PPARGC1A sequence variation and cardiovascular risk-factor levels: a study of the main genetic effects and gene x environment interactions in children from the European Youth Heart Study.

AIMS/HYPOTHESIS: The PPARGC1A gene coactivates multiple nuclear transcription factors involved in cellular energy metabolism and vascular stasis. In the present study, we genotyped 35 tagging polymorphisms to capture all common PPARGC1A nucleotide sequence variations and tested for association with metabolic and cardiovascular traits in 2,101 Danish and Estonian boys and girls from the European Youth Heart Study, a multicentre school-based cross-sectional cohort study. METHODS: Fasting plasma glucose concentrations, anthropometric variables and blood pressure were measured. Habitual physical activity and aerobic fitness were objectively assessed using uniaxial accelerometry and a maximal aerobic exercise stress test on a bicycle ergometer, respectively. RESULTS: In adjusted models, nominally significant associations were observed for BMI (rs10018239, p = 0.039), waist circumference (rs7656250, p = 0.012; rs8192678 [Gly482Ser], p = 0.015; rs3755863, p = 0.02; rs10018239, beta = -0.01 cm per minor allele copy, p = 0.043), systolic blood pressure (rs2970869, p = 0.018) and fasting glucose concentrations (rs11724368, p = 0.045). Stronger associations were observed for aerobic fitness (rs7656250, p = 0.005; rs13117172, p = 0.008) and fasting glucose concentrations (rs7657071, p = 0.002). None remained significant after correcting for the number of statistical comparisons. We proceeded by testing for gene x physical activity interactions for the polymorphisms that showed nominal evidence of association in the main effect models. None of these tests was statistically significant. CONCLUSIONS/INTERPRETATION: Variants at PPARGC1A may influence several metabolic traits in this European paediatric cohort. However, variation at PPARGC1A is unlikely to have a major impact on cardiovascular or metabolic health in these children.

The C(K, X) spaces for compact metric spaces K and X with a uniformly convex maximal factor

In this paper, we prove that if a Banach space X contains some uniformly convex subspace in certain geometric position, then the C(K, X) spaces of all X-valued continuous functions defined on the compact metric spaces K have exactly the same isomorphism classes that the C(K) spaces. This provides a vector-valued extension of classical results of Bessaga and Pelczynski (1960) [2] and Milutin (1966) [13] on the isomorphic classification of the separable C(K) spaces. As a consequence, we show that if 1 < p < q < infinity then for every infinite countable compact metric spaces K(1), K(2), K(3) and K(4) are equivalent: (a) C(K(1), l(p)) circle plus C(K(2), l(q)) is isomorphic to C(K(3), l(p)) circle plus (K(4), l(q)). (b) C(K(1)) is isomorphic to C(K(3)) and C(K(2)) is isomorphic to C(K(4)). (C) 2011 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Anisotropy of the effective Lande factor in AlxGa1-x As parabolic quantum wells under applied magnetic fields

The anisotropy of the effective Lande factor in Al(x)Gal(1-x)As parabolic quantum wells under magnetic fields is theoretically investigated. The non-parabolicity and anisotropy of the conduction band are taken into account through the Ogg-McCombe Hamiltonian together with the cubic Dresselhaus spin-orbit term. The calculated effective g factor is larger when the magnetic field is applied along the growth direction. As the well widens, its anisotropy increases sharply and then decreases slowly. For the considered field strengths, the anisotropy is maximum for a well width similar to 50 angstrom. Moreover, this anisotropy increases with the field strength and the maximum value of the aluminum concentration within the quantum well. (C) 2010 Elsevier B.V. All rights reserved.

Severidade e controle da bacteriose foliar em mudas de Eucalyptus urophyla x Eucalyptus grandis em função do nível tecnológico do viveiro

Pós-graduação em Ciência Florestal - FCA

Evaluation via multivariate techniques of scale factor variability in the rietveld method applied to quantitative phase analysis with X ray powder diffraction

ABSTRACT: The present work uses multivariate statistical analysis as a form of establishing the main sources of error in the Quantitative Phase Analysis (QPA) using the Rietveld method. The quantitative determination of crystalline phases using x ray powder diffraction is a complex measurement process whose results are influenced by several factors. Ternary mixtures of Al2O3, MgO and NiO were prepared under controlled conditions and the diffractions were obtained using the Bragg-Brentano geometric arrangement. It was possible to establish four sources of critical variations: the experimental absorption and the scale factor of NiO, which is the phase with the greatest linear absorption coefficient of the ternary mixture; the instrumental characteristics represented by mechanical errors of the goniometer and sample displacement; the other two phases (Al2O3 and MgO); and the temperature and relative humidity of the air in the laboratory. The error sources excessively impair the QPA with the Rietveld method. Therefore it becomes necessary to control them during the measurement procedure.

