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1.
Biomed Chromatogr ; 33(2): e4376, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30168866

RESUMO

Shuang Huang Lian Injection (SHLI) has been used in China for over 30 years as an effective and widely used Chinese herbal prescription to treat acute respiratory infectious. SHLI has, however, caused many severe anaphylactoid reactions. It is important to identify the potential anaphylactoid components of SHLI. Spectrum-effect relationships were used to explore potentially anaphylactoid components. Based on the original herbal formula, honeysuckle, Fructus Forsythiae and Radix Scutellariae extracts were prepared and combined in appropriate proportions. The preparations were then injected into the caudal vein of rats to obtain in vivo serum samples for pharmacological evaluation and fingerprint analysis. The release rate of ß-hexosaminidase from RBL-2H3 cells and plasma histamine level was used as the pharmacological index. Chromatographic fingerprint analysis identified 22 common peaks. Regression analysis and correlation analysis were used to calculate the relationships between the peaks and the pharmacological effects and identified peaks 5, 6, 11, 12 and 17 as likely anaphylactoid agents. The correlated peaks were identified by comparing the fingerprints with in vitro samples and reference standard samples and the structure was identified by UPLC-TOF-MS. This study established a prospective method to clarify the anaphylactoid components in SHLI, which would provide guidances for screening anaphylactoid components in other traditional Chinese medicine injections.


Assuntos
Antígenos de Plantas/análise , Medicamentos de Ervas Chinesas/análise , Anafilaxia , Animais , Antígenos de Plantas/química , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/química , Feminino , Hexosaminidase B/sangue , Hexosaminidase B/metabolismo , Histamina/sangue , Lonicera/química , Masculino , Espectrometria de Massas , Ratos , Ratos Wistar
2.
Neuroreport ; 29(11): 962-967, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29847465

RESUMO

Sandhoff disease (SD) is a genetic disorder caused by a mutation of the ß-subunit gene ß-hexosaminidase B (HexB) in humans, which results in the massive accumulation of the ganglioside GM2 and related glycosphingolipids in the nervous system. SD causes progressive neurodegeneration and changes in white matter in human infants. An animal model of SD has been established, Hexb-deficient (Hexb) mice, which shows abnormalities resembling the severe phenotype found in human infants. Previously, we reported that the activation state of microglia caused astrogliosis in the early stage of Hexb mouse development. To study how the symptoms of SD develop, we explored the difference in gene expression between 4-week-old Hexb and Hexb mouse cerebral cortices by microarray analysis. The data indicated not only the upregulation of immune system-related genes but also the downregulation of myelin-related genes in the 4-week-old Hexb mouse cerebral cortices. To test the correlation between inflammation and dysmyelination, we generated double-knockout mice of Hexb and the Fc receptor γ gene (Fcrγ), which is a regulator of autoimmune responses. Dysmyelination recovered in these double-knockout mice. The number of oligodendrocyte progenitors, which expressed platelet-derived growth factor receptor-α, did not change in the 2-week-old mouse brain. These results indicate that microglial activation plays an important role in the myelination process, without influencing the number of oligodendrocyte progenitors, in the development of Hexb mice.


Assuntos
Gliose/metabolismo , Hexosaminidase B/farmacologia , Microglia/efeitos dos fármacos , Bainha de Mielina/metabolismo , Doença de Sandhoff/metabolismo , Animais , Modelos Animais de Doenças , Hexosaminidase B/metabolismo , Camundongos Knockout , Microglia/metabolismo , Regulação para Cima
3.
Hum Mol Genet ; 27(6): 954-968, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29325092

RESUMO

Sandhoff disease (SD) is a rare inherited disorder caused by a deficiency of ß-hexosaminidase activity which is fatal because no effective treatment is available. A mouse model of Hexb deficiency reproduces the key pathognomonic features of SD patients with severe ubiquitous lysosomal dysfunction, GM2 accumulation, neuroinflammation and neurodegeneration, culminating in death at 4 months. Here, we show that a single intravenous neonatal administration of a self-complementary adeno-associated virus 9 vector (scAAV9) expressing the Hexb cDNA in SD mice is safe and sufficient to prevent disease development. Importantly, we demonstrate for the first time that this treatment results in a normal lifespan (over 700 days) and normalizes motor function assessed by a battery of behavioral tests, with scAAV9-treated SD mice being indistinguishable from wild-type littermates. Biochemical analyses in multiple tissues showed a significant increase in hexosaminidase A activity, which reached 10-15% of normal levels. AAV9 treatment was sufficient to prevent GM2 and GA2 storage almost completely in the cerebrum (less so in the cerebellum), as well as thalamic reactive gliosis and thalamocortical neuron loss in treated Hexb-/- mice. In summary, this study demonstrated a widespread protective effect throughout the entire CNS after a single intravenous administration of the scAAV9-Hexb vector to neonatal SD mice.


