RESUMO
Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all.
RESUMO
BACKGROUND: Lesch-Nyhan disease (LND; OMIM 300322), caused by virtually absent hypoxanthine-guanine phosphoribosyltransferase activity, in its classic form is characterised by hyperuricemia, variable cognitive impairment, severe motor disorder and a characteristic behavioural disorder (Lesch-Nyhan Behavior, LNB), typically described as self-injurious behavior (SIB) and "self-mutilation." This work focuses on the latter aspect with the aim of exploring and broadening it. METHODS: The participant observation method was used to follow three children diagnosed with LND individually, in different contexts of daily life, always with their usual restraints and in the presence of a caregiver. RESULTS: 60 observational sessions, for over 90 total hours, led to the description of 292 LNBs, interfering with different aspects of life. Harmful behaviors could be classified into different categories, based on the life aspect affected and type of harm provoked. Antecedent conditions, consequent reactions, and emotions of the child and different management of the caregiver were recorded for each LNB. We confirmed that patients normally feel pain. Most common emotional reactions are regret and shock. As a consequence of a LNB, increased anxiety was always recorded, never satisfaction. Caregiver strategies most commonly used to stop the LNB and preventing recurrences are reported and discussed. CONCLUSIONS: We are proposing a wider LNB description, beyond the classical Self-injurious behavior (SIB), stating that it is widespread and pervasive, involving every facet of the patients' life. Caregivers and operators should be aware that they might face different LNBs, and have to recognize them to find the better way to manage patients.
RESUMO
Lesch-Nyhan disease (LND) is a rare X-linked genetic disorder, with complete hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficiency, uric acid (UA), hypoxanthine and xanthine accumulation, and a devastating neurologic syndrome. UA excess, causing renal failure, is commonly decreased by xanthine oxidoreductase (XOR) inhibitors, such as allopurinol, yielding a xanthine and hypoxanthine increase. Xanthine accumulation may result in renal stones, while hypoxanthine excess seems involved in the neurological disorder. Inhibition of purine nucleoside phosphorylase (PNP) represents a different strategy for lowering urate. PNP catalyzes the cleavage of purine ribo- and d-ribo-nucleosides into ribose/deoxyribose phosphate and free bases, starting catabolism to uric acid. Clinical trials demonstrated that PNP inhibitors, initially developed as anticancer drugs, lowered UA in some gouty patients, in association or not with allopurinol. The present study tested the reliability of an analogue of immucillin-G (C1a), a PNP inhibitor, as a therapy for urate, hypoxanthine, and xanthine excess in LND patients by blocking hypoxanthine production upstream. The therapeutic aim is to limit the administration of XOR inhibitors to LND patients by supplying the PNP inhibitor in low doses, avoiding d-nucleoside toxicity. We report studies conducted in primary cultures of skin fibroblasts from controls and LND patients grown in the presence of the PNP inhibitor. Cell viability, oxypurine release in culture medium, and endocellular nucleotide pattern have been monitored in different growth conditions (inhibitor concentration, time, added inosine). Our results demonstrate effective PNP inhibition by low inhibitor concentration, with reduced hypoxanthine release, and no appreciable toxicity in control or patient cells, suggesting a new therapeutic strategy for LND hyperuricemia.
