RESUMO
Background and purpose: MAPKs are among the most relevant signalling pathways involved in coordinating cell responses to different stimuli. This group includes p38MAPKs, constituted by 4 different proteins with a high sequence homology: MAPK14 (p38α), MAPK11 (p38ß), MAPK12 (p38γ) and MAPK13 (p38δ). Despite their high similarity, each member shows unique expression patterns and even exclusive functions. Thus, analysing protein-specific functions of MAPK members is necessary to unequivocally uncover the roles of this signalling pathway. Here, we investigate the possible role of MAPK11 in the cell response to ionizing radiation (IR). Materials and methods: We developed MAPK11/14 knockdown through shRNA and CRISPR interference gene perturbation approaches and analysed the downstream effects on cell responses to ionizing radiation in A549, HCT-116 and MCF-7 cancer cell lines. Specifically, we assessed IR toxicity by clonogenic assays; DNA damage response activity by immunocytochemistry; apoptosis and cell cycle by flow cytometry (Annexin V and propidium iodide, respectively); DNA repair by comet assay; and senescence induction by both X-Gal staining and gene expression of senescence-associated genes by RT-qPCR. Results: Our findings demonstrate a critical role of MAPK11 in the cellular response to IR by controlling the associated senescent phenotype, and without observable effects on DNA damage response, apoptosis, cell cycle or DNA damage repair. Conclusion: Our results highlight MAPK11 as a novel mediator of the cellular response to ionizing radiation through the control exerted onto IR-associated senescence.
RESUMO
CASE REPORT: We describe the case of a patient who presented with progressive and bilateral loss of vision. She had been treated with tamoxifen for 13 years. We performed fluorescein angiography and optical coherence tomography in order to study the macula. DISCUSSION: Loss of visual acuity related to tamoxifen maculopathy may be caused either by retinal nerve fibre atrophy or macular oedema. Macular findings obtained by fluorescein angiography and optical coherence tomography are complementary.
Assuntos
Antineoplásicos Hormonais/efeitos adversos , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/diagnóstico , Tamoxifeno/efeitos adversos , Tomografia de Coerência Óptica , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
A chimeric expression vector which encoded for a molecule encompassing the extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) and the intracellular domain of the ret kinase (EGFR/ret chimera) was generated. Upon ectopic expression in mammalian cells, the EGFR/ret chimera was correctly synthesized and transported to the cell surface, where it was shown capable of binding EGF and transducing an EGF-dependent signal intracellularly. Thus, the EGFR/ret chimera allows us to study the biological effects and biochemical activities of the ret kinase under controlled conditions of activation. Comparative analysis of the growth-promoting activity of the EGFR/ret chimera expressed in fibroblastic or hematopoietic cells revealed a biological phenotype clearly distinguishable from that of the EGFR, indicating that the two kinases couple with mitogenic pathways which are different to some extent. Analysis of biochemical pathways implicated in the transduction of mitogenic signals also evidenced significant differences between the ret kinase and other receptor tyrosine kinases. Thus, the sum of our results indicates the existence of a ret-specific pathway of mitogenic signaling.
Assuntos
Proteínas de Drosophila , Receptores ErbB/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/fisiologia , Células 3T3/citologia , Células 3T3/metabolismo , Animais , Sequência de Bases , Divisão Celular/genética , Divisão Celular/fisiologia , Linhagem Celular , DNA/genética , Receptores ErbB/genética , Proteínas Ativadoras de GTPase , Humanos , Inositol/metabolismo , Camundongos , Dados de Sequência Molecular , Fosfolipídeos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/genética , Transformação GenéticaRESUMO
OBJECTIVE: The aim of the study was to compare the results with those of a previous study by the same author in 1993 when 741 type II diabetic patients were recruited. We determined the prevalence of diabetic retinopathy and the impact of the new diagnostic criteria and stricter control of diabetes on the results obtained. METHODS: The study sample was obtained by hazard selection of 741 type II diabetic patients, from the total diabetic patients visited in the interval between January 1 and December 1 in 2005. RESULTS: We observed a decrease in the prevalence of diabetic retinopathy between the two studies. In the first study the incidence was 39.41% while in the present study it was 27.55%. The diabetic macular edema prevalence was similar in both studies (7.15% in the past and 7.90% in the present study). There was also a decrease in the number of blind patients (11.20% in 1993 and 4.90% in the current study). The number of patients treated with laser photocoagulation increased (13.49% in the current study as compared to 6.20% in the previous study). Statistic analysis revealed the risk factors for retinopathy: diabetes mellitus duration, elevated HbA1C levels and the need for insulin treatment. CONCLUSIONS: A better control of diabetes mellitus may lead us to observe an increase in visual acuity, and a better control of diabetic retinopathy. The incidence of diabetic retinopathy certainly decreased between the study periods; however the overall incidence of diabetes in the community has increased during the last few years, making firm conclusions difficult.
