RESUMO
BACKGROUND: Currently there are no disease-modifying treatments for Parkinson's disease dementia (PDD), a condition linked to aggregation of the protein α-synuclein in subcortical and cortical brain areas. One of the leading genetic risk factors for Parkinson's disease is being a carrier in the gene for ß-Glucocerebrosidase (GCase; gene name GBA1). Studies in cell culture and animal models have shown that raising the levels of GCase can decrease levels of α-synuclein. Ambroxol is a pharmacological chaperone for GCase and is able to raise the levels of GCase and could therefore be a disease-modifying treatment for PDD. The aims of this trial are to determine if Ambroxol is safe and well-tolerated by individuals with PDD and if Ambroxol affects cognitive, biochemical, and neuroimaging measures. METHODS: This is a phase II, single-centre, double-blind, randomized placebo-controlled trial involving 75 individuals with mild to moderate PDD. Participants will be randomized into Ambroxol high-dose (1050 mg/day), low-dose (525 mg/day), or placebo treatment arms. Assessments will be undertaken at baseline, 6-months, and 12-months follow up times. Primary outcome measures will be the Alzheimer's disease Assessment Scale-cognitive subscale (ADAS-Cog) and the ADCS Clinician's Global Impression of Change (CGIC). Secondary measures will include the Parkinson's disease Cognitive Rating Scale, Clinical Dementia Rating, Trail Making Test, Stroop Test, Unified Parkinson's disease Rating Scale, Purdue Pegboard, Timed Up and Go, and gait kinematics. Markers of neurodegeneration will include MRI and CSF measures. Pharmacokinetics and pharmacodynamics of Ambroxol will be examined through plasma levels during dose titration phase and evaluation of GCase activity in lymphocytes. DISCUSSION: If found effective and safe, Ambroxol will be one of the first disease-modifying treatments for PDD. TRIAL REGISTRATION: ClinicalTrials.gov NCT02914366, 26 Sep 2016/retrospectively registered.
Assuntos
Ambroxol/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Projetos de Pesquisa , Idoso , Encéfalo/efeitos dos fármacos , Demência/etiologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Parkinson/psicologiaRESUMO
BACKGROUND: Recent clinical whole exome sequencing (WES) cohorts have identified unanticipated multiple genetic diagnoses in single patients. However, the frequency of multiple genetic diagnoses in families is largely unknown. AIMS: We set out to identify the rate of multiple genetic diagnoses in probands and their families referred for analysis in two national research programs in Canada. MATERIALS & METHODS: We retrospectively analyzed WES results for 802 undiagnosed probands referred over the past 5 years in either the FORGE or Care4Rare Canada WES initiatives. RESULTS: Of the 802 probands, 226 (28.2%) were diagnosed based on mutations in known disease genes. Eight (3.5%) had two or more genetic diagnoses explaining their clinical phenotype, a rate in keeping with the large published studies (average 4.3%; 1.4 - 7.2%). Seven of the 8 probands had family members with one or more of the molecularly diagnosed diseases. Consanguinity and multisystem disease appeared to increase the likelihood of multiple genetic diagnoses in a family. CONCLUSION: Our findings highlight the importance of comprehensive clinical phenotyping of family members to ultimately provide accurate genetic counseling.
Assuntos
Sequenciamento do Exoma , Família , Estudos de Associação Genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Canadá/epidemiologia , Pré-Escolar , Consanguinidade , Feminino , Doenças Genéticas Inatas/epidemiologia , Testes Genéticos , Genótipo , Humanos , Masculino , Mutação , Linhagem , Fenótipo , Estudos Retrospectivos , Irmãos , Sequenciamento do Exoma/métodosRESUMO
Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, and pancytopenia or agammaglobulinemia. It can sometimes resemble neonatal leukemia or severe combined immunodeficiency disease. Diagnosis of TC deficiency is suspected based on megaloblastic anemia, elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or by molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Based on the data presented, current practice appears to favour treatment of individuals with TC deficiency by intramuscular injections of hydroxy- or cyanocobalamin. In most cases presented, at least weekly injections (1 mg IM) were necessary to ensure optimal treatment. Most centres adjusted the treatment regimen based on monitoring CBC, total plasma homocysteine, plasma and urine methylmalonic acid, as well as, clinical status. Finally, continuing IM treatment into adulthood appears to be beneficial.
