The effect of recombinant human (rh) cytokines, interleukin-1 alpha (IL- 1 alpha), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL- 4), granulocyte/macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), monocyte/macrophage colony stimulating factor (M-CSF), interferon-alpha (IF-alpha), interferon-gamma (IF-gamma), and the tumor necrosis factor-alpha (TNF- alpha) on differentiation and function of metachromatic cells (MCS) was studied. Among all cytokines tested, rh interleukin-3 (rhIL-3) selectively induced a significant formation of MCS (IL-3: 1.1 +/- 0.6 x 10(5) v control: 0.02 +/- 0.15 x 10(5) MCS/mL suspension) and dose dependent increase in formation of intracellular histamine (IL-3, 100 U/mL: 95 +/- 23 ng/mL v control: 1.8 +/- 0.8 ng/mL) in a bone marrow suspension culture system (analyzed on day 14 of culture). Besides MCS, formation of eosinophils was observed in this culture system in the continuous presence of rhIL-3, whereas IL-3 pulse-stimulation for three hours and subsequent exposure to control medium induced growth of MCS but not of eosinophils. By combined immunofluorescence/toluidine blue staining, MCS were found to express a cell surface marker profile that corresponds to the immunological phenotype of peripheral blood basophils (MY-7(CD13)+, VIM12(CD11b)+, VIM2+, MAX1-, MAX24- and YB5B8- ). Furthermore, cultured MCS expressed surface membrane receptors for IgE and could be triggered for nontoxic histamine release by a monoclonal anti-IgE antibody. To evaluate a possible influence of IL-3 on basophil function, studies were extended to freshly obtained blood basophils (healthy volunteers, n = 3). However, like all other cytokines tested, rhIL-3 failed to induce basophil histamine release. Taken together, our studies demonstrate that IL-3 is a differentiation factor for human basophils.
lsocitrate lyase (ICL) was assayed during batch cultivations of Ashbya gossypii on soybean oil or glucose as carbon source. On soybean oil, a correlation between enzyme activity and riboflavin synthesis was observed. On glucose as carbon source, riboflavin overproduction started in the late growth phase when glucose was exhausted. ICL activity appeared in parallel and reached a maximum of 041 U (mg protein)-'. This suggested synthesis of vitamin B, from the intracellular reserve fat. ICL specific activity correlated with the enzyme concentration detected by specific antibodies. Itaconate, an efficient inhibitor of ICL, was used as an antimetabolite to screen mutants with enhanced ICL activity. Cultivations of an itaconate-resistant mutant on soybean oil revealed a 15 O/ o increase in enzyme specific activity and a 25-fold increase in riboflavin yield compared to the wild-type. On the other hand, growth experiments on glucose resulted in an eightfold increase in riboflavin yield but showed a 33% reduction in ICL specific activity compared to the wild-type grown on the same medium. These results support the idea of an ICL bottleneck in the riboflavin overproducer A. gossypii when plant oil is used as the substrate.
A specific isocitrate lyase (ICL) activity of 0.17 U (mg protein)-' was detected in cultures of the riboflavin-producing fungus Ashbya gossmii during growth on soybean oil. Enzyme activity was not detectable during growth on glucose [ < 0905 U (mg protein)-'], indicating a regulation. The enzyme was purified 108-fold by means of ammonium sulphate fractionation, gel filtration and cation-exchange chromatography. SDS-PAGE of the purified protein showed a homogeneous band with an M, of 66000. The M, of 254 000 determined by gel-f iltration chromatography indicated a tetrameric structure of the native protein. The enzyme was found to have a pH optimum for the isocitrate cleavage of 79, and the K,,, for thmo-DL-isocitrate was determined as 550 pM. Enzyme activity was Mg2+-dependent. In regulation studies ICL was weakly inhibited by central metabolites. A concentration of 10 mM phosphoenolpyruvate or 6-phosphogluconate revealed a residual activity of more than 40%. On the other hand, oxalate (K,: 4 pM) and itaconate (K,: 170 pM) showed a strong inhibition and may therefore be interesting as antimetabolites.
Patients with antiphospholipid syndrome (APS) are at high risk of developing venous and arterial thromboembolism (TE). The role of platelets in the pathogenesis of these prothrombotic conditions is not yet fully understood. The aim of this study was to gain mechanistic insights into the role of platelets in APS by comparing the platelet proteome between lupus anticoagulant (LA)-positive patients with (LA+ TE+) and without a history of TE (LA+ TE−) and healthy controls. The platelet proteome of 47 patients with LA, 31 with a history of TE and 16 without thrombotic history, and 47 healthy controls was analyzed by two-dimensional differential in-gel electrophoresis and mass spectrometry to identify disease-related proteins. Afterward, selected LA-related platelet proteins were validated by western blot and ELISA. Alterations of 25 proteins were observed between the study groups. STRING pathway analysis showed that LArelated protein profiles were involved in platelet activation, aggregation, and degranulation. For example, protein disulfide isomerase family members, enzymes that promote thrombosis, were upregulated in platelets and plasma of LA+ TE+ patients. Leukocyte elastase inhibitor (SERPINB1), an antagonist of neutrophil extracellular trap (NET) formation, was decreased in platelets of LA+ TE+ patients compared to healthy controls. Additionally, citrullinated histone H3, a NET-specific marker, was increased in plasma of LA+ TE+ patients. These findings suggest that decreased platelet SERPINB1 levels favor prothrombotic NETosis, especially in LA+ TE+ patients. Our findings reveal protein abundance changes connected to altered platelet function in LA-positive patients, thus suggesting a pathogenic role of platelets in thrombotic complications in APS.
Alzheimer’s disease (AD), a multifactorial neurodegenerative condition caused by genetic and environmental factors, is diagnosed using neuropsychological tests and brain imaging; molecular diagnostics are not routinely applied. Studies have identified AD-specific cerebrospinal fluid (CSF) biomarkers but sample collection requires invasive lumbar puncture. To identify AD-modulated proteins in easily accessible blood platelets, which share biochemical signatures with neurons, we compared platelet lysates from 62 AD, 24 amnestic mild cognitive impairment (aMCI), 13 vascular dementia (VaD), and 12 Parkinson’s disease (PD) patients with those of 112 matched controls by fluorescence two-dimensional differential gel electrophoresis in independent discovery and verification sets. The optimal sum score of four mass spectrometry (MS)-identified proteins yielded a sensitivity of 94 % and a specificity of 89 % (AUC = 0.969, 95 % CI = 0.944–0.994) to differentiate AD patients from healthy controls. To bridge the gap between bench and bedside, we developed a high-throughput multiplex protein biochip with great potential for routine AD screening. For convenience and speed of application, this array combines loading control-assisted protein quantification of monoamine oxidase B and tropomyosin 1 with protein-based genotyping for single nucleotide polymorphisms (SNPs) in the apolipoprotein E and glutathione S-transferase omega 1 genes. Based on minimally invasive blood drawing, this innovative protein biochip enables identification of AD patients with an accuracy of 92 % in a single analytical step in less than 4 h.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-014-1341-8) contains supplementary material, which is available to authorized users.
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