RESUMO
The urothelium covering the luminal surface of the urinary bladder has developed an efficient permeability barrier that protects it against the back-flow of toxins eliminated in the urine. The subapical endocytic vesicles containing the urinary bladder fluid phase are formed during the micturition cycle by endocytosis processes of the superficial cells. In normal conditions, the permeability barrier of the endocytic vesicles blocks the passage of the fluid phase to the cellular cytoplasm and the fluid is recycled to the bladder lumen. The aim of this work was to investigate the alteration of the endocytic vesicle membrane permeability barrier to toxins such as iAs (inorganic arsenic) administered in drinking water. By using an induced endocytosis model and the fluorescence requenching technique, it is shown that the exposure of rats to ingestion of water containing iAs not only induced pre-cancerous morphological changes, but allowed the differential leakage of an endocytosed fluorescent marker, HPTS, and its quencher, DPX, (hydroxypyrene-1,3,6-trisulfonic acid and p-xylene-bis-pyridinium bromide, respectively) out of the vesicular lumen. The leakage of the cationic DPX was almost complete, while the release of the anionic HPTS molecule was partial and higher in arsenic-treated-rats than in controls. Such membrane alteration would allow the toxins to elude the permeability barrier and to leak out of the endocytic vesicles, thus establishing a "bypass" to the permeability barrier. The retention of As in the urinary bladder, assessed by synchrotron radiation X-ray fluorescence spectrometry (SR-µXRF), was lower than the kidney accumulation of arsenic previously observed by our group and was accompanied by altered concentrations of K, Ca, Fe, Cu and Zn, all ions related to cellular metabolism. The results support the hypothesis that low amounts of endocytosed As can accumulate in the interior of the urothelial superficial cells and initiate the cytotoxic effects reflected in the morphological alterations observed.
Assuntos
Arsênio/toxicidade , Ácidos Graxos/metabolismo , Lesões Pré-Cancerosas/metabolismo , Vesículas Transportadoras/metabolismo , Bexiga Urinária/metabolismo , Urotélio/metabolismo , Animais , Arsênio/administração & dosagem , Arsênio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Masculino , Lesões Pré-Cancerosas/induzido quimicamente , Lesões Pré-Cancerosas/patologia , Ratos , Ratos Wistar , Vesículas Transportadoras/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/patologia , Urotélio/efeitos dos fármacos , Urotélio/patologiaRESUMO
Chronic toxicity of arsenic resulting from drinking water is a health problem encountered in humans, especially in South America and Asia, where a correlation between oxidative stress, tumor promotion, and arsenic exposure has been observed. Differential solvent extraction (petroleum ether (PE); dichloromethane (DCM); methanol (OL) and water (W)) was performed to compare the protective (antioxidant) activity of five Argentinian medicinal plants on arsenite-induced oxidative stress in Vero cells, assayed by hydroperoxide measurement. The results were analyzed using ANOVA followed by the LSD Fisher test. The data showed that arsenite was a pro-oxidant agent which acts in a time-dose-dependent manner. Extracts from Eupatorium buniifolium (PE), Lantana grisebachii (PE, W), Mandevilla pentlandiana (PE, W), and Sebastiania commersoniana (DCM, OL, W) prevented the formation of both aqueous and lipid hydroperoxides, but Heterothalamus alienus only impeded lipid ones. Therefore, antioxidant extracts are potentially beneficial and may have a protective activity against arsenite-induced renal injury. Among these, the aqueous extract of L. grisebachii may represent the most suitable preparation for humans since the traditional usage of this plant in popular medicine is through consumption of tea.
Assuntos
Antioxidantes/farmacologia , Arsenitos/toxicidade , Rim/efeitos dos fármacos , Extratos Vegetais/farmacologia , Plantas Medicinais/química , Compostos de Sódio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Argentina , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Rim/metabolismo , Medicina Tradicional , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Solventes/química , Células Vero , Água/químicaRESUMO
Chronic toxic effects of arsenic resulting from drinking water are a human health problem, especially in South-America and Asia. Arsenic is capable of influencing various cellular processes, causing adverse effects, including cancer. Although the exact mechanism of the action is not known, a correlation between oxidative stress, tumour promotion and arsenic exposure has been observed. We examined the effects of silymarin and quercetin, in counteracting oxidative stress produced by acute or sub-chronic sodium arsenite exposure. The stress responses to arsenite included an increase in the heat shock protein 70 kDa expression, lipid peroxidation assayed by conjugated dienes measure, and gamma-glutamyl-transpeptidase activity. We found that all these stress responses were eliminated by silymarin and quercetin in acute experiments. Both flavonoids diminished the conjugated dienes formation during sub-chronic cultures. Our results suggest that these antioxidant flavonoids, which may be easily incorporated into the diet, may afford a protective effect against arsenite-induced cytotoxicity.
