A proteomic approach for the characterization of C677T mutation of the human gene methylenetetrahydrofolate reductase

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate (CH2H4folate) to methyltetrahydrofolate (CH3H4folate). The C677T mutation is a common polymorphism of the human enzyme that leads to the replacement of Ala222Val, thermolability of MTHFR, and mild elevation of plasma homocysteine levels. A mild hyperhomocysteinemia is known to be risk factor for cardiovascular and thrombotic diseases, ischemic stroke, neural tube defects, late on-set dementia, and pregnancy complications. Human plasma of subjects carrying the C677T mutation in the MTHFR gene has been investigated for their protein pattern in order to identify novel molecular hallmarks. 2-D analysis of the plasma protein allowed the identification of a specific pattern associated with the TT mutant genotype. Noteworthy, we found one spot shifted to a more basic pI in mutant individuals, and MS identification corresponded to vitamin D-binding protein (DBP or group component (Gc) globulin). MS/MS peptide sequencing allowed to discriminate different allelic variants in the investigated clinical groups. These data confirmed by molecular genetic analysis highlight the novel association between the C677T MTHFR genotype with the Gc2 polymorphism of the DBP. Moreover, we found a quantitative reduction of Apolipoprotein A-I in mutant individuals, which was associated, in previous studies by others to an increased cardiovascular risk.     

ZD4054, a potent endothelin receptor A antagonist, inhibits ovarian carcinoma cell proliferation

Endothelin-1 (ET-1) is present at high concentrations in ovarian cancer ascites and is overexpressed in primary and metastatic ovarian carcinomas. In these tumors, the presence of ET-1 correlates with tumor grade, enhanced neovascularization, and with vascular endothelial growth factor (VEGF) expression. ET-1 acts as an autocrine factor selectively through ET(A) receptor (ET(A)R), predominantly expressed in ovarian carcinoma cells resulting in increased VEGF production and VEGF-mediated angiogenic effects. Previous results demonstrated that in ovarian carcinoma cells, activation of the ET-1/ET(A)R axis promotes cell proliferation, neovascularization, and invasion, which are the principal hallmarks of tumor progression. The present study was designed to investigate the in vitro effects of trans, trans-2(4-methoxydhenyl)-4-(1-3-benzodiazol-5-yl)-1-(dibutylaminocarbonylmethyl)-pyrrolidine-3-carboxylic acid (ZD4054), an orally active specific ET(A)R antagonist, on the ET-1-induced mitogenic effect in OVCA 433 and HEY ovarian carcinoma cell lines secreting ET-1 and expressing ET(A)R and ET(B)R mRNA. We show that ET(A)R blockade by ZD4054 inhibits ET-1-induced mitogenic effects, while the ET(B)R antagonist, BQ 788, is ineffective. In conclusion, our data demonstrate that ZD4054 is capable in inhibiting the proliferative activity of ET-1, indicating that this specific ET(A)R antagonist may be a potential candidate in developing novel treatment of ovarian carcinoma.     

Endothelin-1 is required during epithelial to mesenchymal transition in ovarian cancer progression

In a range of human cancers, tumorigenesis is promoted by activation of the endothelin A receptor (ET(A)R)/endothelin-1 (ET-1) axis. ET-1 and ET(A)R are overexpressed in primary and metastatic ovarian carcinomas, and high levels of ET-1 are detectable in patient ascites, suggesting that ET-1 may promote tumor dissemination. Moreover, in these tumors, engagement of ET(A) receptor by ET-1 triggers tumor growth, survival, angiogenesis, and invasiveness. Thus, ET-1 enhances the secretion of matrix metalloproteinases, disrupts intercellular communications, and stimulates cell migration and invasion. Therefore, we investigated the role of the ET-1/ET(A)R autocrine axis in promoting epithelial to mesenchymal transition (EMT) in ovarian tumor cells, a key event in cancer metastasis, in which epithelial cells depolarize, disassemble cell-cell contacts, and adopt an invasive phenotype. Here, we examine the potential role of ET-1 in regulating cell morphology and behavior and epithelial and mesenchymal proteins employing an in vitro 3-D culture system. We found that in 3-D serum-free collagen I gel cultures, HEY and OVCA 433 ovarian carcinoma cells undergo fibroblast-like morphologic changes between 3 and 5 days of ET-1 treatment. In these cells, ET-1 induces loss of adherens and tight-junction protein expression, E-cadherin, beta-catenin, and zonula occludens-1, and gain of N-cadherin and vimentin expression. These results confirm the ability of ET-1 to promote EMT, a metastable process involving sustained loss of epithelial markers and gain of mesenchymal markers. Collectively, these findings provide evidence of a critical role for the ET-1/ET(A)R axis during distinct steps of ovarian carcinoma progression, thus underlining this axis as a potential target in the treatment of ovarian cancer.     

