RESUMEN

Light-triggered redox activity of small (d = 2 nm) GdYVO4:Eu3+ nanoparticles (NPs) in aqueous solutions and lipid suspensions is reported. It has been revealed that depending on pre-treatment conditions (exposure to UV light or storage in the dark) the same NPs exhibit pro- or anti-oxidant properties. Pro-/anti-oxidant activity in aqueous solutions was evaluated by UV-vis spectroscopy using probe molecules for hydroxyl radicals (·OH) and superoxide anions (O2•-). Lipid oxidation under the effect of NPs has been also analyzed. Multi-functional GdYVO4:Eu3+ NPs are assumed to be a new theranostic agent in cancer therapy, which exhibit fluorescent properties, triggered redox activity and drug-carrier ability.

RESUMEN

Tumor development is the consequence of expanding the population of low differentiated cells with unlimited self-maintenance potential, i.e. cancer stem cells (CSCs). Application of new forms of nanocomposites capable of binding to CSCs and inducing the tumor destruction is perspective direction for treating this pathology. There have been developed the methods of obtaining hybrid nanocomplexes containing rare-earth orthovanadates GdYVO4:Eu³âº, cholesterol and luminescent dye Dil. By immune fluorescence method using monoclonal antibodies to CD44, CD24, CD117 and Sca-1 markers there has been established the change in the ratio of tumor progenitors of various differentiation levels in a general pool of Ehrlich carcinoma (EC) after treatment with hybrid nanocomplexes. Essential reduction in the concentration of the most tumorogenic CD44high cells with simultaneous rise in the number of CD117⁺-cells resulted in an increased index of CD44high/CD117⁺ ratio. It has been demonstrated that application of hybrid nanocomplexes suppressed the tumor growth almost by 80%. The value of cooperative interactions of the cells with different phenotype signs in tumor sites has been proved. The index of CD44high/CD117⁺ ratio can be used as one of diagnostic and prognostic parameters of development and inactivation rate of tumor process when using different types of anti-tumor therapy.

Asunto(s)

Biomarcadores de Tumor/genética , Carcinoma de Ehrlich/tratamiento farmacológico , Receptores de Hialuranos/genética , Nanocápsulas/uso terapéutico , Células Madre Neoplásicas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-kit/genética , Animales , Anticuerpos Monoclonales/química , Antineoplásicos , Biomarcadores de Tumor/metabolismo , Carbocianinas/química , Carcinoma de Ehrlich/genética , Carcinoma de Ehrlich/mortalidad , Carcinoma de Ehrlich/patología , Colesterol/química , Europio/química , Femenino , Gadolinio/química , Expresión Génica , Receptores de Hialuranos/metabolismo , Luminiscencia , Ratones , Ratones Endogámicos BALB C , Nanocápsulas/química , Trasplante de Neoplasias , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Proteínas Proto-Oncogénicas c-kit/metabolismo , Análisis de Supervivencia , Vanadatos/química

RESUMEN

Early events in individual hepatocytes of rat activated by adrenaline (10(-6)M) and phenylephrine (10(-5)M) have been investigated by quantitative image microfluorometry and microspectrofluorometry. Cationic DiOC2 and anionic SqSC4 probes have been used for image analysis and transmembrane potential (ΔΨ p) estimation in real-time studies. Fluorescence spectra resulting from the accumulation of dyes in single cells were recorded. Based on the mean fluorescence intensity, the magnitude of ΔΨ p was calculated by Nernst equation adapted for lipophilic cationic probes. DiOC2 has revealed that both hormones induce biphasic hyperpolarization of hepatocytes membrane with α-agonist phenylephrine causing ΔΨ p changes at higher amplitude. The first increase of ΔΨ p within 2 and 5 min (ΔΔΨ p = -8.6 ± 4.2 mV) apparently related to Na(+)/K(+)-ATPase activation by the Ca(2+)-mobilizing hormone. The second peak of hyperpolarization (ΔΔΨ p = -13.2 ± 3.2 mV) between 25 and 30 min, after a transient decrease of ΔΨ p (ΔΔΨ p = 10.9 ± 4.3 mV) over 15 min experiment, probably is mediated by phenylephrine stimulating action on K(+)-channels. K(+) channel blocker (Ba(2+) or 4-aminopyridine) as well as elevating of extracellular K(+) prevented the hyperpolarization. Modulation of PLD-dependent signal transduction pathway by 0.4% butanol had a weak influence on the first increase of ΔΨ p but it abolished the second phase of hyperpolarization. That points to PLD involvement in the ΔΨ p fluctuations mediated by K(+)-channels in response to phenylephrine. Based on SqSC4, fluorescent parameters estimation of relative changes of ΔΨ p revealed similar character of time dependence with two phases of hyperpolarization. Synchronic fluctuation of ΔΨ p determined by oppositely charged probes demonstrate that the quantitative microfluorometry allows to evaluate slight ΔΨ p changes separately from ΔΨ m in non-excitable individual cells at the short-term hormone action.

