Suppression of Myc oncogenic activity by ribosomal protein haploinsufficiency

The Myc oncogene regulates the expression of several components of the protein synthetic machinery, including ribosomal proteins, initiation factors of translation, RNA polymerase III and ribosomal DNA(1,2). Whether and how increasing the cellular protein synthesis capacity affects the multistep process leading to cancer remains to be addressed. Here we use ribosomal protein heterozygote mice as a genetic tool to restore increased protein synthesis in E mu-Myc/+ transgenic mice to normal levels, and show that the oncogenic potential of Myc in this context is suppressed. Our findings demonstrate that the ability of Myc to increase protein synthesis directly augments cell size and is sufficient to accelerate cell cycle progression independently of known cell cycle targets transcriptionally regulated by Myc. In addition, when protein synthesis is restored to normal levels, Myc- overexpressing precancerous cells are more efficiently eliminated by programmed cell death. Our findings reveal a new mechanism that links increases in general protein synthesis rates downstream of an oncogenic signal to a specific molecular impairment in the modality of translation initiation used to regulate the expression of selective messenger RNAs. We show that an aberrant increase in cap- dependent translation downstream of Myc hyperactivation specifically impairs the translational switch to internal ribosomal entry site ( IRES)- dependent translation that is required for accurate mitotic progression. Failure of this translational switch results in reduced mitotic- specific expression of the endogenous IRES- dependent form of Cdk11 ( also known as Cdc21 and PITSLRE)(3-5), which leads to cytokinesis defects and is associated with increased centrosome numbers and genome instability in E mu-Myc/+ mice. When accurate translational control is re- established in E mu-Myc/+ mice, genome instability is suppressed. Our findings demonstrate how perturbations in translational control provide a highly specific outcome for gene expression, genome stability and cancer initiation that have important implications for understanding the molecular mechanism of cancer formation at the post- genomic level.

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. (C) 2011 Elsevier B.V. All rights reserved.

Nitric Oxide-Induced Vasorelaxation in Response to PnTx2-6 Toxin from Phoneutria nigriventer Spider in Rat Cavernosal Tissue

Introduction. Priapism is one of several symptoms observed in accidental bites by the spider Phoneutria nigriventer. The venom of this spider is comprised of many toxins, and the majority has been shown to affect excitable ion channels, mainly sodium (Na+) channels. It has been demonstrated that PnTx2-6, a peptide extracted from the venom of P. nigriventer, causes erection in anesthetized rats and mice. Aim. We investigated the mechanism by which PnTx2-6 evokes relaxation in rat corpus cavernosum. Main Outcome Measures. PnTx2-6 toxin potentiates nitric oxide (NO)-dependent cavernosal relaxation. Methods. Rat cavernosal strips were incubated with bretylium (3 x 10-5 M) and contracted with phenylephrine (PE; 10-5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 4 minutes of incubation with PnTx2-6 (10-8 M). The effect of PnTx2-6 on relaxation induced by EFS was also tested in the presence of atropine (10-6 M), a muscarinic receptor antagonist, N-type Ca2+ channel blockers (omega-conotoxin GVIA, 10-6 M) and sildenafil (3 x 10-8 M). Technetium99m radiolabeled PnTx2-6 subcutaneous injection was administrated in the penis. Results. Whereas relaxation induced by SNP was not affected by PnTx2-6, EFS-induced relaxation was significantly potentiated by this toxin as well as PnTx2-6 plus SNP. This potentiating effect was further increased by sildenafil, not altered by atropine, however was completely blocked by the N-type Ca2+ channels. High concentrated levels of radiolabeled PnTx2-6 was specifically found in the cavernosum tissue, suggesting PnTx2-6 is an important toxin responsible for P. nigriventer spider accident-induced priapism. Conclusion. We show that PnTx2-6 slows Na+ channels inactivation in nitrergic neurons, allowing Ca2+ influx to facilitate NO/cGMP signalling, which promotes increased NO production. In addition, this relaxation effect is independent of phosphodiesterase enzyme type 5 inhibition. Our data displays PnTx2-6 as possible pharmacological tool to study alternative treatments for erectile dysfunction. Nunes KP, Cordeiro MN, Richardson M, Borges MN, Diniz SOF, Cardoso VN, Tostes R, De Lima ME, Webb RC, and Leite R. Nitric oxide-induced vasorelaxation in response to PnTx2-6 toxin from Phoneutria nigriventer spider in rat cavernosal tissue. J Sex Med 2010;7:3879-3888.