Quantitative proteomic comparison of rat mitochondria from muscle, heart, and liver

Mitochondria, through oxidative phosphorylation, are the primary source of energy production in all tissues under aerobic conditions. Although critical to life, energy production is not the only function of mitochondria, and the composition of this organelle is tailored to meet the specific needs of each cell type. As an organelle, the mitochondrion has been a popular subject for proteomic analysis, but quantitative proteomic methods have yet to be applied to tease apart subtle differences among mitochondria from different tissues or muscle types. Here we used mass spectrometry-based proteomics to analyze mitochondrial proteins extracted from rat skeletal muscle, heart, and liver tissues. Based on 689 proteins identified with high confidence, mitochondria from the different tissues are qualitatively quite similar. However, striking differences emerged from the quantitative comparison of protein abundance between the tissues. Furthermore we applied similar methods to analyze mitochondrial matrix and intermembrane space proteins extracted from the same mitochondrial source, providing evidence for the submitochondrial localization of a number of proteins in skeletal muscle and liver. Several proteins not previously thought to reside in mitochondria were identified, and their presence in this organelle was confirmed by protein correlation profiling. Hierarchical clustering of microarray expression data provided further evidence that some of the novel mitochondrial candidates identified in the proteomic survey might be associated with mitochondria. These data reveal several important distinctions between mitochondrial and submitochondrial proteomes from skeletal muscle, heart, and liver tissue sources. Indeed approximately one-third of the proteins identified in the soluble fractions are associated predominantly to one of the three tissues, indicating a tissue-dependent regulation of mitochondrial proteins. Furthermore a small percentage of the mitochondrial proteome is unique to each tissue.     

BMP-dependent activation of caspase-9 and caspase-8 mediates apoptosis in pulmonary artery smooth muscle cells

Germ line mutations in the bone morphogenetic protein (BMP) receptor type II (BMPRII) gene have been found in >50% of familial idiopathic pulmonary arterial hypertension (IPAH) patients and in 30% of sporadic cases of IPAH. Mutations of BMPRII occur in the extracellular ligand-binding domain, in the cytoplasmic serine/threonine kinase domain, or in the long carboxy terminus domain of unknown function. In this study, we demonstrate that BMPs promote apoptotic cell death in normal human pulmonary artery smooth muscle cells (PASMCs) by activation of caspases-3, -8, and -9, cytochrome c release, and downregulation of Bcl-2. Normal PASMCs expressing a kinase domain mutant or a carboxy-terminal domain deletion mutant of BMPRII identified in IPAH patients are resistant to BMP-mediated apoptosis. This dominant-negative effect may act in heterozygous patients and lead to the development of the pulmonary vascular medial hypertrophy found in IPAH patients. Our study also demonstrates an essential role of the carboxy terminus domain of BMPRII in the activation of the apoptotic signaling cascade.     

Effect of IL-1 on the hydrolysis of the tumor antigen epitope gp100(280-288) by fibroblast-expressed enzymes

The role of proinflammatory cytokines in increasing the activity of specific proteases suggests the hypothesis that, by altering the expression of these mediators, adjuvants may modulate the effectiveness of peptides used as vaccines. The possible effect of IL-1 on fibroblast-expressed, peptidases was, thus, investigated by analyzing the degradation of a tumor antigen epitope (gp100(280-288), YLEPGPVTA) in the presence of cultured human fibroblasts. The data obtained indicate an increase of substrate hydrolysis after IL-1 treatment as compared with non-treated controls. Hydrolysis increase was accompanied by defined changes in the population of the by-products formed: specifically, the amount of peptidic by-products increased more than the amount of single amino acids, and the amount of the C-terminal by-products increased more than the amount of their N-terminal counterpart. These data appear to indicate that the positive effect of IL-1 on the activity of substrate-active enzymes is function of modified expression of a number of these enzymes by fibroblasts. From these data it can be inferred that the use of IL-1-inducing adjuvants, increasing the activity of peptidases expressed by bystander cells, may reduce the bio-availability of peptides used for immunization.     

The decline of swimming performance with advancing age: a cross-sectional study

The aim of this cross-sectional study was to measure the swimming parameters-speed (V), stroke frequency (SF), and stroke length (SL)- in 162 male athletes aged 50-90 (divided into 7 age groups, from A to G) participating in the World Master Championships in the 200-m freestyle event, and to analyze the rates and magnitudes of their age-associated declines. The swimmers were video-recorded by 2 digital cameras during the competitions and the swimming parameters related to every 50-m section (lap) and to the entire race (average) subsequently measured or calculated. Lap V and SF decreased in the second and third quarter (11 and 4% on average) and increased (3% on average) in the fourth quarter of the race, whereas lap SL decreased from the first to the last 50-m section. Average V (m.s(-1)) decreased from 1.39 +/- 0.09 (group A) to 0.84 +/- 0.11 (group G); average SL (m) decreased from 2.10 +/- 0.20 (group A) to 1.78 +/- 0.19 (group G); and average SF (cycles.s(-1)) decreased from 0.67 +/- 0.06 (group A) to 0.47 +/- 0.04 (group G). One-way analysis of variance showed significant declines in average V, SL, and SF (p < 0.01) across the 7 groups. The swimming parameters were normalized to the highest values (set equal to 100); thereafter, a linear regression curve was fitted and the regression equations calculated. Decline of SF was about 2.5 times steeper than that of SL. It was highlighted that (a) among the swimming parameters, SL is less affected by the ageing process; (b) SL decreased from group A through group C and thereafter tended to keep steady, whereas the trend for SF was opposite. The results have the potential to give master swimmers and their coaches useful information for training program design.     

The role of the TRAIL/TRAIL receptors system in hematopoiesis and endothelial cell biology

TRAIL is a member of the tumor necrosis factor superfamily that interacts with an unusually complex receptor system, comprising transmembrane (TRAIL-R1, -R2, -R3 and -R4) and soluble (osteoprotegerin) receptors. TRAIL has received considerable attention because of the finding that many cancer cell types are sensitive to TRAIL-induced apoptosis. However, increasing experimental evidence shows that TRAIL exhibits regulatory roles in various normal tissues, as well. Although the best-characterized biological activity of TRAIL is in the homeostatic regulation of the immune system, in this review we have summarized and discussed the physiological function of TRAIL and its receptors, in normal hematopoiesis and vascular physiopathology.