RESUMO
BACKGROUND AND OBJECTIVE: Extracorporeal shock wave therapy has been used in various clinical conditions as a result of its ability to stimulate healing processes in acute and chronic inflammatory states. Orthodontic force application triggers an inflammatory reaction in the periodontal tissue surrounding the involved teeth, resulting in tooth movement. Preliminary work revealed that extracorporeal shock wave therapy increased the expression of the inflammatory cytokines involved. Our aim was to investigate the expression of inflammatory cytokines in the periodontal tissues following orthodontic force induction, with and without shock wave therapy, in experimental rats. MATERIAL AND METHODS: An orthodontic appliance was fabricated and applied between the molars and the incisors of adult Wistar rats. In conjunction with orthodontic force commencement, the rats were treated with a single episode of 1000 shock waves. Every day, during the 3 d of the study, rats were killed and the immunolocalization of RANKL, interleukin (IL)-1ß, IL-6 and tumor necrosis factor-alpha was evaluated. RESULTS: The percentage of the area staining positively for all inflammatory cytokines during the first 2 d decreased statistically significantly more in the shock wave-treated group compared with the nontreated control group. On the first day, the percentage of the area staining positively for IL-1ß and RANKL on the compression side peaked in both groups, with a sequential rise in the number of TRAP-positive cells. CONCLUSION: The induction of shock wave therapy during orthodontic tooth movement influences the expression of different inflammatory cytokines in the tissue and might alter the expected periodontal remodeling rate.
Assuntos
Citocinas/análise , Ondas de Choque de Alta Energia/uso terapêutico , Aparelhos Ortodônticos , Periodonto/imunologia , Técnicas de Movimentação Dentária/instrumentação , Animais , Interleucina-1beta/análise , Ligante RANK/análise , Ratos , Ratos Wistar , Estresse Mecânico , Fator de Necrose Tumoral alfa/análiseRESUMO
Oxidative stress and inflammation play an important role in the catabolism of skeletal muscles. Recently, cigarette smoke (CS) was shown to stimulate muscle catabolism by activation of p38 MAPK and up-regulation of the muscle-specific E3 ubiquitin ligases (E3s) atrogin-1 and MuRF1 which are over-expressed during muscle atrophy. Peroxynitrite (ONOO-), an oxidative ingredient of CS, also produced during oxidative stress and inflammation, was previously shown to induce ubiquitination and degradation of muscle proteins. To investigate the involvement of p38 MAPK and the muscle-specific E3s in ONOO--induced muscle catabolism, C2 myotubes, differentiated from a myoblast cell line, were exposed to ONOO- (25 µM) in a time-dependent manner. Following exposure, degradation of myosin heavy chain (MyHC) and actin, activation of p38 MAPK, and levels of atrogin-1 and MuRF1 were studied by Western blotting. Peak phosphorylation of p38 MAPK was observed at 1 h of ONOO- exposure. ONOO- caused a significant increase in the levels of atrogin-1 and MuRF1. In accordance, a significant decrease in MyHC levels was observed in a time-dependent manner. These findings support previous studies in which the catabolic effects of ONOO- were shown. In addition, ONOO- was demonstrated to induce degradation of muscle proteins by activation of p38 MAPK and up-regulation of the muscle-specific E3s atrogin-1 and MuRF1.