Asunto(s)

Citofotometría , Epinefrina/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hormonas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Fenilefrina/farmacología , Animales , Ratas , Factores de Tiempo

RESUMEN

The influence of tilorone dihydrochloride and its analogues--diphenyl derivatives on the changes of transmembrane potential of mitochondrial membranes of the isolated rat hepatocytes has been estimated. Authors have shown a significant increase in mitochondrial potential thirty minutes after the introduction of the test compounds to the cells using the fluorescent probe JC-1. These results indicate the rapid activation with tilorone and its analog--dihydrochloryde 4,4'-bis-[2-(diethylamino)ethoxy]diphenyl--of the RLR signaling pathway. The final stage of this pathway is the cell production of IFN type I. The authors concluded that there is an increasing of the organelles resistance to the extra/intracellular damaging agents under the influence of the test compounds.

Asunto(s)

Antivirales/farmacología , Hepatocitos/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Tilorona/farmacología , Animales , Bencimidazoles , Carbocianinas , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes , Hepatocitos/citología , Hepatocitos/metabolismo , Interferón gamma/biosíntesis , Masculino , Mitocondrias Hepáticas/metabolismo , Membranas Mitocondriales/metabolismo , Ratas , Ratas Wistar , Tilorona/análogos & derivados

RESUMEN

The resting membrane potential of isolated hepatocytes from 2- and 20-month-old rats and its changes upon activation of cells by adrenaline have been studied by the method of quantitative microfluorimetry using diethyl derivatives of polymethine probes (H-510 and D-307). The potential was estimated by the Nernst equation adapted to lipophilic cationic probes. It was shown using both probes that the transmembrane potential of hepatocytes decreases with age. The microfluorimetry data were confirmed by the results of spectrofluorimetric measurements in a cell suspension. Changes in fluorescence occurring in adrenaline-activated single cells and suspensions were unidirectional, the effect of the hormone on the cells of old animals being less pronounced. The results indicate that the potential of the plasma membrane of individual hepatocytes can be adequately estimated by microfluorimetry, which can be used in metabolic and toxicologic investigations.

Asunto(s)

Agonistas alfa-Adrenérgicos/farmacología , Membrana Celular/metabolismo , Epinefrina/farmacología , Colorantes Fluorescentes/química , Hepatocitos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Animales , Ratas , Ratas Wistar

RESUMEN

The spectral and luminescent properties of the derivatives of polymethine probes based on 3,3'-dialkyloxacarbocyanine bromide (H-510) and 1,1'-dialkyl-3,3, 3',3'-tetramethylindodicarbocyanine bromide (D-307) (where alkyl is ethyl or octadecyl groups) on accumulation in bone marrow and liver cells of rats have been compared. The general regularities in the interaction between the probes and the membrane microenvironment have been revealed for each type of the cells. It was found that the structural features of membranes and the length of the chains of alkyl substitutes essentially affect the redistribution of dye molecules in the binding censers of particular types, as evidenced by changes in the spectral characteristics of probes. It has been shown by microfluorimetry that the accumulation of short-chain derivatives in single cells is representative of the distinctions in the parameters of their functional metabolic state, which coincides with the present view of the heterogeneous structure of these cellular populations. The hormonal action and the influence of incubation without serum on the intensity of cellular processes were accompanied by changes in the fluorescence of cells stained with the ethyl derivatives of the probes H-510 and D-307. This fact allows us to consider them as optical indicators of the cellular activity. The dye D-307 was found to be more resistant to photodestruction than H-510 both in single cells and model system.

