RESUMO
The fabrication of biodegradable 3-D scaffolds enriched with multipotent stem cells seems to be a promising strategy for the repair of irreversibly injured tissues. The fine mechanisms of the interaction of rat mesenchymal stem cells (rMSCs) with a hyaluronan-based scaffold, i.e. HYAFF(R)11, were investigated to evaluate the potential clinical application of this kind of engineered construct. rMSCs were seeded (2 x 10(6) cells cm(-2)) on the scaffold, cultured up to 21 days and analysed using appropriate techniques. Light (LM), scanning (SEM) and transmission (TEM) electron microscopy of untreated scaffold samples showed that scaffolds have a highly porous structure and are composed of 15-microm-thick microfibres having a rough surface. As detected by trypan blue stain, cell adhesion was high at day 1. rMSCs were viable up to 14 days as shown by CFDA assay and proliferated steadily on the scaffold as revealed by MTT assay. LM showed rMSCs in the innermost portions of the scaffold at day 3. SEM revealed a subconfluent cell monolayer covering 40 +/- 10% of the scaffold surface at day 21. TEM of early culture showed rMSCs wrapping individual fibres with regularly spaced focal contacts, whereas confocal microscopy showed polarized expression of CD44 hyaluronan receptor; TEM of 14-day cultures evidenced fibronexus formation. Immunohistochemistry of 21-day cultures showed that fibronectin was the main matrix protein secreted in the extracellular space; decorin and versican were seen in the cell cytoplasm only and type IV collagen was minimally expressed. The expression of CD90, a marker of mesenchymal stemness, was found unaffected at the end of cell culture. Our results show that HYAFF(R)11 scaffolds support the adhesion, migration and proliferation of rMSCs, as well as the synthesis and delivery of extracellular matrix components under static culture conditions without any chemical induction. The high retention rate and viability of the seeded cells as well as their fine modality of interaction with the substrate suggest that such scaffolds could be potentially useful when wide tissue defects are to be repaired as in the case of cartilage repair, wound healing and large vessel replacement.
Assuntos
Ácido Hialurônico , Células-Tronco Mesenquimais/fisiologia , Alicerces Teciduais , Cicatrização , Animais , Materiais Biocompatíveis , Adesão Celular , Movimento Celular , Proliferação de Células , Imunofluorescência , Receptores de Hialuronatos/análise , Imuno-Histoquímica , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Confocal , Ratos , Engenharia Tecidual/métodosRESUMO
A number of human diseases are linked to local reduced oxygen availability. Hypoxemia, the condition in which oxygen partial pressure in blood falls below 40 mmHg, generates a distress which leads the cells in the vascular wall to activate a genetic program inducing a homeostatic response. The effectiveness of this response is conditioned by the degree and duration of the hypoxic stress and depends on the equilibrium among several factors which are worked out mainly in the vascular endothelial cell layer. Among them are vasoconstrictors such as angiotensin II, endothelins, prostaglandins and thromboxans, and vasodilators such as nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factor. A present challenge of the research is understanding the physiological and pathophysiological relevance of the growing body of data collected, disclosing the potential therapeutical application of the basic knowledge in this field.
Assuntos
Vasos Sanguíneos/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Vasos Sanguíneos/citologia , Hipóxia Celular/fisiologia , Humanos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Pressão ParcialRESUMO
Caspase enzymes are a family of cysteine proteases that play a central role in apoptosis. Recently, it has been demonstrated that caspases can be S-nitrosylated and inhibited by nitric oxide (NO). The present report shows that in chick embryo heart cells (CEHC), NO donor molecules such as S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione, spermine-NO or sodium nitroprusside inhibit caspase activity in both basal and staurosporine-treated cells. However, the inhibitory effect of NO donors on caspase activity is accompanied by a parallel cytotoxic effect, that precludes NO to exert its antiapoptotic capability. N-Acetylcysteine (NAC) at a concentration of 10 mM blocks depletion of cellular glutathione and cell death in SNAP-treated CEHC, but it poorly affects the ability of SNAP to inhibit caspase activity. Consequently, in the presence of NAC, SNAP attenuates not only caspase activity but also cell death of staurosporine-treated CEHC. These data show that changes in the redox environment may inhibit NO-mediated toxicity, without affecting the antiapoptotic capability of NO, mediated by inhibition of caspase enzymes. NO may thus be transformed from a killer molecule into an antiapoptotic agent.