Assuntos
Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Ácido Peroxinitroso/farmacologia , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/agonistas , Proteínas Musculares/genética , Cadeias Pesadas de Miosina/antagonistas & inibidores , Cadeias Pesadas de Miosina/genética , Fosforilação , Proteólise , Proteínas Ligases SKP Culina F-Box/genética , Transdução de Sinais , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Cigarette smoke (CS) is an important environmental source of human exposure to a highly toxic and chemically active α,ß-unsaturated aldehyde: acrolein. It is capable of causing protein carbonylation and dysfunction, especially in oral tissues of smokers, constantly exposed to CS toxic constituents. The foremost damage is considered to be cumulative, but even a short exposure can be potentially harmful. The objectives of the current study were to examine the short time and dose effects of direct CS and acrolein exposure on intracellular protein carbonylation in epithelial cells. HaCaT-keratinocytes were exposed to different doses of acrolein and whole phase CS using a unique smoking simulator apparatus that mimics the exposure in smokers. The rate of intracellular protein carbonyl modification was examined 10-60 min after the exposure by Western blot. In addition, the effect of pre-incubation with a thiol scavenger N-acetylcysteine (NAC) was also assessed. We found that intracellular protein carbonyls increased as fast as 10 min after CS exposure and their concentration doubled after 20 min, with a slight elevation afterwards. Also, carbonyl levels increased gradually as CS and acrolein doses were elevated. Addition of 1 mM NAC neutralized part of the damage. We conclude that CS and acrolein intracellular protein carbonylation is dose- and time- dependent. Even a short time exposure to CS and its aldehydic constituents can be potentially harmful.
Assuntos
Acroleína/toxicidade , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Produtos do Tabaco , Poluição por Fumaça de Tabaco/efeitos adversos , Acetilcisteína/farmacologia , Acroleína/química , Aldeídos/química , Aldeídos/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Sequestradores de Radicais Livres/farmacologia , Humanos , Fumar , Poluição por Fumaça de Tabaco/análiseRESUMO
Smokers tend to have lower body mass index, on one hand, and increased abdominal obesity, on the other hand. Also, low levels of lean mass (LM) and bone mineral content (BMC) were found among older smokers compared with non-smokers. This altered body composition and its consequences raise the need for simple and reliable methods for assessment of body composition in smokers. This study aimed to compare body composition assessment by segmental bioelectrical impedance analysis (sBIA) with the reference method, dual energy X-ray absorptiometry (DEXA). Body composition was measured by sBIA (Tanita BC-545) and DEXA (Hologic) in 49 heavy smokers (>15 cigarettes/day, mean age 43.8±12.0). The comparison included correlations and differences between measurements obtained using the two methods as well as the Blande-Altman analysis. Whole-body fat mass (FM) and LM measured by the two methods were found to be highly correlated (r>0.9, p<0.001). Compared with DEXA, sBIA significantly overestimated whole-body LM and BMC (1,126 g and 382 g, respectively, p<0.01). The Bland-Altman analysis revealed a good agreement for whole-body FM and LM, but a poor agreement for BMC. The segmental FM percentage and LM were also highly correlated (r>0.9, p<0.001). However, sBIA significantly overestimated LM of the trunk and legs and underestimated the appendicular FM percentage. Verified by DEXA, sBIA provides reliable measures of whole-body LM, FM, and trunk FM in heavy smokers. A lesser degree of agreement was found for BMC, appendicular LM, and FM.
Assuntos
Composição Corporal/fisiologia , Densidade Óssea/fisiologia , Fumar/metabolismo , Fumar/fisiopatologia , Absorciometria de Fóton , Tecido Adiposo/fisiologia , Adulto , Impedância Elétrica , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
We have previously shown that green tea (GT) drinking combined with vitamin E supplementation reduced plasma protein carbonyls and increased erythrocytes catalase activity in exercising healthy elderly. In the present study we set out to investigate the antioxidative effects of GT drinking in an aging population. We performed an interventional, crossover, controlled prospective trial with 35 healthy elderly subjects (mean age 67.3±4.8 years), supplemented with four daily placebo maltodextrin "tea-bags" for 12 weeks, followed by four 1.5 g daily GT bags for another 12 weeks. Data were obtained at baseline, at the end of the placebo period, and at the end of the GT intervention period. We found that GT did not alter erythrocyte catalase activity. However, it provided protection against 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis which declined by 10.2% (p<0.001). No changes were observed in saliva oral peroxidase enzymes. Nonetheless, saliva total antioxidant capacity increased by 42.0% (p<0.01). Plasma oxidative products, such as protein carbonyls, lipid peroxides and thiobarbituric acid reactive substances (TBARS) were stable throughout the intervention period. We conclude that four daily cups of GT are well tolerated in elderly free living subjects. Our results demonstrate that both erythrocyte resistances to oxidation and saliva antioxidant capacity are improved by GT drinking. The clinical implications of these oxidation modifications require further research.