Assuntos
Hexosaminidase B/farmacologia , Doença de Sandhoff/tratamento farmacológico , Doença de Sandhoff/patologia , Administração Intravenosa , Animais , Animais Recém-Nascidos , Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Gangliosídeo G(M2)/metabolismo , Gangliosídeos/metabolismo , Hexosaminidase B/genética , Hexosaminidase B/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Sandhoff/metabolismo
4.
J Vet Intern Med ; 32(1): 340-347, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29106755

RESUMO

BACKGROUND: GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either ß-hexosaminidase A (Hex-A) and ß-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. OBJECTIVES: To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. ANIMALS: One affected Shiba Inu and a clinically healthy dog. METHODS: Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. RESULTS: A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). CONCLUSIONS AND CLINICAL IMPORTANCE: Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis.


Assuntos
Doenças do Cão/genética , Gangliosidoses GM2/veterinária , Hexosaminidase B/genética , Doença de Sandhoff/veterinária , Animais , Encéfalo/diagnóstico por imagem , Doenças do Cão/diagnóstico por imagem , Doenças do Cão/patologia , Cães , Feminino , Gangliosidoses GM2/diagnóstico por imagem , Gangliosidoses GM2/genética , Imagem por Ressonância Magnética/veterinária , Doença de Sandhoff/diagnóstico por imagem , Doença de Sandhoff/genética , Análise de Sequência de Proteína , Deleção de Sequência
5.
Exp Neurol ; 299(Pt A): 26-41, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28974375

RESUMO

Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal ß-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa-/- mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by ß-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa-/-Neu3-/- mice were healthy at birth, but died at 1.5 to 4.5months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa-/-Neu3-/- mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa-/-Neu3-/- mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa-/-Neu3-/- mice. Thus, the Hexa-/-Neu3-/- mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition.


Assuntos
Gangliosidoses GM2/genética , Hexosaminidase B/genética , Neuraminidase/genética , Doença de Tay-Sachs/genética , Animais , Química Encefálica/genética , Vesículas Citoplasmáticas/patologia , Gangliosidoses GM2/metabolismo , Gliose/genética , Gliose/patologia , Glicoesfingolipídeos/metabolismo , Coxeadura Animal/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuraminidase/deficiência , Neurônios/patologia , Células de Purkinje/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Doença de Tay-Sachs/patologia
6.
J Neuroimmunol ; 306: 55-67, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28385189