Assuntos
Transcobalaminas/deficiência , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Hidroxocobalamina/uso terapêutico , Lactente , Recém-Nascido , Masculino , Mutação , Resultado do Tratamento , Vitamina B 12/uso terapêuticoRESUMO
BACKGROUND: the maternally inherited MTTL1 A3243G mutation in the mitochondrial genome causes MelaS (Mitochondrial encephalopathy lactic acidosis with Stroke-like episodes), a condition that is multisystemic but affects primarily the nervous system. Significant intra-familial variation in phenotype and severity of disease is well recognized. METHODS: retrospective and ongoing study of an extended family carrying the MTTL1 A3243G mutation with multiple symptomatic individuals. tissue heteroplasmy is reviewed based on the clinical presentations, imaging studies, laboratory findings in affected individuals and pathological material obtained at autopsy in two of the family members. RESULTS: there were seven affected individuals out of thirteen members in this three generation family who each carried the MTTL1 A3243G mutation. the clinical presentations were varied with symptoms ranging from hearing loss, migraines, dementia, seizures, diabetes, visual manifestations, and stroke like episodes. three of the family members are deceased from MelaS or to complications related to MelaS. CONCLUSIONS: the results of the clinical, pathological and radiological findings in this family provide strong support to the current concepts of maternal inheritance, tissue heteroplasmy and molecular pathogenesis in MelaS. neurologists (both adult and paediatric) are the most likely to encounter patients with MelaS in their practice. genetic counselling is complex in view of maternal inheritance and heteroplasmy. newer therapeutic options such as arginine are being used for acute and preventative management of stroke like episodes.
Assuntos
Encéfalo/patologia , Genes Mitocondriais/genética , Síndrome MELAS/genética , Músculo Esquelético/patologia , RNA de Transferência de Leucina/genética , Adolescente , Adulto , Criança , Demência/genética , Demência/fisiopatologia , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Feminino , Perda Auditiva/genética , Perda Auditiva/fisiopatologia , Humanos , Síndrome MELAS/patologia , Síndrome MELAS/fisiopatologia , Masculino , Pessoa de Meia-Idade , Transtornos de Enxaqueca/genética , Transtornos de Enxaqueca/fisiopatologia , Mutação , Linhagem , Fenótipo , Estudos Retrospectivos , Convulsões/genética , Convulsões/fisiopatologia , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/fisiopatologia , Transtornos da Visão/genética , Transtornos da Visão/fisiopatologia , Adulto JovemRESUMO
N-acetyl-glutamate synthase (NAGS) deficiency is a rare autosomal recessive urea cycle disorder (UCD) that uncommonly presents in adulthood. Adult presentations of UCDs include; confusional episodes, neuropsychiatric symptoms and encephalopathy. To date, there have been no detailed neurological descriptions of an adult onset presentation of NAGS deficiency. In this review we examine the clinical presentation and management of UCDs with an emphasis on NAGS deficiency. An illustrative case is provided. Plasma ammonia levels should be measured in all adult patients with unexplained encephalopathy, as treatment can be potentially life-saving. Availability of N-carbamylglutamate (NCG; carglumic acid) has made protein restriction largely unnecessary in treatment regimens currently employed. Genetic counselling remains an essential component of management of NAGS.
Assuntos
Encefalopatias/etiologia , Distúrbios Congênitos do Ciclo da Ureia/complicações , Adulto , Aminoácido N-Acetiltransferase , Amônia/sangue , Encefalopatias/mortalidade , Encefalopatias/terapia , Feminino , Humanos , Masculino , Redes e Vias Metabólicas/fisiologia , Distúrbios Congênitos do Ciclo da Ureia/mortalidade , Distúrbios Congênitos do Ciclo da Ureia/terapiaRESUMO
Mevalonate kinase deficiency is a rare autosomal recessively inherited organic aciduria with a complex multi-systemic phenotype. We describe two deceased patients with clinically severe mevalonate kinase (MK) deficiency confirmed by MK mutation analysis. The phenotype in our patients ranged from neonatal hydrops in the first patient to severe failure to thrive, hepatosplenomegaly, recurrent febrile episodes and lymphadenopathy in the second. Both infants excreted relatively low amounts of mevalonic acid intermittently.
