Role of Rab5 in insulin receptor-mediated endocytosis and signaling.

Activated insulin receptors recruit various intracellular proteins leading to signal generation and endocytic trafficking. Although activated receptors are rapidly internalized into the endocytic compartment and subsequently degraded in lysosomes, the linkage between insulin receptor signaling and endocytosis is not well understood. This study utilizes both overexpression and depletion of Rab5 proteins to show that they play a critical role in both insulin-stimulated fluid phase and receptor-mediated endocytosis. Specifically, Rab5:WT and Rab5:Q79L (a GTP-hydrolysis defective mutant) enhance both types of endocytosis in response to insulin, while Rab5:S34N (a GTP-binding defective mutant) has the opposite effect. Morphological analysis indicates that both Rab5 and insulin receptor are found on early endosomes, but not at the plasma membrane. In addition, expression of Rab5:WT and Rab5:Q79L enhance both Erk1/2 and Akt activation without affecting JN- and p38-kinase activities, while the expression of Rab5:S34N blocks both Erk1/2 and Akt activation. Consistent with these observations, DNA synthesis is also altered by the expression of Rab5:S34N. Taken together, these results demonstrate that Rab5 is required for insulin receptor membrane trafficking and signaling.

Rin1 regulates insulin receptor signal transduction pathways.

Rin1 is a multifunctional protein containing several domains, including Ras binding and Rab5 GEF domains. The role of Rin1 in insulin receptor internalization and signaling was examined by expressing Rin1 and deletion mutants in cells utilizing a retrovirus system. Here, we show that insulin-receptor-mediated endocystosis and fluid phase insulin-stimulated endocytosis are enhanced in cells expressing the Rin1:wild type and the Rin1:C deletion mutant, which contain both the Rab5-GEF and GTP-bound Ras binding domains. However, the Rin1:N deletion mutant, which contains both the SH2 and proline-rich domains, blocked insulin-stimulated receptor-mediated and insulin-stimulated fluid phase endocytosis. In addition, the expression of Rin1:delta (429-490), a natural occurring splice variant, also blocked both receptor-mediated and fluid phase endocystosis. Furthermore, association of the Rin1 SH2 domain with the insulin receptor was dependent on tyrosine phosphorylation of the insulin receptor. Morphological analysis indicates that Rin1 co-localizes with insulin receptor both at the cell surface and in endosomes upon insulin stimulation. Interestingly, the expression of Rin1:wild type and both deletion mutants blocks the activation of Erk1/2 and Akt1 kinase activities without affecting either JN or p38 kinase activities. DNA synthesis and Elk-1 activation are also altered by the expression of Rin1:wild type and the Rin1:C deletion mutant. In contrast, the expression of Rin1:delta stimulates both Erk1/2 and Akt1 activation, DNA synthesis and Elk-1 activation. These results demonstrate that Rin1 plays an important role in both insulin receptor membrane trafficking and signaling.

Rab5-activating protein 6, a novel endosomal protein with a role in endocytosis.

Rab GTPases are regulators of membrane trafficking that cycle between active (GTP-bound) and inactive (GDP-bound) states. In this study, we report the identification of a new human Rab5 guanine nucleotide exchange factor (GEF), which we have named RAP6 (Rab5-activating protein 6). RAP6 contains a Rab5 GEF and a Ras GAP domain. We show that the Vps9 domain is sufficient for the interaction of RAP6 with GDP-bound Rab5 and that RAP6 stimulates Rab5 guanine nucleotide exchange. We also find that the Ras GAP domain of RAP6 shows GAP activity for Ras. Immunofluorescence experiments reveal that RAP6 is associated with plasma membrane and small intracellular vesicles that also contain Rab5. Additionally, the overexpression of RAP6 affects both fluid phase and receptor-mediated endocytosis. This study is the first to show that RAP6 is a novel regulator of endocytosis that exhibits GEF activity specific for Rab5 and GAP activity specific for Ras.

Formation of singlet oxygen during farmorubicin oxidation.

The enhanced generation of singlet oxygen (1O2) during oxidation of farmorubicin in the Co(II) + H2O2 system was studied using chemiluminescent, fluorescent and spectrophotometic techniques. The influence of 1O2-quenchers, catalase, superoxide anion radical (O2*-) and hydroxyl radical (HO*) scavengers on the light emission was studied. The spectrophotometric determination of 1O2 was based on bleaching of p-nitrosodimethylaniline caused by an intermediate product of the reaction of 1O2 with imidazole, and was followed by monitoring the decrease in optical density at 440 nm.

