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ABSTRACT: Gaucher disease, a prevalent lysosomal storage disease, is caused by insufficient activity of acid β-glucosidase (GCase) and resultant glucosylceramide accumulation. Recently in Parkinson disease (PD) patients, heterozygous mutations in GCase have been associated with earlier onset and more progressive PD. To understand the pathogenic relationships between GCase variants and Parkinsonism, α-synuclein and ubiquitin distributions and levels in the brains of several mouse models containing GCase variants were evaluated by immunohistochemistry. Progressive α-synuclein and ubiquitin aggregate accumulations were observed in the cortex, hippocampus, basal ganglia, brainstem, and some cerebellar regions between 4 and 24 weeks in mice that were homozygous for GCase [D409H (9H) or V394L (4L)] variants and also had a prosaposin hypomorphic (PS-NA) transgene. In 4L/PS-NA and 9H/PS-NA mice, this was coincident with progressive neurological manifestations and brain glucosylceramide accumulation. Ultrastructural studies showed electron dense inclusion bodies in neurons and axons of 9H/PS-NA brains. α-synuclein aggregates were also observed in ventricular, brainstem, and cerebellar regions of older mice (>42-weeks) with the GCase variant (D409H/D409H) without overt neurological disease. In a chemically induced GCase deficiency, α-synuclein aggregates and glucosylceramide accumulation also occurred. These studies demonstrate a relationship between glucosylceramide accumulation and α-synuclein aggregates, and implicate glucosylceramide accumulation as risk factor for the α-synucleinopathies.
Molecular Genetics and Metabolism 12/2010; 102(4):436-47. · 3.19 Impact Factor
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T M Cox,
J M F G Aerts,
N Belmatoug,
M D Cappellini,
S vom Dahl,
J Goldblatt, G A Grabowski,
C E M Hollak,
P Hwu,
M Maas,
A M Martins,
P K Mistry,
G M Pastores,
A Tylki-Szymanska,
J Yee,
N Weinreb
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ABSTRACT: Enzyme replacement was introduced as treatment for non-neuronopathic Gaucher disease more than 15 years ago. To ensure the best use of this costly ultra-orphan agent, a systematic disease management approach has been proposed by an international panel; this includes the development, by consensus, of achievable treatment goals. Here we critically review these goals and monitoring guidelines and incorporate emerging experience of the disease in the therapeutic era, as well as contemporary clinical research. This review makes recommendations related specifically to the management of pregnancy; the appropriate use of splenectomy and bisphosphonate treatment; the relevance of biochemical markers to disease monitoring; and the use of semi-quantitative methods for assessing bone marrow infiltration. In addition, we identify key areas for development, including the requirement for a validated index of disease severity; the need to correlate widely used biomarkers with long-term disease outcomes, and the desirability of establishing agreed standards for monitoring of bone disease particularly in infants and children with Gaucher disease.
Journal of Inherited Metabolic Disease 07/2008; 31(3):319-36. · 3.58 Impact Factor
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Skeletal Radiology 04/2008; 37(3):185-8. · 1.54 Impact Factor
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ABSTRACT: Prosaposin has a central role in intracellular glycosphingolipid catabolism and also has extracellular functions. This locus is regulated temporally and spatially. The highest mRNA expression occurs in the central nervous system (CNS) and reproductive system. In vitro, the CNS-expressed proteins Sp4 and RORalpha bind to Sp1 and RORE sites within a 310-bp fragment directly upstream of the transcription start site. These transcription factors exhibit negative cooperativity in vitro for prosaposin expression. Mice deficient in RORalpha and Sp4 (Staggerer [Sg(-/-)] and Sp4 knockout [Sp4 KO], respectively) containing selected prosaposin promoter deletion transgenes were used in comparative expression studies to evaluate this negative cooperativity in vivo. Constructs containing the RORE or Sp1/U cluster alone were independently stimulatory. Deletion of the Sp1/U site led to a decrease in reporter activity only in the cerebellum of Sg(-/-) mice. The deletion of RORE and Sp1/U sites did alter the increase of reporter activity in the brain and eye, but not in the spinal cord, of Sg(-/-) mice. These results indicate that Sp4 and RORalpha play minor and major roles, respectively, in regional expression of the prosaposin locus in the brain, whereas expression in the spinal cord is independent of RORalpha.
