RESUMO
BACKGROUND: The RTS,S/AS01 vaccine targets the circumsporozoite protein of Plasmodium falciparum and has partial protective efficacy against clinical and severe malaria disease in infants and children. We investigated whether the vaccine efficacy was specific to certain parasite genotypes at the circumsporozoite protein locus. METHODS: We used polymerase chain reaction-based next-generation sequencing of DNA extracted from samples from 4985 participants to survey circumsporozoite protein polymorphisms. We evaluated the effect that polymorphic positions and haplotypic regions within the circumsporozoite protein had on vaccine efficacy against first episodes of clinical malaria within 1 year after vaccination. RESULTS: In the per-protocol group of 4577 RTS,S/AS01-vaccinated participants and 2335 control-vaccinated participants who were 5 to 17 months of age, the 1-year cumulative vaccine efficacy was 50.3% (95% confidence interval [CI], 34.6 to 62.3) against clinical malaria in which parasites matched the vaccine in the entire circumsporozoite protein C-terminal (139 infections), as compared with 33.4% (95% CI, 29.3 to 37.2) against mismatched malaria (1951 infections) (P=0.04 for differential vaccine efficacy). The vaccine efficacy based on the hazard ratio was 62.7% (95% CI, 51.6 to 71.3) against matched infections versus 54.2% (95% CI, 49.9 to 58.1) against mismatched infections (P=0.06). In the group of infants 6 to 12 weeks of age, there was no evidence of differential allele-specific vaccine efficacy. CONCLUSIONS: These results suggest that among children 5 to 17 months of age, the RTS,S vaccine has greater activity against malaria parasites with the matched circumsporozoite protein allele than against mismatched malaria. The overall vaccine efficacy in this age category will depend on the proportion of matched alleles in the local parasite population; in this trial, less than 10% of parasites had matched alleles. (Funded by the National Institutes of Health and others.).
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/prevenção & controle , Plasmodium falciparum/genética , África , Feminino , Variação Genética , Humanos , Lactente , Malária Falciparum/imunologia , Malária Falciparum/parasitologia , Masculino , Resultado do TratamentoRESUMO
AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.
Assuntos
Glicemia/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hepatócitos/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Peptídeos/farmacologia , Receptores de Glucagon/agonistas , Redução de Peso/efeitos dos fármacos , Animais , Peso Corporal/efeitos dos fármacos , Células CHO , Linhagem Celular , Cricetulus , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Macaca fascicularis , Camundongos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , RatosRESUMO
We investigated how glycerol, urea, glucose and a GKA influence kinetics and stability of wild-type and mutant GK. Glycerol and glucose stabilized GK additively. Glycerol barely affected the TF spectra of all GKs but decreased k(cat), glucose S(0.5) and K(D) values and ATP K(M) while leaving cooperativity unchanged. Glycerol sensitized all GKs to GKA as shown by TF. Glucose increased TF of GKs without influence of glycerol on the effect. Glycerol and GKA affected kinetics and binding additively. The activation energies for thermal denaturation of GK were a function of glucose with K(D)s of 3 and 1mM without and with glycerol, respectively. High urea denatured wild type GK reversibly at 20 and 60°C and urea treatment of irreversibly heat denatured GK allowed refolding as demonstrated by TF including glucose response. We concluded: Glycerol stabilizes GK indirectly without changing the folding structure of the apoenzyme, by restructuring the surface water of the protein, whereas glucose stabilizes GK directly by binding to its substrate site and inducing a compact conformation. Glucose or glycerol (alone or combined) is unable to prevent irreversible heat denaturation above 40°C. However, urea denatures GK reversibly even at 60°C by binding to the protein backbone and directly interacting with hydrophobic side chains. It prevents irreversible aggregation allowing complete refolding when urea is removed. This study establishes the foundation for exploring numerous instability mutants among the more than 600 variant GKs causing diabetes in animals and humans.
