Separation of molecular species of glucosylceramide by high performance liquid chromatography of their benzoyl derivatives. (1/509)

The method of separation of glucosylceramide by HPLC was reported. Glucosylceramide was perbenzoylated and separated on a packed muBondapack C18 column, using methanol as eluting solvent. The pattern obtained by HPLC closely resembled that obtained by GLC of the TMS-glucosylceramide, and reflected the molecular species of fatty acid components. This method is reproducible, and sensitive as GLC. This method also can be used for analysis of higher glycolipids.  (+info)

Immunosurveillance of alglucerase enzyme therapy for Gaucher patients: induction of humoral tolerance in seroconverted patients after repeat administration. (2/509)

Alglucerase, a macrophage-targeted enzyme replacement therapy for Gaucher disease, has been successfully used for several years to improve clinical symptoms and reverse disease progression. As part of an immunosurveillance program, 1,122 Gaucher patients were monitored for antibody response to glucocerebrosidase, the active component of alglucerase. Seroconversion was detected in 142 patients (12.8%) by enzyme-linked immunosorbent assay (ELISA) and confirmed by radioimmunoprecipitation. The majority (75%) of the seroconverted population had no detectable levels of circulating inhibitory antibody as assessed by in vitro inhibition of enzymatic activity of the therapeutic molecule. Of the remaining patients with putative inhibitory antibodies, the majority had only low levels of serum inhibitory activity, which was transient. A very small number of patients were identified as developing true neutralizing antibodies, as defined by the development of antibodies that impacted clinical efficacy. Many of the patient antibody responses were also diminished with time. Eighty-two of the 142 seroconverted patients have stopped producing antibody to the molecule and appear tolerized. The mean time for humoral tolerization was 28 months from initiation of therapy. Of 64 seroconverted patients followed for at least 30 months of therapy, the tolerization rate was 93%. These results show that although 12.8% of the patients on therapy developed antibodies to the molecule, 90% of these patients became tolerized over time.  (+info)

Non-pseudogene-derived complex acid beta-glucosidase mutations causing mild type 1 and severe type 2 gaucher disease. (3/509)

Gaucher disease is an autosomal recessive inborn error of glycosphingolipid metabolism caused by the deficient activity of the lysosomal hydrolase, acid beta-glucosidase. Three phenotypically distinct subtypes result from different acid beta-glucosidase mutations encoding enzymes with absent or low activity. A severe neonatal type 2 variant who presented with collodion skin, ichthyosis, and a rapid neurodegenerative course had two novel acid beta-glucosidase alleles: a complex, maternally derived allele, E326K+L444P, and a paternally inherited nonsense mutation, E233X. Because the only other non-pseudogene-derived complex allele, D140H+E326K, also had the E326K lesion and was reported in a mild type 1 patient with a D140H+E326K/K157Q genotype, these complex alleles and their individual mutations were expressed and characterized. Because the E233X mutation expressed no activity and the K157Q allele had approximately 1% normal specific activity based on cross-reacting immunologic material (CRIM SA) in the baculovirus system, the residual activity in both patients was primarily from their complex alleles. In the type 1 patient, the D140H+E326K allele was neuroprotective, encoding an enzyme with a catalytic efficiency similar to that of the N370S enzyme. In contrast, the E326K+L444P allele did not have sufficient activity to protect against the neurologic manifestations and, in combination with the inactive E233X lesion, resulted in the severe neonatal type 2 variant. Thus, characterization of these novel genotypes with non-pseudogene-derived complex mutations provided the pathogenic basis for their diverse phenotypes.  (+info)

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice. (4/509)

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.  (+info)

A comparison of the pharmacological properties of carbohydrate remodeled recombinant and placental-derived beta-glucocerebrosidase: implications for clinical efficacy in treatment of Gaucher disease. (5/509)

The objective of these studies was to characterize the macrophage mannose receptor binding and pharmacological properties of carbohydrate remodeled human placental-derived and recombinant beta-glucocerebrosidase (pGCR and rGCR, respectively). These are similar but not identical molecules that were developed as enzyme replacement therapies for Gaucher disease. Both undergo oligosaccharide remodeling during purification to expose terminal mannose sugar residues. Competitive binding data indicated carbohydrate remodeling improved targeting to mannose receptors over native enzyme by two orders of magnitude. Mannose receptor dissociation constants (Kd) for pGCR and rGCR were each 13 nmol/L. At 37 degrees C, 95% of the total macrophage binding was mannose receptor specific. In vivo, pGCR and rGCR were cleared from circulation by a saturable pathway. The serum half-life (t1/2) was 3 minutes when less than saturable amounts were injected intravenously (IV) into mice. Twenty minutes postdose, beta-glucocerebrosidase activity increased over endogenous levels in all tissues examined. Fifty percent of the injected activity was recovered. Ninety-five percent of recovered activity was in the liver. Parenchymal cells (PC), Kupffer cells (KC), and liver endothelium cells (LEC) were responsible for 75%, 22%, and 3%, respectively, of the hepatocellular uptake of rGCR and for 76%, 11%, and 12%, respectively, of the hepatocellular uptake of pGCR. Both molecules had poor stability in LEC and relatively long terminal half-lives in PC (t1/2 = 2 days) and KC (t1/2 = 3 days).  (+info)