Time- and dose-related regional fluxes of tissue-type plasminogen activator in anesthetized endotoxemic pigs

BACKGROUND: Acute endotoxinemia elicits an early fibrinolytic response. This study analyzes the effects of the dose and duration of endotoxin infusion on arterial levels of tissue-type plasminogen activator (tPA) and pulmonary, mesenteric and hepatic plasma tPA fluxes. METHODS: Pigs were randomized to receive an acute, high-dose (for 6 h, n=13, high ETX) or a prolonged, low-dose (for 18 h, n=18, low ETX) infusion of endotoxin or saline vehicle alone (for 18 h, n=14, control). All animals were fluid resuscitated to maintain a normodynamic circulation. Systemic and regional blood flows were measured and arterial, pulmonary arterial, portal and hepatic venous blood samples were analyzed to calculate regional net fluxes of tPA. Plasma tumor necrosis factor (TNF-alpha) levels were analyzed. RESULTS: Mesenteric tPA release and hepatic uptake increased maximally at 1.5 h in ETX groups related to dose. Maximal mesenteric tPA release [high ETX 612 (138-1185) microg/min/kg, low ETX 72 (32-94) microg/min/kg, median+/-interquartile range] and hepatic tPA uptake [high ETX -1549 (-1134 to -2194) microg/min/kg, low ETX -153 (-105 to -307) microg/min/kg] correlated to TNF-alpha levels. Regional tPA fluxes returned to baseline levels at 6 h in both ETX groups and also remained low during sustained low ETX. No changes were observed in control animals. CONCLUSIONS: Endotoxemia induces an early increase in mesenteric tPA release and hepatic tPA uptake related to the severity of endotoxemia. The time patterns of changes in mesenteric and hepatic tPA fluxes are similar in acute high-dose endotoxemia and sustained low-dose endotoxemia.

Role of the transcription factor activator protein-2alpha in prostate carcinogenesis

Activator protein 2α (AP-2) is a transcription factor known to play a crucial role in the progression of malignant melanoma, colorectal carcinoma, and breast cancer. Several AP-2 target genes are known to be deregulated in prostate cancer, therefore, we hypothesize that loss AP-2 expression plays a causal role in prostate carcinogenesis. Immunofluorescent staining for AP-2 of 30 radical prostatectomy specimens demonstrated that while AP-2 was highly expressed in normal prostate epithelium, its expression was lost in most cases of high grade prostatic intraepithelial neoplasia (PIN), and all cases of prostate cancer studied. Additional analyses demonstrated that AP-2 was associated with normal luminal differentiation and it was not expressed in the basal cell layer. In cell lines, AP-2 was strongly expressed in immortalized normal prostate epithelial cells, whereas low expression was observed in the LNCaP, LNCaP-LN3, and PC3M-LN4 prostate cancer cell lines. Transfection of the highly tumorigenic and metastatic cell line PC3M-LN4 with the AP-2 gene significantly decreased tumor growth in the prostate of nude mice (p = 0.032) and inhibited metastases to the lymph nodes. Moreover, transfection of the low tumorigenic, low metastatic cell line LNCaP-LN3 with full length AP-2; resulted in complete inhibition of tumor incidence in the AP-2 transfectants (0/19) vs. neo control (10/16). A potential mechanism for this loss of tumorigenicity was the modulation of gene expression in prostate cancer cells that mimicked the normal phenotype. Analysis of differential expression between neo control- and AP-2-transfected cells in vitro and in tumors demonstrated low VEGF expression in AP-2 transfectants. We further demonstrated that AP-2 acted as a transcriptional repressor of the VEGF promoter by binding to a GC-rich region located between −88 and −66. This region contains an AP-2 consensus element overlapping two Sp1 consensus elements. We found that Sp3 and AP-2 bound to this region in a mutually exclusive manner to promote activation or repression. Increased VEGF expression has been observed in high grade PIN and in prostate cancer. Here we provide evidence that this early molecular change could be a result of loss of AP-2 expression in the prostatic epithelium. ^

Specificity in transforming growth factor β-induced transcription of the plasminogen activator inhibitor-1 gene: Interactions of promoter DNA, transcription factor μE3, and Smad proteins

Transforming growth factor β (TGF-β) regulates a broad range of biological processes, including cell growth, development, differentiation, and immunity. TGF-β signals through its cell surface receptor serine kinases that phosphorylate Smad2 or Smad3 proteins. Because Smad3 and its partner Smad4 bind to only 4-bp Smad binding elements (SBEs) in DNA, a central question is how specificity of TGF-β-induced transcription is achieved. We show that Smad3 selectively binds to two of the three SBEs in PE2.1, a TGF-β-inducible fragment of the plasminogen activator inhibitor-1 promoter, to mediate TGF-β-induced transcription; moreover, a precise 3-bp spacer between one SBE and the E-box, a binding site for transcription factor μE3 (TFE3), is essential for TGF-β-induced transcription. Whereas an isolated Smad3 MH1 domain binds to TFE3, TGF-β receptor-mediated phosphorylation of full-length Smad3 enhances its binding to TFE3. Together, these studies elucidate an important mechanism for specificity in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene.

