Avaliação da teneurina-2 (Ten-2) nos astrócitos do córtex cerebral de ratos adultos após lesão mecânica. Estudo imuno-histoquímico e de expressão gênica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural insights regarding an insecticidal Talisia esculenta protein and its biotechnological potential for Diatraea saccharalis larval control

Talisin is a seed-storage protein from Talisia esculenta that presents lectin-like activities, as well as proteinase-inhibitor properties. The present study aims to provide new in vitro and in silico biochemical information about this protein, shedding some light on its mechanistic inhibitory strategies. A theoretical three-dimensional structure of Talisin bound to trypsin was constructed in order to determine the relative interaction mode. Since the structure of non-competitive inhibition has not been elucidated, Talisin-trypsin docking was carried out using Hex v5.1, since the structure of non-competitive inhibition has not been elucidated. The predicted non-coincidence of the trypsin binding site is completely different from that previously proposed for Kunitz-type inhibitors, which demonstrate a substitution of an Arg(64) for the Glu(64) residue. Data, therefore, provide more information regarding the mechanisms of non-competitive plant proteinase inhibitors. Bioassays with Talisin also presented a strong insecticide effect on the larval development of Diatraea saccharalis, demonstrating LD50 and ED50 of ca. 2.0% and 1.5%, respectively. (C) 2011 Elsevier Inc. All rights reserved.

Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats

Costa-Silva JH, Zoccal DB, Machado BH. Chronic intermittent hypoxia alters glutamatergic control of sympathetic and respiratory activities in the commissural NTS of rats. Am J Physiol Regul Integr Comp Physiol 302: R785-R793, 2012. First published December 28, 2011; doi:10.1152/ajpregu.00363.2011.-Sympathetic overactivity and altered respiratory control are commonly observed after chronic intermittent hypoxia (CIH) exposure. However, the central mechanisms underlying such neurovegetative dysfunctions remain unclear. Herein, we hypothesized that CIH (6% O-2 every 9 min, 8 h/day, 10 days) in juvenile rats alters glutamatergic transmission in the commissural nucleus tractus solitarius (cNTS), a pivotal site for integration of peripheral chemoreceptor inputs. Using an in situ working heart-brain stem preparation, we found that L-glutamate microinjections (1, 3, and 10 mM) into the cNTS of control rats (n = 8) evoked increases in thoracic sympathetic nerve (tSN) and central vagus nerve (cVN) activities combined with inhibition of phrenic nerve (PN) activity. Besides, the ionotropic glutamatergic receptor antagonism with kynurenic acid (KYN; 250 mM) in the cNTS of control group (n = 7) increased PN burst duration and frequency. In the CIH group (n = 10), the magnitude of L-glutamate-induced cVN excitation was smaller, and the PN inhibitory response was blunted (P < 0.05). In addition, KYN microinjections into the cNTS of CIH rats (n = 9) did not alter PN burst duration and produced smaller increases in its frequency compared with controls. Moreover, KYN microinjections into the cNTS attenuated the sympathoexcitatory response to peripheral chemoreflex activation in control but not in CIH rats (P < 0.05). These functional CIH-induced alterations were accompanied by a significant 10% increase of N-methyl-D-aspartate receptor 1 (NMDAR1) and glutamate receptor 2/3 (GluR2/3) receptor subunit density in the cNTS (n = 3-8, P < 0.05), evaluated by Western blot analysis. These data indicate that glutamatergic transmission is altered in the cNTS of CIH rats and may contribute to the sympathetic and respiratory changes observed in this experimental model.

INHIBITION OF OVIPOSITION BY NEEM EXTRACT: A BEHAVIORAL PERSPECTIVE FOR THE CONTROL OF THE MEDITERRANEAN FRUIT FLY (DIPTERA: TEPHRITIDAE)

The behavioral inhibitory effect of methanol extracts from neem leaves (Azadirachta indica A. Juss) at different concentrations (0, 10,000, 18,000, 32,000 and 56,000 ppm) was evaluated using naive and experienced medflies (Ceratitis capitata (Wied.)) ovipositing on the fruits of grape cv. 'Italia'. The grapes were immersed in the specific treatments and were exposed for 24 h to 3 pairs of female and male medflies, both nave and experienced, in a choice test. At concentrations >= 18,000 ppm, the extract that was applied to the grapes inhibited oviposition. The previous experience with treated grapes did not affect the response of the medflies. This study is the first step toward the application of the behavior control of the medflies as a tool in grape vineyards. The potential for using oviposition inhibitory in behavioral control of C. capitata are discussed.

