/análise , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência/veterinária , Vitelogeninas/biossíntese , Vitelogeninas/efeitos dos fármacos , Água/análiseRESUMO
Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3beta (GSK-3beta), and beta-catenin through different binding sites and downregulates beta-catenin. GSK-3beta-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3beta- and beta-catenin- but not APC-binding sites, did not enhance GSK-3beta-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, beta-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3beta in the presence of Axin. Consistent with these in vitro observations, expression of beta-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is effectively phosphorylated by GSK-3beta and that beta-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3beta. Taken together, these results suggest that GSK-3beta-dependent phosphorylation of APC can be modulated by beta-catenin and PP2A complexed with Axin.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Genes APC , Fosfoproteínas Fosfatases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteínas Repressoras , Transativadores , Proteína da Polipose Adenomatosa do Colo , Animais , Proteína Axina , Sítios de Ligação , Células COS , Chlorocebus aethiops , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Células L , Substâncias Macromoleculares , Camundongos , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteína Fosfatase 2 , Proteínas Recombinantes de Fusão/biossíntese , beta CateninaRESUMO
Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Ligação ao GTP/fisiologia , Proteínas ras/fisiologia , Animais , Células COS , Membrana Celular/metabolismo , Proteínas de Ligação ao GTP/genética , Lipossomos/metabolismo , Fosfolipídeos/fisiologia , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Fator ral de Troca do Nucleotídeo Guanina , Proteínas rap de Ligação ao GTP , Proteínas ras/genéticaRESUMO
When Axin, a negative regulator of the Wnt signaling pathway, was expressed in COS cells, it coeluted with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, and adenomatous polyposis coli protein (APC) in a high molecular weight fraction on gel filtration column chromatography. In this fraction, GSK-3beta, beta-catenin, and APC were co-precipitated with Axin. Although beta-catenin was detected in the high molecular weight fraction in L cells on gel filtration column chromatography, addition of conditioned medium expressing Wnt-3a to the cells increased beta-catenin in the low molecular weight fraction. However, Wnt-3a-dependent accumulation of beta-catenin was greatly inhibited in L cells stably expressing Axin. Axin also suppressed Wnt-3a-dependent activation of Tcf-4 which binds to beta-catenin and acts as a transcription factor. These results suggest that Axin forms a complex with GSK-3beta, beta-catenin, and APC, resulting in the stimulation of the degradation of beta-catenin and that Wnt-3a induces the dissociation of beta-catenin from the Axin complex and accumulates beta-catenin.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas/fisiologia , Proteínas Repressoras , Transativadores , Proteína da Polipose Adenomatosa do Colo , Animais , Proteína Axina , Células COS/metabolismo , Cromatografia em Gel , Meios de Cultivo Condicionados , Quinase 3 da Glicogênio Sintase , Quinases da Glicogênio Sintase , Células L/metabolismo , Camundongos , Proteínas/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta CateninaRESUMO
Hemorrhagic factors a and b were isolated from Trimeresurus mucrosquamatus venom by Sephadex G-100, CM-Sephadex C-50 and DEAE-Sephacel column chromatographies. The hemorrhagic factors were homogeneous, as established by a single band on acrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Molecular weights of 15 000 and 27 000 were found for hemorrhagic factors a and b, respectively. Factor a possesses proteolytic activity hydrolyzing the His(10)-Leu(11), Tyr(16)-Leu(17) and Arg(22)-Gly(23) bonds of oxidized insulin B chain, whereas, factor b hydrolyzed only the Ala(14)-Leu(15) bond. Hemorrhagic activity of these hemorrhagic factors was inhibited by ethylenediaminetetraacetic acid, 1,10-phenanthroline or p-chloromercuribenzoate, but not by soybean trypsin inhibitor or diisopropyl fluorophosphate. The hemorrhagic factors were injected into the skin of the back of albino rabbits, and the minimum hemorrhagic dose of factors a and b was 1.7 and 2.3 micrograms, respectively. These purified hemorrhagic factors were not lethal at 15 micrograms/g in mice. Factor a hydrolyzed the B beta chain of fibrinogen, while factor b hydrolyzed the A alpha chain. Hemorrhagic factor a was shown to differ immunologically from factor b. Factors a and b produced systemic hemorrhage in internal organs such as the heart and stomach of mice. Moreover, factor b produced hemorrhage in the liver.