Assuntos
Apoptose/fisiologia , Inibidores de Caspase , Coração/fisiologia , Óxido Nítrico/fisiologia , Acetilcisteína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Glutationa/análogos & derivados , Glutationa/farmacologia , Coração/embriologia , Óxido Nítrico/antagonistas & inibidores , Nitrocompostos/farmacologia , Penicilamina/análogos & derivados , Penicilamina/farmacologia , Estaurosporina/farmacologiaRESUMO
We have developed a rapid and precise method for glutathione quantitation by capillary electrophoresis, that allows a low amount of both redox forms to be measured. Small fragments of rat heart or liver tissues (20 mg wet weight) and the corresponding mitochondria (1 mg protein) were homogenized in 1% perchloric acid and the acid-soluble phase ultrafiltered by centrifugation with a microconcentrator (Mr cut-off 3000 Da). The analysis was performed at a constant temperature (28 degrees C) using a Beckman P/ACE System 2100, equipped with a UV absorbance detector set to 200 nm. The limit of quantitation in heart tissue was 1.8 microM for GSH and 1.2 microM for GSSG. Myocardial concentrations of GSH and GSSG were 8.1 +/- 2.6 and 0.45 +/- 0.15 (nmol/mg protein +/- S.D.), respectively. The ratio of GSH to GSSG was 17.8 +/- 1.3 for heart tissue, whereas it was much higher (>100) in the mitochondria. An oxidative stress decreased the myocardial tissue GSH/GSSG ratio, indicating that the CE analysis of both glutathione forms is also a useful method to study biological redox modification.
Assuntos
Glutationa/análise , Mitocôndrias/química , Animais , Eletroforese Capilar , Indicadores e Reagentes , Masculino , Mitocôndrias Cardíacas/química , Mitocôndrias Hepáticas/química , Oxirredução , Ratos , Ratos Wistar , Espectrofotometria Ultravioleta , UltracentrifugaçãoRESUMO
Nitric oxide synthases (NOS) are different widely expressed enzymes which produce the molecular messenger nitric oxide. The neuronal isoform of NOS (nNOS) is involved in several processes of the cell metabolism, most of which are, at present, not fully understood (neurotransmission, smooth muscle motility, myoblast and myocyte biology and others). In skeletal muscle nNOS is present mainly at the plasmalemma, where it is attached to the dystrophin-related proteins; in fact, in pathologies involving dystrophin, nNOS is altered as well. We report that in aged rats the nNOS amount in skeletal muscle increases both in the soluble and microsomal fractions and that an additional intracytoplasmic localisation appears.
Assuntos
Envelhecimento/fisiologia , Músculo Esquelético/enzimologia , Óxido Nítrico Sintase/metabolismo , Animais , Fracionamento Celular , Citoplasma/enzimologia , Citoplasma/metabolismo , Imunofluorescência , Imuno-Histoquímica , Masculino , Microssomos/enzimologia , Microssomos/metabolismo , Músculo Esquelético/citologia , Ratos , Ratos WistarRESUMO
Ceramide has emerged as a novel lipid mediator in cell growth and apoptosis. In difluoromethylornithine-resistant L1210 cells stimulated to growth from quiescence, the cell-permeant analogues of ceramide N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide) inhibited the induction of ornithine decarboxylase (ODC) activity with IC50 of 8.3 and 1.5 microM respectively. This effect was strictly related to the ability to inhibit cell growth and [3H]thymidine incorporation. The suppression of cell growth was also associated with apoptosis. The addition of bacterial sphingomyelinase resulted in a significant, but limited, reduction of ODC induction and [3H]thymidine incorporation. Bacterial lipopolysaccharide, which may act as a ceramide analogue, also inhibited the induction of the enzyme. Moreover, C6-ceramide largely prevented the accumulation of ODC mRNA and its precursor, ODC heterogeneous nuclear RNA, that accompanied the induction of ODC activity. A slight increase in ODC turnover was also observed. The DNA-binding activity of some transcription factors known to bind and transactivate the ODC gene was investigated by gel mobility-shift assay under the same experimental conditions. However, only the binding of Myc/Max was negatively affected by the treatment with C6-ceramide. Furthermore, the amount of immunoreactive c-Myc, which increased after stimulation of the cells to growth, was strongly reduced by C6-ceramide. These results suggest that the inhibition of c-Myc and ODC expression may be early events in the response of leukaemia cells to ceramide.