Assuntos
Antioxidantes/farmacologia , Intoxicação por Arsênico/prevenção & controle , Arsenitos/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Quercetina/farmacologia , Silimarina/farmacologia , Compostos de Sódio/toxicidade , Animais , Western Blotting , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Interações Medicamentosas , Proteínas de Choque Térmico HSP70/metabolismo , Peróxidos Lipídicos/metabolismo , gama-Glutamiltransferase/metabolismoRESUMO
UNLABELLED: We have previously shown that a single i.p. injection of nitrosomethylurea (NMU) in 3-day-old rats orally treated with the pesticide mancozeb (MZ), the flavonoid quercetin (Q) or in combination (MZ-Q) induces hyperplasia, atypical acinar cell proliferation and carcinoma in situ (CIS) in the pancreas. This work studies the effect of oral administration of phenobarbital (PB) on this model of pancreatic carcinogenesis. The animals were fed on a diet supplemented by MZ or/and Q from the 10th day of pregnancy, thorough lactation and as pups after weaning until being sacrificed at week 24. Saline injection with non-supplemented diet was used for the control group (SAL). The experimental groups were (1) SAL (control), (2) SAL-PB, (3) NMU, (4) NMU-PB, (5) MZ-NMU, (6) MZ-NMU-PB, (7) Q-NMU, (8) Q-NMU-PB, (9) MZ-Q-NMU and (10) MZ-Q-NMU-PB. Acinar cell hyperplasia was found in all groups of NMU-treated rats. Dysplastic foci (DYS) were seen in groups 3-10 at the following percentages: 19, 48, 71, 27, 71, 35, 100 and 30, respectively. CIS were recorded in groups 4 to 10 at percentages: 4, 36, 13, 11, 0, 16, 5, respectively. CONCLUSION: Although PB, Q or MZ given alone enhance DYS lesions in NMU-treated rats, the MZ/Q/PB combined treatments may increase (mainly in males) or decrease (mainly in female) the DYS and CIS proportion. Because PB, MZ and Q influence P450 enzymes, we suggest that these enzymes play a role in the carcinogenesis process.
Assuntos
Carcinógenos/farmacologia , Carcinoma in Situ/induzido quimicamente , Fungicidas Industriais/toxicidade , Maneb/toxicidade , Neoplasias Pancreáticas/induzido quimicamente , Fenobarbital/farmacologia , Quercetina/farmacologia , Zineb/toxicidade , Alquilantes/toxicidade , Animais , Animais Recém-Nascidos , Carcinoma in Situ/patologia , Modelos Animais de Doenças , Interações Medicamentosas , Quimioterapia Combinada , Feminino , Hiperplasia/induzido quimicamente , Hiperplasia/patologia , Exposição Materna , Troca Materno-Fetal , Metilnitrosoureia/toxicidade , Neoplasias Pancreáticas/patologia , Gravidez , Ratos , Ratos WistarRESUMO
Argentina is a country with both rich floral biodiversity and cultural diversity. Traditional herbal medicines are important in the health care of most people, and rely heavily on the use of indigenous plants. An ethnobotanical survey of the "Sierra de Comechingones" made over a 26-year period (1979-2005), indicated that 65 families and 149 different genuses were used in traditional medicines. The use of these medicines was observed to be widespread and prevalent over orthodox medicine. Medicinal native plants from this mountain range make up 31% of the total Argentina medicinal native flora. In addition, there are 15 endemic species that grow only in the region. The botanical name, popular uses, parts utilized, as well as the distribution of these medicinal plants from the "Sierra de Comechingones", Argentina, were summarized. Previous reports on phytochemical and biological activities in relation to cancer, antimicrobials and pesticides were also included.
