Antagonism of fenfluramine-induced hyperthermia: a measure of central serotonin inhibition.

Fenfluramine produced a rapid hyperthermic effect in rats housed in a warm (26–28 °C) environment. Drugs with known activity on serotonin-mediated pathways were the most effective antagonists of this hyperthermia. Among several anti-psychotic agents, clozapine was unique by virtue of its potent and specific antagonism of fenfluramine-induced hyperthermia. It is suggested that this serotonin blocking activity of clozapine may help to account for the lack of extrapyramidal symptoms (EPS) following administration of the drug to man.     

Integration of life cycle assessment into a decision support system for selecting sustainable road asphalt pavement mixtures prepared with waste

The International Journal of Life Cycle Assessment - A challenge that the road paving sector is facing concerns the achievement of highly performing bituminous asphalt mixture solutions that do not...     

Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

Introduction

Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs.

Methods

Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of α_vβ₃ integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis.

Results

Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and α_vβ₃ integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and α_vβ₃ integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed.

Conclusions

Because of the critical role of fibrillin-1 in sequestering the latent form of TGF-β in the extracellular matrix, its decreased deposition by B-MVECs challenged with SSc sera might contribute to dermal fibrosis. In SSc, CYC treatment might limit fibrosis through the maintenance of physiologic fibrillin-1 synthesis and deposition by B-MVECs.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Proteoglycan fragmentation and respiratory mechanics in mechanically ventilated healthy rats.

This research investigated whether stretching of lung tissue due to increased positive alveolar pressure swings during mechanical ventilation (MV) at various tidal volumes (V(T)) might affect the composition and/or structure of the glycosaminoglycan (GAG) components of pulmonary extracellular proteoglycans. Experiments were performed in 30 healthy rats: 1) anesthetized and immediately killed (controls, C-0); 2) anesthetized and spontaneously breathing for 4 h (C-4h); and 3) anesthetized, paralyzed, and mechanically ventilated for 4 h with air at 0-cmH(2)O end-expiratory pressure and V(T) of 8 ml/kg (MV-1), 16 ml/kg (MV-2), 24 ml/kg (MV-3), or 32 ml/kg (MV-4), adjusting respiratory rates at a minute ventilation of 270 ml/min. Compared with C-0 and C-4h, a significant reduction of dynamic and static compliance of the respiratory system and of the lung was observed only in MV-4, while extravascular lung water significantly increased in MV-3 and MV-4, but not in MV-1 and MV-2. However, even in MV-1, MV induced a significant fragmentation of pulmonary GAGs. Extraction of covalently bound GAGs and wash out of loosely bound or fragmented GAGs progressively increased with increasing V(T) and was associated with increased expression of local (matrix metalloproteinase-2) and systemic (matrix metalloproteinase-9) activated metalloproteases. We conclude that 1) MV, even at "physiological" low V(T), severely affects the pulmonary extracellular architecture, exposing the lung parenchyma to development of ventilator-induced lung injury; and 2) respiratory mechanics is not a reliable clinical tool for early detection of lung injury.     

Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.     

Oestrogen-induced expression of a novel liver-specific aspartic proteinase in Danio rerio (zebrafish)

Aspartic proteinases are a group of endoproteolytic proteinases active at acidic pH and characterized by the presence of two aspartyl residues in the active site. They include related paralogous proteins such as cathepsin D, cathepsin E and pepsin. Although extensively investigated in mammals, aspartic proteinases have been less studied in other vertebrates. In a previous work, we cloned and sequenced a DNA complementary to RNA encoding an enzyme present in zebrafish liver. The sequence resulted to be homologous to a novel form of aspartic proteinase firstly described by us in Antarctic fish. In zebrafish, the gene encoding this enzyme is expressed only in the female liver, in contrast with cathepsin D that is expressed in all the tissues examined independently of the sex. For this reason we have termed the new enzyme liver-specific aspartic proteinase (LAP).Northern blot analyses indicate that LAP gene expression is under hormonal control. Indeed, in oestrogen-treated male fish, cathepsin D expression was not enhanced in the various tissues examined, but the LAP gene product appeared exclusively in the liver. Our results provide evidence for an oestrogen-induced expression of LAP gene in liver. We postulate that the sexual dimorphic expression of the LAP gene may be related to the reproductive process.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

"Healthy Men" and High Mortality: Contributions from a Population-Based Study for the Gender Paradox Discussion

Background

Inequalities between men and women in morbidity and mortality show a contrast, which has been called gender paradox. Most studies evaluating this paradox were conducted in high-income countries and, until now, few investigations have been performed in Brazil. This study aims to estimate the magnitude of inequalities between adult men and women in several dimensions: demographic and socioeconomic, health behaviors, morbidity, use of health services and mortality.

Methods

The data were obtained from population-based household survey carried out in Campinas (Campinas Health Survey 2008/09) corresponding to 957 people, and data from the Mortality Information System (MIS) between 2009 and 2011. Prevalences and prevalence ratios were analyzed in order to verify the differences between men and women regarding socioeconomic and demographic variables, health behaviors, morbidities and consultations in the last two weeks. Mortality rates and the ratio between coefficients considering the underlying causes of death were calculated.

Results

Women had a greater disadvantage in socioeconomic indicators, chronic diseases diagnosed by a health professional and referred health problems as well as make more use of health services, while men presented higher frequency of most unhealthy behaviors and excessive mortality for all causes investigated.

Conclusions

The findings contribute to the discussion of gender paradox and demonstrate the need to employ health actions that consider the differences between men and women in the various health dimensions analyzed. The premature male mortality from preventable causes was outstanding, making clear the need for more effective prevention and health promotion directed to this segment of the population.     

CD and NMR conformational studies of a peptide encompassing the Mid Loop interface of Ship2–Sam

The lipid phosphatase Ship2 is a protein that intervenes in several diseases such as diabetes, cancer, neurodegeneration, and atherosclerosis. It is made up of a catalytic domain and several protein docking modules such as a C‐terminal Sam (Sterile alpha motif) domain. The Sam domain of Ship2 (Ship2–Sam) binds to the Sam domains of the EphA2 receptor (EphA2–Sam) and the PI3K effector protein Arap3 (Arap3–Sam). These heterotypic Sam–Sam interactions occur through formation of dimers presenting the canonical “Mid Loop/End Helix” binding mode. The central region of Ship2–Sam, spanning the C‐terminal end of α2, the α3 and α4 helices together with the α2α3 and α3α4 interhelical loops, forms the Mid Loop surface that is needed to bind partners Sam domains. A peptide encompassing most of the Ship2–Sam Mid Loop interface (Shiptide) capable of binding to both EphA2–Sam and Arap3–Sam, was previously identified. Here we investigated the conformational features of this peptide, through solution CD and NMR studies in different conditions. These studies reveal that the peptide is highly flexible in aqueous buffer, while it adopts a helical conformation in presence of 2,2,2‐trifluoroethanol. The discovered structural insights and in particular the identification of a helical motif, may lead to the design of more constrained and possibly cell permeable Shiptide analogs that could work as efficient antagonists of Ship2–Sam heterotypic interactions and embrace therapeutic applications. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1088–1098, 2014.