Reactive oxygen intermediates mediate angiotensin II-induced c-Jun.c-Fos heterodimer DNA binding activity and proliferative hypertrophic responses in myogenic cells

Angiotensin II (Ang-II) receptor engagement activates many immediate early response genes in both vascular smooth muscle cells and cardiomyocytes whether a hyperplastic or hypertrophic response is taking place. Although the signaling pathways stimulated by Ang-II in different cell lines have been widely characterized, the correlation between the generation of different second messengers and specific physiological responses remains relatively unexplored. In this study, we report how in both C2C12 quiescent myoblasts and differentiated myotubes Ang-II significantly stimulates AP1-driven transcription and c-Jun.c-Fos heterodimer DNA binding activity. Using a set of different protein kinase inhibitors, we could demonstrate that Ang-II-induced increase in AP1 binding is not mediated by the cAMP-dependent pathway and that both protein kinase C and tyrosine kinases are involved. The observation that in quiescent myoblasts Ang-II increase of AP1 binding and induction of DNA synthesis and, in differentiated myotubes, Ang-II stimulation of protein synthesis are abolished by the cysteine-derivative and glutathione precursor N-acetyl-L-cysteine strongly suggests a role for reactive oxygen intermediates in the intracellular transduction of Ang-II signals for immediate early gene induction, cell proliferation, and hypertrophic responses

Universal Behavior of Lyapunov Exponents in Unstable Systems

We calculate the Lyapunov exponents in a classical molecular dynamics framework. The system is composed of few hundreds particles interacting either through Yukawa (Nuclear) or Slater-Kirkwood (Atomic) forces. The forces are chosen to give an Equation of State that resembles the nuclear and the atomic $^4He$ Equation Of State respectively near the critical point for liquid-gas phase transition. We find the largest fluctuations for an initial "critical temperature". The largest Lyapunov exponents $\lambda$ are always positive and can be very well fitted near this "critical temperature" with a functional form $\lambda\propto |T-T_c|^{-\omega}$, where the exponent $\omega=0.15$ is independent of the system and mass number. At smaller temperatures we find that $\lambda\propto T~ ^{0.4498}$, a universal behavior characteristic of an order to chaos transition.Comment: 11 pages, RevTeX, 3 figures not included available upon reques

Hsp60 Is Actively Secreted by Human Tumor Cells

Background: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likel

Monarda fistulosa hydrolate as antimicrobial agent in artificial media for the in vitro rearing of the tachinid parasitoid Exorista larvarum

Exorista larvarum (L.) (Diptera: Tachinidae), a larval parasitoid of Lepidoptera, can be reared from egg to fecund adult on artificial media composed of crude components. The standard in vitro culture is performed in 24-well plastic rearing plates. Exorista larvarum eggs, removed from superparasitized larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae), are individually placed in the wells, each containing a cotton swab soaked in liquid medium. The plates are then sealed until parasitoid puparium formation. To avoid contamination by microorganisms, the artificial medium is routinely supplemented with 0.01% solution of gentamicin. Experiments were carried out to assess whether this broad-spectrum antibiotic may be replaced with hydrolate of Monarda fistulosa L. (Lamiaceae), which was selected due to its high in vitro activity against pathogenic microorganisms for humans and plants. The hydrolate was either supplemented to the artificial medium (0.5% wt/wt) (first experiment) or placed in an empty well (200 \ub5l) of the rearing plate, to be supplied as saturated air due to evaporation (second experiment). In both experiments, a standard medium with gentamicin and an antimicrobial-free medium were maintained as positive and negative controls, respectively. In the first experiment, in the hydrolate-supplemented medium fewer E. larvarum completed egg-to-adult development than in the standard medium, but significantly more parasitoids developed from egg to adult compared to the antimicrobial-free medium. No significant difference was found between the numbers of eggs laid by the females obtained from the standard medium vs. those from the hydrolate-supplemented medium. In the second experiment, the hydrolate-saturated air significantly decreased E. larvarum egg hatching, puparium formation, and female fecundity compared to the standard medium. In perspective, M. fistulosa hydrolate supplemented to the artificial media for E. larvarum may be considered as a promising candidate to replace the gentamicin solution, as suggested also by the microbiological analyses of the media, performed at various growth stages of the parasitoid in a separate trial. Conversely, the hydrolate-saturated air treatment was deemed unsuitable

REACTIVE OXYGEN INTERMEDIATES (ROIS) ARE INVOLVED IN THE INTRACELLULAR TRANSDUCTION OF ANGIOTENSIN-II SIGNAL IN C2C12 CELLS

Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10-5 M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation

Effect of Human Natural Killer and gammadelta T Cells on the Growth of Human Atologous Melanoma Xenografts in SCID Mice.

