Atypical GM1 ganglioside accumulation in a case of juvenile amaurotic idiocy

The brain and liver from a 7-year-old Japanese girl with juvenile amaurotic idiocy were examined neuropathologically and biochemically. Visceromegaly and skeletal abnormalities were absent. Nerve cells in the central nervous system were swollen and contained fine fat granules. Electronmicroscopically, there were large numbers of irregular bodies in the perikarya and these corresponded to the curvilinear and membranous cytoplasmic bodies.Lipid analysis of the brain revealed that GM₁ ganglioside was increased in the parietal and occipital areas, while the frontal lobe showed a normal ganglioside pattern. N-Acetyl neuraminic acid (NANA) content in all areas was not elevated. Determinations of β-galactosidase activity were within normal ranges. The liver had no accumulation of GM₁ ganglioside and showed a normal β-galactosidase activity. These unusual findings in GM₁ gangliosidosis were discussed.     

A Bicyclic 1-Deoxygalactonojirimycin Derivative as a Novel Pharmacological Chaperone for GM1 Gangliosidosis

Lysosomal β-galactosidase (β-Gal) deficiency causes a group of disorders that include neuronopathic GM₁ gangliosidosis and non-neuronopathic Morquio B disease. We have previously proposed the use of small molecule ligands of β-Gal as pharmacological chaperones (PCs) for the treatment of GM₁ gangliosidosis brain pathology. Although it is still under development, PC therapy has yielded promising preclinical results in several lysosomal diseases. In this study, we evaluated the effect of bicyclic 1-deoxygalactonojirimycin (DGJ) derivative of the sp²-iminosugar type, namely 5N,6S-(N′-butyliminomethylidene)-6-thio-1- deoxygalactonojirimycin (6S-NBI-DGJ), as a novel PC for human mutant β-Gal. In vitro, 6S-NBI-DGJ had the ability to inhibit the activity of human β-Gal in a competitive manner and was able to protect this enzyme from heat-induced degradation. Computational analysis supported that the rigid glycone bicyclic core of 6S-NBI-DGJ binds to the active site of the enzyme, with the aglycone N′-butyl substituent, in a precise E-orientation, located at a hydrophobic region nearby. Chaperone potential profiling indicated significant increases of enzyme activity in 24 of 88 β-Gal mutants, including four common mutations. Finally, oral administration of 6S-NBI-DGJ ameliorated the brain pathology of GM₁ gangliosidosis model mice. These results suggest that 6S-NBI-DGJ is a novel PC that may be effective on a broad range of β-Gal mutants.     

Mucolipidosis I: Studies of sialidase activity and a prenatal diagnosis

The characteristics of the sialidase (N-acetyl-α-neuraminidase) of human leukocytes, fibroblasts and amniotic fluid cell cultures were determined with a radioactive assay method utilizing neuramin-[³H]actitol as the enzyme substrate. Fibroblast cultures from patients with the inherited sialidase deficiency diseases including mucolipidosis I, sialidosis I and sialidosis II, juvenile type have less than 10% of normal sialidase activity using either this substrate, 2-(3′-methoxyphenyl)-N-acetyl-α-neuraminic acid, or 2′-(4-methylumbelliferyl)-Nacetyl-α-neuraminic acid. The total sialic acid content of fibroblasts and leukocytes from mucolipidosis I and sialidosis I patients is greatly elevated; this parameter is useful in establishing a diagnosis of sialidase deficiency. The sialic acid content of sialidosis II, juvenile type, with coexistent sialidase and β-galactosidase deficiencies, is only slightly elevated above normal levels. A patient with mucolipidosis I has 16% of normal neuramin-[³H]lactitol sialidase activity in his peripheral leukocytes. His parents were clearly distinguished from the normal range using leukocyte enzyme levels and a maternal aunt was identified as a possible carrier. The presence of this enzyme in amniotic fluid cell cultures, both fibroblastic and mixed cell type, makes possible the prenatal detection of these diseases. A pregnancy from a family at risk for having a child with mucolipidosis I was monitored by amniocentesis and subsequent sialidase measurement of the amniotic fluid cell culture.     

Activity of beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in human rectal mucosa

Activity of β-galactosidase, β-glucuronidase and N-acetl-β-glucosaminidase was determined by biopsy specimens of rectal mucosa of control subjects, cystinotic children and their parents.The studied enzymes exhibited maximal activity at pH 5.0, 4.0 and 4.5, respectively. Apparent K_m values using P-nitrophenyl-β-galactoside, p-nitrophenyl-β-glucuronide and p-nitropheny-β-glucosaminide were found to be 0.52 mM, 0.70 mM, and 0.67 mM.The activity of all three enzymes was found to be closely correlated in the 11 subjects of the control group. The values found in parents and their cystinotic children fit into these correlations, but show higher scatter of data caused by the fact that values of β-galactosidase were found to be higher and of β-glucuronidase and N-acetyl-glucosaminidase lower in the group of parents than in the other two groups.     