Anti-inflammatory activity and possible mechanism of extract from Mikania laevigata in carrageenan-induced peritonitis

Objectives The aim was to test the potential use of an extract of Mikania laevigata (popularly known in Brazil as guaco), made from leaves harvested in different months of the year, oil neutrophil migration after all inflammatory Stimulus and investigate the underlying molecular mechanisms. Methods We examined the effect of guaco on vascular permeability and leucocyte function in carrageenan-induced peritonitis in mice. Key findings Our results demonstrated that guaco extract administered subcutaneously (3 mg/kg) decreased the vascular permeability and also leucocyte rolling and adhesion to the inflamed tissues by a mechanism dependent on nitric oxide. Specifically, inhibitors of nitric oxide synthase remarkably abrogated the guaco extract-mediated suppression of neutrophil migration to the inflammatory site. In addition, guaco extract-mediated suppression of neutrophil migration appeared to be dependent on the production of the cytokines interleukin-1 beta and tumour necrosis factor-alpha. One of the major constituents of the guaco extract, coumarin, was able to inhibit the neutrophil migration towards the inflammatory focus. Conclusions In conclusion the anti-inflammatory effect induced by guaco extract may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.

Coupling Between Respiratory and Sympathetic Activities as a Novel Mechanism Underpinning Neurogenic Hypertension

Enhanced sympathetic outflow to the heart and resistance vessels greatly contributes to the onset and maintenance of neurogenic hypertension. There is a consensus that the development of hypertension (clinical and experimental) is associated with an impairment of sympathetic reflex control by arterial baroreceptors. More recently, chronic peripheral chemoreflex activation, as observed in obstructive sleep apnea, has been proposed as another important risk factor for hypertension. In this review, we present and discuss recent experimental evidence showing that changes in the respiratory pattern, elicited by chronic intermittent hypoxia, play a key role in increasing sympathetic activity and arterial pressure in rats. This concept parallels results observed in other models of neurogenic hypertension, such as spontaneously hypertensive rats and rats with angiotensin II-salt-induced hypertension, pointing out alterations in the central coupling of respiratory and sympathetic activities as a novel mechanism underlying the development of neurogenic hypertension.

Further Study of the Psychometric Qualities of a Brief Screening Tool for Social Phobia (MINI-SPIN) Applied to Clinical and Nonclinical Samples

PURPOSE. The purpose of this study was to further assess the psychometric qualities of the Mini-Social Phobia Inventory (MS) to screen for social anxiety disorder (SAD). DESIGN AND METHODS. The MS and other self- and clinician-rated scales for anxiety and social anxiety were applied in 2,314 university students and in samples of SAD patients (n = 88) and nonpatients (n = 90). FINDINGS. The MS revealed adequate discriminative validity, internal consistency (alpha = 0.49-0.73), convergent validity with the Social Phobia Inventory, Brief Social Phobia Scale, and Self-Statements During Public Speaking Scale and convergent and divergent validity with the Beck Anxiety Inventory. PRACTICE IMPLICATIONS. The MS has shown to be a fast and efficient screening instrument for SAD in different cultures and contexts.

Disruption of sarcolemmal dystrophin and beta-dystroglycan may be a potential mechanism for myocardial dysfunction in severe sepsis

Evidence from our laboratory has shown alterations in myocardial structure in severe sepsis/septic shock. The morphological alterations are heralded by sarcolemmal damage, characterized by increased plasma membrane permeability caused by oxidative damage to lipids and proteins. The critical importance of the dystrophin-glycoprotein complex (DGC) in maintaining sarcolemmal stability led us to hypothesize that loss of dystrophin and associated glycoproteins could be involved in early increased sarcolemmal permeability in experimentally induced septic cardiomyopathy. Male C57Bl/6 mice were subjected to sham operation and moderate (MSI) or severe (SSI) septic injury induced by cecal ligation and puncture (CLP). Using western blot and immunofluorescence, a downregulation of dystrophin and beta-dystroglycan expression in both severe and moderate injury could be observed in septic hearts. The immunofluorescent and protein amount expressions of laminin-alpha 2 were similar in SSI and sham-operated hearts. Consonantly, the evaluation of plasma membrane permeability by intracellular albumin staining provided evidence of severe injury of the sarcolemma in SSI hearts, whereas antioxidant treatment significantly attenuated the loss of sarcolemmal dystrophin expression and the increased membrane permeability. This study offers novel and mechanistic data to clarify subcellular events in the pathogenesis of cardiac dysfunction in severe sepsis. The main finding was that severe sepsis leads to a marked reduction in membrane localization of dystrophin and beta-dystroglycan in septic cardiomyocytes, a process that may constitute a structural basis of sepsis-induced cardiac depression. In addition, increased sarcolemmal permeability suggests functional impairment of the DGC complex in cardiac myofibers. In vivo observation that antioxidant treatment significantly abrogated the loss of dystrophin expression and plasma membrane increased permeability supports the hypothesis that oxidative damage may mediate the loss of dystrophin and beta-dystroglycan in septic mice. These abnormal parameters emerge as therapeutic targets and their modulation may provide beneficial effects on future cardiovascular outcomes and mortality in sepsis. Laboratory Investigation (2010) 90, 531-542; doi: 10.1038/labinvest.2010.3; published online 8 February 2010

