Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide.

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.

Investigation of antimalarial activity, cytotoxicity and action mechanism of piperazine derivatives of betulinic acid.

OBJECTIVES: To semisynthesise piperazine derivatives of betulinic acid to evaluate antimalarial activity, cytotoxicity and action mechanism. METHODS: The new derivatives were evaluated against the CQ-sensitive Plasmodium falciparum 3D7 strain by flow cytometry (FC) using YOYO-1 as stain. Cytotoxicity of 4a and 4b was performed with HEK293T cells for 24 and 48 h by MTT assay. The capability of compound 4a to modulate Ca(2+) in the trophozoite stage was investigated. The trophozoites were stained with Fluo4-AM and analysed by spectrofluorimetry. Effect on mitochondrial membrane potential (ΔΨm) was tested for 4a by FC with DiOC6 (3) as stain. For ß-haematin assay, 4a was incubated for 24 h with reagents such as haemin, and the fluorescence was measured by FlexStation at an absorbance of 405 nm. RESULTS: Antimalarial activity of 4a and 4b was IC50 = 1 and 4 µm, respectively. Compound 4a displayed cytotoxicity with IC50 = 69 and 29 µm for 24 and 48 h, respectively, and 4b was not cytotoxic at the tested concentrations. Addition of 4a leads to an increase in cytosolic Ca(2+) . We have measured ΔΨm after treating parasites with the compound. Data on Figure 4a show that mitochondria were not affected. The action mechanism for 4a, inhibition of ß-haematin formation (17%), was lower than CQ treatment (83%; IC50 = 3 mm). CONCLUSION: Compound 4a showed excellent antimalarial activity, and its action mechanism is involved in Ca(2+) pathway(s).

Ras, Rac1, and phosphatidylinositol-3-kinase (PI3K) signaling in nitric oxide induced endothelial cell migration.

The small GTP-binding proteins Ras and Rac1 are molecular switches exchanging GDP for GTP and converting external signals in response to a variety of stimuli. Ras and Rac1 play an important role in cell proliferation, cell differentiation, and cell migration. Rac1 is directly involved in the reorganization and changes in the cytoskeleton during cell motility. Nitric oxide (NO) stimulates the Ras - ERK1/2 MAP kinases signaling pathway and is involved in the interaction between Ras and the phosphatidyl-inositol-3 Kinase (PI3K) signaling pathway and cell migration. This study utilizes bradykinin (BK), which promotes endogenous production of NO, in an investigation of the role of NO in the activation of Rac1 in rabbit aortic endothelial cells (RAEC). NO-derived from BK stimulation of RAEC and incubation of the cells with the s-nitrosothiol S-nitrosoglutathione (GSNO) activated Rac1. NO-derived from BK stimulation promoted RAEC migration over a period of 12 h. The use of RAEC permanently transfected with the dominant negative mutant of Ras (Ras(N17)) or with the non-nitrosatable mutant of Ras (Ras(C118S)); and the use of specific inhibitors of: Ras, PI3K, and Rac1 resulted in inhibition of NO-mediated Rac1 activation. BK-stimulated s-nitrosylation of Ras in RAEC mediates Rac1 activation and cell migration. Inhibition of NO-mediated Rac1 activation resulted in inhibition of endothelial cell migration. In conclusion, the NO indirect activation of Rac1 involves the direct participation of Ras and PI3K in the migration of endothelial cells stimulated with BK.

Endothelium-derived nitric oxide (NO) activates the NO-epidermal growth factor receptor-mediated signaling pathway in bradykinin-stimulated angiogenesis.

