Pandemic ICU triage challenge and medical ethics.

The COVID-19 pandemic has made unprecedented global demands on healthcare in general and especially the intensive care unit (ICU). the virus is spreading out of control. To this day, there is no clear, published directive for doctors regarding the allocation of ICU beds in times of scarcity. This means that many doctors do not feel supported by their government and are afraid of the medicolegal consequences of the choices they have to make. Consequently, there has been no transparent discussion among professionals and the public. The thought of being at the mercy of absolute arbitrariness leads to fear among the population, especially the vulnerable groups.

[Total parenteral nutrition for ileus in the palliative phase]. / Totale parenterale voeding bij ileus in de palliatieve fase.

Total parenteral nutrition may be considered for a carefully selected group of palliative patients with ileus. Predictive factors include a strong desire to live, low burden of disease other than the ileus and expected increase in quality of life as a result of starting with total parenteral nutrition. Therapy compliance is required and a strong social network is desirable. Close collaboration between hospital and general practitioners and frequent reviews of the palliative care plan are also required for success.

[Immunotherapy for cancer: from medical breakthrough to application in daily practice]. / Immuuntherapie voor kanker.

Immunotherapy induces a response against cancer by activating the immune system. Examples are therapies with checkpoint inhibitors, oncolytic viruses and chimeric antigen receptor T-cells (CAR T-cells). These therapies have, due to their rapid development, found their way to daily practice. For some patients with metastatic disease immunotherapy has led to significant long-term survival. Currently, there is a shift in the treatment with checkpoint inhibitors towards the (neo)adjuvant setting. Treatments with CAR T-cells seem particularly effective in haematological malignancies. Oncolytic viruses are used in the treatment for melanoma, but presently only on a limited scale. Only a limited number of patients benefit from immunotherapy. There remain many challenges for the future, most importantly the optimal use of treatment, recognition and treatment of side effects, determining the optimal duration of treatment and the increasing healthcare costs.

Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular ß-glucosidases.

The membrane lipid glucosylceramide (GlcCer) is continuously formed and degraded. Cells express two GlcCer-degrading ß-glucosidases, glucocerebrosidase (GBA) and GBA2, located in and outside the lysosome, respectively. Here we demonstrate that through transglucosylation both GBA and GBA2 are able to catalyze in vitro the transfer of glucosyl-moieties from GlcCer to cholesterol, and vice versa. Furthermore, the natural occurrence of 1-O-cholesteryl-ß-D-glucopyranoside (GlcChol) in mouse tissues and human plasma is demonstrated using LC-MS/MS and (13)C6-labeled GlcChol as internal standard. In cells, the inhibition of GBA increases GlcChol, whereas inhibition of GBA2 decreases glucosylated sterol. Similarly, in GBA2-deficient mice, GlcChol is reduced. Depletion of GlcCer by inhibition of GlcCer synthase decreases GlcChol in cells and likewise in plasma of inhibitor-treated Gaucher disease patients. In tissues of mice with Niemann-Pick type C disease, a condition characterized by intralysosomal accumulation of cholesterol, marked elevations in GlcChol occur as well. When lysosomal accumulation of cholesterol is induced in cultured cells, GlcChol is formed via lysosomal GBA. This illustrates that reversible transglucosylation reactions are highly dependent on local availability of suitable acceptors. In conclusion, mammalian tissues contain GlcChol formed by transglucosylation through ß-glucosidases using GlcCer as donor. Our findings reveal a novel metabolic function for GlcCer.

α-Synuclein dimerization in erythrocytes of Gaucher disease patients: correlation with lipid abnormalities and oxidative stress.

Several observations suggest that disturbed homeostasis of α-Synuclein (α-Syn) may provide a link between Gaucher disease (GD) and Parkinson's disease (PD). We recently reported increased dimerization of α-Syn in the red blood cell (RBC) membrane of patients with GD. Several studies indicate a crucial relationship between lipids, oxidative stress and α-Syn status. Here we investigated the relationship between the observed increased dimerization of α-Syn in the cell membranes of RBCs, cells devoid of lysosomes and lacking lysosomal enzyme synthesis, and the lipid abnormalities and oxidative stress already described in GD. Correlation studies showed that in GD the α-Syn dimer/monomer ratio is positively correlated with the levels of glucosylceramide (GlcCer) and the glucosylceramide/ceramide (GlcCer/Cer) ratio and negatively with the levels of malonyldialdehyde (MDA) and plasmalogens. In conclusion, we have shown that the increased tendency of α-Syn to form dimers in the RBC membrane of patients with GD, is correlated with both the level of lipids, including GlcCer, the primary lipid abnormality in GD, and the increased oxidative stress observed in this disorder. The study of other tissues, and in particular brain, will be important in order to elucidate the significance of these findings regarding the link between GD and PD.

