Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.

New Interaction Partners For Nek4.1 And Nek4.2 Isoforms: From The Dna Damage Response To Rna Splicing.

Neks are serine-threonine kinases that are similar to NIMA, a protein found in Aspergillus nidulans which is essential for cell division. In humans there are eleven Neks which are involved in different biological functions besides the cell cycle control. Nek4 is one of the largest members of the Nek family and has been related to the primary cilia formation and in DNA damage response. However, its substrates and interaction partners are still unknown. In an attempt to better understand the role of Nek4, we performed an interactomics study to find new biological processes in which Nek4 is involved. We also described a novel Nek4 isoform which lacks a region of 46 amino acids derived from an insertion of an Alu sequence and showed the interactomics profile of these two Nek4 proteins. Isoform 1 and isoform 2 of Nek4 were expressed in human cells and after an immunoprecipitation followed by mass spectrometry, 474 interacting proteins were identified for isoform 1 and 149 for isoform 2 of Nek4. About 68% of isoform 2 potential interactors (102 proteins) are common between the two Nek4 isoforms. Our results reinforce Nek4 involvement in the DNA damage response, cilia maintenance and microtubule stabilization, and raise the possibility of new functional contexts, including apoptosis signaling, stress response, translation, protein quality control and, most intriguingly, RNA splicing. We show for the first time an unexpected difference between both Nek4 isoforms in RNA splicing control. Among the interacting partners, we found important proteins such as ANT3, Whirlin, PCNA, 14-3-3ε, SRSF1, SRSF2, SRPK1 and hNRNPs proteins. This study provides new insights into Nek4 functions, identifying new interaction partners and further suggests an interesting difference between isoform 1 and isoform 2 of this kinase. Nek4 isoform 1 may have similar roles compared to other Neks and these roles are not all preserved in isoform 2. Besides, in some processes, both isoforms showed opposite effects, indicating a possible fine controlled regulation.

Jnk And Ikkβ Phosphorylation Is Reduced By Glucocorticoids In Adipose Tissue From Insulin-resistant Rats.

Peripheral insulin resistance (IR) is one of the main side effects caused by glucocorticoid (GC)-based therapies, and the molecular mechanisms of GC-induced IR are not yet fully elucidated. Thus, we aimed to investigate the effects of dexamethasone treatment on the main components of insulin and inflammatory signaling in the adipose tissue of rats. Male Wistar rats received daily injections of dexamethasone (1mg/kg body weight (b.w.), intraperitoneally (i.p.)) for 5 days (DEX), whereas control rats received saline (CTL). The metabolic status was investigated, and the epididymal fat fragments were collected for lipolysis and western blot analyses. The DEX rats became hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, compared with the CTL rats (P<0.05). The basal glycerol release in the fat fragments was 1.5-fold higher in the DEX rats (P<0.05). The phosphorylation of protein kinase B (PKB) at ser(473) decreased by 44%, whereas, the phosphorylation of insulin receptor substrate (IRS)-1 at ser(307) increased by 93% in the adipose tissue of the DEX rats after an oral bolus of glucose (P<0.05). The basal phosphorylation of c-jun-N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B (IKKβ) proteins was reduced by 46% and 58%, respectively, in the adipose tissue of the DEX rats (P<0.05). This was paralleled with a significant reduction (47%) in the glucocorticoid receptor (GR) protein content in the adipose tissue of the DEX rats (P<0.05). The insulin-resistant status of rats induced by dexamethasone administration have PKB and IRS-1 activity attenuated in epididymal fat without increases in the phosphorylation of the proinflammatory signals JNK and IKKβ.

Análise de biomarcadores sanguíneos em estímulos anaeróbios máximos no nado crawl

Universidade Estadual de Campinas . Faculdade de Educação Física

1. Jogos Escolares Brasileiros da Confederação Brasileira de Desportos para Cegos : um estudo de caso

Universidade Estadual de Campinas . Faculdade de Educação Física

Effects of light-curing time on the cytotoxicity of a restorative composite resin on odontoblast-like cells

