Metabolic changes related to the IDH1 mutation in gliomas preserve TCA-cycle activity: An investigation at the protein level.

The discovery of the IDH1 R132H (IDH1 mut) mutation in low-grade glioma and the associated change in function of the IDH1 enzyme has increased the interest in glioma metabolism. In an earlier study, we found that changes in expression of genes involved in the aerobic glycolysis and the TCA cycle are associated with IDH1 mut. Here, we apply proteomics to FFPE samples of diffuse gliomas with or without IDH1 mutations, to map changes in protein levels associated with this mutation. We observed significant changes in the enzyme abundance associated with aerobic glycolysis, glutamate metabolism, and the TCA cycle in IDH1 mut gliomas. Specifically, the enzymes involved in the metabolism of glutamate, lactate, and enzymes involved in the conversion of α-ketoglutarate were increased in IDH1 mut gliomas. In addition, the bicarbonate transporter (SLC4A4) was increased in IDH1 mut gliomas, supporting the idea that a mechanism preventing intracellular acidification is active. We also found that enzymes that convert proline, valine, leucine, and isoleucine into glutamate were increased in IDH1 mut glioma. We conclude that in IDH1 mut glioma metabolism is rewired (increased input of lactate and glutamate) to preserve TCA-cycle activity in IDH1 mut gliomas.

Transcription profiling associated with life cycle of Anopheles gambiae.

Complex biological events occur during the developmental process of the mosquito Anopheles gambiae (Giles). Using cDNA expression microarrays, the expression patterns of 13,440 clones representing 8,664 unique transcripts were revealed from six different developmental stages: early larvae (late third instar/early fourth instar), late larvae (late fourth instar), early pupae (< 30 min after pupation), late pupae (after tanning), and adult female and male mosquitoes (24 h postemergence). After microarray analysis, 560 unique transcripts were identified to show at least a fourfold up- or down-regulation in at least one developmental stage. Based on the expression patterns, these gene products were clustered into 13 groups. In total, eight genes were analyzed by quantitative real-time polymerase chain reaction to validate microarray results. Among 560 unique transcripts, 446 contigs were assigned to respective genes from the An. gambiae genome. The expression patterns and annotations of the genes in the 13 groups are discussed in the context of development including metabolism, transport, protein synthesis and degradation, cellular processes, cellular communication, intra- or extra-cellular architecture maintenance, response to stress or immune-related defense, and spermatogenesis.

A software application for comparing large numbers of high resolution MALDI-FTICR MS spectra demonstrated by searching candidate biomarkers for glioma blood vessel formation.

BACKGROUND: A Java application is presented, which compares large numbers (n > 100) of raw FTICR mass spectra from patients and controls. Two peptide profile matrices can be produced simultaneously, one with occurrences of peptide masses in samples and another with the intensity of common peak masses in all the measured samples, using the peak- and background intensities of the raw data. In latter way, more significantly differentially expressed peptides are found between groups than just using the presence or absence in samples of common peak masses. The software application is tested by searching angiogenesis related proteins in glioma by comparing laser capture micro dissected- and enzymatic by trypsin digested tissue sections. RESULTS: By hierarchical clustering of the presence-absence matrix, it appears that proteins, such as hemoglobin alpha and delta subunit, fibrinogen beta and gamma chain precursor, tubulin specific chaperone A, epidermal fatty acid binding protein, neutrophil gelatinase-associated lipocalin precursor, peptidyl tRNA hydrolase 2 mitochondrial precursor, placenta specific growth hormone, and zinc finger CCHC domain containing protein 13 are significantly different expressed in glioma vessels. The up-regulated proteins in the glioma vessels with respect to the normal vessels determined by the Wilcoxon-Mann-Whitney test on the intensity matrix are vimentin, glial fibrillary acidic protein, serum albumin precursor, annexin A5, alpha cardiac and beta actin, type I cytoskeletal 10 keratin, calcium binding protein p22, and desmin. Peptide masses of calcium binding protein p22, Cdc42 effector protein 3, fibronectin precursor, and myosin-9 are exclusively present in glioma vessels. Some peptide fragments of non-muscular myosin-9 at the C-terminus are strongly up-regulated in the glioma vessels with respect to the normal vessels. CONCLUSION: The less rigorous than in general used commercial propriety software de-isotope algorithm results in more mono-isotopic peptide masses and consequently more proteins. Centroiding of peptide masses takes place by taking the average over more spectra in the profile matrix. Cytoskeleton proteins and proteins involved in the calcium signaling pathway seem to be most up-regulated in glioma vessels. The finding that peptides at the C-terminus of myosin-9 are up-regulated could be ascribed to splicing or fragmentation by proteases.