Nitric oxide (NO) is involved in angiogenesis and stimulates the EGF-R signaling pathway. Stimulation of different endothelial cell lines with bradykinin (BK) activates the endothelial NO synthase (eNOS) and promotes EGF-R tyrosine phosphorylation. Increase in NO production correlated with enhanced phosphorylation of tyrosine residues and S-nitrosylation of the EGF-R. NO-mediated stimulatory effects on tyrosine phosphorylation of the EGF-R, where cGMP independent. Inhibition of soluble guanylyl cyclase followed by BK stimulation of human umbilical vein endothelial cells (HUVECs) did not change tyrosine phosphorylation levels of EGF-R. BK-stimulation of HUVEC promoted S-nitrosylation of the phosphatase SHP-1 and of p21Ras. Phosphorylation and activation of the ERK1/2 MAP kinases mediated by BK was dependent on the activation of the B2 receptor, of the EGF-R, and of p21 Ras. Inhibition of BK-stimulated S-nitrosylation prevented the activation of the ERK1/2 MAP kinases. Furthermore, activated ERK1/2 MAP kinases inhibited internalization of EGF-R by phosphorylating specific Thr residues of its cytoplasmic domain. BK-induced proliferation of endothelial cells was partially inhibited by the NOS inhibitor (L-NAME) and by the MEK inhibitor (PD98059). BK stimulated the expression of vascular endothelial growth factor (VEGF). VEGF expression was dependent on the activation of the EGF-R, the B2 receptor, p21Ras, and on NO generation. A Matrigel®-based in vitro assay for angiogenesis showed that BK induced the formation of capillary-like structures in HUVEC, but not in those cells expressing a mutant of the EGF-R lacking tyrosine kinase activity. Additionally, pre-treatment of BK-stimulated HUVEC with L-NAME, PD98059, and with SU5416, a specific inhibitor of VEGFR resulted in inhibition of in vitro angiogenesis. Our findings indicate that BK-mediated angiogenesis in endothelial cells involves the induction of the expression of VEGF associated with the activation of the NO/EGF-R/p21Ras/ERK1/2 MAP kinases signaling pathway.

Two series of new semisynthetic triterpene derivatives: differences in anti-malarial activity, cytotoxicity and mechanism of action.

BACKGROUND: The discovery and development of anti-malarial compounds of plant origin and semisynthetic derivatives thereof, such as quinine (QN) and chloroquine (CQ), has highlighted the importance of these compounds in the treatment of malaria. Ursolic acid analogues bearing an acetyl group at C-3 have demonstrated significant anti-malarial activity. With this in mind, two new series of betulinic acid (BA) and ursolic acid (UA) derivatives with ester groups at C-3 were synthesized in an attempt to improve anti-malarial activity, reduce cytotoxicity, and search for new targets. In vitro activity against CQ-sensitive Plasmodium falciparum 3D7 and an evaluation of cytotoxicity in a mammalian cell line (HEK293T) are reported. Furthermore, two possible mechanisms of action of anti-malarial compounds have been evaluated: effects on mitochondrial membrane potential (ΔΨm) and inhibition of ß-haematin formation. RESULTS: Among the 18 derivatives synthesized, those having shorter side chains were most effective against CQ-sensitive P. falciparum 3D7, and were non-cytotoxic. These derivatives were three to five times more active than BA and UA. A DiOC(6)(3) ΔΨm assay showed that mitochondria are not involved in their mechanism of action. Inhibition of ß-haematin formation by the active derivatives was weaker than with CQ. Compounds of the BA series were generally more active against P. falciparum 3D7 than those of the UA series. CONCLUSIONS: Three new anti-malarial prototypes were obtained from natural sources through an easy and relatively inexpensive synthesis. They represent an alternative for new lead compounds for anti-malarial chemotherapy.

Biological evaluation of hydroxynaphthoquinones as anti-malarials.

BACKGROUND: The hydroxynaphthoquinones have been extensively investigated over the past 50 years for their anti-malarial activity. One member of this class, atovaquone, is combined with proguanil in Malarone®, an important drug for the treatment and prevention of malaria. METHODS: Anti-malarial activity was assessed in vitro for a series of 3-alkyl-2-hydroxy-1,4-naphthoquinones (N1-N5) evaluating the parasitaemia after 48 hours of incubation. Potential cytotoxicity in HEK293T cells was assessed using the MTT assay. Changes in mitochondrial membrane potential of Plasmodium were measured using the fluorescent dye Mitrotracker Red CMXROS. RESULTS: Four compounds demonstrated IC50s in the mid-micromolar range, and the most active compound, N3, had an IC50 of 443 nM. N3 disrupted mitochondrial membrane potential, and after 1 hour presented an IC50ΔΨmit of 16 µM. In an in vitro cytotoxicity assay using HEK 293T cells N3 demonstrated no cytotoxicity at concentrations up to 16 µM. CONCLUSIONS: N3 was a potent inhibitor of mitochondrial electron transport, had nanomolar activity against cultured Plasmodium falciparum and showed minimal cytotoxicity. N3 may serve as a starting point for the design of new hydroxynaphthoquinone anti-malarials.

