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1.
Hum Mol Genet ; 22(12): 2435-50, 2013 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-23446636

RESUMO

Individual saposin A (A-/-) and saposin B (B-/-)-deficient mice show unique phenotypes caused by insufficient degradation of myelin-related glycosphingolipids (GSLs): galactosylceramide and galactosylsphingosine and sulfatide, respectively. To gain insight into the interrelated functions of saposins A and B, combined saposin AB-deficient mice (AB-/-) were created by knock-in point mutations into the saposins A and B domains on the prosaposin locus. Saposin A and B proteins were undetectable in AB-/- mice, whereas prosaposin, saposin C and saposin D were expressed near wild-type (WT) levels. AB-/- mice developed neuromotor deterioration at >61 days and exhibited abnormal locomotor activity and enhanced tremor. AB-/- mice (~96 days) lived longer than A-/- mice (~85 days), but shorter than B-/- mice (~644 days). Storage materials were observed in Schwann cells and neuronal processes by electron microscopy. Accumulation of p62 and increased levels of LC3-II were detected in the brainstem suggesting altered autophagy. GSL analyses by (liquid chromatography) LC/MS identified substantial increases in lactosylceramide in AB-/- mouse livers. Sulfatide accumulated, but galactosylceramide remained at WT levels, in the AB-/- mouse brains and kidneys. Brain galactosylsphingosine in AB-/- mice was ~68% of that in A-/- mice. These findings indicate that combined saposins A and B deficiencies attenuated GalCer-ß-galactosylceramidase and GM1-ß-galactosidase functions in the degradation of lactosylceramide preferentially in the liver. Blocking sulfatide degradation from the saposin B deficiency diminished galactosylceramide accumulation in the brain and kidney and galctosylsphingosine in the brain. These analyses of AB-/- mice continue to delineate the tissue differential interactions of saposins in GSL metabolism.


Assuntos
Glicoesfingolipídeos/metabolismo , Doenças do Sistema Nervoso/metabolismo , Saposinas/deficiência , Animais , Encéfalo/metabolismo , Feminino , Galactosilceramidase/metabolismo , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/psicologia , Especificidade de Órgãos , Fenótipo , Saposinas/genética , beta-Galactosidase/metabolismo
2.
Mol Genet Metab ; 112(3): 229-41, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24837159

RESUMO

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TG) and cholesteryl esters (CE) in lysosomes. Mutations of the LIPA gene lead to Wolman disease (WD) and cholesterol ester storage disease (CESD). The disease hallmarks include hepatosplenomegaly and extensive storage of CE and/or TG. The effects of intravenous investigational enzyme therapy (ET) on survival and efficacy were evaluated in Lipa knock out, lal-/- mice with advanced disease using recombinant human LAL (rhLAL). Comparative ET was conducted with lower doses (weekly, 0.8 and 3.2mg/kg) beginning at 16 weeks (study 1), and with higher dose (10mg/kg) in early (8-weeks), middle (16-weeks) and late (24-weeks) disease stages (study 2). In study 1, rhLAL extended the life span of lal-/- mice in a dose dependent manner by 52 (0.8 mg/kg) or 94 (3.2mg/kg) days. This was accompanied by partial correction of cholesterol and TG levels in spleen and liver. In study 2, the high dose resulted in a significant improvement in organ size (liver, spleen and small intestine) and tissue histology as well as significant decreases in cholesterol and TG in all three groups. In the treated livers and spleens the cholesterol and TG levels were reduced to below treatment initiation levels indicating a reversal of disease manifestations, even in advanced disease. ET diminished liver fibrosis and macrophage proliferation. These results show that LAL deficiency can be improved biochemically and histopathologically by various dosages of ET, even in advanced disease.


