Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors

Lima GA, Anhe GF, Giannocco G, Nunes MT, Correa-Giannella ML, Machado UF. Contractile activity per se induces transcriptional activation of SLC2A4 gene in soleus muscle: involvement of MEF2D, HIF-1a, and TR alpha transcriptional factors. Am J Physiol Endocrinol Metab 296: E132-E138, 2009. First published October 28, 2008; doi: 10.1152/ajpendo.90548.2008.-Skeletal muscle is a target tissue for approaches that can improve insulin sensitivity in insulin-resistant states. In muscles, glucose uptake is performed by the GLUT-4 protein, which is encoded by the SLC2A4 gene. SLC2A4 gene expression increases in response to conditions that improve insulin sensitivity, including chronic exercise. However, since chronic exercise improves insulin sensitivity, the increased SLC2A4 gene expression could not be clearly attributed to the muscle contractile activity per se and/or to the improved insulin sensitivity. The present study was designed to investigate the role of contractile activity per se in the regulation of SLC2A4 gene expression as well as in the participation of the transcriptional factors myocyte enhancer factor 2D (MEF2D), hypoxia inducible factor 1a (HIF-1a), and thyroid hormone receptor-alpha (TR alpha). The performed in vitro protocol excluded the interference of metabolic, hormonal, and neural effects. The results showed that, in response to 10 min of electrically induced contraction of soleus muscle, an early 40% increase in GLUT-4 mRNA (30 min) occurred, with a subsequent 65% increase (120 min) in GLUT-4 protein content. EMSA and supershift assays revealed that the stimulus rapidly increased the binding activity of MEF2D, HIF-1a, and TR alpha into the SLC2A4 gene promoter. Furthermore, chromatin immunoprecipitation assay confirmed, in native nucleosome, that contraction induced an approximate fourfold (P < 0.01) increase in MEF2D and HIF-1a-binding activity. In conclusion, muscle contraction per se enhances SLC2A4 gene expression and that involves MEF2D, HIF-1a, and TR alpha transcription factor activation. This finding reinforces the importance of physical activity to improve glycemic homeostasis independently of other additional insulin sensitizer approaches.

Do the Effects of Pentoxifylline on the Inflammatory Process and Pancreatic Infection Justify Its Use in Acute Pancreatitis?

Severe acute pancreatitis is associated with high morbidity and mortality rates. At the present time, no specific therapy has been shown to be uniformly effective in reducing morbidity and mortality in this disease. The aim of this study was to determine the effects of pentoxifylline on the pancreatic and systemic inflammatory process, pancreatic infection, and mortality rate in severe acute pancreatitis in rats. Methods: One hundred and twenty male Wistar rats were divided into 3 groups: sham, pancreatitis, and pentoxifylline (acute pancreatitis induction plus administration of 25 mg/kg pentoxifylline). Inflammatory response was measured by histological studies, inflammatory cytokine production (IL-6, IL-10, and TNF-alpha), and mortality rate. Pancreatic infection was evaluated by bacterial cultures expressed in colony-forming units per gram. Results: Pentoxifylline-treated animals had a statistically significant reduction of inflammatory cytokine levels, pancreatic histological damage, occurrence of bacterial translocation and pancreatic infection (p < 0.05), associated with a significant reduction in mortality rate. Conclusions: Pentoxifylline administration in this experimental model of acute pancreatitis reduces local and systemic inflammatory responses and decreases the pancreatic infection and the mortality rate. Copyright (C) 2009 S. Karger AG, Basel and IAP

IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis. The Journal of Immunology, 2009, 182: 7846-7854.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron-and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Human alveolar bone cell proliferation, expression of osteoblastic phenotype, and matrix mineralization on porous titanium produced by powder metallurgy

Objective: This study aimed at investigating the influence of the porous titanium (Ti) structure on the osteogenic cell behaviour. Materials and methods: Porous Ti discs were fabricated by the powder metallurgy process with the pore size typically between 50 and 400 mm and a porosity of 60%. Osteogenic cells obtained from human alveolar bone were cultured until subconfluence and subcultured on dense Ti (control) and porous Ti for periods of up to 17 days. Results: Cultures grown on porous Ti exhibited increased cell proliferation and total protein content, and lower levels of alkaline phosphatase (ALP) activity than on dense Ti. In general, gene expression of osteoblastic markers-runt-related transcription factor 2, collagen type I, alkaline phosphatase, bone morphogenetic protein-7, and osteocalcin was lower at day 7 and higher at day 17 in cultures grown on porous Ti compared with dense Ti, a finding consistent with the enhanced growth rate for such cultures. The amount of mineralized matrix was greater on porous Ti compared with the dense one. Conclusion: These results indicate that the porous Ti is an appropriate substrate for osteogenic cell adhesion, proliferation, and production of a mineralized matrix. Because of the three-dimensional environment it provides, porous Ti should be considered an advantageous substrate for promoting desirable implant surface-bone interactions.