Luminescence investigations of redox cycling of adriamycin.

The light emission from the adriamycin + Co2+ + H2O2 system has been studied. Chemiluminescence, fluorescence and absorption spectra were measured. The fluorescence spectra were time-dependent exhibiting maxima at 555, 590 and 645 nm. The chemiluminescence spectra consist of four bands with maxima at around 460-500, 550-580, 640 and 700 nm. Free radical reaction inhibitors, (1)O2-quenchers and catalase inhibited the light emission indicating that hydroxyl radical, superoxide anion radical and singlet oxygen are generated during the redox cycling of adriamycin. Chemiluminescence studies revealed that adriamycin undergoes chemiexcitation under our experimental conditions.

Antioxidant activity of 4-flavonil-1,4-dihydropyridine derivatives.

The effect of 4-flavonil-1,4-dihydropyridine derivatives on a chemical system involving a superoxide radical anion was tested using the chemiluminescence and spectrophotometry methods. All tested compounds enhanced the light emission from the system. The obtained results indicated that the tested derivatives may catalyze the conversion of superoxide radicals, thus showing superoxide dismutase activity.

The effect of thymol and its derivatives on reactions generating reactive oxygen species.

The effects of thymol (TOH), thymoquinone (TQ) and dithymoquinone (TQ2) on the reactions generating reactive oxygen species (ROS) such as superoxide anion radical (O2*-), hydroxyl radical (HO*) and singlet oxygen (1O2) were tested using the chemiluminescence (CL) and spectrophotometry methods. All tested compounds acted as scavengers of various ROS. The rate constant of 1O2-dimols quenching by thymol was calculated.

Enhancing effect of carazolol on chemiluminescence accompanying decomposition of hydrogen peroxide in the presence of copper ions.

The aim of this study was to investigate the effect of carazolol on the generation of the oxygen species with respect to chemiluminescence (CL) from the copper ion/H2O2 system. The reactions are carried out in buffered aqueous solution. The quantum yield of the CL from the copper ion/H2O2 system is increased by a factor of 2(-10) in the presence of carazolol. The CL spectra from the carazolol/copper ion/H2O2 system are spread through the full visible region and four emission bands are shown at maxima 480-500, 580, 640 and 700 nm. The excitation spectrum obtained from the above mentioned system shows the main maxima at around 273, 310 and 340 nm (lambda obs = 365 nm). The fluorescence emission spectrum exhibits only one maximum at 365 nm (lambda exc = 340 nm) corresponding to carazolol. The light emission decreases in the presence of several biologically important compounds which are typical scavengers of oxygen radicals and singlet oxygen (1O2). The results demonstrate that carazolol enhances the production of oxygen radicals and 1O2 under our experimental conditions. Generation of 1O2 was additionally confirmed using ESR spin trapping and spectrophotometry.

Chemiluminescence accompanying oxidation of bopindolol.

The oxidation of bopindolol using the Co-ethylenediaminetetraacetic acid (EDTA) + H2O2 system was investigated by spectrophotometric, chemiluminescence, and fluorescence methods. The effects of oxygen free radicals scavengers and 1O2 quenchers on the light emission were measured. The obtained results show the electronically excited products of the bopindolol degradation are involved in the oxidation process.

Effects of catechols on free radical formation by chemotherapeutic agents (adriamycin, farmorubicin, and mitomycin).

The aim of the present study was to investigate the effects of biologically important catechols on the cytotoxicity of adriamycin, farmorubicin, and mitomycin C with respect to hydroxyl radical production. Catecholamines (adrenalin, noradrenaline, dopamine) and DOPA enhance the generation of hydroxyl radicals by chemotherapeutic antibiotics. Measures were done using a deoxyribose assay, in presence of the Co(II) + H2O2 system. Catalase and hydroxyl radical scavengers (mannitol, thiourea, cysteine, glutathione, L-lactic dehydrogenase) inhibited the deoxyribose damage caused by the drugs.

Toxicology of cupric salts on honeybees. V. Gluconate and sulfate action on gut alkaline and acid phosphatases.