Lysosomal enzymes in cerebral atrophy

In seven patients with cerebral atrophy due to pre-senile dementia and/or cerebrovascular disease, the activity of acid phosphatase in lumbar cerebrospinal fluid (CSF) was higher (p < 0.05) than in six controls. The activity of arylsulphatase and β-galactosidase in CSF was the same in the two groups. In the serum, the activities of acid phosphatase and arylsulphatase were the same in the two groups but the activity of β-galactosidase was lower (p < 0.02) in patients with cerebral atrophy.     

The use of galactosylceramides with uniform fatty acids as substrates in the diagnosis and carrier detection of Krabbe disease

Cerebroside-β-galactosidase (galactosylceramidase EC 3.2.1.4.6) activity was studied using galactosylceramides of uniform fatty acid composition. The highest activity and the best discrimination between patients with Krabbe disease and controls were found with N-nervonoylgalactosylsphingosine (C 24: 1-cerebroside). As a general rule cerebrosides with a monoenoic fatty acid gave higher activity and better discrimination than the corresponding cerebroside with a saturated fatty acid, the differences being largest for the cerebrosides with the longest fatty acids. In two methods the C 24: 1 cerebroside was used as substrate in the assay of the cerebroside-β-galactosidase activity in leukocytes from 12 Krabbe patients, 14 parents and 22 controls. In a third method lactosylceramide prepared from mammalian brain gangliosides was used as substrate. With all three methods the residual activity in the leukocytes of the Krabbe patients did not exceed 5%, there was no tendency for overlap between the activities of the patients and those of the obligate carriers, and the values of half the carriers fell within the range for the controls.     

Substrate inhibition of lysosomal hydrolases: α-Galactosidase A and β-glucocerebrosidase

ObjectiveWe have investigated the kinetics of α-galactosidase A and β-glucocerebrosidase deficient in Fabry and Gaucher diseases, respectively.Design and methodsWe have performed spectrofluorymetric measurements of the activity of enzymes using a derivative of 4-methylumbelliferone as a substrate and a human T-cell line as a source of enzymes.ResultsWe have observed the substrate inhibition effect, which is related to temperature.ConclusionsThe diagnostic procedures for Fabry and Gaucher diseases used now in laboratory practice neglect temperature-dependent substrate inhibition, which may significantly reduce the sensitivity of enzyme activity determinations.     

Human erythrocyte phenol O-methyltransferase: Radiochemical microassay and biochemical properties

A radiochemical microassay for the determination of phenol O-methyltransferase (PMT) activity in human red blood cell membranes has been developed. Acetaminophen was used as the substrate. The apparent Michaelis-Menten (K_M) value for acetaminophen was 21.2 × 10^?3 M. The apparent K_M value for S-adenosyl-L-methionine, a co-substrate for the reaction, was 4.8 × 10^?6M, and the pH optimum of the reaction was approximately 9.0 with four different buffer systems. Phenol was also tested as a substrate and had an apparent K_m value of 2.0 × 10^?3 M. Human erythrocyte (RBC) membrane PMT activity did not have the biochemical characteristics of catechol O-methyltransferase, another RBC membrane methyltransferase enzyme activity. Blood samples obtained from 212 randomly selected adult white subjects had a mean activity of 134.5 ± 41.5 pmol of p-acetanisidide formed per mg protein per hour (mean ± S.D.). Activities varied from 44 to 282 units. There were no differences in the mean activities of samples from men and women. Experiments in which mixtures of “low” and “high” activity RBC membrane preparations were assayed for PMT provided no evidence that the variations in enzyme activity were due to the presence of endogenous PMT activators or inhibitors. RBC membrane PMT activity in blood from 9 patients with renal failure, a pathological state in which there are elevated circulating levels of phenols, was found to be significantly decreased with average activity of 76.2 ± 9.7 (mean ± S.E.M., P < 0.001).     

