RESUMO
Cornelia de Lange syndrome (CdLS) is a clinically heterogeneous disorder characterized by typical facial dysmorphism, cognitive impairment and multiple congenital anomalies. Approximately 75% of patients carry a variant in one of the five cohesin-related genes NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Herein we report on the clinical and molecular characterization of 11 patients carrying 10 distinct variants in HDAC8. Given the high number of variants identified so far, we advise sequencing of HDAC8 as an indispensable part of the routine molecular diagnostic for patients with CdLS or CdLS-overlapping features. The phenotype of our patients is very broad, whereas males tend to be more severely affected than females, who instead often present with less canonical CdLS features. The extensive clinical variability observed in the heterozygous females might be at least partially associated with a completely skewed X-inactivation, observed in seven out of eight female patients. Our cohort also includes two affected siblings whose unaffected mother was found to be mosaic for the causative mutation inherited to both affected children. This further supports the urgent need for an integration of highly sensitive sequencing technology to allow an appropriate molecular diagnostic, genetic counseling and risk prediction.
Assuntos
Síndrome de Cornélia de Lange/genética , Face/anormalidades , Assimetria Facial/genética , Histona Desacetilases/genética , Mutação , Proteínas Repressoras/genética , Sequência de Aminoácidos , Sequência de Bases , Criança , Síndrome de Cornélia de Lange/patologia , Assimetria Facial/patologia , Fácies , Feminino , Aconselhamento Genético , Genótipo , Humanos , Masculino , Fenótipo , Fatores de Risco , Análise de Sequência de DNA/métodos , Homologia de Sequência de Aminoácidos , Índice de Gravidade de Doença , Inativação do Cromossomo XRESUMO
The disorders caused by mutations in genes encoding subunits and accessory proteins of cohesin complex are collectively termed as cohesinopathies. The best known cohesinopathy is Cornelia de Lange Syndrome (CdLS), which is a multisystem developmental disorder characterized by facial dysmorphism, limb malformations, growth and cognitive impairment. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), are responsible for â¼ 70% of CdLS cases. We describe a 16-year-old boy with facial dysmorphism, growth retardation, intellectual disability, hirsutism and small hands, who has a small Supernumerary Marker Chromosome (sSMC) present in mosaic form. sSMC is composed of two duplicated segments encompassing 17 genes including SMC1A gene, at the regions Xp11.22 and Xp11.21q11.1. Clinical comparison between our patient with a previously reported individual with a SMC1A duplication and four male carriers of similar sSMC reported in databases, suggest that they all share clinical features related to cohesinopathies. Although our patient does not have the classical CdLS craniofacial phenotype, he has pre and postnatal growth retardation, intellectual disability and mild musculoskeletal anomalies, features commonly seen in patients with cohesinopathies.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Síndrome de Cornélia de Lange/genética , Deficiência Intelectual/genética , Adolescente , Cromossomos Humanos X , Síndrome de Cornélia de Lange/fisiopatologia , Genes Duplicados , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Fenótipo , CoesinasRESUMO
We demonstrated previously that acetylated tubulin inhibits plasma membrane Ca(2+)-ATPase (PMCA) activity in plasma membrane vesicles (PMVs) of rat brain through a reversible interaction. Dissociation of the PMCA/tubulin complex leads to restoration of ATPase activity. We now report that, when the enzyme is reconstituted in phosphatidylcholine vesicles containing acidic or neutral lipids, tubulin not only loses its inhibitory effect but is also capable of activating PMCA. This alteration of the PMCA-inhibitory effect of tubulin was dependent on concentrations of both lipids and tubulin. Tubulin (300µg/ml) in combination with acidic lipids at concentrations >10%, increased PMCA activity up to 27-fold. The neutral lipid diacylglycerol (DAG), in combination with 50µg/ml tubulin, increased PMCA activity >12-fold, whereas tubulin alone at high concentration (≥300µg/ml) produced only 80% increase. When DAG was generated in situ by phospholipase C incubation of PMVs pre-treated with exogenous tubulin, the inhibitory effect of tubulin on PMCA activity (ATP hydrolysis, and Ca(2+) transport within vesicles) was reversed. These findings indicate that PMCA is activated independently of surrounding lipid composition at low tubulin concentrations (<50µg/ml), whereas PMCA is activated mainly by reconstitution in acidic lipids at high tubulin concentrations. Regulation of PMCA activity by tubulin is thus dependent on both membrane lipid composition and tubulin concentration.
