Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

The peripheral pro-nociceptive state induced by repetitive inflammatory stimuli involves continuous activation of protein kinase A and protein kinase C epsilon and its Na(v)1.8 sodium channel functional regulation in the primary sensory neuron

In the present study, the participation of the Na(v)1.8 sodium channel was investigated in the development of the peripheral pro-nociceptive state induced by daily intraplantar injections of PGE(2) in rats and its regulation in vivo by protein kinase A (PKA) and protein kinase C epsilon (PKC epsilon) as well. In the prostaglandin E(2) (PGE(2))-induced persistent hypernociception, the Na(v)1.8 mRNA in the dorsal root ganglia (DRG) was up-regulated. The local treatment with dipyrone abolished this persistent hypernociception but did not alter the Na(v)1.8 mRNA level in the DRG. Daily intrathecal administrations of antisense Na(v)1.8 decreased the Na(v)1.8 mRNA in the DRG and reduced ongoing persistent hypernociception. once the persistent hypernociception had been abolished by dipyrone, but not by Na(v)1.8 antisense treatment, a small dose of PGE(2) restored the hypernociceptive plateau. These data show that, after a period of recurring inflammatory stimuli, an intense and prolonged nociceptive response is elicited by a minimum inflammatory stimulus and that this pro-nociceptive state depends on Na(v)1.8 mRNA up-regulation in the DRG. in addition, during the persistent hypernociceptive state, the PKA and PKC epsilon expression and activity in the DRG are up-regulated and the administration of the PKA and PKC epsilon inhibitors reduce the hypernociception as well as the Na(v)1.8 mRNA level. In the present study, we demonstrated that the functional regulation of the Na(v)1.8 mRNA by PKA and PKC epsilon in the primary sensory neuron is important for the development of the peripheral pro-nociceptive state induced by repetitive inflammatory stimuli and for the maintenance of the behavioral persistent hypernociception. (C) 2008 Elsevier Inc. All rights reserved.

Selective activation of the rectus abdominis muscle during low-intensity and fatiguing tasks

In order to understand the potential selective activation of the rectus abdominis muscle, we conducted two experiments. In the first, subjects performed two controlled isometric exercises: the curl up (supine trunk raise) and the leg raise (supine bent leg raise) at low intensity (in which only a few motor units are recruited). In the second experiment, subjects performed the same exercises, but they were required to maintain a certain force level in order to induce fatigue. We recorded the electromyographic (EMG) activities of the lower and upper portions of the rectus abdominis muscle during the exercises and used spatial-temporal and frequency analyses to describe muscle activation patterns. At low-intensity contractions, the ratio between the EMG intensities of the upper and lower portions during the curl up exercise was significantly larger than during the leg raise exercise (p = 0.02). A cross-correlation analysis indicated that the signals of the abdominal portions were related to each other and this relation did not differ between the tasks (p = 0.12). In the fatiguing condition, fatigue for the upper portion was higher than for the lower portion during the curl up exercise (p = 0.008). We conclude that different exercises evoked, to a certain degree, individualized activation of each part of the rectus abdominis muscle, but different portions of the rectus abdominis muscle contributed to the same task, acting like a functional unit. These results corroborate the relevance of varying exercise to modify activation patterns of the rectus abdominis muscle.

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C) ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.

On the functional integration between postural and supra-postural tasks on the basis of contextual cues and task constraint

In order to evaluate the effects of uncertainty about direction of mechanical perturbation and supra-postural task constraint on postural control young adults had their upright stance perturbed while holding a tray in a horizontal position Stance was perturbed by moving forward or backward a supporting platform contrasting situations of certainty versus uncertainty of direction of displacement Increased constraint on postural stability was Imposed by a supra-postural task of equilibrating a cylinder on the tray Performance was assessed through EMG of anterior leg muscles angular displacement of the main Joints involved in the postural reactions and displacement of the tray Results showed that both certainty on the direction of perturbation and Increased supra-postural task constraint led to decreased angular displacement of the knee and the hip Furthermore combination of certainty and high supra-postural task constraint produced shorter latency of muscular activation Such postural responses were paralleled by decreased displacement of the tray Thesi results suggest a functional integration between the tasks with central set priming reactive postural responses from contextual cues and Increased stability demand (C) 2010 Elsevier B V All rights reserved