DNA and Cell Biology 01/2002; 20(12):781-9. · 2.07 Impact Factor
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ABSTRACT: The hydrolysis of glucosylceramide (GC) to ceramide and glucose requires the action of the lysosomal enzyme, acid beta-glucosidase (GCase), encoded by gba in the mouse. Gaucher disease, an autosomal recessive disorder, results from the inherited deficiency of this enzyme. Although enzyme activity is present in all mammalian tissues, the patterns of mRNA expression have not been explored. In situ hybridization analyses of mouse embryonic, newborn, and adult tissues were conducted to evaluate the spectrum of gba mRNA expression. Signals were present in all tissues and cell types. Distinct patterns of differential expression were identified in specific tissues and cell types, and at defined developmental stages. Differential expression was first observed around E14 in the intestinal tract, kidneys, skeletal system, and skin. At E18, moderate intensity signals were in adipocytes of brown fat and pancreatic cells. Differential expression remained in skin, bone, and the GI tract postnatally. In the postnatal and adult animals increasing expression was observed throughout the CNS, esophageal epithelium, intestinal villi, pancreas, and thymus and lymph node capsular cells. These tissue-, cell-, and developmental stage-specific variations of the gba mRNA level indicate major developmentally regulated changes in the expression pattern of gba in the late gestational period and postnatally.
Molecular Genetics and Metabolism 01/2002; 74(4):426-34. · 3.19 Impact Factor
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ABSTRACT: Lysosomal acid lipase (LAL) is the critical enzyme for the hydrolysis of the triglycerides (TG) and cholesteryl esters (CE) delivered to lysosomes. Its deficiency produces two human phenotypes, Wolman disease (WD) and cholesteryl ester storage disease (CESD). A targeted disruption of the LAL locus produced a null (lal( -/-)) mouse model that mimics human WD/CESD. The potential for enzyme therapy was tested using mannose terminated human LAL expressed in Pichia pastoris (phLAL), purified, and administered by tail vein injections to lal( -/-) mice. Mannose receptor (MR)-dependent uptake and lysosomal targeting of phLAL were evidenced ex vivo using competitive assays with MR-positive J774E cells, a murine monocyte/macrophage line, immunofluorescence and western blots. Following (bolus) IV injection, phLAL was detected in Kupffer cells, lung macrophages and intestinal macrophages in lal( -/-) mice. Two-month-old lal( -/-) mice received phLAL (1.5 U/dose) or saline injections once every 3 days for 30 days (10 doses). The treated lal( -/-) mice showed nearly complete resolution of hepatic yellow coloration; hepatic weight decreased by approximately 36% compared to PBS-treated lal( -/-) mice. Histologic analyses of numerous tissues from phLAL-treated mice showed reductions in macrophage lipid storage. TG and cholesterol levels decreased by approximately 50% in liver, 69% in spleen and 50% in small intestine. These studies provide feasibility for LAL enzyme therapy in human WD and CESD.