Assuntos
Ceramidas/farmacologia , Eflornitina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc , Leucemia L1210/genética , Ornitina Descarboxilase/genética , Animais , Permeabilidade da Membrana Celular , Leucemia L1210/enzimologia , Leucemia L1210/patologia , Camundongos , Células Tumorais CultivadasRESUMO
Trimetazidine (TMZ) added in vitro to isolated cardiac mitochondria at concentrations 10-100 microM in the presence of 25-100 nM extramitochondrial Ca2+ increased Ca2+ uptake and matrix Ca2+ concentration. This effect was less evident in the presence of physiologically Na+ and Mg2+ extramitochondrial concentrations since only 100 microM TMZ was able to increase mitochondrial Ca2+ entry in the presence of 100 nM Ca2+. The drug stimulated a Ca(2+)-cooperative effect on mitochondrial Ca2+ transport, but did not modify the rate of Ca2+ egress stimulated by 10 mM NaCl. An increase in mitochondrial Ca2+ level produced by TMZ enhanced oxoglutarate dehydrogenase activity and then ATP synthesis, particularly when 50 nM extramitochondrial Ca2+ was used. These data suggest that a possible cardiac mechanism of action of TMZ at mitochondrial level could support ATP synthesis by elevating the mitochondrial Ca2+ level.
Assuntos
Cálcio/metabolismo , Mitocôndrias Cardíacas/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Trimetazidina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Complexo Cetoglutarato Desidrogenase/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Magnésio/farmacologia , Masculino , Mitocôndrias Cardíacas/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Ratos Wistar , Sódio/farmacologia , Vasodilatadores/farmacologiaRESUMO
In quiescent thymocytes, mitochondrial de-energization was not correlated to apoptotic death. In fact, thymocytes treated with oligomycin, a highly specific inhibitor of ATP synthase, alone or with atractyloside to block ATP translocation from the cytoplasm, were alive, even if their mitochondria were depolarized, as revealed by flow cytometry after Rhodamine 123 staining. Furthermore, oligomycin was a powerful inhibitor of apoptosis induced in rat thymocytes by dexamethasone and, to a lesser extent, by the calcium ionophore A23187 and etoposide, but was without effect when apoptosis was induced by staurosporine, and increased cell death in mitogen-treated thymocytes. The inhibition of apoptosis was confirmed by morphological criteria, inhibition of inter-nucleosomal DNA fragmentation and inhibition of the loss of membrane integrity. The anti-apoptotic effect of oligomycin in cells treated with A23187 or etoposide was correlated to the inhibition of protein synthesis, while inhibition of apoptosis induced by dexamethasone, already evident at an oligomycin concentration of 10 ng/ml, was instead strictly correlated to the effect exerted on the cellular ATP level. Thymocyte apoptosis triggered by dexamethasone was blocked or delayed by inhibitors of respiratory-chain uncouplers, inhibitors of ATP synthase and antioxidants: a lasting protection from dexamethasone-induced apoptosis was always correlated to a drastic and rapid reduction in ATP level (31-35% of control), while a delay in the death process was characterized by a moderate decrease in ATP (73-82% of control). Oligomycin inhibited the specific binding of radioactive corticosteroid to thymocyte nuclei, confirming the inhibitory effect of ATP depletion on glucocorticoid binding and suggesting that ATP depletion is a common mediator of the anti-apoptotic action of different effectors in glucocorticoid-induced apoptosis. In conclusion, the reported data indicate that ATP may act as a cellular modulator of some forms of apoptosis, depending on the death trigger, and that in quiescent cells the de-energization of mitochondria is not necessarily linked to apoptosis.