Assuntos
Inibidores Enzimáticos/farmacologia , Hiperuricemia/tratamento farmacológico , Síndrome de Lesch-Nyhan/tratamento farmacológico , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Pirimidinonas/farmacologia , Pirróis/farmacologia , Alopurinol/farmacologia , Células Cultivadas , Humanos , Hiperuricemia/metabolismo , Hipoxantina/farmacologia , Hipoxantina Fosforribosiltransferase/metabolismo , Síndrome de Lesch-Nyhan/metabolismo , Purinas/metabolismo , Reprodutibilidade dos Testes , Ácido Úrico/metabolismo , Xantina/farmacologiaRESUMO
OBJECTIVES: Two methods are described for homogentisic acid (HGA) determination in dried urine spots (DUS) on paper from Alkaptonuria (AKU) patients, devised for quick early diagnosis. AKU is a rare autosomal recessive disorder caused by deficiency of homogentisate 1,2-dioxygenase, yielding in accumulation of HGA. Its massive excretion causes urine darkening by exposure to air or alkalinization, and is a diagnostic marker. The deposition of polymers produced after HGA oxidation within the connective tissues causes ochronotic arthritis, a degenerative joint disease manifesting in adulthood and only rarely in childhood. No early diagnosis is usually accomplished, awareness following symptom development. DESIGN AND METHODS: Two methods were designed for HGA determination in DUS: (1) a rapid semi-quantitative reliable method based on colour development in alkali and quantification by comparison with dried paper spots from HGA solutions of known concentration and (2) a quantitative and sensitive HPLC-linked method, previously devised for purine and pyrimidine analysis in urine and plasma. RESULTS: Colour intensity developed by DUS after alkali addition was proportional to HGA concentration, and calculated amounts were in good agreement with quantitative analysis performed by RP-HPLC on DUS and on urines as such. CONCLUSIONS: DUS, often used for different diagnostic purpose, are easily prepared and safely delivered. The simple and quick colour method proposed provides reliable HGA assessment and is fit for large screening. HGA concentration determined in 10 AKU patient DUS by both methods 1 and 2 was in agreement with direct urine assay and in the range reported by literature.A reliable HGA quantification based on colour development in paper urine spots is validated by HPLC-linked HGA quantification, and proposed as a quick diagnostic tool for AKU patients.
Assuntos
Gastroenteropatias/genética , Encefalomiopatias Mitocondriais/genética , Timidina Fosforilase/genética , Timidina Fosforilase/fisiologia , Acidentes de Trânsito , Sequência de Aminoácidos , Encéfalo/patologia , DNA/genética , Eritrócitos/enzimologia , Feminino , Gastroenteropatias/enzimologia , Gastroenteropatias/patologia , Humanos , Imageamento por Ressonância Magnética , Encefalomiopatias Mitocondriais/enzimologia , Encefalomiopatias Mitocondriais/patologia , Dados de Sequência Molecular , Mutação , Parestesia/etiologia , Elongação Traducional da Cadeia Peptídica/genética , Elongação Traducional da Cadeia Peptídica/fisiologia , Adulto JovemRESUMO
We report a 45-day-old male infant who presented with anuric renal failure and fluid overload due to urate nephropathy consequent upon hyperuricemia with hyperuricosuria. His maternal uncle had undergone renal transplantation for chronic renal failure secondary to uric acid nephrolithiasis. The levels of hypoxanthine phosphoribosyl transferase (HPRT) and adenine phosphoribosyl transferase activity in the baby were found to be quantitatively normal. However, when the HPRT activity was measured at low substrate concentrations [phosphoribosyl pyrophosphate (PRPP) and hypoxanthine] and compared with usual assay conditions, the HPRT activity at lower PRPP was less in the propositus, suggesting altered enzyme kinetics. Apparent K (m(PRPP)) and V (max), but not K (m(hypoxanthine)), were then found to be higher in the propositus than the control range. This is the first case of urate nephropathy secondary to altered enzyme kinetics presenting as early as 45 days. Uric acid nephropathy should be considered in the differential diagnosis of unexplained acute kidney injury in infants. In such cases, quantitative tests for HPRT enzyme activity may not be sufficient and altered enzyme kinetics should also be investigated.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Hiperuricemia/complicações , Hipoxantina Fosforribosiltransferase/metabolismo , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Ácido Úrico/toxicidade , Ácido Úrico/urina , Evolução Fatal , Humanos , Lactente , Cinética , MasculinoRESUMO
Purines and pyrimidines, regarded for a long time merely as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, have attracted increasing attention after genetically determined aberrations in their metabolism were linked to a range of symptoms from hyperuricemia and immunodeficiency to neurological disorders. The pathogenesis of such disorders involves cell or mitochondrial damage, but the molecular mechanisms underlying symptoms is often unclear. H. Anne Simmonds made major contributions to the metabolic, clinical, and molecular aspects of these disorders and the Purine Research Laboratory, which she established in London, became the world center for clinical and experimental studies in the field. We owe her gratitude not only for this direct contribution but also for her enthusiasm for purine and pyrimidine research that she transmitted to generations of scientists. Our research in this field stemmed from expertise in pyridine metabolism and its connection with purines, and from clinical involvement with biochemical diagnosis of enzyme deficiencies. We joined H. Anne Simmonds in studying the biochemical basis of altered NAD content in erythrocytes of PNP- and HPRT-deficient patients, discovering some alterations in NAD synthesis and breakdown.