Assuntos
Cálcio/metabolismo , Membrana Celular/enzimologia , Lipídeos de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Encéfalo/metabolismo , Química Encefálica/fisiologia , Membrana Celular/química , Transporte de Íons/fisiologia , Lipídeos de Membrana/química , Proteínas do Tecido Nervoso/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Ratos , Tubulina (Proteína)/química , Fosfolipases Tipo C/químicaRESUMO
BACKGROUND: Cornelia de Lange Syndrome (CdLS) is a rare congenital disorder characterized by typical facial features, growth failure, limb abnormalities, and gastroesophageal dysfunction that may be caused by mutations in several genes that disrupt gene regulation early in development. Symptoms in individuals with CdLS suggest that the peripheral nervous system (PNS) is involved, yet there is little direct evidence. METHOD: Somatic nervous system was evaluated by conventional motor and sensory nerve conduction studies and autonomic nervous system by heart rate variability, sympathetic skin response and sudomotor testing. CdLS Clinical Score and genetic studies were also obtained. RESULTS: Sympathetic skin response and sudomotor test were pathological in 35% and 34% of the individuals with CdLS, respectively. Nevertheless, normal values in large fiber nerve function studies. CONCLUSIONS: Autonomic nervous system (ANS) dysfunction is found in many individuals with Cornelia de Lange Syndrome, and could be related to premature aging.
Assuntos
Síndrome de Cornélia de Lange , Sistema Nervoso Autônomo , Proteínas de Ciclo Celular/genética , Síndrome de Cornélia de Lange/genética , Humanos , Mutação/genética , FenótipoRESUMO
Cornelia de Lange syndrome (CdLS) is a rare disease affecting multiple organs and systems during development. Mutations in the cohesin loader, NIPBL/Scc2, were first described and are the most frequent in clinically diagnosed CdLS patients. The molecular mechanisms driving CdLS phenotypes are not understood. In addition to its canonical role in sister chromatid cohesion, cohesin is implicated in the spatial organization of the genome. Here, we investigate the transcriptome of CdLS patient-derived primary fibroblasts and observe the downregulation of genes involved in development and system skeletal organization, providing a link to the developmental alterations and limb abnormalities characteristic of CdLS patients. Genome-wide distribution studies demonstrate a global reduction of NIPBL at the NIPBL-associated high GC content regions in CdLS-derived cells. In addition, cohesin accumulates at NIPBL-occupied sites at CpG islands potentially due to reduced cohesin translocation along chromosomes, and fewer cohesin peaks colocalize with CTCF.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Síndrome de Cornélia de Lange/genética , Genoma Humano , Transcriptoma/genética , Diferenciação Celular/genética , Cromatina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Estabilidade Proteica , CoesinasRESUMO
To evaluate a method for preventing the nephrotoxicity caused by the high renal accumulation of radiolabeled or toxin-conjugated small immunoproteins used for cancer therapy, we conjugated humanized anti-Tac Fab fragments with various numbers of glycolate molecules [glycolated Fab fragments (glyco-Fabs)] and separated the conjugates by means of ion-exchange columns into three fractions, depending on their isoelectric points (pIs). We evaluated the biodistribution, pharmacokinetics, and catabolism in normal nude mice of nonglycolated Fab (pI > or = 9.3) and three different preparations of glyco-Fab, including strongly anionic glyco-Fab (sa-glyco-Fab: pI < or = 4.5), mildly anionic glyco-Fab (pI = 4.5-7), and mildly cationic glyco-Fab (pI = 7-9.3). In addition, the biodistributions of 125I-labeled sa-glyco-Fab and 131I-labeled nonglycolated Fab were evaluated in normal nude mice coinjected with 50 mg of L-lysine and/or 1 microg of furosemide and in a control group without coinjection. We then evaluated the serial biodistribution of 125I-labeled sa-glyco-Fab (4 microCi/1 microg) and 131I-labeled nonglycolated Fab (5 microCi/1 microg) in Tac antigen-positive (ATAC4) and -negative (A431) tumor-bearing nude mice with s.c. tumor xenografts derived from Tac antigen-positive ATAC4 cells and receptor-negative A431 cells. These animals were coinjected with 30 mg of lysine i.v. and 30 mg of lysine i.p. 15 min after the radiolabeled Fab injection. To evaluate the biodistribution data and study scintigraphic imaging, we performed serial scintigraphy on normal and tumor-bearing mice with all four 131I-labeled preparations. 