Assuntos
Apoenzimas/química , Ativadores de Enzimas/química , Glucoquinase/química , Glucose/química , Glicerol/química , Ureia/química , Trifosfato de Adenosina/química , Regulação Alostérica , Apoenzimas/genética , Estabilidade Enzimática , Escherichia coli/genética , Glucoquinase/genética , Humanos , Cinética , Modelos Moleculares , Mutação , Pressão Osmótica , Conformação Proteica , Dobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Temperatura , Termodinâmica , Água/químicaRESUMO
Epidemiological data and genetic studies indicate that certain forms of human epilepsy are inherited. Based on the similarity between the human and mouse genomes, mouse models of epilepsy could facilitate the discovery of genes associated with epilepsy syndromes. Here, we report an insertional murine mutation that inactivates a novel gene and results in whole body jerks, generalized clonic seizures, and epileptic brain activity in transgenic mice. The gene, named jerky, encodes a putative 41.7 kD protein displaying homology to a number of nuclear regulatory proteins, suggesting that perhaps the jerky protein is able to bind DNA.
Assuntos
Proteínas de Ligação a DNA/genética , Epilepsia Tônico-Clônica/genética , Genes , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Modelos Animais de Doenças , Epilepsia Tônico-Clônica/fisiopatologia , Manobra Psicológica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes Neurológicos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Linhagem , Proteínas de Ligação a RNARESUMO
MAOA and MAOB are key iso-enzymes that degrade biogenic and dietary amines. MAOA preferentially oxidizes serotonin (5-hydroxytryptamine, or 5-HT) and norepinephrine (NE), whereas MAOB preferentially oxidizes beta-phenylethylamine (PEA). Both forms can oxidize dopamine (DA). A mutation in MAOA results in a clinical phenotype characterized by borderline mental retardation and impaired impulse control. X-chromosomal deletions which include MAOB were found in patients suffering from atypical Norrie's disease, which is characterized by blindness and impaired hearing. Reduced MAOB activity has been found in type-II alcoholism and in cigarette smokers. Because most alcoholics smoke, the effects of alcohol on MAOB activity remain to be determined. Here we show that targetted inactivation of MAOB in mice increases levels of PEA but not those of 5-HT, NE and DA, demonstrating a primary role for MAOB in the metabolism of PEA. PEA has been implicated in modulating mood and affect. Indeed, MAOB-deficient mice showed an increased reactivity to stress. In addition, mutant mice were resistant to the neurodegenerative effects of MPTP, a toxin that induces a condition reminiscent of Parkinson's disease.
Assuntos
Monoaminoxidase/deficiência , Fenetilaminas/metabolismo , Estresse Fisiológico/enzimologia , Animais , Sequência de Bases , Monoaminas Biogênicas/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Primers do DNA/genética , Feminino , Humanos , Intoxicação por MPTP , Masculino , Camundongos , Camundongos Knockout , Monoaminoxidase/genética , Atividade Motora/genética , Atividade Motora/fisiologia , Oxirredução , Reação em Cadeia da Polimerase , Mapeamento por Restrição , Estresse Fisiológico/genética , Estresse Fisiológico/fisiopatologia , Cromossomo X/genéticaRESUMO
Deficiency in monoamine oxidase A (MAOA), an enzyme that degrades serotonin and norepinephrine, has recently been shown to be associated with aggressive behavior in men of a Dutch family. A line of transgenic mice was isolated in which transgene integration caused a deletion in the gene encoding MAOA, providing an animal model of MAOA deficiency. In pup brains, serotonin concentrations were increased up to ninefold, and serotonin-like immunoreactivity was present in catecholaminergic neurons. In pup and adult brains, norepinephrine concentrations were increased up to twofold, and cytoarchitectural changes were observed in the somatosensory cortex. Pup behavioral alterations, including trembling, difficulty in righting, and fearfulness were reversed by the serotonin synthesis inhibitor parachlorophenylalanine. Adults manifested a distinct behavioral syndrome, including enhanced aggression in males.
Assuntos
Agressão/fisiologia , Encéfalo/metabolismo , Monoaminoxidase/deficiência , Norepinefrina/metabolismo , Serotonina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Modelos Animais de Doenças , Dopamina/metabolismo , Feminino , Interferon beta/genética , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Transgênicos , Dados de Sequência Molecular , Deleção de SequênciaRESUMO
Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.