Assuntos
Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Comportamento Cooperativo , Eritrócitos/enzimologia , História do Século XX , Humanos , Redes e Vias Metabólicas , Modelos Biológicos , Purinas/sangue , Pirimidinas/sangueRESUMO
Xanthinuria is a rare autosomal recessive disorder associated with a deficiency of xanthine oxidoreductase (XOR), which normally catalyzes the conversion of hypoxanthine to uric acid. The effects of this deficit are an elevated concentration of hypoxanthine and xanthine in the blood and urine, hypouricemia, and hypouricuria. The deficit in XOR can be isolated (type I xanthinuria) or associated with a deficit in aldehyde oxidase (type II xanthinuria) and sulfite oxidase (type III xanthinuria). While the first two variants have a benign course, are often asymptomatic (20%), and clinically indistinguishable, type III xanthinuria is a harmful form that leads to infant death due to neurological damage. The clinical symptoms (kidney stones, CKD, muscle and joint pain, peptic ulcer) are the result of the accumulation of xanthine, which is highly insoluble, in the body fluids. We describe a case of type I xanthinuria in a 52-year-old woman who presented with hypouricemia, hypouricuria and kidney stones. The diagnosis was based on purine catabolite levels in urine and serum measured by 3 nonroutine methods: high-pressure liquid chromatography, mass spectrometry, and magnetic resonance imaging. To identify the type of xanthinuria the allopurinol test was used. We believe that these tests will facilitate the diagnosis of xantinuria especially in asymptomatic patients without the need for a biopsy of the liver or intestines, which is useful only for scientific purposes.
Assuntos
Hipoxantinas , Erros Inatos do Metabolismo da Purina-Pirimidina/complicações , Erros Inatos do Metabolismo da Purina-Pirimidina/diagnóstico , Ácido Úrico , Xantina/urina , Alopurinol/urina , Biomarcadores/sangue , Biomarcadores/urina , Cromatografia Líquida de Alta Pressão , Feminino , Humanos , Hipertensão/etiologia , Hipoxantinas/sangue , Hipoxantinas/urina , Imageamento por Ressonância Magnética , Espectrometria de Massas , Pessoa de Meia-Idade , Nefrolitíase/etiologia , Valor Preditivo dos Testes , Erros Inatos do Metabolismo da Purina-Pirimidina/sangue , Erros Inatos do Metabolismo da Purina-Pirimidina/dietoterapia , Erros Inatos do Metabolismo da Purina-Pirimidina/urina , Sensibilidade e Especificidade , Resultado do Tratamento , Ácido Úrico/sangue , Ácido Úrico/urina , Xantina/sangueRESUMO
Brain relies on circulating nucleosides, mainly synthesised de novo in the liver, for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, and pyrimidine sugar- and lipid-conjugates. Essentially, the paths of nucleoside salvage in the brain include a two step conversion of inosine and guanosine to IMP and GMP, respectively, and a one step conversion of adenosine, uridine, and cytidine, to AMP, UMP, and CMP, respectively. With the exception of IMP, the other four nucleoside monophosphates are converted to their respective triphosphates via two successive phosphorylation steps. Brain ribonucleotide reductase converts nucleoside diphosphates to their deoxy counterparts. The delicate qualitative and quantitative balance of intracellular brain nucleoside triphosphates is maintained by the relative concentrations of circulating nucleosides, the specificity and the K(m) values of the transport systems and of cytosolic and mitochondrial nucleoside kinases and 5'-nucleotidases, and the relative rates of nucleoside triphosphate extracellular release. A cross talk between extra- and intra-cellular nucleoside metabolism exists, in which released nucleoside triphosphates, utilised as neuroactive signals, are catabolised by a membrane bound ectonucleotidase cascade system to their respective nucleosides, which are uptaken into brain cytosol, and converted back to nucleoside triphosphates by the salvage enzymes. Finally, phosphorolysis of brain nucleosides generates pentose phosphates, which are utilised for nucleoside interconversion, 5-phosphoribosyl-1-pyrophosphate synthesis, and energy repletion. This review focuses on these aspects of brain nucleoside metabolism, with the aim of giving a comprehensive picture of the metabolic network of nucleosides in normoxic conditions, with some hints on the derangements in anoxic/ischemic conditions.
