RESUMO
This study aimed to examine the evidence of direct interaction among actin, myosin and phosphatidylinositol 3-kinase (PI3K) in the polarisation and formation of the tetraspore germ tube of Gelidium floridanum. After release, tetraspores were exposed to cytochalasin B, latrunculin B, LY294002 and BDM for a period of 6 h. In control samples, formation of the germ tube occurred after the experimental period, with cellulose formation and elongated chloroplasts moving through the tube region in the presence of F-actin. In the presence of cytochalasin B, an inhibitor of F-actin, latrunculin B, an inhibitor of G-actin, and BDM, a myosin inhibitor, tetraspores showed no formation of the germ tube or cellulose. Spherical-shaped chloroplasts were observed in the central region with a few F-actin filaments in the periphery of the cytoplasm. Tetraspores treated with LY294002, a PI3K inhibitor, showed no formation of the tube at the highest concentrations. Polarisation of cytoplasmic contents did not occur, only cellulose formation. It was concluded that F-actin directs the cell wall components and contributes to the maintenance of chloroplast shape and elongation during germ tube formation. PI3K plays a fundamental role in signalling for the asymmetric polarisation of F-actin. Thus, F-actin regulates the polarisation and germination processes of tetraspores of G. floridanum.
Assuntos
Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Rodófitas/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Parede Celular/metabolismo , Cloroplastos/metabolismo , Cromonas/farmacologia , Citocalasinas , Diacetil/análogos & derivados , Diacetil/farmacologia , Morfolinas/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase , Estruturas Vegetais/crescimento & desenvolvimento , Estruturas Vegetais/metabolismo , Rodófitas/efeitos dos fármacos , Rodófitas/crescimento & desenvolvimento , Tiazolidinas/farmacologiaRESUMO
Diacetyl is a flavoring that imparts a buttery flavor to foods, but the use or exposure to diacetyl has been related to some diseases. We investigated the effect of oral intake of diacetyl in male and female C57/Bl mice. We performed a target metabolomics assay using ultraperformance liquid chromatography paired with triple quadrupole mass spectrometry (UPLC-MS/MS) for the determination and quantification of plasmatic metabolites. We observed alterations in metabolites present in the urea and tricarboxylic acid (TCA) cycles. Peroxynitrite plasmatic levels were evaluated by a colorimetric method, final activity of superoxide dismutase (SOD) was evaluated by an enzymatic method, and mouse behavior was evaluated. Majority of the assay showed differences between control and treatment groups, as well as between genders. This may indicate the involvement of sex hormones in the regulation of a normal metabolic profile, and the implication of sex differences in metabolite disease response.
Assuntos
Diacetil/farmacologia , Aromatizantes/farmacologia , Metabolômica , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Feminino , Masculino , Camundongos , Espectrometria de Massas em TandemRESUMO
OBJECTIVES: The purpose of this study was to evaluate the effect of two additives: propionaldehyde (aldehyde) and 2,3-butanedione (diketone) on the properties of Bis-GMA diluted with TEGDMA and the synthesized Bis-GMA analogs, propoxylated Bis-GMA (CH(3)Bis-GMA) and propoxylated fluorinated Bis-GMA (CF(3)Bis-GMA). METHODS: Nine experimental comonomers were prepared combining Bis-GMA and TEGDMA, CH(3)Bis-GMA, CF(3)Bis-GMA, with aldehyde (32mol%) and diketone (32mol%). Photopolymerization was effected by using Camphorquinone (0.2wt%) and N,N-dimethyl-p-toluidine (0.2wt%). Experimental comonomer viscosity (Brookfield viscometer), polymerization shrinkage (gravimetrically), degree of conversion (FT-IR) and contact angles (contact angle goniometer) were determined. Comonomer and copolymer T(g)s (DSC and Fox equation) were also evaluated. Data were analyzed by one-way ANOVA and Tukey test (alpha=0.05). RESULTS: Bis-GMA/CH(3)Bis-GMA and Bis-GMA/CF(3)Bis-GMA with additives exhibited lower viscosities (p<0.01). Inclusion of additives into the comonomer systems did not produce significant increase in polymerization shrinkage (p>0.05). A significant increase in degree of conversion was shown for Bis-GMA/TEGDMA and Bis-GMA/CH(3)Bis-GMA with additives (p<0.01). Additives reduced contact angle and comonomer T(g) values, whereas the corresponding copolymers with additives showed an increase in T(g). SIGNIFICANCE: Use of novel comonomer systems with the addition of aldehyde and diketone functional groups would improve dental resin composite properties.