Assuntos
Envelhecimento/sangue , Antioxidantes/metabolismo , Eritrócitos/metabolismo , Saliva/química , Chá , Idoso , Amidinas/farmacologia , Células Cultivadas , Estudos Cross-Over , Eritrócitos/efeitos dos fármacos , Feminino , Humanos , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Oxidantes/farmacologia , Oxirredução , Peroxidases/metabolismo , Estudos Prospectivos , Carbonilação Proteica , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Oxidative stress (OS) is common in inflammatory conditions and may be important in atopic dermatitis (AD) etiology. The aim of this project was to study the involvement of oxidation in FSL-1 (deacylated lipoprotein)-triggered signaling pathways leading to AD-typical cytokine expression in HaCaT keratinocytes. HaCaT keratinocytes, pretreated with the inhibitor to OS N-acetylcysteine (NAC), were exposed to FSL-1, a stimulator of AD-related cytokines. Cytokines expression was studied by real time polymerase chain reaction (PCR); nuclear factor-kappa B (NF-κB) and p38 mitogen activated protein kinase (MAPK) activities were studied by western blotting; and the oxidative state of cells was determined by the dichlorofluorescein (DCF) assay. We found that endogenous OS in keratinocytes appeared 4 h after FSL-1 administration. OS activated NF-κB, but not p38 MAPK, and the inhibition of OS reduced FSL-1 induced interleukin (IL) 33, thymic stromal lymphopoietin (TSLP) and TNFα mRNA expression. We conclude that FSL-1 triggers an OS reaction in HaCaT keratinocytes, which is probably a secondary event affecting the expression of specific AD typical cytokines, possibly through the NF-κB pathways. This role of OS in the inflammatory response in AD is worth further investigating.
Assuntos
Dermatite Atópica/tratamento farmacológico , Diglicerídeos/farmacologia , Queratinócitos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Humanos , NF-kappa B/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The objective of this study was to investigate the relationship between smoking history expressed by pack-years, metabolic and inflammatory markers, parameters of body composition (BC) and muscle strength among heavy smokers. A detailed smoking history was obtained from 49 heavy smokers (age = 44 ± 12, pack-years = 31 ± 23). Blood samples were analyzed for levels of glucose, lipids, liver enzymes and C-reactive protein (CRP). Anthropometric measurements included waist circumference and assessment of BC by dual energy X-ray absorptiometry (DEXA) and bioelectrical impedance analysis (BIA). Muscle strength was assessed by handgrip dynamometry and predicted one-repetition maximum (p1RM) tests. Positive correlations were found between pack-years of smoking, fasting glucose, alkaline phosphatase and CRP levels. Pack-years were also positively correlated with waist circumference, body mass index (BMI), whole-body and trunk fat mass measured by both DEXA and BIA. A negative correlation was found between pack-years of smoking and muscle strength measured by p1RM for the leg press exercise. After adjustment for age, sex and BMI, a positive correlation remained between pack-years of smoking and CRP levels. In conclusion, after controlling for possible confounders, smoking history was found to be positively associated with CRP levels among heavy smokers.