RESUMO

Sandhoff disease is an inherited lysosomal storage disease, resulting from the deficiency of lysosomal ß-hexosaminidase A and B enzyme activity. The Hexb-/- mouse model recapitulates human disease and leads to fatal neurodegeneration and neuroinflammation. IL-15 is important for the proliferation of NK, NK T, and CD8+ cytotoxic/memory T cells. In order to determine how changes to IL-15-dependent immune cell populations would alter the course of Sandhoff disease in mice, we generated a Hexb-/-Il-15-/- double knockout mouse and used motor behaviour tests, analyzed peripheral blood and brain leukocyte immunophenotypes, cytokine secretion, as well as examined markers of microgliosis, astrogliosis and apoptosis. Hexb-/-Il-15-/- mice had an accelerated neurodegenerative phenotype, and reached the humane endpoint at 118±3.5d, compared to Hexb-/- mice (127±2.2d). The performance of Hexb-/-Il-15-/- mice declined earlier than Hexb-/- mice on the rotarod and righting reflex motor behaviour tests. Hexb-/- mice had a significantly higher prevalence of pro-inflammatory monocytes in the blood relative to C57BL/6 mice, but this was unaltered by IL-15 deficiency. The prevalence of NK cells and CD8+ T cells in Il-15-/- and Hexb-/-Il-15-/- mice was decreased compared to wild type and Hexb-/- mice. While Hexb-/- mice displayed an increase in the prevalence of CD4+ and CD8+ T cells in brain leukocytes compared to C57BL/6 mice, there was a decrease in CD8+ T cells in Hexb-/-Il-15-/- compared to Hexb-/- mice. In addition, circulating IL-17 and IL-10 levels were significantly higher in Hexb-/-Il-15-/- mice, suggesting heightened inflammation compared to Hexb-/- mice. Interestingly, astrogliosis levels were significantly reduced in the cerebellum of Hexb-/-Il-15-/- mice compared to Hexb-/- mice while microgliosis was not affected in brains of Hexb-/-Il-15-/- mice. Our study demonstrated that IL-15 depletion dramatically reduced numbers of NK and CD8+ T cells as well as astrocytes but accelerated disease progression in Sandhoff mice. These results pointed to interactions between NK/CD8+ T cells and astrogliosis and potentially a protective role for NK/CD8+ T cells and/or astrocytes during disease progression. This observation supports the notion that expanding the IL-15-dependent NK and CD8+ T cells populations with IL-15 therapy may have therapeutic benefits for Sandhoff disease.


Assuntos
Linfócitos T CD8-Positivos/patologia , Doenças Cerebelares/etiologia , Gliose/terapia , Células Matadoras Naturais/patologia , Doença de Sandhoff/complicações , Doença de Sandhoff/mortalidade , Animais , Antígenos CD/metabolismo , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Doenças Cerebelares/genética , Doenças Cerebelares/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/genética , Proteína Glial Fibrilar Ácida/metabolismo , Hexosaminidase B/genética , Hexosaminidase B/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Células Matadoras Naturais/metabolismo , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Doença de Sandhoff/genética
7.
Annu Rev Med ; 68: 445-458, 2017 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-28099085

RESUMO

Several proteins that are mutated in lysosomal storage diseases are linked to neurodegenerative disease. This review focuses on some of these lysosomal enzymes and transporters, as well as current therapies that have emerged from the lysosomal storage disease field. Given the deeper genetic understanding of lysosomal defects in neurodegeneration, we explore why some of these orphan disease drug candidates are also attractive targets in subpopulations of individuals with neurodegenerative disease.


Assuntos
Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/genética , Lisossomos/enzimologia , Doenças Neurodegenerativas/genética , Proteínas/genética , Acetilglucosaminidase/genética , Proteínas Amiloidogênicas/metabolismo , Autofagia , Proteínas de Transporte/genética , Endocitose , Glucosilceramidase/genética , Hexosaminidase A/genética , Hexosaminidase B/genética , Humanos , Doenças por Armazenamento dos Lisossomos/complicações , Doenças por Armazenamento dos Lisossomos/metabolismo , Glicoproteínas de Membrana/genética , Proteínas/metabolismo , ATPases Translocadoras de Prótons/genética , Esfingomielina Fosfodiesterase/genética
8.
Brain Dev ; 39(2): 171-176, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27697305

RESUMO

INTRODUCTION: Hepatosplenomegaly is often present in infantile Sanshoff disease. However, cardiac involvement is extremely uncommon. CASE REPORT: We describe a 14-month-old female baby who exhibited mitral regurgitation and cardiomegaly at the age of 2months, dilation of the left atrium and left ventricle at age of 6months, followed by regression of developmental milestones after an episode of minor infection at age of 14months. Brain magnetic resonance imaging revealed signal changes over the bilateral thalami, bilateral cerebral white matter and left putamen. An examination of the fundus showed presence of cherry-red spots in both macular areas. The lysosomal enzymatic activities showed a marked reduction of ß-hexosaminidase B (HEXB) activity. Two novel mutations of HEXB gene were identified. One of the mutations was a c.1538 T>C mutation, which predicted a p.L513P amino acid substitution of leucine to proline; the other was a c.299+5 G>A mutation, which was a splice site mutation. CONCLUSION: Cardiac involvement might occur prior to neurological symptoms in infantile Sandhoff disease, and it should be included in the differential diagnoses of metabolic cardiomyopathies in the infantile stage.