Models of passive and active dendrite motoneuron pools and their differences in muscle force control

Motoneuron (MN) dendrites may be changed from a passive to an active state by increasing the levels of spinal cord neuromodulators, which activate persistent inward currents (PICs). These exert a powerful influence on MN behavior and modify the motor control both in normal and pathological conditions. Motoneuronal PICs are believed to induce nonlinear phenomena such as the genesis of extra torque and torque hysteresis in response to percutaneous electrical stimulation or tendon vibration in humans. An existing large-scale neuromuscular simulator was expanded to include MN models that have a capability to change their dynamic behaviors depending on the neuromodulation level. The simulation results indicated that the variability (standard deviation) of a maintained force depended on the level of neuromodulatory activity. A force with lower variability was obtained when the motoneuronal network was under a strong influence of PICs, suggesting a functional role in postural and precision tasks. In an additional set of simulations when PICs were active in the dendrites of the MN models, the results successfully reproduced experimental results reported from humans. Extra torque was evoked by the self-sustained discharge of spinal MNs, whereas differences in recruitment and de-recruitment levels of the MNs were the main reason behind torque and electromyogram (EMG) hysteresis. Finally, simulations were also used to study the influence of inhibitory inputs on a MN pool that was under the effect of PICs. The results showed that inhibition was of great importance in the production of a phasic force, requiring a reduced co-contraction of agonist and antagonist muscles. These results show the richness of functionally relevant behaviors that can arise from a MN pool under the action of PICs.

A possible new mechanism for the control of miRNA expression in neurons

The control of gene expression by miRNAs has been widely investigated in different species and cell types. Following a probabilistic rather than a deterministic regimen, the action of these short nucleotide sequences on specific genes depends on intracellular concentration,which in turn reflects the balance between biosynthesis and degradation. Recent studies have described the involvement of XRN2, an exoribonuclease, in miRNA degradation and PAPD4, an atypical poly(A) polymerase, in miRNA stability. Herein, we examined the expression of XRN2 and PAPD4 in developing and adult rat hippocampi. Combining bioinformatics and real-time PCR,we demonstrated that XRN2 and PAPD4 expression is regulated by the uncorrelated action of transcription factors, resulting in distinct gene expression profiles during development. Analyses of nuclei position and nestin labeling revealed that both proteins progressively accumulated during neuronal differentiation, and that they are weakly expressed in immature neurons and absent in glial and endothelial cells. Despite the differences in subcellular localization, both genes were concurrently identified within identical neuronal subpopulations, including specific inhibitory interneurons. Thus, we cope with a singular circumstance in biology: an almost complete intersected expression of functional-opposed genes, reinforcing that their antagonistically driven actions on miRNAs “make sense” if simultaneously present at the same cells. Considering that the transcriptome in the nervous system is finely tuned to physiological processes, it was remarkable that miRNA stability-related genes were oncurrently identified in neurons that play essential roles in cognitive functions such as memory and learning. In summary, this study reveals a possible new mechanism for the control of miRNA expression.

Biofilms on exposed monumental stones: mechanism of formation and development of new control methods

Within the stone monumental artefacts artistic fountains are extremely favorable to formation of biofilms, giving rise to biodegradation processes related with physical-chemical and visual aspect alterations, because of their particular exposure conditions. Microbial diversity of five fountains (two from Spain and three from Italy) was investigated. It was observed an ample similarity between the biodiversity of monumental stones reported in literature and that one found in studied fountains. Mechanical procedures and toxic chemical products are usually employed to remove such phototrophic patinas. Alternative methods based on natural antifouling substances are recently experimented in the marine sector, due to their very low environmental impact and for the bio settlement prevention on partially immersed structures of ships. In the present work groups of antibiofouling agents (ABAs) were selected from literature for their ability to interfere, at molecular level, with the microbial communication system “quorum sensing”, inhibiting the initial phase of biofilm formation. The efficacy of some natural antibiofoulants agents (ABAs) with terrestrial (Capsaicine - CS, Cinnamaldehyde - CI) and marine origin (Zosteric Acid - ZA, poly-Alkyl Pyridinium Salts – pAPS and Ceramium botryocarpum extract - CBE), incorporated into two commercial coatings (Silres BS OH 100 - S and Wacker Silres BS 290 - W) commonly used in stone conservation procedures were evaluated. The formation of phototrophic biofilms in laboratory conditions (on Carrara marble specimens and Sierra Elvira stone) and on two monumental fountains (Tacca’s Fountain 2 - Florence, Italy and Fountain from Patio de la Lindaraja - Alhambra Palace, Granada, Spain) has been investigated in the presence or absence of these natural antifouling agents. The natural antibiofouling agents, at tested concentrations, demonstrated a certain inhibitory effect. The silane-siloxane based silicone coating (W) mixing with ABAs was more suitable with respect to ethyl silicate coating (S) and proved efficacy against biofilm formation only when incompletely cured. The laboratory results indicated a positive action in inhibiting the patina formation, especially for poly-alkyl pyridinium salts, zosteric acid and cinnamaldehyde, while on site tests revealed a good effect for zosteric acid.

