BackgroundAllergy diagnosis by determination of allergen-specific IgE is complicated by clinically irrelevant IgE, of which the most prominent example is IgE against cross-reactive carbohydrate determinants (CCDs) that occur on allergens from plants and insects. Therefore, CCDs cause numerous false-positive results. Inhibition of CCDs has been proposed as a remedy, but has not yet found its way into the routine diagnostic laboratory. We sought to provide a simple and affordable procedure to overcome the CCD problem.MethodsSerum samples from allergic patients were analysed for allergen-specific IgEs by different commercial tests (from Mediwiss, Phadia and Siemens) with and without a semisynthetic CCD blocker with minimized potential for nonspecific interactions that was prepared from purified bromelain glycopeptides and human serum albumin.ResultsTwenty two per cent of about 6000 serum samples reacted with CCD reporter proteins. The incidence of anti-CCD IgE reached 35% in the teenage group. In patients with anti-CCD IgE, application of the CCD blocker led to a clear reduction in read-out values, often below the threshold level. A much better correlation between laboratory results and anamnesis and skin tests was achieved in many cases. The CCD blocker did not affect test results where CCDs were not involved.ConclusionEliminating the effect of IgEs directed against CCDs by inhibition leads to a significant reduction in false-positive in vitro test results without lowering sensitivity towards relevant sensitizations. Application of the CCD blocker may be worthwhile wherever natural allergen extracts or components are used.

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.DOI: http://dx.doi.org/10.7554/eLife.07736.001

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SummaryIn the ascomycete fungus Aspergillus nidulans, the transcriptional activation of nitrate assimilating genes (niiA, niaD) depends on the cooperativity between a general nitrogen status-sensing regulator (the GATA factor AreA) and a pathway-specific activator (the Zn-cluster regulator NirA). Because nitrate assimilation leads to intracellular ammonium formation, it is difficult to determine the individual contributions of NirA and AreA in this complex activation/ inactivation process. In an attempt to find a suitable marker for the nitrogen status sensed by AreA, we determined the intracellular free amino acid levels on different nitrogen growth conditions. We show that the amount of glutamine (Gln) inversely correlates with all known AreA activities. We find that AreA mediates chromatin remodelling by increasing histone H3 acetylation, a process triggered by transcriptional activation and, independently of transcription, by nitrogen starvation. NirA also participates in the chromatin opening process during nitrate induction but its function is not related to histone acetylation. This chromatin remodelling function of NirA is dispensable only in nitrogen-starved cells, conditions that lead to elevated AreA chromatin occupancy and histone H3 hyperacetylation. Continuous nitrate assimilation leads to self-nitrogen metabolite repression but nitrate-activated NirA is partially compensating for lowered AreA activities under these conditions.

The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)8COOH3 and 3H-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a non-radioactive HPLC method using pyridylaminated Man3-GlcNAc2 and Man5GlcNAc2 as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man3GlcNAc2 contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor. Compared to the enzyme from the human hepatoma cell line HepG2, insect cell GlcNAc-T I exhibited a much higher preference for the Man5 substrate. The GlcNAc-T I from Mb-0503 cells had apparent Km and Vmax values for pyridylaminated Man3- and Man5GlcNAc2 of 2.15 and 0.21 mM, and of 3.4 and 11.4 nmol/h/mg of cell protein, respectively. When Man5GlcNAc2 was used as the acceptor substrate, the levels of GlcNAc-T I activity in the four insect cell lines ranged between 7.5 and 14.7 nmol/h/mg of cell protein, and thus were comparable to that of HepG2 cells. Evidence is presented for the dependence of lepidopteran fucosyltransferase on the presence of terminal N-acetylglucosamine.(ABSTRACT TRUNCATED AT 250 WORDS)