A dynamic role for HDAC7 in MEF2-mediated muscle differentiation

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7, MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

Proteomic analysis reveals that 14-3-3 sigma in downregulated in human breast cancer cells

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, me have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3 sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 mere the same in both normal and transformed cells. The data support the idea that 14-3-3 sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

Proteomics of breast cancer for marker discovery and signal pathway profiling

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

A novel 14-kilodalton protein interacts with the mitogen-activated protein kinase scaffold MP1 on a late endosomal/lysosomal compartment

We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro. The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.

Cloning and functional analysis of the Sry-related HMG box gene, Sox18

The Sox gene family (Sry like HMG box gene) is characterised by a conserved DNA sequence encoding a domain of approximately 80 amino acids which is responsible for sequence specific DNA binding. We initially published the identification and partial cDNA sequence of murine Sox18, a new member of this gene family, isolated from a cardiac cDNA library. This sequence allowed us to classify Sox18 into the F sub-group of Sox proteins, along with Sox7 and Sox17. Recently, we demonstrated that mutations in the Sox18 activation domain underlie cardiovascular and hair follicle defects in the mouse mutation, ragged (Ra) (Pennisi et al., 2000. Mutations in Sox18 underlie cardiovascular and hair follicle defecs in ragged mice. Nat. Genet. 24, 434-437). Ra homozygotes lack vibrissae and coat hairs, have generalised oedema and an accumulation of chyle in the peritoneum. Here we have investigated the genomic sequences encoding Sox18. Screening of a mouse genomic phage library identified four overlapping clones, we sequenced a 3.25 kb XbaI fragment that defined the entire coding region and approximately 1.5 kb of 5' flanking sequences. This identified (i) an additional 91 amino acids upstream of the previously designated methionine start codon in the original cDNA, and (ii);ln intron encoded within the HMG box/DNA binding domain in exactly the same position as that found in the Sox5, -13 and -17 genes. The Sox18 gene encodes a protein of 468 aa. We present evidence that suggests HAF-2, the human HMG-box activating factor-2 protein, is the orthologue of murine Sox18. HAF-2 has been implicated in the regulation of the Human IgH enhancer in a B cell context. Random mutagenesis coupled with GAL4 hybrid analysis in the activation domain between amino acids 252 and 346, of Sox18, implicated the phosphorylation motif, SARS, and the region between amino acid residues 313 and 346 as critical components of Sox18 mediated transactivation. Finally, we examined the expression of Sox18 in multiple adult mouse tissues using RT-PCR. Low-moderate expression was observed in spleen, stomach, kidney, intestine, skeletal muscle and heart. Very abundant expression was detected in lung tissue. (C) 2001 Elsevier Science B.V. All rights reserved.

Predicting the HER2 status of breast cancer from basic histopathology data: an analysis of 1500 breast cancers as part of the HER2000 International Study

The tests that are currently available for the measurement of overexpression of the human epidermal growth factor-2 (HER2) in breast cancer have shown considerable problems in accuracy and interlaboratory reproducibility. Although these problems are partly alleviated by the use of validated, standardised 'kits', there may be considerable cost involved in their use. Prior to testing it may therefore be an advantage to be able to predict from basic pathology data whether a cancer is likely to overexpress HER2. In this study, we have correlated pathology features of cancers with the frequency of HER2 overexpression assessed by immunohistochemistry (IHC) using HercepTest (Dako). In addition, fluorescence in situ hybridisation (FISH) has been used to re-test the equivocal cancers and interobserver variation in assessing HER2 overexpression has been examined by a slide circulation scheme. Of the 1536 cancers, 1144 (74.5%) did not overexpress HER2. Unequivocal overexpression (3+ by IHC) was seen in 186 cancers (12%) and an equivocal result (2+ by IHC) was seen in 206 cancers (13%). Of the 156 IHC 3+ cancers for which complete data was available, 149 (95.5%) were ductal NST and 152 (97%) were histological grade 2 or 3. Only 1 of 124 infiltrating lobular carcinomas (0.8%) showed HER2 overexpression. None of the 49 'special types' of carcinoma showed HER2 overexpression. Re-testing by FISH of a proportion of the IHC 2+ cancers showed that only 25 (23%) of those assessable exhibited HER2 gene amplification, but 46 of the 47 IHC 3+ cancers (98%) were confirmed as showing gene amplification. Circulating slides for the assessment of HER2 score showed a moderate level of agreement between pathologists (kappa 0.4). As a result of this study we would advocate consideration of a triage approach to HER-2 testing. Infiltrating lobular and special types of carcinoma may not need to be routinely tested at presentation nor may grade 1 NST carcinomas in which only 1.4% have been shown to overexpress HER2. Testing of these carcinomas may be performed when HER2 status is required to assist in therapeutic or other clinical/prognostic decision-making. The highest yield of HER2 overexpressing carcinomas is seen in the grade 3 NST subgroup in which 24% are positive by IHC. (C) 2003 Elsevier Science Ltd. All rights reserved.