Improving adoptive T cell therapies of cancer by ectopic expression of miR181a to repress inhibitory phosphatases

Adoptive T cell therapy using antigen-specific T lymphocytes is a powerful immunotherapeutic approach against cancer. Nevertheless, many T cells against tumor-antigens exhibit only weak anti-tumoral response. To overcome this barrier it is necessary to improve the potency and anti-tumoral efficacy of these T cells. Activation and activity of T cells are tightly controlled to inhibit unwanted T cell responses and to reduce the risk of autoimmunity. Both are regulated by extrinsic signals and intrinsic mechanisms which suppress T cell activation. The intrinsic mechanisms include the expression of phosphatases that counteract the activation-inducing kinases. Modifying the expression of these phosphatases allows the targeted modulation of T cell reactivity. MicroRNAs (miRNAs) are regulatory small noncoding RNA molecules that control gene expression by targeting messenger RNAs in a sequence specific manner. Gene-specific silencing plays a key role in diverse biological processes, such as development, differentiation, and functionality. miR181a has been shown to be highly expressed in immature T cells that recognize low-affinity antigens.rnThe present study successfully shows that ectopic expression of miR181a is able to enhance the sensitivity of both murine and human T cells. In CD4+ T helper cells as well as in CD8+ cytotoxic T cells the overexpression of miR181a leads to downregulation of multiple phosphatases involved in the T cell receptor signaling pathway. Overexpression of miR181a in human T cells achieves a co-stimulatory independent activation and has an anti-apoptotic effect on CD4+ T helper cells. Additionally, increasing the amount of miR181a enhances the cytolytic activity of murine CD8+ TCRtg T cells in an antigen-specific manner.rnTo test miR181a overexpressing T cells in vivo, a mouse tumor model using a B cell lymphoma cell line (A20-HA) expressing the Influenza hemagglutinin (Infl.-HA) antigen was established. The expression of model antigens in tumor cell lines enables targeted elimination of tumors using TCRtg T cells. The transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ T cells alone has no positive effect neither on tumor control nor on survival of A20-HA tumor-bearing mice. In contrast, the co-transfer of miR181a overexpressing Infl.-HA TCRtg CD8+ and CD4+ T cells leads to improved tumor control and prolongs survival of A20-HA tumor-bearing mice. This effect is characterized by higher amounts of effector T cells and the expansion of Infl.-HA TCRtg CD8+ T cells.rnAll effects were achieved by changes in expression of several genes including molecules involved in T cell differentiation, activation, and regulation, cytotoxic effector molecules, and receptors important for the homing process of T cells in miR181a overexpressing T cells. The present study demonstrates that miR181a is able to enhance the anti-tumoral response of antigen-specific T cells and is a promising candidate for improving adoptive cell therapy.

Do statins reduce the risk of aneurysm development? A case-control study

Recent studies in rats have demonstrated that statins may have an inhibitory effect on intracranial aneurysm (IA) development. The purpose of this study was to assess whether long-term statin use is associated with a reduced risk of IA formation in humans.

Inhibitory regulation of dendritic activity in vivo

The spatiotemporal control of neuronal excitability is fundamental to the inhibitory process. We now have a wealth of information about the active dendritic properties of cortical neurons including axonally generated sodium action potentials as well as local sodium spikelets generated in the dendrites, calcium plateau spikes, and NMDA spikes. All of these events have been shown to be highly modified by the spatiotemporal pattern of nearby inhibitory input which can drastically change the output firing mode of the neuron. This means that particular populations of interneurons embedded in the neocortical microcircuitry can more precisely control pyramidal cell output than has previously been thought. Furthermore, the output of any given neuron tends to feed back onto inhibitory circuits making the resultant network activity further dependent on inhibition. Network activity is therefore ultimately governed by the subcellular microcircuitry of the cortex and it is impossible to ignore the subcompartmentalization of inhibitory influence at the neuronal level in order to understand its effects at the network level. In this article, we summarize the inhibitory circuits that have been shown so far to act on specific dendritic compartments in vivo.

Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections

Imatinib mesylate (imatinib) is a potent inhibitor of defined tyrosine kinases (TKs) and is effective in the treatment of malignancies characterized by constitutive activation of these TKs such as chronic myeloid leukemia and gastrointestinal stromal tumors. TKs also play an important role in T-cell receptor (TCR) signal transduction. Inhibitory as well as stimulating effects of imatinib on T cells and dendritic cells have been described. Here, we analyzed the effects of imatinib treatment on antiviral immune responses in vivo. Primary cytotoxic T-cell (CTL) responses were not impaired in imatinib-treated mice after infection with lymphocytic choriomeningitis virus (LCMV) or after immunization with a tumor cell line expressing LCMV glycoprotein (LCMV-GP). Similarly, neutralizing antibody responses to vesicular stomatitis virus (VSV) were not affected. In contrast, secondary expansion of LCMV-specific memory CTLs was reduced in vitro and in vivo, resulting in impaired protection against reinfection. In addition, imatinib treatment delayed the onset of diabetes in a CTL-induced diabetes model. In summary, imatinib treatment in vivo selectively inhibits the expansion of antigen-experienced memory CTLs without affecting primary T- or B-cell responses. Therefore, imatinib may be efficacious in the suppression of CTL-mediated immunopathology in autoimmune diseases without the risk of acquiring viral infections.

Medroxyprogesterone abrogates the inhibitory effects of estradiol on vascular smooth muscle cells by preventing estradiol metabolism

Sequential conversion of estradiol (E) to 2/4-hydroxyestradiols and 2-/4-methoxyestradiols (MEs) by CYP450s and catechol-O-methyltransferase, respectively, contributes to the inhibitory effects of E on smooth muscle cells (SMCs) via estrogen receptor-independent mechanisms. Because medroxyprogesterone (MPA) is a substrate for CYP450s, we hypothesized that MPA may abrogate the inhibitory effects of E by competing for CYP450s and inhibiting the formation of 2/4-hydroxyestradiols and MEs. To test this hypothesis, we investigated the effects of E on SMC number, DNA and collagen synthesis, and migration in the presence and absence of MPA. The inhibitory effects of E on cell number, DNA synthesis, collagen synthesis, and SMC migration were significantly abrogated by MPA. For example, E (0.1micromol/L) reduced cell number to 51+/-3.6% of control, and this inhibitory effect was attenuated to 87.5+/-2.9% by MPA (10 nmol/L). Treatment with MPA alone did not alter any SMC parameters, and the abrogatory effects of MPA were not blocked by RU486 (progesterone-receptor antagonist), nor did treatment of SMCs with MPA influence the expression of estrogen receptor-alpha or estrogen receptor-beta. In SMCs and microsomal preparations, MPA inhibited the sequential conversion of E to 2-2/4-hydroxyestradiol and 2-ME. Moreover, as compared with microsomes treated with E alone, 2-ME formation was inhibited when SMCs were incubated with microsomal extracts incubated with E plus MPA. Our findings suggest that the inhibitory actions of MPA on the metabolism of E to 2/4-hydroxyestradiols and MEs may negate the cardiovascular protective actions of estradiol in postmenopausal women receiving estradiol therapy combined with administration of MPA.

A MIF haplotype is associated with the outcome of patients with severe sepsis: a case control study

BACKGROUND: Macrophage migration inhibitory factor (MIF) plays an important regulatory role in sepsis. In the promoter region a C/G single nucleotide polymorphism (SNP) at position -173 (rs755622) and a CATT5-8 microsatellite at position -794 are related to modified promoter activity. The purpose of the study was to analyze their association with the incidence and outcome of severe sepsis. METHODS: Genotype distributions and allele frequencies in 169 patients with severe sepsis, 94 healthy blood donors and 183 postoperative patients without signs of infection or inflammation were analyzed by real time PCR and Sequence analysis. All included individuals were Caucasians. RESULTS: Genotype distribution and allele frequencies of severe sepsis patients were comparable to both control groups. However, the genotype and allele frequencies of both polymorphisms were associated significantly with the outcome of severe sepsis. The highest risk of dying from severe sepsis was detectable in patients carrying a haplotype with the alleles -173 C and CATT7 (p = 0.0005, fisher exact test, RR = 1,806, CI: 1.337 to 2.439). CONCLUSION: The haplotype with the combination of the -173 C allele and the -794 CATT7 allele may not serve as a marker for susceptibility to sepsis, but may help identify septic patients at risk of dying.

