45 results on '"Ruijter GJ"'
Search Results
2. Effect of Anti-Iduronate 2-Sulfatase Antibodies in Patients with Mucopolysaccharidosis Type II Treated with Enzyme Replacement Therapy.
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Vollebregt AAM, Hoogeveen-Westerveld M, Ruijter GJ, van den Hout H, Oussoren E, van der Ploeg AT, and Pijnappel WWMP
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- Antibodies, Enzyme Replacement Therapy methods, Glycosaminoglycans urine, Humans, Phenotype, Iduronate Sulfatase genetics, Iduronate Sulfatase therapeutic use, Mucopolysaccharidosis II drug therapy, Mucopolysaccharidosis II genetics
- Abstract
Objective: To assess the relationship between anti-Iduronate 2-sulfatase (IDS) antibodies, IDS genotypes, phenotypes and their impact in patients with enzyme replacement therapy (ERT)-treated Mucopolysaccharidosis type II., Study Design: Dutch patients treated with ERT were analyzed in this observational cohort study. Antibody titers were determined by enzyme-linked immunosorbent assay. Neutralizing effects were measured in fibroblasts. Pharmacokinetic analysis of ERT was combined with immunoprecipitation. Urinary glycosaminoglycans were measured using mass spectrometry and dimethylmethylene blue., Results: Eight of 17 patients (47%) developed anti-IDS antibodies. Three patients with the severe, neuronopathic phenotype, two of whom did not express IDS protein, showed sustained antibodies for up to 10 years of ERT. Titers of 1:5120 or greater inhibited cellular IDS uptake and/or intracellular activity in vitro. In 1 patient who was neuronopathic with a titer of 1:20 480, pharmacokinetic analysis showed that all plasma recombinant IDS was antibody bound. This finding was not the case in 2 patients who were not neuronopathic with a titer of 1:1280 or less. Patients with sustained antibody titers showed increased urinary glycosaminoglycan levels compared with patients with nonsustained or no-low titers., Conclusions: Patients with the neuronopathic form and lack of IDS protein expression were most at risk to develop sustained anti-IDS antibody titers, which inhibited IDS uptake and/or activity in vitro, and the efficacy of ERT in patients by lowering urinary glycosaminoglycan levels., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.) more...
- Published
- 2022
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3. Genotype-phenotype relationship in mucopolysaccharidosis II: predictive power of IDS variants for the neuronopathic phenotype.
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Vollebregt AAM, Hoogeveen-Westerveld M, Kroos MA, Oussoren E, Plug I, Ruijter GJ, van der Ploeg AT, and Pijnappel WWMP
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- Adolescent, Adult, Child, Child, Preschool, Cohort Studies, Educational Status, Epilepsy enzymology, Epilepsy genetics, Epilepsy psychology, Genetic Association Studies, Glycosaminoglycans urine, Humans, Immunoblotting, Intelligence, Mass Spectrometry, Middle Aged, Mucopolysaccharidosis II drug therapy, Mucopolysaccharidosis II metabolism, Netherlands, Phenotype, Young Adult, Genetic Variation, Glycoproteins genetics, Glycoproteins metabolism, Mucopolysaccharidosis II genetics, Mucopolysaccharidosis II psychology
- Abstract
Aim: Mucopolysaccharidosis type II (MPS II) is caused by variants in the iduronate-2-sulphatase gene (IDS). Patients can be either neuronopathic with intellectual disability, or non-neuronopathic. Few studies have reported on the IDS genotype-phenotype relationship and on the molecular effects involved. We addressed this in a cohort study of Dutch patients with MPS II., Method: Intellectual performance was assessed for school performance, behaviour, and intelligence. Urinary glycosaminoglycans were quantified by mass spectrometry. IDS variants were analysed in expression studies for enzymatic activity and processing by immunoblotting., Results: Six patients had a non-neuronopathic phenotype and 11 a neuronopathic phenotype, three of whom had epilepsy. Total deletion of IDS invariably resulted in the neuronopathic phenotype. Phenotypes of seven known IDS variants were consistent with the literature. Expression studies of nine variants were novel and showed impaired IDS enzymatic activity, aberrant intracellular processing, and elevated urinary excretion of heparan sulphate and dermatan sulphate irrespective of the MPS II phenotype., Interpretation: We speculate that very low or cell-type-specific IDS residual activity is sufficient to prevent the neuronal phenotype of MPS II. Whereas the molecular effects of IDS variants do not distinguish between MPS II phenotypes, the IDS genotype is a strong predictor., (© 2017 Mac Keith Press.) more...
- Published
- 2017
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4. Paroxysmal exercise-induced dystonia within the phenotypic spectrum of ECHS1 deficiency.
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Olgiati S, Skorvanek M, Quadri M, Minneboo M, Graafland J, Breedveld GJ, Bonte R, Ozgur Z, van den Hout MC, Schoonderwoerd K, Verheijen FW, van IJcken WF, Chien HF, Barbosa ER, Chang HC, Lai SC, Yeh TH, Lu CS, Wu-Chou YH, Kievit AJ, Han V, Gdovinova Z, Jech R, Hofstra RM, Ruijter GJ, Mandemakers W, and Bonifati V more...
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- Adolescent, Enoyl-CoA Hydratase genetics, Exercise, Humans, Male, Pedigree, Dystonic Disorders genetics, Dystonic Disorders physiopathology, Enoyl-CoA Hydratase deficiency
- Abstract
Background: ECHS1 encodes a mitochondrial enzyme involved in the degradation of essential amino acids and fatty acids. Recently, ECHS1 mutations were shown to cause a new severe metabolic disorder presenting as Leigh or Leigh-like syndromes. The objective of this study was to describe a family with 2 siblings affected by different dystonic disorders as a resulting phenotype of ECHS1 mutations., Methods: Clinical evaluation, MRI imaging, genome-wide linkage, exome sequencing, urine metabolite profiling, and protein expression studies were performed., Results: The first sibling is 17 years old and presents with generalized dystonia and severe bilateral pallidal MRI lesions after 1 episode of infantile subacute metabolic encephalopathy (Leigh-like syndrome). In contrast, the younger sibling (15 years old) only suffers from paroxysmal exercise-induced dystonia and has very mild pallidal MRI abnormalities. Both patients carry compound heterozygous ECHS1 mutations: c.232G>T (predicted protein effect: p.Glu78Ter) and c.518C>T (p.Ala173Val). Linkage analysis, exome sequencing, cosegregation, expression studies, and metabolite profiling support the pathogenicity of these mutations. Expression studies in patients' fibroblasts showed mitochondrial localization and severely reduced levels of ECHS1 protein. Increased urinary S-(2-carboxypropyl)cysteine and N-acetyl-S-(2-carboxypropyl)cysteine levels, proposed metabolic markers of this disorder, were documented in both siblings. Sequencing ECHS1 in 30 unrelated patients with paroxysmal dyskinesias revealed no further mutations., Conclusions: The phenotype associated with ECHS1 mutations might be milder than reported earlier, compatible with prolonged survival, and also includes isolated paroxysmal exercise-induced dystonia. ECHS1 screening should be considered in patients with otherwise unexplained paroxysmal exercise-induced dystonia, in addition to those with Leigh and Leigh-like syndromes. Diet regimens and detoxifying agents represent potential therapeutic strategies. © 2016 International Parkinson and Movement Disorder Society., (© 2016 International Parkinson and Movement Disorder Society.) more...
- Published
- 2016
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5. A Multiplex Assay for the Diagnosis of Mucopolysaccharidoses and Mucolipidoses.
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Langereis EJ, Wagemans T, Kulik W, Lefeber DJ, van Lenthe H, Oussoren E, van der Ploeg AT, Ruijter GJ, Wevers RA, Wijburg FA, and van Vlies N
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- Adolescent, Adult, Aged, Child, Child, Preschool, Chromatography, High Pressure Liquid methods, Dermatan Sulfate urine, Diagnosis, Differential, Heparitin Sulfate urine, Humans, Infant, Infant, Newborn, Keratan Sulfate urine, Middle Aged, Mucolipidoses urine, Mucopolysaccharidoses urine, Sensitivity and Specificity, Tandem Mass Spectrometry methods, Young Adult, Dermatan Sulfate isolation & purification, Heparitin Sulfate isolation & purification, Keratan Sulfate isolation & purification, Mucolipidoses diagnosis, Mucopolysaccharidoses diagnosis
- Abstract
Introduction: Diagnosis of the mucopolysaccharidoses (MPSs) generally relies on an initial analysis of total glycosaminoglycan (GAG) excretion in urine. Often the dimethylmethylene blue dye-binding (DMB) assay is used, although false-negative results have been reported. We report a multiplexed diagnostic test with a high sensitivity for all MPSs and with the potential to identify patients with I-cell disease (ML II) and mucolipidosis III (ML III)., Methods: Urine samples of 100 treatment naive MPS patients were collected and analyzed by the conventional DMB assay and a multiplex assay based on enzymatic digestion of heparan sulfate (HS), dermatan sulfate (DS) and keratan sulfate (KS) followed by quantification by LC-MS/MS. Specificity was calculated by analyzing urine samples from a cohort of 39 patients suspected for an inborn error of metabolism, including MPSs., Results: The MPS cohort consisted of 18 MPS I, 16 MPS II, 34 MPS III, 10 MPS IVA, 3 MPS IVB, 17 MPS VI and 2 MPS VII patients. All 100 patients were identified by the LC-MS/MS assay with typical patterns of elevation of HS, DS and KS, respectively (sensitivity 100%). DMB analysis of the urine was found to be in the normal range in 10 of the 100 patients (sensitivity 90%). Three out of the 39 patients were identified as false-positive, resulting in a specificity of the LS-MS/MS assay of 92%. For the DMB this was 97%. All three patients with MLII/MLIII had elevated GAGs in the LC-MS/MS assay while the DMB test was normal in 2 of them., Conclusion: The multiplex LC-MS/MS assay provides a robust and very sensitive assay for the diagnosis of the complete spectrum of MPSs and has the potential to identify MPS related disorders such as MLII/MLIII. Its performance is superior to that of the conventional DMB assay. more...
- Published
- 2015
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6. First two unrelated cases of isolated sedoheptulokinase deficiency: A benign disorder?