Accuracy and Reproducibility in Quantification of Plasma Protein Concentrations by Mass Spectrometry without the Use of Isotopic Standards.

BACKGROUND: Quantitative proteomic analysis with mass spectrometry holds great promise for simultaneously quantifying proteins in various biosamples, such as human plasma. Thus far, studies addressing the reproducible measurement of endogenous protein concentrations in human plasma have focussed on targeted analyses employing isotopically labelled standards. Non-targeted proteomics, on the other hand, has been less employed to this end, even though it has been instrumental in discovery proteomics, generating large datasets in multiple fields of research. RESULTS: Using a non-targeted mass spectrometric assay (LCMSE), we quantified abundant plasma proteins (43 mg/mL-40 ug/mL range) in human blood plasma specimens from 30 healthy volunteers and one blood serum sample (ProteomeXchange: PXD000347). Quantitative results were obtained by label-free mass spectrometry using a single internal standard to estimate protein concentrations. This approach resulted in quantitative results for 59 proteins (cut off ≥11 samples quantified) of which 41 proteins were quantified in all 31 samples and 23 of these with an inter-assay variability of ≤ 20%. Results for 7 apolipoproteins were compared with those obtained using isotope-labelled standards, while 12 proteins were compared to routine immunoassays. Comparison of quantitative data obtained by LCMSE and immunoassays showed good to excellent correlations in relative protein abundance (r = 0.72-0.96) and comparable median concentrations for 8 out of 12 proteins tested. Plasma concentrations of 56 proteins determined by LCMSE were of similar accuracy as those reported by targeted studies and 7 apolipoproteins quantified by isotope-labelled standards, when compared to reference concentrations from literature. CONCLUSIONS: This study shows that LCMSE offers good quantification of relative abundance as well as reasonable estimations of concentrations of abundant plasma proteins.

Mass spectrometric quantification of glucosylsphingosine in plasma and urine of type 1 Gaucher patients using an isotope standard.

Deficiency of glucocerebrosidase (GBA) leads to Gaucher disease (GD), an inherited disorder characterised by storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. Recently, we reported marked increases of deacylated GlcCer, named glucosylsphingosine (GlcSph), in plasma of GD patients. To improve quantification, [5-9] (13)C5-GlcSph was synthesised for use as internal standard with quantitative LC-ESI-MS/MS. The method was validated using plasma of 55 GD patients and 20 controls. Intra-assay variation was 1.8% and inter-assay variation was 4.9% for GlcSph (m/z 462.3). Plasma GlcSph levels with the old and new methods closely correlate (r=0.968, slope=1.038). Next, we analysed GlcSph in 24h urine samples of 30 GD patients prior to therapy. GlcSph was detected in the patient samples (median 1.20nM, range 0.11-8.92nM), but was below the limit of quantification in normal urine. Enzyme replacement therapy led to a decrease of urinary GlcSph of GD patients, coinciding with reductions in plasma GlcSph and markers of Gaucher cells (chitotriosidase and CCL18). In analogy to globotriaosylsphingsone in urine of Fabry disease patients, additional isoforms of GlcSph differing in structure of the sphingosine moiety were identified in GD urine samples. In conclusion, GlcSph can be sensitively detected by LC-ESI-MS/MS with an internal isotope standard. Abnormalities in urinary GlcSph are a hallmark of Gaucher disease allowing biochemical confirmation of diagnosis.

Inhibition of UDP-glucosylceramide synthase in mice prevents Gaucher disease-associated B-cell malignancy.