The PfNF-YB transcription factor is a downstream target of melatonin and cAMP signalling in the human malaria parasite Plasmodium falciparum.

Plasmodium falciparum causes the most severe form of malaria and is responsible for the majority of deaths worldwide. The mechanism of cell cycle control within intra-erythrocytic stages has been examined as a potential means of a promising way to identifying how to stop parasite development in red blood cells. Our group determined that melatonin increases parasitemia in P. falciparum and P. chabaudi through a complex signalling cascade. In vertebrates, melatonin controls the expression of transcription factors, leading us to postulate rather that the indoleamine would affect PfNF-YB expression in human malaria parasites. We show here that PfNF-YB transcription factor is highly expressed and colocalized in the nucleus in mature parasites during intra-erythrocytic stages, thus suggesting an important role in cell division. Moreover, we demonstrate for the first time that melatonin and cAMP modulate the PfNF-YB transcription factor expression in P. falciparum at erythrocytic stages. In addition, PfNF-YB is found to be more ubiquitinated in the presence of melatonin. Finally, the proteasome inhibitor bortezomib is able to modulate PfNF-YB expression as well. Taken together, our dada reinforce the role played by melatonin in the cell cycle control of P. falciparum and point this indolamine as a target to develop new antimalarial drugs.

Synthesis and antiplasmodial activity of betulinic acid and ursolic acid analogues.

More than 40% of the World population is at risk of contracting malaria, which affects primarily poor populations in tropical and subtropical areas. Antimalarial pharmacotherapy has utilised plant-derived products such as quinine and artemisinin as well as their derivatives. However, worldwide use of these antimalarials has caused the spread of resistant parasites, resulting in increased malaria morbidity and mortality. Considering that the literature has demonstrated the antimalarial potential of triterpenes, specially betulinic acid (1) and ursolic acid (2), this study investigated the antimalarial activity against P. falciparum chloroquine-sensitive 3D7 strain of some new derivatives of 1 and 2 with modifications at C-3 and C-28. The antiplasmodial study employed flow cytometry and spectrofluorimetric analyses using YOYO-1, dihydroethidium and Fluo4/AM for staining. Among the six analogues obtained, compounds 1c and 2c showed excellent activity (IC50 = 220 and 175 nM, respectively) while 1a and b demonstrated good activity (IC50 = 4 and 5 µM, respectively). After cytotoxicity evaluation against HEK293T cells, 1a was not toxic, while 1c and 2c showed IC50 of 4 µM and a selectivity index (SI) value of 18 and 23, respectively. Moreover, compound 2c, which presents the best antiplasmodial activity, is involved in the calcium-regulated pathway(s).

The low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine, promotes cell cycle progression in rabbit aortic endothelial cells.

S-Nitrosylation reactions are considered to be a major mechanism by which NO-related bioactivities are regulated in vivo. In the present study, we show the effects of the low molecular weight S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), on cell cycle progression of rabbit aortic endothelial cells (RAEC). SNAP at low concentrations (0.1mM) stimulated the p21Ras-ERK1/2 MAP kinase signaling pathway. Activation of this signaling pathway was strongly inhibited in cells stably transfected with S-nitrosylation insensitive p21Ras (p21(Ras (C118S))). Furthermore, the SNAP-induced effects on cell cycle progression were eliminated in RAEC expressing N17Ras, a negative dominant mutant of p21Ras. Upon stimulation with SNAP, ERK1/2 MAP kinases become phosphorylated and translocate to the nucleus promoting the phosphorylation of the transcription factor Elk1. Synthesis of Cyclin D1 and stimulation of the cyclin-dependent kinases cdk4 and cdk6 resulted in the phosphorylation of the nuclear protein Rb and its dissociation from the E2F family of transcription factors. Cells then pass the restriction point in the late G1 phase. Cyclins E and A were expressed as the cell cycle progressed through the S phase upon stimulation with SNAP. Further transition in the cell cycle from the G2 to M phase was evidenced by the G2/M peak found in a histogram of the cell-phase distribution in SNAP-treated RAEC. These observations suggest that low molecular weight S-nitrosothiols may promote cell cycle progression possibly through the transnitrosation of p21Ras, and activation of the Ras-ERK1/2 MAP kinases signaling pathway.