Assuntos
Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Hexosaminidase B/genética , Mutação , Doença de Sandhoff/genética , Doença de Sandhoff/fisiopatologia , Encéfalo/diagnóstico por imagem , Cardiomiopatias/diagnóstico por imagem , Análise Mutacional de DNA , Diagnóstico Diferencial , Ecocardiografia , Feminino , Hexosaminidase B/metabolismo , Humanos , Lactente , Imagem por Ressonância Magnética , Linhagem , Doença de Sandhoff/diagnóstico por imagem
9.
J Control Release ; 199: 156-67, 2015 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-25481446

RESUMO

Millions of Americans suffer from dry eye disease, and there are few effective therapies capable of treating these patients. A decade ago, an abundant protein component of human tears was discovered and named lacritin (Lacrt). Lacrt has prosecretory activity in the lacrimal gland and mitogenic activity at the corneal epithelium. Similar to other proteins placed on the ocular surface, the durability of its effect is limited by rapid tear turnover. Motivated by the rationale that a thermo-responsive coacervate containing Lacrt would have better retention upon administration, we have constructed and tested the activity of a thermo-responsive Lacrt fused to an elastin-like polypeptide (ELP). Inspired from the human tropoelastin protein, ELP protein polymers reversibly phase separate into viscous coacervates above a tunable transition temperature. This fusion construct exhibited the prosecretory function of native Lacrt as illustrated by its ability to stimulate ß-hexosaminidase secretion from primary rabbit lacrimal gland acinar cells. It also increased tear secretion from non-obese diabetic (NOD) mice, a model of autoimmune dacryoadenitis, when administered via intra-lacrimal injection. Lacrt ELP fusion proteins undergo temperature-mediated assembly to form a depot inside the lacrimal gland. We propose that these Lacrt ELP fusion proteins represent a potential therapy for dry eye disease and the strategy of ELP-mediated phase separation may have applicability to other diseases of the ocular surface.


Assuntos
Síndromes do Olho Seco/tratamento farmacológico , Elastina/uso terapêutico , Glicoproteínas/uso terapêutico , Proteínas/uso terapêutico , Actinas/metabolismo , Animais , Dacriocistite/imunologia , Preparações de Ação Retardada , Elastina/química , Feminino , Glicoproteínas/química , Hexosaminidase B/metabolismo , Temperatura Alta , Humanos , Técnicas In Vitro , Aparelho Lacrimal/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Cultura Primária de Células , Proteínas/química , Coelhos , Proteínas Recombinantes de Fusão , Lágrimas/metabolismo , Transcitose
10.
Toxicol Lett ; 229(1): 198-209, 2014 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-24910985

RESUMO

Carbon nanotubes (CNT) are environmental challenges to the respiratory and gastrointestinal mucosa, and to the dermal immune system. Mast cells (MC) are pro-inflammatory immunocytes that reside at these interfaces with the environment. Mast cells are sources of pro-inflammatory mediators (histamine, serotonin, matrix-active proteases, eicosanoids, prostanoids, cytokines and chemokines), which are released in a calcium-dependent manner following immunological challenge or physico-chemical stimulation. Since C-60 fullerenes, which share geometry with CNT, are suppressive of mast cell-driven inflammatory responses, we explored the effects of unmodified SWCNT aggregates on mast cell signaling pathways, phenotype and pro-inflammatory function. We noted SWCNT suppression of antigen-induced signalling pathways and pro-inflammatory degranulation responses. Mast cells recognize unmodified SWCNT by remodeling the plasma membrane, disaggregating the cortical actin cytoskeleton and relocalizing clathrin. Clathrin was also identified as a component of an affinity-purified 'interactome' isolated from MC using an SWCNT affinity matrix for mast cell lysates. Together, these data are consistent with the ability of SWCNT to suppress mast cell pro-inflammatory function via a novel recognition mechanism.


