EXPRESSION OF GENES RELATED TO MYOSTATIN SIGNALING DURING RAT SKELETAL MUSCLE LONGITUDINAL GROWTH

In this study we investigated the gene expression of proteins related to myostatin (MSTN) signaling during skeletal muscle longitudinal growth. To promote muscle growth, Wistar male rats were submitted to a stretching protocol for different durations (12, 24, 48, and 96 hours). Following this protocol, soleus weight and length and sarcomere number were determined. In addition, expression levels of the genes that encode MSTN, follistatin isoforms 288 and 315 (FLST288 and FLST315), follistatin-like 3 protein (FLST-L3), growth and differentiation factor-associated protein-1 (GASP-1), activin IIB receptor (ActIIB), and SMAD-7 were determined by real-time polymerase chain reaction. Prolonged stretching increased soleus weight, length, and sarcomere number. In addition, MSTN gene expression was increased at 12-24 hours, followed by a decrease at 96 hours when compared with baseline values. FLST isoforms, FLST-L3, and GASP-1 mRNA levels increased significantly over all time-points. ActIIB gene expression decreased quickly at 12-24 hours. SMAD-7 mRNA levels showed a late increase at 48 hours, which peaked at 96 hours. The gene expression pattern of inhibitory proteins related to MSTN signaling suggests a strong downregulation of this pathway in response to prolonged stretching. Muscle Nerve 40: 992-999, 2009

Effect of eccentric exercise velocity on akt/mtor/p70(s6k) signaling in human skeletal muscle

It has been suggested that muscle tension plays a major role in the activation of intracellular pathways for skeletal muscle hypertrophy via an increase in mechano growth factor (MGF) and other downstream targets. Eccentric exercise (EE) imposes a greater amount of tension on the active muscle. In particular, high-speed EE seems to exert an additional effect on muscle tension and, thus, on muscle hypertrophy. However, little is known about the effect of EE velocity on hypertrophy signaling. This study investigated the effect of acute EE-velocity manipulation on the Akt/mTORCI/p70(S6K) hypertrophy pathway. Twenty subjects were assigned to either a slow (20 degrees.s(-1); ES) or fast EE (210 degrees.s(-1); EF) group. Biopsies were taken from vastus lateralis at baseline (B), immediately after (T1), and 2 h after (T2) the completion of 5 sets of 8 repetitions of eccentric knee extensions. Akt, mTOR, and p70(S6K) total protein were similar between groups, and did not change postintervention. Further, Akt and p70(S6K) protein phosphorylation were higher at T2 than at B for ES and EF. MGF messenger RNA was similar between groups, and only significantly higher at T2 than at B in ES. The acute manipulation of EE velocity does not seem to differently influence intracellular hypertrophy signaling through the Akt/mTORCI/p70S6K pathway.

Double disruption of alpha(2A)- and alpha(2C)-adrenoceptors results in sympathetic hyperactivity and high-bone-mass phenotype

Evidence demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via beta(2)-adrenoceptor (beta(2)-AR) signaling. Here we show that female mice with chronic sympathetic hyperactivity owing to double knockout of adrenoceptors that negatively regulate norepinephrine release, alpha(2A)-AR and alpha(2C)-AR(alpha(2A)/alpha(2C)-ARKO), present an unexpected and generalized phenotype of high bone mass with decreased bone resorption and increased formation. In alpha(2A)/alpha(2C)-ARKO versus wild-type (WT) mice, micro-computed tomographic (mu CT) analysis showed increased, better connected, and more plate-shaped trabeculae in the femur and vertebra and increased cortical thickness in the vertebra, whereas biomechanical analysis showed increased tibial and femoral strength. Tibial mRNA expression of tartrate-resistant acid phosphatase (TRACP) and receptor activator of NF-kappa B (RANK), which are osteoclast-related factors, was lower in knockout (KO) mice. Plasma leptin and brain mRNA levels of cocaine amphetamine-regulated transcript (CART), which are factors that centrally affect bone turnover, and serum levels of estradiol were similar between mice strains. Tibial beta(2)-AR mRNA expression also was similar in KO and WT littermates, whereas alpha(2A)-, alpha(2B)- and alpha(2C)-AR mRNAs were detected in the tibia of WT mice and in osteoblast-like MC3T3-E1 cells. By immunohistochemistry, we detected alpha(2A)-, alpha(2B)-, alpha(2C)- and beta(2)-ARs in osteoblasts, osteoclasts, and chondrocytes of 18.5-day-old mouse fetuses and 35-day-old mice. Finally, we showed that isolated osteoclasts in culture are responsive to the selective alpha(2)-AR agonist clonidine and to the nonspecific alpha-AR antagonist phentolamine. These findings suggest that beta(2)-AR is not the single adrenoceptor involved in bone turnover regulation and show that alpha(2)-AR signaling also may mediate the SNS actions in the skeleton. (c) 2011 American Society for Bone and Mineral Research.

Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats

DA SILVA, A. S. R., J. R. PAULI, E. R. ROPELLE, A. G. OLIVEIRA, D. E. CINTRA, C. T. DE SOUZA, L. A. VELLOSO, J. B. C. CARVALHEIRA, and M. J. A. SAAD. Exercise Intensity, Inflammatory Signaling, and Insulin Resistance in Obese Rats. Med. Sci. Sports Exerc., Vol. 42, No. 12, pp. 2180-2188, 2010. Purpose: To evaluate the effects of intensity of exercise on insulin resistance and the expression of inflammatory proteins in the skeletal muscle of diet-induced obese (DIO) rats after a single bout of exercise. Methods: In the first exercise protocol, the rats swam for two 3-h bouts, separated by a 45-min rest period (with 6 h in duration-DIO + EXE), and in the second protocol, the rats were exercised with 45 min of swimming at 70% of the maximal lactate steady state-MLSS (DIO + MLSS). Results: Our data demonstrated that both protocols of exercise increased insulin sensitivity and increased insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate 1 and serine phosphorylation of protein kinase B in the muscle of DIO rats by the same magnitude. In parallel, both exercise protocols also reduced protein tyrosine phosphatase 1B activity and insulin receptor substrate 1 serine phosphorylation, with concomitant reduction in c-jun N-terminal kinase and I kappa B kinase activities in the muscle of DIO rats in a similar fashion. Conclusions: Thus, our data demonstrate that either exercise protocols with low intensity and high volume or exercise with moderate intensity and low volume represents different strategies to restore insulin sensitivity with the same efficacy.

Effects of glutamine on the nuclear factor-kappaB signaling pathway of murine peritoneal macrophages

The aim of this study was to evaluate the effect of glutamine on the expression of proteins involved in the nuclear factor-kappaB (NF-kappa B) signaling pathway of murine peritoneal macrophages. Since glutamine is essential for the normal functioning of macrophages, it was hypothesized that in vitro glutamine supplementation would increase NF-kappa B activation. Peritoneal macrophages were pretreated with glutamine (0, 0.6, 2 and 10 mM) before incubation with lipopolysaccharide (LPS), and the effects of glutamine on the production of tumor necrosis factor-alpha and on the expression and activity of proteins involved in the NF-kappa B signaling pathway were studied by an enzyme linked immuno-sorbent assay, Western blotting, and an electrophoretic mobility shift assay. Glutamine treatment (2 and 10 mM) increased the activation of NF-kappa B in LPS-stimulated peritoneal macrophages (P < 0.05). In non-stimulated cells, glutamine treatment (2 and 10 mM) significantly reduced I kappa B-alpha protein expression (P < 0.05). Glutamine modulates NF-kappa B signaling pathway by reducing the level of I kappa B-alpha, leading to an increase in NF-kappa B within the nucleus in peritoneal macrophages.

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1 beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from Cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. in THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells. (C) 2008 Elsevier B.V. All rights reserved.

Human Airway Epithelial Cell Responses to Neisseria lactamica and Purified Porin via Toll-Like Receptor 2-Dependent Signaling

The human airway epithelium is constantly exposed to microbial products from colonizing organisms. Regulation of Toll-like receptor (TLR) expression and specific interactions with bacterial ligands is thought to mitigate exacerbation of inflammatory processes induced by the commensal flora in these cells. The genus Neisseria comprises pathogenic and commensal organisms that colonize the human nasopharynx. Neisseria lactamica is not associated with disease, but N. meningitidis occasionally invades the host, causing meningococcal disease and septicemia. Upon colonization of the airway epithelium, specific host cell receptors interact with numerous Neisseria components, including the PorB porin, at the immediate bacterial-host cell interface. This major outer membrane protein is expressed by all Neisseria strains, regardless of pathogenicity, but its amino acid sequence varies among strains, particularly in the surface-exposed regions. The interaction of Neisseria PorB with TLR2 is essential for driving TLR2/TLR1-dependent cellular responses and is thought to occur via the porin`s surface-exposed loop regions. Our studies show that N. lactamica PorB is a TLR2 ligand but its binding specificity for TLR2 is different from that of meningococcal PorB. Furthermore, N. lactamica PorB is a poor inducer of proinflammatory mediators and of TLR2 expression in human airway epithelial cells. These effects are reproduced by whole N. lactamica organisms. Since the responsiveness of human airway epithelial cells to colonizing bacteria is in part regulated via TLR2 expression and signaling, commensal organisms such as N. lactamica would benefit from expressing a product that induces low TLR2-dependent local inflammation, likely delaying or avoiding clearance by the host.