Assuntos
Mapeamento Cromossômico , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Glucoquinase/genética , Obesidade/complicações , Transgenes/fisiologia , Animais , Glicemia/análise , Gorduras na Dieta/administração & dosagem , Glucagon/sangue , Glucoquinase/metabolismo , Insulina/sangue , Fígado/enzimologia , Camundongos , Camundongos Transgênicos/genética , Obesidade/sangue , Obesidade/etiologia , RNA Mensageiro/metabolismo , Valores de ReferênciaRESUMO
PURPOSE: To survey patients with dominant retinitis pigmentosa (RP) for mutations in the RP1 gene to determine the spectrum of dominant mutations in this gene, to estimate the proportion of dominant RP caused by this gene, and to determine whether the clinical features of patients with RP1 mutations differ from features of those with rhodopsin mutations. METHODS: A set of 241 patients who did not have mutations in the rhodopsin gene (based on previous work) formed the basis for the study. Of these patients, 117 had also been previously evaluated and were found not to carry mutations in the RDS gene. The single-strand conformation polymorphism (SSCP) method was used to search for sequence variants, which were then directly sequenced. The relatives of selected patients were recruited for segregation analyses. Clinical evaluations of patients included a measurement of Snellen visual acuity, final dark adaptation thresholds, visual fields, and ERGs. Clinical data were compared with those obtained earlier from a study of 128 patients with dominant rhodopsin mutations. RESULTS: Of the 241 patients, all were screened for the most common RP1 mutation (Arg677Ter), and 10 patients were found to have this mutation. In addition, an evaluation of a subset of 189 patients in whom the entire coding sequence was evaluated revealed the following mutations: Gln679Ter (1 case), Gly723Ter (2 cases), Glu729(1-bp del) (1 case), Leu762(5-bp del) (2 cases), and Asn763(4-bp del) (1 case). All of these mutations cosegregated with RP in the families of the index patients. Nine missense mutations that were each found in six or fewer patients were encountered. The segregation of eight of these was evaluated in the respective patients' families, and only one segregated with dominant RP. This cosegregating missense change was in cis with the nonsense mutation Gln679Ter. Although patients with RP1 mutations had, on average, slightly better visual acuity than patients with rhodopsin mutations, there was no statistically significant difference in final dark-adaptation thresholds, visual field diameters, or cone electroretinogram (ERG) amplitudes. Comparably aged patients with RP1 mutations had visual function that varied by approximately two orders of magnitude, based on visual fields and ERG amplitudes. CONCLUSIONS: Dominant RP1 alleles typically have premature nonsense codons occurring in the last exon of the gene and would be expected to encode mutant proteins that are only approximately one third the size of the wild-type protein, suggesting that a dominant negative effect rather than haploinsufficiency is the mechanism leading to RP caused by RP1 mutations. On average, patients with RP1 mutations have slightly better visual acuity than patients with dominant rhodopsin mutations; otherwise, they have similarly severe disease. The wide range in severity among patients with RP1 mutations indicates that other genetic or environmental factors modulate the effect of the primary mutation.
Assuntos
Proteínas do Olho/genética , Mutação da Fase de Leitura , Mutação de Sentido Incorreto , Retinose Pigmentar/genética , Adolescente , Adulto , Idoso , Adaptação à Escuridão , Eletrorretinografia , Feminino , Genes Dominantes , Humanos , Masculino , Proteínas Associadas aos Microtúbulos , Pessoa de Meia-Idade , Linhagem , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Retinose Pigmentar/fisiopatologia , Rodopsina/genética , Análise de Sequência de DNA , Acuidade Visual , Campos VisuaisRESUMO
Serotonin (5-hydroxytryptamine; 5-HT) mediates many central and peripheral nervous system functions by its interaction with specific neuronal receptors. Here we report the genomic structure of the human 5-HT2 receptor. The SacI-EcoRI restriction fragment of rat 5-HT2 receptor cDNA was used as a probe to identify and isolate two positive clones of 8.5 and 7.0 kb from an EcoRI restriction digest of a chromosome 13 specific EcoRI fragment lambda-phage human genomic library. Subcloning and sequencing of these fragments showed the 8.5 kb fragment (designated lambda SE-5) contained the first two exons of the 5-HT2 receptor gene. The 7.0 kb insert (lambda SE-2) contained an incomplete third exon. A HindIII-EcoRI fragment of this insert was used as a probe to isolate a 9.0 kb clone (lambda SH-2), which contained the entire third exon, from a chromosome 13 specific HindIII-fragment lambda-phage human genomic library. The isolation of these three clones (lambda SE-5, lambda SE-2 and lambda SH-2) shows that the human 5-HT2 receptor gene consists of three exons separated by two introns and spans over 20 kb. The deduced amino acid sequence of the human, mouse and rat 5-HT2 receptors are highly conserved and all three share a 90% sequence similarity.