Properties of beta-glucosidase in cultured skin fibroblasts from controls and patients with Gaucher disease. (6/509)

Membrane-bound beta-glucosidase from cultured skin fibroblasts can be solubilized in an active form by treatment of membrane preparations with a mixture of Triton X-100 and sodium taurocholate. Several properties of the solubilized enzyme have been studied in fibroblasts from normal, healthy individuals and from 14 patients with different clinical forms of Gaucher disease. The patients studied were classified as follows: group 1 consisted of 10 chronic patients, all (with one exception) of Ashkenazi Jewish origin; group 2 consisted of three black American patients with severe visceral symptoms, manifest from early childhood, but with no apparent neurological involvement; and group 3 consisted of a single white patient with the classical infantile form of the disease. Specific beta-glucosidase activity ranged from 6.6% to 16.5% mean control value in group 1 patients and from 4.1% to 5.8% in groups 2 and 3. When compared with the enzyme from control fibroblasts, the enzyme from chronic Gaucher patients (group 1) was more rapidly inactivated at 50 degrees C, had an altered pH curve, was less effectively inhibited by deoxycorticosterone-beta-glucoside, and was more effectively inhibited by deoxycorticosterone. The enzyme from patients in groups 2 and 3 was qualitatively indistinguishable from the control enzyme in terms of these parameters. No differences in Km (4-methylumbelliferyl-beta-glucoside) or sedimentation coefficient were found between the beta-glucosidases from control and Gaucher cells. The results demonstrate that cells from Ashkenazi Jewish patients with the chronic form of Gaucher disease contain a structurally altered form of beta-glucosidase. This enzyme differs both from normal beta-glucosidase and from the residual enzyme in patients of different ethnic origin and with clinically more severe forms of the disease.  (+info)

Enzyme therapy in Gaucher disease type 2: an autopsy case. (7/509)

A Japanese patient with Gaucher disease type 2 was treated with enzyme therapy, alglucerase, from 7 to 22 months of age. Whereas hematologic parameters were normalized and hepatosplenomegaly was alleviated, no improvement in neurologic symptoms occurred, and the patient died of respiratory failure at age 22 months. Postmortem examination revealed massive intra-alveolar infiltration of Gaucher cells in lungs and in the central nervous system, i.e., the presence of Gaucher cells in the perivascular Virchow-Robins spaces in the cortex and deep white matter and extensive lamilar necrosis with reactive proliferation of blood vessels and macrophage infiltration of the cerebral cortex. It is suggested that enzyme therapy, with thus far recommended dose, does not prevent long-term respiratory and central nervous system involvement in severe varients of Gaucher disease.  (+info)

Accuracy of preimplantation diagnosis of single-gene disorders by polar body analysis of oocytes. (8/509)

PURPOSE: A number of pitfalls in single-cell DNA analysis, including undetected DNA contamination, undetected allele drop out, and preferential amplification, may lead to misdiagnosis in preimplantation genetic diagnosis of single-gene disorders. METHODS: Preimplantation genetic diagnosis was performed by sequential first and second polar body analysis of oocytes in 26 couples at risk for having children with various single-gene disorders. Mutant genes were amplified simultaneously with linked polymorphic markers, and only embryos resulting from the mutation-free oocytes predicted by polar body analysis with confirmation by polymorphic marker testing were transferred back to patients. RESULTS: Overall 529 oocytes from 48 clinical cycles (26 patients) were tested, resulting in the transfer of 106 embryos in 44 clinical cycles. As many as 46 (9.6%) instances of allele dropout were observed, the majority (96%) of which were detected. Seventeen unaffected pregnancies were established, of which nine resulted in the birth of an unaffected child, and the rest are ongoing. CONCLUSIONS: A high accuracy of preimplantation genetic diagnosis of single-gene disorders is achieved by application of sequential analysis of the first and second polar body and multiplex polymerase chain reaction.  (+info)