Assuntos
Deficiência de Mevalonato Quinase/diagnóstico , Fenótipo , Evolução Fatal , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Deficiência de Mevalonato Quinase/genética , Deficiência de Mevalonato Quinase/metabolismo , Ácido Mevalônico/metabolismo , MutaçãoRESUMO
It is unusual for inborn errors of metabolism to be considered in the investigative work-up of pancytopenia. We report a family in which the proband presented with failure to thrive at 2 months of age and subsequent bone marrow failure. A previous sibling had died at 7 months of age with suspected leukaemia. Haematological findings in the proband were significant for pancytopenia, and bone marrow aspiration showed dysplastic changes in all cell lineages. Urinary organic acid analysis revealed elevated methylmalonic acid. The synthesis of transcobalamin II (transcobalamin, TC) by cultured fibroblasts was markedly reduced, confirming the diagnosis of TC deficiency. The proband and his younger asymptomatic sister (also found to have TC deficiency) were homozygous for R399X (c.1195C>T), a novel mutation resulting in the loss of the C- terminal 29 amino acids of TC, a highly conserved region. Response to parenteral vitamin B(12) in the proband was dramatic. At 6 years 3 months of age, physical examination is normal and developmental level is age appropriate. His sister is clinically asymptomatic and is also developing normally. Propionylcarnitine concentrations were not elevated in the newborn screening cards from the proband and sister, but that was for specimens retrieved from storage after 7 years and 5 years, respectively. Inherited and acquired cobalamin disorders should both be considered in the differential diagnosis of bone marrow failure syndromes in young children. Early detection of the metabolic causes of bone marrow failure can ensure prompt recovery in some cases involving the vitamin B(12) pathway.
Assuntos
Doenças da Medula Óssea/etiologia , Erros Inatos do Metabolismo/diagnóstico , Transcobalaminas/deficiência , Biomarcadores/sangue , Biomarcadores/urina , Doenças da Medula Óssea/sangue , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/terapia , Exame de Medula Óssea , Células Cultivadas , Criança , Desenvolvimento Infantil , Pré-Escolar , Análise Mutacional de DNA , Insuficiência de Crescimento/sangue , Insuficiência de Crescimento/etiologia , Feminino , Fibroblastos/metabolismo , Predisposição Genética para Doença , Humanos , Lactente , Masculino , Erros Inatos do Metabolismo/sangue , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/terapia , Mutação , Pancitopenia/sangue , Pancitopenia/etiologia , Linhagem , Fenótipo , Transcobalaminas/genética , Resultado do Tratamento , Vitamina B 12/administração & dosagemRESUMO
Polyisoprenoid alcohols consisting of 9 or more isoprene units are present in all living cells. They can be fully unsaturated (polyprenols) or alpha-saturated (dolichol). Dolichol forms may have additional saturation at or near the omega-end. Some species contain ony dolichol or only polyprenols while others have nearly equal amounts of both types. Some polyisoprenoid alcohols consist entirely of trans isoprene units but most, including dolichol, contain both trans and cis units. Considerable advances in lipid methodology have occurred since the first review of polyisoprenoid alcohols by Hemming in 1974. For example, direct analysis of both dolichol and Dol-P by HPLC has replaced earlier methods which were often both insensitive and inaccurate. The availability of radiolabeled dolichol and polyprenols has facilitated studies concerning the metabolism and distribution of these compounds. Those studies suggest that only a small portion of the dolichol present in cells is likely to be involved in glycosylation. Polyisoprenoid alcohols are usually present at a family of homologues where each differs in size by one isoprene unit. Little or no size related specificity has been observed for any reaction involving dolichol or polyisoprenol intermediates. The overall length of polyisoprenoid alcohols may, however, affect the manner in which these compounds influence the physical and biochemical properties of membranes. Studies on the biosynthetic pathway leading from cis, trans Pol-PP by phosphatase action. The formation of the dolichol backbone from a polyprenol requires the action of an additional enzyme, an alpha-saturase. This enzyme does not always act at the level of a single common substrate, since Pol-PP, Pol-P, and polyprenol all appear to be utilized as substrates. The major product of the de novo pathway differs among different species. Dol-P would appear to be the most energy efficient end-product since it can participate directly in glycoprotein formation. Most often, however, Dol-P is not the major product of metabolic labeling experiments. In some cases, dolichol is formed so that rephosphorylation is required to provide Dol-P for participation in glycoprotein formation. The kinase responsible for this phosphorylation appears to bypass the considerable stores of dolichol present in tissues (i.e. sea urchin eggs) in favor of dolichol derived directly from de novo synthesis. Although HMGR is a major regulatory component of the pathway leading to polyisoprenoid alcohols and cholesterol, control is most often not co-ordinated.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Diterpenos/metabolismo , Dolicóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool) , Pirofosfatases , Terpenos/metabolismo , Álcoois/metabolismo , Animais , Humanos , Monoéster Fosfórico Hidrolases/metabolismo , Fosfotransferases/metabolismo , Frações Subcelulares/metabolismo , Distribuição TecidualRESUMO