Assuntos
Aldeídos/farmacologia , Bis-Fenol A-Glicidil Metacrilato/química , Diacetil/farmacologia , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Viscosidade/efeitos dos fármacosRESUMO
Entamoeba histolytica manifests its pathogenicity through several cellular processes triggered by external stimuli that activate signal transduction pathways. The intense secretory activity resulting from stimulation is not correlated with a typical endoplasmic reticulum (ER) or Golgi organization, and little is known in this parasite about endocytic/exocytic pathways. The interactions of trophozoites with fibronectin (FN) and cultured mammalian cells, which elicit secretory activities, were chosen to study mechanisms that regulate cytoplamic traffic. Results showed that Brefeldin A (BFA) induced redistribution of the vesicular network recognized by antibodies against amoebic proteins PDI and ERD2. Furthermore, BFA diminished traffic to the plasma membrane of the beta1 integrin-like FN receptor and the heavy subunit of the Gal/GalNAc lectin, required for adhesion to FN and target cells, respectively. However, BFA did not prevent thiol-proteinase secretion or inhibit the traffic of de novo synthesized proteinases. These data suggest that two distinct transport systems occur in E. histolytica, one similar to classical membrane protein transport and another independent of BFA and inducible by external stimuli. Actin-myosin contractility of the cortical cytoskeleton seems necessary for the final release of exported proteinases and the proper function of the surface proteins involved in adhesion.
Assuntos
Brefeldina A/farmacologia , Diacetil/análogos & derivados , Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Exocitose/efeitos dos fármacos , Actinas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Cisteína Endopeptidases/metabolismo , Diacetil/farmacologia , Cães , Entamoeba histolytica/efeitos dos fármacos , Glicoproteínas/metabolismo , Integrina alfa5beta1/metabolismo , Lectinas/metabolismo , Proteínas de Membrana/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/metabolismoRESUMO
Recently cytochrome c has been mentioned as an important mediator in the events of cellular oxidative stress and apoptosis. To investigate the influence of charged interfaces on the conformation of cytochrome c, the CD and magnetic circular dichroic behavior of ferric and ferrous cytochrome c in homogeneous medium and in phosphatidylcholine/phosphatidylethanolamine/cardiolipin and dicetylphosphate liposomes was studied in the 300-600 and 200-320 nm wavelength region. EPR spectra demonstrate that the association of cytochrome c with membranes promotes alterations of the crystal field symmetry and spin state of the heme Fe(3+). The studies also include the effect of P(i), NaCl, and CaCl(2). Magnetic circular dichroism and CD results show that the interaction of both ferrous and ferric cytochrome c with charged interfaces promotes conformational changes in the alpha-helix content, tertiary structure, and heme iron spin state. Moreover, the association of cytochrome c with different liposomes is sensitive to the heme iron valence state. The more effective association with membranes occurs with ferrous cytochrome c. Dicetylphosphate liposomes, as a negatively charged membrane model, promoted a more pronounced conformational modification in the cytochrome c structure. A decrease in the lipid/protein association is detected in the presence of increasing amounts of CaCl(2), NaCl, and P(i), in response to the increase of the ionic strength.
Assuntos
Grupo dos Citocromos c/química , Grupo dos Citocromos c/metabolismo , Heme/metabolismo , Ferro/metabolismo , Lipossomos/metabolismo , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Dicroísmo Circular , Diacetil/análogos & derivados , Diacetil/farmacologia , Espectroscopia de Ressonância de Spin Eletrônica , Heme/química , Cavalos , Ferro/química , Lipossomos/química , Miocárdio/química , Compostos Organofosforados/farmacologia , Concentração Osmolar , Estrutura Secundária de Proteína , Sais/farmacologia , Eletricidade EstáticaRESUMO
Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e < or = 5 microM) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin-myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10 min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction where Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid( an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin-myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]3 poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.
