Heinz Troxler

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patients fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.

High levels of anti-inflammatory and pro-resolving lipid mediators lipoxins and resolvins and declining docosahexaenoic acid levels in human milk during the first month of lactation

BackgroundThe fatty acid mixture of human milk is ideal for the newborn but little is known about its composition in the first few weeks of lactation. Of special interest are the levels of long-chain PUFAs (LCPUFAs), since these are essential for the newborn’s development. Additionally, the LCPUFAs arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are precursors for lipid mediators which regulate inflammation.MethodsWe determined the composition of 94 human milk samples from 30 mothers over the first month of lactation for fatty acids using GC-MS and quantified lipid mediators using HPLC-MS/MS.ResultsOver the four weeks period, DHA levels decreased, while levels of γC18:3 and αC18:3 steadily increased. Intriguingly, we found high concentrations of lipid mediators and their hydroxy fatty acid precursors in human milk, including pro-inflammatory leukotriene B4 (LTB4) and anti-inflammatory and pro-resolving lipoxin A4 (LXA4), resolvin D1 (RvD1) and resolvin E1 (RvE1). Lipid mediator levels were stable with the exception of two direct precursors.ConclusionsElevated levels of DHA right after birth might represent higher requirements of the newborn and the high content of anti-inflammatory and pro-resolving lipid mediators and their precursors may indicate their role in neonatal immunity and may be one of the reasons for the advantage of human milk over infant formula.

Specific Citrullination Causes Assembly of a Globular S100A3 Homotetramer A PUTATIVE Ca2+ MODULATOR MATURES HUMAN HAIR CUTICLE

S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.

Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities.

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.

Mass Spectrometric Analysis of Human Transferrin in Different Body Fluids

Abstract In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

Metabolic Profiling of Hearts Exposed to Sevoflurane and Propofol Reveals Distinct Regulation of Fatty Acid and Glucose Oxidation CD36 and Pyruvate Dehydrogenase as Key Regulators in Anesthetic-induced Fuel Shift

Background:Myocardial energy metabolism is a strong predictor of postoperative cardiac function. This study profiled the metabolites and metabolic changes in the myocardium exposed to sevoflurane, propofol, and Intralipid and investigated the underlying molecular mechanisms. Methods:Sevoflurane (2 vol%) and propofol (10 and 100 &mgr;m) in the formulation of 1% Diprivan® (AstraZeneca Inc., Mississauga, ON, Canada) were compared for their effects on oxidative energy metabolism and contractility in the isolated working rat heart model. Intralipid served as a control. Substrate flux through the major pathways for adenosine triphosphate generation in the heart, that is, fatty acid and glucose oxidation, was measured using [3H]palmitate and [14C]glucose. Biochemical analyses of nucleotides, acyl-CoAs, ceramides, and 32 acylcarnitine species were used to profile individual metabolites. Lipid rafts were isolated and used for Western blotting of the plasma membrane transporters CD36 and glucose transporter 4. Results:Metabolic profiling of the hearts exposed to sevoflurane and propofol revealed distinct regulation of fatty acid and glucose oxidation. Sevoflurane selectively decreased fatty acid oxidation, which was closely related to a marked reduction in left ventricular work. In contrast, propofol at 100 &mgr;m but not 10 &mgr;m increased glucose oxidation without affecting cardiac work. Sevoflurane decreased fatty acid transporter CD36 in lipid rafts/caveolae, whereas high propofol increased pyruvate dehydrogenase activity without affecting glucose transporter 4, providing mechanisms for the fuel shifts in energy metabolism. Propofol increased ceramide formation, and Intralipid increased hydroxy acylcarnitine species. Conclusions:Anesthetics and their solvents elicit distinct metabolic profiles in the myocardium, which may have clinical implications for the already jeopardized diseased heart.

Coordinated balancing of muscle oxidative metabolism through PGC-1α increases metabolic flexibility and preserves insulin sensitivity.

The peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) enhances oxidative metabolism in skeletal muscle. Excessive lipid oxidation and electron transport chain activity can, however, lead to the accumulation of harmful metabolites and impair glucose homeostasis. Here, we investigated the effect of over-expression of PGC-1α on metabolic control and generation of insulin desensitizing agents in extensor digitorum longus (EDL), a muscle that exhibits low levels of PGC-1α in the untrained state and minimally relies on oxidative metabolism. We demonstrate that PGC-1α induces a strictly balanced substrate oxidation in EDL by concomitantly promoting the transcription of activators and inhibitors of lipid oxidation. Moreover, we show that PGC-1α enhances the potential to uncouple oxidative phosphorylation. Thereby, PGC-1α boosts elevated, yet tightly regulated oxidative metabolism devoid of side products that are detrimental for glucose homeostasis. Accordingly, PI3K activity, an early phase marker for insulin resistance, is preserved in EDL muscle. Our findings suggest that PGC-1α coordinately coactivates the simultaneous transcription of gene clusters implicated in the positive and negative regulation of oxidative metabolism and thereby increases metabolic flexibility. Thus, in mice fed a normal chow diet, over-expression of PGC-1α does not alter insulin sensitivity and the metabolic adaptations elicited by PGC-1α mimic the beneficial effects of endurance training on muscle metabolism in this context.