Clonal B-cell proliferation is a frequent manifestation of Gaucher disease - a sphingolipidosis associated with a high risk of multiple myeloma and non-Hodgkin lymphoma. Gaucher disease is caused by genetic deficiency of acid ß-glucosidase, the natural substrates of which (ß-d-glucosylceramide and ß-d-glucosylsphingosine) accumulate, principally in macrophages. Mice with inducible deficiency of ß-glucosidase [Gba(tm1Karl/tm1Karl)Tg(MX1-cre)1Cgn/0] serve as an authentic model of human Gaucher disease; we have recently reported clonal B-cell proliferation accompanied by monoclonal serum paraproteins and cognate tumours in these animals. To explore the relationship between B-cell malignancy and the biochemical defect, we treated Gaucher mice with eliglustat tartrate (GENZ 112638), a potent and selective inhibitor of the first committed step in glycosphingolipid biosynthesis. Twenty-two Gaucher mice received 300 mg/kg of GENZ 112638 daily for 3-10 months from 6 weeks of age. Plasma concentrations of ß-d-glucosylceramide and the unacylated glycosphingolipid, ß-d-glucosylsphingosine, declined. After administration of GENZ 112638 to Gaucher mice for 3-10 months, serum paraproteins were not detected and there was a striking reduction in the malignant lymphoproliferation: neither lymphomas nor plasmacytomas were found in animals that had received the investigational agent. In contrast, 14 out of 60 Gaucher mice without GENZ 112638 treatment developed these tumours; monoclonal paraproteins were detected in plasma from 18 of the 44 age-matched mice with Gaucher disease that had not received GENZ 112638. Long-term inhibition of glycosphingolipid biosynthesis suppresses the development of spontaneous B-cell lymphoma and myeloma in Gaucher mice.

Gaucher disease: plasmalogen levels in relation to primary lipid abnormalities and oxidative stress.

Plasmalogens represent a unique class of phospholipids. Reduced red blood cell plasmalogen levels in Gaucher disease patients were reported, correlating to total disease burden. The relation between plasmalogen abnormalities in Gaucher disease patients and primary glycosphingolipid abnormalities, malonyldialdehyde levels, an indicator of lipid peroxidation, and the total antioxidant status was further investigated. Significant reduction of C16:0 and C18:0 plasmalogens in red blood cells of Gaucher disease patients was confirmed. In parallel, a significant increase in the glucosylceramide/ceramide ratio in red blood cell membranes, as well as an average 200-fold increase in plasma glucosylsphingosine levels was observed. Red blood cell malonyldialdehyde levels were significantly increased in patients, whereas their total antioxidant status was significantly reduced. A negative correlation between plasmalogen species and glucosylceramide, ceramide, glucosylceramide/ceramide ratio, glucosylsphingosine and malonyldialdehyde, significant for the C16:0 species and all the above parameters with the exception of malonyldialdehyde levels, was found along with a positive non-significant correlation with the total antioxidant status. Our results indicate that increased lipid peroxidation and reduced total antioxidant status exist in Gaucher disease patients. They demonstrate a clear link between plasmalogen levels and the primary glycolipid abnormalities characterizing the disorder and an association with the increased oxidative stress observed in Gaucher disease patients.

Quantification of globotriaosylsphingosine in plasma and urine of fabry patients by stable isotope ultraperformance liquid chromatography-tandem mass spectrometry.