Assuntos
Trifosfato de Adenosina/metabolismo , Anti-Inflamatórios/farmacologia , Apoptose/efeitos dos fármacos , Dexametasona/farmacologia , Linfócitos T/patologia , Administração Tópica , Animais , Citometria de Fluxo , Glucocorticoides , Masculino , Ratos , Ratos Wistar , Linfócitos T/metabolismoRESUMO
A particular mitochondrial DNA (mtDNA) deletion, the so-called "common deletion", accumulates progressively with age in human post-mitotic cells. We investigated the presence of age-related mtDNA deletions in mouse heart and, according to the free-radical theory of aging, the potential involvement of reactive oxygen species (ROS). Hearts from young, adult and old male Balb/c mice were homogenized and centrifuged in order to discard nuclear DNA. The supernatant was then utilized to prepare mtDNA by SDS-proteinase K digestion. The presence of a mtDNA4236 deletion was estimated by PCR analysis, by separating the amplificated segment on agarose gel. The incidence of the mtDNA4236 deletion was 16%, 28% and 78% in young, adult and old mice, respectively. 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of DNA oxidation, was also determined by HPLC-electrochemical analysis. 8-OH-dG was not detectable in young mice, while its concentrations (moles 8-OH-dG/10(6) moles dG; mean +/- SD) were 59.0 +/- 1.41 and 31.0 +/- 4.24 (p < 0.02) in adult and old mice, respectively. These data indicate that a mtDNA4236 deletion is progressively associated with aging in mouse hearts, and that oxidative damage to mtDNA is greater in middle-aged than senescent animals.
Assuntos
Envelhecimento/fisiologia , DNA Mitocondrial/genética , Desoxiguanosina/análogos & derivados , Deleção de Genes , Camundongos/genética , Mitocôndrias Cardíacas/genética , 8-Hidroxi-2'-Desoxiguanosina , Animais , Sequência de Bases , Desoxiguanosina/genética , Masculino , Camundongos Endogâmicos BALB CRESUMO
In difluoromethylornithine-resistant L1210 cells stimulated to grow from quiescence, haloperidol caused an early and dose-dependent inhibition of the induction of ornithine decarboxylase (ODC) activity, with an IC50 of 3.5 microM. This effect was accompanied by a reduction in the ODC mRNA level and inhibition of cell growth. Other sigma ligands of different chemical classes inhibited the induction of ODC activity, whereas sulpiride, a dopamine antagonist devoid of sigma-binding affinity, was ineffective. These results indicate that the inhibition of ODC expression may be an early event involved in the antiproliferative response of leukemia cells to haloperidol.
Assuntos
Eflornitina/farmacologia , Haloperidol/farmacologia , Ornitina Descarboxilase/biossíntese , Ornitina Descarboxilase/genética , Animais , Antineoplásicos/farmacologia , Antipsicóticos/farmacologia , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Indução Enzimática/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Leucemia L1210/enzimologia , Leucemia L1210/genética , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The observation of a case of intestinal intussusception caused by lipoma of the colon prompted the authors to review the literature on the subject and to examine the main characteristics of lipoma of the colon which represent the most frequent benign neoplasia of the large intestine after adenomatous polyps. Lipomas of the colon are localised in 90% of cases at the submucous level, are usually solitary, of varying size and may be sessile or pedunculated. They are almost always asymptomatic; only when they are of a reasonable size do they become manifest following alterations of the alveus, rectorrhagia, abdominal pain or the occupation of the colic lumen by the mass, or intestinal intussusception caused by the progression of the pedunculated lipoma. This difficult diagnosis may be aided by colonscopy with biopsy and dual contrast opaque enema. The prognosis of the disease depends on the presence or absence of complications and, in the case of the former, on early diagnosis and treatment. Lipoma of the colon of less than 2 cm may be electively removed endoscopically, those greater than 2 cm by laparotomy or laparoscopy. In emergency cases, it is advisable to perform a more or less extensive resection of the colon depending on the size of the tumour. In the case reported by the authors, an intussusception manouevre was first performed followed by left segmentary colectomy.