m.3291T > C mutation in the MT-TL1 gene has been infrequently encountered in association with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), however remains poorly characterized from a clinical perspective. In the following report we describe in detail the phenotypic features, long term follow up (> 7 years) and management in a Caucasian family with MELAS due to the m.3291T > C mutation and review the literature on m.3291T > C mutation. The clinical phenotype in the proposita included overlapping features of MELAS, MERRF (Myoclonic epilepsy and ragged-red fiber syndrome), MNGIE (Mitochondrial neurogastrointestinal encephalopathy), KSS (Kearns-Sayre Syndrome) and CPEO (Chronic progressive external ophthalmoplegia).
RESUMO
Intrinsic factor deficiency (OMIM #261000, IFD) is a rare inherited disorder of vitamin B12 metabolism due to mutations in the gastric intrinsic factor (GIF) gene.We report three individuals from an Old Order Mennonite community who presented with B12 deficiency. Two cases are siblings born to consanguineous parents and the third case is not known to be closely related. The older male sib presented at 4 years with gastrointestinal symptoms, listlessness, and pallor. He had pancytopenia with megaloblastic anemia. Serum B12 was 61 (198-615 pmol/L). Methylmalonic aciduria was present. C3 was elevated on acylcarnitine profile. Homocysteine was high at 16.7 (5.0-12.0 umol/L). His asymptomatic female sibling was also found to have B12 deficiency. Genetic testing for methylmalonic aciduria (MMAA), transcobalamin deficiency (TCN2), and Imerslund-Gräsbeck syndrome (AMN) showed no mutation in both siblings. The third patient, a 34-year-old woman, had presented in infancy with a diagnosis of pernicious anemia. Mutation analysis of GIF revealed compound heterozygosity for a c.79+1G>A substitution and a c.973delG deletion in all three individuals. Oral or parenteral vitamin B12 has led to complete recovery of clinical parameters and vitamin B12 levels. Newborn screening samples on the siblings revealed normal methylcitrate, C3, and C3/C2 ratios thus indicating no disruption of propionic or methylmalonic acid metabolism.A high index of suspicion should be maintained if children present with megaloblastic anemia since GIF deficiency is a treatable disorder and newborn screening may not be able to detect this condition.
RESUMO
Morquio disease (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) activity. Patients commonly present in early infancy with growth failure, spondyloepiphyseal dysplasia, corneal opacification, and keratan sulfaturia, but milder forms have been described. We report on a patient who grew normally until age 5 years. Her keratan sulfaturia was not detected until adolescence, and she now has changes restricted largely to the axial skeleton. She has experienced only mildly impaired vision. At age 22, thin-layer chromatography of purified glycosaminoglycans showed some keratan sulfaturia. GALNS activity in fibroblast homogenate supernatants was 20 +/- 5% of controls (as compared to 5 +/- 3% of controls in severe MPS IVA, P < .003). Kinetic analysis of residual fibroblast GALNS activity in patient and parents revealed decreased K(m) and increased Vmax in the mother and daughter, but not in the father, compatible with compound heterozygosity. GALNS exons were amplified from patient genomic DNA and screened by SSCP. Two missense mutations, a C to T transition at position 335 (predicting R94C) and a T to G transversion at position 344 (predicting F97V), were found on sequencing an abnormally migrating exon 3 amplicon. Digestion of the amplicon with FokI and AccI restriction enzymes (specific for the R94C and F97V mutations, respectively) confirmed heterozygosity. In fibroblast transfection experiments, heterozygous R94C and F97V mutants independently expressed as severe and mild GALNS deficiency, respectively. We interpret these findings to indicate that our patient bears heteroallelic GALNS missense mutations, leading to GALNS deficiency and mild MPS IVA. Our findings expand the clinical and biochemical phenotype of MPS IVA, but full delineation of the genotype-phenotype relationship requires further study of native and transfected mutant cell lines.