Assuntos
Retinopatia Diabética/epidemiologia , Idoso , Glicemia/análise , Estudos Transversais , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/epidemiologia , Retinopatia Diabética/terapia , Feminino , Hemoglobinas Glicadas/análise , Humanos , Hipertensão/epidemiologia , Hipoglicemiantes/uso terapêutico , Incidência , Insulina/uso terapêutico , Fotocoagulação a Laser , Masculino , Pessoa de Meia-Idade , Papiledema/epidemiologia , Papiledema/etiologia , Prevalência , Índice de Gravidade de Doença , Espanha/epidemiologia , Acuidade VisualRESUMO
OBJECTIVE: To describe intra- and post-operative complications, as well as the evolution of the surgical technique in first 4years of work of a novice retina surgeon, and evaluate minimal learning time required to reduce its complications, deciding which pathologies should still be referred to higher level hospitals, until further experience may be achieved. METHODS: A study was conducted on patients that had undergone vitreoretinal surgery by a novice surgeon in Tarragona between 23rd October 2007 and 31st December 2011. The primary diagnosis, surgeon learning time, surgical technique, intra-operative and post-operative complications were recorded. RESULTS: A total of 247 surgeries were studied. The percentage of use of 20G and 23G calibres during the time, marks a change towards trans-conjunctival surgery from the ninth trimester (98 surgeries). Surgical complications decreased towards twelfth trimester (130 surgeries) with an increase in the previous months. CONCLUSIONS: The shift towards 23G technique around 100 surgeries is interpreted as greater comfort and safety by the surgeon. Increased surgical complications during the following months until its decline around 130 surgeries can be interpreted as an 'overconfidence'. It is arguable that the learning curve is slower than what the surgeon believes. An individual analysis of the complications and surgical outcomes is recommended to ascertain the status of the learning curve.
Assuntos
Curva de Aprendizado , Cirurgia Vitreorretiniana , Atitude do Pessoal de Saúde , Bolsas de Estudo , Humanos , Complicações Intraoperatórias/epidemiologia , Complicações Intraoperatórias/etiologia , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Descolamento Retiniano/cirurgia , Estudos Retrospectivos , Espanha , Cirurgiões/psicologia , Suturas , Vitrectomia/métodos , Vitrectomia/estatística & dados numéricos , Cirurgia Vitreorretiniana/métodos , Cirurgia Vitreorretiniana/estatística & dados numéricos , Hemorragia Vítrea/cirurgiaRESUMO
Purified amino-terminal Src homology 2 (SH2) domains of GAP, PLCgamma1 and the p85alpha subunit of PI 3-kinase, as well as the carboxy-terminal SH2 domain of the latter protein and the unique SH2 domain of Grb2, were injected into full grown, stage VI Xenopus laevis oocytes. None of the injected domains showed any effect when injected alone, nor did they affect the rate of GVBD induced by progesterone, an adenylate cyclase-dependent process. On the other hand, the unique Grb2 SH2 domain and all N-terminal SH2 domains injected inhibited to various degrees the rate of insulin-induced GVBD, a tyrosine kinase dependent pathway. Interestingly, and in contrast to the behavior shown by the N-terminal domain of the same molecule, the C-terminal SH2 domain of p85 did not inhibit, but slightly accelerated the rate of GVBD induced by insulin. Furthermore, whereas the Grb SH2 domain and all N-terminal SH2 domains tested failed to co-operate with normal Ras protein to induce GVBD, the C-terminal SH2 domain of p85alpha exhibited significant synergy when coinjected with normal Ras protein, indicating that the C- and N-terminal SH2 domains of p85alpha exert opposite (positive and negative, respectively) regulatory roles in the control of oocyte insulin/Ras signaling pathways. Our results demonstrate that the purified, isolated SH2 domains retain structural and functional specificity and that Xenopus oocytes constitute an useful biological system to analyse their functional role in tyrosine kinase signaling pathways.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Insulina/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Oócitos/fisiologia , Transdução de Sinais , Domínios de Homologia de src/fisiologia , Animais , Feminino , Proteína Adaptadora GRB2 , Proteínas Ativadoras de GTPase , Insulina/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Meiose/efeitos dos fármacos , Microinjeções , Proteína Oncogênica p21(ras)/isolamento & purificação , Proteína Oncogênica p21(ras)/farmacologia , Oócitos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases , Fosfolipase C gama , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Progesterona/metabolismo , Progesterona/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Xenopus laevis/metabolismo , Proteínas Ativadoras de ras GTPaseRESUMO