Asunto(s)

Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Membrana Celular/química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacocinética , Hepatocitos/química , Hepatocitos/citología , Animales , Fotoblanqueo , Ratas , Ratas Wistar

RESUMEN

The incorporation of the fluorescent probe 3,3-dialkyloxacarbocyanine bromide H-510 (where alkyl is ethyl- (C2), nonyl- (C9), or octadecyl (C18) groups) into cells of different kind has been explored. It has been revealed that the length of alkyl chains significantly influences the dynamics and mechanisms of accumulation of the probe by the cells. It has been found by microfluorimetry that all probe species have similar spectral characteristics in bone marrow cells, indicating that all probes, independently on their lipophilic properties, are incorporated into micelle-like structures formed probably by cell phospholipids. Spectroscopy experiments have shown that, in hepatocytes, the fluorescent probes 3,3-diethyloxacarbocyanine bromide (H-510/C2) and 3,3-dinonyloxacarbocyanine bromide (H-510/C9) are mainly accumulated in weakly polar media (nonpolar and weakly polar lipids of these cells). The luminescence maximum of the H-510/C18 probe in hepatocytes is blue-shifted and coincides with that in an albumin solution. We suppose that the incorporation of the probe into cells occurs by endocytosis when the probe binds to surface proteins.

Asunto(s)

Células de la Médula Ósea/metabolismo , Endocitosis/fisiología , Colorantes Fluorescentes/química , Hepatocitos/metabolismo , Fosfolípidos/metabolismo , Albúminas/química , Animales , Células de la Médula Ósea/química , Células de la Médula Ósea/citología , Células Cultivadas , Hepatocitos/química , Hepatocitos/citología , Micelas , Ratas , Ratas Wistar , Espectrometría de Fluorescencia

RESUMEN

Maturation of spermatozoa represents a formation of competent cell with capacity to fertilize. For the purpose of this review the morphological and functional changes of nucleus, plasmatic and nuclear membranes, tail region as well as the change of localisation and activity of enzymes have been described to illustrate the epididymis stage of maturation. Particular attention has been paid to the role of active oxygen species in the cell transformation, pro- and antioxidant balance during the maturation and to the systems controlling gametes control. The possible ways of influence on the some events of spermatozoa maturation are shown.

Asunto(s)

Maduración del Esperma , Espermatozoides , Animales , Humanos , Masculino , Espermatozoides/enzimología , Espermatozoides/metabolismo , Espermatozoides/fisiología

RESUMEN

The review is devoted to peculiarities of structure, regulation and interaction of enzyme systems of thyroid hormone synthesis. The structural and functional differences between enzymes of the thyroid gland, and nonthyroid homologues making a family of enzymes are considered. The contribution of various signal systems, including those related to generation of oxygen forms, to regulation of the thyroid hormone synthesis estimates. A problem on influence of the oxidation-reduction status of thyroid cells on their functional activity is also discussed. The role of disregulation of separate parts of thyroid hormonopoesis in pathogenesis of thyroid disease is marked.

Asunto(s)

Yoduro Peroxidasa , NADPH Oxidasas , Glándula Tiroides , Hormonas Tiroideas/biosíntesis , Animales , Transporte Biológico , Humanos , Peróxido de Hidrógeno/metabolismo , Yoduro Peroxidasa/genética , Yoduro Peroxidasa/metabolismo , Yoduro Peroxidasa/fisiología , Yodo/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , NADPH Oxidasas/fisiología , Simportadores/metabolismo , Glándula Tiroides/enzimología , Glándula Tiroides/inmunología , Glándula Tiroides/metabolismo , Hormonas Tiroideas/química , Hormonas Tiroideas/genética

RESUMEN

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP2 labeled with [14C]linoleic acid and [3H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [14C]DAG, total inositol phosphates, [3H]inositol 1,4,5-trisphosphate (Ins1,4,5P3) and [3H]inositol 1,4-bisphosphate (Ins1,4P2). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [14C]DAG and total [3H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [14C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of 14C-labeled fatty acids did not change. The L-thyroxine-induced accumulation of [3H]Ins1,4,5P3 and [3H]Ins1,4P2 did not depend on the presence of calcium ions in the incubation medium of the cells.