Assuntos
Doença de Wolman/metabolismo , Doença de Wolman/patologia , Animais , Peso Corporal , Modelos Animais de Doenças , Terapia de Reposição de Enzimas , Feminino , Metabolismo dos Lipídeos , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Fenótipo , Esterol Esterase/administração & dosagem , Resultado do Tratamento , Doença de Wolman/tratamento farmacológico , Doença de Wolman/genética , Doença de Wolman/mortalidade , Doença de Wolman
3.
Hum Mol Genet ; 19(6): 1088-97, 2010 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-20047948

RESUMO

Gaucher disease is caused by defective acid beta-glucosidase (GCase) function. Saposin C is a lysosomal protein needed for optimal GCase activity. To test the in vivo effects of saposin C on GCase, saposin C deficient mice (C-/-) were backcrossed to point mutated GCase (V394L/V394L) mice. The resultant mice (4L;C*) began to exhibit CNS abnormalities approximately 30 days: first as hindlimb paresis, then progressive tremor and ataxia. Death occurred approximately 48 days due to neurological deficits. Axonal degeneration was evident in brain stem, spinal cord and white matter of cerebellum accompanied by increasing infiltration of the brain stem, cortex and thalamus by CD68 positive microglial cells and activation of astrocytes. Electron microscopy showed inclusion bodies in neuronal processes and degenerating cells. Accumulation of p62 and Lamp2 were prominent in the brain suggesting the impairment of autophagosome/lysosome function. This phenotype was different from either V394L/V394L or C-/- alone. Relative to V394L/V394L mice, 4L;C* mice had diminished GCase protein and activity. Marked increases (20- to 30-fold) of glucosylsphingosine (GS) and moderate elevation (1.5- to 3-fold) of glucosylceramide (GC) were in 4L;C* brains. Visceral tissues had increases of GS and GC, but no storage cells were found. Neuronal cells in thick hippocampal slices from 4L;C* mice had significantly attenuated long-term potentiation, presumably resulting from substrate accumulation. The 4L;C* mouse mimics the CNS phenotype and biochemistry of some type 3 (neuronopathic) variants of Gaucher disease and is a unique model suitable for testing pharmacological chaperone and substrate reduction therapies, and investigating the mechanisms of neuronopathic Gaucher disease.


Assuntos
Doença de Gaucher/enzimologia , Glucosilceramidase/genética , Glucosilceramidas/metabolismo , Proteínas Mutantes/metabolismo , Doenças do Sistema Nervoso/complicações , Psicosina/análogos & derivados , Saposinas/deficiência , Substituição de Aminoácidos/genética , Animais , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Cromatografia Líquida , Modelos Animais de Doenças , Doença de Gaucher/genética , Doença de Gaucher/patologia , Doença de Gaucher/fisiopatologia , Inflamação/complicações , Inflamação/patologia , Potenciação de Longa Duração/fisiologia , Longevidade , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Espectrometria de Massas , Camundongos , Doenças do Sistema Nervoso/enzimologia , Doenças do Sistema Nervoso/patologia , Doenças do Sistema Nervoso/fisiopatologia , Fenótipo , Psicosina/metabolismo , Saposinas/metabolismo
4.
Hum Mol Genet ; 19(4): 634-47, 2010 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-20015957

RESUMO

Saposins A, B, C and D are derived from a common precursor, prosaposin (psap). The few patients with saposin C deficiency develop a Gaucher disease-like central nervous system (CNS) phenotype attributed to diminished glucosylceramide (GC) cleavage activity by acid beta-glucosidase (GCase). The in vivo effects of saposin C were examined by creating mice with selective absence of saposin C (C-/-) using a knock-in point mutation (cysteine-to-proline) in exon 11 of the psap gene. In C-/- mice, prosaposin and saposins A, B and D proteins were present at near wild-type levels, but the saposin C protein was absent. By 1 year, the C-/- mice exhibited weakness of the hind limbs and progressive ataxia. Decreased neuromotor activity and impaired hippocampal long-term potentiation were evident. Foamy storage cells were observed in dorsal root ganglion and there was progressive loss of cerebellar Purkinje cells and atrophy of cerebellar granule cells. Ultrastructural analyses revealed inclusions in axonal processes in the spinal cord, sciatic nerve and brain, but no excess of multivesicular bodies. Activated microglial cells and astrocytes were present in thalamus, brain stem, cerebellum and spinal cord, indicating regional pro-inflammatory responses. No storage cells were found in visceral organs of these mice. The absence of saposin C led to moderate increases in GC and lactosylceramide (LacCer) and their deacylated analogues. These results support the view that saposin C has multiple roles in glycosphingolipid (GSL) catabolism as well as a prominent function in CNS and axonal integrity independent of its role as an optimizer/stabilizer of GCase.