Recent evidence has suggested that the Vav oncoprotein may function as a hematopoietic-specific GTP exchange factor for the Ras superfamily of proteins. However, transformation of NIH3T3 fibroblast cells by Vav is morphologically distinct from that induced by activated Ras oncogenes, suggesting that the two oncoproteins induce separate signal transduction pathways which promote transformation. To address this issue, the effects of dominant negative mutants of H-ras and proto-Vav (proto-VavR695L, a mutation in the VavSH2 domain) were tested on Vav- and Ras-induced transformation. These mutants partially inhibited both Vav- and Ras-induced transformation, suggesting that they may induce a common downstream signaling pathway which potentiates transformation. As an independent measure of Vav function we also tested the ability of the purified protein encoded by VavSH2 to influence Germinal Vesicle Breakdown (GVBD) during Xenopus oocyte maturation. Microinjection of the VavSH2 protein alone, but not mutant VavR695L SH2 protein, was sufficient to induce GVBD and accelerated maturation induced by normal Ras, suggesting that in this system as well Vav and Ras signals overlap through a common effector. A key target of multiple signalling pathways is c-Myc. Dominant negative versions of c-Myc totally abolished morphologic transformation of NIH3T3 cells by both Vav and Ras oncogenes. These results suggest that distinct, but overlapping, signalling pathways are induced by Vav and Ras and that fibroblast cell transformation by either oncogene requires c-Myc functions.
Assuntos
Proteínas de Ciclo Celular , Transformação Celular Neoplásica , Genes myc/fisiologia , Genes ras/fisiologia , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes/fisiologia , Células 3T3 , Animais , Feminino , Camundongos , Mutação , Proteínas Proto-Oncogênicas c-vav , Xenopus laevis , Domínios de Homologia de srcRESUMO
Two human hSos1 isoforms (Isf I and Isf II; Rojas et al., Oncogene 12, 2291-2300, 1996) defined by the presence of a distinct 15 amino acid stretch in one of them, were compared biologically and biochemically using representative NIH3T3 transfectants overexpressing either one. We showed that hSos1-Isf II is significantly more effective than hSos1-Isf I to induce proliferation or malignant transformation of rodent fibroblasts when transfected alone or in conjunction with normal H-Ras (Gly12). The hSos1-Isf II-Ras cotransfectants consistently exhibited higher saturation density, lower cell-doubling times, increased focus-forming activity and higher ability to grow on semisolid medium and at low serum concentration than their hSos1-Isf I-Ras counterparts. Furthermore, the ratio of GTP/GDP bound to cellular p21ras was consistently higher in the hSos1-Isf II-transfected clones, both under basal and stimulated conditions. However, no significant differences were detected in vivo between Isf I- and Isf II-transfected clones regarding the amount, stability and subcellular localization of Sos1-Grb2 complex, or the level of hSos1 phosphorylation upon cellular stimulation. Interestingly, direct Ras guanine nucleotide exchange activity assays in cellular lysates showed that Isf II transfectants consistently exhibited about threefold higher activity than Isf I transfectants under basal, unstimulated conditions. Microinjection into Xenopus oocytes of purified peptides corresponding to the C-terminal region of both isoforms (encompassing the 15 amino acid insertion area and the first Grb2-binding motif) showed that only the Isf II peptide, but not its corresponding Isf I peptide, was able to induce measurable rates of meiotic maturation, and synergyzed with insulin, but not progesterone, in induction of GVBD. Our results suggest that the increased biological potency displayed by hSos1-Isf II is due to higher intrinsic guanine nucleotide exchange activity conferred upon this isoform by the 15 a.a. insertion located in proximity to its Grb2 binding region.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteína Adaptadora GRB2 , Fatores de Troca do Nucleotídeo Guanina , Humanos , Camundongos , Dados de Sequência Molecular , Mutagênese Insercional , Isoformas de Proteínas , Proteínas/genética , Transfecção , Transformação Genética , Fatores ras de Troca de Nucleotídeo Guanina , Proteínas ras/metabolismoRESUMO
