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At least 541 records · Page 30Linked to original sources
PubMed1990

GM2-gangliosidosis. Clinical and biochemical aspects of four cases.

We discuss four cases of GM2-gangliosidosis. In one of them the biochemical diagnostic confirmation was difficult. This case revealed striking discrepancies between the results of different methods of enzyme assay. The hexosaminidase A determination based on pH inactivation is not always reliable; assay with natural substrate may be necessary. However, the results with the newly developed substrate 4-MU-GlcNac-6-SO4 are promising and it seems to be a good alternative to the traditional (pH or heat) inactivation procedures. The deficiency can be shown in leukocytes, plasma and fibroblasts with the 6-sulfated substrate. The carrier state seems better reflected in plasma hexosaminidase A than in leukocyte hexaminidase A levels.

Adult↗
PubMed1984

High- and low-uptake forms of beta-hexosaminidase in human platelets. Selective retention of the high-uptake form during stimulation with thrombin.

Human platelets are rich in beta-hexosaminidase and other acid hydrolases contained in organelles (lysosomes) distinct from alpha-granules and dense granules. Incubation of platelets with bovine or human thrombin (100 U/ml for 5 min at 37 degrees C) induces the secretion of 100% of the contents of alpha- and dense granules, but only 40-60% of total beta-hexosaminidase from lysosomes. Both isozymes Hex A and Hex B are secreted in the same proportion as found intracellularly. There is no selective recapture or plasma membrane binding by platelets of secreted beta-hexosaminidase. The secreted enzyme is of the low-uptake type, i.e., it is poorly recognized by the phosphomannosyl receptor-mediated uptake mechanism of fibroblasts, while the retained enzyme is a 3-fold higher uptake form. Preincubation of platelets with NH4Cl (10 mM, 2 h), followed by thrombin stimulation, results in secretion of all beta-hexosaminidase as a low-uptake form. The data support the hypothesis that there are secretory and nonsecretory forms of lysosomes. The secretory lysosomes would contain low-uptake forms of hydrolases in addition to acid phosphatase, while the nonsecretory lysosomes would contain high-uptake hydrolases and be acid phosphatase-deficient. Conditions where the contents of both lysosomal populations were released together, i.e., amine treatment followed by thrombin induction, or extraction of unstimulated cells, would result in the exposure of high-uptake phosphomannosylated hydrolases released from one population of lysosomes to acid phosphatase released from the second population of lysosomes with their subsequent conversion to low-uptake forms.

Blood Platelets↗
PubMed1985

Soluble sphingomyelinase from human urine as antigen for obtaining anti-sphingomyelinase antibodies.

A soluble form of lysosomal sphingomyelinase was partially purified from human urine using concanavalin A-Sepharose 4B, Sephadex G-100 and octyl-Sepharose 4B chromatography. The octyl-Sepharose 4B eluate was used to immunise a rabbit. The antiserum obtained was able to precipitate about 70% of the sphingomyelinase activity present in urine from control subjects. Both the immunoprecipitable and non-precipitable activities were found to be deficient in urine from patients with Niemann-Pick disease Type A and Type B. In contrast, both activities were present in urine from patients with Niemann-Pick disease Type C. The antiserum was able to precipitate about 80% of the sphingomyelinase activity present in an aqueous extract of placenta.

Animals↗
PubMed1985

The effect of detergents on immunoprecipitability of lysosomal sphingomyelinase.

Antibodies raised against the soluble form of acid sphingomyelinase from human urine and placenta are able to precipitate about 70% of the sphingomyelinase activity present in preparations of urinary sphingomyelinase. In contrast, no precipitation of sphingomyelinase activity occurs in detergent-containing preparations from placenta or splenic membranes. The formation of immune complexes between the antibodies and urinary sphingomyelinase is inhibited if detergents are added. With the non-ionic detergent Triton X-100 significant inhibition occurs only above the critical micellar concentration of the detergent. With the anionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (Chaps) substantial inhibition is already observed below the critical micellar concentration of the detergent.

Animals↗
PubMed1994

Characterization of the murine beta-hexosaminidase (HEXB) gene.

The murine HEXB gene, encoding the beta-subunit of the lysosomal hydrolase, beta-hexosaminidase, was isolated from a mouse cosmid library as a single cosmid clone. The entire gene spans 22 kb, considerably less than the 40 kb spanned by its human counterpart. It is highly homologous to the human gene. The 14 intron-exon junctions are entirely conserved, although the intron sequences diverge rapidly. Upstream of the coding region, a 1.3 kb segment was sequenced and shown to function as a promoter when fused with a reporter gene and expressed in monkey COS-7 cells. A short sequence (100 bp), near the start of the coding region, exhibits strong homology to the human HEXB promoter. Analysis of the tissue distribution of the HEXB mRNA in 129/Sv male mice revealed up to 28-fold tissue-specific variations in transcript levels. The kidney and the epididymis had the highest mRNA levels consistent with past surveys of enzyme activity.