BACKGROUND: Biochemical markers that accurately reflect the severity and progression of disease in patients with Fabry disease and their response to treatment are urgently needed. Globotriaosylsphingosine, also called lysoglobotriaosylceramide (lysoGb3), is a promising candidate biomarker. METHODS: We synthesized lysoGb3 and isotope-labeled [5,6,7,8,9] (13)C5-lysoGb3 (internal standard). After addition of the internal standard to 25 µL plasma or 400 µL urine from patients with Fabry disease and healthy controls, samples were extracted with organic solvents and the lysoGb3 concentration was quantified by UPLC-ESI-MS/MS (ultraperformance liquid chromatography-electrospray ionization-tandem mass spectrometry). Calibration curves were constructed with control plasma and urine supplemented with lysoGb3. In addition to lysoGb3, lyso-ene-Gb3 was quantified. Quantification was achieved by multiple reaction monitoring of the transitions m/z 786.4 > 282.3 [M+H](+) for lysoGb3, m/z 791.4 > 287.3 [M+H](+) for [5,6,7,8,9] (13)C5-lysoGb3, and 784.4 > 280.3 [M+H](+) for lyso-ene-Gb3. RESULTS: The mean (SD) plasma lysoGb3 concentration from 10 classically affected Fabry hemizygotes was 94.4 (25.8) pmol/mL (range 52.7-136.8 pmol/mL), from 10 classically affected Fabry heterozygotes 9.6 (5.8) pmol/mL (range 4.1-23.5 pmol/mL), and from 20 healthy controls 0.4 (0.1) pmol/mL (range 0.3-0.5 pmol/mL). Lyso-ene-Gb3 concentrations were 10%-25% of total lysoGb3. The urine concentration of lysoGb3 was 40-480 times lower than in corresponding plasma samples. Lyso-ene-Gb3 concentrations in urine were comparable or even higher than the corresponding lysoGb3 concentrations. CONCLUSIONS: This assay for the quantification of lysoGb3 and lyso-ene-Gb3 in human plasma and urine samples will be an important tool in the diagnosis of Fabry disease and for monitoring the effect of enzyme replacement therapy in patients with Fabry disease.

Fingolimod attenuates ceramide-induced blood-brain barrier dysfunction in multiple sclerosis by targeting reactive astrocytes.

Alterations in sphingolipid metabolism are described to contribute to various neurological disorders. We here determined the expression of enzymes involved in the sphingomyelin cycle and their products in postmortem brain tissue of multiple sclerosis (MS) patients. In parallel, we investigated the effect of the sphingosine-1 receptor agonist Fingolimod (Gilenya(®)) on sphingomyelin metabolism in reactive astrocytes and determined its functional consequences for the process of neuro-inflammation. Our results demonstrate that in active MS lesions, marked by large number of infiltrated immune cells, an altered expression of enzymes involved in the sphingomyelin cycle favors enhanced ceramide production. We identified reactive astrocytes as the primary cellular source of enhanced ceramide production in MS brain samples. Astrocytes isolated from MS lesions expressed enhanced mRNA levels of the ceramide-producing enzyme acid sphingomyelinase (ASM) compared to astrocytes isolated from control white matter. In addition, TNF-α treatment induced ASM mRNA and ceramide levels in astrocytes isolated from control white matter. Incubation of astrocytes with Fingolimod prior to TNF-α treatment reduced ceramide production and mRNA expression of ASM to control levels in astrocytes. Importantly, supernatants derived from reactive astrocytes treated with Fingolimod significantly reduced transendothelial monocyte migration. Overall, the present study demonstrates that reactive astrocytes represent a possible additional cellular target for Fingolimod in MS by directly reducing the production of pro-inflammatory lipids and limiting subsequent transendothelial leukocyte migration.

Glycosphingolipids and insulin resistance.

Glycosphingolipids are structural membrane components, residing largely in the plasma membrane with their sugar-moieties exposed at the cell's surface. In recent times a crucial role for glycosphingolipids in insulin resistance has been proposed. A chronic state of insulin resistance is a rapidly increasing disease condition in Western and developing countries. It is considered to be the major underlying cause of the metabolic syndrome, a combination of metabolic abnormalities that increases the risk for an individual to develop Type 2 diabetes, obesity, cardiovascular disease, polycystic ovary syndrome and nonalcoholic fatty liver disease. As discussed in this chapter, the evidence for a direct regulatory interaction of glycosphingolipids with insulin signaling is still largely indirect. However, the recent finding in animal models that pharmacological reduction of glycosphingolipid biosynthesis ameliorates insulin resistance and prevents some manifestations of metabolic syndrome, supports the view that somehow glycosphingolipids act as critical regulators, Importantly, since reductions in glycosphingolipid biosynthesis have been found to be well tolerated, such approaches may have a therapeutic potential.

Elevated plasma glucosylsphingosine in Gaucher disease: relation to phenotype, storage cell markers, and therapeutic response.

Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, leads to prominent glucosylceramide accumulation in lysosomes of tissue macrophages (Gaucher cells). Here we show glucosylsphingosine, the deacylated form of glucosylceramide, to be markedly increased in plasma of symptomatic nonneuronopathic (type 1) Gaucher patients (n = 64, median = 230.7 nM, range 15.6-1035.2 nM; normal (n = 28): median 1.3 nM, range 0.8-2.7 nM). The method developed for mass spectrometric quantification of plasma glucosylsphingosine is sensitive and robust. Plasma glucosylsphingosine levels correlate with established plasma markers of Gaucher cells, chitotriosidase (ρ = 0.66) and CCL18 (ρ = 0.40). Treatment of Gaucher disease patients by supplementing macrophages with mannose-receptor targeted recombinant glucocerebrosidase results in glucosylsphingosine reduction, similar to protein markers of Gaucher cells. Since macrophages prominently accumulate the lysoglycosphingolipid on glucocerebrosidase inactivation, Gaucher cells seem a major source of the elevated plasma glucosylsphingosine. Our findings show that plasma glucosylsphingosine can qualify as a biomarker for type 1 Gaucher disease, but that further investigations are warranted regarding its relationship with clinical manifestations of Gaucher disease.

Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies.

A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

The cytosolic ß-glucosidase GBA3 does not influence type 1 Gaucher disease manifestation.

GBA3, also known as cytosolic ß-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a ß-glucosidase degrading glucosylceramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence the clinical manifestation of Gaucher disease. Recombinant GBA3 was found to hydrolyze artificial substrates such as 4-methylumbelliferyl-ß-D-glucoside and C6-NBD-glucosylceramide, but hydrolysis of naturally occurring lipids like glucosylceramide and glucosylsphingosine was hardly detected. Consistent with this, inhibition of GBA3 in cultured cells using a novel inhibitor (alpha-1-C-nonyl-DIX) did not result in an additional increase in glucosylceramide as compared to GBA inhibition alone. Examination of the GBA3 gene led to the identification of a common substitution in its open reading frame (1368T→A), resulting in a truncated GBA3 protein missing the last α-helix of its (ß/α)(8) barrel. Both recombinant 1368A GBA3 and 1368A enzyme from spleen of a homozygous individual were found to be inactive. Amongst non-neuronopathic (type 1) Gaucher disease patients, we subsequently identified individuals being wild-type, heterozygous, or homozygous for the GBA3 1368T→A mutation. No correlation was observed between GBA3 1368A/T haplotypes and severity of type 1 Gaucher disease manifestation. In conclusion, GBA3 does not seem to modify type 1 Gaucher disease manifestation.

Reduction of elevated plasma globotriaosylsphingosine in patients with classic Fabry disease following enzyme replacement therapy.

Fabry disease is treated by two-weekly infusions with α-galactosidase A, which is deficient in this X-linked globotriaosylceramide (Gb3) storage disorder. Elevated plasma globotriaosylsphingosine (lysoGb3) is a hallmark of classical Fabry disease. We investigated effects of enzyme replacement therapy (ERT) on plasma levels of lysoGb3 and Gb3 in patients with classical Fabry disease treated with agalsidase alfa at 0.2mg/kg, agalsidase beta at 0.2mg/kg or at 1.0mg/kg bodyweight. Each treatment regimen led to prominent reductions of plasma lysoGb3 in Fabry males within 3 months (P=0.0313), followed by relative stability later on. Many males developed antibodies against α-galactosidase A, particularly those treated with agalsidase beta. Patients with antibodies tended towards smaller correction in plasma lysoGb3 concentration, whereas treatment with high dose agalsidase beta allowed a reduction comparable to patients without antibodies. Pre-treatment plasma lysoGb3 concentrations of Fabry females were relatively low. In all females and with each treatment regimen, ERT gave reduction or stabilisation of plasma lysoGb3. Our investigation revealed that ERT of Fabry patients reduces plasma lysoGb3, regardless of the recombinant enzyme used. This finding shows that ERT can correct a characteristic biochemical abnormality in Fabry patients.

Role of the synthase domain of Ags1p in cell wall alpha-glucan biosynthesis in fission yeast.