Assuntos
Membrana Celular/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Nanotubos de Carbono/toxicidade , Receptores de IgE/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sequência de Aminoácidos , Western Blotting , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Membrana Celular/ultraestrutura , Clatrina/metabolismo , Citoesqueleto/efeitos dos fármacos , Fulerenos/toxicidade , Hexosaminidase B/metabolismo , Humanos , Imuno-Histoquímica , Mastócitos/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Receptores de IgE/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
11.
Zhonghua Er Ke Za Zhi ; 52(4): 313-6, 2014 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-24915922

RESUMO

OBJECTIVE: To explore the clinical features and molecular mutation of HEXB gene in a case with juvenile Sandhoff disease. METHOD: We retrospectively reviewed the clinical, neuroimaging and biochemical findings in this Chinese child with juvenile Sandhoff disease. Hexosaminidase A and hexosaminidase A & B activities were measured in blood leukocytes by fluorometric assay. HEXB gene molecular analysis was performed by PCR and direct sequencing. RESULT: The 9-year-old boy was admitted for psychomotor regression. He presented slowly progressive gait disorder and dysarthria during the last three years. Cranial MRI revealed a marked cerebellar atrophy with normal intensity in the thalamus and basal ganglia. Brain MRS showed normal in the thalamus and basal ganglia. Hexosaminidase A was 69.5 (mg·h) [normal controls 150-360 nmol/(mg·h)], hexosaminidase A & B activity was 119 nmol/(mg·h)[normal controls 600-3 500 nmol/(mg·h)], confirming the diagnosis of Sandhoff disease. The patient was a compound heterozygote for a novel deletion mutation c.1404delT (p. P468P fsX62) and a reported mutation c.1509-26G>A. CONCLUSION: The clinical features of juvenile Sandhoff disease include ataxia, dysarthria and cerebellar atrophy. The enzyme assay and molecular analysis of HEXB gene can confirm the diagnosis of Sandhoff disease. The novel mutation c.1404delT(p. P468P fsX62) is a disease-related mutation.


Assuntos
Mutação , Doença de Sandhoff/diagnóstico , Doença de Sandhoff/genética , Cadeia beta da beta-Hexosaminidase/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Ataxia Cerebelar/diagnóstico , Ataxia Cerebelar/enzimologia , Ataxia Cerebelar/genética , Criança , Análise Mutacional de DNA , Heterozigoto , Hexosaminidase A/sangue , Hexosaminidase A/metabolismo , Hexosaminidase B/sangue , Hexosaminidase B/metabolismo , Humanos , Leucócitos/enzimologia , Imagem por Ressonância Magnética , Masculino , Radiografia , Estudos Retrospectivos , Doença de Sandhoff/enzimologia
12.
Otolaryngol Pol ; 68(1): 20-4, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24484945

RESUMO

UNLABELLED: Nasal polyps and hypertrophic lower nasal conchae are common disorders of nasal cavity. The majority of etiopathogenetic theories indicate inflammatory background of polyps and hypertrophic concha. N-acetyl-ß-D-hexosaminidase and ß-glucuronidase are lysosomal exoglycosidases revealing accelerated activity in inflammatory processes. AIM: The aim of the study was to evaluate the catabolism of glycoconjugates in nasal polyps and hypertrophic nasal concha basing on the activity of N-acetyl-ß-D-hexosaminidase (HEX) and ß-glucuronidase (GLU). MATERIAL AND METHODS: Material consisted of nasal polyps taken from 40 patients during polypectomy in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) and hypertrophic lower nasal conchae taken from 20 patients during mucotomy. The activity of HEX, HEX A, HEX B and GLU in supernatant of homogenates of nasal polyps and hypertrophic lower nasal concha tissues has been estimated using colorimetric method. RESULTS: Statistically significant decrease has been observed in concentration of the activity (per 1mg of tissue) of HEX (p<0.05), HEX B (p<0.001) and specific activity (per 1mg of protein) of HEX B (p<0.001) in nasal polyps tissue in comparison to hypertrophic lower nasal conchae tissue. CONCLUSIONS: Decrease in the activity and specific activity concentration of the majority of examined lysosomal exoglycosidases (increasing in inflammations) in comparison to hypertrophic lower nasal conchae suggests electrolytes disorders and questions the inflammatory background of nasal polyps.