125I-labeled mildly cationic glyco-Fab and 131I-labeled nonglycolated Fab had similar distributions, except in the kidney. However, both 125I-labeled anionic glyco-Fab preparations showed significantly different distributions from both cationic Fabs in the blood, liver, lung, and spleen. Renal accumulation of all four radiolabeled Fab preparations increased significantly as the pI increased (P < 0.01). In addition, the intact fraction of Fab excreted into urine increased as pI decreased. Therefore, the glomerular filtration depended on whether the charge on the Fab was positive or negative. The proportion of Fab reabsorbed by the proximal tubules increased as pI increased. 125I-labeled sa-glyco-Fab and 125I-labeled mildly anionic glyco-Fab showed a similar distribution in the blood and all organs except the kidney. Lysine led to an additional blocking effect on proximal tubular uptake of both sa-glyco-Fab and nonglycolated Fab. Addition of furosemide yielded only a small effect when used with lysine. With lysine, the sa-glyco-Fab:nonglycolated Fab estimated integral radioactivity ratios were 4.7 and 0.7 in the ATAC4 tumor and in the kidney, respectively. The use of anionic fragments, which may be used in conjunction with lysine, represents a promising approach that may help decrease the renal toxicity of other small fragments, the molecular weights of which range from Mr 40,000 to 70,000, and, thereby, allow higher doses of radiation to the tumor.
Assuntos
Fragmentos Fab das Imunoglobulinas/metabolismo , Rim/metabolismo , Receptores de Interleucina-2/imunologia , Animais , Feminino , Furosemida/farmacologia , Glicosilação , Radioisótopos do Iodo , Ponto Isoelétrico , Lisina/farmacologia , Camundongos , Camundongos Nus , Radioimunodetecção , Distribuição TecidualRESUMO
In this brief review the antioxidative actions of melatonin are summarized and they are discussed relative to several models of oxidative neurotoxicity. Melatonin is a ubiquitously acting antioxidant. It has been shown to scavenge the hydroxyl radical, peroxyl radical, singlet oxygen and the peroxynitrite anion; secondarily, it also scavenges the superoxide anion radical. In addition, melatonin reportedly stimulates a number of antioxidative enzymes including glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase. On the other hand, melatonin inhibits the pro-oxidative enzyme nitric oxide synthase. Besides these actions which help to resist oxidative damage, melatonin prevents membrane rigidity, reduces polymorphonuclear cell infiltration into damaged tissue, limits the adhesion of leucocytes to endothelial cells, thereby increasing blood flow and reducing edema. Some or all of these actions may have been operative in the experimental models of oxidative neurotoxicity that were improved by melatonin treatment. In brief, melatonin has been found to protect the CNS from beta-amyloid toxicity, experimental models of Parkinsonism, excitotoxicity, nitric oxide toxicity, aminolevulinic acid, lipopolysaccharide, hyperbaric hyperoxia, L-cysteine, cyanide and ischemia/reperfusion injury.
RESUMO
Cytotoxic lipid peroxides such as 4-hydroxy-2-nonenal (HNE) are produced when cells are exposed to toxic chemicals. However, the mechanism by which HNE induces cell death has been poorly understood. In this study, we investigated the molecular mechanism of HNE-induced apoptosis in PC12 cells by measuring the activities of the mitogen-activated protein (MAP) kinases involved in early signal transduction pathways. Within 15-30 min after HNE treatment, c-Jun N-terminal protein kinase (JNK) was maximally activated, before returning to control level after 1 h post-treatment. In contrast, activities of extracellular signal regulated kinase (ERK) and p38 MAP kinase remained unchanged from their basal levels. SEK1, an upstream kinase of JNK, was also activated (phosphorylated) within 5 min after HNE treatment and remained activated for up to 60 min. Marked activation of the JNK pathway through SEK1 was demonstrated by the transient transfection of cDNA for wild type SEK1 and JNK into COS-7 cells. Furthermore, significant reductions in JNK activation and HNE-induced cell death were observed when the dominant negative mutant of SEK1 was co-transfected with JNK. Pretreatment of PC12 cells with a survival promoting agent, 8-(4-chlorophenylthio)-cAMP, prevented both the HNE-induced JNK activation and apoptosis. Nonaldehyde, a nontoxic aldehyde, caused neither apoptosis nor JNK activation. Pretreatment of PC12 cells with SB203580, a specific inhibitor of p38 MAP kinase, had no effect on HNE-induced apoptosis. All these data suggest that the HNE-mediated apoptosis of PC12 cells is likely to be mediated through the selective activation of the SEK1-JNK pathway without activation of ERK or p38 MAP kinase.