Assuntos
Sistema Nervoso Central/metabolismo , Doença de Gaucher/metabolismo , Glucosilceramidase/metabolismo , Saposinas/deficiência , Animais , Comportamento Animal , Sistema Nervoso Central/citologia , Sistema Nervoso Central/enzimologia , Modelos Animais de Doenças , Feminino , Doença de Gaucher/enzimologia , Doença de Gaucher/genética , Doença de Gaucher/fisiopatologia , Glucosilceramidase/genética , Glicoesfingolipídeos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células de Purkinje/metabolismo
5.
Hum Mol Genet ; 17(15): 2345-56, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18480170

RESUMO

Saposin B derives from the multi-functional precursor, prosaposin, and functions as an activity enhancer for several glycosphingolipid (GSL) hydrolases. Mutations in saposin B present in humans with phenotypes resembling metachromatic leukodystrophy. To gain insight into saposin B's physiological functions, a specific deficiency was created in mice by a knock-in mutation of an essential cysteine in exon 7 of the prosaposin locus. No saposin B protein was detected in the homozygotes (B-/-) mice, whereas prosaposin, and saposins A, C and D were at normal levels. B-/- mice exhibited slowly progressive neuromotor deterioration and minor head tremor by 15 months. Excess hydroxy and non-hydroxy fatty acid sulfatide levels were present in brain and kidney. Alcian blue positive (sulfatide) storage cells were found in the brain, spinal cord and kidney. Ultrastructural analyses showed lamellar inclusion material in the kidney, sciatic nerve, brain and spinal cord tissues. Lactosylceramide (LacCer) and globotriaosylceramide (TriCer) were increased in various tissues of B-/- mice supporting the in vivo role of saposin B in the degradation of these lipids. CD68 positive microglial cells and activated GFAP positive astrocytes showed a proinflammatory response in the brains of B-/- mice. These findings delineate the roles of saposin B for the in vivo degradation of several GSLs and its primary function in maintenance of CNS function. B-/- provide a useful model for understanding the contributions of this saposin to GSL metabolism and homeostasis.


Assuntos
Encéfalo/metabolismo , Glucosilceramidase/metabolismo , Glicoesfingolipídeos/metabolismo , Saposinas/fisiologia , Medula Espinal/metabolismo , Animais , Encéfalo/ultraestrutura , Feminino , Homozigoto , Rim/metabolismo , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Mutação Puntual , Saposinas/genética , Medula Espinal/ultraestrutura
6.
Sci Rep ; 9(1): 5571, 2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30944381

RESUMO

Gaucher disease is caused by mutations in GBA1 encoding acid ß-glucosidase (GCase). Saposin C enhances GCase activity and protects GCase from intracellular proteolysis. Structure simulations indicated that the mutant GCases, N370S (0 S), V394L (4L) and D409V(9V)/H(9H), had altered function. To investigate the in vivo function of Gba1 mutants, mouse models were generated by backcrossing the above homozygous mutant GCase mice into Saposin C deficient (C*) mice. Without saposin C, the mutant GCase activities in the resultant mouse tissues were reduced by ~50% compared with those in the presence of Saposin C. In contrast to 9H and 4L mice that have normal histology and life span, the 9H;C* and 4L;C* mice had shorter life spans. 9H;C* mice developed significant visceral glucosylceramide (GC) and glucosylsphingosine (GS) accumulation (GC¼GS) and storage macrophages, but lesser GC in the brain, compared to 4L;C* mice that presents with a severe neuronopathic phenotype and accumulated GC and GS primarily in the brain. Unlike 9V mice that developed normally for over a year, 9V;C* pups had a lethal skin defect as did 0S;C* mice resembled that of 0S mice. These variant Gaucher disease mouse models presented a mutation specific phenotype and underscored the in vivo role of Saposin C in the modulation of Gaucher disease.