Identification of glioma neovascularization-related proteins by using MALDI-FTMS and nano-LC fractionation to microdissected tumor vessels.

The identification of angiogenesis-related proteins is important for the development of new antiangiogenic therapies, and such proteins are potential new biomarkers for gliomas. The aim of this study was to identify proteins that are exclusively present in glioma neovasculature and not in the vasculature of normal brain. We combined advanced proteomics techniques to compare the expression profiles of microdissected blood vessels from glioma with blood vessels of normal control brain samples. We measured the enzymatic generated peptide profiles from these microdissected samples by MALDI-FTMS. Subsequently, the samples were fractionated by nano-LC prior to MALDI-TOF/TOF. This combined approach enabled us to identify four proteins that appeared to be exclusively expressed in the glioma blood vessels. Two of these proteins, fibronectin and colligin 2, were validated on tissue sections using specific antibodies. We found that both proteins are present in active angiogenesis in glioma, other neoplasms, and reactive conditions in which neoangiogenesis takes place. This work proves that gel-free mass spectrometric techniques can be used on relatively small numbers of cells generated by microdissection procedures to successfully identify differentially expressed proteins.

The influence of wind and locomotor activity on surface temperature and energy expenditure of the Eastern house finch (Carpodacus mexicanus) during cold stress.

We investigated the extent to which exercise-generated heat compensates for regulatory thermogenesis of Eastern house finches (Carpodacus mexicanus Müller) exposed to ambient temperatures (Ta) and convective conditions typical of that which birds experience in nature while perched in the open or foraging on the ground. We addressed the hypothesis that resting and active birds exposed to similar net convective conditions will exhibit similar surface temperatures (Ts) and metabolic energy expenditures. To test this hypothesis, resting birds were exposed to a wind speed equivalent to the treadmill speed (0.5 m s-1) for a hopping bird (active). Ts of resting birds in no wind, resting birds exposed to wind, and active birds were measured with infrared thermography at Ta between 0 degrees and 25 degrees C. Metabolic heat production was estimated from measures of respiratory gases at Ta between -5 degrees and 25 degrees C. For resting birds in no wind, resting birds in wind, and active birds, Ts decreased with decreasing Ta. The effects of variation in Ta on Ts depended on activity level (F=3.91, df=2,40, P=0.0280). The regression relationship of Ts on Ta, however, did not differ significantly between resting birds exposed to wind and active birds (F=0.12, df=2,40, P=0.8865), whereas the slope was lower and intercept higher for resting birds in no wind compared with those of resting birds exposed to wind and active birds combined (F=20.96, df=2,42, P<0.0001). Metabolic heat production for resting birds exposed to wind and active birds increased with decreasing Ta. Average metabolic heat production of resting (46.01 mW g-1+/-10.60 SD) and active birds (47.63 mW g-1+/-8.76 SD) exposed to similar net convective conditions did not differ significantly (F=3.87, df=1,44, P=0.0556). These results support our hypothesis and provide evidence that exercise generated compensates for thermostatic requirements at Ta just below thermoneutrality, which resembles conditions under which house finches naturally forage. We conclude that the compensation of exercise-generated heat for regulatory thermogenesis may occur more frequently under natural environmental conditions than implied by most previous investigators and can result in considerable energy savings for birds living in cold environments.