Assuntos
Aldeídos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , AMP Cíclico/análogos & derivados , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aldeídos/administração & dosagem , Animais , AMP Cíclico/farmacologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Fármacos Neuroprotetores/farmacologia , Células PC12 , Fosforilação , Piridinas/farmacologia , Ratos , Transdução de Sinais , Tionucleotídeos/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Oxysterols in cultured bovine aortic smooth muscle cells and in the monocyte-like cell line U937 were analyzed by gas-liquid chromatography. The following products were detected: 7 alpha- and 7 beta-hydroxycholesterol, 7-ketocholesterol, cholesterol-alpha-epoxide, cholesterol-beta-epoxide, cholestanetriol, and 20- and 25-hydroxycholesterol. The proportions of oxidized cholesterol varied from 1 to 3% of total cholesterol. The concentrations of oxysterols were lower when the smooth muscle cells were maintained in a vitamin E-enriched medium, and were higher in U937 cells when the cells were activated with phorbol myristate acetate. The cell oxysterol content appears to be regulated by factors that inhibit or enhance free radical generation. The concentrations of oxysterols found in cells may serve as an indicator of the extent of lipid peroxidation.
Assuntos
Monócitos/química , Músculo Liso Vascular/química , Esteróis/análise , Animais , Bovinos , Células Cultivadas , Colesterol/análise , Músculo Liso Vascular/citologia , Oxirredução , Acetato de Tetradecanoilforbol/farmacologia , Vitamina E/farmacologiaRESUMO
BACKGROUND: The aim of this study was to investigate the outcome of preperitoneal repair using laparoscopic (TEP) and open (OPM) approach in recurrent inguinal hernia. METHODS: We performed a prospective controlled nonrandomized clinical study in 188 patients with 207 recurrent inguinal hernias over a period of 5 years. TEP repair was employed for 86 repairs, and OPM was used in 121 procedures. The main outcome measurements were: recurrence rate, operating time, hospital stay, and postoperative complications. RESULTS: There were three recurrences (1.7%). Two in the OPM group (1.8%) and one (1.3%) in the TEP group [ P=NS (not significant)]. The TEP procedure was faster than OPM for unilateral repair (40.8 vs 46.3 min) (P<0.001). Postoperative complications were more frequent in the OPM group (23.9%) than the TEP group (13.9%) ( P=NS). Hospital stay was significantly shorter in the TEP group (1.2 vs 3.9 days) (P<0.001). CONCLUSIONS: Preperitoneal approach (open or laparoscopic) seems to be a good option in recurrent inguinal hernia when these procedures are done by experienced surgeons.
Assuntos
Hérnia Inguinal/cirurgia , Laparoscopia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Complicações Pós-Operatórias , Recidiva , ReoperaçãoRESUMO
3-Hydroxy-3-methylglutaric aciduria is a human autosomal recessive metabolic disorder that usually appears within the first year of life. The causes of this aciduria are lethal mutations in the gene encoding for 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL). HL is a mitochondrial matrix enzyme that catalyzes the last step of ketogenesis and leucine catabolism. This gene has been mapped to chromosome 1 at locus 1pter-p33 and its genomic organisation comprises 9 exons whose sizes vary between 64-678 bp. The human cDNA sequence was reported in 1993 with the first genetic study of two Acadian-French Canadian siblings. To date, 24 mutations in 36 patients have been described; most of them are single-base substitutions causing amino acid replacements and a variety of splicing defects. In the population studied two mutations appear predominant: g.122GA (8 patients and 15 alleles) frequent in Saudi Arabia, and g.109GT (6 patients and 12 alleles), prevalent in Spain. At least seven mutations are clustered in the second half of exon 2 affecting aminoacids E37, R41 and D42 and conforming a possible hot spot. The genotype-phenotype correlation is difficult to establish since the probands received different treatments, and the onset of an acute episode frequently depends on external factors such as fasting or acute illness.