Src kinase activation by nitric oxide promotes resistance to anoikis in tumour cell lines.

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.

Organização da informação para o domínio prevenção e controle de câncer: relato de experiência / Organization of information for the cancer prevention and control domain: experience report / Organización de la información para el dominio de prevención y control del cáncer: reporte de experiencia

O trabalho apresenta o relato de organização da Biblioteca Virtual em Saúde (BVS) Prevenção e Controle de Câncer, mediante estrutura de categorias de assunto adequada à sua especificidade, apresenta os princípios envolvidos na construção e os métodos explorados para alcançar o desenvolvimento do modelo conceitual para o domínio. Aponta a utilização de sua estrutura no atendimento ao usuário para concluir sua pesquisa e construir sua estratégia de busca, confirmando, assim que a organização do modelo de conceitos descreve e fornece informações que servem de referência para a construção de estratégias de buscae recuperação da informação.

The paper presents the organization report of the Virtual Health Library (VHL) Cancer Prevention and Control, through a structure of subject categories appropriate toits specificity, presents the principles involved in the construction and the methods explored to achieve the development of the conceptual model for the domain. It points out the use of its structure in the service to the user to complete its research and build its search strategy, thus confirming that the organization of the concept model describes and provides information that serve as a reference for the construction of search strategies and information retrieval.

El artículo presenta el informe de organización de la Biblioteca Virtual en Salud (BVS) Prevención y Control del Cáncer, a través de una estructura de categorías temáticas adecuada a su especificidad, presenta los principios involucrados en la construcción y los métodos explorados para lograr el desarrollo del modelo conceptual para el dominio. Señala el uso de su estructura en el servicio al usuario para completar su investigación y construir su estrategia de búsqueda, confirmando así que la organización del modelo conceptual describe y brinda información que sirve de referencia para la construcción de estrategias de búsqueda e recuperación de información.

Plasmodium falciparum GPCR-like receptor SR25 mediates extracellular K+ sensing coupled to Ca2+ signaling and stress survival.

The malaria parasite Plasmodium falciparum is exposed, during its development, to major changes of ionic composition in its surrounding medium. We demonstrate that the P. falciparum serpentine-like receptor PfSR25 is a monovalent cation sensor capable of modulating Ca2+ signaling in the parasites. Changing from high (140 mM) to low (5.4 mM) KCl concentration triggers [Ca2+]cyt increase in isolated parasites and this Ca2+ rise is blocked either by phospholipase C (PLC) inhibition or by depleting the parasite's internal Ca2+ pools. This response persists even in the absence of free extracellular Ca2+ and cannot be elicited by addition of Na+, Mg2+ or Ca2+. However, when the PfSR25 gene was deleted, no effect on [Ca2+]cyt was observed in response to changing KCl concentration in the knocked out (PfSR25 -) parasite. Finally, we also demonstrate that: i) PfSR25 plays a role in parasite volume regulation, as hyperosmotic stress induces a significant decrease in parasite volume in wild type (wt), but not in PfSR25 - parasites; ii) parasites lacking PfSR25 show decreased parasitemia and metacaspase gene expression on exposure to the nitric oxide donor sodium nitroprusside (SNP) and iii), compared to PfSR25 - parasites, wt parasites showed a better survival in albumax-deprived condition.

Genome-wide analysis of the human malaria parasite Plasmodium falciparum transcription factor PfNF-YB shows interaction with a CCAAT motif.