Assuntos
Glucuronidase/metabolismo , Hexosaminidase A/metabolismo , Hexosaminidase B/metabolismo , Pólipos Nasais/enzimologia , Conchas Nasais/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hipertrofia/enzimologia , Masculino , Pessoa de Meia-Idade , Conchas Nasais/patologia , Adulto Jovem
13.
Mol Neurobiol ; 50(1): 159-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24356898

RESUMO

Sphingolipidoses are inherited genetic diseases due to mutations in genes encoding proteins involved in the lysosomal catabolism of sphingolipids. Despite a low incidence of each individual disease, altogether, the number of patients involved is relatively high and resolutive approaches for treatment are still lacking. The chaperone therapy is one of the latest pharmacological approaches to these storage diseases. This therapy allows the mutated protein to escape its natural removal and to increase its quantity in lysosomes, thus partially restoring the metabolic functions. Sandhoff disease is an autosomal recessive inherited disorder resulting from ß-hexosaminidase deficiency and characterized by large accumulation of GM2 ganglioside in brain. No enzymatic replacement therapy is currently available, and the use of inhibitors of glycosphingolipid biosynthesis for substrate reduction therapy, although very promising, is associated with serious side effects. The chaperone pyrimethamine has been proposed as a very promising drug in those cases characterized by a residual enzyme activity. In this review, we report the effect of pyrimethamine on the recovery of ß-hexosaminidase activity in cultured fibroblasts from Sandhoff patients.


Assuntos
Fibroblastos/efeitos dos fármacos , Hexosaminidase B/metabolismo , Chaperonas Moleculares/farmacologia , Pirimetamina/farmacologia , Doença de Sandhoff/tratamento farmacológico , Fibroblastos/enzimologia , Humanos , Chaperonas Moleculares/uso terapêutico , Pirimetamina/uso terapêutico , Doença de Sandhoff/enzimologia
14.
Pol Merkur Lekarski ; 34(203): 259-62, 2013 May.
Artigo em Polonês | MEDLINE | ID: mdl-23894776

RESUMO

UNLABELLED: Parenteral nutrition entails numerous metabolic complications resulting from food bypass of the gastrointestinal tract. Up to now have not been established all complications of parenteral nutrition, despite intensive research and clinical observations. Knowledge of the biochemical changes resulting from parenteral nutrition is essential to effective prevention, early detection and effective treatment of the metabolic disorders induced by parenteral nutrition. The aim of the study was to evaluate the catabolism of glycoconjugates of parenterally fed patients, reflected by the activity of N-acetyl-beta-D-hexosaminidase (HEX): HEX A and HEX B isoenzymes in serum and urine. MATERIAL AND METHODS: Samples of blood and urine were collected from 23 patients: before intravenous alimentation, at start, as well as of fifth and tenth day of parenteral nutrition. The activity of HEX A and HEX B in serum and urine was determined by the colorimetric method of Zwierz et al. as modified by Marciniak et al. The activity of urinary HEXA and HEX B has been calculated per 1 mg of creatinine. RESULTS: The activity of serum HEXA significantly decreased at fifth day, in comparison to the activity before parenteral alimentation, and significantly increased at tenth day of parenteral nutrition. The activity of HEX B in serum increased significantly at fifth and tenth day of the parenteral nutrition. CONCLUSIONS: Parenteral nutrition alter the catabolism of glycoconjugates, reflected by significant changes in serum HEX A and HEX B activities. Urine was the not appropriate material to evaluate the catabolism of glycoconjugates in view of HEX A and HEX B activities.


Assuntos
Hexosaminidase A/sangue , Hexosaminidase A/urina , Hexosaminidase B/sangue , Hexosaminidase B/urina , Nutrição Parenteral , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Doenças Metabólicas/etiologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/prevenção & controle , Pessoa de Meia-Idade , Nutrição Parenteral/efeitos adversos , Adulto Jovem
15.
Biosci Biotechnol Biochem ; 77(3): 497-504, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23470754

RESUMO

Lysosomal hexosaminidases are glycosyl hydrolases that remove the terminal hexosamine residues of glycoconjugates. Though mammalian hexosaminidases are well characterized, the biochemical nature of these enzymes among invertebrates remains elusive. In this study, we purified two thermostable N-acetyl ß-D-hexosaminidases (hex A and B) to homogeneity from soluble extracts of whole Unio animal tissue by a combination of chromatographic procedures. Purified hex A and hex B migrated as a single protein species on native PAGE and exhibited enzyme activity. However on SDS-PAGE, hex A dissociated into two subunits of molecular masses about 75 kDa and 30 kDa respectively, while hex B showed a molecular mass of 40 kDa. Hex A and B were recognized by the affinity purified mannose 6-phosphate receptor 46 on ligand blot analysis. This specific interaction was similar to what is known for the vertebrate receptors and lysosomal enzymes. The enzymes showed different K(M) values with respect to the substrates p-nitrophenyl N-acetyl-ß-D-glucosaminide and p-nitrophenyl N-acetyl-ß-D-galactosaminide. The enzymes were thermally stable up to 80 °C and showed pH optima between 5.0 and 6.0. This is the first report on the purification of two forms of hexosaminidases from Unio.