Involvement of human chromosome 17 in the control of the metastatic phenotype in melanoma

Fusion of nonmetastatic murine melanoma K1735 C19H cells with metastatic human melanoma A375 C15N cells resulted in a hybrid (termed H7) which was highly metastatic in a nude mouse model. The H7 hybrid retained chromosome 17 as the sole intact human chromosome in the cell. A lung bioassay showed that the K1735 C19H cells were present in the lungs of nude mice with s.c. tumors, yet at 6-weeks after tumor resection, no cells remained in the lung and therefore did not form lung metastases. Examination of various phenotypic properties such as in vivo and in vitro growth demonstrated that phenotypically the H7 hybrid was most like the K1735 C19H cell line except for its metastatic ability. In contrast the H7 hybrid cells containing single or multiple copies of human chromosome 17 with a point mutation at codon 249 (arg-gly) of the p53 gene, readily formed lung metastases. A plasmid containing the human p53 from the H7 hybrid and four other contructs with mutations at codon 143 (val-arg), 175 (arg-his), 249 (arg-ser) and 273 (arg-his) were transfected into K1735 C19H cells. K1735 C19H cells expressing human p53 genes with mutations at codons 249, both the arg-ser mutation and the mutation from the H7 hybrid and 273 produced significantly more lung metastases.^ In vitro assays demonstrated that responses to various cytotoxic and DNA damaging agents varied with the presence of mutant p53 and with the type of agent used. When cultured in mouse lung-conditioned medium, the K1735 C19H cell line was growth-inhibited, while cells containing a mutant human p53 (either on the whole chromosome 17, as in the H7 hybrid cells or from a stably transfected construct) were growth stimulated. Western blot analysis of lung-conditioned media derived from either 6-month or 15-month old mice has detected high levels of soluble Fas ligand in the medium from older animals. Comparison of the levels of Fas receptor on the K1735 C19H cell line and the H7 hybrid were almost identical, but 50% of the K1735 C19H cells were killed in the presence of anti-Fas antibody as opposed to 7% of the H7 hybrid cells. The growth-inhibitory effects of the lung-conditioned medium on the K1735 C19H cells were abrogated by coculture with Fas-Fc, which competes with the Fas ligand for receptor binding. Growth-inhibition of the K1735 C19H was 54% when cultured in 60 $\mu$g/0.2 ml lung-conditioned medium and a control Fc, with only 9% inhibition in 60 $\mu$g/0.2 ml lung-conditioned medium and Fas-Fc. Growth of the H7 cells and K1735 C19H cells transfected with various mutant human p53 genes were unchanged by the presence of either the control Fc or the Fas-Fc. This indicates that the presence of human chromosome 17, and mutant p53 in part protects the cells from Fas:Fas ligand induced apoptosis, and allows the growth of lung metastases. ^

An fMRI-Study on context-specificity of inhibitory functions in alcohol-dependency: Preliminary results

Introduction: Alcohol-dependency is a common disease with many negative consequences in the daily life. A typical symptom of alcoholic-patients is the persistent and uncontrollable desire to consume alcohol. Inspite of different treatments, alcohol-dependency has a relapse rate of about 85%. This high rate is facilitated by a dysfunction of cognitive control-processes. In order to understand this disease sustaining factor, the present study investigated the neurophysiological correlates of inhibition of alcoholic-patients in a neutral as well as an alcohol-related context. Methods: A total of 18 participants, (9 alcohol-dependent-patients (age range: 27-62 years), 9 healthy controls (age range: 29-60 years)) have been measured with functional magnetic resonance imaging while they participated in an alcohol-specific Go/NoGo-Task. Neurophysiological correlates of inhibition in an alcohol-related as well as a neutral context were compared in both groups. Results: When comparing correct stop-trials in alcohol-related to neutral context, only alcohol-dependent patients showed significant hyperactivation in frontal regions (superior and medial gyrus frontalis, anterior gyrus cinguli, gyrus paracentralis and the gyrus praecentralis). No significant differences were found in any of the behavioral analyses. Discussion: These preliminary results thus indicate that successful inhibition in a drug-related context demands additional resources in patients. Especially the hyperactivation of the anterior gyrus cinguli might be important because of its involvement in decision-processes. In the absent of deficits in behavioral data, this suggests that alcohol-dependent patients need more neuronal activity to achieve the same performance-level like healthy controls.