Assuntos
Encéfalo/fisiologia , Nucleosídeos de Purina/metabolismo , Nucleosídeos de Pirimidina/metabolismo , Animais , Homeostase/fisiologia , Humanos , Cinética , Redes e Vias Metabólicas , Camundongos , Proteínas de Transporte de Nucleosídeos/fisiologia , Via de Pentose Fosfato/fisiologia , Fosforribosil Pirofosfato/metabolismo , Fosfotransferases/metabolismo , Coelhos , Ratos , Ribonucleotídeo Redutases/metabolismo , Transdução de SinaisRESUMO
Purines and pyrimidines, regarded for a long time only as building blocks for nucleic acid synthesis and intermediates in the transfer of metabolic energy, gained increasing attention since genetically determined aberrations in their metabolism were associated clinically with various degrees of mental retardation and/or unexpected and often devastating neurological dysfunction. In most instances the molecular mechanisms underlying neurological symptoms remain undefined. This suggests that nucleotides and nucleosides play fundamental but still unknown roles in the development and function of several organs, in particular central nervous system. Alterations of purine and pyrimidine metabolism affecting brain function are spread along both synthesis (PRPS, ADSL, ATIC, HPRT, UMPS, dGK, TK), and breakdown pathways (5NT, ADA, PNP, GCH, DPD, DHPA, TP, UP), sometimes also involving pyridine metabolism. Explanations for the pathogenesis of disorders may include both cellular and mitochondrial damage: e.g. deficiency of the purine salvage enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase are associated to the most severe pathologies, the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to impairment of mitochondrial functions. This review gathers the presently known inborn errors of purine and pyrimidine metabolism that manifest neurological syndromes, reporting and commenting on the available hypothesis on the possible link between specific enzymatic alterations and brain damage. Such connection is often not obvious, and though investigated for many years, the molecular basis of most dysfunctions of central nervous system associated to purine and pyrimidine metabolism disorders are still unexplained.
Assuntos
Doenças do Sistema Nervoso/metabolismo , Neurônios/metabolismo , Purinas/metabolismo , Pirimidinas/metabolismo , Adenosina Desaminase/deficiência , Adenosina Desaminase/metabolismo , Adenilossuccinato Liase/deficiência , Adenilossuccinato Liase/metabolismo , Agamaglobulinemia/metabolismo , Animais , Transtorno Autístico , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Feminino , Humanos , Hipoxantina Fosforribosiltransferase/deficiência , Masculino , Camundongos , Doenças do Sistema Nervoso/fisiopatologia , Neurônios/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Purina-Núcleosídeo Fosforilase/deficiência , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Ratos , Ribose-Fosfato Pirofosfoquinase/deficiência , Ribose-Fosfato Pirofosfoquinase/metabolismo , Imunodeficiência Combinada Severa/metabolismoRESUMO
Adenine phosphoribosyltransferase (APRT) deficiency, a rare inborn error inherited as an autosomic recessive trait, presents with 2,8-dihydroxyadenine (2,8-DHA) crystal nephropathy. We describe clinical, biochemical and molecular findings in a renal transplant recipient with renal failure, 2,8-DHA stones and no measurable erythrocyte APRT activity. Homozygous C > G substitution at -3 in the splicing site of exon 2 (IVS2 -3 c > g) was found in the APRT gene. The patient's asymptomatic brother was heterozygous for such mutation, and his APRT activity was 23% of controls. A splicing alteration leading to incorrect gene transcription and virtually absent APRT activity is seemingly associated with the newly identified mutation.