Locus de Control y Ira

La ira se ha relacionado con atribuciones externas en distintos modelos atribucionales, sin embargo, desde los planteamientos de la teoría del “locus de control” (Rotter, 1966) la respuesta emocional de ira no ha sido objeto de estudio con la frecuencia que sería deseable (aunque sí la conducta agresiva). El presente trabajo explora las relaciones del locus de control externo con la emoción de ira, así como con la expresión y control de la misma. Para ello se utilizó una muestra de 177 estudiantes universitarios (27 hombres y 150 mujeres) a los que se evaluó mediante los instrumentos siguientes: Escala de Rotter (Rotter, 1966, versión española de Pérez García, 1984); el Inventario de Expresión de Ira Estado/Rasgo -STAXI-de Spielberger (1988, 1991), versión española de Spielberger, Miguel-Tobal, Cano-Vindel y Casado, 1992); y el Inventario de Control, Defensa y Expresión de Emociones -CDE-(Cano Vindel y Miguel Tobal, 1996). Se realizó un análisis correlacional entre las distintas escalas de los instrumentos y diferencias de medias para dos grupos extremos (altos y bajos) en locus de control (equiparados en la variable sexo). Nuestros resultados muestran una relación positiva y significativa entre el locus de control externo y la respuesta emocional de ira, así como una tendencia a expresar externamente la ira y a controlarla menos en sujetos con atribuciones de causalidad externas. Conocer los procesos atribucionales asociados a la ira y a su afrontamiento permitirá una intervención más eficaz sobre la misma.

Participación de los gobiernos regionales en la definición de políticas públicas en Chile: el caso del gobierno regional de Los Lagos y la política nacional de acuicultura

El artículo aporta evidencia respecto de la participación de los gobiernos regionales en la definición de políticas públicas en Chile a través del caso del gobierno regional de Los Lagos. Se evalúa la participación de los principales actores regionales en la definición de políticas públicas como indicador del grado de centralización en el diseño político institucional en Chile. La metodología utilizada fue el análisis documental relacionado con el caso presentado, el conocimiento del estado del arte respecto del análisis de políticas públicas como disciplina y la descentralización, posteriormente se contrastaron las hipótesis que guiaron el estudio a través del análisis de discurso aplicado a las entrevistas de los actores que estuvieron involucrados en la definición de la Política Nacional de Acuicultura.

Interactions between physical and psychosocial demands of work associated to low back pain

OBJECTIVE: To examine the interaction between physical and psychosocial demands of work associated to low back pain. METHODS: Cross-sectional study carried out in a stratified proportional random sample of 577 plastic industry workers in the metropolitan area of the city of Salvador, Northeast Brazil in 2002. An anonymous standard questionnaire was administered in the workplace by trained interviewers. Physical demands at work were self-rated on a 6-point numeric scale, with anchors at each end of the scale. Factor analysis was carried out on 11 physical demand variables to identify underlying factors. Psychosocial work demands were measured by demand, control and social support questions. Multivariate analysis was performed using the likelihood ratio test. RESULTS: The factor analysis identified two physical work demand factors: material handling (factor 1) and repetitiveness (factor 2). The multiple logistic regression analysis showed that factor 1 was positively associated with low back pain (OR=2.35, 95% CI 1.50;3.66). No interaction was found between physical and psychosocial work demands but both were independently associated to low back pain. CONCLUSIONS: The study found independent effects of physical and psychosocial work demands on low back pain prevalence and emphasizes the importance of physical demands especially of material handling involving trunk bending forward and trunk rotation regardless of age, gender, and body fitness.