Assuntos
Venenos de Crotalídeos/análise , Venenos de Crotalídeos/isolamento & purificação , Animais , Cromatografia , Venenos de Crotalídeos/metabolismo , Dipeptídeos/metabolismo , Eletroforese , Fibrinogênio/metabolismo , Hemorragia/induzido quimicamente , Camundongos , Peso Molecular , Necrose/induzido quimicamente , Especificidade por SubstratoRESUMO
We examined the effect of PGE1 on the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA induced by tumor necrosis factor-alpha (TNF-alpha) in human mesangial cells, because PAI-1 is one of major factors for the progression of glomerulosclerosis. The expression of PAI-1 mRNA was increased after stimulation with TNF-alpha, and it was diminished by pre-incubation with PGE1. Next, we examined the effect of PGE1 on the phosphorylation of mitogen activated protein kinase (MAPK) family and Akt. TNF-alpha activated the phosphorylation of p44/42 MAPK, p38 MAPK, SAPK/JNK and Akt in mesangial cells. PGE1 inhibited the TNF-alpha induced phosphorylation of SAPK/JNK and Akt, but not p44/42 MAPK and p38 MAPK. The TNF-alpha induced expression of PAI-1 mRNA was not affected by PD98059, an inhibitor of MEK, SB203580, an inhibitor of p38 MAPK, nor LY294002, an inhibitor of PI3 K. However, DMAP, an inhibitor of SAPK/JNK, inhibited the expression of PAI-1 mRNA, suggesting that the TNF-alpha induced expression of PAI-1 mRNA is regulated by the SAPK/JNK dependent pathway in human mesangial cells. By the incubation with H8, an inhibitor of PKA, the inhibitory effect of PGE1 on the expression of PAI-1 mRNA was abolished, suggesting that PGE1 inhibited the PAI-1 mRNA expression via the PKA pathway. Our results suggest that the inhibition of PAI-1 synthesis by PGE1 in human mesangial cells may have therapeutic implications for glomerulosclerosis such as occurs in diabetic nephropathy.
Assuntos
Alprostadil/farmacologia , Nefropatias Diabéticas/imunologia , Mesângio Glomerular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Fator de Necrose Tumoral alfa/farmacologia , Northern Blotting , Western Blotting , AMP Cíclico/metabolismo , Interações Medicamentosas , Mesângio Glomerular/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidores de Proteínas Quinases/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
As a first step toward understanding estrogen's role in neurodevelopment, a PCR cloning strategy was used to isolate complementary DNAs encoding two distinct cytochrome P450 aromatase isoforms in adult zebrafish (Danio rerio) brain and ovary (termed P450aromB and P450aromA, respectively). Sequence and phylogenetic analysis showed that the zebrafish P450arom forms are orthologs of previously identified cyp19b and cyp19a genes in goldfish. On Northern blots, a single 4.4-kb transcript of the P450aromB subtype was identified in brain, and a 2.1-kb transcript of the P450aromA subtype in ovary, but RT-PCR showed a degree of overlapping expression. Both messenger RNA (mRNA) forms were detected in unfertilized eggs and 1.5 hpf (cleavage stage) embryos but declined by 12 hpf, indicating maternal transfer. A secondary rise in mRNAs between 12-24 hpf indicated the onset of embryonic cyp19b and -a transcription. Both mRNA species accumulated progressively to 120 hpf (early larval stage), but the relative magnitude and pattern of change was isoform specific. Estradiol (E(2,) 1 microM) advanced and amplified the developmentally programmed accumulation of P450aromB mRNA, and ICI164.384 decreased expressed levels, implying blockade of an endogenous estrogen mediated regulatory component. Conversely, E(2) had no effect or decreased P450aromA mRNA. The early embryonic expression of P450aromB and P450aromA isoforms, and differences in developmental programming and estrogen regulation, imply independent regulatory mechanisms and unique functions during major morphogenetic and differentiative events.
Assuntos
Envelhecimento/metabolismo , Aromatase/metabolismo , Encéfalo/enzimologia , Embrião não Mamífero/metabolismo , Estrogênios/fisiologia , Isoenzimas/metabolismo , Ovário/enzimologia , Peixe-Zebra/metabolismo , Sequência de Aminoácidos/genética , Animais , Aromatase/genética , Encéfalo/embriologia , DNA Complementar/genética , Estradiol/farmacologia , Feminino , Isoenzimas/genética , Larva/metabolismo , Dados de Sequência Molecular , Ovário/embriologia , Óvulo/metabolismo , Filogenia , RNA Mensageiro/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/crescimento & desenvolvimentoRESUMO
The novel gene named PSX1, encoding a new protopectinase with the polymethoxygalacturonase activity, was isolated from Trichosporon penicillatum. Nucleotide sequencing revealed that the PSX1 gene is composed of 1080 bases (360 amino acids, 38,747 Da). The N-terminal amino acid sequences of the open reading frame correspond to a signal peptide and propeptide processed by a Kex2-like proteinase. Mature PPase SX1 was composed of 334 amino acids (36,121 Da). PPase SX1 produced by a S. cerevisiae transformant harboring the PSX1 gene degraded methoxylated polygalacturonic acid as a substrate, but not degraded unmethoxylated polygalacturonic acid.