Human Molecular Genetics 09/2001; 10(16):1639-48. · 7.64 Impact Factor
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ABSTRACT: Lysosomal acid lipase (LAL) is essential for the hydrolysis of triglycerides (TG) and cholesteryl esters (CE) in lysosomes. A mouse model created by gene targeting produces no LAL mRNA, protein, or enzyme activity. The lal-/- mice appear normal at birth, survive into adulthood, and are fertile. Massive storage of TG and CE is observed in adult liver, adrenal glands, and small intestine. The age-dependent tissue and gross progression in this mouse model are detailed here. Although lal-/- mice can be bred to give homozygous litters, they die at ages of 7 to 8 months. The lal-/- mice develop enlargement of a single mesenteric lymph node that is full of stored lipids. At 6;-8 months of age, the lal-/- mice have completely absent inguinal, interscapular, and retroperitoneal white adipose tissue. In addition, brown adipose tissue is progressively lost. The plasma free fatty acid levels are significantly higher in lal-/- mice than age-matched lal+/+ mice, and plasma insulin levels were more elevated upon glucose challenge. Energy intake was also higher in lal-/- male mice, although age-matched body weights were not significantly altered from age-matched lal+/+ mice. Early in the disease course, hepatocytes are the main storage cell in the liver; by 3;-8 months, the lipid-stored Kupffer cells progressively fill the liver. The involvement of macrophages throughout the body of lal-/- mice provide evidence for a critical nonappreciated role of LAL in cellular cholesterol and fatty acid metabolism, adipocyte differentiation, and fat mobilization.
The Journal of Lipid Research 05/2001; 42(4):489-500. · 5.56 Impact Factor
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ABSTRACT: Wolman disease is a lethal lysosomal storage disease due to deficiency of lysosomal acid lipase (LAL). Wolman disease is characterized by pronounced hepatic involvement while neurological symptoms are uncommon, making Wolman disease an attractive candidate for liver-directed gene therapy. This study was performed to test the effects of gene replacement in fibroblasts lacking LAL, using a recombinant adenovirus encoding the human LAL cDNA (AdhLAL). Human fibroblasts from a Wolman disease patient were infected with AdhLAL and showed a dose-dependent increase in LAL protein and activity up to 5-fold above levels in control fibroblasts. Furthermore, 72 hr after infection with AdhLAL there was a dose-dependent correction of the severe lipid storage phenotype of Wolman disease fibroblasts. Electron microscopy confirmed significant correction of the lysosomal lipid storage in AdhLAL-infected Wolman disease fibroblasts at the ultrastructural level. Intravenous injection of AdhLAL into wild-type mice resulted in a 13.5-fold increase in hepatic LAL activity, and overexpression of LAL was not associated with toxic side effects. These data demonstrate high-level lysosomal expression of recombinant LAL in vitro and in vivo and show the feasibility of gene therapeutic strategies for the treatment of Wolman disease.
Human Gene Therapy 03/2001; 12(3):279-89. · 4.22 Impact Factor
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Progress in Nucleic Acid Research and Molecular Biology 02/2001; 66:203-39. · 0.31 Impact Factor
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ABSTRACT: The expression of prosaposin is temporally and spatially regulated at the transcriptional and post-translational levels. In vitro, the mouse prosaposin promoter contains functional RORE [retinoic acid-receptor-related orphan receptor alpha subunit (RORalpha)-binding element], Sp1 and U (unknown) sites within 310 bp directly 5' to the transcription start site and additional elements within 2400 bp 5' to the transcription start site. To elucidate promoter regions important to tissue-preferential expression in vivo, transgenic mice were created with 5'-flanking deletions of the prosaposin gene fused to a luciferase reporter. Nearly exclusive expression was observed in cerebrum, cerebellum and eyes of adult transgenic mice containing constructs with 234-310 bp of 5'-flanking DNA. This central nervous system (CNS) expression was due to the presence of RORE and overlapping Sp1 sites in this region. Internal deletion of RORE and the Sp1 cluster from the longer constructs with 2400 bp of 5'-flanking DNA significantly diminished expression in the CNS. The appearance of substantial visceral tissue (e.g. liver, spleen, lung, kidney, thymus and heart) expression was obtained with transgenic mice bearing constructs with 742-2400 bp of 5'-flanking DNA. The cellular localization of luciferase reporter-gene expression from these constructs corresponded closely with that for prosaposin. These results define important CNS and visceral regulatory regions in the promoter in vivo and may be sufficient to account for the majority of prosaposin's tissue-preferential expression.