This in vitro study evaluated the cytotoxicity of an experimental restorative composite resin subjected to different light-curing regimens. METHODS: Forty round-shaped specimens were prepared and randomly assigned to four experimental groups (n=10), as follows: in Group 1, no light-curing; in Groups 2, 3 and 4, the composite resin specimens were light-cured for 20, 40 or 60 s, respectively. In Group 5, filter paper discs soaked in 5 µL PBS were used as negative controls. The resin specimens and paper discs were placed in wells of 24-well plates in which the odontoblast-like cells MDPC-23 (30,000 cells/cm²) were plated and incubated in a humidified incubator with 5% CO2 and 95% air at 37ºC for 72 h. The cytotoxicity was evaluated by the cell metabolism (MTT assay) and cell morphology (SEM). The data were analyzed statistically by Kruskal-Wallis and Mann-Whitney tests (p<0.05). RESULTS: In G1, cell metabolism decreased by 86.2%, indicating a severe cytotoxicity of the non-light-cured composite resin. On the other hand, cell metabolism decreased by only 13.3% and 13.5% in G2 and G3, respectively. No cytotoxic effects were observed in G4 and G5. In G1, only a few round-shaped cells with short processes on their cytoplasmic membrane were observed. In the other experimental groups as well as in control group, a number of spindle-shaped cells with long cytoplasmic processes were found. CONCLUSION: Regardless of the photoactivation time used in the present investigation, the experimental composite resin presented mild to no toxic effects to the odontoblast-like MDPC-23 cells. However, intense cytotoxic effects occurred when no light-curing was performed.

Effect of drying on the reproducibility of DIAGNOdent to detect caries-like lesions

The aim of this study was to verify the drying effect on the reproducibility of DIAGNOdent (Dd) devices to detect caries-like lesions. Three areas were created in each of the 34 bovine incisors: sound (S), demineralized (DE) and remineralized (RE). One examiner measured each area with two Dd devices (denominated X and Y), twice under humid, and twice under dry condition. Intra-rater agreement according each device and inter-device agreement were estimated by kappa statistics (k). Intra-rater agreement for device Y was substantial under humid (k DE=0.68 and k RE+S=0.68) and dry condition (k DE=0.64 and k RE+S=0.67). For device X, it was substantial under humid condition (k DE=0.57 and k RE+S=0.49), and it was almost perfect after air drying (k DE=1.0 and kRE+S=1.0). Inter-device agreement was slight (k =0.17) under humid condition, and it was substantial under dry condition (k =0.62). As reproducibility increased under dry condition, drying is advised to detect caries-like lesions on free smooth surfaces when different devices are used.

Differential regulation of PGC-1 alpha expression in rat liver and skeletal muscle in response to voluntary running

Background: The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1 alpha. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1 alpha protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism. Methods: Two groups of male Wistar rats (2 Mo of age, 188.82 +/- 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1 alpha protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations. Results: Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean +/- SE) of 4.102 +/- 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1 alpha protein expression increased significantly from a 1.11 +/- 0.12 in the sedentary rats to 1.74 +/- 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1 alpha protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1 alpha protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle. Conclusion: These data suggest that PGC-1 alpha most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.

Acute reversible inactivation of the bed nucleus of stria terminalis induces antidepressant-like effect in the rat forced swimming test

Background: The bed nucleus of stria terminalis (BNST) is a limbic forebrain structure involved in hypothalamo-pituitary-adrenal axis regulation and stress adaptation. Inappropriate adaptation to stress is thought to compromise the organism's coping mechanisms, which have been implicated in the neurobiology of depression. However, the studies aimed at investigating BNST involvement in depression pathophysiology have yielded contradictory results. Therefore, the objective of the present study was to investigate the effects of temporary acute inactivation of synaptic transmission in the BNST by local microinjection of cobalt chloride (CoCl(2)) in rats subjected to the forced swimming test (FST). Methods: Rats implanted with cannulae aimed at the BNST were submitted to 15 min of forced swimming (pretest). Twenty- four hours later immobility time was registered in a new 5 min forced swimming session (test). Independent groups of rats received bilateral microinjections of CoCl(2) (1 mM/100 nL) before or immediately after pretest or before the test session. Additional groups received the same treatment and were submitted to the open field test to control for unspecific effects on locomotor behavior. Results: CoCl(2) injection into the BNST before either the pretest or test sessions reduced immobility in the FST, suggesting an antidepressant-like effect. No significant effect of CoCl(2) was observed when it was injected into the BNST immediately after pretest. In addition, no effect of BNST inactivation was observed in the open field test. Conclusion: These results suggest that acute reversible inactivation of synaptic transmission in the BNST facilitates adaptation to stress and induces antidepressant-like effects.