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Wamelink MM, Ramos RJ, van den Elzen AP, Ruijter GJ, Bonte R, Diogo L, Garcia P, Neves N, Nota B, Haschemi A, Tavares de Almeida I, and Salomons GS
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- Anemia complications, Anemia genetics, Arthrogryposis genetics, Child, Preschool, Cholestasis complications, Cholestasis genetics, Codon, Nonsense, Consanguinity, Female, Heptoses metabolism, Humans, Hypoglycemia complications, Hypoglycemia genetics, Male, Phenotype, Sugar Phosphates metabolism, Pentose Phosphate Pathway genetics, Phosphotransferases (Alcohol Group Acceptor) deficiency, Phosphotransferases (Alcohol Group Acceptor) genetics, Transcription Factors deficiency, Transcription Factors genetics
- Abstract
We present the first two reported unrelated patients with an isolated sedoheptulokinase (SHPK) deficiency. The first patient presented with neonatal cholestasis, hypoglycemia, and anemia, while the second patient presented with congenital arthrogryposis multiplex, multiple contractures, and dysmorphisms. Both patients had elevated excretion of erythritol and sedoheptulose, and each had a homozygous nonsense mutation in SHPK. SHPK is an enzyme that phosphorylates sedoheptulose to sedoheptulose-7-phosphate, which is an important intermediate of the pentose phosphate pathway. It is questionable whether SHPK deficiency is a causal factor for the clinical phenotypes of our patients. This study illustrates the necessity of extensive functional and clinical workup for interpreting a novel variant, including nonsense variants. more...
- Published
- 2015
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7. Genetic basis of alpha-aminoadipic and alpha-ketoadipic aciduria.
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Hagen J, te Brinke H, Wanders RJ, Knegt AC, Oussoren E, Hoogeboom AJ, Ruijter GJ, Becker D, Schwab KO, Franke I, Duran M, Waterham HR, Sass JO, and Houten SM
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- 2-Aminoadipic Acid urine, Adipates urine, Adolescent, Adult, Amino Acid Metabolism, Inborn Errors diagnosis, Amino Acid Metabolism, Inborn Errors metabolism, Child, Preschool, Female, Humans, Infant, Newborn, Ketoglutarate Dehydrogenase Complex, Ketone Oxidoreductases deficiency, Ketone Oxidoreductases metabolism, Male, Young Adult, 2-Aminoadipic Acid metabolism, Adipates metabolism, Amino Acid Metabolism, Inborn Errors genetics, Ketone Oxidoreductases genetics
- Abstract
Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex. more...
- Published
- 2015
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8. Newborn screening for hunter disease: a small-scale feasibility study.
- Author
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Ruijter GJ, Goudriaan DA, Boer AM, Van den Bosch J, Van der Ploeg AT, Elvers LH, Weinreich SS, and Reuser AJ
- Abstract
Hunter disease (Mucopolysaccharidosis type II, MPS II) is an X-linked lysosomal storage disorder caused by deficiency of iduronate-2-sulfatase (IDS). Two main therapies have been reported for MPS II patients: enzyme-replacement therapy (ERT) and hematopoietic stem-cell transplantation (HSCT). Both treatment modalities have been shown to improve some symptoms, but the results with regard to cognitive functioning have been poor. Early initiation of therapy, i.e., before neurological symptoms have manifested, may alter cognitive outcome. The need for early identification makes Hunter disease a candidate for newborn screening (NBS). Our objective was to explore the use of a fluorometric assay that could be applicable for high-throughput analysis of IDS activity in dried blood spots (DBS). The median IDS activity in DBS samples from 1,426 newborns was 377 pmol/punch/17 h (range 78-1111). The IDS activity in one sample was repeatedly under the cutoff value (set at 20% of the median value), which would imply a recall rate of 0.07%. A sample from a clinically diagnosed MPS II individual, included in each 96-well test plate, had IDS activities well below the 20% cutoff value. Coefficients of variation in quality control samples with low, medium, and high IDS activities (190, 304, and 430 pmol/punch/17 h, respectively) were 12% to 16%. This small-scale pilot study shows that newborn screening for Hunter disease using a fluorometric assay in DBS is technically feasible with a fairly low recall rate. NBS may allow for identification of infants with Hunter disease before clinical symptoms become evident enabling early intervention. more...
- Published
- 2014
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9. Residual α-L-iduronidase activity in fibroblasts of mild to severe Mucopolysaccharidosis type I patients.
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Oussoren E, Keulemans J, van Diggelen OP, Oemardien LF, Timmermans RG, van der Ploeg AT, and Ruijter GJ
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- Cell Line, Early Diagnosis, Humans, Hymecromone metabolism, Infant, Newborn, Mucopolysaccharidosis I enzymology, Mucopolysaccharidosis I metabolism, Mucopolysaccharidosis I pathology, Mutation, Fibroblasts enzymology, Hymecromone analogs & derivatives, Iduronidase metabolism, Mucopolysaccharidosis I diagnosis
- Abstract
Three major clinical subgroups are usually distinguished in Mucopolysaccharidosis type I: Hurler (MPS IH, severe presentation), Hurler-Scheie (MPS IH/S, intermediate) and Scheie (MPS IS, mild). To facilitate treatment with hematopoietic stem-cell transplantation, early diagnosis is important for MPS IH patients. Although screening for MPS I in newborns would allow detection at an early age, it may be difficult to predict the phenotype on the basis of the genotype in these infants. Extra diagnostic tools are thus required. Based on the hypothesis that distinct MPS I phenotypes may result from differences in residual α-l-iduronidase (IDUA) activity, we modified the common IDUA assay using the substrate 4-methylumbelliferyl-α-l-iduronide to allow quantification of low IDUA activity in MPS I fibroblasts. Enzyme incubation was performed with high protein concentrations at different time points up to 8h. Mean residual IDUA activity was 0.18% (range 0-0.6) of the control value in MPS IH fibroblasts (n=5); against 0.27% (range 0.2-0.3) in MPS IH/S cells (n=3); and 0.79% (range 0.3-1.8) in MPS IS fibroblasts (n=5). These results suggest that residual IDUA activity and severity of the MPS I phenotype are correlated. Two MPS IS patients with rare (E276K/E276K) or indefinite (A327P/unknown) IDUA genotypes had residual IDUA activity in the MPS IS range, illustrating the usefulness of our approach. IDUA(E276K) was very unstable at 37°C, but more stable at 23°C, suggesting thermal instability. We conclude that this procedure for determining residual IDUA activity in fibroblasts of MPS I patients may be helpful to predict MPS I phenotype., (Copyright © 2013 Elsevier Inc. All rights reserved.) more...
- Published
- 2013
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10. Up to five years experience with 11 mucopolysaccharidosis type VI patients.
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Brands MM, Oussoren E, Ruijter GJ, Vollebregt AA, van den Hout HM, Joosten KF, Hop WC, Plug I, and van der Ploeg AT
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- Adolescent, Child, Child, Preschool, Female, Follow-Up Studies, Humans, Male, Mucopolysaccharidosis VI enzymology, Mucopolysaccharidosis VI genetics, N-Acetylgalactosamine-4-Sulfatase genetics, N-Acetylgalactosamine-4-Sulfatase metabolism, Prospective Studies, Respiratory Function Tests, Young Adult, Enzyme Replacement Therapy, Glycosaminoglycans metabolism, Mucopolysaccharidosis VI physiopathology, Mucopolysaccharidosis VI therapy, N-Acetylgalactosamine-4-Sulfatase therapeutic use
- Abstract
Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is a rare progressive metabolic disorder characterized by coarse facial features, hepatosplenomegaly, restrictive pulmonary function, cardiac abnormalities and stiff joints. The disease is caused by a deficiency of the lysosomal enzyme N-acetyl galactosamine 4-sulfatase which leads to glycosaminoglycan (GAG) storage in various tissues. It presents as a clinical spectrum with varying disease progressions and severities. While the phases I/II/III studies proved the effectiveness of enzyme-replacement therapy (ERT) with recombinant human arylsulfatase B, long-term data are still scarce. Over treatment periods ranging from 1.3 to 5.4 years, this prospective open-label follow-up study in 11 Dutch mucopolysaccharidosis type VI patients (age 2-18 years) showed that ERT had significant positive effects on cardiac-wall diameters (IVSd and LVMI), left and right shoulder flexions (p<0.001), liver size and spleen size (p<0.001), urinary GAG excretion (p<0.001), and the scales of quality of life (motor functioning and body functioning). ERT did not affect cardiac valve regurgitation or hearing function; HRQoL decreased slightly in two domains ('anxiety' and 'negative emotions'), and patients with the rapid and slow progressive forms of the disease differed with regard to baseline GAG excretion and GAG decrease during treatment. In conclusion, ERT had an effect on several clinical parameters. This effect was established in an open cohort of young mucopolysaccharidosis type VI patients., (Copyright © 2013 Elsevier Inc. All rights reserved.) more...
- Published
- 2013
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11. Mucopolysaccharidosis type VI phenotypes-genotypes and antibody response to galsulfase.