Assuntos
Doenças do Colo/etiologia , Neoplasias do Colo/etiologia , Intussuscepção/etiologia , Lipoma/complicações , Adulto , Humanos , MasculinoRESUMO
Biochemical and structural changes occurring in the myocardium with aging are mainly resulting from the association of a general tissue atrophy with the hypertrophy of the remaining myocytes. Whilst hypertrophy seems to be a compensatory process to the loss of cardiomyocytes and to a mild systolic hypertensive condition that accompanies elderly people, atrophy should be the modification more closely related to aging 'per se.' In support to the free radical theory of aging, several signs of oxidative damage have been shown in the aged heart, such as lipofuscin accumulation, decreased phospholipid unsaturation index, greater formation of both hydrogen peroxide and 8-hydroxy-2'deoxyguanosine. As a compensatory reaction, the activities of the main oxygen-radical scavenger enzymes are stimulated in the mitochondria of aged rat heart. Endothelium-mediated vasoregulation is more susceptible to oxidative stress in aged with respect to young rats, suggesting that also the vasculature can be negatively influenced by the oxygen free radicals generated during aging. The possible primary role of oxygen free radicals in the development of myocardial atrophy is also discussed.
Assuntos
Envelhecimento/fisiologia , Fenômenos Fisiológicos Cardiovasculares , Oxigênio/fisiologia , Acetilcolina/farmacologia , Animais , DNA/metabolismo , Relação Dose-Resposta a Droga , Radicais Livres/metabolismo , Miocárdio/metabolismo , Oxirredução , Estresse Oxidativo , Ratos , SístoleRESUMO
In difluoromethylornithine resistant L1210 cells stimulated to growth from quiescence, the selective kappa-opioidergic agonist trans-(+/-)-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneaceta mid e (U-50488H) caused a dose dependent inhibition of the induction of ODC activity, with a half-maximal effect at about 1 microM. U-50488H also provoked reduction of ODC mRNA level and increase of ODC turnover, as well as inhibition of cell growth. U-69593, another kappa-selective agonist, was only slightly effective. The action of U-50488H on ODC induction was not blocked by naloxone, beta-chlornaltrexamine or by the kappa-selective opioid antagonists Mr1452 and nor-binaltorphimine (nBNI). Actually Mr1452 and nBNI exerted some inhibitory effect. Furthermore, the separated enantiomers (+) and (-) of U-50488H were similarly effective. The (-)cis-(1S,2R)-U50488 stereoisomer, exhibiting low affinity for kappa and high affinity for sigma receptors and carbetapentane, another sigma ligand, also inhibited ODC induction, although less effectively than U-50488H. None of several other opioid ligands tested had significant effects on ODC induction. In conclusion, the inhibition of ODC expression by U-50488H does not involve classical, enantiospecific opioid receptors; rather, these results suggest the involvement of a distinct site of action linked to inhibition of lymphoid cell proliferation.