The Penicillium chrysogenum aclA gene encodes a broad-substrate-specificity acyl-coenzyme A ligase involved in activation of adipic acid, a side-chain precursor for cephem antibiotics

Activation of the cephalosporin side-chain precursor to the corresponding CoA-thioester is an essential step for its incorporation into the P-lactam backbone. To identify an acyl-CoA ligase involved in activation of adipate, we searched in the genome database of Penicillium chrysogenum for putative structural genes encoding acyl-CoA ligases. Chemostat-based transcriptome analysis was used to identify the one presenting the highest expression level when cells were grown in the presence of adipate. Deletion of the gene renamed aclA, led to a 32% decreased specific rate of adipate consumption and a threefold reduction of adipoyl-6-aminopenicillanic acid levels, but did not affect penicillin V production. After overexpression in Escherichia coli, the purified protein was shown to have a broad substrate range including adipate. Finally, protein-fusion with cyan-fluorescent protein showed co-localization with microbody-borne acyl-transferase. Identification and functional characterization of aclA may aid in developing future metabolic engineering strategies for improving the production of different cephalosporins. (C) 2009 Elsevier Inc. All rights reserved.

Endogenous cannabinoids induce fever through the activation of CB(1) receptors

Background and purpose: The effects of centrally administered cannabinoids on body core temperature (Tc) and the contribution of endogenous cannabinoids to thermoregulation and fever induced by lipopolysaccharide (LPS) (Sigma Chem. Co., St. Louis, MO, USA) were investigated. Experimental approach: Drug-induced changes in Tc of male Wistar rats were recorded over 6 h using a thermistor probe (Yellow Springs Instruments 402, Dayton, OH, USA) inserted into the rectum. Key results: Injection of anandamide [(arachidonoylethanolamide (AEA); Tocris, Ellisville, MO, USA], 0.01-1 mu g i.c.v. or 0.1-100 ng intra-hypothalamic (i.h.), induced graded increases in Tc (peaks 1.5 and 1.6 degrees C at 4 h after 1 mu g i.c.v. or 10 ng i.h.). The effect of AEA (1 mu g, i.c.v.) was preceded by decreases in tail skin temperature and heat loss index (values at 1.5 h: vehicle 0.62, AEA 0.48). Bell-shaped curves were obtained for the increase in Tc induced by the fatty acid amide hydrolase inhibitor [3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate (Cayman Chemical Co., Ann Arbor, MI, USA) (0.001-1 ng i.c.v.; peak 1.9 degrees C at 5 h after 0.1 ng) and arachidonyl-2-chloroethylamide (ACEA; Tocris) (selective CB(1) agonist; 0.001-1 mu g i.c.v.; peak 1.4 degrees C 5 h after 0.01 mu g), but (R,S)-(+)-(2-Iodo-5-nitrobenzoyl)-[1-(1-methyl-piperidin-2-ylmethyl)-1H-indole-3-yl] methanone (Tocris) (selective CB(2) agonist) had no effect on Tc. AEA-induced fever was unaffected by i.c.v. pretreatment with 6-Iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indole-3-yl](4-methoxyphenyl) methanone (Tocris) (selective CB(2) antagonist), but reduced by i.c.v. pretreatment with N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; Tocris) (selective CB(1) antagonist). AM251 also reduced the fever induced by ACEA or LPS. Conclusions and implications: The endogenous cannabinoid AEA induces an integrated febrile response through activation of CB(1) receptors. Endocannabinoids participate in the development of the febrile response to LPS constituting a target for antipyretic therapy.