Assuntos
Condroitina Sulfatases/genética , Mucopolissacaridose IV/enzimologia , Mucopolissacaridose IV/genética , Mutação , Adolescente , Adulto , Idade de Início , Alelos , Sequência de Aminoácidos , Northern Blotting , Southern Blotting , Células Cultivadas , Criança , Condroitina Sulfatases/metabolismo , Opacidade da Córnea/genética , Feminino , Fibroblastos/enzimologia , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/urina , Quadril/diagnóstico por imagem , Quadril/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Dados de Sequência Molecular , Linhagem , Pelve/anormalidades , Pelve/diagnóstico por imagem , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Gravidez , Radiografia , Coluna Vertebral/patologiaRESUMO
The concentration of dolichol has been determined in various human tissues obtained at autopsy. The highest levels ( 3000 microgram/g wet weight) were found in testes. Liver and several other endocrine tissues contained about 1000 microgram/g. Lower levels were present in other tissues examines. Only a small proportion of the total dolichol in human tissues was esterified to fatty acids.
Assuntos
Diterpenos/metabolismo , Dolicóis/metabolismo , Ésteres/metabolismo , Ácidos Graxos/metabolismo , Humanos , Distribuição TecidualRESUMO
Dolichol from human liver was shown by reverse-phase high pressure liquid chromatography to consist of a series of homologues ranging in length from 17 to 23 isoprene units. The two major components, corresponding to 19 and 20 isoprene units, respectively, were isolated and identified by mass spectrometry. Dolichyl palmitate, synthesized from liver dolichol, showed an identical series of peaks with longer retention times. Attempts to chromatography dolichyl phosphate under similar conditions were unsuccessful. Dolichol from uterine tissue and several other human tissues showed a shift toward shorter chain length, with a predominance of homologues containing 18 and 19 isoprene units.
Assuntos
Diterpenos/análise , Dolicóis/análise , Fígado/análise , Útero/análise , Cromatografia Líquida de Alta Pressão/métodos , Feminino , Humanos , Espectrometria de Massas , Relação Estrutura-AtividadeAssuntos
Cistinose/genética , Etnicidade/genética , Glicoproteínas , Proteínas de Membrana/genética , Mutação de Sentido Incorreto/genética , Sistemas de Transporte de Aminoácidos Neutros , Sequência de Bases , Cristianismo , Cistinose/epidemiologia , Análise Mutacional de DNA , Éxons/genética , Feminino , Frequência do Gene/genética , Testes Genéticos , Homozigoto , Humanos , Lactente , Masculino , Proteínas de Membrana Transportadoras , Ontário/epidemiologia , Deleção de Sequência/genéticaRESUMO
Glycogen storage disease, type II (GSDII; Pompe disease; acid maltase deficiency) is an autosomal recessive disease caused by mutations of the GAA gene that lead to deficient acid alpha-glucosidase enzyme activity and accumulation of lysosomal glycogen. Although measurement of acid alpha-glucosidase enzyme activity in fibroblasts remains the gold standard for the diagnosis of GSDII, analysis of the GAA gene allows confirmation of clinical or biochemical diagnoses and permits predictive and prenatal testing of individuals at risk of developing GSDII. We have developed a clinical molecular test for the detection of GAA mutations based on cycle sequencing of the complete coding region. GAA exons 2-20 are amplified in six independent PCR using intronic primers. The resulting products were purified and sequenced. Preliminary studies using this protocol were conducted with DNA from 21 GSDII-affected individuals from five centers across Canada. In total, 41 of 42 mutations were detected (96.7% detection rate). Mutations spanned intron 1 through exon 19 and included nine novel mutations. Haplotype analysis of recurrent mutations further suggested that three of these mutations are likely to have occurred independently at least twice. Additionally, we report the identification of the c.-32-13T>G GAA mutation in an individual with infantile variant GSDII, despite reports of this mutation being associated almost exclusively with late-onset forms of the disease. The development of a clinical molecular test provides an important tool for the management and counseling of families and individuals with GSDII, and has provided useful information about the GAA mutation spectrum in Canada.