Assuntos
Biomarcadores/metabolismo , Composição Corporal/efeitos dos fármacos , Inflamação/metabolismo , Força Muscular/efeitos dos fármacos , Fumar/efeitos adversos , Fumar/metabolismo , Absorciometria de Fóton , Adulto , Biomarcadores/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dinamômetro de Força MuscularRESUMO
Exposure to cigarette smoke (CS) and cigarette smoking have been shown to promote catabolism of skeletal muscle. Previous studies and recent findings from our laboratory have demonstrated the involvement of the ubiquitin proteasome system and the muscle-specific E3 ubiquitin ligases MAFbx/atrogin-1 and MuRF1 in CS induced skeletal muscle catabolism. The essential amino acid leucine is a known anticatabolic agent that improves skeletal muscle metabolism in various atrophic conditions. To examine the protective effect of leucine and proteasome inhibition in CS induced muscle catabolism, C2 myotubes, from an in vitro skeletal muscle cell line, were exposed to CS in the presence or absence of leucine and a proteasome inhibitor, MG132. Diameter of myotubes, levels of the main contractile proteins - myosin heavy chain and actin, expression of MAFbx/atrogin-1 and MuRF1 were studied by microscopy, Western blotting, and qPCR. Leucine pretreatment prevented the CS-induced reduction in diameter of myotubes and degradation of myosin heavy chain by suppressing the upregulation of MAFbx/atrogin-1 and MuRF1. MG132 also attenuated the CS-induced decrease in diameter of myotubes and degradation of myosin heavy chain. Our findings demonstrate that supplementation with the essential amino acid leucine and inhibition of the proteasome may protect skeletal muscle from CS induced catabolism.
Assuntos
Aminoácidos Essenciais/química , Leucina/química , Leupeptinas/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Fumaça/efeitos adversos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Metabolismo , Camundongos , Cadeias Pesadas de Miosina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Produtos do Tabaco/efeitos adversosRESUMO
INTRODUCTION: Nitration of tyrosine and tyrosine-containing proteins and their roles in pathophysiology have just recently been reviewed. Despite low yields of tyrosine modifications, nitration of tyrosine residues may inactivate important proteins. Nitrotyrosine can be formed by various nitrating agents, including peroxynitrite. Thus, the occurrence of nitrotyrosine-containing proteins in vivo should be regarded as a general indication of tissue damage induced by reactive nitrogen species such as peroxynitrite. This strongly suggests that peroxynitrite could be formed in vivo under certain pathophysiological conditions. OBJECTIVE: Our aim in this study was to elucidate the effect of cigarette smoke (CS) on nitrotyrosine formation in saliva proteins. METHODS: We exposed saliva to CS, in vitro, and used Western Blotting (WB) and monoclonal anti-nitrotyrosine antibody to assess the level of saliva protein nitration. RESULTS: As saliva contains extensive amounts of nitrites, it was no surprise that at basal levels, saliva proteins, albumin, and α-amylase all were already nitrated. The WB also revealed that with continuous exposure to CS the tyrosine nitration of both albumin and α-amylase is declining significantly after 3 h. A quite similar effect was seen after exposure to aldehydes, but to a less extent as compared to CS. Exposure of nitrotyrosine-modified bovine serum albumin (BSA-N) to aldehydes, produced a similar effect, meaning a decrease in tyrosine nitration. CONCLUSIONS: These findings might be explained by the possible ability of CS aldehydes to reduce protein-bound nitro group to an amine. Another proposed mechanism is that CS unsaturated aldehydes react with proteins mainly through Michael addition reaction; leading to the generation of stable aldehyde-protein adducts (APA). Thus, it may react with nitro groups of saliva proteins, like albumin or α-amylase, to generate APA, which ultimately, may not be recognized by our antibody. Another possible mechanism, is interaction between the aldehyde group with the hydroxyl group of the 3-nitrotyrosine, forming a hemiacetal, which is not recognized by the antibody. This mechanism might explain the difference in the denitration effects caused by the saturated aldehyde acetaldehyde, which exists in large amounts in CS, and unsaturated aldehydes. Therefore, it is possible that the main player in the CS smoke denitration effect on salivary proteins is the aldehyde group and not the double bond of unsaturated aldehydes.