Little is known about transcription factor regulation during the Plasmodium falciparum intraerythrocytic cycle. In order to elucidate the role of the P. falciparum (Pf)NF-YB transcription factor we searched for target genes in the entire genome. PfNF-YB mRNA is highly expressed in late trophozoite and schizont stages relative to the ring stage. In order to determine the candidate genes bound by PfNF-YB a ChIP-on-chip assay was carried out and 297 genes were identified. Ninety nine percent of PfNF-YB binding was to putative promoter regions of protein coding genes of which only 16% comprise proteins of known function. Interestingly, our data reveal that PfNF-YB binding is not exclusively to a canonical CCAAT box motif. PfNF-YB binds to genes coding for proteins implicated in a range of different biological functions, such as replication protein A large subunit (DNA replication), hypoxanthine phosphoribosyltransferase (nucleic acid metabolism) and multidrug resistance protein 2 (intracellular transport).

Adduction laryngeal dystonia: proposal and evaluation of nasofibroscopy.

UNLABELLED: Dystonias are organic central motor processing disorders characterized by involuntary muscular contractions or incontrollable spasms induced by task-specific movements. Adduction laryngeal dystonias present with important speech impairments, with inappropriate spasms and abrupt voice breaks. The diagnosis is based on clinical features, evaluation by a speech therapist and transnasal fiber optic laryngoscopy. AIM: Our objective is to propose and evaluate a task-oriented transnasal fiber optic laryngoscopy protocol, which shows the spasms, and propose maneuvers that reduce or make them disappear, in order to facilitate the diagnosis. METHODS: transversal study. Analysis of the transnasal fiber optic laryngoscopy records of 15 patients with adductor laryngeal dystonia using the proposed protocol. RESULTS: most of the speech and non-vocal tasks allowed us to identify the spasms and reduce or make them disappear. We propose the exclusion of two of the maneuvers that dont bring new data to the evaluation. CONCLUSION: the protocol was useful for the evaluation of the patients, showing changes in muscle behavior in the structure under investigation.

Signaling transcript profile of the asexual intraerythrocytic development cycle of Plasmodium falciparum induced by melatonin and cAMP.

According to the World Health Organization (WHO), Plasmodium falciparum is the deadliest parasite among all species. This parasite possesses the ability to sense molecules, including melatonin (MEL) and cAMP, and modulate its cell cycle accordingly. MEL synchronizes the development of this malaria parasite by activating several cascades, including the generation of the second messenger cAMP. Therefore, we performed RNA sequencing (RNA-Seq) analysis in P. falciparum erythrocytic stages (ring, trophozoite and schizont) treated with MEL and cAMP. To investigate the expression profile of P. falciparum genes regulated by MEL and cAMP, we performed RNA-Seq analysis in three P. falciparum strains (control, 3D7; protein kinase 7 knockout, PfPK7-; and PfPK7 complement, PfPK7C). In the 3D7 strain, 38 genes were differentially expressed upon MEL treatment; however, none of the genes in the trophozoite (T) stage PfPK7- knockout parasites were differentially expressed upon MEL treatment for 5 hours compared to untreated controls, suggesting that PfPK7 may be involved in the signaling leading to differential gene expression. Moreover, we found that MEL modified the mRNA expression of genes encoding membrane proteins, zinc ion-binding proteins and nucleic acid-binding proteins, which might influence numerous functions in the parasite. The RNA-Seq data following treatment with cAMP show that this molecule modulates different genes throughout the intraerythrocytic cycle, namely, 75, 101 and 141 genes, respectively, in the ring (R), T and schizont (S) stages. Our results highlight P. falciparum's perception of the external milieu through the signaling molecules MEL and cAMP, which are able to drive to changes in gene expression in the parasite.

Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses.

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration.

Tyrosine phosphorylation in nitric oxide-mediated signaling events.