Asunto(s)

Calcio/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Fosfatidilinositoles/metabolismo , Tiroxina/farmacología , Animales , Radioisótopos de Carbono , Células Cultivadas , Diglicéridos/metabolismo , Masculino , Ratas , Ratas Wistar , Tritio

RESUMEN

L-thyroxine action on GPI and phosphatidylinositol (PI) metabolism in the liver have been investigated in 3- and 24-month-old Wistar rats. PI and GPI were labeled by [14C]acetate Na in vivo and [14C]glucose in vitro. Aging caused a significant decrease in basal PI and GPI levels and reduced [14C]glucose incorporation into GPI of liver. The addition of exogenous PI stimulated the [14C]GPI formation (about 2-3 fold) in 24-month-old rat liver. Thyroxine injection (200 micrograms/100 g weight) to young rats induced triphasic alteration in GPI content in the liver. We observed the marked violation in the thyroxine-mediated GPI-metabolism in the old rats liver. These results indicate that thyroid hormones regulate GPI metabolism in rat liver.

Asunto(s)

Envejecimiento , Regulación de la Expresión Génica , Glicosilfosfatidilinositoles/metabolismo , Hígado/efectos de los fármacos , Tiroxina/farmacología , Acetatos/metabolismo , Animales , Glucosa/metabolismo , Hígado/metabolismo , Fosfatidilinositoles/metabolismo , Ratas , Ratas Wistar

RESUMEN

BACKGROUND: Numerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC) is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNgamma in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T4, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells. RESULTS: The results obtained provide the first demonstration of phospholipase C, phospholipase D and protein kinase C nongenomic activation and diacylglycerol (DAG) accumulation by L-T4 in liver cells. The experiments were performed in either the [14C]CH3COOH-labeled rat liver slices or isolated hepatocytes pre-labeled by [14C]oleic acid. L-T4 activates the DAG production in a concentration- and time-dependent manner. DAG formation in stimulated cells is biphasic and short-lived event: there is an initial, rapid rise in DAG concentration and then a slower accumulation that can be sustained for a few minutes. The early phase of L-T4 generated DAG only is accompanied by phosphatidylinositol 4,5-bisphosphate level decrease and inositol 1,4,5-trisphosphate formation while the second phase is abolished by PKC inhibitor l,(5-isoquinolinesulphonyl)2methylpiperasine dihydrochloride (H7) and propranolol. The second phase of DAG production is accompanied by free choline release, phosphatidylcholine content drop and phosphatidylethanol (Peth) formation. Inhibitor of phospholipase-C-dependent phosphoinositide hydrolysis, neomycin sulfate, reduced the Peth as well as the DAG response to L-T4. CONCLUSIONS: The present data have indicated the DAG signaling in thyroid hormone-stimulated liver cells. L-thyroxine activates a dual phospholipase pathway in a sequential and synchronized manner: phospholipase C initiates the DAG formation, and PKC mediates the integration of phospholipase D into the signaling response during the sustained phase of agonist stimulation.

Asunto(s)

Hígado/enzimología , Fosfolipasa D/metabolismo , Transducción de Señal , Tiroxina/farmacología , Fosfolipasas de Tipo C/metabolismo , Animales , Membrana Celular/química , Membrana Celular/metabolismo , Colina/metabolismo , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática , Genoma , Glicerofosfolípidos/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Hepatocitos/metabolismo , Cinética , Hígado/citología , Hígado/efectos de los fármacos , Masculino , Fosfatidilcolinas/metabolismo , Fosfolípidos/análisis , Proteína Quinasa C/fisiología , Ratas , Ratas Wistar

RESUMEN

The age specificity of the regulation by thyroid hormones of 1,2-diacylglycerol production in rat liver has been studied. It was found that L-thyroxine-stimulation of the 3-month old rats liver cells resulted in a rapid rise in 1,2-diacylglycerol concentration in hepatocytes and simultaneous degradation of phospholipids. The endogenous phosphatidylcholine and phosphatidylethanolamine are the sources of 1,2-diacylglycerol in a liver. Under the action of hormone liver cells of young rats may product 1,2-diacylglycerol from exogenous 1-acyl, 2-[14C]arachidonyl-phosphatidylethanolamine. Thyroxine had no effect on de novo 1,2-diacylglycerols formation and their release from triacylglycerol. In liver cells of elder rats, 1,2-diacylglycerol and individual phospholipids content are unaffected by hormones.