Assuntos
Actinas/fisiologia , Cálcio/farmacologia , Tamanho Celular/efeitos dos fármacos , Miosinas/fisiologia , 2,4-Dinitrofenol/farmacologia , Animais , Linhagem Celular/citologia , Linhagem Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Desoxiglucose/farmacologia , Diacetil/análogos & derivados , Diacetil/farmacologia , Cães , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Immunoblotting , Rim , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Inibidores da Síntese de Ácido Nucleico/farmacologia , Ácido Okadáico/farmacologiaRESUMO
10 and 30 mM 2,3-butanedione monoxime (BDM) applied extracellularly to voltage-clamped frog skeletal muscle twitch fibres suppressed both Ca2+ release flux and intramembranous charge movement. Both effects could be clearly separated. The early peak of the Ca2+ release flux was suppressed at every test voltage. The steady level attained at the end of a 100 ms clamp depolarization was relatively spared for lower depolarizing pulses, but was as suppressed as the peak at voltages above -20 mV. The intramembranous charge movement was affected mainly in the I gamma component. The drug had a distinct effect on the kinetics of the intramembranous charge movement current around the threshold for Ca2+ release. The three kinetic components of I gamma were simultaneously affected. For more positive depolarizations where the kinetic effect was not evident, the oxime had no significant effect on the charge moved. Under conditions in which I gamma was absent (i.e. stretched fibres, intracellular solutions containing 6 to 10 mM BAPTA), treatment with 10 mM BDM had a small, not significant suppressive effect on the maximum charge moved (Qmax), while it affected Ca2+ release significantly. When 10 mM BDM was applied in the presence of 0.2 mM tetracaine, the local anaesthetic-resistant Ca2+ release flux was not further suppressed by the oxime.
Assuntos
Reativadores da Colinesterase/farmacologia , Diacetil/análogos & derivados , Contração Muscular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Anestésicos Locais/farmacologia , Animais , Cálcio/metabolismo , Quelantes/farmacologia , Diacetil/farmacologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica , Cinética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Técnicas de Patch-Clamp , Rana catesbeiana , Sarcômeros/efeitos dos fármacos , Sarcômeros/metabolismo , Retículo Sarcoplasmático/metabolismo , Tetracaína/farmacologiaRESUMO
The effect of 20 mM extracellularly applied 2,3-Butanedione monoxime (BDM) on L-type Ca2+ channel charge movement current was studied in whole-cell voltage-clamped guinea-pig ventricular myocytes. Intramembraneous charge movement in response to depolarizing pulses (charge 1), was reduced after the application of BDM. The effect was more pronounced at the OFF of the charge transient (41%) than at the ON (7%). The steady-state availability curve of charge 1 was shifted to the left; the magnitude of the voltage shift was similar to the shift in Ca2+ current availability. Charge movement recorded in the negative voltage range (charge 2) after conditioning depolarizing pulses of different duration, was increased by BDM. For a 300-ms conditioning pulse charge 2 measured during a negative test pulse increased 40% (in Ba2+ external solution) or 35% (in Ca2+ external solution). These results show that BDM promotes voltage-dependent inactivation of L-type Ca2+ channels in parallel with charge interconversion between intramembranous charges 1 and 2. Mechanistically they are consistent either with dephosphorylation or a dihydropyridine-like action, but argue against open channel block as the mechanism of the effect of the drug.
Assuntos
Canais de Cálcio/metabolismo , Diacetil/análogos & derivados , Ativação do Canal Iônico/efeitos dos fármacos , Miocárdio/metabolismo , Animais , Bário/metabolismo , Bário/farmacologia , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Diacetil/farmacologia , Condutividade Elétrica , Cobaias , Coração/efeitos dos fármacos , Miocárdio/citologiaRESUMO
1. The role of histidine on the decarboxylation of porphyrinogens of 7-, 6-, and 5-COOH III brought about by porphyrinogen carboxy-lyase (PCL) was studied. 2. For this purpose hepatic PCL from normal and hexachlorobenzene (HCB) treated rats were modified with diethylpyrocarbonate. 3. The results indicated that the enzyme from both normal and porphytic animals had histidine at the binding sites of all the porphyrinogens assayed. 4. Comparative studies between the enzyme from normal and porphyric rats suggested that in vivo HCB treatment affected the active site for the decarboxylation of 7-, 6- and 5-COOH porphyrinogens III at histidine residues. 5. On the other hand arginine modification by 2,3-butanedione treatment altered 5-COOH porphyrinogen III decarboxylation for both enzymes. However this amino acid was not involved at the binding site of this substrate.
Assuntos
Carboxiliases/metabolismo , Fígado/enzimologia , Animais , Arginina/química , Sítios de Ligação , Carboxiliases/química , Descarboxilação , Diacetil/farmacologia , Feminino , Hexaclorobenzeno/toxicidade , Histidina/química , Porfirias/induzido quimicamente , Porfirias/enzimologia , Porfirinogênios/química , Ratos , Ratos WistarRESUMO
Calcium-activated phosphoenolpyruvate carboxykinase from Escherichia coli is not inactivated by a number of sulfhydryl-directed reagents [5,5'-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N'-(5-sulfo-1-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn2+, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5'-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30 mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.