Some aspects of putative nontarget effects of cupric ions systemically fed to honeybees against their parasite mite Varroa jacobsoni have been investigated on the host phosphatases. The alkaline and acid forms extracted from the guts of worker bees exhibited substrate-inhibition features. Upon detailed kinetic analysis, cupric organic salts indicate activation effects at concentrations of about 1 mM. Concentrations up to 10 mM (alkaline form) and 25 mM (acid form) induced no important changes, except a partial quenching of the substrate-inhibition process, characterized by a wide increase in the constant of apparent inhibitory binding of substrate to the enzyme-substrate complex. Partial purification gave a single alkaline form with quite similar kinetic behavior in the absence of natural ions as in crude extracts. Cupric gluconate and sulfate demonstrated similar patterns, except an increase of the apparent Hill coefficient by sulfate only. The substrate constant of acid phosphatases was decreased at high cupric gluconate doses while its maximum velocity was biphasically increased (with observed maximum at 1 mM), resulting in a sustained activation. Chemiluminescence studies revealed that cupric ion activation is counteracted by oxygen radicals generated by cupric ions and also, in vitro, by the artificial substrate para-nitrophenylphosphate. The para-nitrophenol molecules released from the reaction are therefore responsible for biphasic effects selectively observed with gluconate salts. In apicultural practice, neither blockade of activity nor dramatic changes are to be expected at doses administered to bees against the parasite.

The extra-weak chemiluminescence generated during oxidation of some tetracycline antibiotics. II. Peroxidation.

This study was undertaken to establish the mechanism of chemiluminescence during the oxidation reaction of tetracycline antibiotics in the presence of molecular oxygen and H2O2. The spectral distribution of chemiluminescence and fluorescence and the quantum yields of chemiluminescence were measured. The chemiluminescence spectrum measured with cut-off filters revealed one broad blue-green band and bands with maxima at 580, 640 and 700 nm. The bands at 580, 640 and 700 nm were similar to those observed for singlet molecular oxygen. The effect of superoxide radical, hydroxyl radical inhibitors, singlet oxygen quenchers and D2O as solvent on the light emission was also studied. The formation of singlet oxygen during the oxidation of the investigated tetracyclines was also checked using the spectrophotometric method based on the bleaching of p-nitrosodimethylaniline. A mechanism for the reactions generating electronically excited compounds is proposed.

The extra-weak chemiluminescence generated during oxidation of some tetracycline antibiotics. 1. Autoxidation.

Chemiluminescence (CL) appearing during autoxidation of tetracycline (TC) antibiotics has been studied. The CL spectrum consists of four emission bands with maxima at 520, 585, 640 and 700 nm. The bands at 585, 640 and 700 nm are similar to those observed for singlet molecular oxygen (1O2). The effect of 1O2 quenchers and free radical reaction inhibitors on the light emission is also reported. The data support the concept that, during the autoxidation of TCs, cytotoxic oxygen species such as HO., O2.-, H2O2 and 1O2 are formed.

Cytotoxic effect of xanthotoxol (8-hydroxypsoralen) on TCTC cells in vitro.

The effect of xanthotoxol (8-hydroxypsoralen) on proliferation of TCTC cells in vitro has been studied. Xanthotoxol at concentrations of 5 to 50 micrograms/ml inhibited the growth of cells. In cultures with xanthotoxol, decreased amount of cell protein, mitotic index, and decreased ability to form a colony, were observed. Moreover, xanthotoxol disturbed mitoses elevating the number of mitotic cells in the telophase stage. An increase of giant and multinuclear cells was also found. On the basis of these results it can be concluded, that 8-hydroxypsoralen which in comparison with other psoralens is not sensitive to photostimulation, inhibits the cell proliferation anyway. This fact shows that the mechanism of the psoralens activity is to some extent independent from the photostimulation.

The formation of singlet oxygen during oxidation of catechol amines as detected by infrared chemiluminescence and spectrophotometric method.

1 delta g----3 sigma-g near infrared chemiluminescence from the peroxidation reaction of dopa, dopamine, noradrenaline and adrenaline was measured. The spectrophotometric method based on the bleaching of p-nitrosodimethylaniline was applied to check the generation of singlet oxygen during oxidation of the above-mentioned catecholamines.

Evidence for the generation of singlet molecular oxygen during dopa and dopamine peroxidation.

Participation of singlet molecular oxygen (1O2) in peroxidation of dopa and dopamine was studied by measurements of chemiluminescence spectra, the influence of solvents with various lifetime of O2 (1 delta g) and O2 (1 delta g)-quenchers on quantum yield of chemiluminescence. A decrease of absorption and fluorescence intensities of 1,3-diphenylizobenzofuran (DPBF) was also studied in the presence of dopa and dopamine oxidized with alkaline H2O2 as a criterion for the involvement of 1O2.