Biochemical Journal 01/2001; 352 Pt 2:549-56. · 4.90 Impact Factor
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ABSTRACT: Gaucher disease (GD) is associated with mutations at the acid beta-glucosidase (GCase) locus and the resultant defective activity of the enzyme product. GCase is a membrane-associated glycoprotein that requires detergents for extraction and phospholipid interfaces for full catalytic activity. Normal human fibroblasts and overexpressing transgenic cell lines were used to evaluate the intracellular disappearance, degradation, and secretion of human GCase, including GD fibroblasts and C2C12 cells transduced with MFG-GCase retrovirus and CHO cells stably transfected with the tetracycline transactivation conditional expression system (tet-CHO-GCase). Compared to HF, the disappearance of GCase from the transgenic cells was 12-30 times greater, and had degradative and secretory components. In tet-CHO-GCase cells the majority of GCase was secreted. Intracellular degradation occurred in compartments sensitive to monensin and brefeldin A, and the ALLN or leupeptin protease inhibitors, i.e., ER, Golgi, and lysosomes. In tet-CHO-GCase cells, GCase degradation and secretion rates were inversely related to expression level. Saponin permeabilization analyses of tet-CHO-GCase cells showed that a majority of GCase was soluble, with a rapid disappearance via secretion and degradation. A progressively increasing proportion of GCase became saponin insoluble with a t(1/2) = 2-3 h. Intracellular saponin-soluble and -insoluble GCases were degraded with t(1/2) approximately 2 and 14 h, respectively. Confocal microscopy showed colocalization of glycosylated or unglycosylated GCase with LAMP-2, an integral lysosomal membrane protein, to vesicular bodies. These studies show that GCase secretion was N-linked glycosylation dependent, whereas sorting to and membrane attachment in the lysosome were N-linked glycosylation independent.
Molecular Genetics and Metabolism 09/2000; 70(4):281-94. · 3.19 Impact Factor
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ABSTRACT: Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, an inherited metabolic prototype for enzyme and gene therapy. An 80-kDa mammalian cytoplasmic protein (TCP80/NF90) was discovered to interact with the GCase mRNA coding region and inhibit its translation in vitro and ex vivo. Human TCP80/NF90 is identical to NF90, an IL-2 enhancer protein, and MPP4, an M-phase phosphoprotein. The interaction of recombinant TCP80/NF90 with GCase mRNA was evaluated using the baculovirus/Sf9 insect cell system since these cells lack this protein. Purified recombinant and isolated mammalian cytoplasmic TCP80/NF90 had identical functions including binding of coding regions of selected RNAs and inhibition of their in vitro translation. Individual baculoviruses containing the human TCP80/NF90 cDNA (vSf9/TCP80) and GCase cDNA (vSf9/GCase) were used to co-infect Sf9 cells. The presence of preformed TCP80/NF90 significantly (>87%) inhibited wild-type GCase mRNA translation in these cells, but baculovirus containing a mutant GCase did not. Sf9 cells co-infected with vSf9/TCP80 showed a major reduction of GCase RNA polysome association. These results show that the multifunctional protein, TCP80/NF90, can function in vivo as a translation inhibitory protein and include alterations of mRNA binding to polysomes as a component of its mechanism of action.