Assuntos
Hexosaminidase A/isolamento & purificação , Hexosaminidase A/metabolismo , Hexosaminidase B/isolamento & purificação , Hexosaminidase B/metabolismo , Lisossomos/enzimologia , Unio/citologia , Unio/enzimologia , Animais , Hexosaminidase A/química , Hexosaminidase B/química , Concentração de Íons de Hidrogênio , Cinética , Manosefosfatos/metabolismo , Solubilidade , Temperatura
16.
Mol Genet Metab ; 108(1): 70-5, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23266199

RESUMO

GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of ß-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a ß subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two ß subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins.


Assuntos
Modelos Animais de Doenças , Doenças do Cão/genética , Gangliosidoses GM2/genética , Hexosaminidase B/genética , Mutação de Sentido Incorreto , Animais , Sequência de Bases , Sondas de DNA , Cães , Feminino , Masculino , Linhagem , Reação em Cadeia da Polimerase
17.
Pol Arch Med Wewn ; 122(11): 551-6, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23111620

RESUMO

INTRODUCTION: Changes in the structure of membrane glycoconjugates and activity of glycosidases and proteases are important in tumor formation. OBJECTIVES: The aim of the study was to compare the specific activity of lysosomal exoglycosidases: N-acetyl-ß-D-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), ß-D-galactosidase (GAL), α-fucosidase (FUC), and α-mannosidase (MAN) with the activity of cathepsin D (CD) in serum, urine, and carcinoma tissue of patients with colon adenocarcinoma. PATIENTS AND METHODS: The specific activity of HEX, HEX A, HEX B, GAL, FUC, MAN, and CD was assayed in serum, urine, and carcinoma tissue of 12 patients with colon adenocarcinoma. RESULTS: Lysosomal exoglycosidases and CD have similar specific activity in colon adenocarcinoma tissue and urine, which is higher than their activity in serum (with the exception of the highest specific activity of CD in urine). A positive correlation was observed between the specific activity of CD and that of HEX, HEX A, FUC, and MAN in the carcinoma tissue and urine as well as between CD and GAL in the urine of patients with colon adenocarcinoma. Negative correlations were observed between protein levels and the specific activity of HEX, HEX A, FUC, MAN, and CD in the carcinoma tissue and urine, and between protein levels and GAL in urine. CONCLUSIONS: Increased degradation and remodeling of glycoconjugates in the colon adenocarcinoma tissue is reflected by increased specific activity of exoglycosidases and CD. The results suggest a strong effect of exoglycosidase action on tissue degradation and a potential role of exoglycosidases in the initiation of proteolysis.


Assuntos
Adenocarcinoma/enzimologia , Biomarcadores Tumorais/metabolismo , Catepsina D/metabolismo , Neoplasias do Colo/enzimologia , Lisossomos/metabolismo , Adenocarcinoma/metabolismo , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/metabolismo , Feminino , Hexosaminidase A/metabolismo , Hexosaminidase B/metabolismo , Humanos , Isoenzimas/metabolismo , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Sensibilidade e Especificidade , Soro/metabolismo , alfa-Manosidase/metabolismo , beta-Galactosidase/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo
18.
Gene ; 506(1): 25-30, 2012 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-22789865

