Vascular Response Of Ruthenium Tetraamines In Aortic Ring From Normotensive Rats.

Background: Ruthenium (Ru) tetraamines are being increasingly used as nitric oxide (NO) carriers. In this context, pharmacological studies have become highly relevant to better understand the mechanism of action involved. Objective: To evaluate the vascular response of the tetraamines trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO) (cyclan)](PF6)2, and trans-[RuII(NH3)4(4-acPy)(NO)]3+. Methods: Aortic rings were contracted with noradrenaline (10-6 M). After voltage stabilization, a single concentration (10-6 M) of the compounds was added to the assay medium. The responses were recorded during 120 min. Vascular integrity was assessed functionally using acetylcholine at 10-6 M and sodium nitroprusside at 10-6 M as well as by histological examination. Results: Histological analysis confirmed the presence or absence of endothelial cells in those tissues. All tetraamine complexes altered the contractile response induced by norepinephrine, resulting in increased tone followed by relaxation. In rings with endothelium, the inhibition of endothelial NO caused a reduction of the contractile effect caused by pyridine NO. No significant responses were observed in rings with endothelium after treatment with cyclan NO. In contrast, in rings without endothelium, the inhibition of guanylate cyclase significantly reduced the contractile response caused by the pyridine NO and cyclan NO complexes, and both complexes caused a relaxing effect. Conclusion: The results indicate that the vascular effect of the evaluated complexes involved a decrease in the vascular tone induced by norepinephrine (10-6 M) at the end of the incubation period in aortic rings with and without endothelium, indicating the slow release of NO from these complexes and suggesting that the ligands promoted chemical stability to the molecule. Moreover, we demonstrated that the association of Ru with NO is more stable when the ligands pyridine and cyclan are used in the formulation of the compound.Fundamento: As tetra-aminas de rutênio cada vez mais se destacam como carreadoras da molécula de óxido nítrico. Desse modo, estudos farmacológicos tornam-se altamente relevantes, afim de melhor compreender o mecanismo de ação envolvido. Objetivo: Avaliar a resposta vascular das tetra-aminas trans-[RuII(NH3)4(Py)(NO)]3+, trans-[RuII(Cl)(NO)(Cyclan)](PF6)2 e trans-[RuII(NH3)4(4-acPy)(NO)]3+. Métodos: Anéis de aorta foram pré-contraídos com noradrenalina (10-6M). Após estabilização da tensão, concentração única (10-6M) dos compostos foi adicionada ao banho de incubação. As respostas foram registradas ao longo de 120 minutos. A integridade vascular foi avaliada funcionalmente (acetilcolina 10-6M; nitroprussiato de sódio 10-6M) e histologicamente Resultados: A análise histológica confirmou a presença ou não de células endoteliais nos tecidos analisados. Todos os complexos alteraram a resposta contrátil induzida pela noradrenalina, resultando em aumento de tônus seguido de efeito relaxante. Em anéis com endotélio, a inibição do óxido nítrico endotelial causou redução do efeito contrátil da piridina óxido nítrico. Não foram observadas respostas significativas em anéis com endotélio referente ao composto cyclan óxido nítrico. Por outro lado, em anéis sem endotélio, a inibição da guanilato ciclase reduziu significativamente a resposta contrátil dos complexos piridina óxido nítrico e cyclan óxido nítrico, levando ambos os compostos a um efeito relaxante. Conclusão: Os resultados obtidos demonstram que o efeito vascular dos complexos avaliados apresentaram diminuição no tônus vascular induzido pela noradrenalina (10-6M) ao final do tempo de incubação, em anéis com e sem endotélio, indicando liberação lenta da molécula de óxido nítrico do composto estudado e sugerindo que os ligantes causaram estabilidade química à molécula. Demonstramos que a ligação rutênio óxido nítrico é mais estável quando utilizamos os ligantes piridina e cyclan para a formulação do composto.

Tissue Microarray Is A Reliable Method For Immunohistochemical Analysis Of Pleomorphic Adenoma.