Molecular Genetics and Metabolism 06/2000; 70(2):106-15. · 3.19 Impact Factor
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ABSTRACT: Acid beta-glucosidase (GCase) is the enzyme deficient in Gaucher disease, a prototypical inherited metabolic error for enzyme and gene therapy. An 80-kDa cytoplasmic protein, termed TCP80, was found to inhibit GCase mRNA translation in mammalian cells by binding to RNA-coding regions. The TCP80 cDNA was cloned by screening an expression library with the GCase-coding region RNA. The cDNA sequence was nearly identical to those for M-phase phosphoprotein (MPP4; 99%) and for the IL-2 enhancer binding protein (NF90; 96%). Expression of the carboxy-terminal third, TCP30, showed it to be an RNA-binding protein that bound to a 184-nt fragment of GCase-coding sequence near the 5' end of the mature mRNA. When added to reactions, a large molar excess of TCP30 diminished the translation inhibition of GCase RNA by cytoplasmic TCP80. TCP50, expressed from the NH(2)-terminal two-thirds of TCP80, did not bind to nor inhibit the translation of GCase RNA. Reconstitution of in vitro translation inhibition of GCase RNA required intact human TCP80 heterologously expressed in insect cells. Time course analyses show that TCP80 functions at the initiation phase of GCase mRNA translation, probably by inhibiting its binding to polysomes. Seven additional RNAs were isolated by specific binding to TCP30 including those for aldolase B, complement protein 8 gamma-subunit, fibronectin receptor beta1, ABL, lactate dehydrogenase A, fibrinogen gamma-chain, and peroxisomal proliferator-activated receptor alpha. In vitro translation of their RNAs was inhibited by TCP80. These studies show that TCP80 has RNA-binding (TCP30) and inhibitory (TCP50) domains that function to modulate translation of several mRNAs. TCP80 is likely identical to MPP4 and NF90, but has previously undescribed roles in cellular function.
Molecular Genetics and Metabolism 12/1999; 68(4):441-54. · 3.19 Impact Factor
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ABSTRACT: Acid alpha-glucosidase (GAA) cleaves the alpha1-4 and alpha1-6 glycosidic linkages of glycogen and related alpha-glucosyl substrates within lysosomes. Its deficiency results in glycogen storage disease type II (GSDII) variants including Pompe disease. To gain insight into the tissue patterns of involvement by glycogen storage in GSDII, GAA mRNA expression in mouse tissues was evaluated by Northern blot and in situ hybridization analyses. Extensive temporal and spatial variation of GAA mRNA was observed. During preterm maturation, GAA mRNA levels of whole mice progressively increased as assessed by Northern analysis. By in situ hybridization with GAA antisense mRNA, low signals were detected in most tissues throughout gestation. However, increased expression in specific cell types of different tissues was observed beginning at 16 days post coitum in developing brain neurons, primitive inner ear cells, and seminiferous tubular epithelium. In adult mice, whole-organ GAA mRNA levels were highest in brain, moderate in heart, liver, and skeletal muscle, and lowest in the series kidney > lung > testis > spleen. By in situ hybridization, the highest-intensity signals were in neurons of the central and peripheral nervous systems whereas neuroglial cells had only low-level signal. Signals of moderate intensity were in cardiomyocytes whereas low signals were in hepatocytes and skeletal muscle myocytes and very low in cells of the lungs, thymus, pancreas, spleen, and adrenal glands. However, testicular Sertoli cells and kidney tubular epithelial cells had significant signals even though surrounding cells had very low signals. The discrete temporal and spatial variations of GAA mRNA during development indicate different physiological roles for this enzyme in various cell types and developmental stages.
American Journal Of Pathology 04/1999; 154(4):1089-96. · 4.89 Impact Factor
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ABSTRACT: Prosaposin is a multifunctional protein that encodes four glycoproteins, named saposins A, B, C and D. They participate in the catabolism of glycosphingolipids in lysosomes. When secreted, intact prosaposin may function as a neuritogenic factor. Human and mouse prosaposin displayed similar temporal and spatial regulation of expression. To gain insight into the transcriptional regulation of this locus, the 5' region was characterized from the human prosaposin gene. The putative human promoter was shown to be TATA-less, i.e. it belonged to the TATA-less housekeeping gene family. The transcription initiation sites were localized to -23, -27, -31 and -83bp 5' to ATG, compared to -87 and -94bp in the mouse. In SK-N-SH neuroblastoma cells, positive regulatory elements were detected -343 to -813bp upstream of ATG. A negative regulatory region existed between -813 and -2500bp using SK-N-SH, H441 and NS20Y cells. EMSA and DNA-footprint analysis showed that Sp1 and Sp3 are involved in human prosaposin gene regulation. Compared to the mouse promoter, the human promoter is missing a Sp1 cluster within a 310-bp upstream segment, and has AP-1, Oct-1 and two RORalpha sites that are protected from DNaseI by selected nuclear extracts.