Assuntos
Nicotiana/efeitos adversos , Proteínas e Peptídeos Salivares/metabolismo , Fumaça/efeitos adversos , Tirosina/análogos & derivados , Tirosina/metabolismo , Aldeídos/metabolismo , Feminino , Glutationa/metabolismo , Humanos , MasculinoRESUMO
AIM: This study was designed to analyze serum oxidative stress (OS) levels in healthy young individuals performing a routine maximal aerobic exercise and to evaluate the correlation between OS levels and physiological parameters. METHODS: Serum OS levels were studied by thermochemiluminescence (TCL) parameters at rest and following maximal aerobic exercise in 85 healthy young subjects. Levels were measured by a real time on line TCL assay (higher TCL-Ratio and TCL-H3 = lower OS level). RESULTS: Aerobic capacity had no effect on baseline OS levels. Post-exercise OS levels correlated with maximal oxygen uptake (V.O(2max)) (P<0.005), delta V.O(2) (V.O(2max)- V.O(2)rest) (P<0.005), anaerobic threshold (VTH) (P<0.01), and total oxygen uptake (especially O(2) after VTH), (P<0.005). TCL-Ratio was related to total running time (P<0.01), as well. Post-exercise OS levels for the whole study group did not vary from baseline values. However, individuals with higher fitness level (V.O(2max) >percentile 60) had significantly lower values of TCL-H3 (P=0.04) and tended to have lower TCL-Ratio, indicating they had elevated OS levels. In a multivariate analysis OS level was most affected by V.O(2) after VTH (anaerobic phase of the test) (P=0.003; adjusted odds ratio of 3.41, 95% confidence interval: 1.55-7.48). CONCLUSIONS: In conclusion, acute incremental exercise to maximal performance does not cause alterations in serum oxidant levels of healthy young individuals. In healthy individuals performing maximal aerobic exercise, OS levels correlate with maximal aerobic power.
Assuntos
Tolerância ao Exercício/fisiologia , Exercício Físico/fisiologia , Músculo Esquelético/fisiologia , Oxidantes/sangue , Estresse Oxidativo/fisiologia , Consumo de Oxigênio/fisiologia , Adulto , Teste de Esforço , Feminino , Humanos , Masculino , Adulto JovemRESUMO
The increase in life expectancy, along with the accompanying ongoing increase in the proportion and absolute numbers of nonagenarians and centenarians have set forth the curiosity regarding the question of the quality of health in very old age. Studies on that issue have pointed to the fact that the very old people are actually healthier than originally predicted on the basis of the earlier studies on aging. Current efforts are thus invested in elucidating the possible basis of health in the very old people, as well as better understanding of potential causes of frailty and common diseases in old age. This review recounts on the various aspects underlying evidence-based recommendations for healthy life in old age. We focus on the genetic and non-genetic bases of aging and longevity, and the various directions towards the promotion of health, both via avoiding, or eliminating risk factors and deleterious effects, as well as conducting healthy lifestyle - in terms of proper nutrition and physical exercise. Next, we touch upon preventive medicine, particularly as related to vaccination, with a note also on the need for a reasonable use of medications. In addition, we report about the developing area of regenerative medicine and its potential in relation to the prevention of damage and possible strategies towards tissue repair in cases of age-related degenerative processes.