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Brands MM, Hoogeveen-Westerveld M, Kroos MA, Nobel W, Ruijter GJ, Özkan L, Plug I, Grinberg D, Vilageliu L, Halley DJ, van der Ploeg AT, and Reuser AJ
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- Adolescent, Child, Child, Preschool, Enzyme-Linked Immunosorbent Assay, Female, Genotype, Humans, Immunoprecipitation, Infant, Male, Mucopolysaccharidosis VI immunology, Mutagenesis, Site-Directed, Phenotype, Recombinant Proteins immunology, Antibody Formation immunology, Mucopolysaccharidosis VI genetics, Mucopolysaccharidosis VI pathology, N-Acetylgalactosamine-4-Sulfatase immunology
- Abstract
Background: Mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome; MPS VI) is an autosomal recessive lysosomal storage disorder in which deficiency of N-acetylgalactosamine 4-sulfatase (arylsulfatase B; ARSB) leads to the storage of glycosaminoglycans (GAGs) in connective tissue. The genotype-phenotype correlation has been addressed in several publications but the picture is not complete. Since 2007, enzyme-replacement therapy (ERT) has been available for patients with MPS VI in the Netherlands. The purpose of our study was to learn more about the genotype-phenotype correlations in MPS VI and the antibody response to ERT with galsulfase (recombinant human arylsulfatase B)., Methods: We identified ARSB mutations in 12 patients and used site-directed mutagenesis to study their effect. Antibody levels to galsulfase were measured using ELISA and a semi-quantitative immunoprecipitation method. We assessed the in vitro inhibitory effect of antibodies on galsulfase uptake and their effect on clinical outcome., Results: Five patients had a rapidly progressive phenotype and seven a slowly progressive phenotype. In total 9 pathogenic mutations were identified including 4 novel mutations (N301K, V332G, A237D, and c.1142 + 2 T > C) together composing 8 pathogenic genotypes. Most mutations appeared not to affect the synthesis of ARSB (66 kD precursor), but to hamper its maturation (43 kD ARSB). Disease severity was correlated with urinary GAG excretion. All patients developed antibodies to galsulfase within 26 weeks of treatment. It was demonstrated that these antibodies can inhibit the uptake of galsulfase in vitro., Conclusions: The clinical phenotypes and the observed defects in the biosynthesis of ARSB show that some of the mutations that we identified are clearly more severe than others. Patients receiving galsulfase as enzyme-replacement therapy can develop antibodies towards the therapeutic protein. Though most titers are modest, they can exceed a level at which they potentially affect the clinical outcome of enzyme-replacement therapy. more...
- Published
- 2013
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12. Rapid ultraperformance liquid chromatography-tandem mass spectrometry assay for a characteristic glycogen-derived tetrasaccharide in Pompe disease and other glycogen storage diseases.
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Sluiter W, van den Bosch JC, Goudriaan DA, van Gelder CM, de Vries JM, Huijmans JG, Reuser AJ, van der Ploeg AT, and Ruijter GJ
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- Adolescent, Adult, Age Factors, Aged, Child, Child, Preschool, Chromatography, Liquid, Glycogen Storage Disease Type II urine, Glycogen Storage Disease Type III urine, Glycogen Storage Disease Type IV urine, Humans, Infant, Infant, Newborn, Maltose analogs & derivatives, Maltose urine, Middle Aged, Reference Values, Spectrometry, Mass, Electrospray Ionization, Tandem Mass Spectrometry, Young Adult, Glycogen metabolism, Glycogen Storage Disease urine, Oligosaccharides urine
- Abstract
Background: Urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc₄) is increased in various clinical conditions associated with increased turnover or storage of glycogen, making Glc₄ a potential biomarker for glycogen storage diseases (GSD). We developed an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay to detect Glc₄ in urine without interference of the Glc₄ isomer maltotetraose (M₄)., Methods: Urine samples, diluted in 0.1% ammonium hydroxide containing the internal standard acarbose, were filtered, and the filtrate was analyzed by UPLC-MS/MS., Results: We separated and quantified acarbose, M₄, and Glc₄ using the ion pairs m/z 644/161, 665/161, and 665/179, respectively. Response of Glc₄ was linear up to 1500 μmol/L and the limit of quantification was 2.8 μmol/L. Intra- and interassay CVs were 18.0% and 18.4% (10 μmol/L Glc₄), and 10.5% and 16.2% (200 μmol/L Glc₄). Glc₄ in control individuals (n = 116) decreased with increasing age from a mean value of 8.9 mmol/mol to 1.0 mmol/mol creatinine. M₄ was present in 5% of urine samples. Mean Glc₄ concentrations per age group in untreated patients with Pompe disease (GSD type II) (n = 66) were significantly higher, ranging from 39.4 to 10.3 mmol/mol creatinine (P < 0.001-0.005). The diagnostic sensitivity of Glc₄ for GSD-II was 98.5% and the diagnostic specificity 92%. Urine Glc₄ was also increased in GSD-III (8 of 9), GSD-IV (2 of 3) and GSD-IX (6 of 10) patients., Conclusions: The UPLC-MS/MS assay of Glc₄ in urine was discriminative between Glc₄ and M₄ and confirmed the diagnosis in >98% of GSD-II cases. more...
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- 2012
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13. Bone, joint and tooth development in mucopolysaccharidoses: relevance to therapeutic options.
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Oussoren E, Brands MM, Ruijter GJ, der Ploeg AT, and Reuser AJ
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- Animals, Humans, Bone and Bones cytology, Joints growth & development, Mucopolysaccharidoses physiopathology, Mucopolysaccharidoses therapy, Tooth growth & development
- Abstract
The mucopolysaccharidoses (MPS) are prominent among the lysosomal storage diseases. The intra-lysosomal accumulation of glycosaminoglycans (GAGs) in this group of diseases, which are caused by several different enzyme deficiencies, induces a cascade of responses that affect cellular functions and maintenance of the extra-cellular matrix. Against the background of normal tissue-specific processes, this review summarizes and discusses the histological and biochemical abnormalities reported in the bones, joints, teeth and extracellular matrix of MPS patients and animal models. With an eye to the possibilities and limitations of reversing the pathological changes in the various tissues, we address therapeutic challenges, and present a model in which the cascade of pathologic events is depicted in terms of primary and secondary events., (2011 Elsevier B.V. All rights reserved.) more...
- Published
- 2011
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14. Prenatal screening of sialic acid storage disease and confirmation in cultured fibroblasts by LC-MS/MS.
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van den Bosch J, Oemardien LF, Srebniak MI, Piraud M, Huijmans JG, Verheijen FW, and Ruijter GJ
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- Amniotic Fluid chemistry, Calibration, Cells, Cultured, Chromatography, Liquid methods, Chromatography, Liquid standards, Female, Humans, Pregnancy, Pregnancy Trimester, Second metabolism, Pregnancy Trimester, Second urine, Pregnancy Trimester, Third metabolism, Pregnancy Trimester, Third urine, Prenatal Diagnosis instrumentation, Prenatal Diagnosis standards, Reproducibility of Results, Sialic Acid Storage Disease pathology, Tandem Mass Spectrometry standards, Urinalysis methods, Fibroblasts cytology, Fibroblasts pathology, Prenatal Diagnosis methods, Sialic Acid Storage Disease diagnosis, Tandem Mass Spectrometry methods
- Abstract
Sialic acid storage disease (SASD) is an inborn error resulting from defects in the lysosomal membrane protein sialin. The SASD phenotypical spectrum ranges from a severe presentation, infantile sialic acid storage disease (ISSD) which may present as hydrops fetalis, to a relatively mild form, Salla disease. Screening for SASD is performed by determination of free sialic acid (FSA) in urine or amniotic fluid supernatant (AFS). Subsequent diagnosis of SASD is performed by quantification of FSA in cultured fibroblasts and by mutation analysis of the sialin gene, SLC17A5. We describe simple quantitative procedures to determine FSA as well as conjugated sialic acid in AFS, and FSA in cultured fibroblasts, using isotope dilution ((13)C(3)-sialic acid) and multiple reaction monitoring LC-ESI-MS/MS. The whole procedure can be performed in 2-4 h. Reference values in AFS were 0-8.2 μmol/L for 15-25 weeks of gestation and 3.2-12.0 μmol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 μmol/L demonstrating that this method is able to discriminate ISSD pregnancies from normal ones. The method was also validated for determination of FSA in fibroblast homogenates. FSA in SASD fibroblasts (ISSD; 20-154 nmol/mg protein, intermediate SASD; 12.9-15.1 nmol/mg, Salla disease; 5.9-7.4 nmol/mg) was clearly elevated compared to normal controls (0.3-2.2 nmol/mg). In conclusion, we report simple quantitative procedures to determine FSA in AFS and cultured fibroblasts improving both prenatal diagnostic efficacy for ISSD as well as confirmatory testing in cultured fibroblasts following initial screening in urine or AFS. more...
- Published
- 2011
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15. Hemoglobin precipitation greatly improves 4-methylumbelliferone-based diagnostic assays for lysosomal storage diseases in dried blood spots.
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Oemardien LF, Boer AM, Ruijter GJ, van der Ploeg AT, de Klerk JB, Reuser AJ, and Verheijen FW
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- Adolescent, Adult, Case-Control Studies, Clinical Enzyme Tests, Enzyme Assays, Enzymes blood, Fluorescence, Fractional Precipitation, Humans, Hymecromone blood, Hymecromone chemistry, Indicators and Reagents, Infant, Leukocytes enzymology, Lysosomal Storage Diseases blood, Trichloroacetic Acid chemistry, Hemoglobins analysis, Hymecromone analogs & derivatives, Lysosomal Storage Diseases diagnosis
- Abstract
Derivatives of 4-methylumbelliferone (4MU) are favorite substrates for the measurement of lysosomal enzyme activities in a wide variety of cell and tissue specimens. Hydrolysis of these artificial substrates at acidic pH leads to the formation of 4-methylumbelliferone, which is highly fluorescent at a pH above 10. When used for the assay of enzyme activities in dried blood spots the light emission signal can be very low due to the small sample size so that the patient and control ranges are not widely separated. We have investigated the hypothesis that quenching of the fluorescence by hemoglobin leads to appreciable loss of signal and we show that the precipitation of hemoglobin with trichloroacetic acid prior to the measurement of 4-methylumbelliferone increases the height of the output signal up to eight fold. The modified method provides a clear separation of patients' and controls' ranges for ten different lysosomal enzyme assays in dried blood spots, and approaches the conventional leukocyte assays in outcome quality., (Copyright © 2010 Elsevier Inc. All rights reserved.) more...
- Published
- 2011
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16. Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations.