Natural killer (NK) cells were first identified for their ability to kill tumor cells of different origin in vitro. Similarly, gammadelta T lymphocytes display strong cytotoxic activity against various tumor cell lines. However, the ability of both the NK and gammadelta cells to mediate natural immune response against human malignant tumors in vivo is still poorly defined. Severe combined immunodeficient (SCID) mice have been successfully engrafted with human tumors. In this study, the antitumor effect of local as well as of systemic treatments based on NK cells or Vdelta1 or Vdelta2 gamma/delta T lymphocytes against autologous melanoma cells was investigated in vivo. The results show that all three of the populations were effective in preventing growth of autologous human melanomas when both tumor and lymphoid cells were s.c. inoculated at the same site. However, when lymphoid cells were infused i.v., only NK cells and Vdelta1 gamma/delta T lymphocytes could either prevent or inhibit the s.c. growth of autologous melanoma. Accordingly, both NK cells and Vdelta1 gammadelta T lymphocytes could be detected at the s.c. tumor site. In contrast, Vdelta2 gammadelta T lymphocytes were only detectable in the spleen of the SCID mice. Moreover, NK cells maintained their inhibitory effect on tumor growth even after discontinuation of the treatment. Indeed they were present at the tumor site for a longer period. These data support the possibility to exploit NK cells and Vdelta1 gammadelta T lymphocytes in tumor immunotherapy. Moreover, our study emphasizes the usefulness of human tumor/SCID mouse models for preclinical evaluation of immunotherapy protocols against human tumors

Barbed reposition pharyngoplasty (BRP) in obstructive sleep apnea treatment: state of the art

Purpose: In this paper, we perform a systematic review that discusses the state of the art and evolution on the barbed reposition pharyngoplasty (BRP) in the velo-pharyngeal surgery. Clinical evidence and published outcomes of this surgical technique are reported and discussed. Materials and methods: We performed a systematic review of the current literature through the analysis of the last 10 years of literature on barbed palate surgery. Study design, number of patients enrolled, inclusion criteria, pre- and posttreatment outcomes (AHI, ODI), surgical success rate, follow-up time and complication has been collected and reported. Results: 15 studies for a total of 1531 patients, out of which 1061 underwent barbed reposition pharyngoplasty. Five trials were uncontrolled prospective studies (215 patients, 14% of total), nine were retrospective studies (1266 patients, 82,6% of total), and one randomized prospective clinical trial (RCT) (50 patients, 3,32% of total). All analyzed studies reported good outcomes after BRP surgery. Average preoperative values of AHI and ODI reduced in all studies considered with a significative statistical difference between preoperative and postoperative values (p < 0.05 in all cases). The postoperative surgical success rate ranged between 65.4 and 93% of cases. There were no significant intra-operative or post-operative complications in all studies considered in this systematic review. Conclusions: Barbed reposition pharyngoplasty has proven to be an easy to learn, quick, safe and effective new palatopharyngeal procedure, that can be used in a single level surgery or as a part of multilevel procedures

Effects of protein-secretion inhibitors on Hsp60 secretion by tumor cells.

<p><b>A</b>) Hsp60 and Hsp70 detected by Western blotting in: (<b>a</b>) immunoprecipitates from conditioned media from untreated (Unt) and inhibitor-treated H292 tumor cells; and (<b>b</b>) whole-cell lysates from H292 cells. The inhibitors are listed on top of the respective lanes. Histograms to the right represent the levels of the Hsps in immunoprecipitates determined in three separate experiments as mean percentages +/− SD of arbitrary units (AU) obtained with NIH image J 1.40 analysis software. * and ** significantly different from untreated control, p<0.005 and p<0.001, respectively. The two inhibitors (listed below the bars) significantly decreased secretion of Hsp60 and Hsp70. Also, the data from whole-cell lysates show that the protein-secretion inhibitors had no detectable effect on Hsp levels inside the cells. <b>B</b>) Hsp60 levels secreted by the H292 tumor cells before and after exposure for 1 hour, followed by a 4 hours recovery period, to protein-secretion inhibitors measured by ELISA in: (<b>a</b>) conditioned media; and (<b>b</b>) exosomes. Histograms represent Hsp60 levels expressed as pg of protein normalized for mL normalised for 10⁶ cells. Data represent mean +/− SD of three different experiments in duplicate. * Significantly different from untreated control, p<0.005. The results, which are in agreement with those obtained by Western blotting, show that the inhibitors tested significantly reduced secretion of Hsp60 by the H292 tumor cells.</p