We compared structure, expression and functional properties of two hSos1 cDNA isoforms (IsfI and Isf II) isolated, respectively, from human fetal brain and adult skeletal muscle libraries. IsfI and IsfII nucleotide sequences differ only by the presence in IsfII of an inframe 45 hp insertion located near the first proline-rich motif required for Grb2 binding. Some human tissues express only one isoform whereas others express different proportions of both in fetal and adult stages. In vitro binding assays and in vivo functional studies showed that MI exhibits significantly higher Grb2 binding affinity and biological activity than IsfI. These results suggest that functionally different hSos1 isoforms, with differential tissue expression and distribution, play important regulatory roles in the mechanisms controlling Ras activation in different tissues and/or developmental stages.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Química Encefálica , DNA Complementar/química , Músculo Esquelético/química , Fragmentos de Peptídeos/química , Proteínas/química , Proteínas/metabolismo , Adulto , Sequência de Bases , Feto , Proteína Adaptadora GRB2 , Regulação da Expressão Gênica , Genes ras/genética , Glutationa Transferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Transfecção , Leveduras/enzimologia , beta-Galactosidase/biossíntese , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
A protein that catalyzes the decoloration of dopachrome has been partially purified from B16 mouse melanoma tumors. The enzyme is preferentially associated to the melanosomes, but it is also found in the microsomal and cytosolic fractions of cellular homogenates. The protein is clearly different from tyrosinase, and should be related to the dopachrome oxidoreductase (Barber et al. (1984) J. Invest. Dermatol. 83, 145-149) and the dopachrome conversion factor (Korner and Pawelek (1980) J. Invest. Dermatol. 75, 192-195) since the reaction product of dopachrome conversion is 5,6-dihydroxyindole-2-carboxylic acid. The protein appears to have an oligomeric structure, with a molecular mass slightly higher than 300 kDa estimated by gel filtration, whereas the molecular mass of the monomer might be approx. 46 kDa estimated by SDS-PAGE electrophoresis. Its Km for dopachrome is around 100 microM. The enzyme is competitively inhibited by indoles and is unaffected by metal chelators. It also has the ability to increase the amount of melanin formed from L-tyrosine by melanoma tyrosinase, and therefore, cannot be considered an 'indole blocking factor' as was suggested for the related dopachrome oxidoreductase. Since the reaction catalyzed by the enzyme is a tautomeric shift on dopachrome, we would propose dopachrome tautomerase (EC 5.3.2.3) as the most precise and informative name.
Assuntos
Catecol Oxidase/isolamento & purificação , Oxirredutases Intramoleculares , Melaninas/biossíntese , Melanoma Experimental/enzimologia , Animais , Catecol Oxidase/metabolismo , Linhagem Celular , Cromatografia , Cromatografia em Gel , Durapatita , Feminino , Hidroxiapatitas , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Peso Molecular , Frações Subcelulares/enzimologiaRESUMO
The effect of cysteine and glutathione on mammalian melanogenesis has been studied. It has been shown that their action is mediated by two different mechanisms. (a) The reaction of the thiol groups with dopaquinone after the tyrosinase-catalyzed oxidation of tyrosine and dopa. This mechanism leads to the formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments, phaeomelanins instead of eumelanins. This fact might produce an inhibition of melanogenesis due to the slower rate of chemical reactions involved in the polymerization of such thiol-conjugates when compared to that of indoles. (b) The direct interaction between the sulfhydryl compounds and the tyrosinase active site. This interaction may regulate the activity of the enzyme. It is shown that Harding-Passey mouse melanoma tyrosinase is more sensitive to sulfhydryl compounds than mushroom tyrosinase. Cysteine always produces an inhibition of the tyrosinase hydroxylase and dopa oxidase activities of melanoma tyrosinase, this inhibition becoming greater as the cysteine concentration increases. On the other hand, glutathione produces an activation of the tyrosine hydroxylase activity below 3 mM and an inhibition at higher concentrations. The limit between the enzymatic activation and inhibition appears at glutathione concentrations similar to the physiological levels of this compound found in melanocytes. Although the switch from eumelanogenesis to phaeomelanogenesis occurs at much lower concentrations of glutathione, taking into account these data it is discussed that this sulfhydryl compound may regulate not only the type but also the amount of melanin formed inside melanocytes.