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Activation of beta(2)-adrenergic receptors hastens relaxation and mediates phosphorylation of phospholamban, troponin I, and C-protein in ventricular myocardium from patients with terminal heart failure

Background-Catecholamines hasten cardiac relaxation through beta-adrenergic receptors, presumably by phosphorylation of several proteins, but it is unknown which receptor subtypes are involved in human ventricle. We assessed the role of beta(1)- and beta(2)-adrenergic receptors in phosphorylating proteins implicated in ventricular relaxation. Methods and Results-Right ventricular trabeculae, obtained from freshly explanted hearts of patients with dilated cardiomyopathy (n=5) or ischemic cardiomyopathy (n=5), were paced at 60 bpm. After measurement of the contractile and relaxant effects of epinephrine (10 mu mol/L) or zinterol (10 mu mol/L), mediated through beta(2)-adrenergic receptors, and of norepinephrine (10 mu mol/L), mediated through beta(1)-adrenergic receptors, tissues were freeze clamped. We assessed phosphorylation of phospholamban, troponin I, and C-protein, as well as specific phosphorylation of phospholamban at serine 16 and threonine 17, Data did not differ between the 2 disease groups and were therefore pooled. Epinephrine, zinterol, and norepinephrine increased contractile force to approximately the same extent, hastened the onset of relaxation by 15+/-3%, 5+/-2%, and 20+/-3%, respectively, and reduced the time to half-relaxation by 26+/-3%, 21+/-3%, and 37+/-3%. These effects of epinephrine, zinterol, and norepinephrine were associated with phosphorylation (pmol phosphate/mg protein) of phospholamban 14+/-3, 12+/-4, and 12+/-3, troponin I 40+/-7, 33+/-7, and 31+/-6; and C-protein 7.2+/-1.9, 9.3 +/- 1.4, and 7.5 +/- 2.0. Phosphorylation of phospholamban occurred at both Ser16 and Thr17 residues through both beta(1)- and beta(2)-adrenergic receptors. Conclusions-Norepinephrine and epinephrine hasten human ventricular relaxation and promote phosphorylation of implicated proteins through both beta(1)- and beta(2)-adrenergic receptors, thereby potentially improving diastolic function.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (h beta c) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3R alpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type h beta c exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type h beta c, despite failing to undergo IL-3-stimulated h beta e tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and h beta c tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein

Background-In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Methods and Results-Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 2-1/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC50 [mol/L] NE 5.5+/-0.1, n=12; -EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC50 [mol/L] NE 5.8+/--0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC50 [mol/L] NE 5.7+/-0.2: EPI 5.8+/-0.1), Ventricular membranes from Fallot infants, labeled with (-)-[I-125]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Conclusions-Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Eye design and color signaling in a stomatopod crustacean Gonodactylus smithii

Many species of stomatopod crustaceans have multiple spectral classes of photoreceptors in their retinas. Behavioral evidence also indicates that stomatopods are capable of discriminating objects by their spectral differences alone, Most animals use only two to four different types of photoreceptors in their color vision systems, typically with broad sensitivity functions, but the stomatopods apparently include eight or more narrowband photoreceptor classes for color recognition. It is also known that stomatopods use several colored body regions in social interactions. To examine why stomatopods may be so 'concerned' with color, we measured the absorption spectra of visual pigments and intrarhabdomal filters, and the reflectance spectra from different parts of the bodies of several individuals of the gonodactyloid stomatopod species, Gonodactylus smithii. We then applied a model of multiple dichromatic channels for color encoding to examine whether the finely tuned color vision was specifically co-evolved with their complex color signals. Although the eye design of stomatopods seems suitable for detecting color signals of their own, the detection of color signals from other animals, such as reef fishes, can be enhanced as well. Color vision in G. smithii is therefore not exclusively adapted to detect its own color signals, but the spectral tuning of some photoreceptors (e.g. midband Rows 2 and 3) enhances the contrast of certain color signals to a large enough degree to make co-evolution between color vision and these rather specific color signals likely. Copyright (C) 2000 S. Karger AG, Basel.

Semiochemicals and social signaling in the wild European rabbit in Australia: I. Scent profiles of chin gland secretion from the field

The European rabbit (Oryctolagus cuniculus) uses the secretion of the chin gland in the maintenance of social status. Previous work has concentrated on secretion collected directly from the animal. In this study, the analysis was conducted by collecting scent marks made by free-ranging animals. Scent marks were found to be concentrated at the center of the area controlled by a social group, and at the boundaries between two adjacent social groups. Only the mark from dominant animals could be identified. Marks were also collected from the skin of rabbits, where they had been placed by the dominant individual. The mark found on the head of a subordinate animal may, in the future, be used to identify the dominant animal of the social group, who placed the mark.