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8 +/- 25.2 nM and 167.39 +/- 60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a KD of 55.4 +/- 15.9 nM to laminin and of 290.8 +/- 11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Serotonin receptors are involved in the spinal mediation of descending facilitation of surgical incision-induced increase of Fos-like immunoreactivity in rats

Background: Descending pronociceptive pathways may be implicated in states of persistent pain. Paw skin incision is a well-established postoperative pain model that causes behavioral nociceptive responses and enhanced excitability of spinal dorsal horn neurons. The number of spinal c-Fos positive neurons of rats treated intrathecally with serotonin, noradrenaline or acetylcholine antagonists where evaluated to study the descending pathways activated by a surgical paw incision. Results: The number of c-Fos positive neurons in laminae I/II ipsilateral, lamina V bilateral to the incised paw, and in lamina X significantly increased after the incision. These changes: remained unchanged in phenoxybenzamine-treated rats; were increased in the contralateral lamina V of atropine-treated rats; were inhibited in the ipsilateral lamina I/II by 5-HT(1/2B/2C) (methysergide), 5-HT(2A) (ketanserin) or 5-HT(1/2A/2C/5/6/7) (methiothepin) receptors antagonists, in the ipsilateral lamina V by methysergide or methiothepin, in the contralateral lamina V by all the serotonergic antagonists and in the lamina X by LY 278,584, ketanserin or methiothepin. Conclusions: We conclude: (1) muscarinic cholinergic mechanisms reduce incision-induced response of spinal neurons inputs from the contralateral paw; (2) 5-HT(1/2A/2C/3) receptors-mediate mechanisms increase the activity of descending pathways that facilitates the response of spinal neurons to noxious inputs from the contralateral paw; (3) 5-HT(1/2A/2C) and 5-HT(1/2C) receptors increases the descending facilitation mechanisms induced by incision in the ipsilateral paw; (4) 5-HT(2A/3) receptors contribute to descending pronociceptive pathways conveyed by lamina X spinal neurons; (5) alpha-adrenergic receptors are unlikely to participate in the incision-induced facilitation of the spinal neurons.

The Monocarboxylate Transporter 8 and L-Type Amino Acid Transporters 1 and 2 Are Expressed in Mouse Skeletons and in Osteoblastic MC3T3-E1 Cells

Background: Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. Methods: The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. Results: MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. Conclusions: This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

Normal and defective mariner-like elements in Rhynchosciara species (Sciaridae, Diptera)

Mariner-like elements are widely present in diverse organisms. These elements constitute a large fraction of the eukaryotic genome; they transpose by a ""cut-and-paste"" mechanism with their own transposase protein. We found two groups of mobile elements in the genus Rhynchosciara. PCR using primers designed from R. americana transposons (Ramar1 and Ramar2) were the starting point for this comparative study. Genomic DNA templates of four species: R. hollaenderi, R. millerii, R. baschanti, and Rhynchosciara sp were used and genomic sequences were amplified, sequenced and the molecular structures of the elements characterized as being putative mariner-like elements. The first group included the putative full-length elements. The second group was composed of defective mariner elements that contain a deletion overlapping most of the internal region of the transposase open reading frame. They were named Rmar1 (type 1) and Rmar2 (type 2), respectively. Many conserved amino acid blocks were identified, as well as a specific D,D(34) D signature motif that was defective in some elements. Based on predicted transposase sequences, these elements encode truncated proteins and are phylogenetically very close to mariner-like elements of the mauritiana subfamily. The inverted terminal repeat sequences that flanked the mariner-like elements are responsible for their mobility. These inverted terminal repeat sequences were identified by inverse PCR.

Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.

Thermodynamic origin of cooperativity in actomyosin interactions: The coupling of short-range interactions with actin bending stiffness in an Ising-like model

We present Monte Carlo simulations for a molecular motor system found in virtually all eukaryotic cells, the acto-myosin motor system, composed of a group of organic macromolecules. Cell motors were mapped to an Ising-like model, where the interaction field is transmitted through a tropomyosin polymer chain. The presence of Ca(2+) induces tropomyosin to block or unblock binding sites of the myosin motor leading to its activation or deactivation. We used the Metropolis algorithm to find the transient and the equilibrium states of the acto-myosin system composed of solvent, actin, tropomyosin, troponin, Ca(2+), and myosin-S1 at a given temperature, including the spatial configuration of tropomyosin on the actin filament surface. Our model describes the short- and long-range cooperativity during actin-myosin binding which emerges from the bending stiffness of the tropomyosin complex. We found all transition rates between the states only using the interaction energy of the constituents. The agreement between our model and experimental data also supports the recent theory of flexible tropomyosin.