Sex-specific effect of the thermolabile C677T mutation in the methylenetetrahydrofolate reductase gene on angiographically assessed coronary artery disease in Brazilians

Hyperhomocysteinemia is associated with increased coronary artery disease (CAD) risk. Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the metabolism of homocysteine and presents a common mutation (C677T) that leads to a thermolabile enzyme, mild hyperhomocysteinemia, and increased CAD risk. The C677T MTHFR mutation was studied in 772 subjects (480 Caucasian Brazilians and 292 African Brazilians) who underwent coronary angiography at the hemodynamic center of the Santa Izabel Hospital in Salvador, Bahia State, Brazil. The 677T allele frequency was increased in Caucasian Brazilians (28.1%) compared to the frequency observed in African Brazilians (18.3%; p < 0.001). In Caucasian Brazilians the frequency of the 677T homozygous genotype was increased in CAD cases (10.4%) compared to control subjects (1.4%; p = 0.014) in males but not in females. In African Brazilians the mutation was not associated with CAD in either sex. The multivariate logistic regression analysis of all the samples shows that the 677T homozygous interaction with sex was a significant CAD predictor, independent of other classical risk factors and ethnic group. The odds ratio associated with male 677T homozygotes was increased 9.2-fold (p = 0.021) compared to the 677C carriers. The present study suggests that the C677T MTHFR mutation is associated with increased CAD risk in a sex-dependent manner in Brazilians.     

Mismatch between uniform increase in cardiac glucose uptake and regional contractile dysfunction in pacing-induced heart failure

Increased glucose utilization and regional differences in contractile function are well-known alterations of the failing heart and play an important pathophysiological role. We tested whether, similar to functional derangement, changes in glucose uptake develop following a regional pattern. Heart failure was induced in 13 chronically instrumented minipigs by pacing the left ventricular (LV) free wall at 180 beats/min for 3 wk. Regional changes in contractile function and stress were assessed by magnetic resonance imaging, whereas regional flow and glucose uptake were measured by positron emission tomography utilizing, respectively, the radiotracers [(13)N]ammonia and (18)F-deoxyglucose. In heart failure, LV end-diastolic pressure was 20 +/- 4 mmHg, and ejection fraction was 35 +/- 4% (all P < 0.05 vs. control). Sustained pacing-induced dyssynchronous LV activation caused a more pronounced decrease in LV systolic thickening (7.45 +/- 3.42 vs. 30.62 +/- 8.73%, P < 0.05) and circumferential shortening (-4.62 +/- 1.0 vs. -7.33 +/- 1.2%, P < 0.05) in the anterior/anterior-lateral region (pacing site) compared with the inferoseptal region (opposite site). Conversely, flow was reduced significantly by approximately 32% compared with control and was lower in the opposite site region. Despite these nonhomogeneous alterations, regional end-systolic wall stress was uniformly increased by 60% in the failing LV. Similar to wall stress, glucose uptake markedly increased vs. control (0.24 +/- 0.004 vs. 0.07 +/- 0.01 micromol x min(-1) x g(-1), P < 0.05), with no significant regional differences. In conclusion, high-frequency pacing of the LV free wall causes a dyssynchronous pattern of contraction that leads to progressive cardiac failure with a marked mismatch between increased glucose uptake and regional contractile dysfunction.     

Metabolism of vitamin D in the human choriocarcinoma cell line JEG-3

Calcitriol is an antiproliferative prodifferentiating secosteroid that exerts a protective role for some kinds of cancer. Alterations in 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1) activity have been found in some tumor cells, but there are no studies performed in human choriocarcinoma. In the present work, calcitriol production and CYP27B1 gene regulation were studied in the human choriocarcinoma cell line JEG-3, and compared with normal human syncytiotrophoblasts (hS) in culture. In JEG-3 cells, secretion of [(3)H]calcitriol was significantly less (P<0.001) than in hS (45+/-17fmol/mg protein versus 174+/-87fmol/mg protein, respectively; n=8). CYP27B1 mRNA was similar in both JEG-3 and hS cells; but the protein was detected only in hS extracts. In contrast to the hS, JEG-3 CYP27B1 gene expression was not regulated by calcitriol or by a cAMP analogue. Our results indicate that in JEG-3 cells calcitriol production is diminished due to CYP27B1 dysregulation and low protein content, and suggest that hyperproliferation could be a consequence of these alterations.     