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Valstar MJ, Neijs S, Bruggenwirth HT, Olmer R, Ruijter GJ, Wevers RA, van Diggelen OP, Poorthuis BJ, Halley DJ, and Wijburg FA
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- Adolescent, Adult, Behavioral Symptoms etiology, Cells, Cultured, Child, Child, Preschool, Cohort Studies, DNA Mutational Analysis, Epilepsy etiology, Female, Fibroblasts, Hearing Disorders etiology, Humans, Hydrolases genetics, Kaplan-Meier Estimate, Male, Middle Aged, Mucopolysaccharidosis III complications, Mucopolysaccharidosis III enzymology, Mucopolysaccharidosis III genetics, Mucopolysaccharidosis III pathology, Mutation genetics, Pregnancy, Regression Analysis, Severity of Illness Index, Skin pathology, Sleep Wake Disorders etiology, Vision Disorders etiology, Young Adult, Genetic Association Studies, Genotype, Phenotype
- Abstract
Objective: Mucopolysaccharidosis (MPS) IIIA (Sanfilippo syndrome type A) is a lysosomal storage disorder caused by deficiency of the enzyme sulfamidase. Information on the natural course of MPS IIIA is scarce, but is much needed in view of emerging therapies., Methods: Clinical history and molecular defects of all 110 MPS IIIA patients identified by enzymatic studies in the Netherlands were collected and included in this study., Results: First clinical signs, mainly consisting of delayed speech development and behavioral problems, were noted between the ages of 1 and 6 years. Other symptoms included sleeping and hearing problems, recurrent upper airway infections, diarrhea, and epilepsy. The clinical course varied remarkably and could be correlated with the molecular defects. The frequent pathogenic mutations p.R245H, p.Q380R, p.S66W, and c.1080delC were associated with the classical severe phenotype. Patients compound heterozygous for the p.S298P mutation in combination with 1 of the mutations associated with the classical severe phenotype had a significantly longer preservation of psychomotor functions and a longer survival. Two patients homozygous for the p.S298P mutation, and 4 patients from 3 families heterozygous for 3 missense variants not reported previously (p.T421R, p.P180L, and p.L12Q), showed a remarkably attenuated phenotype., Interpretation: We report the natural history and mutational analysis in a large unbiased cohort of MPS IIIA patients. We demonstrate that the clinical spectrum of MPS IIIA is much broader than previously reported. A significant genotype-phenotype correlation was established in this cohort. more...
- Published
- 2010
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17. Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations.
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Valstar MJ, Bertoli-Avella AM, Wessels MW, Ruijter GJ, de Graaf B, Olmer R, Elfferich P, Neijs S, Kariminejad R, Suheyl Ezgü F, Tokatli A, Czartoryska B, Bosschaart AN, van den Bos-Terpstra F, Puissant H, Bürger F, Omran H, Eckert D, Filocamo M, Simeonov E, Willems PJ, Wevers RA, Niermeijer MF, Halley DJ, Poorthuis BJ, and van Diggelen OP more...
- Subjects
- Adolescent, Adult, Child, Child, Preschool, DNA Mutational Analysis, Female, Humans, Male, Mutation genetics, Phenotype, Young Adult, Mucopolysaccharidosis III genetics, Sulfatases deficiency, Sulfatases genetics
- Abstract
Mucopolysaccharidosis III D (Sanfilippo disease type D, MPS IIID) is a rare autosomal recessive lysosomal storage disorder previously described in only 20 patients. MPS IIID is caused by a deficiency of N-acetylglucosamine-6-sulphate sulphatase (GNS), one of the enzymes required for the degradation of heparan sulphate. So far only seven mutations in the GNS gene have been reported. The clinical phenotype of 12 new MPS IIID patients from 10 families was studied. Mutation analysis of GNS was performed in 16 patients (14 index cases). Clinical signs and symptoms of the MPS IIID patients appeared to be similar to previously described patients with MPS III. Early development was normal with onset of behavioral problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication and subsequent deterioration of motor functions. Sequence analysis of the coding regions of the gene encoding GNS (GNS) resulted in the identification of 15 novel mutations: 3 missense mutations, 1 nonsense mutation, 4 splice site mutations, 3 frame shift mutations, 3 large deletions and 1 in-frame small deletion. They include the first missense mutations and a relatively high proportion of large rearrangements, which warrants the inclusion of quantitative techniques in routine mutation screening of the GNS gene., ((c) 2010 Wiley-Liss, Inc.) more...
- Published
- 2010
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18. Sanfilippo syndrome: a mini-review.
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Valstar MJ, Ruijter GJ, van Diggelen OP, Poorthuis BJ, and Wijburg FA
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- Acetylglucosaminidase genetics, Acetyltransferases genetics, Adolescent, Adult, Animals, Child, Child, Preschool, Genetic Predisposition to Disease, Humans, Hydrolases genetics, Incidence, Infant, Mucopolysaccharidosis III diagnosis, Mucopolysaccharidosis III genetics, Mucopolysaccharidosis III mortality, Mucopolysaccharidosis III therapy, Phenotype, Prognosis, Sulfatases genetics, Time Factors, Young Adult, Acetylglucosaminidase deficiency, Acetyltransferases deficiency, Heparitin Sulfate metabolism, Hydrolases deficiency, Lysosomes enzymology, Mucopolysaccharidosis III enzymology, Sulfatases deficiency
- Abstract
Mucopolysaccharidosis type III (MPS III, Sanfilippo syndrome) is an autosomal recessive disorder, caused by a deficiency in one of the four enzymes involved in the lysosomal degradation of the glycosaminoglycan heparan sulfate. Based on the enzyme deficiency, four different subtypes, MPS IIIA, B, C, and D, are recognized. The genes encoding these four enzymes have been characterized and various mutations have been reported. The probable diagnosis of all MPS III subtypes is based on increased concentration of heparan sulfate in the urine. Enzymatic assays in leukocytes and/or fibroblasts confirm the diagnosis and allow for discrimination between the different subtypes of the disease. The clinical course of MPS III can be divided into three phases. In the first phase, which usually starts between 1 and 4 years of age, a developmental delay becomes apparent after an initial normal development during the first 1-2 years of life. The second phase generally starts around 3-4 years and is characterized by severe behavioural problems and progressive mental deterioration ultimately leading to severe dementia. In the third and final stage, behavioural problems slowly disappear, but motor retardation with swallowing difficulties and spasticity emerge. Patients usually die at the end of the second or beginning of the third decade of life, although survival into the fourth decade has been reported. Although currently no effective therapy is yet available for MPS III, several promising developments raise hope that therapeutic interventions, halting the devastating mental and behavioural deterioration, might be feasible in the near future. more...
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- 2008
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19. Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands.
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Ruijter GJ, Valstar MJ, van de Kamp JM, van der Helm RM, Durand S, van Diggelen OP, Wevers RA, Poorthuis BJ, Pshezhetsky AV, and Wijburg FA
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- Acetyltransferases chemistry, Adolescent, Adult, Age of Onset, Child, Child, Preschool, DNA genetics, DNA Mutational Analysis, Female, Genotype, Humans, Infant, Male, Middle Aged, Models, Molecular, Mucopolysaccharidosis III classification, Mucopolysaccharidosis III physiopathology, Mutation, Missense, Netherlands, Phenotype, Acetyltransferases deficiency, Acetyltransferases genetics, Mucopolysaccharidosis III enzymology, Mucopolysaccharidosis III genetics, Mutation
- Abstract
Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo C syndrome) is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). We performed a clinical study on 29 Dutch MPS IIIC patients and determined causative mutations in the recently identified HGSNAT gene. Psychomotor development was reported to be normal in all patients during the first year of life. First clinical signs were usually noted between 1 and 6 years (mean 3.5 years), and consisted of delayed psychomotor development and behavioral problems. Other symptoms included sleeping and hearing problems, recurrent infections, diarrhoea and epilepsy. Two sisters had attenuated disease and did not have symptoms until the third decade. Mean age of death was 34 years (range 25-48). Molecular analysis revealed mutations in both alleles for all patients except one. Altogether 14 different mutations were found: two splice site mutations, one frame shift mutation due to an insertion, three nonsense mutations and eight missense mutations. Two mutations, p.R344C and p.S518F, were frequent among probands of Dutch origin representing 22.0% and 29.3%, respectively, of the mutant alleles. This study demonstrates that MPS IIIC has a milder course than previously reported and that both severity and clinical course are highly variable even between sibs, complicating prediction of the clinical phenotype for individual patients. A clear phenotype-genotype correlation could not be established, except that the mutations p.G262R and p.S539C were only found in two sisters with late-onset disease and presumably convey a mild phenotype. more...
- Published
- 2008
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20. Congenital disorder of glycosylation type Ia presenting with hydrops fetalis.
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van de Kamp JM, Lefeber DJ, Ruijter GJ, Steggerda SJ, den Hollander NS, Willems SM, Matthijs G, Poorthuis BJ, and Wevers RA
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- Codon, Nonsense, Fatal Outcome, Female, Ferritins blood, Frameshift Mutation, Glycoproteins metabolism, Heart Defects, Congenital genetics, Humans, Hydrops Fetalis diagnostic imaging, Hypoalbuminemia congenital, Hypoalbuminemia genetics, Infant, Newborn, Isoelectric Focusing, Male, Mutagenesis, Insertional, Mutation, Missense, Pericardial Effusion congenital, Phosphotransferases (Phosphomutases) deficiency, Thrombocytopenia congenital, Thrombocytopenia genetics, Transferrin analysis, Ultrasonography, Prenatal, Abnormalities, Multiple genetics, Glycosylation, Hydrops Fetalis genetics, Phosphotransferases (Phosphomutases) genetics, Protein Processing, Post-Translational genetics
- Abstract
There is a growing awareness that inborn errors of metabolism can be a cause of non-immune hydrops fetalis. The association between congenital disorders of glycosylation (CDG) and hydrops fetalis has been based on one case report concerning two sibs with hydrops fetalis and CDG-Ik. Since then two patients with hydrops-like features and CDG-Ia have been reported. Two more unrelated patients with CDG-Ia who presented with hydrops fetalis are reported here, providing definite evidence that non-immune hydrops fetalis can be caused by CDG-Ia. The presence of congenital thrombocytopenia and high ferritin levels in both patients was remarkable. These might be common features in this severe form of CDG. Both patients had one severe mutation in the phosphomannomutase 2 gene, probably fully inactivating the enzyme, and one milder mutation with residual activity, as had the patients reported in literature. The presence of one severe mutation might be required for the development of hydrops fetalis. CDG-Ia should be considered in the differential diagnosis of hydrops fetalis and analysis of PMM activity in chorionic villi or amniocytes should also be considered. more...
- Published
- 2007
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21. Selection and characterisation of a xylitol-derepressed Aspergillus niger mutant that is apparently impaired in xylitol transport.
