Mascot file parsing and quantification (MFPaQ), a new software to parse, validate, and quantify proteomics data generated by ICAT and SILAC mass spectrometric analyses: application to the proteomics study of membrane proteins from primary human endothelial cells

Proteomics strategies based on nanoflow (nano-) LC-MS/MS allow the identification of hundreds to thousands of proteins in complex mixtures. When combined with protein isotopic labeling, quantitative comparison of the proteome from different samples can be achieved using these approaches. However, bioinformatics analysis of the data remains a bottleneck in large scale quantitative proteomics studies. Here we present a new software named Mascot File Parsing and Quantification (MFPaQ) that easily processes the results of the Mascot search engine and performs protein quantification in the case of isotopic labeling experiments using either the ICAT or SILAC (stable isotope labeling with amino acids in cell culture) method. This new tool provides a convenient interface to retrieve Mascot protein lists; sort them according to Mascot scoring or to user-defined criteria based on the number, the score, and the rank of identified peptides; and to validate the results. Moreover the software extracts quantitative data from raw files obtained by nano-LC-MS/MS, calculates peptide ratios, and generates a non-redundant list of proteins identified in a multisearch experiment with their calculated averaged and normalized ratio. Here we apply this software to the proteomics analysis of membrane proteins from primary human endothelial cells (ECs), a cell type involved in many physiological and pathological processes including chronic inflammatory diseases such as rheumatoid arthritis. We analyzed the EC membrane proteome and set up methods for quantitative analysis of this proteome by ICAT labeling. EC microsomal proteins were fractionated and analyzed by nano-LC-MS/MS, and database searches were performed with Mascot. Data validation and clustering of proteins were performed with MFPaQ, which allowed identification of more than 600 unique proteins. The software was also successfully used in a quantitative differential proteomics analysis of the EC membrane proteome after stimulation with a combination of proinflammatory mediators (tumor necrosis factor-alpha, interferon-gamma, and lymphotoxin alpha/beta) that resulted in the identification of a full spectrum of EC membrane proteins regulated by inflammation.     

Discharge�Ccalcium concentration relationships in streams of the Amazon and Cerrado of Brazil: soil or land use controlled

Stream discharge?Cconcentration relationships are indicators of terrestrial ecosystem function. Throughout the Amazon and Cerrado regions of Brazil rapid changes in land use and land cover may be altering these hydrochemical relationships. The current analysis focuses on factors controlling the discharge?Ccalcium (Ca) concentration relationship since previous research in these regions has demonstrated both positive and negative slopes in linear log₁₀discharge?Clog₁₀Ca concentration regressions. The objective of the current study was to evaluate factors controlling stream discharge?CCa concentration relationships including year, season, stream order, vegetation cover, land use, and soil classification. It was hypothesized that land use and soil class are the most critical attributes controlling discharge?CCa concentration relationships. A multilevel, linear regression approach was utilized with data from 28 streams throughout Brazil. These streams come from three distinct regions and varied broadly in watershed size (<1 to >10⁶ ha) and discharge (10^?5.7?C10^3.2 m³ s^?1). Linear regressions of log₁₀Ca versus log₁₀discharge in 13 streams have a preponderance of negative slopes with only two streams having significant positive slopes. An ANOVA decomposition suggests the effect of discharge on Ca concentration is large but variable. Vegetation cover, which incorporates aspects of land use, explains the largest proportion of the variance in the effect of discharge on Ca followed by season and year. In contrast, stream order, land use, and soil class explain most of the variation in stream Ca concentration. In the current data set, soil class, which is related to lithology, has an important effect on Ca concentration but land use, likely through its effect on runoff concentration and hydrology, has a greater effect on discharge?Cconcentration relationships.     

YphC and YsxC GTPases assist the maturation of the central protuberance,GTPase associated region and functional core of the 50S ribosomal subunit

YphC and YsxC are GTPases in Bacillus subtilis that facilitate the assembly of the 50S ribosomal subunit, however their roles in this process are still uncharacterized. To explore their function, we used strains in which the only copy of the yphC or ysxC genes were under the control of an inducible promoter. Under depletion conditions, they accumulated incomplete ribosomal subunits that we named 45S_YphC and 44.5S_YsxC particles. Quantitative mass spectrometry analysis and the 5–6 Å resolution cryo-EM maps of the 45S_YphC and 44.5S_YsxC particles revealed that the two GTPases participate in the maturation of the central protuberance, GTPase associated region and key RNA helices in the A, P and E functional sites of the 50S subunit. We observed that YphC and YsxC bind specifically to the two immature particles, suggesting that they represent either on-pathway intermediates or that their structure has not significantly diverged from that of the actual substrate. These results describe the nature of these immature particles, a widely used tool to study the assembly process of the ribosome. They also provide the first insights into the function of YphC and YsxC in 50S subunit assembly and are consistent with this process occurring through multiple parallel pathways, as it has been described for the 30S subunit.     

