Degradation of D-2-hydroxyglutarate in the presence of isocitrate dehydrogenase mutations

D-2-Hydroxyglutarate (D-2-HG) is regarded as an oncometabolite. It is found at elevated levels in certain malignancies such as acute myeloid leukaemia and glioma. It is produced by a mutated isocitrate dehydrogenase IDH1/2, a low-affinity/high-capacity enzyme. Its degradation, in contrast, is catalysed by the high-affinity/low-capacity enzyme D-2-hydroxyglutarate dehydrogenase (D2HDH). So far, it has not been proven experimentally that the accumulation of D-2-HG in IDH mutant cells is the result of its insufficient degradation by D2HDH. Therefore, we developed an LC-MS/MS-based enzyme activity assay that measures the temporal drop in substrate and compared this to the expression of D2HDH protein as measured by Western blot. Our data clearly indicate, that the maximum D-2-HG degradation rate by D2HDH is reached in vivo, as v(max) is low in comparison to production of D-2-HG by mutant IDH1/2. The latter seems to be limited only by substrate availability. Further, incubation of IDH wild type cells for up to 48 hours with 5 mM D-2-HG did not result in a significant increase in either D2HDH protein abundance or enzyme activity

Detecting early signs of heat and drought stress in Phoenix dactylifera (date palm)

Plants adapt to the environment by either long-term genome evolution or by acclimatization processes where the cellular processes and metabolism of the plant are adjusted within the existing potential in the genome. Here we studied the adaptation strategies in date palm, Phoenix dactylifera, under mild heat, drought and combined heat and drought by transcriptomic and metabolomic profiling. In transcriptomics data, combined heat and drought resembled heat response, whereas in metabolomics data it was more similar to drought. In both conditions, soluble carbohydrates, such as fucose, and glucose derivatives, were increased, suggesting a switch to carbohydrate metabolism and cell wall biogenesis. This result is consistent with the evidence from transcriptomics and cis-motif analysis. In addition, transcriptomics data showed transcriptional activation of genes related to reactive oxygen species in all three conditions (drought, heat, and combined heat and drought), suggesting increased activity of enzymatic antioxidant systems in cytosol, chloroplast and peroxisome. Finally, the genes that were differentially expressed in heat and combined heat and drought stresses were significantly enriched for circadian and diurnal rhythm motifs, suggesting new stress avoidance strategies.Peer reviewe

Stanniocalcin 1 is overexpressed in multipotent mesenchymal stromal cells from acute myeloid leukemia patients

Objectives: Multipotent mesenchymal stromal cells (MSC) play a pivotal role in the bone marrow (BM) niche. Stanniocalcin 1 (STC1) secreted by MSC has been demonstrated to promote the survival of neoplastic cells and was suggested a marker for minimal residual disease of acute myeloid leukemia (AML). Therefore, we evaluated the expression of STC1 in MSC from AML patients (MSCAML) compared to MSC from healthy donors (MSCHD). Methods: Liquid culture assays of MSCAML and MSCHD were performed to compare expansion capacity. Gene expression profiles of MSCAML vs. MSCHD were established. Secretion of STC1 was tested by ELISA in MSCAML vs. MSCHD and expression of STC1 in AML- vs. HD-BM by immunohistochemistry. In addition, co-cultures of AML cells on MSC were initiated and ultrastructural intercellular communication patterns were investigated. Finally, the effect of blocking STC1 on AML cells was evaluated. Results: MSCAML showed significant decreased expansion capacity compared to MSCHD. Gene analysis revealed marked overexpression of STC1 in MSCAML. ELISA and immunohistochemical findings confirmed this observation. Electron microscopy analysis showed reciprocal stimulation between AML cells and MSC. Blockade of STC1 did not significantly affect AML cell proliferation and apoptosis. Discussion: Characteristics of MSC differ depending on whether they originate from AML patients or from HD. STC1 was mostly overexpressed in MSCAML compared to MSCHD. In vitro blockade of STC1, however, was not associated with AML cell proliferation and apoptosis. Conclusion: Differences in expression levels of glycoproteins from MSCAML compared to MSCHD not necessarily assume that these molecules are niche-relevant in leukemic disease

Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of glioma cells

The epidermal growth factor receptor (EGFR, ErbB1) is frequently dysregulated in a variety of solid human tumors, including malignant glioma. EGFR expression has been associated with disease progression, resistance to standard therapies and poor survival. The application of small interfering RNAs (siRNAs) has become an effective and highly specific tool to modulate gene expression, and a wide range of oncogenes have been silenced successfully. Here we show the siRNA-mediated down-regulation of EGFR in two established glioma cell lines with different EGFR expression levels (U373 MG, LN18). The expression of EGFR mRNA and protein was down-regulated by 70-90%. However, siRNA treatment had no inhibitory effect on cell proliferation, migration and activation status of EGFR-coupled signaling cascades. In accordance with these results, gene expression analysis with microarrays revealed only small, albeit specific changes in expression patterns. In conclusion, these data indicate that the specific down-regulation of EGFR might not be sufficient for a single agent therapeutic approach in malignant glioma

Chondroitin sulfate disaccharide stimulates microglia to adopt a novel regulatory phenotype

A disaccharide degradation product of chondrotin sulfate proteoglycan-disaccharide (CSPG-DS) has been implicated previously in the inhibition of neurodegeneration by influencing microglia activation. In this study, genome-wide microarray analysis was used to identify specific gene expression profiles of CSPG-DS-stimulated BV-2 microglia-like cells. Gene products involved in phagocytosis, detoxification, migration, immune regulation, and antigen presentation were found to be altered significantly. These findings were replicated and compared with IFN-gamma-stimulated primary microglia using real-time quantitative RT-PCR validation. Importantly, a unique transcriptional phenotype with anti-inflammatory and IFN-gamma counter-regulatory properties partially related to alternatively activated macrophages was identified. Using functional cell assays, we found that CSPG-DS-stimulated microglia possess increased phagocytic capacity but lack direct cytotoxic effects such as secretion of NO. Furthermore, conditioned media from CSPG-DS-treated microglia did not diminish the viability or cause apoptosis of cultured photoreceptor cells and partially rescued these cells from IFN-gamma-induced apoptosis. Taken together, our data provide a unique transcript dataset and important in vitro findings about the functional properties of CSPG-DS-activated microglia. These might be starting points to explore the in vivo role of CSPG-DS as a bioactive microglia regulator and its potential, therapeutic application in immune-related, neurodegenerative disorders

Validation of microarray-based resequencing of 93 worldwide mitochondrial genomes

The human mitochondrial genome consists of a multicopy, circular dsDNA molecule of 16,569 base pairs. It encodes for 13 proteins, two ribosomal genes, and 22 tRNAs that are essential in the generation of cellular ATP by oxidative phosphorylation in eukaryotic cells. Germline mutations in mitochondrial DNA (mtDNA) are an important cause of maternally inherited diseases, while somatic mtDNA mutations may play important roles in aging and cancer. mtDNA polymorphisms are also widely used in population and forensic genetics. Therefore, methods that allow the rapid, inexpensive and accurate sequencing of mtDNA are of great interest. One such method is the Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 (MitoChip v.2.0) (Santa Clara, CA). A direct comparison of 93 worldwide mitochondrial genomes sequenced by both the MitoChip and dideoxy terminator sequencing revealed an average call rate of 99.48% and an accuracy of > or =99.98% for the MitoChip. The good performance was achieved by using in-house software for the automated analysis of additional probes on the array that cover the most common haplotypes in the hypervariable regions (HVR). Failure to call a base was associated mostly with the presence of either a run of > or =4 C bases or a sequence variant within 12 bases up- or downstream of that base. A major drawback of the MitoChip is its inability to detect insertions/deletions and its low sensitivity and specificity in the detection of heteroplasmy. However, the vast majority of haplogroup defining polymorphism in the mtDNA phylogeny could be called unambiguously and more rapidly than with conventional sequencing