The cell wall is important for maintenance of the structural integrity and morphology of fungal cells. Besides beta-glucan and chitin, alpha-glucan is a major polysaccharide in the cell wall of many fungi. In the fission yeast Schizosaccharomyces pombe, cell wall alpha-glucan is an essential component, consisting mainly of (1,3)-alpha-glucan with approximately 10% (1,4)-linked alpha-glucose residues. The multidomain protein Ags1p is required for alpha-glucan biosynthesis and is conserved among cell wall alpha-glucan-containing fungi. One of its domains shares amino acid sequence motifs with (1,4)-alpha-glucan synthases such as bacterial glycogen synthases and plant starch synthases. Whether Ags1p is involved in the synthesis of the (1,4)-alpha-glucan constituent of cell wall alpha-glucan had remained unclear. Here, we show that overexpression of Ags1p in S. pombe cells results in accumulation of (1,4)-alpha-glucan. To determine whether the synthase domain of Ags1p is responsible for this activity, we overexpressed Ags1p-E1526A, which carries a mutation in a putative catalytic residue of the synthase domain, but observed no accumulation of (1,4)-alpha-glucan. Compared with wild-type Ags1p, this mutant Ags1p showed a markedly reduced ability to complement the cell lysis phenotype of the temperature-sensitive ags1-1 mutant. Therefore, we conclude that, in S. pombe, the production of (1,4)-alpha-glucan by the synthase domain of Ags1p is important for the biosynthesis of cell wall alpha-glucan.

Role of the alpha-glucanase Agn2p in ascus-wall endolysis following sporulation in fission yeast.

During sporulation in the ascomyceteous fungus Schizosaccharomyces pombe, diploid cells undergo differentiation into asci containing four haploid ascospores, which are highly resistant to environmental stresses. Although the morphogenetic processes involved in ascospore formation have been studied extensively, little is known about the molecular mechanism that ensures the release of mature ascospores from the ascus, allowing their dispersal into the environment. Recently, we identified Agn2p as the paralogue of the characterized endo-(1,3)-alpha-glucanase Agn1p, and observed that asci deleted for agn2 are defective in ascospore dispersal. Here, we focus on the cellular and biochemical functions of Agn2p. By placing agn2 under the control of an inducible promoter, we show that expression of agn2 is required for the efficient release of ascospores from their asci. Furthermore, we characterize the enzyme activity of purified recombinant Agn2p and show that Agn2p, like Agn1p, is an endo-(1,3)-alpha-glucanase that produces predominantly (1,3)-alpha-glucan pentasaccharides. Finally, we demonstrate that exogenous addition of purified Agn2p liberated the ascospores from asci deleted for agn2. We propose that Agn2p participates in the endolysis of the ascus wall by hydrolysing its (1,3)-alpha-glucan, thereby assisting in the release of ascospores.

Mechanism of action of the endo-(1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum.

(1-->3)-alpha-glucanases catalyze the hydrolysis of fungal cell wall (1-->3)-alpha-glucan, and function during cell division of yeasts containing this cell wall component or act in mycoparasitic processes. Here, we characterize the mechanism of action of the (1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum. We observed that MutAp releases predominantly beta-glucose upon hydrolysis of crystalline (1-->3)-alpha-glucan, indicating inversion of the anomeric configuration. After having identified (1-->3)-alpha-glucan tetrasaccharide as the minimal substrate for MutAp, we showed that reduced (1-->3)-alpha-glucan pentasaccharide is cleaved into a trisaccharide and a reduced disaccharide, demonstrating that MutAp displays endo-hydrolytic activity. We propose a model for the catalytic mechanism of MutAp, whereby the enzyme breaks an intrachain glycosidic linkage of (1-->3)-alpha-glucan, and then continues its hydrolysis towards the non-reducing end by releasing beta-glucose residues in a processive manner.

Transcription regulation of the alpha-glucanase gene agn1 by cell separation transcription factor Ace2p in fission yeast.

During the final stage of the cell division cycle in the fission yeast Schizosaccharomyces pombe, transcription factor Ace2p activates expression of genes involved in the separation of newly formed daughter cells, such as agn1+, which encodes the alpha-glucanase Agn1p. The agn1 promoter contains three copies of the nucleotide sequence motif CCAGCC, whose presence seems to correlate with Ace2p-mediated transcription activation. Here, we describe a simple plate-based assay utilizing as a reporter the secreted glucoamylase of Arxula adeninivorans to investigate the function of this motif. We show that not all three repeats, but only the two most proximal to the transcription start point, act as an upstream activating sequence (UAS). Finally, we demonstrate that this UAS is essential for agn1 promoter activity in vivo.