Role of Cytoskeleton Proteins in the Morphological Changes During Apoptotic Cell Death of Cerebellar Granule Neurons

Cytoskeleton proteins are substrates for proteases and further apoptotic death. We evaluated the participation of cytoskeleton in morphological changes during cell death induced by two apoptotic conditions, potassium deprivation (K5) and staurosporine, in cerebellar granule neurons (CGC). We found that K5 induced somatic damage, but neurites were relatively preserved, which corresponded to the reorganization of actin and α-tubulin in neurites. Staurosporine (STS) induced an early alteration of neurites with reorganization of cytoskeleton proteins in somas. Caspase inhibitor ZVAD totally inhibited STS-induced α-tubulin reorganization and partially blocked STS-induced actin reorganization. α-tubulin and actin reorganization induced by K5 was affected by ZVAD. Calpain inhibitor (IC1) did not affect α-tubulin or actin reorganization induced by STS, K5 or ionomycin. Neither ZVAD, nor IC1 changed α-tubulin or actin levels upon K5 treatment. STS increased α-tubulin and actin levels, but neither ZVAD nor IC1 changed α-tubulin levels upon STS treatment. In contrast, ZVAD reduced the STS-induced increase of actin. These results suggest that CGC cytoskeleton proteins undergo a differential expression and reorganization depending on the apoptotic condition.     

Digoxin Suppresses Pyruvate Kinase M2-Promoted HIF-1α Transactivation in Steatohepatitis

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Rapid up-regulation of alpha4 integrin-mediated leukocyte adhesion by transforming growth factor-beta1

The alpha4 integrins (alpha4beta1 and alpha4beta7) are cell surface heterodimers expressed mostly on leukocytes that mediate cell-cell and cell-extracellular matrix adhesion. A characteristic feature of alpha4 integrins is that their adhesive activity can be subjected to rapid modulation during the process of cell migration. Herein, we show that transforming growth factor-beta1 (TGF-beta1) rapidly (0.5-5 min) and transiently up-regulated alpha4 integrin-dependent adhesion of different human leukocyte cell lines and human peripheral blood lymphocytes (PBLs) to their ligands vascular cell adhesion molecule-1 (VCAM-1) and connecting segment-1/fibronectin. In addition, TGF-beta1 enhanced the alpha4 integrin-mediated adhesion of PBLs to tumor necrosis factor-alpha-treated human umbilical vein endothelial cells, indicating the stimulation of alpha4beta1/VCAM-1 interaction. Although TGF-beta1 rapidly activated the small GTPase RhoA and the p38 mitogen-activated protein kinase, enhanced adhesion did not require activation of both signaling molecules. Instead, polymerization of actin cytoskeleton triggered by TGF-beta1 was necessary for alpha4 integrin-dependent up-regulated adhesion, and elevation of intracellular cAMP opposed this up-regulation. Moreover, TGF-beta1 further increased cell adhesion mediated by alpha4 integrins in response to the chemokine stromal cell-derived factor-1alpha. These data suggest that TGF-beta1 can potentially contribute to cell migration by dynamically regulating cell adhesion mediated by alpha4 integrins.     

Pharmacological reactivity in cricetus aureus uterus (author's transl)]

The motility of the isolated Cricetus auratus uterus was studied and compared to that of other species. Oxitocyn, epinephrine, norepinephrine, histamine, 5-hydroxy-tryptamine and acetylcholine were used as spasmogen agents. There was not contractil response with epinephrine or nor-epinephrine. Histamine reduced basal tonus. There was contraction with acetylcholine, oxytocin and 5-hydroxy-tryptamine. Cricetus auratus uterus appeared more sensitive when the contraction was registered by the isometric method. No taquifilaxy was produced by 5-hydroxy-tryptamine, as opposed to such effect in rat uterus. The Cricetus auratus uterus has, therefore, shown similar reactivity to that of rat, but different from rabbit and guinea-pig.     

Somatotype and physical performance in a sample of university students from Madrid

A sample of 303 Madrid Complutense University students (100 males and 203 females), aged 21–29 years has been studied in order to establish the relationship between somatotype components and physical work performance. Since particular interest is focused on a possible sexual difference in that relationship, males and females were analyzed separately. Results prove the high correlation of test scores implying muscularity (hand grips, pulling strength) with the mesomorphic component of the somatotype, mainly in males. Variability in tests relative to physical totype, mainly in males. Variability in tests relative to physical fitness is mainly explained by differences in endomorphy, although regarding the step test, ectomorphy is also a factor to be taken into account in females, as well as the pulling strength in males.     

High‐throughput analysis of vocalizations reveals sex‐specific changes in Fmr1 mutant pups

There have been several reports that individuals with Fragile X syndrome (FXS) and animal models of FXS have communication deficits. The present study utilized two different call classification taxonomies to examine the sex‐specificity of ultrasonic vocalization (USV) production on postnatal day (PD8) in the FVB strain of Fmr1 knockout (KO) mice. One classification protocol requires the investigator to score each call by hand, while the other protocol uses an automated algorithm. Results using the hand‐scoring protocol indicated that male Fmr1 KO mice exhibited longer calls (P = .03) than wild types on PD8. Male KOs also produced fewer complex, composite, downward, short and two‐syllable call‐types, as well as more frequency steps and chevron call‐types. Female heterozygotes exhibited no significant changes in acoustic or temporal aspects of calls, yet showed significant changes in call‐type production proportions across two different classification taxonomies (P < .001). They exhibited increased production of harmonic and frequency steps calls, as well as fewer chevron, downward and short calls. According to the second high‐throughput analysis, female heterozygotes produced significantly fewer single‐type and more multiple‐type syllables, unlike male KOs that showed no changes in these aspects of syllable production. Finally, we correlated both scoring methods and found a high level of correlation between the two methods. These results contribute further knowledge of sex differences in USV calling behavior for Fmr1 heterozygote and KO mice and provide a foundation for the use of high‐throughput analysis of neonatal USVs.