SNP dependent modulation of circulating miRNAs from the miR25/93/106 cluster in patients undergoing weight loss

Background: Diet induced weight loss represents an intervention for obesity to prevent associated diseases. However there is considerable inter-individual variation. Single nucleotide polymorphisms (SNPs) and plasma miRNA might be contributing factors. We therefore hypothesized that changes in the miRNA pattern during weight loss depend on the SNP genotype. Methods: Plasma miRNA profiles from 12 patients were determined before and after a three month weight loss intervention by Illumina sequencing. 46 further patients were analyzed by qPCR. SNP genotypes were determined on the Sequenom iPLEX platform. Results: Samples before and after weight loss were analyzed by miRNA-seq and delta miRNA levels ranked according to p-value. Levels of miRNAs 25, 93 and 106 that are expressed from a common genomic cluster were reduced after weight loss. Those results were substantiated in a qPCR analysis of 46 additional patients. This is in accordance with mouse data showing a functional involvement of this cluster in obesity. Correlation of the changes in miRNA abundance with SNP genotypes revealed a statistical association of all three miRNAs with known obesity susceptibility SNPs. Conclusion: Diet induced weight loss leads to SNP dependent modulation of miRNAs from the miR 25/93/106 gene cluster in humans

Novel role for SLPI in MOG-induced EAE revealed by spinal cord expression analysis

<p>Abstract</p> <p>Background</p> <p>Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte protein (MOG) in female <it>Dark Agouti </it>(DA) rats is a chronic demyelinating animal model of multiple sclerosis (MS). To identify new candidate molecules involved in the evolution or repair of EAE-lesions we used Affymetrix oligonucleotide microarrays to compare the spinal cord transcriptome at the peak of EAE, during remission and at the first relapse with healthy DA rats.</p> <p>Methods</p> <p>Untreated DA rats and DA rats immunised with MOG protein were sacrificed at defined time points. Total RNA was isolated from spinal cord tissue and used for hybridization of Affymetrix rat genome arrays RG U34 A-C. Selected expression values were confirmed by RealTime PCR.</p> <p>Adult neural stem cells were incubated with recombinant secretory leukocyte protease inhibitor (SLPI). Proliferation was assessed by BrdU incorporation, cyclin D1 and HES1 expression by RealTime PCR, cell differentiation by immunofluorescence analysis and IkappaBalpha degradation by Western blot.</p> <p>Results</p> <p>Among approximately 26,000 transcripts studied more than 1,100 were differentially regulated. Focussing on functional themes, we noticed a sustained downregulation of most of the transcripts of the cholesterol biosynthesis pathway. Furthermore, we found new candidate genes possibly contributing to regenerative processes in the spinal cord. Twelve transcripts were solely upregulated in the recovery phase, including genes not previously associated with repair processes. Expression of SLPI was upregulated more than hundredfold during EAE attack. Using immunohistochemistry, SLPI was identified in macrophages, activated microglia, neuronal cells and astrocytes. Incubation of adult neural stem cells (NSC) with recombinant SLPI resulted in an increase of cell proliferation and of differentiation towards oligodendrocytes. These processes were paralleled by an upregulation of the cell-cycle promotor <it>cyclin D1 </it>and a suppression of the cell differentiation regulator HES1. Finally, SLPI prevented the degradation of IkappaBalpha, which may explain the suppression of the cell differentiation inhibitor HES1 suggesting a possible mechanism of oligodendroglial differentiation.</p> <p>Conclusion</p> <p>We identified novel features of gene expression in the CNS during EAE, in particular the suppression of genes of cholesterol biosynthesis and a strong upregulation of SLPI, a gene which is for the first time associated with autoimmune inflammation. The capacity of SLPI to increase proliferation of adult NSC and of oligodendroglial differentiation suggests a novel role for SLPI in the promotion of tissue repair, beyond its known functions in the prevention of tissue damages by protease inhibition damage and modulation of inflammatory reactions.</p