Quantification of phenylalanine hydroxylase activity by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry

BACKGROUNDnResidual phenylalanine hydroxylase (PAH) activity is the key determinant for the phenotype severity in phenylketonuria (PKU) patients and correlates with the patients genotype. Activity of in vitro expressed mutant PAH may predict the patients phenotype and response to tetrahydrobiopterin (BH(4)), the cofactor of PAH.nnnMETHODSnA robust LC-ESI-MSMS PAH assay for the quantification of phenylalanine and tyrosine was developed. We measured PAH activity a) of the PAH mutations p.Y417C, p.I65T, p.R261Q, p.E280A, p.R158Q, p.R408W, and p.E390G expressed in eukaryotic COS-1 cells; b) in different cell lines (e.g. Huh-7, Hep3B); and c) in liver, brain, and kidney tissue from wild-type and PKU mice.nnnRESULTSnThe PAH assay was linear for phenylalanine and tyrosine (r(2)≥0.99), with a detection limit of 105 nmol/L for Phe and 398 nmol/L for Tyr. Intra-assay and inter-assay coefficients of variation were <5.3% and <6.2%, respectively, for the p.R158Q variant in lower tyrosine range. Recovery of tyrosine was 100%. Compared to the wild-type enzyme, the highest PAH activity at standard conditions (1 mmol/L L-Phe; 200 μmol/L BH(4)) was found for the mutant p.Y417C (76%), followed by p.E390G (54%), p.R261Q (43%), p.I65T (33%), p.E280A (15%), p.R158Q (5%), and p.R408W (2%). A relative high PAH activity was found in kidney (33% of the liver activity), but none in brain.nnnCONCLUSIONSnThis novel method is highly sensitive, specific, reproducible, and efficient, allowing the quantification of PAH activity in different cells or tissue extracts using minimum amounts of samples under standardized conditions.

Enhanced glucose uptake via GLUT4 fuels recovery from calcium overload after ischaemia-reperfusion injury in sevoflurane- but not propofol-treated hearts.

BACKGROUNDnSo far, no study has explored the effects of sevoflurane, propofol, and Intralipid on metabolic flux rates of fatty acid oxidation (FOX) and glucose oxidation (GOX) in hearts exposed to ischaemia-reperfusion.nnnMETHODSnIsolated paced working rat hearts were exposed to 20 min of ischaemia and 30 min of reperfusion. Peri-ischaemic sevoflurane (2 vol%) and propofol (100 µM) in the formulation of 1% Diprivan(®) were assessed for their effects on oxidative energy metabolism and intracellular diastolic and systolic Ca(2+) concentrations. Substrate flux was measured using [(3)H]palmitate and [(14)C]glucose and [Ca(2+)] using indo-1AM. Western blotting was used to determine the expression of the sarcolemmal glucose transporter GLUT4 in lipid rafts. Biochemical analyses of nucleotides, ceramides, and 32 acylcarnitines were also performed.nnnRESULTSnSevoflurane, but not propofol, improved the recovery of left ventricular work (P=0.008) and myocardial efficiency (P=0.008) compared with untreated ischaemic hearts. This functional improvement was accompanied by reduced increases in post-ischaemic diastolic and systolic intracellular Ca(2+) concentrations (P=0.008). Sevoflurane, but not propofol, increased GOX (P=0.009) and decreased FOX (P=0.019) in hearts exposed to ischaemia-reperfusion. GLUT4 expression was markedly increased in lipid rafts of sevoflurane-treated hearts (P=0.016). Increased GOX closely correlated with reduced Ca(2+) overload. Intralipid alone decreased energy charge and increased long-chain and hydroxyacylcarnitine tissue levels, whereas sevoflurane decreased toxic ceramide formation.nnnCONCLUSIONSnEnhanced glucose uptake via GLUT4 fuels recovery from Ca(2+) overload after ischaemia-reperfusion in sevoflurane- but not propofol-treated hearts. The use of a high propofol concentration (100 µM) did not result in similar protection.

Improvement of dolichol-linked oligosaccharide biosynthesis by the squalene synthase inhibitor zaragozic acid.

The majority of congenital disorders of glycosylation (CDG) are caused by defects of dolichol (Dol)-linked oligosaccharide assembly, which lead to under-occupancy of N-glycosylation sites. Most mutations encountered in CDG are hypomorphic, thus leaving residual activity to the affected biosynthetic enzymes. We hypothesized that increased cellular levels of Dol-linked substrates might compensate for the low biosynthetic activity and thereby improve the output of protein N-glycosylation in CDG. To this end, we investigated the potential of the squalene synthase inhibitor zaragozic acid A to redirect the flow of the polyisoprene pathway toward Dol by lowering cholesterol biosynthesis. The addition of zaragozic acid A to CDG fibroblasts with a Dol-P-Man synthase defect led to the formation of longer Dol-P species and to increased Dol-P-Man levels. This treatment was shown to decrease the pathologic accumulation of incomplete Dol pyrophosphate-GlcNAc2Man5 in Dol-P-Man synthase-deficient fibroblasts. Zaragozic acid A treatment also decreased the amount of truncated protein N-linked oligosaccharides in these CDG fibroblasts. The increased cellular levels of Dol-P-Man and possibly the decreased cholesterol levels in zaragozic acid A-treated cells also led to increased availability of the glycosylphosphatidylinositol anchor as shown by the elevated cell-surface expression of the CD59 protein. This study shows that manipulation of the cellular Dol pool, as achieved by zaragozic acid A addition, may represent a valuable approach to improve N-linked glycosylation in CDG cells.