Assuntos
Doença de Gaucher/genética , Glucosilceramidase/genética , Mutação/genética , Saposinas/deficiência , beta-Glucosidase/genética , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Glucosilceramidas/genética , Camundongos , Camundongos Endogâmicos C57BL , Fenótipo
7.
BMC Neurosci ; 9: 76, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18673548

RESUMO

BACKGROUND: Prosaposin encodes, in tandem, four small acidic activator proteins (saposins) with specificities for glycosphingolipid (GSL) hydrolases in lysosomes. Extensive GSL storage occurs in various central nervous system regions in mammalian prosaposin deficiencies. RESULTS: Our hypomorphic prosaposin deficient mouse, PS-NA, exhibited 45% WT levels of brain saposins and showed neuropathology that included neuronal GSL storage and Purkinje cell loss. Impairment of neuronal function was observed as early as 6 wks as demonstrated by the narrow bridges tests. Temporal transcriptome microarray analyses of brain tissues were conducted with mRNA from three prosaposin deficient mouse models: PS-NA, prosaposin null (PS-/-) and a V394L/V394L glucocerebrosidase mutation combined with PS-NA (4L/PS-NA). Gene expression alterations in cerebrum and cerebellum were detectable at birth preceding the neuronal deficits. Differentially expressed genes encompassed a broad spectrum of cellular functions. The number of down-regulated genes was constant, but up-regulated gene numbers increased with age. CCAAT/enhancer-binding protein delta (CEBPD) was the only up-regulated transcription factor in these two brain regions of all three models. Network analyses revealed that CEBPD has functional relationships with genes in transcription, pro-inflammation, cell death, binding, myelin and transport. CONCLUSION: These results show that: 1) Regionally specific gene expression abnormalities precede the brain histological and neuronal function changes, 2) Temporal gene expression profiles provide insights into the molecular mechanism during the GSL storage disease course, and 3) CEBPD is a candidate regulator of brain disease in prosaposin deficiency to participate in modulating disease acceleration or progression.


Assuntos
Encefalopatias/genética , Proteína delta de Ligação ao Facilitador CCAAT/genética , Perfilação da Expressão Gênica , Saposinas/deficiência , Fatores Etários , Animais , Encefalopatias/fisiopatologia , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/fisiologia , Cerebelo/patologia , Cerebelo/fisiopatologia , Cérebro/patologia , Cérebro/fisiopatologia , Análise por Conglomerados , Modelos Animais de Doenças , Regulação para Baixo/genética , Genes Reguladores , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Análise em Microsséries/métodos , Análise em Microsséries/estatística & dados numéricos , Atividade Motora/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saposinas/genética , Regulação para Cima/genética
8.
Trends Mol Med ; 10(12): 599-606, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15567330

RESUMO

Pyocyanin (PCN) is a blue redox-active secondary metabolite that is produced by Pseudomonas aeruginosa. PCN is readily recovered in large quantities in sputum from patients with cystic fibrosis who are infected by P. aeruginosa. Despite in vitro studies demonstrating that PCN interferes with multiple cellular functions, its importance during clinical infection is uncertain. This is partially caused by the difficulty in defining the contribution of PCN among the numerous virulence factors produced by P. aeruginosa during infection. In addition, few cellular pathways that are affected by PCN are known. This review briefly highlights recent advances that might clarify the role of PCN in P. aeruginosa pathogenesis.


Assuntos
Fibrose Cística/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/metabolismo , Animais , Sequência Conservada , Evolução Molecular , Humanos , Camundongos , Pneumonia/metabolismo , Pneumonia/microbiologia , Infecções por Pseudomonas/tratamento farmacológico , Piocianina/antagonistas & inibidores , Piocianina/genética , Virulência , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
9.
PLoS One ; 10(3): e0118771, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25822147

RESUMO

Gaucher disease (GD) is caused by insufficient activity of acid ß-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD, induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs, NPCs, and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition, GD2 neurons showed increased α-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons, but intriguingly, those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes, sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings, providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge, this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease.