Assuntos
Meglutol/urina , Mutação , Oxo-Ácido-Liases/deficiência , Oxo-Ácido-Liases/genética , Processamento Alternativo , Cromossomos Humanos Par 1 , Análise Mutacional de DNA , Éxons , Genótipo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/urina , Leucina/metabolismo , Mitocôndrias/enzimologia , Fenótipo , Polimorfismo GenéticoRESUMO
The gastrointestinal stromal tumor with extragastrointestinal location are very infrequent. We often diagnose them when they show a big size. Their bening or malignant nature is difficult to fix. The best histological parameters to evaluate their prognosis are a high cellularity, the tumor-like necrosis presence and having more than two mitosis per fifty high-power fields. We introduce an asyntomatic patient's case by a routine echographical control for chronic hepatitis by C virus, that has been diagnosed of a mesentery tumor. The patient has been treated surgically. The inmunohistological study of the tumor had confirmed a stromal gastrointestinal tumor. The showed case's analysis and the considered bibliography suggest some clinical discoveries characteristic of this entity. The histogenesis of these neoplasias are examined and made up to date and the usefulness of the new medication to control check the tumor-like progress is emphasized.
Assuntos
Mesentério , Neoplasias Peritoneais , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Peritoneais/diagnóstico , Neoplasias Peritoneais/terapiaRESUMO
Mitochondrial HMG-CoA synthase deficiency is an inherited metabolic disorder caused by a defect in the enzyme that regulates the formation of ketone bodies. Patients present with hypoketotic hypoglycaemia, encephalopathy and hepatomegaly, usually precipitated by an intercurrent infection or prolonged fasting. The diagnosis may easily be missed as previously reported results of routine metabolic investigations, urinary organic acids and plasma acylcarnitines may be nonspecific or normal, and a high index of suspicion is required to proceed to further confirmatory tests. We describe a further acute case in which the combination of urinary organic acids, low free carnitine and changes in the plasma acylcarnitine profile on carnitine supplementation were very suggestive of a defect in ketone synthesis. The diagnosis of mitochondrial HMG-CoA synthase deficiency was confirmed on genotyping, revealing two novel mutations: c.614G > A (R188H) and c.971T > C (M307T). A further sibling, in whom the diagnosis had not been made acutely, was also found to be affected. The possible effects of these mutations on enzyme activity are discussed.
Assuntos
Hidroximetilglutaril-CoA Sintase/deficiência , Erros Inatos do Metabolismo/diagnóstico , Doenças Mitocondriais/diagnóstico , Carnitina/análogos & derivados , Carnitina/sangue , Carnitina/farmacologia , Análise Mutacional de DNA , Genótipo , Heterozigoto , Humanos , Lactente , Masculino , MutaçãoRESUMO
3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine metabolism. The disease is caused by mutations in the gene coding for 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL). To date 26 different mutations have been described. A (betaalpha)(8) TIM barrel structure has been proposed for the protein, and almost all missense mutations identified so far localize in the beta sheets that define the inside cavity. We report an Italian patient who bears homozygously a novel HL mutation, c.608G > A (p. G203E) in beta sheet six. A structural model of the mutated protein suggests that glutamic acid 203 impedes catalysis by blocking the entrance to the inner cavity of the enzyme. Loss of functionality has been confirmed in expression studies in E. coli, which demonstrate that the G203E mutation completely abolishes enzyme activity. Beta sheet six and beta sheet two are the two protein regions that accumulate most missense mutations, indicating their importance in enzyme functionality. A model for the mechanism of enzyme function is proposed.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Meglutol/urina , Mutação de Sentido Incorreto , Oxo-Ácido-Liases/química , Oxo-Ácido-Liases/genética , Sequência de Aminoácidos , Animais , Criança , Análise Mutacional de DNA , Humanos , Masculino , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The analysis of insulin receptors in erythrocytes demands a relatively small blood sample, which justifies the interest in its use as an index of the cellular capacity for binding hormone. In order to establish criteria for normalcy, the capacity of erythrocytes for binding in vitro insulin labelled with 125I before increasing concentrations of cold insulin (from 0.5 to 10(3) ng/ml), was studied in a group of 41 healthy men and another of 35 women with normal menstrual cycles. In the female group the study was carried out in three different days of the same cycle (days 3, 12 and 21). The binding capacity in the male was higher than in the female (p less than 0.05) in the follicular phase (days 3 and 12) as well as in the luteal phase (day 21) and, among women, it was higher in the follicular phase than in the luteal one (p less than 0.05). The results indicate that progesterone, as well as prolactin and glucagon, may play an important role in the binding capacity of insulin to its receptor. To make the values comparable, it is suggested that blood extraction in women be carried out during the first five days of the cycle.