In this chapter, we provide an overview of nitric oxide (NO)-tyrosine phosphorylation signal transduction pathways, integrating them with the cyclic guanosine monophosphate (cGMP) and S-nitrosylation-mediated pathways that are triggered by NO. The second half of this chapter includes a description of the methods that our laboratory has used extensively to characterize the mechanisms involved in signaling events mediated by this pathway. These include assays for detecting protein tyrosine phosphorylation, tyrosine phosphorylation of the epidermal growth factor (EGF) receptor, phosphorylation of the ERK1/2 mitogen-activated protein (MAP) kinases, transfection of cells with modified forms of p21Ras, and an assay of p21Ras.

c-Myc protein is stabilized by fibroblast growth factor 2 and destabilized by ACTH to control cell cycle in mouse Y1 adrenocortical cells.

ACTH is the hormone known to control adrenal cortex function and maintenance in the intact animal but, in culture, it inhibits proliferation of adrenocortical cells from different mammalian species, a puzzle that has remained unsolved for nearly 30 years. In this paper we compare ACTH and fibroblast growth factor 2 (FGF2) antagonistic effects on the cell cycle in the Y1 cell line, a functional lineage of mouse adreno-cortical tumor cells. This cell line displays chronic high levels of c-Ki-Ras-GTP, high active constitutive levels of phosphatidylinositol 3-OH kinase/Protein Kinase B (PI3K/AKT) and low constitutive basal expression of c-Myc, which accounts for a minor deregulation of the cell cycle. In G0/G1-arrested Y1 cells, over-expression of the dominant negative mutant HaRasN17 drastically reduces c-Ki-Ras-GTP levels, eliminating basal c-Myc expression and basal S phase entry. PI3K/Akt seems to be the downstream pathway from c-Ki-ras for deregulation of c-Myc basal expression, since wortmannin abolishes c-Myc expression in serum-starved, G0/G1-arrested Y1 cells. FGF2 is a strong mitogen for Y1 cells, promoting -- in a manner dependent on the MEK/ERK pathway -- c-myc transcription induction, c-Myc protein stabilization and S phase entry in G0/G1-arrested Y1 cells. On the other hand, ACTH causes c-Myc protein destabilization, partially blocking S phase entry induced by FGF2, by a process dependent on the cAMP/protein kinase A (PKA) pathway. The whole pathway activated by ACTH to destabilize c-Myc protein in Y1 cells might comprise the following steps: ACTH receptor -->cAMP/PKA --> Akt deactivation -->GSK3 activity liberation --> c-Myc Thr58 phosphorylation. We demonstrate that c-Myc regulation is a central key in the cell cycle control by these factors, since enforced expression of c-Myc through the MycER chimera abrogates the ACTH inhibitory effect over FGF2-induced S phase entry.

S-nitrosoglutathione and endothelial nitric oxide synthase-derived nitric oxide regulate compartmentalized ras S-nitrosylation and stimulate cell proliferation.

AIMS: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. RESULTS: Ras signaling pathway stimulation by 50 and/or 100 µM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 µM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras. INNOVATION: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. CONCLUSION: S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.

Pain in popular singers.

PURPOSE: To identify and characterize the presence of body pains in popular singers, to observe the differences in the reported pains according to gender, and to relate with data regarding vocal behavior and usage in this population. METHODS: A self-explanatory questionnaire was applied to 100 popular singers (50 men and 50 women), in order to collect information about personal identification, voice use and presence of pains. Pains were divided into two groups: proximal pains (TMA, tongue, throat, nape, shoulders, neck, and pain during speech), and distal pains (arms, back/column, chest, hands, ear, and headache). RESULTS: The mean value of pain presence referred by popular singers was 2.9. There was no difference in reported pain according to gender. Predominant pains were on the throat (66%), during speech (41%) and on the neck (35%), all classified as proximal to the larynx. The least predominant pains were in arms, hands and chest (4%), all classified as distal pains. CONCLUSION: Popular singers reported presence of body pains mainly proximal to the larynx. There is no difference in reported pain according to gender. The presence of body pain is related to the presence of voice disorders, the need to stop singing, the absence of vocal training, and search for professional advice (otolaryngologists and speech-language pathologist) due to vocal problems. These data justify the investigation and attention to body pain symptoms by the professionals responsible for the treatment of this population.