Assuntos
Medicina Tradicional , Extratos Vegetais/farmacologia , Plantas Medicinais , Animais , Anti-Infecciosos/farmacologia , Antifúngicos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Argentina , Etnobotânica , Humanos , Inseticidas/farmacologiaRESUMO
We have previously shown that in rat pups intracranially injected with a single dose of apotransferrin (aTf), there is an early oligodendroglial cell OLGc differentiation. The expression of the mRNAs of myelin basic proteins and of 2',3' cyclic nucleotide 3'-phosphodiesterase and the amount of the corresponding proteins, as well as myelin glycolipids and phospholipids, were significantly increased in these animals at 10 and 17 days of age. Microtubules and myelin basic proteins appear to be closely associated in OLGc and it has been shown that the mRNAs of myelin basic proteins are concentrated in the OLGc processes. The aim of this work was to clarify if the accelerated myelination produced by aTf could be linked to changes in certain cytoskeletal elements present in the myelin fraction such as tubulin, actin, and different microtubule-associated proteins (MAPs). A significant increase in the expression of the mRNA of tubulin and actin was observed in the brain of the aTf-treated animals. Several MAPs, particularly MAP 1B and stable tubule only peptide as well as actin and tubulin, were markedly increased in the Triton X-100 insoluble pellet obtained from the myelin fraction of these animals. The changes that we have previously described in the myelin of aTf intracranially injected rats, could be the consequence of its action on the cytoskeletal network of the OLGc. An enlargement of this structure would result in a more efficient and faster movement of the different components that are normally transported to the myelin by the cytoskeleton of this cell.
Assuntos
Apoproteínas/farmacologia , Proteínas do Citoesqueleto/genética , Citoesqueleto/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Básica da Mielina/genética , Oligodendroglia/fisiologia , Transferrina/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , Animais , Animais Recém-Nascidos , Apoproteínas/administração & dosagem , Citoesqueleto/metabolismo , Glicolipídeos/metabolismo , Microinjeções , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Fosfolipídeos/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Ratos , Ratos Wistar , Transcrição Gênica/efeitos dos fármacos , Transferrina/administração & dosagemRESUMO
The aim of this study was to analyze the N-terminal post-translational incorporation of arginine into cytosolic proteins from cultured cells and the in vitro incorporation of arginine into soluble proteins of PC12 cells after serum deprivation. Arginine incorporation was measured in the presence of protein synthesis inhibitors. None of the inhibitors used affected significantly the arginylation reaction while the novo synthesis of protein was reduced by 98%. Under these conditions, we found that of the total [14C]arginine incorporated into the proteins, around 20% to 40% was incorporated into the N-terminal position of soluble proteins by a post-translational mechanism. These results suggest that this post-translational aminoacylation may be a widespread reaction in neuronal and non-neuronal cells. We also found that in PC12 cells, the in vitro post-translational arginylation was 60% higher in apoptotic cells with respect to control cells. These findings suggest that the post-translational arginylation of proteins may be involved in programmed cell death.
Assuntos
Arginina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Apoptose , Química Encefálica , Radioisótopos de Carbono , Células Cultivadas , Embrião de Galinha , Fragmentação do DNA , Células PC12 , RatosRESUMO
We have previously reported the posttranslational addition of [14C]-arginine in the N-terminus of several soluble rat brain proteins. One of these proteins was identified as the microtubule-associated protein, the stable tubule only polypeptide (STOP). However, despite the fact that the biological significance of arginylation is not completely understood, some evidence associates it with proteolysis via the ubiquitin pathway. Since this degradative via is exacerbated as a response to stress, we studied in vitro the posttranslational [14C]-arginylation of cytosolic brain proteins of rats subjected to hyperthermia in vivo. Immediately after subjecting the animals to hyperthermia, a minor reduction (16%) in the acceptor capacity of [14C]-arginine into proteins was observed in comparison with animals maintained at 28 degrees C. However, in the animals allowed to recover for 3 h, an increase (46%) in the arginylation was observed concomitantly with a significant accumulation of the heat shock protein (70 kDa; hsp 70) when compared to the control animals. These findings suggest that the posttranslational arginylation of proteins participate in the heat shock response. The STOP protein of the soluble brain fraction of control animals, which in Western blot appears as a doublet band (125 and 130 kDa, respectively), is seen, after the hyperthermic treatment, as a single band of 125 kDa. The amount of 125 kDa protein, as well as the in vitro incorporation of [14C]-arginine, increases after hyperthermia in comparison with control animals. Following hyperthermic treatment, we observed a decrease in the amount of in vivo [35S]-methionine-labeled brain proteins. We speculate that, as observed for STOP protein, the increase in the degradation of protein that occurs in hyperthermia, would produce an increase in the amount of arginine acceptor proteins.