Assuntos
Fibroblastos/citologia , Doença de Gaucher/patologia , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Neurônios/citologia , Células Cultivadas , Fibroblastos/metabolismo , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Glucosilceramidas/metabolismo , Humanos , Potenciais da Membrana , Células-Tronco Neurais/metabolismo , Neurogênese , Neurônios/metabolismo , Neurônios/fisiologia , Psicosina/análogos & derivados , Psicosina/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
10.
PLoS One ; 6(4): e19037, 2011 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-21533102

RESUMO

The pharmacological chaperone, isofagomine (IFG), enhances acid ß-glucosidase (GCase) function by altering folding, trafficking, and activity in wild-type and Gaucher disease fibroblasts. The in vivo effects of IFG on GCase activity, its substrate levels, and phenotype were evaluated using a neuronopathic Gaucher disease mouse model, 4L;C* (V394L/V394L + saposin C-/-) that has CNS accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) as well as progressive neurological deterioration. IFG administration to 4L;C* mice at 20 or 600 mg/kg/day resulted in life span extensions of 10 or 20 days, respectively, and increases in GCase activity and protein levels in the brain and visceral tissues. Cerebral cortical GC and GS levels showed no significant reductions with IFG treatment. Increases of GC or GS levels were detected in the visceral tissues of IFG treated (600 mg/kg/day) mice. The attenuations of brain proinflammatory responses in the treated mice were evidenced by reductions in astrogliosis and microglial cell activation, and decreased p38 phosphorylation and TNFα levels. Terminally, axonal degeneration was present in the brain and spinal cord from untreated and treated 4L;C* mice. These data demonstrate that IFG exerts in vivo effects by enhancing V394L GCase protein and activity levels, and in mediating suppression of proinflammation, which led to delayed onset of neurological disease and extension of the life span of 4L;C* mice. However, this was not correlated with a reduction in the accumulation of lipid substrates.


Assuntos
Modelos Animais de Doenças , Doença de Gaucher/tratamento farmacológico , Imino Piranoses/uso terapêutico , Animais , Glicosídeo Hidrolases/antagonistas & inibidores , Imino Piranoses/farmacologia , Inflamação/tratamento farmacológico , Camundongos , Fosforilação , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
11.
PLoS One ; 4(10): e7320, 2009 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-19809509

RESUMO

Gaucher disease is a lysosomal storage disease caused by mutations in acid beta-glucosidase (GCase) leading to defective hydrolysis and accumulation of its substrates. Two L-type calcium channel (LTCC) blockers-verapamil and diltiazem-have been reported to modulate endoplasmic reticulum (ER) folding, trafficking, and activity of GCase in human Gaucher disease fibroblasts. Similarly, these LTCC blockers were tested with cultured skin fibroblasts from homozygous point-mutated GCase mice (V394L, D409H, D409V, and N370S) with the effect of enhancing of GCase activity. Correspondingly, diltiazem increased GCase protein and facilitated GCase trafficking to the lysosomes of these cells. The in vivo effects of diltiazem on GCase were evaluated in mice homozygous wild-type (WT), V394L and D409H. In D409H homozygotes diltiazem (10 mg/kg/d via drinking water or 50-200 mg/kg/d intraperitoneally) had minor effects on increasing GCase activity in brain and liver (1.2-fold). Diltiazem treatment (10 mg/kg/d) had essentially no effect on WT and V394L GCase protein or activity levels (<1.2-fold) in liver. These results show that LTCC blockers had the ex vivo effects of increasing GCase activity and protein in the mouse fibroblasts, but these effects did not translate into similar changes in vivo even at very high drug doses.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Glucosilceramidase/metabolismo , Animais , Diltiazem/farmacologia , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Homozigoto , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Mutação , Pele/efeitos dos fármacos , Verapamil/farmacologia
12.
Hum Mol Genet ; 16(8): 957-71, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17353235

RESUMO

Saposins (A, B, C and D) are approximately 80 amino acid stimulators of glycosphingolipid (GSL) hydrolases that derive from a single precursor, prosaposin. In both humans and mice, prosaposin/saposin deficiencies lead to severe neurological deficits. The CD-/- mice with saposin C and D combined deficiencies were produced by introducing genomic point mutations into a critical cysteine in each of these saposins. These mice develop a severe neurological phenotype with ataxia, kyphotic posturing and hind limb paralysis. Relative to prosaposin null mice ( approximately 30 days), CD-/- mice had an extended life span ( approximately 56 days). Loss of Purkinje cells was evident after 6 weeks, and storage bodies were present in neurons of the spinal cord, brain and dorsal root ganglion. Electron microscopy showed well-myelinated fibers and axonal inclusions in the brain and sciatic nerve. Marked accumulations of glucosylceramides and alpha-hydroxy ceramides were present in brain and kidney. Minor storage of lactosylceramide (LacCer) was observed when compared with tissues from the prosaposin null mice, suggesting a compensation in LacCer degradation by saposin B for the saposin C deficiency. Skin fibroblasts and tissues from CD-/- mice showed an increase of intracellular prosaposin, impaired prosaposin secretion, deficiencies of saposins C and D and decreases in saposins A and B. In addition, the deficiency of saposin C in CD-/- mice resulted in cellular decreases of acid beta-glucosidase activity and protein. This CD null mouse model provides a tool to explore the in vivo functional interactions of saposins in GSL metabolism and lysosomal storage diseases, and prosaposin's physiological effects.