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van de Vondervoort PJ, de Groot MJ, Ruijter GJ, and Visser J
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- Alanine metabolism, Aspergillus niger growth & development, Biological Transport, Gene Expression Regulation, Fungal, Gluconates metabolism, Aspergillus niger genetics, Aspergillus niger metabolism, Mutation, Xylitol metabolism
- Abstract
Aspergillus niger is known for its biotechnological applications, such as the use of xylanase enzyme for the degradation of hemicellulose. Depending on culture conditions, several polyols may also be accumulated, such as xylitol during D: -xylose oxidation. Also during industrial fermentation of xylose for the production of fuel ethanol by recombinant yeast, xylitol is a by-product. We studied xylitol metabolism by isolating mutants that have impaired xylitol-mediated repression. Genetic and biochemical characterisation revealed that one of these mutants was affected not only in xylitol-mediated carbon repression, but also had impaired xylitol transport. more...
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- 2006
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22. Persistent 5-oxoprolinuria with normal glutathione synthase and 5-oxoprolinase activities.
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Ruijter GJ, Mourad-Baars PE, Ristoff E, Onkenhout W, and Poorthuis BJ
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- Child, Humans, Male, Glutathione Synthase metabolism, Pyroglutamate Hydrolase metabolism, Pyrrolidonecarboxylic Acid urine
- Abstract
5-Oxoprolinuria is primarily associated with inborn errors of the gamma-glutamyl cycle. In addition, transient 5-oxoprolinuria has been reported to occur in a variety of conditions, such as prematurity and malnutrition, and during medication. We report an unusual case of permanent 5-oxoprolinuria. The patient presented 3 days after birth with acidosis, and metabolic screening revealed massive excretion of 5-oxoproline. Following recovery, growth and psychomotor development were normal, but 5-oxoprolinuria persisted. Primary defects in the gamma-glutamyl cycle were ruled out since glutathione synthase and 5-oxoprolinase activities were normal. All known secondary causes of 5-oxoprolinuria were also excluded, leaving the basis of the permanent 5-oxoprolinuria in this patient unresolved. more...
- Published
- 2006
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23. Metabolic control analysis of Aspergillus niger L-arabinose catabolism.
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de Groot MJ, Prathumpai W, Visser J, and Ruijter GJ
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- Aldehyde Reductase isolation & purification, Aldehyde Reductase metabolism, Aspergillus niger enzymology, Biotechnology, Kinetics, Models, Biological, Sugar Alcohol Dehydrogenases isolation & purification, Sugar Alcohol Dehydrogenases metabolism, Xylose metabolism, Arabinose metabolism, Aspergillus niger metabolism
- Abstract
A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins. more...
- Published
- 2005
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24. Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger.
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R Poulsen B, Nøhr J, Douthwaite S, Hansen LV, Iversen JJ, Visser J, and Ruijter GJ
- Subjects
- Aspergillus niger enzymology, Aspergillus niger genetics, Cloning, Molecular, DNA Primers, Glucosephosphate Dehydrogenase genetics, Least-Squares Analysis, Models, Biological, Molecular Sequence Data, Phosphogluconate Dehydrogenase genetics, Polymerase Chain Reaction, Recombinant Proteins metabolism, Regression Analysis, Transketolase genetics, Aspergillus niger metabolism, NADP metabolism, Pentose Phosphate Pathway physiology
- Abstract
Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two- to ninefold as a result of 13-fold overproduction of 6-phosphogluconate dehydrogenase. Although overproduction of glucose 6-phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild-type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra- and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms. more...
- Published
- 2005
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25. External quality assurance programme for enzymatic analysis of lysosomal storage diseases: a pilot study.
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Ruijter GJ, Boer M, Weykamp CW, de Vries R, van den Berg I, Janssens-Puister J, Niezen-Koning K, Wevers RA, Poorthuis BJ, and van Diggelen OP
- Subjects
- Blood metabolism, Clinical Laboratory Techniques, Glycogen Storage Disease Type II diagnosis, Glycogen Storage Disease Type II enzymology, Humans, Leukocytes enzymology, Leukocytes metabolism, Lysosomal Storage Diseases diagnosis, Lysosomes metabolism, Pilot Projects, Quality Control, Reproducibility of Results, Specimen Handling, Temperature, Time Factors, alpha-Galactosidase metabolism, beta-Galactosidase metabolism, Lysosomal Storage Diseases enzymology
- Abstract
Inborn errors of metabolism are rare and laboratories performing diagnostic tests in this field must participate in external quality assurance (EQA) schemes to demonstrate their competence and also to maintain sufficient experience with patient material. EQA schemes for metabolite analyses are available (ERNDIM), but corresponding EQA schemes for enzyme analyses are nonexistent. In this paper we describe a pilot study on lysosomal enzyme testing by four centres in The Netherlands. Quantitative aspects of EQA were studied by interlaboratory comparison of activities of six lysosomal enzymes in a series of buffy coat samples. Interlaboratory variance was enormous. To reduce variance caused by methodological differences, participants reported enzyme activities relative to mean normal values. Beta-D-Galactosidase activities compared well between the participating laboratories (average interlaboratory CV 13%), but for other enzymes large differences were observed, e.g. sphingomyelinase (average CV 38%). Diagnostic proficiency was tested with cultured fibroblasts. In 45 out of a total of 48 tests (12 cell lines, 4 participants) the correct diagnosis was accomplished on the basis of merely biochemical investigations, i.e. without clinical data of the patients. In a survey using blood of a late-onset Pompe disease patient, less conclusive results were obtained. A stable enzyme source was developed for easy distribution. Most lysosomal enzymes were stable upon lyophilization of leukocyte homogenates and during subsequent storage of the freeze-dried material at room temperature, in particular when cryolyoprotectant was added. Shipment of such lyophilized samples is simple and cheap and ideal for an EQA scheme. Our study shows that an EQA programme for enzymatic testing of lysosomal storage diseases is necessary to accomplish reliable diagnostic procedures for lysosomal storage diseases. We recommend that EQA for lysosomal enzymes be implemented through ERNDIM. more...
- Published
- 2005
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26. Isolation of a fluffy mutant of Aspergillus niger from chemostat culture and its potential use as a morphologically stable host for protein production.
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van de Vondervoort PJ, Poulsen BR, Ruijter GJ, Schuleit T, Visser J, and Iversen JJ
- Subjects
- Aspergillus niger metabolism, Bioreactors, Culture Media, Genetic Techniques, Mutation, Recombinant Proteins biosynthesis, Temperature, Aspergillus niger genetics, Fungal Proteins biosynthesis
- Abstract
Chemostat cultivation of Aspergillus niger and other filamentous fungi is often hindered by the spontaneous appearance of morphologic mutants. Using the Variomixing bioreactor and applying different chemostat conditions we tried to optimize morphologic stability in both ammonium- and glucose-limited cultures. In most cultivations mutants with fluffy (aconidial) morphology became dominant. From an ammonium-limited culture, a fluffy mutant was isolated and genetically characterized using the parasexual cycle. The mutant contained a single morphological mutation, causing an increased colony radial growth rate. The fluffy mutant was subjected to transformation and finally conidiospores from a forced heterokaryon were shown to be a proper inoculum for fluffy strain cultivation., (Copyright 2004 Wiley Periodicals, Inc.) more...
- Published
- 2004
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27. Aspergillus niger mstA encodes a high-affinity sugar/H+ symporter which is regulated in response to extracellular pH.
- Author
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Vankuyk PA, Diderich JA, MacCabe AP, Hererro O, Ruijter GJ, and Visser J
- Subjects
- Aspergillus niger cytology, Aspergillus niger metabolism, Biological Transport, Carbohydrate Metabolism, Cloning, Molecular, Fungal Proteins metabolism, Gene Deletion, Hydrogen-Ion Concentration, Membrane Proteins metabolism, Monosaccharide Transport Proteins metabolism, Phenotype, Repressor Proteins metabolism, Saccharomyces cerevisiae metabolism, Symporters, Transcription Factors metabolism, Aspergillus niger genetics, Fungal Proteins genetics, Gene Expression Regulation, Fungal, Membrane Proteins genetics, Monosaccharide Transport Proteins genetics
- Abstract
A sugar-transporter-encoding gene, mstA, which is a member of the major facilitator superfamily, has been cloned from a genomic DNA library of the filamentous fungus Aspergillus niger. To enable the functional characterization of MSTA, a full-length cDNA was expressed in a Saccharomyces cerevisiae strain deficient in hexose uptake. Uptake experiments using 14C-labelled monosaccharides demonstrated that although able to transport D-fructose ( K(m), 4.5+/-1.0 mM), D-xylose ( K(m), 0.3+/-0.1 mM) and D-mannose ( K(m), 60+/-20 microM), MSTA has a preference for D-glucose (K(m), 25+/-10 microM). pH changes associated with sugar transport indicate that MSTA catalyses monosaccharide/H+ symport. Expression of mstA in response to carbon starvation and upon transfer to poor carbon sources is consistent with a role for MSTA as a high-affinity transporter for D-glucose, D-mannose and D-xylose. Northern analysis has shown that mstA is subject to CreA-mediated carbon catabolite repression and pH regulation mediated by PacC. A. niger strains in which the mstA gene had been disrupted are phenotypically identical with isogenic reference strains when grown on 0.1-60 mM D-glucose, D-mannose, D-fructose or D-xylose. This indicates that A. niger possesses other transporters capable of compensating for the absence of MSTA. more...
- Published
- 2004
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28. Characterization of nerolidol biotransformation based on indirect on-line estimation of biomass concentration and physiological state in batch cultures of Aspergillus niger.