Depression in Parkinson`s disease: Convergence from voxel-based morphometry and functional magnetic resonance imaging in the limbic thalamus

Depression is the most frequent psychiatric disorder in Parkinson`s disease (PD). Although evidence Suggests that depression in PD is related to the degenerative process that underlies the disease, further studies are necessary to better understand the neural basis of depression in this population of patients. In order to investigate neuronal alterations underlying the depression in PD, we studied thirty-six patients with idiopathic PD. Twenty of these patients had the diagnosis of major depression disorder and sixteen did not. The two groups were matched for PD motor severity according to Unified Parkinson Disease Rating Scale (UPDRS). First we conducted a functional magnetic resonance imaging (fMRI) using an event-related parametric emotional perception paradigm with test retest design. Our results showed decreased activation in the left mediodorsal (MD) thalamus and in medial prefrontall cortex in PD patients with depression compared to those without depression. Based upon these results and the increased neuron count in MD thalamus found in previous studies, we conducted a region of interest (ROI) guided voxel-based morphometry (VBM) study comparing the thalamic volume. Our results showed an increased volume in mediodorsal thalamic nuclei bilaterally. Converging morphological changes and functional emotional processing in mediodorsal thalamus highlight the importance of limbic thalamus in PD depression. In addition this data supports the link between neurodegenerative alterations and mood regulation. (C) 2009 Elsevier Inc. All rights reserved.

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

The impact of functional connectivity changes on support vector machines mapping of fMRI data

Functional magnetic resonance imaging (fMRI) is currently one of the most widely used methods for studying human brain function in vivo. Although many different approaches to fMRI analysis are available, the most widely used methods employ so called ""mass-univariate"" modeling of responses in a voxel-by-voxel fashion to construct activation maps. However, it is well known that many brain processes involve networks of interacting regions and for this reason multivariate analyses might seem to be attractive alternatives to univariate approaches. The current paper focuses on one multivariate application of statistical learning theory: the statistical discrimination maps (SDM) based on support vector machine, and seeks to establish some possible interpretations when the results differ from univariate `approaches. In fact, when there are changes not only on the activation level of two conditions but also on functional connectivity, SDM seems more informative. We addressed this question using both simulations and applications to real data. We have shown that the combined use of univariate approaches and SDM yields significant new insights into brain activations not available using univariate methods alone. In the application to a visual working memory fMRI data, we demonstrated that the interaction among brain regions play a role in SDM`s power to detect discriminative voxels. (C) 2008 Elsevier B.V. All rights reserved.

Influence of ankle functional instability on the ankle electromyography during landing after volleyball blocking

The purpose of this study was to describe, interpret and compare the EMG activation patterns of ankle muscles - tibialis anterior (TA), peroneus longus (PL) and gastrocnemius lateralis (GL) - in volleyball players with and without ankle functional instability (FI) during landing after the blocking movement. Twenty-one players with FI (IG) and 19 controls (CG) were studied. The cycle of movement analyzed was the time period between 200 ms before and 200 ms after the time of impact determined by ground reaction forces. The variables were analyzed for two different phases: pre-landing (200 ms before impact) and post-landing (200 ms after impact). The RMS values and the timing of onset activity were calculated for the three studied muscles, in both periods and for both groups. The co-activation index for TA and PL, TA and GL were also calculated. Individuals with FI presented a lower RMS value pre-landing for PL (CG = 43.0 perpendicular to 22.0; IG = 26.2 perpendicular to 8.4, p < 0.05) and higher RMS value post-landing (CG = 47.5 perpendicular to 13.3; IG = 55.8 perpendicular to 21.6, p < 0.10). Besides that, in control group PL and GL activated first and simultaneously, and TA presented a later activation, while in subjects with FI all the three muscles activated simultaneously. There were no significant differences between groups for co-activation index. Thus, the rate of contraction between agonist and antagonist muscles is similar for subjects with and without FI but the activation individually was different. Volleyball players with functional instability of the ankle showed altered patterns of the muscles that play an important role in the stabilization of the foot-ankle complex during the performance of the blocking movement, to the detriment of the ligament complex, and this fact could explain the usual complaints in these subjects. (C) 2007 Elsevier Ltd. All rights reserved.

Abnormal Left and Right Amygdala-Orbitofrontal Cortical Functional Connectivity to Emotional Faces: State Versus Trait Vulnerability Markers of Depression in Bipolar Disorder