To determine the most adequate number and size of tissue microarray (TMA) cores for pleomorphic adenoma immunohistochemical studies. Eighty-two pleomorphic adenoma cases were distributed in 3 TMA blocks assembled in triplicate containing 1.0-, 2.0-, and 3.0-mm cores. Immunohistochemical analysis against cytokeratin 7, Ki67, p63, and CD34 were performed and subsequently evaluated with PixelCount, nuclear, and microvessel software applications. The 1.0-mm TMA presented lower results than 2.0- and 3.0-mm TMAs versus conventional whole section slides. Possibly because of an increased amount of stromal tissue, 3.0-mm cores presented a higher microvessel density. Comparing the results obtained with one, two, and three 2.0-mm cores, there was no difference between triplicate or duplicate TMAs and a single-core TMA. Considering the possible loss of cylinders during immunohistochemical reactions, 2.0-mm TMAs in duplicate are a more reliable approach for pleomorphic adenoma immunohistochemical study.

Monte Carlo Simulation Of The Response Functions Of Cdte Detectors To Be Applied In X-ray Spectroscopy.

In this work, the energy response functions of a CdTe detector were obtained by Monte Carlo (MC) simulation in the energy range from 5 to 160keV, using the PENELOPE code. In the response calculations the carrier transport features and the detector resolution were included. The computed energy response function was validated through comparison with experimental results obtained with (241)Am and (152)Eu sources. In order to investigate the influence of the correction by the detector response at diagnostic energy range, x-ray spectra were measured using a CdTe detector (model XR-100T, Amptek), and then corrected by the energy response of the detector using the stripping procedure. Results showed that the CdTe exhibits good energy response at low energies (below 40keV), showing only small distortions on the measured spectra. For energies below about 80keV, the contribution of the escape of Cd- and Te-K x-rays produce significant distortions on the measured x-ray spectra. For higher energies, the most important correction is the detector efficiency and the carrier trapping effects. The results showed that, after correction by the energy response, the measured spectra are in good agreement with those provided by a theoretical model of the literature. Finally, our results showed that the detailed knowledge of the response function and a proper correction procedure are fundamental for achieving more accurate spectra from which quality parameters (i.e., half-value layer and homogeneity coefficient) can be determined.

Low-level Laser Therapy To The Mouse Femur Enhances The Fungicidal Response Of Neutrophils Against Paracoccidioides Brasiliensis.

Neutrophils (PMN) play a central role in host defense against the neglected fungal infection paracoccidioidomycosis (PCM), which is caused by the dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is of major importance, especially in Latin America, and its treatment relies on the use of antifungal drugs. However, the course of treatment is lengthy, leading to side effects and even development of fungal resistance. The goal of the study was to use low-level laser therapy (LLLT) to stimulate PMN to fight Pb in vivo. Swiss mice with subcutaneous air pouches were inoculated with a virulent strain of Pb or fungal cell wall components (Zymosan), and then received LLLT (780 nm; 50 mW; 12.5 J/cm2; 30 seconds per point, giving a total energy of 0.5 J per point) on alternate days at two points on each hind leg. The aim was to reach the bone marrow in the femur with light. Non-irradiated animals were used as controls. The number and viability of the PMN that migrated to the inoculation site was assessed, as well as their ability to synthesize proteins, produce reactive oxygen species (ROS) and their fungicidal activity. The highly pure PMN populations obtained after 10 days of infection were also subsequently cultured in the presence of Pb for trials of protein production, evaluation of mitochondrial activity, ROS production and quantification of viable fungi growth. PMN from mice that received LLLT were more active metabolically, had higher fungicidal activity against Pb in vivo and also in vitro. The kinetics of neutrophil protein production also correlated with a more activated state. LLLT may be a safe and non-invasive approach to deal with PCM infection.

Altered Urinary Sodium Excretion Response After Central Cholinergic And Adrenergic Stimulation Of Adult Spontaneously Hypertensive Rats.