Assuntos
Membrana Eritrocítica/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Caracteres Sexuais , Adulto , Membrana Eritrocítica/ultraestrutura , Feminino , Hormônios/sangue , Humanos , Masculino , Ciclo Menstrual/sangue , Fatores de TempoRESUMO
Renal disfunctions which appear in the chronic respiratory insufficient patient are analysed, as well as the participation of the arterial blood hypoxemia in their genesis. Renal clearances of Na, K, Cl, Ca, Mg and Pi, and those of urea and creatinine, were lower in 36 patients having chronic hypoxemia than in 15 normosemic controls, showing significant statistical differences for Na, K, Cl, Ca and urea. The correlations between the clearances of these substances and the pO2 arterial blood levels had a greater statistical significance than can be established with pCO2 or [H+] levels. Thus, the existence of a causal dependency between renal disfunction and hypoxemia may be deduced.
Assuntos
Eletrólitos/metabolismo , Rim/fisiopatologia , Insuficiência Respiratória/fisiopatologia , Gasometria , Doença Crônica , Humanos , Insuficiência Respiratória/sangueRESUMO
Acetaminophen (AAP), a widely used analgesic drug, can damage various organs when taken in large doses. In this study, we investigate whether AAP causes cell damage by altering the early signaling pathways associated with cell death and survival. AAP caused time- and concentration-dependent apoptosis and DNA fragmentation of C6 glioma cells used as a model. AAP activated c-Jun N-terminal protein kinase (JNK) by 5.3-fold within 15 min. The elevated JNK activity persisted for up to 4 h before it returned to the basal level at 8 h. In contrast, activities of other mitogen-activated protein (MAP) kinases and the level of Akt phosphorylation in the cell survival pathway remained unchanged throughout the treatment. Wortmannin, an inhibitor of phosphatidylinositol-3 kinase, or SB203580, an inhibitor of p38 MAP kinase, did not reduce AAP-induced toxicity, indicating that these enzymes do not play a major role in cell toxicity. AAP-induced apoptosis was preceded by the sequential elevation of the pro-apoptotic Bax protein, cytochrome c release, and caspase-3 activity. Treatment with caspase inhibitor benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (Z-DEVD-FMK) significantly reduced AAP-induced caspase-3 activation and cytotoxicity. Transfection of cDNA for the dominant-negative mutant JNK-KR or stress-activated protein kinase kinase-1 Lys-->Arg mutant (SEK1-KR), an immediate upstream kinase of JNK, significantly reduced AAP-induced JNK activation and cell death rate. The noncytotoxic analog of AAP, 3-hydroxyacetanilide, neither increased JNK activity nor caused apoptosis. Pretreatment with YH439, an inhibitor of CYP2E1 gene transcription, markedly reduced CYP2E1 mRNA, protein content, and activity, as well as the rate of AAP-induced JNK activation and cell death. These data indicate that AAP can cause cell damage by activating the JNK-related cell death pathway, providing a new mechanism for AAP-induced cytotoxicity.
Assuntos
Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Apoptose , Glioma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Acetanilidas/farmacologia , Analgésicos/farmacologia , Androstadienos/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Grupo dos Citocromos c/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Imidazóis/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Piridinas/farmacologia , Ratos , Tiazóis/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Wortmanina , Proteína X Associada a bcl-2RESUMO
Serum concentrations of Na+, K+ and Cl- are studied, as well as the elimination of these ions in urine, in patients suffering from chronic respiratory insufficiency, being classified in two groups according to the level of hypoxemia: group A (PO2 less than 6.66 KPa) and group B (PO2 less than 8 KPa). A third group C of healthy patients with analogous anthropological characteristics has served as a control group. The concentrations of serum of the three ions are noticeably similar in the three groups, but the daily elimination of Na+, K+ and Cl- is less in those suffering from respiratory insufficiency than in those of the control group, with significant statistical differences in all cases except with K+ in those suffering from pronounced hypoxemia. On analysing the correlation between the rates of elimination of ions in urine, with the plasmatic values of PO2, PCO2 and [H+] of all the patients studied, the highest values of the Pearson coefficient are found on correlating the elimination of ions with the partial pressures of oxygen, therefore suggesting that hypoxia could be the main motor inducing metabolic changes.