RESUMO
This review is devised to gather the presently known inborn errors of purine metabolism that manifest neurological pediatric syndromes. The aim is to draw a comprehensive picture of these rare diseases, characterized by unexpected and often devastating neurological symptoms. Although investigated for many years, most purine metabolism disorders associated to psychomotor dysfunctions still hide the molecular link between the metabolic derangement and the neurological manifestations. This basically indicates that many of the actual functions of nucleosides and nucleotides in the development and function of several organs, in particular central nervous system, are still unknown. Both superactivity and deficiency of phosphoribosylpyrophosphate synthetase cause hereditary disorders characterized, in most cases, by neurological impairments. The deficiency of adenylosuccinate lyase and 5-amino-4-imidazolecarboxamide ribotide transformylase/IMP cyclohydrolase, both belonging to the de novo purine synthesis pathway, is also associated to severe neurological manifestations. Among catabolic enzymes, hyperactivity of ectosolic 5'-nucleotidase, as well as deficiency of purine nucleoside phosphorylase and adenosine deaminase also lead to syndromes affecting the central nervous system. The most severe pathologies are associated to the deficiency of the salvage pathway enzymes hypoxanthine-guanine phosphoribosyltransferase and deoxyguanosine kinase: the former due to an unexplained adverse effect exerted on the development and/or differentiation of dopaminergic neurons, the latter due to a clear impairment of mitochondrial functions. The assessment of hypo- or hyperuricemic conditions is suggestive of purine enzyme dysfunctions, but most disorders of purine metabolism may escape the clinical investigation because they are not associated to these metabolic derangements. This review may represent a starting point stimulating both scientists and physicians involved in the study of neurological dysfunctions caused by inborn errors of purine metabolism with the aim to find novel therapeutical approaches.
Assuntos
Encefalopatias Metabólicas Congênitas/metabolismo , Encefalopatias Metabólicas Congênitas/fisiopatologia , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Erros Inatos do Metabolismo da Purina-Pirimidina/fisiopatologia , Purinas/metabolismo , Fatores Etários , Encefalopatias Metabólicas Congênitas/patologia , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/fisiopatologia , Criança , Pré-Escolar , Enzimas/metabolismo , Humanos , Lactente , Recém-Nascido , Redes e Vias Metabólicas/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Nucleotídeos/metabolismo , Erros Inatos do Metabolismo da Purina-Pirimidina/patologiaRESUMO
Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype.
Assuntos
Encefalomiopatias Mitocondriais/genética , Encefalomiopatias Mitocondriais/fisiopatologia , Timidina Fosforilase/deficiência , Idade de Início , Encéfalo/patologia , Encéfalo/fisiopatologia , Células Cultivadas , Feminino , Fibroblastos/enzimologia , Humanos , Pessoa de Meia-Idade , Encefalomiopatias Mitocondriais/sangue , Mutação , Mutação de Sentido Incorreto , Nervos Periféricos/fisiopatologia , Fenótipo , Timidina/sangue , Timidina Fosforilase/genéticaRESUMO
Lesch-Nyhan disease (LND), a genetic disorder associated with motor and psychiatric disturbance and self-injurious behaviour (SIB) is caused by a complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT). The connection between enzyme deficiency and neurological involvement is still unclear. Evidence exists for a role of basal ganglia dysfunction with decreased dopamine and excess serotonin striatal content. In this study, we investigate the role of serotonin receptor 2C (HTR2C) in the brains of HPRT gene knock-out mice, a model of LND. HTR2C expression is analyzed by real-time polymerase chain reaction (PCR) using SYBR-green detection methods. The percentage of edited HTR2C mRNA was determined by direct sequencing of amplification products of the region containing the editing sites. We found a 55% increase in the expression of HTR2C gene but no significant difference in mRNA editing levels between knock-out and control mice. The above alteration found in HPRT-deficient mice is similar to those found in other animal models used to study aggressive and self-injurious behaviour.