Assuntos
Catecol Oxidase/metabolismo , Cisteína/farmacologia , Glutationa/farmacologia , Melaninas/biossíntese , Monofenol Mono-Oxigenase/metabolismo , Animais , Melanoma/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Melanogenesis can be divided into two phases. The first one involves two tyrosinase-catalyzed oxidations from tyrosine to dopaquinone and a very fast chemical step leading to dopachrome. The second phase, from dopachrome to melanin, can proceed spontaneously through several incompletely known reactions. However, some metal transition ions and protein factors different from tyrosinase might regulate the reaction rate and determine the structure and relative concentrations of the intermediates. The study of the effects of some divalent metal ions (Zn, Cu, Ni and Co) on some steps of the melanogenesis pathway has been approached using different radiolabeled substrates. Zn(II) inhibited tyrosine hydroxylation whereas Ni(II) and Co(II) were activators. Ni(II), Cu(II) and Co(II) accelerated chemical reactions from dopachrome but inhibited its decarboxylation. Dopachrome tautomerase also decreased decarboxylation. When metal ions and this enzyme act together, the inhibition of decarboxylation was greater than that produced by each agent separately, but amount of carboxylated units incorporated to the melanin was not higher than the amount incorporated in the presence of only cations. The amount of total melanin formed from tyrosine was increased by the presence of both agents. The action of Zn(II) was different from other ions also in the second phase of melanogenesis, and its effect on decarboxylation was less pronounced. Since tyrosine hydroxylation is the rate-limiting step in melanogenesis, Zn(II) inhibited the pathway. This ion seems to be the most abundant cation in mammalian melanocytes. Therefore, under physiological conditions, the regulatory role of metal ions and dopachrome tautomerase does not seem to be mutually exclusive, but rather complementary.
Assuntos
Melaninas/biossíntese , Melanoma Experimental/metabolismo , Animais , Cátions Bivalentes , Di-Hidroxifenilalanina/metabolismo , Feminino , Cinética , Masculino , Melanoma Experimental/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Monofenol Mono-Oxigenase/metabolismo , Tirosina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
A vis-a-vis comparison between the effects of dopachrome tautomerase (DCT) and metal ions, e.g., cupric ions, on the kinetics and mode of rearrangement of dopachrome has been carried out under appropriate analytical conditions. The enzyme-promoted reaction is highly stereospecific for L-dopachrome, is unaffected by metal chelators and has an optimal pH around 6.8. By contrast, the kinetics of dopachrome rearrangement catalysed by cupric ions are not dependent on the stereochemistry of the substrate, are affected by EDTA and are not influenced by the pH of the medium in the range between 5-7.5. Both cupric ions and DCT catalyse the rearrangement of dopachrome to give 5,6-dihydroxyindole-2-carboxylic acid (DICA) rather than 5,6-dihydroxyindole (DI). However, at comparable activity, the ratio of formation DICA/DI is significantly higher in the enzyme-catalysed than in the metal-catalysed reaction. These results provide an improved background to look into the mode of action of DCT and metal ions, enabling a clear cut differentiation between the effects of the two factors when both are present in biological extracts.