Assuntos
Ceramidas/metabolismo , Doença de Gaucher/genética , Glucosilceramidas/metabolismo , Saposinas/genética , Animais , Células Cultivadas , Doença de Gaucher/mortalidade , Doença de Gaucher/patologia , Regulação da Expressão Gênica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteína Básica da Mielina/genética , Fenótipo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Saposinas/metabolismo
13.
Cell Microbiol ; 8(7): 1121-33, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16819965

RESUMO

Pseudomonas aeruginosa (PA) is a major pathogen causing morbidity and ultimately mortality in patients afflicted with cystic fibrosis (CF) lung disease. One important virulence factor, pyocyanin (PCN), is a blue, redox-active compound that is secreted in such copious amounts by PA in the CF lungs that it determines the colour of expectorated sputum. In this study, we discovered that physiological concentrations of PCN inactivate the airway epithelial vacuolar ATPase, resulting in reduced expression and trafficking of the cystic fibrosis transmembrane conductance regulator in cultured lung and primary nasal epithelial cells. Our study supports the notion that PCN contributes significantly to the pathogenesis of CF and other bronchiectasis patients infected by PA.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/microbiologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/farmacologia , Mucosa Respiratória/microbiologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Canais de Cálcio Tipo L/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Células Cultivadas , Regulador de Condutância Transmembrana em Fibrose Cística/análise , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Transporte Proteico/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Canais de Sódio/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismo
14.
Proc Natl Acad Sci U S A ; 100(24): 14315-20, 2003 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-14605211

RESUMO

Pseudomonas aeruginosa produces copious amounts of the redoxactive tricyclic compound pyocyanin that kills competing microbes and mammalian cells, especially during cystic fibrosis lung infection. Cross-phylum susceptibility to pyocyanin suggests the existence of evolutionarily conserved physiological targets. We screened a Saccharomyces cerevisiae deletion library to identify presumptive pyocyanin targets with the expectation that similar targets would be conserved in humans. Fifty S. cerevisiae targets were provisionally identified, of which 60% have orthologous human counterparts. These targets encompassed major cellular pathways involved in the cell cycle, electron transport and respiration, epidermal cell growth, protein sorting, vesicle transport, and the vacuolar ATPase. Using cultured human lung epithelial cells, we showed that pyocyanin-mediated reactive oxygen intermediates inactivate human vacuolar ATPase, supporting the validity of the yeast screen. We discuss how the inactivation of V-ATPase may negatively impact the lung function of cystic fibrosis patients.


Assuntos
Pseudomonas aeruginosa/patogenicidade , Piocianina/toxicidade , Apoptose/efeitos dos fármacos , Linhagem Celular , Farmacorresistência Fúngica , Transporte de Elétrons/efeitos dos fármacos , Genes Bacterianos , Genes Fúngicos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Estresse Oxidativo/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo
15.
Infect Immun ; 72(7): 4275-8, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15213173

RESUMO

Pseudomonas aeruginosa secretes copious amounts of the redox-active phenazine, pyocyanin (PCN), during cystic fibrosis lung infection. PCN has been shown to interfere with a variety of cellular processes in cultured lung epithelial cells. Here, by using two respiratory tract models of infection, we demonstrate that PCN mediates tissue damage and necrosis during lung infection.


Assuntos
Pneumopatias/metabolismo , Pulmão/microbiologia , Pseudomonas aeruginosa/patogenicidade , Piocianina/metabolismo , Animais , Pulmão/patologia , Pneumopatias/microbiologia , Camundongos , Mutação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Piocianina/genética
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