Human trisomy 21 fibroblasts rescue methotrexate toxic effect after treatment with 5-methyl-tetrahydrofolate and 5-formyl-tetrahydrofolate

Trisomy 21 causes Down syndrome (DS), the most common human genetic disorder and the leading genetic cause of intellectual disability. The alteration of one-carbon metabolism was described as the possible metabolic cause of the intellectual disability development in subjects with DS. One of the biochemical pathways involved in the one-carbon group transfer is the folate cycle. The cytotoxic drug methotrexate (MTX) is a folic acid (FA) analogue which inhibits the activity of dihydrofolate reductase enzyme involved in the one-carbon metabolic cycle. Trisomy 21 cells are more sensitive to the MTX effect than euploid cells, and in 1986 Jérôme Lejeune and Coll. demonstrated that MTX was twice as toxic in trisomy 21 lymphocytes than in control cells. In the present work, the rescue effect on MTX toxicity mediated by FA and some of its derivatives, tetrahydrofolate (THF), 5-formyl-THF, and 5-methyl-THF, in both normal and trisomy 21 skin fibroblast cells, was evaluated. A statistically significant rescue effect was obtained by 5-formyl-THF, 5-methyl-THF, and their combination, administered together with MTX. In conclusion, trisomy 21 fibroblast cell lines showed a good response to the rescue effects of 5-formyl-THF and 5-methyl-THF on the MTX toxicity almost as normal cell lines.     

Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.     

A trait-based approach to assess the vulnerability of European aquatic insects to climate change

Aquatic insects are the dominant taxon group in most freshwater ecosystems. As temperature is the main driver of their life cycle development, metabolic activity, and geographic distribution, these macroinvertebrates are particularly suitable for large scale and comparative studies of freshwater community responses to climate change. A dataset of bio-ecological traits of 1,942 Ephemeroptera, Plecoptera, and Trichoptera (EPT) taxa was used to analyze (1) the relationships among traits, (2) the potential vulnerability of EPT species to climate change, and (3) the geographical occurrence patterns of these potentially endangered species at the scale of European ecoregions. By means of a fuzzy correspondence analysis (FCA), two gradients emerged: (1) a longitudinal gradient, describing successive upstream–downstream features, and (2) a biogeographical gradient, separating endemic and micro-endemic species from widely distributed taxa. Moreover, aquatic insects of southern European ecoregions emerged as those most endangered in terms of potential vulnerability to climate change. Comparative multi-taxon studies provide important new insights into freshwater ecosystem functioning and responses to climate change, and could be the first step toward developing integrative monitoring or assessment tools (e.g., trait-based indicators at the species level) by means of non-arbitrary statistical methods.     

A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model

We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF–RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis.     

Modulation of the Chromatin Phosphoproteome by the Haspin Protein Kinase

Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr³ of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg² and Lys⁴ adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys⁴ of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser¹³⁷ of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser¹³⁷ resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser¹³⁷ might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.Eukaryotic protein kinases (ePK)¹ constitute a large family of enzymes that coordinate virtually any cellular processes by the phosphorylation of their target proteins at specific sites (1, 2). Active kinases often modulate the activity of other enzymes, including other kinases, thus amplifying and extending an initial signal that affect sometimes thousands of proteins (3). This creates a highly complex network of feedback and forward loops where multiple kinases can mutually influence each other''s activity. Kinases adopt three molecular strategies to select and specifically phosphorylate their substrates in the crowded environment of a cell (2). First, tight control of cellular kinase localization assures that only proteins present in the close proximity of the kinase can be phosphorylated; second, the kinase specific activity can be regulated via post-translational modifications or the recruitment of cofactor molecules; and third, the recognition of specific consensus motifs on substrates ensures that phosphorylation only occurs at the intended site or sites (2).The Haspin kinase is a member of the ePK family that structurally diverges from most ePKs (1, 4). The Haspin kinase domain displays structural features that have never been observed in other ePK family members (5, 6). Specifically, the possibility of activation loop phosphorylation, a frequent regulatory mechanisms to control kinase activity, is absent in Haspin (5). Haspin is characterized by an active conformation that is stabilized by a hydrophobic lock of the helix αC inducing a stable S conformation of the structurally unique activation segment. These specific structural features also create a structurally diverse substrate binding site comprising a highly electronegative cleft for the histone H3 basic tails (5). Interestingly, the recognition of H3 has been shown to be modulated by methylation at H3 residue Lys⁴, thus coupling Haspin activity with epigenetic mechanisms of chromatin regulation (5). Histone H3 that is phosphorylated at Thr³ is so far the only well-characterized Haspin substrate (7). H3Thr³ phosphorylation (H3Thr^3ph) is required for the localization of Aurora B at the centromere (8–10). Inactivation of Haspin catalytic activity by ATP mimetic inhibitors induces Aurora B centromeric delocalization, leading to a loss of phosphorylation in chromatin associated Aurora B substrates (11, 12). To date, apart from this well-characterized centromeric function of Haspin activity, the broader cellular functions of the kinase and the phosphorylation events that control these remain essentially unknown.In this study, we used an integrated biochemical, proteomic, pharmacologic, and structural biology approach to study the Haspin kinase, its substrates and the cellular consequences of its activity. Specifically, we determined a new mode of kinase substrate binding and identified a Haspin kinase substrate recognition motif. We identified 3964 phosphorylation sites in chromatin-associated proteins, quantified their response to Haspin inhibition, and verified the mitotic phosphorylation of MacroH2A Ser¹³⁷ (13) as directly dependent by Haspin activity. Altogether, our data suggest that Haspin regulates the phosphorylation of proteins involved in mechanisms that control gene expression, including the modifications of histones, and provide evidence for novel molecular effects of Haspin activity on mitotic chromatin.