Amino Acid Sequence↗
PubMed1996

Topology of glycosphingolipid degradation.

Glycosphingolipids (GSLs) form cell-type-specific patterns on the surface of eukaryotic cells. Degradation of plasma-membrane-derived GSLs in the lysosomes after internalization through the endocytic pathway is achieved through the concerted actions of hydrolysing enzymes and sphingolipid activator proteins. The latter are proteins necessary for the degradation of GSLs possessing short oligosaccharide chains. Some activator proteins bind to GSLs and form water-soluble complexes, which lift out of the membrane and give the water-soluble hydrolysing enzymes access to the regions of the GSL that would otherwise be obscured by the membrane. The inherited deficiency of both lysosomal hydrolases and sphingolipid activator proteins gives rise to sphingolipid storage diseases. An analysis of these diseases suggests a new model for the topology of endocytosis and lysosomal digestion, which is discussed in this article.

Journal Article↗
PubMed2005

Evaluation of cases of severe statin-related transaminitis within a large health maintenance organization.

PURPOSE: To describe the rate, potential causes, symptoms, time to onset, and time to resolution of severe transaminitis associated with increased 3-hydroxy-3-methylglutaryl coenzyme reductase inhibitor ("statin") usage in a large group model health maintenance organization. SUBJECTS AND METHODS: Health plan members 18 years of age and older, not receiving chemotherapy, who had received at least 1 statin prescription between January 1, 1997, and December 31, 2001 were eligible. All eligible patients with an alanine aminotransferase greater than 10 times the upper limit of normal at any time during the study period were identified using computerized laboratory records. Medical records were subsequently reviewed in order to determine whether the elevation was attributable to statin therapy. RESULTS: Alanine aminotransferase had never been measured in 2334 of 25334 (9%) of eligible patients. In the remaining 23000 patients, 62 (0.3%) were identified with an alanine aminotransferase greater than 10 times the upper limit of normal during the 5-year study period. Of these, 17 (0.1% of 23000 patients) had severe transaminitis deemed directly attributable to statin use. All except 4 of these 17 cases were associated with drug interactions. In 16 cases, transaminitis resolved upon statin discontinuation. CONCLUSIONS: In the observed study sample, statin-related severe transaminitis occurred infrequently. These findings support less frequent liver enzyme monitoring for most patients on statins. Continued monitoring remains warranted for patients on concomitant medications or those with comorbid conditions.

Adult↗
PubMed2005

Chemokines bind to sulfatides as revealed by surface plasmon resonance.

Chemokines bind to sulfated cell surface glycosaminoglycans and thereby modulate signaling mediated by G-protein-coupled seven-transmembrane domain chemokine receptors. Similar to glycosaminoglycans, sulfated oligosaccharides are also exposed on the cell surface by sulfatides, a class of glycosphingolipids. We have now identified sulfated glycosphingolipids (sulfatides) as novel binding partners for chemokines. Using surface plasmon resonance (SPR), the binding of proinflammatory and homeostatic chemokines to glycosphingolipids, in particular sulfatides, was investigated. Chemokines were immobilized while glycosphingolipids or additional phospholipids incorporated into liposomes were applied as soluble analytes. A specific affinity of the chemokines MCP-1/CCL2, IL-8/CXCL8, SDF-1alpha/CXCL12, MIP-1alpha/CCL3 and MIP-1beta/CCL4 to the sulfatides SM4s, SM3, SM2a and SB2, SB1a was detected. No significant interactions with the chemokines were observed for gangliosides, neutral glycosphingolipids or phospholipids. Chemokine receptors have been associated with the detergent-insoluble fraction supposed to contain 'rafts', i.e., glycosphingolipid enriched microdomains of the cell surface. Accordingly, the data suggest that early chemokine receptor signaling may take place in the vicinity of sulfated glycosphingolipids on the cell surface, whereby these sulfatides could modulate the chemokine receptor-mediated cell activation signal.

Animals↗
PubMed2006

Sphingolipid metabolism diseases.