- Author
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Hrdlicka PJ, Sørensen AB, Poulsen BR, Ruijter GJ, Visser J, and Iversen JJ
- Subjects
- Algorithms, Aspergillus niger cytology, Aspergillus niger isolation & purification, Biotransformation, Cell Culture Techniques methods, Cell Division physiology, Computer Simulation, Online Systems, Aspergillus niger growth & development, Aspergillus niger metabolism, Bioreactors microbiology, Cell Count methods, Models, Biological, Sesquiterpenes metabolism
- Abstract
Biotransformation of the sesquiterpenoid trans-nerolidol by Aspergillus niger has previously been investigated as a method for the formation of 12-hydroxy-trans-nerolidol, a precursor in the synthesis of the industrially interesting flavor alpha-sinensal. We characterized biotransformations of cis-nerolidol, trans-nerolidol, and a commercially available cis/trans-nerolidol mixture in repeated batch cultures of A. niger grown in computer-controlled bioreactors. On-line quantification of titrant addition in pH control allowed characterization of (1) maximal specific growth rate in exponential growth phases, (2) exponential induction of acid formation in postexponential phases, (3) inhibition of organic acid formation after nerolidol addition, and (4) exponential recovery from this inhibition. Addition of a (+/-)-cis/trans-nerolidol mixture during exponential or postexponential phase to cultures grown in minimal medium at high dissolved oxygen tension (above 50% air saturation), to cultures at low dissolved oxygen tension (5% air saturation), or to cultures grown in rich medium demonstrated that the physiological state before nerolidol addition had a major influence on biotransformation. The maximal molar yield of 12-hydroxy-trans-nerolidol (9%) was obtained by addition of a (+/-)-cis/trans-nerolidol mixture to the culture in the postexponential phase at high dissolved oxygen tension in minimal medium. Similar yields were obtained in rich medium, where the rate of biotransformation was doubled. more...
- Published
- 2004
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29. Mannitol is required for stress tolerance in Aspergillus niger conidiospores.
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Ruijter GJ, Bax M, Patel H, Flitter SJ, van de Vondervoort PJ, de Vries RP, vanKuyk PA, and Visser J
- Subjects
- Binding Sites genetics, Cell Death physiology, Cells, Cultured, DNA-Binding Proteins metabolism, Energy Metabolism physiology, Fungal Proteins metabolism, Genes, Regulator genetics, Molecular Sequence Data, Mutation genetics, Oxidative Stress physiology, Phosphoric Monoester Hydrolases metabolism, Promoter Regions, Genetic genetics, Sugar Alcohol Dehydrogenases genetics, Sugar Alcohol Dehydrogenases isolation & purification, Transcription Factors metabolism, Aspergillus niger metabolism, Mannitol metabolism, Spores, Fungal metabolism
- Abstract
D-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungus Aspergillus niger and makes up 10 to 15% of the dry weight. A number of physiological functions have been ascribed to mannitol, including serving as a reserve carbon source, as an antioxidant, and to store reducing power. In this study, we cloned and characterized the A. niger mpdA gene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. The mpdA promoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression of mpdA in sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated in A. niger. Inactivation of mpdA abolished mannitol biosynthesis in growing mycelium and reduced the mannitol level in conidiospores to 30% that in the wild type, indicating that MPD and mannitol 1-phosphate phosphatase form the major metabolic pathway for mannitol biosynthesis in A. niger. The viability of spores after prolonged storage and germination kinetics were normal in an mpdA null mutant, indicating that mannitol does not play an essential role as a reserve carbon source in A. niger conidia. However, conidiospores of a DeltampdA strain were extremely sensitive to a variety of stress conditions, including high temperature, oxidative stress and, to a lesser extent, freezing and lyophilization. Since mannitol supplied in the medium during sporulation repaired this deficiency, mannitol appears to be essential for the protection of A. niger spores against cell damage under these stress conditions. more...
- Published
- 2003
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30. Glycerol dehydrogenase, encoded by gldB is essential for osmotolerance in Aspergillus nidulans.
- Author
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de Vries RP, Flitter SJ, van de Vondervoort PJ, Chaveroche MK, Fontaine T, Fillinger S, Ruijter GJ, d'Enfert C, and Visser J
- Subjects
- Aspergillus nidulans genetics, Aspergillus nidulans physiology, Cell Division, Gene Silencing, Glucose metabolism, Glycerol metabolism, Molecular Sequence Data, NADP metabolism, Osmolar Concentration, Sodium Chloride metabolism, Sugar Alcohols metabolism, Aspergillus nidulans enzymology, Fungal Proteins genetics, Fungal Proteins metabolism, Sugar Alcohol Dehydrogenases genetics, Sugar Alcohol Dehydrogenases metabolism
- Abstract
We have characterized the Aspergillus nidulans gldB gene encoding a NADP+-dependent glycerol dehydrogenase. A basal expression level was observed for gldB, which increased significantly under conditions of hyper-osmotic shock (1 M NaCl). Growth of strains in which gldB was disrupted was severely reduced on plates containing 1% glucose and 1 M NaCl, but these strains were able to grow on plates containing 1 M NaCl and 1% glycerol, arabitol, mannitol or erythritol. Uptake of these polyols compensated for the inability of the gldB disruptants to produce glycerol. Presence of 1% glucose in these plates prevented growth restoration by all the polyols tested with the exemption of glycerol, indicating that uptake of mannitol, arabitol and erythritol is subject to glucose repression, whereas uptake of glycerol is significantly less or not repressed. No intracellular glycerol dehydrogenase activity could be detected in the gldB disruption strains. Intracellular glycerol levels in these strains were strongly decreased compared to wild type, whereas intracellular mannitol, erythritol and arabitol levels were increased. Conidia of the gldB disruption strain did not accumulate glycerol upon germination in glucose media with or without 1 M NaCl and germ tube emergence was significantly delayed in this strain in the presence of 1 M NaCl in comparison to the wild type. These data indicate that gldB is essential for osmotolerance in A. nidulans and that the pathways for glycerol biosynthesis under osmotic stress differ between yeast and filamentous fungi. more...
- Published
- 2003
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31. Onset of carbon catabolite repression in Aspergillus nidulans. Parallel involvement of hexokinase and glucokinase in sugar signaling.
- Author
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Flipphi M, van de Vondervoort PJ, Ruijter GJ, Visser J, Arst HN Jr, and Felenbok B
- Subjects
- Acetate-CoA Ligase metabolism, Animals, Aspergillus nidulans genetics, Endo-1,4-beta Xylanases, Ethanol metabolism, Fructose metabolism, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Glucokinase genetics, Glucose metabolism, Hexokinase genetics, Phosphorylation, Repressor Proteins genetics, Repressor Proteins metabolism, Signal Transduction physiology, Xylans metabolism, Xylose metabolism, Xylosidases genetics, Xylosidases metabolism, Aspergillus nidulans enzymology, Carbon metabolism, Energy Metabolism physiology, Glucokinase metabolism, Hexokinase metabolism
- Abstract
The role of hexose phosphorylating enzymes in the signaling of carbon catabolite repression was investigated in the filamentous fungus Aspergillus nidulans. A d-fructose non-utilizing, hexokinase-deficient (hxkA1, formerly designated frA1) strain was utilized to obtain new mutants lacking either glucokinase (glkA4) or both hexose kinases (hxkA1/glkA4). d-Glucose and d-fructose phosphorylation is completely abolished in the double mutant, which consequently cannot grow on either sugar. The glucokinase single mutant exhibits no nutritional deficiencies. Three repressible diagnostic systems, ethanol utilization (alcA and alcR genes), xylan degradation (xlnA), and acetate catabolism (facA), were analyzed in these hexose kinase mutants at the transcript level. Transcriptional repression by d-glucose is fully retained in the two single kinase mutants, whereas the hexokinase mutant is partially derepressed for d-fructose. Thus, hexokinase A and glucokinase A compensate each other for carbon catabolite repression by d-glucose in the single mutants. In contrast, both d-glucose and d-fructose repression are severely impaired for all three diagnostic systems in the double mutant. Unlike the situation in Saccharomyces cerevisiae, the hexose phosphorylating enzymes play parallel roles in glucose repression in A. nidulans. more...
- Published
- 2003
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32. Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.
- Author
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Hesse SJ, Ruijter GJ, Dijkema C, and Visser J
- Subjects
- Antimetabolites pharmacology, Aspergillus niger chemistry, Carbohydrate Metabolism, Carbonyl Cyanide m-Chlorophenyl Hydrazone pharmacology, Citric Acid metabolism, Computer Systems, Energy Metabolism, Homeostasis, Intracellular Fluid chemistry, Ionophores pharmacology, Nuclear Magnetic Resonance, Biomolecular, Sodium Azide pharmacology, Vacuoles chemistry, Aspergillus niger physiology, Hydrogen-Ion Concentration
- Abstract
Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively). more...
- Published
- 2002
- Full Text
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33. The Aspergillus niger D-xylulose kinase gene is co-expressed with genes encoding arabinan degrading enzymes, and is essential for growth on D-xylose and L-arabinose.
- Author
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vanKuyk PA, de Groot MJ, Ruijter GJ, de Vries RP, and Visser J
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Monophosphate metabolism, Aspergillus niger genetics, Aspergillus niger growth & development, Aspergillus niger metabolism, Cloning, Molecular, DNA metabolism, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Mutation genetics, Phosphotransferases (Alcohol Group Acceptor) chemistry, RNA, Messenger genetics, RNA, Messenger metabolism, Spectrometry, Mass, Electrospray Ionization, Transcription, Genetic genetics, Up-Regulation, Arabinose metabolism, Aspergillus niger enzymology, Genes, Fungal genetics, Phosphotransferases (Alcohol Group Acceptor) genetics, Phosphotransferases (Alcohol Group Acceptor) metabolism, Polysaccharides metabolism, Xylose metabolism
- Abstract
The Aspergillus niger D-xylulose kinase encoding gene has been cloned by complementation of a strain deficient in D-xylulose kinase activity. Expression of xkiA was observed in the presence of L-arabinose, L-arabitol and D-xylose. Expression of xkiA is not mediated by XLNR, the xylose-dependent positively-acting xylanolytic regulator. Although the expression of xkiA is subject to carbon catabolite repression, the wide domain regulator CREA is not directly involved. The A. niger D-xylulose kinase was purified to homogeneity, and the molecular mass determined using electrospray ionization mass spectrometry agreed with the calculated molecular mass of 62816.6 Da. The activity of XKIA is highly specific for D-xylulose. Kinetic parameters were determined as Km(D-xylulose) = 0.76 mM and Km(ATP) = 0.061 mM. Increased transcript levels of the genes encoding arabinan and xylan degrading enzymes, observed in the xylulose kinase deficient strain, correlate with increased accumulation of L-arabitol and xylitol, respectively. This result supports the suggestion that L-arabitol may be the specific low molecular mass inducer of the genes involved in arabinan degradation. It also suggests a possible role for xylitol in the induction of xylanolytic genes. Conversely, overproduction of XKIA did not reduce the size of the intracellular arabitol and xylitol pools, and therefore had no effect on expression of genes encoding xylan and arabinan degrading enzymes nor on the activity of the enzymes of the catabolic pathway. more...