Assuntos
Genes , Receptores de Serotonina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Éxons , Proteínas de Ligação ao GTP/fisiologia , Humanos , Íntrons , Camundongos/genética , Dados de Sequência Molecular , Ratos/genética , Receptores de Serotonina/classificação , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Especificidade da EspécieRESUMO
Northern analysis using 32P-labeled subfragments of human liver MAO-A and B cDNA clones detected 5Kb and 3Kb transcripts, respectively in fetal tissues and adult brains. The tissue distribution of these transcripts was determined. Small intestine and placenta express, in addition to the MAO-A 5Kb transcript, a 2Kb transcript determined to lack a 3' flanking region. MAO-A appeared prior to MAO-B in the fetal brain, whereas both MAO-A and B were found in adult brain.
Assuntos
Monoaminoxidase/genética , Northern Blotting , Encéfalo/enzimologia , Encéfalo/ultraestrutura , Sondas de DNA , Feminino , Feto/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Intestino Delgado/enzimologia , Placenta/enzimologia , Gravidez , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Transcrição Gênica , Cromossomo X/enzimologiaRESUMO
The promoter of human monoamine oxidase (MAO) A and B genes have been identified. The core promoter region of MAO A is comprised of two 90 bp repeats each of which contains two Sp1 elements and lacks a TATA box. The MAO B core promoter region contains two sets of overlapping Sp1 sites which flank a CACCC element all upstream of a TATA box. The different organization of the MAO A and B promoters may underlie their different cell and tissue specific expression.
Assuntos
Monoaminoxidase/genética , Regiões Promotoras Genéticas , Sequência de Bases , Deleção de Genes , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico , TATA BoxRESUMO
Monoamine oxidase (MAO) A and B are flavoenzymes that catalyze the oxidative deamination of biogenic and xenobiotic amines. To search for domains that confer substrate and inhibitor selectivities, two chimeric proteins were constructed and expressed in yeast. The kinetic constants and IC50 values were determined for these chimeric enzymes using MAO-A/B selective substrates and inhibitors. Replacement of MAO-A amino acids 161-375 with the corresponding region of MAO-B, termed AB(161-375)A, converted MAO-A catalytic properties to MAO-B like ones. The specificity constants (k(cat)/K(m))for the oxidation of beta-phenylethylamine (PEA) (1.6 x 10(5) s-1 M-1) and benzylamine (2.4 x 10(4) s-1 M-1) by AB (161-375)A were similar to wild-type MAO-B (PEA, 8 x 10(5)s(-1) M(-1); benzylamine, 4.9 x 10(4) s(-1) M(-1). Serotonin (5-HT), a preferred substrate for MAO-A, was not oxidized by AB(161-375)A or wild-type MAO-B. Furthermore, (AB161-375)A was more sensitive to the MAO-B specific inhibitor deprenyl (IC50 2.7 +/- 0.4 x 10(-8) M) than to the MAO-A specific inhibitor clorgyline (IC50 5.4 +/- 0.8 x 10(-7) M). However, the reciprocal chimera in which a MAO-B segment was replaced with the corresponding region of MAO-A, termed ++(+BA152-366B), lacked catalytic activity. The lack of catalytic activity was not due to aberrant expression but rather an inactive protein as demonstrated by Western blot analysis. These results demonstrate that MAO-B amino acids 152-366 contain a domain(s) that confers substrate and inhibitor selectivity.