Human diseases caused by alterations in the metabolism of sphingolipids or glycosphingolipids are mainly disorders of the degradation of these compounds. The sphingolipidoses are a group of monogenic inherited diseases caused by defects in the system of lysosomal sphingolipid degradation, with subsequent accumulation of non-degradable storage material in one or more organs. Most sphingolipidoses are associated with high mortality. Both, the ratio of substrate influx into the lysosomes and the reduced degradative capacity can be addressed by therapeutic approaches. In addition to symptomatic treatments, the current strategies for restoration of the reduced substrate degradation within the lysosome are enzyme replacement therapy (ERT), cell-mediated therapy (CMT) including bone marrow transplantation (BMT) and cell-mediated "cross correction", gene therapy, and enzyme-enhancement therapy with chemical chaperones. The reduction of substrate influx into the lysosomes can be achieved by substrate reduction therapy. Patients suffering from the attenuated form (type 1) of Gaucher disease and from Fabry disease have been successfully treated with ERT.

Humans↗
PubMed2006

Apoptotic vesicles crossprime CD8 T cells and protect against tuberculosis.

CD8 T lymphocytes are important effectors in protective immunity against Mycobacterium tuberculosis. We recently characterized the detour pathway of CD8 T cell activation in tuberculosis mediated by apoptotic vesicles from infected cells that transport mycobacterial antigens to dendritic cells (DCs). Here we demonstrate that apoptotic vesicles from mycobacteria-infected macrophages stimulate CD8 T cells in vivo. Homing of DCs to draining lymph nodes was critically required for effective crosspriming. Subsequent fate of vesicle-associated antigens in recipient DCs was characterized by endosomal mechanisms predominating over proteasomal processing. In addition, vesicle processing depended on the presence of saposins to disintegrate apoptotic membranes. Apoptotic vesicles displayed potent adjuvant activity by stimulating through Toll-like receptors (TLR). Ultimately, vaccination with vesicles from infected cells induced protection against M. tuberculosis infection. Taken together, we propose the detour pathway to represent a genuine immunological mechanism mediating crosspriming of CD8 T cells in vivo and protection against tuberculosis.

Adjuvants, Immunologic↗
PubMed2007

Clinical and molecular analysis of GM2 gangliosidosis in two apparent littermate kittens of the Japanese domestic cat.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.

Animals↗
PubMed2005

Atherosclerotic risk factor control in patients with peripheral arterial disease.

BACKGROUND: The presence of peripheral arterial disease (PAD), even in the absence of overt coronary artery disease (CAD), confers the same relative risk of death from a cardiovascular cause as in patients with a previous cardiovascular event. Current guidelines recommend atherosclerotic risk factor-reduction strategies in PAD patients identical to those in patients with a recent coronary event. The purpose of this study was to determine the status of atherosclerotic risk factor control in patients with PAD. METHODS: We analyzed the records of patients treated at 2 regional clinics serving 92,940 individuals. Full examination, laboratory, and pharmacy data were available for all patients. Pharmacy data were analyzed to determine prescriptions for beta-blocker therapy, angiotensin-converting enzyme inhibitors, and lipid-lowering agents. Lipid control was assessed through fasting lipid data. Glycemic control in diabetics was evaluated by using hemoglobin A 1c levels. RESULTS: We administratively identified 2839 patients with a diagnosis of PAD. The exclusion of 1106 patients with a diagnosis of CAD or validated not to have PAD resulted in a cohort of 1733 patients. Of these, 33.1% (574/1733) were currently receiving beta-blockers, 28.9% (500/1733) were receiving an angiotensin-converting enzyme inhibitor, and 31.3% (543/1733) were receiving a statin. Most patients (92%; 1594/1733) had a recent blood pressure recorded. However, 56% (893/1594) had a systolic blood pressure of 130 mm Hg or higher, 45.5% (726/1594) had a diastolic blood pressure of 80 mm Hg or higher, and 13.6% (217/1594) had a diastolic blood pressure of 90 mm Hg or higher. Screening fasting lipid profiles were found in 62.6% (1085/1733) of patients, 56% (508/912) had a low-density lipoprotein of 100 mg/dL or higher, and 21% (187/912) had a value of more than 130 mg/dL. In patients with diabetes, a hemoglobin A 1c level of 7.0% or higher was found in 54.2% (198/365) of patients. CONCLUSIONS: Despite national consensus of PAD as a CAD equivalent, patients are currently undertreated with regard to atherosclerotic risk factor modification. Until broader recognition of this disease process exists, vascular surgeons must continue to champion medical as well as surgical treatments for these patients.

Adrenergic beta-Antagonists↗
PubMed2006

Effect of a clinical pharmacy service on lipid control in patients with peripheral arterial disease.