Dual role of the nuclear factor of activated T cells insert region in DNA recognition and cooperative contacts to activator protein 1

The transcription factors nuclear factor of activated T cells (NFAT) and activator protein 1 (AP-1) coordinately regulate cytokine gene expression in activated T-cells by binding to closely juxtaposed sites in cytokine promoters. The structural basis for cooperative binding of NFAT and AP-1 to these sites, and indeed for the cooperative binding of transcription factors to composite regulatory elements in general, is not well understood. Mutagenesis studies have identified a segment of AP-1, which lies at the junction of its DNA-binding and dimerization domains (basic region and leucine zipper, respectively), as being essential for protein–protein interactions with NFAT in the ternary NFAT/AP-1/DNA complex. In a model of the ternary complex, the segment of NFAT nearest AP-1 is the Rel insert region (RIR), a feature that is notable for its hypervariability in size and in sequence amongst members of the Rel transcription factor family. Here we have used mutational analysis to study the role of the NFAT RIR in binding to DNA and AP-1. Parallel yeast one-hybrid screening assays in combination with alanine-scanning mutagenesis led to the identification of four amino acid residues in the RIR of NFAT2 (also known as NFATC1 or NFATc) that are essential for cooperativity with AP-1 (Ile-544, Glu-545, Thr-551, and Ile-553), and three residues that are involved in interactions with DNA (Lys-538, Arg-540, and Asn-541). These results were confirmed and extended through in vitro binding assays. We thus conclude that the NFAT RIR plays an essential dual role in DNA recognition and cooperative binding to AP-1 family transcription factors.

Different Tyrosine Autophosphorylation Requirements in Fibroblast Growth Factor Receptor-1 Mediate Urokinase-Type Plasminogen Activator Induction and Mitogenesis

Among the seven tyrosine autophosphorylation sites identified in the intracellular domain of tyrosine kinase fibroblast growth factor receptor-1 (FGFR1), five of them are dispensable for FGFR1-mediated mitogenic signaling. The possibility of dissociating the mitogenic activity of basic FGF (FGF2) from its urokinase-type plasminogen activator (uPA)-inducing capacity both at pharmacological and structural levels prompted us to evaluate the role of these autophosphorylation sites in transducing FGF2-mediated uPA upregulation. To this purpose, L6 myoblasts transfected with either wild-type (wt) or various FGFR1 mutants were evaluated for the capacity to upregulate uPA production by FGF2. uPA was induced in cells transfected with wt-FGFR1, FGFR1-Y463F, -Y585F, -Y730F, -Y766F, or -Y583/585F mutants. In contrast, uPA upregulation was prevented in L6 cells transfected with FGFR1-Y463/583/585/730F mutant (FGFR1–4F) or with FGFR1-Y463/583/585/730/766F mutant (FGFR1–5F) that retained instead a full mitogenic response to FGF2; however, preservation of residue Y730 in FGFR1-Y463/583/585F mutant (FGFR1–3F) and FGFR1-Y463/583/585/766F mutant (FGFR1–4Fbis) allows the receptor to transduce uPA upregulation. Wild-type FGFR1, FGFR1–3F, and FGFR1–4F similarly bind to a 90-kDa tyrosine-phosphorylated protein and activate Shc, extracellular signal-regulated kinase (ERK)2, and JunD after stimulation with FGF2. These data, together with the capacity of the ERK kinase inhibitor PD 098059 to prevent ERK2 activation and uPA upregulation in wt-FGFR1 cells, suggest that signaling through the Ras/Raf-1/ERK kinase/ERK/JunD pathway is necessary but not sufficient for uPA induction in L6 transfectants. Accordingly, FGF2 was able to stimulate ERK1/2 phosphorylation and cell proliferation, but not uPA upregulation, in L6 cells transfected with the FGFR1-Y463/730F mutant, whereas the FGFR1-Y583/585/730F mutant was fully active. We conclude that different tyrosine autophosphorylation requirements in FGFR1 mediate cell proliferation and uPA upregulation induced by FGF2 in L6 cells. In particular, phosphorylation of either Y463 or Y730, dispensable for mitogenic signaling, represents an absolute requirement for FGF2-mediated uPA induction.

Fusion of the transcription factor TFE3 gene to a novel gene, PRCC, in t(X;1)(p11;q21)-positive papillary renal cell carcinomas

The (X;1)(p11;q21) translocation is a recurrent chromosomal abnormality in a subset of human papillary renal cell carcinomas, and is sometimes the sole cytogenetic abnormality present. Via positional cloning, we were able to identify the genes involved. The translocation results in a fusion of the transcription factor TFE3 gene on the X chromosome to a novel gene, designated PRCC, on chromosome 1. Through this fusion, reciprocal translocation products are formed, which are both expressed in papillary renal cell carcinomas. PRCC is ubiquitously expressed in normal adult and fetal tissues and encodes a putative protein of 491 aa with a relatively high content of prolines. No relevant homologies with known sequences at either the DNA or the protein level were found.

PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91phox) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91phox gene revealed a single-base mutation (C → T) at position −53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position −53 of the gp91phox promoter, and the mutation at position −53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position −50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position −56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions −53 and −50 significantly reduced the gp91phox promoter activity; however, the mutation at position −56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91phox promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C → T) at position −52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91phox gene in human neutrophils, monocytes, and B lymphocytes.

Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.