Assuntos
Encéfalo/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Hipoxantina Fosforribosiltransferase/genética , Síndrome de Lesch-Nyhan/genética , Edição de RNA , Receptor 5-HT2C de Serotonina/genética , Receptor 5-HT2C de Serotonina/metabolismo , Animais , Camundongos , Camundongos Knockout , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
The aim of this study is to investigate the role of poly(ADP-ribose) polymerase (PARP), involved in DNA repair and in autoimmune pathologic conditions such as systemic lupus erythematosus (SLE) and both limited systemic sclerosis (lSSc) and diffuse systemic sclerosis (dSSc), to assess its possible implication in the pathogenetic processes. The relationship between PARP activity and the intracellular concentration of its substrate nicotinamide adenine dinucleotide (NAD) is also investigated. Peripheral mononuclear cells (PMC) from controls and patients with SLE, lSSc, and dSSc were irradiated with ultraviolet light (UV) and PARP activity was assayed by a radiochemical method. Pyridine nucleotide concentrations were assayed by a high-performance liquid chromatography-linked method. PARP activity was detectable in nonirradiated cells and showed similar values in all groups. The activity significantly increased after UV irradiation in control, SLE, and lSSc cells, but not in dSSc cells. Irradiated PMC from both SLE and dSSc showed lower enzyme activity with respect to irradiated controls. Higher intracellular NAD content was found in all of the pathologic conditions in comparison to values in the control; this difference was statistically significant in dSSc. Our data demonstrate a lower PARP activity in response to UV damage in PMC from patients affected by the above pathologic conditions compared with controls. An inverse relationship between PARP activity and NAD content was also observed.
Assuntos
Leucócitos Mononucleares/enzimologia , Lúpus Eritematoso Sistêmico/enzimologia , Poli(ADP-Ribose) Polimerases/metabolismo , Escleroderma Sistêmico/enzimologia , Adulto , Idoso , Feminino , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/efeitos da radiação , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/patologia , Masculino , Pessoa de Meia-Idade , NAD/sangue , NAD/metabolismo , Poli(ADP-Ribose) Polimerases/sangue , Esclerodermia Difusa/sangue , Esclerodermia Difusa/enzimologia , Escleroderma Sistêmico/sangue , Escleroderma Sistêmico/patologia , Raios Ultravioleta , Adulto JovemRESUMO
The activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) is virtually absent in Lesch-Nyhan disease (LND), an X-linked genetic disorder characterized by uric acid accumulation and neurodevelopmental dysfunction. The biochemical basis for the neurological and behavioral abnormalities have not yet been completely explained. Prior studies of cells from affected patients have shown abnormalities of NAD metabolism. In the current studies, NAD metabolism was evaluated in HPRT gene knock-out mice. NAD content and the activities of the enzymes required for synthesis and breakdown of this coenzyme were investigated in blood, brain and liver of HPRT(-) and control mice. NAD concentration and enzyme activities were found to be significantly increased in liver, but not in brain or blood of the HPRT(-) mice. These results demonstrate that changes in NAD metabolism occur in response to HPRT deficiency depending on both species and tissue type.