Assuntos
Glicosídeo Hidrolases/biossíntese , Glicosídeo Hidrolases/genética , Polissacarídeo-Liases/biossíntese , Polissacarídeo-Liases/genética , Trichosporon/enzimologia , Trichosporon/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Precursores Enzimáticos/biossíntese , Precursores Enzimáticos/química , Genes Fúngicos , Glicosídeo Hidrolases/química , Dados de Sequência Molecular , Fases de Leitura Aberta , Polissacarídeo-Liases/química , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/química , Saccharomyces cerevisiae , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Vascular smooth muscle cells play a key role in the development of atherosclerosis. Culture of vascular smooth muscle A10 cells with high glucose for 4 weeks enhanced platelet-derived growth factor (PDGF)-induced BrdU incorporation. Since a long period of high glucose incubation was required for the effect, and it was inhibited by co-incubation with azaserine, the role of hexosamine biosynthesis in the development of atherosclerosis in diabetes was studied in A10 cells. Addition of glucosamine to the culture media enhanced PDGF-stimulated BrdU incorporation, and PDGF-induced tyrosine phosphorylation of the PDGF beta-receptor was increased by glucosamine treatment. Of the subsequent intracellular signaling pathways, PDGF-induced PDGF beta-receptor association with PLC gamma was not affected, whereas tyrosine phosphorylation of Shc, subsequent association of Shc with Grb2, and MAP kinase activation were relatively decreased. In contrast, PDGF-induced PDGF beta-receptor association with the p85 regulatory subunit of PI3-kinase and PI3-kinase activation were increased by 20% (P<0.01) and 36% (P<0.01), respectively. The intracellular signaling molecules responsible for the glucosamine effect were further examined using pharmacological inhibitors. Pretreatment with PLC inhibitor (U73122) had negligible effects, and MEK1 inhibitor (PD98059) showed only a slight inhibitory effect on the PDGF-induced BrdU incorporation. In contrast, pretreatment with PI3-kinase inhibitor (LY294002) significantly inhibited glucosamine enhancement of PDGF-induced BrdU incorporation. These findings suggest that glucosamine is involved in the development of atherosclerosis by enhancing PDGF-induced mitogenesis specifically via the PI3-kinase pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Aorta/metabolismo , DNA/biossíntese , Glucosamina/farmacologia , Músculo Liso Vascular/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Azasserina/farmacologia , Bromodesoxiuridina/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Proteína Adaptadora GRB2 , Glucose/farmacologia , Isoenzimas/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Fosfolipase C gama , Fosforilação/efeitos dos fármacos , Proteínas/metabolismo , Ratos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Fosfolipases Tipo C/metabolismo , Tirosina/metabolismoRESUMO
1. 3-Hydroxy-3-methylglutaryl co-enzyme A reductase inhibitors (statins) prevent the progression of atherosclerosis by lowering cholesterol. However, the effect of statins on the synthesis of pro-inflammatory cytokines from endothelial cells has not yet been fully investigated. Here, we examined the effect of pravastatin, one of the statins, on IL-8 synthesis induced by thrombin in human aortic endothelial cells (AoEC) cultured with high glucose concentrations. 2. Pravastatin significantly decreased the IL-8 synthesis induced by thrombin. 3. Pravastatin inhibited the p44/42 MAP kinase activity induced by thrombin, but did not inhibit the p38 MAP kinase activity. 4. Translocation of ras protein from the cytosol to plasma membrane was inhibited by pravastatin. 5. Pravastatin inhibit the activator protein-1 activity, but did not inhibit the activation of IkappaB-alpha. 6. Dominant negative ras inhibited the p44/42 MAP kinase activity induced by PMA. 7. Our results suggest that pravastatin inhibits IL-8 synthesis by blocking the ras-MAP (p44/42) kinase pathway rather than nuclear factor-kappaB. Pravastatin may prevent atherosclerosis not only by lowering cholesterol levels, but also by suppressing IL-8 synthesis in AoEC through the inhibition of p44/42 MAP kinase, and this may be more beneficial in diabetic patients than in non-diabetics.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Glucose/farmacologia , Proteínas I-kappa B , Interleucina-8/biossíntese , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Pravastatina/farmacologia , Trombina/farmacologia , Anticolesterolemiantes/farmacologia , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Western Blotting , Células Cultivadas , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/genética , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidor de NF-kappaB alfa , Fosforilação/efeitos dos fármacos , Proteínas de Plantas , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Fator de Transcrição AP-1/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Proteínas ras/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