Gene 10/1998; 218(1-2):37-47. · 2.34 Impact Factor
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ABSTRACT: Lysosomal acid lipase (LAL) is essential for the hydrolysis of the triglycerides and cholesteryl esters in lysosomes. Its deficiency produces two phenotypes, a severe infantile-onset variant, Wolman disease (WD), and a later onset variant, cholesteryl ester storage disease (CESD). A mouse model with a LAL null mutation was produced by targeting disruption of the mouse gene. Homozygote knockout mice (lal -/lal-) produce no LAL mRNA, protein or enzyme activity. The lal-/lal- mice are born in Mendelian ratios, are normal appearing at birth, and follow normal development into adulthood. However, massive accumulation of triglycerides and cholesteryl esters occurs in several organs. By 21 days, the liver develops a yellow-orange color and is approximately 1.5-2.0x larger than normal. The accumulated cholesteryl esters and triglycerides are approximately 30-fold greater than normal. The lal+/lal- mice have approximately 50% of normal LAL activity and do not show lipid accumulation. Male and female lal-/lal- mice are fertile and can be bred to produce progeny. This mouse model is a phenotypic model of human CESD, and a biochemical and histopathologic mimic of human WD. The lal-/lal- mice provide a model to determine the role of LAL in lipid metabolism and the pathogenesis of its deficiency states.
Human Molecular Genetics 10/1998; 7(9):1347-54. · 7.64 Impact Factor
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ABSTRACT: Acid beta-glucosidase is a lysosomal membrane protein that cleaves the O-beta-D-glucosidic linkage of glucosylceramide and aryl-beta-glucosides. Full activity reconstitution of the pure enzyme requires phospholipids and saposin C, an 80 aa activator protein. The deficiency of the enzyme or activator leads to Gaucher disease. A conformational change of acid beta-glucosidase is shown to accompany activity reconstitution by selected phospholipids or, particularly, phospholipid/saposin C complexes by intrinsic fluorescence spectral shifts, fluorescence quenching, and circular dichroism (CD). Negatively charged phospholipid (NCP) interfaces with unsaturated fatty acid acyl chains (UFAC) induced concordant blue-shifts in tryptophanyl fluorescence spectra and a loss of beta-strand structure by CD. The enzyme required an unsaturated fatty acid acyl chain in proximity (10-11 A) within liposomal membranes for activation, fluorescence blue-shifts, and changes in CD spectra. Activity enhancements were greatest when UFAC and the negatively charged headgroup were present on the same phospholipid. NCPs with UFAC protected the enzyme from fluorescence quenching by aqueous agents (I-, Cs+, acrylamide, TEMPO). Phosphatidylcholine with doxyl spin-labeled fatty acid acyl chains at carbons 7, 10, or 16 quenched enzyme fluorescence only when in NCP/PC liposomes. Saposin C (Trp-free) induced additional activity and fluorescence spectral changes in the enzyme only in the presence of NCP liposomes containing UFA. CD spectral changes indicated saposin C and acid beta-glucosidase interaction only in the presence of NCPs with UFA. These studies show that acid beta-glucosidase requires interfaces composed of NCPs, containing UFAC, for penetration into the outer leaflet of membranes. Furthermore, this interaction induces essential conformational changes for saposin C binding and further enhancement of acid beta-glucosidase catalytic activity.