Assuntos
Inibidores da Monoaminoxidase/farmacologia , Monoaminoxidase/metabolismo , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Clorgilina/farmacologia , Expressão Gênica , Humanos , Isoenzimas/metabolismo , Cinética , Fígado/enzimologia , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Proteínas Recombinantes de Fusão/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae , Selegilina/farmacologiaRESUMO
The search for innovative and clinically-differentiated medicines for the treatment of type 2 diabetes is an active area of research for pharmaceutical companies. The discovery of allosteric Glucokinase (GK) activators in 2003 represents the first time a pharmaceutical agent was used to directly augment the actions of an enzyme by increasing its maximal velocity and substrate affinity. This discovery, coupled with translational medicine which has shown that inactivating and activating GK mutations cause glycemic diseases, has triggered an intensive medicinal chemistry effort in the field of glucokinase activators (GKAs). The antidiabetic effects of GK activators observed in animal models support the notion that these agents act to both augment insulin release from pancreatic beta-cells and suppress hepatic glucose production in the liver. This review describes the unprecedented task of optimizing small molecules in order to affect the appropriate changes in the kinetic parameters of an enzyme. In addition, a pharmacophore model for the various classes of glucokinase activators that have been described in the literature will be presented. Overall, the available data suggests that potent glucokinase activators with the desired effects on the kinetic properties of the enzyme can be designed to achieve strong and persistent antidiabetic effects. GK activators thus represent a promising new treatment for type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucoquinase/metabolismo , Hipoglicemiantes/uso terapêutico , Animais , Diabetes Mellitus Tipo 2/enzimologia , Ativação Enzimática , Humanos , Células Secretoras de Insulina/metabolismo , Cinética , Modelos Moleculares , Conformação ProteicaRESUMO
The enzyme GK (glucokinase), which phosphorylates glucose to form glucose 6-phosphate, serves as the glucose sensor of insulin-producing beta-cells. GK has thermodynamic, kinetic, regulatory and molecular genetic characteristics that are ideal for its glucose sensor function and allow it to control glycolytic flux of the beta-cells as indicated by control-, elasticity- and response-coefficients close to or larger than 1.0. GK operates in tandem with the K(+) and Ca(2+) channels of the beta-cell membrane, resulting in a threshold for glucose-stimulated insulin release of approx. 5 mM, which is the set point of glucose homoeostasis for most laboratory animals and humans. Point mutations of GK cause 'glucokinase disease' in humans, which includes hypo- and hyper-glycaemia syndromes resulting from activating or inactivating mutations respectively. GK is allosterically activated by pharmacological agents (called GK activators), which lower blood glucose in normal animals and animal models of T2DM. On the basis of crystallographic studies that identified a ligand-free 'super-open' and a liganded closed structure of GK, on thermostability studies using glucose or mannoheptulose as ligands and studies showing that mannoheptulose alone or combined with GK activators induces expression of GK in pancreatic islets and partially preserves insulin secretory competency, a new hypothesis was developed that GK may function as a metabolic switch per se without involvement of enhanced glucose metabolism. Current research has the goal to find molecular targets of this putative 'GK-switch'. The case of GK research illustrates how basic science may culminate in therapeutic advances of human medicine.