Assuntos
Hipoxantina Fosforribosiltransferase/metabolismo , Síndrome de Lesch-Nyhan/enzimologia , Fígado/metabolismo , NAD/metabolismo , Análise de Variância , Animais , Proteínas Sanguíneas/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Hipoxantina Fosforribosiltransferase/deficiência , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Purinas/metabolismo , Piridinas/metabolismoRESUMO
Nucleoside monophosphate phosphohydrolases comprise a family of enzymes dephosphorylating nucleotides both in intracellular and extracellular compartments. Members of this family exhibit different sequence, location, substrate specificity and regulation. Besides the ectosolic 5'-nucleotidase, several cytosolic and one mitochondrial enzymes have been described. Nevertheless, researchers refer any AMP-dephosphorylating activity to as 5'-nucleotidase, lacking a more accurate identification. Increase of AMP hydrolysing activity has been associated with neurological and developmental disorders. The identification of the specific enzyme involved in these pathologies would be fundamental for the comprehension of the linkage between the enzyme activity alteration and brain functions. We demonstrate that the described neurological symptoms are associated with increased ectosolic 5'-nucleotidase activity on the basis of radiochemical assays and immunoblotting analysis. Furthermore, present data evidence that the assay conditions normally applied for the determination of cytosolic 5'-nucleotidases activity in crude extracts are affected by the presence of solubilised ectosolic nucleotidase.
Assuntos
5'-Nucleotidase/metabolismo , Monofosfato de Adenosina/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Humanos , HidróliseRESUMO
BACKGROUND: Coffin-Lowry syndrome is a semi-dominant condition characterized by severe psychomotor retardation with facial, hand and skeletal malformations resulting from mutations in RSK2 gene, encoding for a serine/threonine kinase. More than 100 different mutations have been identified to date; however, about 50% of clinically diagnosed patients apparently do not have mutations. In order to exclude that these patients have RSK2 mutations missed by standard mutation detection techniques, a rapid and sensitive assay allowing evaluation of RSK2 activity was needed. METHODS: RSK2 capacity to phosphorylate a synthetic CREB-peptide in basal and PMA-stimulated conditions was evaluated in lymphoblasts from 3 patients with RSK2 mutations and normal controls. RESULTS: Patients RSK2 activity is normal in nonstimulated conditions but fails to grow following stimulation. The evaluation of the stimulated/non-stimulated activity ratio demonstrated a statistically significant impairment in patients. CONCLUSIONS: We have set up an assay which allows the identification of even partial alterations of RSK2 activity and seems to give good results also in females with a balanced X-chromosome inactivation and thus with a presumably normal enzymatic activity in about 50% of cells. Moreover, our data seem to confirm previous reports of a potential direct correlation between the level of RSK2 activity and the severity of cognitive impairment.
Assuntos
Síndrome de Coffin-Lowry/diagnóstico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Linfócitos/enzimologia , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Adulto , Bioensaio , Feminino , Humanos , Masculino , FosforilaçãoRESUMO
BACKGROUND: Lesch-Nyhan disease (LND), an X-linked genetic disease caused by complete deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), is characterized by hyperuricemia and psychiatric disturbance, mainly self-aggressiveness. Literature dates support the hypothesis that dopaminergic deficit and serotonergic excess in the circuit of basal ganglia are responsible for the aggressive behavior. Altered adenosine transport across the membrane of HPRT-deficient lymphocytes has been reported, suggesting adenosine involvement in LND. METHODS: The expression of several genes related to the adenosinergic system (ADORA1A, ADORA2A, ADORA2B) were studied in the brain of the murine model of LND by real time PCR. Nucleotide levels and enzyme activities possibly involved in adenosine release were also measured. RESULTS: Studies performed by real time PCR showed 95% increase in ADORA1A expression, 15% decrease in ADORA2A expression, and no change in ADORA2B expression in knockout mice compared to controls. No significant differences were found in the level of nucleotides or enzyme activities between control and mutant mice. CONCLUSIONS: Our results suggest that adenosine neurotransmission might be involved in the specific neurobehavioral features of LND by increased expression of adenosine A1 receptors.