Assuntos
Cobre/farmacologia , Indolquinonas , Indóis/química , Oxirredutases Intramoleculares , Isomerases/farmacologia , Quinonas/química , Animais , Catálise , Cromatografia Líquida de Alta Pressão , Ácido Edético/farmacologia , Melanoma Experimental/química , Melanoma Experimental/enzimologia , Camundongos , Células Tumorais CultivadasRESUMO
The potential cytotoxicity of the melanogenic intermediates DOPA, (L-3,4-dihydroxyphenylalanine) and DHI (5,6-dihydroxyindole) has long been recognized and exploited as a targeting concept in experimental melanoma therapy. In recent years, however, a novel branchpoint in the melanin biosynthetic pathway has been shown to divert the metabolism of DOPAchrome to a carboxylated derivative termed DHICA (DHI-2-carboxylic acid) rather than to DHI. In order to evaluate the biological implications of this regulatory control, we have reexamined the inherent cytotoxicity of DHICA versus DHI on different cell lines. We found that under the usual conditions of the biological assay, the apparent cytotoxicity of the two indoles reflect their instability in the culture medium, the less stable DHI being generally more toxic than DHICA to melanoma cells and nonmelanocytic cells. Moreover, the observed cytotoxic effects increased with the time of incubation and were markedly reduced by the addition of catalase to the medium, suggesting that they were probably due to the generation of reactive oxygen species (particularly H2O2) during the autoxidation of the melanin precursors outside the cells. To circumvent this problem, we then tested the diacetylated derivatives of DHI and DHICA (DAI and DAICA) which are sufficiently stable until taken up into the cells whereupon they may be converted by endogenous esterases back to the parent indoles. Although DAI proved to be cytotoxic for nonmelanocytic cells, it had no detectable activity on melanoma cells, whereas DAICA showed no effect on any of the cells examined. These results, when combined with other studies, point to a reconsideration of the inherent cytotoxicity of the 5,6-dihydroxyindoles, as well as DOPA, to melanin producing cells.
Assuntos
Melaninas/biossíntese , Precursores de Proteínas/toxicidade , Células 3T3 , Animais , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Di-Hidroxifenilalanina/toxicidade , Peróxido de Hidrogênio/metabolismo , Indóis/toxicidade , Camundongos , Microscopia de Contraste de Fase , Oxirredução , Células Tumorais CultivadasRESUMO
The C1 domains are 50 amino acid sequences present in protein kinase C (PKC) isozymes that are responsible for binding of phorbol esters and the lipid second messenger diacylglycerol (DAG). We found that bacterially expressed C1b domain of PKCdelta induces germinal vesicle breakdown (GVBD) when microinjected into Xenopus laevis oocytes. Injection of the C1b domain of PKCdelta significantly enhanced insulin- but not progesterone-induced maturation. Interestingly, the PKCdelta C1b domain markedly synergized with normal Ras protein to induce oocyte maturation when both proteins were co-injected in oocytes. Our results demonstrate that the purified C1b domain of PKCdelta is sufficient to promote meiotic maturation of X. laevis oocytes probably through activation of components of the insulin/Ras signaling pathway.
Assuntos
Isoenzimas/farmacologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Proteína Quinase C/farmacologia , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Relação Dose-Resposta a Droga , Feminino , Insulina/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Microinjeções , Mutação , Oócitos/crescimento & desenvolvimento , Ésteres de Forbol/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/farmacologia , Progesterona/farmacologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-delta , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Xenopus , Proteínas ras/farmacologia , Domínios de Homologia de src/genéticaRESUMO
It is generally believed that the spinal cord and hindbrain consist of a motor basal plate and a sensory alar plate. We now have molecular markers for these territories. The relationship of migrating branchiomotor neurons to molecularly defined alar and basal domains was examined in the chicken embryo by mapping the expression of cadherin-7 and cadherin-6B, in comparison to genetic markers for ventrodorsal patterning (Otp, Pax6, Pax7, Nkx2.2, and Shh) and motoneuron subpopulations (Phox2b and Isl1). We show cadherin-7 is expressed in a complete radial domain occupying a lateral region of the hindbrain basal plate. The cadherin-7 domain abuts the medial border of Pax7 expression; this common limit defines, or at least approximates, the basal/alar boundary. The hindbrain branchiomotor neurons originate in the medial part of the basal plate, close to the floor plate. Their cadherin-7-positive axons grow into the alar plate and exit the hindbrain close to the corresponding afferent nerve root. The cadherin-7-positive neuronal cell bodies later translocate laterally, following this axonal trajectory, thereby passing through the cadherin-7-positive basal plate domain. Finally, the cell bodies traverse the molecularly defined basal/alar boundary and move into positions within the alar plate. After the migration has ended, the branchiomotor neurons switch expression from cadherin-7 to cadherin-6B. These findings demonstrate that a specific subset of primary motor neurons, the branchiomotor neurons, migrate into the alar plate of the chicken embryo. Consequently, the century-old concept that all primary motor neurons come to reside in the basal plate should be revised.