The ACB technique: a biomagentic tool for monitoring gastrointestinal contraction directly from smooth muscle in dogs

The aim of this paper was to verify whether AC biosusceptometry (ACB) is suitable for monitoring gastrointestinal (GI) contraction directly from smooth muscle in dogs, comparing with electrical recordings simultaneously. All experiments were performed in dogs with magnetic markers implanted under the serosa of the right colon and distal stomach, and their movements were recorded by ACB. Monopolar electrodes were implanted close to the magnetic markers and their electric potentials were recorded by electromyography (EMG). The effects of neostigmine, hyoscine butylbromide and meal on gastric and colonic parameters were studied. The ACB signal from the distal stomach was very similar to EMG; in the colonic recordings, however, within the same low-frequency band, ACB and EMG signals were characterized by simultaneity or a widely changeable frequency profile with time. ACB recordings were capable of demonstrating the changes in gastric and colonic motility determined by pharmacological interventions as well as by feeding. Our results reinforce the importance of evaluating the mechanical and electrical components of motility and show a temporal association between them. ACB and EMG arecomplementary for studying motility, with special emphasis on the colon. ACB offers an accurate method for monitoring in vivo GI motility.

MTA-induced neutrophil recruitment: a mechanism dependent on IL-1 beta, MIP-2, and LTB(4)

Objective. The objective of this study was to investigate the mediators and the resident peritoneal cells involved in the neutrophil migration (NM) induced by mineral trioxide aggregate (MTA) in mice. Study design. MTA (25 mg/cavity) was injected into normal and pretreated peritoneal cavities (PC) with indomethacin (IND), dexamethasone (DEX), BWA4C, U75302, antimacrophage inflammatory protein-2 (MIP-2), and anti-interleukin-1 beta (IL-1 beta) antibodies and the NM was determined. The role of macrophage (MO) and mast cells (MAST) was determined by administration of thioglycollate 3% or 48/80 compound, respectively. The concentration of IL-1 beta and MIP-2 exudates was measured by ELISA. Results. MTA induced dose-and time-dependent NM into mice PC, with the participation of MO and MAST. NM was inhibited by DEX, BWA4C, and U75302, as well as anti-MIP-2 and anti-IL-1 beta antibodies. In the exudates, IL-1 beta and MIP-2 were detected. Conclusions. This study suggests that MTA induces NM via a mechanism dependent on MAST and MO mediated by IL-1 beta, MIP-2, and LTB(4).

Galectin-3 expression: a useful tool in the differential diagnosis of posterior fossa tumors in children

Galectin-3 (Gal-3) is a glycan-binding protein highly expressed in several tumors, including brain neoplasms. This protein has been demonstrated to be correlated with adverse prognosis in some tumor types. However, the role of Gal-3 in pediatric posterior fossa tumors (PPFTs) has not yet been fully addressed. The goals of this study were to evaluate Gal-3 expression in a series of PPFTs and verify whether this expression is related to patient outcome. Gal-3 expression was analyzed by immunohistochemistry in 42 cases of surgically resected primary PPFTs. Surgeries were performed in our institution from January 2003 to December 2006. Tumor samples consisted of 21 pilocytic astrocytomas (PAs), 13 medulloblastomas, 4 ependymomas, 2 diffuse cerebellar astrocytomas, and 2 atypical teratoid/rhabdoid tumors (AT/RTs). All PAs and ependymomas strongly showed Gal-3 expression, whereas no immunostaining was observed in medulloblastomas and diffuse astrocytomas. In AT/RTs, Gal-3 expression was conspicuous but heterogeneous, being mainly observed in rhabdoid cells. Concerning the Gal-3 expressing tumors, no relationship was observed between the degree of expression and patient survival. Gal-3 was strongly expressed in reactive astrocytes, normal endothelial cells, and macrophages in the adjacent non-neoplastic brain parenchyma. Interestingly, the endothelial cells in the tumor bulk of PAs lacked Gal-3 expression. Gal-3 is differentially expressed in PPFTs, but its expression shows no correlation with patient outcome. However, the evaluation of Gal-3 is helpful in establishing a differential diagnosis among PPFTs, especially between PAs and diffuse astrocytomas, and in some circumstances between medulloblastomas and AT/RTs.