RESUMO

The GM2 gangliosidoses are autosomal recessive lysosomal storage diseases caused by a deficiency of the ß-hexosaminidase A enzyme. This enzyme is composed of two polypeptide chains designated the α- and ß- subunits and it interacts with the GM2 activator protein. The HEXA and HEXB genes encode the α-subunit and the ß-subunit, respectively. Mutations in these genes are causative of Tay-Sachs disease (HEXA) and Sandhoff disease (HEXB). We analyzed the complete HEXA gene in 34 Spanish patients with Tay-Sachs disease and the HEXB gene in 14 Spanish patients with Sandhoff disease. We identified 27 different mutations, 14 of which were novel, in the HEXA gene and 14 different mutations, 8 of which unreported until now, in the HEXB gene, and we attempted to correlate these mutations with the clinical presentation of the patients. We found a high frequency of c.459+5G>A (IVS4+5G>A) mutation in HEXA affected patients, 22 of 68 alleles, which represent the 32.4%. This is the highest percentage found of this mutation in a population. All patients homozygous for mutation c.459+5G>A presented with the infantile form of the disease and, as previously reported, patients carrying mutation p.R178H in at least one of the alleles presented with a milder form. In HEXB affected patients, the novel deletion c.171delG accounts for 21.4% of the mutant alleles (6/28). All patients with this deletion showed the infantile form of the disease. The Spanish GM2 gangliosidoses affected patients show a great mutational heterogeneity as seen in other inherited lisosomal diseases in this country.


Assuntos
Hexosaminidase A/genética , Hexosaminidase B/genética , Mutação , Doença de Sandhoff/enzimologia , Doença de Sandhoff/genética , Doença de Tay-Sachs/enzimologia , Doença de Tay-Sachs/genética , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Fibroblastos/enzimologia , Frequência do Gene , Estudos de Associação Genética , Heterozigoto , Hexosaminidase A/sangue , Hexosaminidase A/metabolismo , Hexosaminidase B/sangue , Hexosaminidase B/metabolismo , Homozigoto , Humanos , Lactente , Leucócitos/enzimologia , Masculino , Mutagênese Insercional , Mutação de Sentido Incorreto , Deleção de Sequência , Espanha , Adulto Jovem
19.
Vet J ; 194(3): 412-6, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22766310

RESUMO

GM2 gangliosidosis variant 0 (Sandhoff disease, SD) is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations in the HEXB gene. Toy poodles recently were reported as the second breed of dog with SD. The present paper describes the molecular defect of this canine SD as the first identification of a pathogenic mutation in the canine HEXB gene. Genomic and complementary DNA sequences covering exonic regions of the canine HEXB gene, except exon 1, were analysed using DNA and RNA in an affected dog. A homozygous single base pair deletion of guanine in exon 3 was identified at nucleotide position 283 of the putative open reading frame (c.283delG). This mutation has the potential to cause a frameshift resulting in the alteration of valine at amino acid position 59 to a stop codon (p.V59fsX). Genotyping using the mutagenically separated PCR method demonstrated a correlation between phenotype and genotype in dogs with a pedigree related to the disease and that the mutation was rare in a randomly-selected population of toy poodles. These results strongly suggest that the deletion is pathogenic.


Assuntos
Doenças do Cão/genética , Mutação da Fase de Leitura , Hexosaminidase B/genética , Reação em Cadeia da Polimerase/métodos , Doença de Sandhoff/veterinária , Animais , Sequência de Bases , DNA Complementar/análise , Doenças do Cão/diagnóstico , Doenças do Cão/metabolismo , Cães , Éxons , Hexosaminidase B/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/veterinária , RNA/análise , Doença de Sandhoff/diagnóstico , Doença de Sandhoff/genética , Doença de Sandhoff/metabolismo
20.
J Neurosci ; 32(15): 5223-36, 2012 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-22496568

RESUMO

Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-ß peptide (Aß)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of ß-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aß-like immunoreactivity (iAß-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aß. In addition, we observed increased levels of Aß40 and Aß42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aß (GAß) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aß were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAß-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAß-LIR may be associated with the lysosomal-autophagic turnover of Aß and fragments of APP-containing Aß epitopes. Importantly, intraneuronal GAß immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Gangliosídeos/metabolismo , Hexosaminidase B/genética , Lisossomos/fisiologia , Doença de Sandhoff/patologia , Adulto , Animais , Western Blotting , Química Encefálica/genética , Química Encefálica/fisiologia , Pré-Escolar , Gangliosídeo G(M2)/metabolismo , Humanos , Imuno-Histoquímica , Lactente , Metabolismo dos Lipídeos , Bulbo/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Medula Espinal/metabolismo , Substância Negra/metabolismo , Adulto Jovem , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo
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