Four novel mutations in the β-galactosidase gene identified in infantile type of GM1 gangliosidosis

ObjectivesThe aim of this study is to find out mutations of Turkish GM1 gangliosidosis patients and to make genotype–phenotype correlations.Design and methodsβ-galactosidase activities were measured by using fluorometric substrate. Mutation screening of 16 exons of β-galactosidase gene and mutation detection were done by PCR-SSCP and DNA sequencing, respectively.ResultsFour new mutations, c.188_189insT in exon 2, c.569_570insA in exon 6, p.K142Q in exon 4, p.G190D in exon 6, and one known mutation p.P549L in exon 15, were identified in the β-galactosidase gene in 5 Turkish patients. Mutations in exons 4 and 6 are in the active site and mutation in exon is in the galactose-binding domain of the β-galactosidase gene.ConclusionThis is the first mutational analysis performed in Turkish GM1 gangliosidosis patients and shows the molecular heterogeneity of the disease in Turkish population. All identified mutations result in severe enzyme deficiency and infantile phenotype.     

Dosage de l'activité adénosine désaminasique dans les érythrocytes et les lymphocytes humains

A radiochromatographic method is described for measuring enzymatic activity of adenosine deaminase in human erythrocytes and lymphocytes. [8-¹⁴C]-adenosine is converted into inosine and hypoxanthine; after Chromatographie separation of the products, the radioactivity is determined.The kinetic properties of the enzyme have been studied. The K_m values for the erythrocyte and lymphocyte enzymes are higher as compared with purified deaminase. Optimum conditions for substrate concentration for assay were established.The mean normal activity (± S.E. of mean) is: for erythrocytes, 494 ± 61; nmol min^-1 ml^-1; for lymphocytes, 147 ± 0.18 nmol min^?1 10⁶ cellules.The mean values are higher than that given by other methods working at a lower (non-staurating) substrate concentration.     

Production of cellulase by Trichoderma reesei from pretreated straw and furfural residues

In this study, furfural residues were used as a substrate for cellulase production by the fungi Trichoderma reesei. The results indicated that a low pH and the presence of lignin in the furfural residues have an obvious impact on cellulase production by T. reesei. After pH adjustment, furfural residues could be used for cellulase production by T. reesei, with a higher filter paper activity (FPA) and a higher activity of CMCase compared to that yielded from furfural residues with pH unadjusted. After being washed with 1.6% (w/v) H₂O₂, all of the lignin in the furfural residues was removed, and an FPA of 7.1 FPU ml⁻¹ and a CMCase activity of 3.4 IU ml⁻¹ were obtained in 115 h, while pretreated straw could yield an FPA of 8.0 FPU ml⁻¹ and a CMCase activity of 2.7 IU ml⁻¹ in 160 h. Moreover, after being treated with H₂O₂, furfural residues could be used as an inducer in the production of cellulases. With the treated furfural residues added into the medium at the beginning of cultivation, T. reesei gave the maximum FPA (8.4 FPU ml⁻¹) and CMCase activity (4.8 IU ml⁻¹) at 142 h from pretreated straw, which is relatively high for cellulase production compared to that from most other agricultural wastes reported.

In this study, furfural residues were used as a substrate for cellulase production by the fungi Trichoderma reesei.     

Acid hydrolases in amniotic fluid and chorionic villi at various stages of pregnancy

A study was made at various stages of pregnancy of five acid hydrolases which occur in amniotic fluid and chorionic villi and which are relevant to serious storage disorders.In amniotic fluid β-galactosidase and α-mannosidase decreased moderately towards term, while β-glucosidase decreased markedly. N-Acetyl-β-glucosaminidase and β-glucuronidase were relatively unchanged.In chorionic villi N-acetyl-β-glucosaminidase, β-galactosidase, and α-mannosidase were substantially decreased towards term, while β-glucosidase was unchanged and β-glucuronidase markedly increased.In both amniotic fluid and chorionic villi the enzyme pattern was approximately the same as that found in liver in a previous study.The findings suggest that these enzyme assays might be useful in the diagnosis of inborn errors prenatally by using amniotic fluid, and early postnatally by using chorionic villi.     

Lentivector Transduction Improves Outcomes Over Transplantation of Human HSCs Alone in NOD/SCID/Fabry Mice

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb₃) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34⁺ cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb₃ quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb₃ reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.     

