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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.

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.

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.

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.

Contains fulltext : 89263.pdf (Publisher’s version ) (Closed access) 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. 01 mei 2010

Contains fulltext : 69547.pdf (Publisher’s version ) (Closed access) 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.

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.

Contains fulltext : 53596.pdf (Publisher’s version ) (Closed access) 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.

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.

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.

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.

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. (C) 2004 Wiley Periodicals, Inc.

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.

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.

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.

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.

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.

d-Mannitol is the predominant carbon compound in conidiospores of the filamentous fungusAspergillus nigerand 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 theA. niger mpdAgene, which encodes mannitol 1-phosphate dehydrogenase (MPD), the first enzyme in the mannitol biosynthesis pathway. ThempdApromoter contains putative binding sites for the development-specific transcription factors BRLA and ABAA. Furthermore, increased expression ofmpdAin sporulating mycelium suggests that mannitol biosynthesis is, to a certain extent, developmentally regulated inA. niger. Inactivation ofmpdAabolished 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 inA. niger. The viability of spores after prolonged storage and germination kinetics were normal in anmpdAnull mutant, indicating that mannitol does not play an essential role as a reserve carbon source inA. nigerconidia. However, conidiospores of a ΔmpdAstrain 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 ofA. nigerspores against cell damage under these stress conditions.

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).

The external pH appeared to be the main factor governing oxalic acid production by Aspergillus niger. A glucose-oxidase-negative mutant produced substantial amounts of oxalic acid as long as the pH of the culture was 3 or higher. When pH was decreased below 2, no oxalic acid was formed. The activity of oxaloacetate acetylhydrolase (OAH), the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production. OAH was purified from A. niger and characterized. OAH cleaves oxaloacetate to oxalate and acetate, but A. niger never accumulated any acetate in the culture broth. Since an A. niger acuA mutant, which lacks acetyl-CoA synthase, did produce some acetate, wild-type A. niger is apparently able to catabolize acetate sufficiently fast to prevent its production. An A. niger mutant, prtF28, previously isolated in a screen for strains deficient in extracellular protease expression, was shown here to be oxalate non-producing. The prtF28 mutant lacked OAH, implying that OAH is the only enzyme involved in oxalate production in A. niger. In a traditional citric acid fermentation low pH and absence of Mn2+ are prerequisites. Remarkably, a strain lacking both glucose oxidase (goxC) and OAH (prtF) produced citric acid from sugar substrates in a regular synthetic medium at pH 5 and under these conditions production was completely insensitive to Mn2+.

Summary: Aspergillus niger mutants relieved of carbon repression were isolated from an areA parental strain by selection of colonies that exhibited improved growth on a combination of 4-aminobutanoic acid (GABA) and D-glucose. In addition to derepression of the utilization of GABA as a nitrogen source in the presence of D-glucose, three of the four mutants also showed derepression of L-alanine and L-proline utilization. Transformation of the mutants with the A. niger creA gene, encoding the repressor protein CREA, re-established the areA phenotype on GABA/D-glucose, identifying the mutations as creA d. The creA gene mapped on chromosome IV by linkage analysis and contour-clamped homogeneous electric field hybridization. The creA mutants obtained were used to study the involvement of CREA in repression by D-glucose of arabinases and L-arabinose catabolism in A. niger. In wild-type A. niger, α-L-arabinofuranosidase A, α-L-arabinofuranosidase B, endo-arabinase, L-arabinose reductase and L-arabitol dehydrogenase were induced on L-arabinose, but addition of D-glucose prevented this induction. Repression was relieved to varying degrees in the creA mutants, showing that biosynthesis of arabinases and L-arabinose catabolic enzymes is under control of CREA.

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.

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.

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 mu mol/L for 15-25 weeks of gestation and 3.2-12.0 mu mol/L for 26-38 weeks of gestation. In AFS samples from five fetuses affected with ISSD FSA was 23.9-58.9 mu mol/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.