Assuntos
Predisposição Genética para Doença/genética , Doença de Depósito de Glicogênio Tipo II/genética , alfa-Glucosidases/genética , Alelos , Análise Mutacional de DNA , Doença de Depósito de Glicogênio Tipo II/diagnóstico , Doença de Depósito de Glicogênio Tipo II/enzimologia , Humanos , Mutação , alfa-Glucosidases/deficiênciaRESUMO
Quiescent thymocytes, mitogen-stimulated thymocytes and acute-leukaemic lymphoblasts provide a model for the study of protein glycosylation in quiescent cells, mitotically active non-malignant and malignant cells respectively. The biosynthesis of both complex and high-mannose-type oligosaccharides was monitored by metabolic labelling with [6-3]fucose and [2-3H]mannose. Bio-Gel P6 elution profiles of [6-3H]fucose-labelled glycopeptides showed that quiescent thymocytes and stimulated thymocytes synthesized qualitatively and quantitatively similar glycopeptides; however, higher-molecular-weight glycopeptides were synthesized by the acute-leukaemic lymphoblasts. The amount of [2(-3)H]mannose incorporated into glycopeptide by quiescent thymocytes was less than 10% of that incorporated by stimulated thymocytes. The Bio-Gel P6 elution profile of [2(-3)H]mannose-labelled glycopeptides from acute leukaemic lymphoblasts was qualitatively similar to that of stimulated thymocytes, with about 40% of the radioactivity incorporated into one glycopeptide peak. This glycopeptide was characterized by Bio-Gel P6 and concanavalin A affinity chromatography, radioactive-sugar analysis, sensitivity to alpha-mannosidase and endoglycosidase H and resistance to beta-glucosaminidase as containing a high-mannose oligosaccharide, possible of Man7-8GlcNAc2 structure. Pulse/chase experiments indicated that this high-mannose oligosaccharide was an end product and not a biosynthetic intermediate. It is concluded that higher-molecular-weight fucose-labelled glycopeptides are characteristic of the malignant cell type, and the synthesis of high-mannose oligosaccharide, Man7-8GlcNAc2, in stimulated thymocytes and acute-leukaemic lymphoblasts is associated with mitotically active cells.
Assuntos
Glicoproteínas/biossíntese , Leucemia Linfoide/metabolismo , Linfócitos T/metabolismo , Animais , Cromatografia em Gel , Concanavalina A/farmacologia , Fucose/metabolismo , Leucemia Experimental/metabolismo , Ativação Linfocitária , Manose/metabolismo , Camundongos , Camundongos Endogâmicos AKR , Oligossacarídeos/biossíntese , TrítioRESUMO
Lysosome membranes were isolated, and membrane proteins and glycoproteins were characterized by electrophoresis and lectin probes of nitrocellulose blots. Rat liver lysosomes were isolated on a discontinuous metrizamide gradient and characterized by subcellular marker enzymes. Lysosomes were lysed by hypotonic freeze-thaw shock and membranes were isolated. The release of beta-N-acetylhexosaminidase was used to monitor the disruption of the lysosomes. Proteins of lysosome membranes were analyzed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. There were at least 30 proteins present and several were glycoproteins. Nitrocellulose blots of lysosome membrane proteins were probed with a panel of lectins, including concanavalin A, Ulex europaeus agglutinin I, Lotus tetragonolobus agglutinin, soybean agglutinin, peanut agglutinin, and Ricinus communis agglutinin I. Peanut agglutinin and Ricinus communis agglutinin I binding were also examined after neuramidase treatment of lysosome membranes. Ten proteins bound concanavalin A, and neuraminidase pretreatment revealed six proteins that bound Ricinus communis agglutinin I and three proteins that bound peanut agglutinin. The other lectins tested did not bind to any lysosome membrane proteins. These results indicate that lysosome membranes contain several glycoproteins, some of which contain sialic acid terminating complex oligosaccharides.