Assuntos
Benzenoacetamidas , Eflornitina/farmacologia , Linfócitos/enzimologia , Antagonistas de Entorpecentes/farmacologia , Ornitina Descarboxilase/biossíntese , Pirrolidinas/farmacologia , Receptores Opioides kappa/metabolismo , (trans)-Isômero de 3,4-dicloro-N-metil-N-(2-(1-pirrolidinil)-ciclo-hexil)-benzenoacetamida , Animais , Sequência de Bases , Divisão Celular/efeitos dos fármacos , Primers do DNA/química , Resistência a Medicamentos , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Leucemia L1210 , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Entorpecentes/metabolismo , Entorpecentes/farmacologia , Ornitina Descarboxilase/genética , RNA Mensageiro/análise , Receptores Opioides kappa/agonistas , Estereoisomerismo , Células Tumorais CultivadasRESUMO
Acetylcholine-induced, endothelium-dependent relaxation of norepinephrine-precontracted aortic strips, was severely impaired after exposure to a hypoxanthine/xanthine oxidase reaction generating oxygen radicals. This effect was more evident in aortic strips of aging rats (24 months old) in comparison to young rats (3 months old). The addition of authentic .NO (1 microM) completely relaxed aortic strips exposed to oxidative stress both in young and aging rats. In vitro EPR measurements showed that the .NO signal was reduced by enzymatic O2.- generating reaction. The activity of a partial purified preparation of constitutive NO synthase from rat cerebellum was significantly decreased after exposure to exogenous oxygen radicals. Pretreatment of aortic strips with 100 microM alpha-tocopherol-phosphate, produced a significant improvement of acetylcholine-dependent relaxation in the aortic strips exposed to oxidative stress, particularly in the aged vessel. The content of malondialdehyde in aortic tissue did not change after oxidative stress or alpha-tocopherol pretreatment. Alpha-tocopherol was unable to recover the NO synthase activity depressed in vitro by hypoxanthine/xanthine oxidase reaction. This study confirms that an oxidative stress impairs the endothelium-mediated vasodilation. Alpha-tocopherol pretreatment protects the vessel against this damage. The mechanism of action of alpha-tocopherol is unknown, but seems unrelated to an antioxidant activity.
Assuntos
Envelhecimento/fisiologia , Antioxidantes/farmacologia , Endotélio Vascular/fisiologia , Músculo Liso Vascular/fisiologia , Óxido Nítrico Sintase/metabolismo , Estresse Oxidativo , Vasodilatação , Vitamina E/farmacologia , Acetilcolina/farmacologia , Animais , Aorta , Arginina/metabolismo , Relação Dose-Resposta a Droga , Hipoxantina , Hipoxantinas , Cinética , Masculino , Malondialdeído/análise , Desenvolvimento Muscular , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/crescimento & desenvolvimento , Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , Vasodilatação/efeitos dos fármacos , Xantina OxidaseRESUMO
Internucleosomal DNA fragmentation and cell death induced by dexamethasone in rat thymocytes were inhibited when cells were cultured in 95% N2/5% CO2 atmosphere, in which oxygen was rapidly reduced to under 0.5%. DNA fragmentation was delayed by a less severe hypoxia in 5% oxygen whilst in cell cultured in high oxygen atmosphere (95% O2) cell death was increased. On the other hand, prolonged oxygen deprivation caused an increase of spontaneous apoptotic cell death. Hypoxia also inhibited DNA fragmentation induced by calcium ionophore A23187, but not by topoisomerase inhibitor camptothecin. These data support the hypothesis of the involvement of oxygen reactive species in calcium-mediated apoptosis and suggest a complex role of oxygen in the modulation of programmed cell death.
Assuntos
Apoptose/efeitos dos fármacos , DNA/metabolismo , Dexametasona/farmacologia , Oxigênio/farmacologia , Timo/citologia , Animais , Calcimicina/farmacologia , DNA/efeitos dos fármacos , Eletroforese em Gel de Ágar , L-Lactato Desidrogenase/metabolismo , Masculino , Microscopia Eletrônica , Oxigênio/administração & dosagem , Ratos , Ratos Wistar , Timo/efeitos dos fármacos , Fator de Transcrição AP-1/metabolismo , Proteína Supressora de Tumor p53/fisiologiaAssuntos
Sequestradores de Radicais Livres/uso terapêutico , Radicais Livres/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Vitamina E/uso terapêutico , Animais , Humanos , Peroxidação de Lipídeos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , CoelhosRESUMO
Pathological conditions that cause oxidative stress can affect DNA integrity. The aim of this research was to study the protective effect of spermine against DNA damage induced by an oxygen-radical generating system. Deoxyguanosine and DNA were separately dissolved in phosphate buffer and incubated for 1 h at 40 degrees C in the presence of 50 mM H2O2/10 mM ascorbic acid. Single nucleosides and their products of oxidation were then obtained by enzymatic digestion of DNA. The compounds were separated by micellar electrokinetic capillary chromatography (MECC) with SDS-modified mobile phase and detected at 254 nm. Two major products of DNA oxidation have been identified as derivatives of deoxyguanosine with electrophoretic properties different from 8-hydroxy-2'-deoxyguanosine. When the oxidation of DNA was carried out in the presence of 0.1 mM spermine, the formation of the two by-products of deoxyguanosine was markedly reduced. On the contrary, spermine did not prevent the oxidation of deoxyguanosine alone, suggesting that the polyamine should be bound to the DNA strands to exert its antioxidative effect.