Assuntos
Padronização Corporal/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/metabolismo , Rombencéfalo/citologia , Medula Espinal/citologia , Animais , Caderinas/metabolismo , Embrião de Galinha , Proteínas do Olho , Proteínas Hedgehog , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Interferon Tipo I/metabolismo , Metaloproteínas/metabolismo , Proteínas Nucleares , Fator de Transcrição PAX6 , Fator de Transcrição PAX7 , Fatores de Transcrição Box Pareados , Proteínas da Gravidez/metabolismo , RNA Mensageiro/biossíntese , Proteínas Repressoras , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Medula Espinal/embriologia , Medula Espinal/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-ZebraRESUMO
The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopachrome into 5,6-dihydroxyindole. The advantages of these methods as compared to the previously used procedures are discussed.
Assuntos
Catecol Oxidase/análise , Indolquinonas , Oxirredutases Intramoleculares , Melanoma Experimental/enzimologia , Animais , Indóis/síntese química , Masculino , Camundongos , Quinonas/síntese química , Espectrofotometria UltravioletaRESUMO
This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.
Assuntos
Catecol Oxidase/análise , Melaninas/análise , Melanoma Experimental/enzimologia , Monofenol Mono-Oxigenase/análise , Animais , Radioisótopos de Carbono , Cátions Bivalentes , Linhagem Celular , Cinética , Melaninas/biossíntese , Camundongos , Camundongos Endogâmicos , Monofenol Mono-Oxigenase/metabolismo , Níquel/farmacologia , Técnica de Diluição de Radioisótopos , Hidróxido de Sódio/farmacologia , TrítioRESUMO
INTRODUCTION: Suprachoroidal hemorrhage (SCH) is a dramatic complication of intraocular surgery that can result in total loss of vision. METHODS: The records of eight cases of SCH during cataract surgery were reviewed. Six of eight patients were treated by combined radial sclerotomies for suprachoroidal drainage and vitrectomy. Risk factors, therapeutic strategies, and functional and anatomical results were analyzed. RESULTS: The incidence of SCH was 0.45%. Preoperative visual acuity of all eyes suffering from SCH was limited to the perception of light. Postoperatively, six patients showed an increase in visual acuity greater than 0.1; one patient achieved 0.5. Ocular and general risk factors (ocular hypotony, myopia, Valsalva-type maneuvers, intraoperative systemic hypertension) and surgery complications were analyzed. CONCLUSIONS: In spite of using state-of-the-art surgical techniques, the prognosis of SCH remains serious, with a poorer outcome associated with increasing complications due to hemorrhage. Secondary treatment combining radial sclerotomies and vitrectomy should be performed to minimize the damaging effect of choroidal hemorrhage.
Assuntos
Hemorragia da Coroide , Idoso , Idoso de 80 Anos ou mais , Hemorragia da Coroide/diagnóstico , Hemorragia da Coroide/terapia , Feminino , Humanos , MasculinoRESUMO
PURPOSE: To determine the relationship between microalbuminuria and diabetic retinopathy. METHODS: A prospective 10-year study of 104 younger-onset diabetic patients. The diabetic retinopathy diagnosis was made by fundus retinography, and determination of microalbuminuria was made from urine samples. RESULTS: The incidence of diabetic retinopathy in this group of patients was 39 (37.5%). The epidemiological factors implicated were diabetes duration, higher levels of HbA(1c), male sex, and diastolic arterial hypertension. The incidence of microalbuminuria was 21 patients (20.2%), with high levels of HbA(1c) the epidemiological factor implicated. The association between microalbuminuria and diabetic retinopathy grouped the patients as follows: 56 patients without microalbuminuria or retinopathy, 16 patients who developed microalbuminuria and diabetic retinopathy, 23 patients who developed retinopathy but not microalbuminuria, and nine patients who developed only microalbuminuria. The discriminant analysis showed that the high levels of HbA(1c) were associated with microalbuminuria and diabetes duration and high levels of HbA(1c) were associated with diabetic retinopathy. CONCLUSIONS: In the population studied, microalbuminuria was not a good marker for diabetic retinopathy.