To elucidate the role of nitric oxide (NO) and renin-angiotensin system (RAS) in the development of salt-sensitive hypertension, we investigated the pressor responses and renal histologic changes after long-term inhibition of endogenous NO synthesis in Dahl-Iwai salt-sensitive (DS) and salt-resistant (DR) rats under salt-re-stricted conditions that exaggerate RAS activation. Male DS and DR rats (6 weeks old) were fed with a low-salt (0.3%) diet for 5 weeks. NG-nitro-L-arginine (L-NA; dissolved in 60 mg/L deionized water), an arginine analogue acting as a NO-inhibitor, was also administered for 5 weeks. L-NA administration induced a gradual increase in systolic blood pressure (SBP) in both strains, and the pressor response in DS rats was apparently more enhanced relative to that in DR rats. Urinary nitrate plus nitrite (u-NOx) excretion was decreased by L-NA, with a significant negative correlation between SBP and u-NOx excretion in DS rats but not in DR rats. Plasma renin activity and urinary aldosterone level were significantly increased in L-NA-treated DS rats on week 5. Marked histologic changes with glomerular sclerosis and increased proteinuria and urinary N-acetyl-beta-glucosaminidase excretion were found in L-NA-treated DS rats but not DR rats. Competitive RT-PCR of mRNA extracted from the glomeruli revealed that angiotensin II type 1 receptor (AT1R) mRNA level was significantly lower in DS rats than in DR rats at week 2, and that L-NA administration significantly reduced glomerular AT1R level of DS rats at week 5, possibly because of downregulation. Our results showed that, even under sodium restriction, the pressor response and renal injury induced by chronic NO inhibition were markedly more enhanced in DS rats than in DR rats, which indicates that depletion of NO participates in both the development of hypertension and glomerular injury in DS rats through a potential activation of RAS irrespective of sodium loading. These data suggest that endogenous NO is an essential determinant of salt-sensitive hypertension in DS rats.
Assuntos
Dieta Hipossódica , Hipertensão/etiologia , Óxido Nítrico/fisiologia , Sistema Renina-Angiotensina/fisiologia , Aldosterona/urina , Animais , Pressão Sanguínea/efeitos dos fármacos , Regulação para Baixo , Glucuronidase/urina , Glomérulos Renais , Masculino , Nitratos/urina , Óxido Nítrico/antagonistas & inibidores , Nitritos/urina , Nitroarginina/farmacologia , Proteinúria/urina , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos Dahl , Receptor Tipo 1 de Angiotensina , Receptor Tipo 2 de Angiotensina , Receptores de Angiotensina/análise , Receptores de Angiotensina/genética , Renina/sangue , EscleroseRESUMO
Teleost fish are characterized by exceptionally high levels of neural estrogen biosynthesis when compared with the brains of other vertebrates or to the ovaries of the same fish. Two P450arom mRNAs which derive from separate gene loci (cyp19a and cyp19b) are differentially expressed in brain (b>>a) and ovary (a>>b) and have a different developmental program (b>>a) and estrogen upregulation (b only). A polymerase chain reaction (PCR)-based genomic walking strategy was used to isolate the 5'-flanking regions of the goldfish (Carassius auratus) cyp19 genes. Sequence analysis of the cyp19b gene approximately 1.8 kb upstream of the transcription start site revealed a TATA box at nucleotide (nt) -30, two estrogen responsive elements (EREs; nt -351 and -211) and a consensus binding site (NBRE) for nerve growth factor inducible-B protein (NGFI-B/Nur77) at -286, which includes another ERE half-site. Also present were a sequence at nt -399 (CCCTCCT) required for neural specificity of the zebrafish GATA-2 gene, and 16 copies of an SRY/SOX binding motif. The 5'-flanking region ( approximately 1.0 kb) of the cyp19a gene had TATA (nt -48) and CAAT (nt -71) boxes, a steroidogenic factor-1 (SF-1) binding site (nt -265), eight copies of the SRY/SOX motif, and two copies of a recognition site for binding the arylhydrocarbon receptor (AhR)/AhR nuclear translocator factor (ARNT) heterodimer. Both genes had elements previously identified in the brain specific exon I promoter of the mouse aromatase gene. Cyp19a- and -b/luciferase constructs showed basal promoter activity in aromatase-expressing rodent pituitary (GH3) cells, but differences (a>>b) did not reflect expression in fish pituitary in vivo (b>>a), implying a lack of appropriate cell factors. Consistent with the onset of cyp19b expression in zebrafish embryos, microinjection of a green fluorescent protein (GFP) reporter plasmid into fertilized eggs revealed labeling in neural tissues at 30-48 h post-fertilization (hpf), most prominently in retinal ganglion cells (RGC) and axon-like projections to the optic tectum. Expression of a cyp19a/GFP reporter was not detectable up to 72 hpf. Tandem analysis of cyp19a and cyp19b promoters in living zebrafish embryos can be a useful approach for identifying cis-elements and cellular factors involved in the correct tissue-specific, spatial, temporal and estrogen regulated expression of aromatase genes during CNS and gonadal development.