Background: Amygdala-orbitofrontal cortical (OFC) functional connectivity (FC) to emotional stimuli and relationships with white matter remain little examined in bipolar disorder individuals (BD). Methods: Thirty-one BD (type 1; n = 17 remitted; n = 14 depressed) and 24 age- and gender-ratio-matched healthy individuals (HC) viewed neutral, mild, and intense happy or sad emotional faces in two experiments. The FC was computed as linear and nonlinear dependence measures between amygdala and OFC time series. Effects of group, laterality, and emotion intensity upon amygdala-OFC FC and amygdala-OFC FC white matter fractional anisotropy (FA) relationships were examined. Results: The BD versus HC showed significantly greater right amygdala-OFC FC (p <= .001) in the sad experiment and significantly reduced bilateral amygdala-OFC FC (p = .007) in the happy experiment. Depressed but not remitted female BD versus female HC showed significantly greater left amygdala-OFC FC (p = .001) to all faces in the sad experiment and reduced bilateral amygdala-OFC FC to intense happy faces (p = .01). There was a significant nonlinear relationship (p = .001) between left amygdala-OFC FC to sad faces and FA in HC. In BD, antidepressants were associated with significantly reduced left amygdala-OFC FC to mild sad faces (p = .001). Conclusions: In BD, abnormally elevated right amygdala-OFC FC to sad stimuli might represent a trait vulnerability for depression, whereas abnormally elevated left amygdala-OFC FC to sad stimuli and abnormally reduced amygdala-OFC FC to intense happy stimuli might represent a depression state marker. Abnormal FC measures might normalize with antidepressant medications in BD. Nonlinear amygdala-OFC FC-FA relationships in BID and HC require further study.

Changes in slow and fast alpha bands in subjects submitted to different amounts of functional electrostimulation

Objective: To examine the changes in slow (8-10 Hz)and fast (10-12 Hz) alpha bands of EEG in three groups of subjects submitted to different amounts of functional electrostimulation (FES). Our hypothesis is that different amounts of electrostimulation may cause different patterns of activation in the sensorimotor cortex. In particular, we expect to see an increase in alpha power due to habituation effects. We examine the two bands comprised by alpha rhythm (i.e., slow and fast alpha), since these two sub-rhythms are related to distinct aspects: general energy demands and specific motor aspects, respectively. Methods: The sample was composed of 27 students, both sexes, aging between 25 and 40 years old. The subjects were randomly distributed in three groups: control (n = 9), G24 (n = 9) and G36 (n = 9). A FES equipment (Neuro Compact-2462) was used to stimulate the right index finger extension. Simultaneously, the electroencephalographic signal was acquired. We investigated the absolute power in slow and fast alpha bands in the sensorimotor cortex. Results: The G36 indicated a significant increasing in absolute power values in lower and higher alpha components, respectively, when compared with the control group. Particularly, in the following regions: pre-motor cortex and primary motor cortex. Discussion: FES seems to promote cortical adaptations that are similar to those observed when someone learns a procedural task. FES application in the G36 was more effective in promoting such neural changes. The lower and higher components of alpha rhythms behave differently in their topographical distribution during FES application. These results suggest a somatotopic organization in primary motor cortex which can be represented by the fast alpha component. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Phenotypic and functional characterization of pulmonary macrophages subpopulations after intratracheal injection of Paracoccidioides brasiliensis cell wall components

A shift in the activation of pulmonary macrophages characterized by an increase of IL-1, INF-alpha and IL-6 production has been induced in mice infected with Paracoccidioides brasiliensis. It is still unclear whether a functional shift in the resident alveolar macrophage population would be responsible for these observations due to the expression of cell surface molecules. We investigated pulmonary macrophages by flow cytometry from mice treated with P. brasiliensis derivatives by intratracheal route. In vivo labeling with the dye PKH26GL was applied to characterize newly recruited pulmonary macrophages from the bloodstream. Pulmonary macrophages from mice inflamed with P. brasiliensis derivatives showed a high expression of the surface antigens CD11b/CD18 and CD23 among several cellular markers. The expression of these markers indicated a pattern of activation of a subpopulation characterized as CD11b(+) or CD23(+), which was modulated in vitro by IFN-gamma and IL-4. Analysis of monocytes labelled with PKH26GL demonstrated that CD11b(+) cells did infiltrate the lung exhibiting a proinflammatoni pattern of activation, whereas CD23(+) cells were considered to be resident in the lung. These findings may contribute to better understand the pathology of lung inflammation caused by P. brasiliensis infection. (C) 2010 Elsevier GmbH. All rights reserved.

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. (J Clin Endocrinol Metab 95: 2857-2867, 2010)