Assuntos
Glucoquinase/metabolismo , Glucose/metabolismo , Homeostase , Cristalografia por Raios X , Glucoquinase/antagonistas & inibidores , Glucoquinase/química , Glucoquinase/genética , Humanos , Hipoglicemiantes/farmacologia , Cinética , Mutação Puntual , Conformação ProteicaRESUMO
Monoamine oxidases A/B (EC 1.4.3.4, MAO), flavoenzymes located on the outer mitochondrial membrane, catalyze the oxidative deamination of biogenic amines, such as dopamine, serotonin, and norepinephrine. In this study, we examined whether the H2O2 formed during the two-electron oxidation of tyramine [4-(2-aminoethyl)phenol] (a substrate for monoamine oxidases A/B) may contribute to the intramitochondrial steady-state concentration of H2O2 ([H2O2]ss) and, thus, be involved in the oxidative impairment of mitochondrial matrix components. Supplementation of intact, coupled rat brain mitochondria with benzylamine, beta-phenylethylamine, or tyramine showed initial rates of H2O2 production ranging from 0.4- to 1.6 nmol H2O2/min/mg protein. ESR analysis of the oxidative deamination of tyramine by intact rat brain mitochondria revealed the formation of hydroxyl (HO.) and carbon-centered radical adducts--the latter probably originating by the (HO.-)-mediated oxidation of mannitol. The signals were substantially enhanced upon addition of FeSO4 and were abolished by catalase. The intramitochondrial [H2O2]ss calculated in terms of glutathione peroxidase activity during the metabolism of tyramine was 48-fold higher (7.71 +/- 0.25 x 10(-7) M) than that obtained during the oxidation of succinate via complex II in the presence of antimycin A (1.64 +/- 0.2 x 10(-8) M). Oxidative damage to the brain mtDNA was assessed by single strand breakage. The ratio of nicked DNA for the preparations treated with tyramine and those without the amine was 1.5 +/- 0.29 (n = 4), 2.12 +/- 0.28 (n = 8, P < or = 0.05), and 3.12 +/- 0.69 (n = 3, P < or = 0.05) at 15, 30, and 60 min, respectively . Preincubation of mitochondria with tranylcypromine (trans-2-phenylcyclopropylamine), an inhibitor to MAO A/B, abolished mtDNA oxidative damage. Catalase inhibited mtDNA strand breakage by approximately 60%. Incubation of intact, coupled rat brain mitochondria with chlorodinitrobenzene (CDNB) depleted mitochondrial GSH by 72%. Tyramine-dependent damage of mtDNA was decreased by 68% in CDNB-treated mitochondria (with 28% remaining GSH). The [H2O2]ss was slightly increased in CDNB-treated mitochondria: 1.38- and 1.28-fold increase during the oxidation of succinate in the presence of antimycin A and during the oxidation of tyramine, respectively. These results suggest that the H2O2 generated during the MAO-catalyzed oxidation of biogenic amines and possibly certain neurotransmitters at the outer mitochondrial membrane contributes to the intramitochondrial [H2O2]ss and may cause oxidative damage to mtDNA. This is effected by the intramitochondrial concentration of GSH and might have potential implications for aging and neurodegenerative processes.
Assuntos
DNA Mitocondrial/metabolismo , Mitocôndrias/metabolismo , Monoaminoxidase/metabolismo , Tiramina/metabolismo , Animais , Encéfalo , Compartimento Celular , DNA de Cadeia Simples/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Peróxido de Hidrogênio/metabolismo , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
Monoamine oxidase (MAO) A and B play important roles in the metabolism of biogenic amines. Northern analysis using 32P-labeled subfragments of human liver MAO A and B cDNA clones detected a 5- and a 3-kb transcript, respectively, in most human tissues examined. However, fetal heart and thymus express minute amounts of MAO A transcript, whereas fetal brain, muscle, thymus, spleen, meninges, and placenta express minute amounts of MAO B transcript. Small intestine and placenta express, in addition to the MAO A 5-kb transcript, a 2-kb transcript, which may arise from an alternative polyadenylation site. MAO A and B transcripts are expressed in similar regions of adult human brain. The highest concentrations of these transcripts were located in frontal cortex and locus coeruleus. This study demonstrates the tissue-specific distribution of the MAO genes and will provide insight into the physiological functions of MAO A and B.