Assuntos
Envelhecimento/fisiologia , Envelhecimento/psicologia , Promoção da Saúde/métodos , Nível de Saúde , Qualidade de Vida , Atividades Cotidianas , Idoso de 80 Anos ou mais , Medicina Baseada em Evidências , Exercício Físico/fisiologia , Feminino , Humanos , Expectativa de Vida , Estilo de Vida , Masculino , Fenômenos Fisiológicos da Nutrição/fisiologia , Prevenção Primária , Fatores de RiscoRESUMO
Cigarette smoking is linked to various human disorders. Active and passive smokers suffer from inflammatory diseases of lungs and airways. Smoking-dependent airway inflammation is related to the cytotoxic effects of cigarette smoke (CS) and chronic recruitment of neutrophils and macrophages. NF-kappaB is a key inflammatory, redox-sensitive transcription factor. Its role in CS-induced airway inflammation is unclear. This study investigated CS-induced NF-kappaB activation in human lymphocytes and the possible involvement of oxidative insult in this activation. A method for accurate and reproducible exposure of lymphocytes to CS was developed. The intensity of CS exposure was linearly correlated with nitrite concentration originating from reactive oxygen species in CS. Mild, but not high exposure to CS, induced NF-kappaB in lymphocytes through the increase in oxidative stress and the reduction in the intracellular glutathione levels. These findings may have implications to active as well as to passive smokers, suffering from inflammatory diseases of lungs and airways.
Assuntos
Linfócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Nicotiana , Estresse Oxidativo/efeitos dos fármacos , Fumaça/efeitos adversos , Fumar/metabolismo , Poluição por Fumaça de Tabaco , Adulto , Células Cultivadas , Relação Dose-Resposta a Droga , Glutationa/metabolismo , Humanos , Linfócitos/metabolismo , Pessoa de Meia-Idade , Nitritos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Reprodutibilidade dos TestesRESUMO
Cigarette smoke (CS) is an important source of reactive nitrogen species (RNS). It has been demonstrated that CS constitutes the highest source of exogenous nitric oxide and peroxynitrite to which humans are exposed. NF-kappaB is a key inflammatory, redox-sensitive transcription factor, which role in CS-induced airway inflammation is unclear. Moreover, the role of RNS in the activation of NF-kappaB and in inflammation has remained vague. This study investigated CS-induced NF-kappaB activation in human lymphocytes and assessed the involvement of CS-derived RNS in NF-kappaB activation and their possible biological effects. Mild, but not high, exposure to CS induced NF-kappaB in lymphocytes through IKK activation, I-kappaBalpha degradation, and the reduction in the intracellular glutathione levels. Peroxynitrite, but not NO, mimicked the effects of CS on NF-kappaB. Reduction of intracellular peroxynitrite formation by the inhibition of the mitochondrial respiratory chain resulted in decreased activation of NF-kappaB by CS. NF-kappaB-induced iNOS levels were increased in response to CS. Low levels of CS exposure induced classical NF-kappaB activation pathway in lymphocytes via intracellular formation of peroxynitrite, through a reaction between smoke-derived NO and endogenously produced superoxide. This NF-kappaB activation resulted in inflammatory gene expression, which may contribute to CS-related airway inflammation.
Assuntos
Linfócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Nicotiana , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Fumaça/efeitos adversos , Fumar/metabolismo , Adulto , Células Cultivadas , Relação Dose-Resposta a Droga , Ativação Enzimática , Indução Enzimática , Glutationa/metabolismo , Humanos , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/metabolismo , Linfócitos/enzimologia , Linfócitos/metabolismo , Pessoa de Meia-Idade , Inibidor de NF-kappaB alfa , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Ácido Peroxinitroso/metabolismo , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
Three glycoproteins, designated as F, M and S glycoproteins were identified in the HCIO4-soluble fraction of ascitic fluid of Ehrlich ascites tumor by 8% polyacrylamide disc gel electrophoresis. They were separated and purified as described previously (Reznick, A.Z. and Winzler, R.J. (1973) Fed. Proc. 32, 368 and Reznick, A.Z., Allen, H.J. and Winzler, R.J. (1973) Anal. Biochem. 52, 395-401) and subjected to physical characterization. Several physical properties such as molecular weights, sedimentation and diffusion coefficients, partial specific volumes, Stoke's radii and frictional ratios were determined. The physical parameters of F and S glycoproteins resemble data that have been reported for orosomucoid and haptoglobin-like glycoproteins, respectively. Properties of M glycoprotein could not be associated with a known glycoprotein.