Biochemistry 09/1998; 37(33):11544-54. · 3.42 Impact Factor
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ABSTRACT: Gaucher disease was first described by Philippe Gaucher in his 1882 medical thesis. Gaucher's original concept was of an unusual epithelioma of the spleen. By the early 1900s, Mandelbaum recognized the systemic nature of the disease. Several children with Gaucher disease were described at the turn of the century, but Rusca described a rapidly progressive fatal neurodegenerative type of disease, i.e. type 2, in the 1920s. The 'juvenile' form (type 3) of the disease was described in Sweden in the 1950s. In 1965, the deficient enzyme, acid beta-glucosidase, was discovered and the lysosomal nature of the disease was elucidated. Currently, three variants of Gaucher disease have been defined clinically and are distinguished by the presence and severity of neuronopathic involvement (Table 1). Each of these clinical types has substantial phenotypic variation, but types 1 and 3 have significantly heterogeneous rates of disease progression and degrees of visceral organs involvement. The neuronopathic involvement in type 3 also has substantial variation in the age of onset and disease progression even within relatively isolated communities. An extensive review of the clinical and pathologic involvement by Gaucher disease is available.
Blood Reviews 07/1998; 12(2):115-33. · 5.36 Impact Factor
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ABSTRACT: Prosaposin is the precursor of four low molecular weight sphingolipid-activating proteins (SAPs) or saposins. These four proteins function as intracellular activators of several lysosomal enzymes involved in the degradation of glycosphingolipids, and prosaposin itself has neurite outgrowth effects. Expression of prosaposin is regulated in a temporal and spatial manner with expression in specific brain neurons and visceral cell types. Here a major regulatory fragment was characterized within 310 bp 5' to the transcription start site. Using electrophoretic mobility shift assay (EMSA) and DNA footprinting, members of the Sp family (Sp1, Sp3, and Sp4), the orphan nuclear receptor (RORalpha), and an unknown transcription factor (U; TGGGGGAG) were shown to bind to this region. To evaluate the role of such transcription factor binding sites for this locus, a series of mutant constructs was generated within this region, and their function was evaluated in cultured NS20Y neuroblastoma cells. A 3' Sp1 site, a 5' Sp1/U cluster and the RORalpha binding sites were functional. The data are consistent with a model in which the factors that bind to the Sp1/U cluster and RORE site interact negatively to diminish promoter activity to a background level that is determined primarily by the 3' Sp1 site. These interactions depend on the tissue-specific repertoire of transcription factors leading to differential expression of this locus.
Journal of Biological Chemistry 06/1998; 273(21):13208-16. · 4.77 Impact Factor
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ABSTRACT: The cDNA for acid beta-glucosidase, the Gaucher disease enzyme, was overexpressed in a variety of mammalian cells and in Sf9 insect cells. Whether overexpressed from the MFG-GC retrovirus or the tetracycline transactivator system, there was a large discrepancy between the amounts of mRNA (>750-fold) and acid beta-glucosidase protein (approximately 6- to 14-fold) produced in mammalian cells. This was not observed in Sf9 insect cells. Quantitative evaluation of translation of this mRNA in intact mammalian cells indicated a 55- to 135-fold inefficiency in cell lines compared to normal human skin fibroblasts. In vitro translation efficiency with acid beta-glucosidase mRNAs from overexpressing mammalian or insect cells was similar to that from normal human fibroblasts. A cytoplasmic, heat labile protein was suggested as inhibitory to in vitro translation of these RNAs. North-Western blots and cytoplasmic depletion experiments showed this to be an 80-kDa cytoplasmic mRNA-binding protein that recognized acid beta-glucosidase coding sequences. The cytoplasmic protein was not detected in insect cells. These results implicate acid beta-glucosidase coding sequences and a heat labile cytoplasmic protein in modulating the translation of overexpressed mRNA in transgenic cell lines.
Molecular Genetics and Metabolism 06/1998; 64(2):87-98. · 3.19 Impact Factor