- Published
- 2001
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34. Properties of Aspergillus niger citrate synthase and effects of citA overexpression on citric acid production.
- Author
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Ruijter GJ, Panneman H, Xu D, and Visser J
- Subjects
- Acetyl Coenzyme A metabolism, Amino Acid Sequence, Aspergillus niger genetics, Citrate (si)-Synthase genetics, Citrate (si)-Synthase isolation & purification, Cloning, Molecular, DNA, Complementary chemistry, DNA, Complementary genetics, Gene Expression Regulation, Enzymologic, Gene Expression Regulation, Fungal, Kinetics, Molecular Sequence Data, Oxaloacetates metabolism, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Species Specificity, Transformation, Genetic, Aspergillus niger enzymology, Citrate (si)-Synthase metabolism, Citric Acid metabolism
- Abstract
Using a combination of dye adsorption and affinity elution we purified Aspergillus niger citrate synthase to homogeneity using a single column and characterised the enzyme. An A. niger citrate synthase cDNA was isolated by immunological screening and used to clone the corresponding citA gene. The deduced amino acid sequence showed high similarity to other fungal citrate synthases. After processing upon mitochondrial import, the calculated M(r) of A. niger citrate synthase is 48501, which agrees well with the estimated molecular mass of the purified protein (48 kDa). In addition to an N-terminal mitochondrial import signal, a peroxisomal target sequence (AKL) was found at the C-terminus of the protein. Whether both signals are functional in vivo is not clear. Strains overexpressing citA were made by transformation and cultured under citric acid-producing conditions. Up to 11-fold overproduction of citrate synthase did not increase the rate of citric acid production by the fungus, suggesting that citrate synthase contributes little to flux control in the pathway involved in citric acid biosynthesis by a non-commercial strain. more...
- Published
- 2000
- Full Text
- View/download PDF
35. Measurement of intracellular (compartmental) pH by 31P NMR in Aspergillus niger.
- Author
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Hesse SJ, Ruijter GJ, Dijkema C, and Visser J
- Subjects
- Alginates, Aspergillus niger drug effects, Biocompatible Materials, Buffers, Cell Compartmentation drug effects, Cells, Immobilized, Chelating Agents pharmacology, Citric Acid pharmacology, Glucose analysis, Glucose pharmacology, Glucuronic Acid, Hexuronic Acids, Nitrates analysis, Oxygen pharmacology, Perfusion, Phosphorus Isotopes, Time Factors, Vacuoles metabolism, Aspergillus niger metabolism, Cell Compartmentation physiology, Hydrogen-Ion Concentration, Magnetic Resonance Spectroscopy
- Abstract
31P nuclear magnetic resonance (31P NMR) was used to monitor cytoplasmic and vacuolar pH values in the filamentous fungus Aspergillus niger. To obtain a homogeneous cell sample and to be able to perform long term in vivo NMR measurements A. niger mycelium was kept in a setup that allows perfusion of the cell plug within the NMR tube. Mycelial samples, however, became rapidly clogged during perfusion leading to (partial) anaerobiosis of the plug with subsequent acidification of the cytoplasm. As a result, only short-term NMR measurements (5-10 min) were possible using free mycelium. To increase and to prolong perfusion, A. niger was immobilized in Ca(2+)-alginate beads. Deteriorated spectra recorded under hypoxia could be completely restored in the presence of oxygen. With this system perfusion in the presence of citrate could be maintained for at least 18 h at much higher rates (15 ml min-1 compared with 4 ml min-1 for free mycelium). During this period 31P NMR spectra were highly invariable, indicating approximate steady-state intracellular conditions during long term measurements. Perfusion in the presence of glucose resulted in complete depletion of the vacuolar inorganic phosphate pool within 45 min and yielded a higher pH gradient over the tonoplast than when citrate was used (delta pH = 1.6 and 1.4, respectively). more...
- Published
- 2000
- Full Text
- View/download PDF
36. Characterization of Aspergillus niger phosphoglucose isomerase. Use for quantitative determination of erythrose 4-phosphate.
- Author
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Ruijter GJ and Visser J
- Subjects
- Electrophoresis, Polyacrylamide Gel, Enzyme Inhibitors pharmacology, Gluconates pharmacology, Glucose-6-Phosphate Isomerase antagonists & inhibitors, Hydrogen-Ion Concentration, Kinetics, Aspergillus niger enzymology, Glucose-6-Phosphate Isomerase chemistry, Sugar Phosphates analysis
- Abstract
Phosphoglucose isomerase (PGI) was purified from Aspergillus niger and the in vitro kinetic properties of the enzyme were related to its functioning in vivo. A new assay method was developed to study the forward reaction making use of mannitol 1-P dehydrogenase as the coupling enzyme. In this simple assay system mannitol 1-P dehydrogenase converts fructose 6-P and NADH to mannitol 1-P and NAD+, respectively. At pH 7.5 the Km for glucose 6-P was 0.48 mM, whereas the Km for fructose 6-P was 0.32 mM. The pentose phosphate pathway intermediates 6-phosphogluconate and erythrose 4-P (E4P) were competitive inhibitors of PGI with Ki values of approximately 0.2 mM and 1 microM respectively. In citric acid producing A. niger mycelium inhibition by 6-phosphogluconate is of minor physiological significance (10% inhibition). Since E4P could not be detected by an existing procedure, a novel assay was developed based on the strong inhibition of PGI by E4P. Although the new assay is very sensitive (detection limit 25 pmol), E4P could still not be detected in metabolite extracts indicating that a very low level of E4P is present in the cells. Using in vitro kinetics and concentrations of intracellular metabolites the in vivo activity of PGI was calculated and closely matched the steady state glycolytic flux observed during citric acid production. more...
- Published
- 1999
- Full Text
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37. Cloning and biochemical characterisation of Aspergillus niger hexokinase--the enzyme is strongly inhibited by physiological concentrations of trehalose 6-phosphate.
- Author
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Panneman H, Ruijter GJ, van den Broeck HC, and Visser J
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, Ion Exchange, Cloning, Molecular, DNA, Fungal, Glucokinase metabolism, Glucose metabolism, Hexokinase antagonists & inhibitors, Hexokinase genetics, Hydrogen-Ion Concentration, Kinetics, Molecular Sequence Data, Osmolar Concentration, Phosphorylation, Phylogeny, Substrate Specificity, Trehalose physiology, Aspergillus niger enzymology, Hexokinase metabolism, Sugar Phosphates physiology, Trehalose analogs & derivatives
- Abstract
The Aspergillus niger hexokinase gene hxkA has been cloned by heterologous hybridisation using the Aspergillus nidulans hexokinase gene as a probe. The DNA sequence of the gene was determined, and the deduced amino acid sequence showed significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to those of the budding yeasts. The encoded protein was purified from a multicopy hxkA transformant, and extensively characterised. The hexokinase protein has a molecular mass of 54090, a pI of 4.9 and is a homodimer. D-Glucose, the glucose analogue 2-deoxy-D-glucose, D-fructose, D-mannose and D-glucosamine are phosphorylated by hexokinase, whereas the hexoses D-galactose, L-sorbose, methyl alpha-D-glucoside and the pentoses L-arabinose and D-xylose are not. The enzyme has high affinity for glucose (Km = 0.35 mM at pH 7.5) and for fructose (Km = 2.0 mM at pH 7.5) and is inhibited by ADP. The enzyme is strongly inhibited by physiological concentrations (0.1-0.2 mM) of trehalose 6-phosphate, which may be of importance for in vivo regulation of the enzyme. Inhibition of A. niger hexokinase by trehalose 6-phosphate is competitive towards the sugar substrate (Ki = 0.01 mM). Based on the kinetic constants of hexokinase and glucokinase their relative contribution to in vivo glucose phosphorylation was calculated and found to be strongly dependent on intracellular pH and glucose concentration. At pH 7.5 glucokinase is predominant, whereas at pH 6.5 hexokinase is predominant at glucose concentrations higher than 0.5 mM. Expression of the hexokinase and the glucokinase gene requires active carbon metabolism. Also on carbon sources which are not substrates for hexokinase or glucokinase, clear expression is observed. The hexokinase and glucokinase enzymes are quite stable in vivo. Even in the absence of transcription, active glucokinase and hexokinase remain present in the cells at almost the same level for at least 3-4 h after depletion of the carbon source. more...
- Published
- 1998
- Full Text
- View/download PDF
38. Carbon repression in Aspergilli.
- Author
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Ruijter GJ and Visser J
- Subjects
- Cyclic AMP physiology, Fungal Proteins genetics, Mutation, Repressor Proteins genetics, Aspergillus metabolism, Carbon metabolism, Fungal Proteins physiology, Repressor Proteins physiology
- Abstract
Many microorganisms prefer easily metabolizable carbon sources over alternative, less readily metabolized carbon sources. One of the mechanisms to achieve this is repression of the synthesis of enzymes related to catabolism of the alternative carbon sources, i.e. carbon repression. It is now clear that in Aspergillus nidulans and Aspergillus niger the repressor protein CREA plays a major role in carbon repression. CREA inhibits transcription of many target genes by binding to specific sequences in the promoter of these genes. Unfortunately there is little information on other components of the signalling pathway that triggers repression by CREA. In this review we summarize the current understanding of carbon repression in Aspergilli. more...