Assuntos
Carcinoma de Ehrlich/análise , Glicoproteínas , Animais , Cromatografia em Gel , Camundongos , Peso Molecular , Pressão Osmótica , Conformação Proteica , UltracentrifugaçãoRESUMO
Three glycoprotein bands were identified by polyacrylamide disc gel electrophoresis in the perchloric acid soluble fraction of ascitic fluid of Ehrlich ascites tumor in mice. The three proteins were first separated by a new discontinuous preparative electrophoresis apparatus described previously [1]. They were further purified on Sephadex G-100 and then were subjected to chemical characterization. These glycoproteins were rich in glutamic and aspartic acids and contained the sugar moieties galactose, mannose, fucose, N-acetyl-D-glucosamine and sialic acid. The percent sugar composition ranged from 17.7-37.3% of the total weights of these glycoproteins.
Assuntos
Carcinoma de Ehrlich/análise , Glicoproteínas/análise , Aminoácidos/análise , Animais , Cromatografia em Gel , Eletroforese Descontínua , Feminino , Fucose/análise , Galactose/análise , Manose/análise , Camundongos , Camundongos Endogâmicos ICRRESUMO
Although cardiac endogenous antioxidants have been reported to be oxidized and decreased by ischemia-reperfusion, little is known whether the changes in these antioxidants are correlated with each other in a systematic relationship. In this study, isolated rat hearts were subjected to various periods of ischemia-reperfusion using the Langendorff method, and the content and/or redox status of tissue antioxidants were analyzed. Significant losses in the tissue hydrophilic antioxidants, ascorbate, and glutathione were observed. These losses were dependent on the duration of the reperfusion period (between 0-40 min) but not of ischemia (20-60 min). Marked increases of dehydroascorbate and glutathione disulfide, the oxidized forms of ascorbate and glutathione, respectively, were found during reperfusion, but these changes were not observed during ischemia. These findings indicate that the tissue hydrophilic antioxidants are easily oxidized and may be the first line of antioxidant defenses during reperfusion. Lipophilic antioxidants, like ubiquinol 9 and vitamin E, were not decreased during ischemia-reperfusion using regular buffer; however, if oxidative stress was induced by addition of H2O2 to the buffer solution during reperfusion after 20 min of ischemia, decreases in both the hydrophilic and hydrophobic antioxidants were noticeable. With 100 microM H2O2, the tissue antioxidant decreases were ubiquinol 9 (39%), vitamin E (3%), glutathione (44%) and ascorbate (58%). Only with 500 microM H2O2 treatment were marked decreases in tissue vitamin E (65%) observed; this was associated with almost complete depletion of tissue ubiquinol 9 (95%). These results suggest that prior to the consumption of vitamin E, other antioxidants are depleted and that vitamin E may serve as the ultimate antioxidant, protecting the integrity of cellular membranes. Thus, in this work, cardiac antioxidants were demonstrated to change in a systematically organized relationship under ischemia-reperfusion. This graded utilization of antioxidants supports the redox based antioxidant network concept, found to be present in other biological systems.
Assuntos
Antioxidantes/metabolismo , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica , Animais , Ácido Ascórbico/metabolismo , Ácido Desidroascórbico/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos Sprague-Dawley , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Vitamina E/metabolismoRESUMO
Fructose-1,6-diphosphate aldolase has been purified to homogeneity 62.0 and 58.3 fold from young and old nematodes respectively. The aldolase preparations from young (7 days) and old (35 days) animals are indistinguishable in their electrophoretic mobility, molecular weight of the tetramer (158,000) and monomer (40,000), and Km, although the "old" enzyme is more heat stable than the "young" enzyme. Enzyme from old animals has only about 55% specific activity per mg purified protein of the "young" enzyme and its catalytic activity per unit of enzyme antigen is about 50% of that of the enzyme from young animals. Immunological identity of purified enzyme from old and young animals was established by the Ouchterlony technique by antiserum produced against purified "young" enzyme and antiserum against purified "old" enzyme. Thus, this work shows for the first time that the altered form of an enzyme which appears in senescent animals apparently does not possess extra antigenic sites which are acquired as a function of age.