Assuntos
Arginina/metabolismo , Encéfalo/metabolismo , Hipertermia Induzida , Proteínas dos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Radioisótopos de Carbono , Citosol/metabolismo , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico/biossíntese , Masculino , Metionina/metabolismo , Proteínas dos Microtúbulos/biossíntese , Proteínas dos Microtúbulos/isolamento & purificação , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/isolamento & purificação , Ratos , Ratos Wistar , Radioisótopos de EnxofreRESUMO
The knowledge of brain protein metabolism is important in understanding nervous system brain function. Protein synthesis rates are high in young brain, decline rapidly at adult stages, and thereafter continue falling slowly with age. The breakdown of protein appears to follow a similar rate (1). Protein synthesis and degradation however, are only the two extremes of a complex phenomena which includes a variety of other protein modifications. Proteolytic cleavage is the most common covalent modification of proteins; probably all proteins that have been isolated were modified by proteolysis, since only few are found with the starting amino acid (methionine) attached. This suggests that most proteins were subject to one or more co- and/or posttranslational modifications (2). One of these posttranslational modifications is the arginylation of proteins, described 30 years ago, which now is being recognized as a widespread modification of proteins. In this review, the current status of posttranslational arginylation of brain proteins is discussed.
Assuntos
Arginina/metabolismo , Encéfalo/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Envelhecimento , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas Associadas aos Microtúbulos/metabolismo , Ubiquitinas/metabolismoRESUMO
The beta-amyloid peptide (beta AP1-40) inhibited the in vitro post-translational incorporation of [14C]arginine at the N-terminus of brain soluble proteins and was labelled by the incorporation of [14C]arginine. Addition of arginine at the N-terminal position of beta AP1-40 is predicted to increase the probability of an alpha-helix structure being formed on the first residues with a higher hydrophilic characteristic, increasing the possibility of these residues being exposed to the aqueous environment. Unmodified beta AP1-40 has a low alpha-helix content and a higher probability of beta-turn formation. Accumulation of beta AP1-40 in Alzheimer's disease may therefore be due to a reduced arginylation reaction and consequently to a decrease in its normal degradation by the ubiquitin pathway.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Arginina/química , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , ProbabilidadeRESUMO
Properties so far studied of the 125-kDa 14C-arginylated protein from rat brain show remarkable similarities with those of the STOP (stable tubule only polypeptide) protein. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis the 125-kDa 14C-arginylated protein moves to the same position as the STOP protein. The 125-kDa 14C-arginylated protein was immunoprecipitated by the monoclonal Mab 296 antibody specific for neuronal STOP protein. The 125-kDa 14C-arginylated protein was retained by a calmodulin column like STOP protein. As occurs with the STOP protein, the 125-kDa 14C-arginylated protein is found in higher proportion in cold-stable than in cold-labile microtubules. However, the modified protein associates with microtubules in a lower proportion than the STOP protein. We conclude that the STOP protein incorporates arginine by a posttranslational reaction but that only a small fraction of the STOP protein shows acceptor capacity in vitro.
Assuntos
Arginina/metabolismo , Química Encefálica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Encéfalo/ultraestrutura , Calmodulina/metabolismo , Temperatura Baixa , Eletroforese em Gel de Poliacrilamida , Técnicas de Imunoadsorção , Proteínas Associadas aos Microtúbulos/imunologia , Microtúbulos/metabolismo , RatosRESUMO
The posttranslational incorporation of arginine into proteins catalyzed by arginyl-tRNA protein transferase was determined in vitro in different rat brain regions. The incorporation was found in all the regions studied, although with different specific activities (pmol [14C]arginine incorporated/mg protein). Of the regions studied, hippocampus had the highest specific activity followed by striatum, medulla oblongata, cerebellum, and cerebral cortex. Electrophoretic analysis of the [14C]arginyl proteins from the different regions followed by autoradiography and scanner densitometry showed at least 13 polypeptide bands that were labeled with [14C]arginine. The radioactive bands were qualitatively coincident with protein bands revealed by Coomassie Blue. There were peaks that showed different proportions of labeling in comparison with peaks of similar molecular mass from total brain. Most notable because of their high proportions were those of molecular mass 125 kDa in hippocampus, striatum, and cerebral cortex; 112 and 98 kDa in striatum and cerebellum; and 33 kDa in hippocampus and striatum. In lower proportions than in total brain were the peaks of 33 kDa in medulla oblongata and cerebral cortex and of 125 kDa in medulla oblongata.