Asunto(s)

Factores de Edad , Diglicéridos/metabolismo , Hígado/efectos de los fármacos , Tiroxina/farmacología , Animales , Diglicéridos/biosíntesis , Hígado/metabolismo , Masculino , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Ratas , Ratas Wistar , Tiroxina/administración & dosificación

RESUMEN

L-Thyroxine rapidly stimulated the accumulation of diacylglycerols in isolated hepatocytes and in liver when lipids were prelabeled with [14C]oleic acid or with [14C]CH3COONa. Perfusion of the liver of hypothyroid animals with L-thyroxine-containing solution or incubation of liver fragments with the hormone increased the content of diacylglycerols in the liver cells. The increase in [14C]diacylglycerol level in the liver cells was accompanied by a decrease in the level of [14C]phosphatidylcholine, whereas contents of other 14C-labeled phospholipids, such as phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylinositol (PtdIns), phosphatidylinositol-4-phosphate (PtdIns4P), and phosphatidylinositol-4,5-bis-phosphate (PtdIns(4,5)P2), and of 14C-labeled fatty acids were the same as in the control. The L-thyroxine-induced accumulation of diacylglycerols in hepatocytes was not affected by neomycin but was inhibited by propranolol. Incubation of hepatocytes prelabeled with [14C]oleic acid with L-thyroxine and ethanol (300 mM) was accompanied by generation and accumulation of [14C]phosphatidylethanol that was partially suppressed by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7). L-Thyroxine was responsible for the translocation of protein kinase C from the cytosol into the membrane fraction and for a many-fold activation of the membrane-bound enzyme. D-Thyroxine failed to affect the generation of diacylglycerols in hepatocytes and the activity of protein kinase C.

Asunto(s)

Diglicéridos/metabolismo , Hepatocitos/efectos de los fármacos , Proteína Quinasa C/metabolismo , Tiroxina/farmacología , Animales , Membrana Celular/enzimología , Citosol/enzimología , Activación Enzimática , Hepatocitos/enzimología , Hepatocitos/metabolismo , Masculino , Fosfolípidos/metabolismo , Ratas , Ratas Wistar

RESUMEN

The effects of thyroxine, phorbol esters and sphingosine on incorporation of [14C]linoleic acid into phospholipids, triacylglycerols and cholesterol esters of albino rat liver have been studied. It has been found that phorbol-12-myristate-13-acetate and thyroxine activate the [14C]linoleic acid incorporation into liver cell phospholipids and triacylglycerols. On the other hand, methyl esters of phorbol-12-myristate-13-acetate do not influence, while mercasolyl and sphingosine decrease the fatty acid incorporation into liver glycerolipids. It has been found that sphingosine abolishes the thyroxine effect on glycerolipid activation. It has been shown also that sphingosine, thyroxine and phorbol-12-myristate-13-acetate methyl esters do not influence the cholesterol ester synthesis. However, phorbol-12-myristate-13-acetate enhances the cholesterol ester activation in liver cells twofold.

Asunto(s)

Ácidos Linoleicos/metabolismo , Metabolismo de los Lípidos , Hígado/efectos de los fármacos , Esfingosina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Tiroxina/farmacología , Animales , Radioisótopos de Carbono , Ésteres del Colesterol/metabolismo , Ácido Linoleico , Hígado/metabolismo , Masculino , Ratas , Ratas Wistar

RESUMEN

The activity of phospholipases A1 and A2 in liver cells and liver cell nuclei of rats of various age and the thyroid status of experimental animals have been studied. It was found that phospholipid deacylation and acylation in liver cells is under control of thyroid hormones. Age-specific fluctuations in the activity of the thyroid gland may be one of possible reasons for phospholipase A1 activity and phospholipid level changes in liver nuclei during ontogenesis. Thyroid hormones play an important role in regulation of polyunsaturated fatty acid metabolism and prostaglandin E2 production in the liver.

Asunto(s)

Envejecimiento/fisiología , Núcleo Celular/enzimología , Hígado/enzimología , Fosfolipasas A/metabolismo , Hormonas Tiroideas/fisiología , Animales , Dinoprostona/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Hígado/citología , Masculino , Fosfolipasas A1 , Ratas , Ratas Wistar