Assuntos
Envelhecimento , Frutose-Bifosfato Aldolase , Nematoides/enzimologia , Animais , Fenômenos Químicos , Química , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática , Frutose-Bifosfato Aldolase/isolamento & purificação , Testes de PrecipitinaRESUMO
The administration of 0.2% of the amino acid analogues canavanine and 6-fluorotryptophan to young nematode cultures caused the appearance of inactive but antigenically reactive aldolase molecules at levels comparable to those found in old untreated cultures. Concomitant with the rapid appearance of inactive enzyme molecules a rapid rate of mortality could be detected. Mortality, however, ceased when a massive disappearance of inactive molecules could be observed. Subsequently, mortality resumed at a rate similar to that found in untreated cultures. The NaH14CO3 method of Swick and Ip (J. Biol. Chem., 249 (1974) 6836-6841) was used to estimate the half-life of proteins. The half-life of total soluble proteins in old and intermediate age animals was 4 times and 2.5 times longer, respectively, than that of young nematodes. No differences could be discerned in the half-lives of total proteins between analogue-treated and control animals of young and intermediate ages. However, the rate of disposal of inactive molecules was slower in intermediate age as compared to young animals as it was closely related to the general rate of protein degradation in the different age groups. The results suggest that (a) the proportion of inactive enzyme molecules encountered in old animals is potentially detrimental, (b) the decline in efficiency of the degradation system in older animals may account for the accumulation of altered protein molecules in aging organisms.
Assuntos
Envelhecimento , Nematoides/metabolismo , Proteínas/metabolismo , Animais , Canavanina/farmacologia , Frutose-Bifosfato Aldolase/metabolismo , Temperatura Alta , Nematoides/efeitos dos fármacos , Triptofano/análogos & derivados , Triptofano/farmacologiaRESUMO
CWI mice at the age of 6, 22 and 27 months were subjected to a controlled physical training program for 5 weeks. Changes in specific activity of the enzymes aldolase, superoxide dismutase and catalase of the total hind-leg muscle tissues were followed. During the training period, specific activities of these muscle enzymes exhibited an adaptive increase in the 6 and 22 months groups. In senile (27 months) mice, however, enzyme activities decreased as a consequence of the physical challenge. Total body weight was not altered under these conditions. Immunotitration of extracts with anti-aldolase and anti-creatine kinase antibodies showed no significant differences, indicating that there was no accumulation of disappearance of faulty proteins during aging and physical exercise.
Assuntos
Envelhecimento , Músculos/enzimologia , Condicionamento Físico Animal , Animais , Catalase/metabolismo , Creatina Quinase/metabolismo , Feminino , Frutose-Bifosfato Aldolase/metabolismo , Membro Posterior , Camundongos , Esforço Físico , Superóxido Dismutase/metabolismoRESUMO
The effect of wheel running on the levels of fructose 1,6-bisphosphate aldolase A (EC 4.1.2.13) in striated muscles of young and old mice was compared. Short-term (6-10 weeks) and long-term (over 12 months) regimens were included in the study. The studies were conducted on the CW-1 outbred strain and on the C57/BL inbred strain of mice. It was shown that, in the short-term regimens, old animals of both strains showed either no increase (C57/BL) or a reduction (CW-1) in aldolase activity in hind leg muscles. On the other hand, young animals of both strains showed increases in aldolase activity of 10-20%. In the long-term regimen young and intermediate age animals showed 30-100% increases in aldolase activity in hind leg muscles over control sedentary animals. This adaptive capacity to exercise was not observed in old animals. However, long-term exercise regimen prevented the age-associated decline in aldolase activity found in sedentary animals.