A New Ketolide, HMR 3004, Active against Streptococci Inducibly Resistant to Erythromycin

HMR 3004 is a new hydrazono ketolide characterized by a 3-keto function instead of the cladinose moiety. The effect of this antimicrobial agent on inducible and constitutive macrolide-lincosamide-streptogramin B (MLS_B) resistance was tested in a lacZ reporter system under control of several ermAM-like attenuator variants. For one constitutively resistant Streptococcus agalactiae strain, three inducibly resistant Streptococcus pneumoniae strains, and one inducibly resistant Enterococcus faecalis strain, the attenuators fused with lacZ were cloned into the shuttle plasmid pJIM2246 and the plasmid was introduced into Staphylococcus aureus RN4220. For the wild-type attenuators, HMR 3004 was a very weak inducer, unlike its cladinose counterpart RU 6652 and erythromycin. As expected, for the fusion originating from the constitutively resistant S. agalactiae strain, the level of uninduced β-galactosidase synthesis was high. For one S. pneumoniae attenuator, mutations in the 3′ end of the attenuator that weakened the stem-loop structure that sequesters the ribosome-binding site and start codon for ermAM methylase could explain the high level of uninduced β-galactosidase produced. For streptococci, the activity of HMR 3004 correlated with the basal level of β-galactosidase synthesized. The weak inducer activity of HMR 3004 explained its activity against inducibly MLS_B-resistant S. pneumoniae but did not correlate with the moderate activity of the antibiotic against inducibly resistant E. faecalis.     

Altered levels of tissue gangliosides and glycoproteins in the infantile form of GM1-gangliosidosis

The concentration of phospholipid, cerebroside, and sulfatide was decreased in brain grey and white matter from two cases of infantile GM1-gangliosidosis. The decrease of cerebroside and sulfatide in white matter was especially marked. Gangliosides showed marked elevations in both white and grey matter. GM1 was the predominant ganglioside, accounting for over 60% of the total ganglioside preparation from brain tissue. GM2 and GM3 also showed increases. GM1 was also elevated in the liver and kidney. A marked elevation in the concentration of galactose, mannose, and hexosamine associated with the heteropolysaccharide moieties of the brain glycoproteins was also noted. Presumably, the β-galactosidase that cleaves the terminal galactose from GM1 is equally capable of cleaving β-galactose linkages present in the glycoproteins.     

Catechol oxidation promoted by bridging phenoxo moieties in a bis(μ-phenoxo)-bridged dicopper(ii) complex

A dinuclear copper(ii) complex [Cu₂(papy)₂(CH₃OH)₂] has been synthesized by reaction of one equiv. of Cu(OAc)₂·2H₂O with one equiv. of the tetradentate tripodal ligand H₂papy [N-(2-hydroxybenzyl)-N-(2-picolyl)glycine] and has been characterized by various spectroscopic techniques and its solid state structure has been confirmed by X-ray crystal structure analysis. The single-crystal structure of the complex reveals that the two copper centers are hexa-coordinated and bridged by two O-atoms of the phenoxo moieties. The variable temperature magnetic susceptibility measurement of the complex reveals weak ferromagnetic interactions among the Cu(ii) ions with a J value of 1.1 cm⁻¹. The catecholase activity of the complex has been investigated spectrophotometrically using 3,5-di-tert-butyl catechol as a model substrate in methanol solvent under aerobic conditions. The Michaelis–Menten kinetic treatment has been applied using different excess substrate concentrations. The parameters obtained from the catecholase activity by the complex are K_M 2.97 × 10⁻⁴ M, V_max 2 × 10⁻⁴ M s⁻¹, and k_cat 7.2 × 10³ h⁻¹. A reaction mechanism has been proposed based on experimental findings and theoretical calculations. The catechol substrate binds to dicopper(ii) centers and subsequently two electrons are transferred to the metal centers from the substrate. The bridging phenoxo moieties participate as a Brønsted base by accepting protons from catechol during the catalytic cycle and thereby facilitating the catechol oxidation process.

A bis(μ-phenoxo)-bridged dicopper(ii) complex capable of oxidizing catechol with the highest efficiency amongst any bis(μ-phenoxo)-bridged dicopper(ii) complexes is reported.     

Characterization and properties of alpha-D-mannosidase of human polymorphonuclear leucocytes

Leukocytes isolated from human blood contain strong α-mannosidase activity. This enzyme has a pH optimum of 4.3 and is unstable by preincubation in citrate buffers at pH's close to the optimum pH of incubation. The K_m and V_max values have been determined with p-nitrophenyl-α-mannopyranoside as substrate. They were higher in acetate buffers than in citrate buffers. The enzyme is highly inhibited by d-mannono-(1→5)lactone (K_i= 90 μM), and shows a subcellular distribution and a structure linked latency compatible with its main localization in primary granules or lysosomes. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn²⁺. Other cations such as Ag⁺, Hg²⁺, Co²⁺, Fe²⁺, Fe³⁺, Cu²⁺ and Mn²⁺ accelerate inactivation. Moreover, in an EDTA-treated preparation Zn²⁺ reactivates the enzyme during assay. It is postulated that human polymorpho-nuclear leukocyte α-mannosidase is a dissociable Zn²⁺-protein complex in which Zn²⁺ is essential for enzyme activity.