- Published
- 1997
- Full Text
- View/download PDF
39. Overexpression of phosphofructokinase and pyruvate kinase in citric acid-producing Aspergillus niger.
- Author
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Ruijter GJ, Panneman H, and Visser J
- Subjects
- Aspergillus niger genetics, Cloning, Molecular, Fructosediphosphates analysis, Molecular Sequence Data, Aspergillus niger enzymology, Citric Acid metabolism, Phosphofructokinase-1 biosynthesis, Pyruvate Kinase biosynthesis
- Abstract
Phosphofructokinase and pyruvate kinase were overexpressed in the filamentous fungus Aspergillus niger. Moderate overexpression of these glycolytic enzymes in A. niger N400 (3-5-fold the wild-type level), either individually or simultaneously, did not increase citric acid production by the fungus significantly. Thus, phosphofructokinase and pyruvate kinase do not seem to contribute in a major way to flux control of the metabolism involved in the conversion of glucose to citric acid. Overexpression of phosphofructokinase and pyruvate kinase did not influence the activities of other enzymes in the pathway, nor did it change intermediary metabolite levels. However, in strains overexpressing phosphofructokinase, the level of fructose 2,6-bisphosphate, a positive allosteric effector of phosphofructokinase, was reduced almost 2-fold compared to the wild-type strain. Measurements with purified phosphofructokinase, using substrate, product and effector concentrations found intracellularly, showed that such a reduction in the fructose-2,6-bisphosphate level could decrease the specific activity of phosphofructokinase in the cell significantly. Thus, the fungus seems to adapt to overexpression of phosphofructokinase by decreasing the specific activity of the enzyme through a reduction in the level of fructose 2,6-bisphosphate. more...
- Published
- 1997
- Full Text
- View/download PDF
40. Cloning and biochemical characterisation of an Aspergillus niger glucokinase. Evidence for the presence of separate glucokinase and hexokinase enzymes.
- Author
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Panneman H, Ruijter GJ, van den Broeck HC, Driever ET, and Visser J
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Amino Acid Sequence, Base Sequence, Chemical Phenomena, Chemistry, Physical, Cloning, Molecular, DNA, Fungal genetics, Genes, Fungal, Glucokinase metabolism, Hexokinase metabolism, Hexoses metabolism, Isoelectric Point, Kinetics, Molecular Sequence Data, Molecular Weight, Phosphorylation, Phylogeny, Substrate Specificity, Aspergillus niger enzymology, Aspergillus niger genetics, Glucokinase chemistry, Glucokinase genetics, Hexokinase genetics
- Abstract
The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a pI of 5.2. The enzyme has high affinity for glucose (K(m) 0.063 mM at pH 7.5) and a relatively low affinity for fructose (K(m) 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non-competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcal (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexosephosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6-phosphate. more...
- Published
- 1996
- Full Text
- View/download PDF
41. Characterisation of the Aspergillus nidulans frA1 mutant: hexose phosphorylation and apparent lack of involvement of hexokinase in glucose repression.
- Author
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Ruijter GJ, Panneman H, van den Broeck HC, Bennett JM, and Visser J
- Subjects
- Amino Acid Sequence, Aspergillus nidulans enzymology, Fructose metabolism, Genetic Complementation Test, Glucose metabolism, Molecular Sequence Data, Mutation genetics, Phosphorylation, Trehalose metabolism, Aspergillus nidulans genetics, Hexokinase metabolism, Hexoses metabolism
- Abstract
Hexose phosphorylation was studied in Aspergillus nidulans wild-type and in a fructose non-utilising mutant (frA). The data indicate the presence of at least one hexokinase and one glucokinase in wild-type A. nidulans, while the frA1 mutant lacks hexokinase activity. The A. nidulans gene encoding hexokinase was isolated by complementation of the frA1 mutation. The absence of hexokinase activity in the frA1 mutant did not interfere with glucose repression of the enzymes involved in alcohol and L-arabinose catabolism. This suggest that, unlike the situation in yeast where mutation of hexokinase PII abolishes glucose repression, the A. nidulans hexokinase might not be involved in glucose repression. more...
- Published
- 1996
- Full Text
- View/download PDF
42. An extreme creA mutation in Aspergillus nidulans has severe effects on D-glucose utilization.
- Author
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van der Veen P, Ruijter GJ, and Visser J
- Subjects
- Aspergillus nidulans metabolism, Fungal Proteins metabolism, Mutation, Repressor Proteins metabolism, Sugar Alcohols metabolism, Aspergillus nidulans genetics, Fungal Proteins genetics, Gene Expression Regulation, Fungal, Glucose metabolism, Glycolysis, Repressor Proteins genetics
- Abstract
Aspergillus nidulans wild-type and the extreme carbon catabolite derepressed mutant creAd-30 were characterized with respect to enzyme activities, metabolite concentrations and polyol pools all related to glycolysis, after growth on D-glucose. In the creAd-30 strain the enzymes hexokinase and fructose-6-phosphate reductase showed a two- and threefold increase in activity, respectively, whereas phosphofructokinase and pyruvate kinase activity decreased two- and threefold, respectively, in comparison with the wild-type strain. The most notable changes in metabolite concentrations were that fructose 2,6-bisphosphate and fructose 1,6-bisphosphate showed a 2.5-fold increase, whereas both pyruvate and citrate decreased in the creAd-30. Striking differences were found for the polyol concentrations measured for the two strains tested. Intracellular glycerol and arabitol concentrations were 10-fold higher and erythritol fivefold higher in creAd-30, whereas intracellular trehalose and mannitol were both decreased. The total internal polyol concentration appears to be constant at approximately 700 mumol (g dry wt)-1. All polyols were also detected in high amounts in the culture filtrate of the creAd-30 mutant strain but no extracellular trehalose was found. The overall production of polyols in this strain was therefore much higher than in the wild-type. The high level of polyols produced and the changes in metabolite concentrations in the creAd-30 strain suggest that the differences in enzyme activities result in an altered flow through glycolysis leading to a more rapid formation of polyols which are subsequently secreted. more...
- Published
- 1995
- Full Text
- View/download PDF
43. Analysis of mutations that uncouple transport from phosphorylation in enzyme IIGlc of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system.
- Author
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Ruijter GJ, van Meurs G, Verwey MA, Postma PW, and van Dam K
- Subjects
- Base Sequence, Binding Sites, Biological Transport, Chromosome Mapping, Escherichia coli enzymology, Kinetics, Phosphorylation, Escherichia coli genetics, Genes, Bacterial genetics, Glucose metabolism, Mutation genetics, Phosphoenolpyruvate Sugar Phosphotransferase System genetics
- Abstract
Mutations that uncouple glucose transport from phosphorylation were isolated in plasmid-encoded Escherichia coli enzyme IIGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). The uncoupled enzymes IIGlc were able to transport glucose in the absence of the general phosphoryl-carrying proteins of the PTS, enzyme I and HPr, although with relatively low affinity. Km values of the uncoupled enzymes IIGlc for glucose ranged from 0.5 to 2.5 mM, 2 orders of magnitude higher than the value of normal IIGlc. Most of the mutant proteins were still able to phosphorylate glucose and methyl alpha-glucoside (a non-metabolizable glucose analog specific for IIGlc), indicating that transport and phosphorylation are separable functions of the enzyme. Some of the uncoupled enzymes IIGlc transported glucose with a higher rate and lower apparent Km in a pts+ strain than in a delta ptsHI strain lacking the general proteins enzyme I and HPr. Since the properties of these uncoupled enzymes IIGlc in the presence of PTS-mediated phosphoryl transfer resembled those of wild-type IIGlc, these mutants appeared to be conditionally uncoupled. Sequencing of the mutated ptsG genes revealed that all amino acid substitutions occurred in a hydrophilic segment within the hydrophobic N-terminal part of IIGlc. These results suggest that this hydrophilic loop is involved in binding and translocation of the sugar substrate. more...
- Published
- 1992
- Full Text
- View/download PDF
44. Energetics of glucose uptake in a Salmonella typhimurium mutant containing uncoupled enzyme IIGlc.
- Author
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Ruijter GJ, Postma PW, and van Dam K
- Subjects
- Anaerobiosis, Biological Transport, Active, Mutation, Phosphoenolpyruvate metabolism, Phosphorylation, Salmonella typhimurium genetics, Salmonella typhimurium growth & development, Glucose metabolism, Salmonella typhimurium metabolism
- Abstract
Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP): glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. We have studied the energetics of glucose uptake catalyzed by this uncoupled enzyme IIGlc. The molar growth yields on glucose of two strains cultured anaerobically in glucose-limited chemostat- and batch cultures were compared. Strain PP799 transported and phosphorylated glucose via an intact PTS, while strain PP952 took up glucose exclusively via uncoupled enzyme IIGlc, followed by ATP-dependent phosphorylation by glucokinase. Thus the strains were isogenic except for the mode of uptake and phosphorylation of the growth substrate. PP799 and PP952 exhibited similar YGlc values. Assuming equal YATP values for both strains this result indicated that there were no energetic demands for glucose uptake via uncoupled enzyme IIGlc. more...
- Published
- 1991
- Full Text
- View/download PDF
45. Adaptation of Salmonella typhimurium mutants containing uncoupled enzyme IIGlc to glucose-limited conditions.
- Author
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Ruijter GJ, Postma PW, and van Dam K
- Subjects
- Biological Transport, Carbohydrate Metabolism, Genotype, Kinetics, Phosphoenolpyruvate Sugar Phosphotransferase System genetics, Salmonella typhimurium enzymology, Salmonella typhimurium growth & development, Glucose metabolism, Mutation, Phosphoenolpyruvate Sugar Phosphotransferase System metabolism, Salmonella typhimurium genetics
- Abstract
Uncoupled enzyme IIGlc of the phosphoenolpyruvate (PEP):glucose phosphotransferase system (PTS) in Salmonella typhimurium is able to catalyze glucose transport in the absence of PEP-dependent phosphorylation. As a result of the ptsG mutation, the apparent Km of the system for glucose transport is increased about 1,000-fold (approximately 18 mM) compared with wild-type PTS-mediated glucose transport. An S. typhimurium mutant containing uncoupled enzyme IIGlc as the sole system for glucose uptake was grown in glucose-limited chemostat cultures. Selective pressure during growth in the chemostat resulted in adaptation to the glucose-limiting conditions in two different ways. At first, mutations appeared that led to a decrease in Km value of uncoupled enzyme IIGlc. These results suggested that uncoupled enzyme IIGlc had significant control on the growth rate under glucose-limiting conditions. More efficient glucose uptake enabled a mutant to outgrow its parent and caused a decrease in the steady-state glucose concentration in the chemostat. At very low glucose concentrations (10 microM), mutants arose that contained a constitutively synthesized methyl-beta-galactoside permease. Apparently, further changes in the uncoupled enzyme IIGlc did not lead to a substantial increase in growth rate at very low glucose concentrations. more...
- Published
- 1990
- Full Text
- View/download PDF
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