Assuntos
Encéfalo/enzimologia , Isoenzimas/genética , Monoaminoxidase/genética , Placenta/enzimologia , RNA Mensageiro/análise , Feminino , Feto , Humanos , Especificidade de Órgãos , Gravidez , RNA Mensageiro/genéticaRESUMO
A trout liver monoamine oxidase (MAO) cDNA was cloned by screening a cDNA library with a human MAO-A cDNA probe. The trout MAO cDNA encodes 499 amino acids, with a molecular mass of 56.6 kDa. The deduced amino acid sequence of trout MAO shows 70% and 71% identity with those of human MAO-A and MAO-B, respectively. Trout MAO contains the pentapeptide sequence Ser-Gly-Gly-Cys-Tyr, to which the cofactor FAD is covalently bound. Transient expression of the cDNA in COS-7 cells shows that trout MAO oxidizes both serotonin [5-hydroxytryptamine (5-HT)] and beta-phenylethylamine (PEA), unlike human MAO-A and MAO-B, which oxidize only 5-HT and PEA, respectively. The Km for 5-HT is similar for trout MAO (130 +/- 17 mM) and human MAO-A (68 +/- 4 mM). The Km for PEA is similar for trout MAO (12.5 +/- 2.0 mM) and human MAO-B (1.5 +/- 0.2 mM). When 5-HT is used as a substrate, trout MAO is more sensitive to clorgyline (IC50, 2.8 +/- 0.2 x 10(-8) M) than deprenyl (IC50, 1.0 +/- 0.1 x 10(-6) M), a result similar to the inhibition selectivity of human MAO-A. However, trout MAO is less sensitive to clorgyline than is human MAO-A (IC50, 5.8 +/- 0.1 x 10(-10) M). Trout MAO is less sensitive to deprenyl (IC50, 4.6 +/- 0.3 x 10(-7) M) than is human MAO-B (IC50, 1.4 +/- 0.1 x 10(-9) M) when PEA is used as the substrate. These results indicate that trout MAO displays substrate and inhibitor selectivities that are not identical to those of either MAO-A and -B, and it therefore represents a novel type of MAO. The structure of trout MAO will provide insights into the substrate and inhibitor selectivities of the MAOs.
Assuntos
Fígado/enzimologia , Monoaminoxidase/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Inibidores da Monoaminoxidase/farmacologia , Homologia de Sequência de Aminoácidos , TrutaRESUMO
Monoamine oxidase A and B (MAO A and B) play important roles in the metabolism of biogenic and dietary amines and are encoded by two genes derived from a common ancestral gene. The promoter regions for human MAO A and B genes have been characterized using a series of 5' flanking sequences linked to a human growth hormone reporter gene. When these constructs were transfected into NIH3T3, SHSY-5Y, and COS7 cells, the maximal promoter activity for MAO A was found in a 0.14 kilobase (kb) PvuII/DraII fragment (A0.14) and in a 0.15 kb PstI/NaeI fragment (B0.15) for MAO B. Both fragments are GC-rich, contain potential Sp1 binding sites, and are in the region where the MAO A and B 5' flanking sequences share the highest identity (approximately 60%). However, the organization of the transcription elements is distinctly different between these two promoters. Fragment A0.14 consists of three Sp1 elements, all in reversed orientations, and lacks a TATA box. Two of the Sp1 sites are located within the downstream 90 base pair (bp) direct repeat, and the third is located at the 3' end of the upstream 90 bp direct repeat. Fragment B0.15 contains an Sp1-CACCC-Sp1-TATA structure; deletion of any of these elements reduced promoter activity. Additional Sp1 sites, CACCC elements, CCAAT boxes, and direct repeats (four 30 bp direct repeats in MAO A and two 29 bp direct repeats in MAO B) are found in farther-upstream sequences of both genes (1.27 kb for MAO A and mostly in 0.2 kb for MAO B). Inclusion of these sequences decreased promoter activity. The different promoter organization of MAO A and B genes provides the basis for their different tissue- and cell-specific expression.
Assuntos
Genes , Monoaminoxidase/genética , Regiões Promotoras Genéticas , Células 3T3/fisiologia , Animais , Sequência de Bases , DNA/genética , Humanos , Camundongos , Dados de Sequência Molecular , Monoaminoxidase/química , Sequências Repetitivas de Ácido Nucleico , Estereoisomerismo , TATA BoxRESUMO
Monoamine oxidases A and B [MAOA and MAOB; amine:oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4] play important roles in the metabolism of neuroactive, vasoactive amines and the Parkinsonism-producing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Human MAOA and MAOB genes isolated from X chromosome-specific libraries span at least 60 kilobases, consist of 15 exons, and exhibit identical exon-intron organization. Exon 12 codes for the covalent FAD-binding-site and is the most conserved exon; the MAOA and MAOB exon 12 products share 93.9% peptide identity. These results suggest that MAOA and MAOB are derived from duplication of a common ancestral gene and provide insight on the structural/functional relationship of the enzyme products.