In this study, we hypothesized that blunting of the natriuresis response to intracerebroventricularly (i.c.v.) microinjected cholinergic and adrenergic agonists is involved in the development of hypertension in spontaneously hypertensive rats (SHR). We evaluated the effect of i.c.v. injection of cholinergic and noradrenergic agonists, at increasing concentrations, and of muscarinic cholinergic and α1 and α2-adrenoceptor antagonists on blood pressure and urinary sodium handling in SHR, compared with age-matched Wistar Kyoto rats (WR). We confirmed that CCh and NE microinjected into the lateral ventricle (LV) of conscious rats leads to enhanced natriuresis. This response was associated with increased proximal and post-proximal sodium excretion accompanied by an unchanged rate of glomerular filtration. We showed that cholinergic-induced natriuresis in WR and SHR was attenuated by previous i.c.v. administration of atropine and was significantly lower in the hypertensive strain than in WR. In both groups the natriuretic effect of injection of noradrenaline into the LV was abolished by previous local injection of an α1-adrenoceptor antagonist (prazosin). Conversely, LV α2-adrenoceptor antagonist (yohimbine) administration potentiated the action of noradrenaline. The LV yohimbine pretreatment normalized urinary sodium excretion in SHR compared with age-matched WR. In conclusion, these are, as far as we are aware, the first results showing the importance of interaction of central cholinergic and/or noradrenergic receptors in the pathogenesis of spontaneous hypertension. These experiments also provide good evidence of the existence of a central adrenergic mechanism consisting of α1 and α2-adrenoceptors which works antagonistically on regulation of renal sodium excretion.

Reduction Of Oxidative Damage And Inflammatory Response In The Diaphragm Muscle Of Mdx Mice Using Iron Chelator Deferoxamine.

Oxidative stress and inflammatory processes strongly contribute to pathogenesis in Duchenne muscular dystrophy (DMD). Based on evidence that excess iron may increase oxidative stress and contribute to the inflammatory response, we investigated whether deferoxamine (DFX), a potent iron chelating agent, reduces oxidative stress and inflammation in the diaphragm (DIA) muscle of mdx mice (an experimental model of DMD). Fourteen-day-old mdx mice received daily intraperitoneal injections of DFX at a dose of 150 mg/kg body weight, diluted in saline, for 14 days. C57BL/10 and control mdx mice received daily intraperitoneal injections of saline only, for 14 days. Grip strength was evaluated as a functional measure, and blood samples were collected for biochemical assessment of muscle fiber degeneration. In addition, the DIA muscle was removed and processed for histopathology and Western blotting analysis. In mdx mice, DFX reduced muscle damage and loss of muscle strength. DFX treatment also resulted in a significant reduction of dystrophic inflammatory processes, as indicated by decreases in the inflammatory area and in NF-κB levels. DFX significantly decreased oxidative damage, as shown by lower levels of 4-hydroxynonenal and a reduction in dihydroethidium staining in the DIA muscle of mdx mice. The results of the present study suggest that DFX may be useful in therapeutic strategies to ameliorate dystrophic muscle pathology, possibly via mechanisms involving oxidative and inflammatory pathways.

Didanosine-loaded Chitosan Microspheres Optimized By Surface-response Methodology: A Modified Maximum Likelihood Classification" Approach Formulation For Reverse Transcriptase Inhibitors."

Didanosine-loaded chitosan microspheres were developed applying a surface-response methodology and using a modified Maximum Likelihood Classification. The operational conditions were optimized with the aim of maintaining the active form of didanosine (ddI), which is sensitive to acid pH, and to develop a modified and mucoadhesive formulation. The loading of the drug within the chitosan microspheres was carried out by ionotropic gelation technique with sodium tripolyphosphate (TPP) as cross-linking agent and magnesium hydroxide (Mg(OH)2) to assure the stability of ddI. The optimization conditions were set using a surface-response methodology and applying the Maximum Likelihood Classification, where the initial chitosan concentration, TPP and ddI concentration were set as the independent variables. The maximum ddI-loaded in microspheres (i.e. 1433mg of ddI/g chitosan), was obtained with 2% (w/v) chitosan and 10% TPP. The microspheres depicted an average diameter of 11.42μm and ddI was gradually released during 2h in simulated enteric fluid.

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.

A Leucine-rich Diet Modulates The Tumor-induced Down-regulation Of The Mapk/erk And Pi3k/akt/mtor Signaling Pathways And Maintains The Expression Of The Ubiquitin-proteasome Pathway In The Placental Tissue Of Nmri Mice.

Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process.

Application Of Laser Microdissection Icp-ms For High Resolution Elemental Mapping In Mouse Brain Tissue: A Comparative Study With Laser Ablation Icp-ms.

Mapping of elements in biological tissue by laser induced mass spectrometry is a fast growing analytical methodology in life sciences. This method provides a multitude of useful information of metal, nonmetal, metalloid and isotopic distribution at major, minor and trace concentration ranges, usually with a lateral resolution of 12-160 µm. Selected applications in medical research require an improved lateral resolution of laser induced mass spectrometric technique at the low micrometre scale and below. The present work demonstrates the applicability of a recently developed analytical methodology - laser microdissection associated to inductively coupled plasma mass spectrometry (LMD ICP-MS) - to obtain elemental images of different solid biological samples at high lateral resolution. LMD ICP-MS images of mouse brain tissue samples stained with uranium and native are shown, and a direct comparison of LMD and laser ablation (LA) ICP-MS imaging methodologies, in terms of elemental quantification, is performed.

Magnetic resonance imaging in the evaluation of patients with aseptic meningoencephalitis and connective tissue disorders

OBJECTIVE: To describe the role of magnetic resonance imaging (MRI) in the evaluation of patients with chronic and recurrent aseptic meningitis.METHOD: A retrospective study of five patients with aseptic meningoencefalitis diagnosed by clinical and CSF findings. CT scans showed without no relevant findings. RESULTS: MRI showed small multifocal lesions hyperintense on T2 weighted images and FLAIR, with mild or no gadolinium enhancement, mainly in periventricular and subcortical regions. Meningoencephalitis preceded the diagnosis of the underlying disease in four patients (Behçet´s disease or systemic lupus erythematosus). After the introduction of adequate treatment for the rheumatic disease, they did not present further symptoms of aseptic meningoencephalitis. CONCLUSION: Aseptic meningoencephalitis can be an early presentation of an autoimmune disease. It is important to emphasize the role of MRI in the diagnosis and follow-up of these patients.

Tissue culture of Cecropia glaziovii Sneth (urticaceae): vegetative micropropagation and plant regeneration from callus

Cecropia glaziovii is a tree with used in Brazilian popular medicine. Methods allowing the clonal propagation of this species are of great interest for superior genotype multiplication and perpetuation. For this reason, we examined the effect of different culture media and different types of explants on adventitious shoot regeneration from callus and buds of C. glaziovii. Leaves, petioles and stipules obtained from aseptically grown seedlings or from pre-sterilized plants were used to initiate cultures. Adventitious shoot regeneration was achieved when apical and axillary buds were inoculated on gelled Murashige & Skoog (MS) medium supplemented with 6-benzylaminopurine alone (BAP) (1.0, 5.0 or 10.0 mg L-1) or combined with -naphthalene acetic acid (NAA) (1.0 or 2.0 mg L-1), after 40 days of culture. Best callus production was obtained after 30 days of petioles' culture on gelled MS medium with 2,4 dichlorophenoxyacetic acid (2,4-D) (5.0 mg L-1) combined with BAP (1.0 mg L-1). Successful shoot regeneration from callus was achieved when MS medium supplemented with zeatin (ZEA) (0.1 mg L-1) alone or combined with 2,4-D (1.0 or 5.0 mg L-1) was inoculated with friable callus obtained from petioles. All shoots were rooted by inoculation on MS medium supplemented with indole-3-acetic acid (IAA) (1.0 mg L-1). Rooted plants transferred to potting soil were successfully established. All in vitro regenerated plantlets showed to be normal, without morphological variations, being also identical to the source plant. Our study has shown that C. glaziovii can be propagated by tissue culture methods, allowing large scale multiplication of superior plants for pharmacological purposes.

Paramentros fisiologicos, motores e morfologicos de bailarinas classicas

Universidade Estadual de Campinas . Faculdade de Educação Física

Pulp tissue from primary teeth: new source of stem cells

SHED (stem cells from human exfoliated deciduous teeth) represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.