Assuntos
Dano ao DNA/efeitos dos fármacos , Estresse Oxidativo , Espermina/química , Ácido Ascórbico , Adutos de DNA/biossíntese , DNA de Cadeia Simples/química , Desoxiguanosina/química , Desoxirribonuclease I/química , Exonucleases/química , Peróxido de Hidrogênio/química , Espectrofotometria UltravioletaRESUMO
This study investigated the biosynthesis of ubiquinone in isolated and perfused hearts of young and aged rats exposed to ischemia and reperfusion. A first group of hearts was used to determine the changes in coenzyme Q9 (CoQ9) and coenzyme Q10 (CoQ10) concentrations at mitochondrial and microsomal level after 30 min of ischemia (98% reduction of the preischemic flow) and 60 min of reperfusion. A second group was utilized to evaluate the rate of CoQ9 and CoQ10 biosynthesis in the membranes by dissolving two ubiquinone precursors, p-OH-[U-14C]benzoate and mevalonolactone, in the perfusion buffer. The hearts were aerobically perfused for 60 min in the presence of the precursors either immediately after the equilibration period or following 30 min ischemia. The young rat hearts showed a 30% reduction in the mitochondrial levels of CoQ9 after ischemia and reperfusion with respect to the preischemic values (P < 0.05 and P < 0.01, respectively). On the contrary, the mitochondrial CoQ9 content was not modified under these conditions in the aged hearts. At the end of reperfusion, the biosynthesis of mitochondrial CoQ9 and CoQ10 was higher in the young rats (P < 0.05), and lower in the aged rats (P < 0.05), with respect to the aerobic perfusion. In both young and aged rats minor changes in CoQ9 concentrations and biosynthesis were observed at microsomal level. These results indicate that myocardial reperfusion decreases the mitochondrial content of ubiquinone and stimulates CoQ9 biosynthesis in young rats but not in aged rats.
Assuntos
Envelhecimento/metabolismo , Coração/crescimento & desenvolvimento , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica , Miocárdio/metabolismo , Ubiquinona/biossíntese , Aclimatação , Animais , Técnicas In Vitro , Cinética , Masculino , Microssomos/metabolismo , Mitocôndrias Cardíacas/metabolismo , Parabenos/metabolismo , Ratos , Ratos Wistar , Valores de ReferênciaRESUMO
8-Hydroxydeoxyguanosine (8-OH-dG) is widely recognized as a marker of DNA oxidation. Until now, 8-OH-dG has been measured by high-performance liquid chromatography or by gas chromatography-mass spectrometry. A method is reported that detects oxidative derivatives of deoxynucleosides by micellar electrokinetic capillary chromatography. Single-stranded DNA was incubated in the presence of 50 mM hydrogen peroxide-10 mM ascorbic acid and hydrolysed by enzymatic digestion. The order of electrophoretic mobilities of deoxynucleosides was dC > dA > T > dG > 8-OH-dG. 8-OH-dG was determined by introducing a laboratory-prepared internal standard. Two additional major oxidative derivatives were identified by comparing the electropherogram of the oxidized DNA with that of the oxidized standard deoxyguanosine.