OBJECTIVE: Our group and others have previously established that patients with peripheral artery disease (PAD) are significantly undertreated with respect to overall cardiovascular risk factor management, despite national guidelines to the contrary. In an effort to maximize risk factor control in our patients with PAD, we established a pharmacist-managed, physician-monitored algorithmic approach to the outpatient management of lipids in patients with PAD. The purpose of this study was to determine the effect of this service on lipid screening and control in patients with PAD. METHODS: We analyzed the records of patients treated at a large, group-model, not-for-profit regional managed care system serving approximately 405,000 members. An electronic medical record provided full examination, laboratory, and pharmacy data for all patients. Pharmacy data were analyzed to determine prescriptions for lipid-lowering agents. Lipid control was assessed through fasting lipid data. Patients with validated PAD and the absence of clinical coronary artery disease (CAD) were offered the service between May 2003 and September 2004 and followed up for a minimum of 6 months. RESULTS: We administratively identified 5159 active patients with a diagnosis of PAD. Of these, 1075 could be validated with a noninvasive arterial study. The exclusion of 384 patients with a diagnosis of CAD resulted in a cohort of 691 patients. Of these, 90 patients were enrolled in the lipid service (study group), and 601 received standard care. Mean follow-up was 17.1 months. Screening fasting lipid profiles were found in 95.6% (86/90) of patients in the study group and only 66.9% (402/601) of the standard care patients (P < .0001). Low-density lipoprotein cholesterol (LDL-C) control was improved in the pharmacist-managed group, with 79.1% (68/86) achieving an LDL-C of less than 100 mg/dL in comparison to the standard care group (54.8% [219/400]; P < .0001). An LDL-C value of more than 130 mg/dL was noted in 1.2% and 14.0% (56/400) in the treatment and control groups, respectively (P < .001). Statin use was present in 51.9% (312/601) of the control group patients and 84.4% (76/90) of the pharmacist-managed group (P < .001). CONCLUSIONS: Despite national consensus of PAD as a CAD equivalent, patients are currently undertreated with regard to atherosclerotic risk factor modification. Initiation of a pharmacist-managed, physician-monitored lipid service provides improved compliance with national guidelines.

Aged↗
PubMed2003

Toxicity of glucosylsphingosine (glucopsychosine) to cultured neuronal cells: a model system for assessing neuronal damage in Gaucher disease type 2 and 3.

Patients with Gaucher disease have been classified as type 1 nonneuronopathic, type 2 acute neuronopathic, and type 3 chronic neuronopathic phenotypes. Increased quantities of glucocerebroside and glucosylsphingosine (glucopsychosine) are present in the brain of type 2 and type 3 Gaucher patients. Galactosylsphingosine has previously been shown to be neurotoxic in globoid cell leukodystrophy (Krabbe disease). To determine whether glucosylsphingosine is also neurotoxic, we examined its effect on cultured cholinergic neuron-like LA-N-2 cells. When these cells were exposed to 1, 5, or 10 microM glucosylsphingosine for a period of 18 h, they became shriveled, neurite outgrowth was suppressed, and the activities of the lysosomal enzymes glucocerebrosidase, sphingomyelinase, and beta-galactosidase were reduced in a dose-dependent manner. Acetylcholine in cells exposed to glucosylsphingosine also declined. Cells switched to glucosylsphingosine-free medium partially recovered. The data suggest that accumulation of glucosylsphingosine contributes to neuronal dysfunction and destruction in patients with neuronopathic Gaucher disease.

Acetylcholine↗
PubMed2004

Specific downregulation and mistargeting of the lipid raft-associated protein MAL in a glycolipid storage disorder.

Metachromatic leukodystrophy (MLD) is a lysosomal lipid storage disease caused by arylsulfatase A deficiency. In MLD patients the sphingolipid sulfatide increasingly accumulates leading to progressive demyelination. We have analysed arylsulfatase A-deficient mice, a MLD mouse model, and we show that accumulation of sulfatide is not restricted to the lysosomal compartment but also occurs in myelin itself. Although, this sulfatide storage did not affect the overall composition of most myelin proteins, it specifically caused a severe reduction of MAL. This demonstrates a regulatory link between sulfatide accumulation and MAL expression and indicates the existence of regulatory mechanisms between lipid and myelin protein synthesis in oligodendrocytes. In addition, in cultured renal epithelial cells, sulfatide accumulation diverts MAL to the late endosomal/lysosomal compartment and thus also affects the intracellular distribution of MAL. The specific reduction and mistargeting of MAL protein as a reaction to sulfatide overload may contribute to the pathogenic mechanisms in metachromatic leukodystrophy.

Animals↗
PubMed2004

Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.

The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule.

Chromatography↗