Biological activity of metal-edds (ethylenediaminedisuccinate) complexes in K562 and PBMC cells

The effect of S,S-ethylenediaminedisuccinic acid (edds) on the quenching of metal-catalyzed (metal = Mn, Fe, Co, Ni, Cu, Zn) oxidation of ascorbic acid was tested in vitro via oxidation of the fluorescent probe 1,2,3-dihydrorhodamine dihydrochloride. The pro-oxidant activity of iron was not fully suppressed, even at a four-fold molar excess of the ligand. The effect of serum on the toxicity to peripheral blood mononuclear cells (PBMC) and K562 cells was investigated. The cytotoxic effect of Fe-edds was abrogated in the presence of Trolox or serum proteins. The probable pathways of cell toxicity were investigated through blocking of the monocarboxylate transporters (MCT) in association with cell cycle studies by flow cytometry. Cells treated with metal complexes and alpha-cyano-4-hydroxycinnamic acid, a known MCT inhibitor, showed recovery of viability, suggesting that MCT proteins may be involved in the internalization of metal-edds complexes. The free acid induced cell cycle arrest in G0/G1 (PBMC) and S (K562) phases, suggesting direct DNA damage or interference in DNA replication.

Central chemoreceptors and neural mechanisms of cardiorespiratory control

The arterial partial pressure (P CO2) of carbon dioxide is virtually constant because of the close match between the metabolic production of this gas and its excretion via breathing. Blood gas homeostasis does not rely solely on changes in lung ventilation, but also to a considerable extent on circulatory adjustments that regulate the transport of CO2 from its sites of production to the lungs. The neural mechanisms that coordinate circulatory and ventilatory changes to achieve blood gas homeostasis are the subject of this review. Emphasis will be placed on the control of sympathetic outflow by central chemoreceptors. High levels of CO2 exert an excitatory effect on sympathetic outflow that is mediated by specialized chemoreceptors such as the neurons located in the retrotrapezoid region. In addition, high CO2 causes an aversive awareness in conscious animals, activating wake-promoting pathways such as the noradrenergic neurons. These neuronal groups, which may also be directly activated by brain acidification, have projections that contribute to the CO2-induced rise in breathing and sympathetic outflow. However, since the level of activity of the retrotrapezoid nucleus is regulated by converging inputs from wake-promoting systems, behavior-specific inputs from higher centers and by chemical drive, the main focus of the present manuscript is to review the contribution of central chemoreceptors to the control of autonomic and respiratory mechanisms.

Engagement of multifocal neural circuits during recall of autobiographical happy events

Happy emotional states have not been extensively explored in functional magnetic resonance imaging studies using autobiographic recall paradigms. We investigated the brain circuitry engaged during induction of happiness by standardized script-driven autobiographical recall in 11 healthy subjects (6 males), aged 32.4 ± 7.2 years, without physical or psychiatric disorders, selected according to their ability to vividly recall personal experiences. Blood oxygen level-dependent (BOLD) changes were recorded during auditory presentation of personal scripts of happiness, neutral content and negative emotional content (irritability). The same uniform structure was used for the cueing narratives of both emotionally salient and neutral conditions, in order to decrease the variability of findings. In the happiness relative to the neutral condition, there was an increased BOLD signal in the left dorsal prefrontal cortex and anterior insula, thalamus bilaterally, left hypothalamus, left anterior cingulate gyrus, and midportions of the left middle temporal gyrus (P < 0.05, corrected for multiple comparisons). Relative to the irritability condition, the happiness condition showed increased activity in the left insula, thalamus and hypothalamus, and in anterior and midportions of the inferior and middle temporal gyri bilaterally (P < 0.05, corrected), varying in size between 13 and 64 voxels. Findings of happiness-related increased activity in prefrontal and subcortical regions extend the results of previous functional imaging studies of autobiographical recall. The BOLD signal changes identified reflect general aspects of emotional processing, emotional control, and the processing of sensory and bodily signals associated with internally generated feelings of happiness. These results reinforce the notion that happiness induction engages a wide network of brain regions.

Induction of Zenk protein expression within the nucleus taeniae of the amygdala of pigeons following tone and shock stimulation

In this study, we evaluated the expression of the Zenk protein within the nucleus taeniae of the pigeon’s amygdala (TnA) after training in a classical aversive conditioning, in order to improve our understanding of its functional role in birds. Thirty-two 18-month-old adult male pigeons (Columba livia), weighing on average 350 g, were trained under different conditions: with tone-shock associations (experimental group; EG); with shock-alone presentations (shock group; SG); with tone-alone presentations (tone group; TG); with exposure to the training chamber without stimulation (context group; CG), and with daily handling (naive group; NG). The number of immunoreactive nuclei was counted in the whole TnA region and is reported as density of Zenk-positive nuclei. This density of Zenk-positive cells in the TnA was significantly greater for the EG, SG and TG than for the CG and NG (P < 0.05). The data indicate an expression of Zenk in the TnA that was driven by experience, supporting the role of this brain area as a critical element for neural processing of aversive stimuli as well as meaningful novel stimuli.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Unconventional neurotransmitters, neurodegeneration and neuroprotection

Neurotransmitters are also involved in functions other than conventional signal transfer between nerve cells, such as development, plasticity, neurodegeneration, and neuroprotection. For example, there is a considerable amount of data indicating developmental roles for the glutamatergic, cholinergic, dopaminergic, GABA-ergic, and ATP/adenosine systems. In this review, we discuss the existing literature on these "new" functions of neurotransmitters in relation to some unconventional neurotransmitters, such as the endocannabinoids and nitric oxide. Data indicating both transcriptional and post-transcriptional modulation of endocannabinoid and nitrinergic systems after neural lesions are discussed in relation to the non-conventional roles of these neurotransmitters. Knowledge of the roles of neurotransmitters in brain functions other than information transfer is critical for a more complete understanding of the functional organization of the brain and to provide more opportunities for the development of therapeutical tools aimed at minimizing neuronal death.

Hypothyroidism decreases proinsulin gene expression and the attachment of its mRNA and eEF1A protein to the actin cytoskeleton of INS-1E cells

The actions of thyroid hormone (TH) on pancreatic beta cells have not been thoroughly explored, with current knowledge being limited to the modulation of insulin secretion in response to glucose, and beta cell viability by regulation of pro-mitotic and pro-apoptotic factors. Therefore, the effects of TH on proinsulin gene expression are not known. This led us to measure: a) proinsulin mRNA expression, b) proinsulin transcripts and eEF1A protein binding to the actin cytoskeleton, c) actin cytoskeleton arrangement, and d) proinsulin mRNA poly(A) tail length modulation in INS-1E cells cultured in different media containing: i) normal fetal bovine serum - FBS (control); ii) normal FBS plus 1 µM or 10 nM T3, for 12 h, and iii) FBS depleted of TH for 24 h (Tx). A decrease in proinsulin mRNA content and attachment to the cytoskeleton were observed in hypothyroid (Tx) beta cells. The amount of eEF1A protein anchored to the cytoskeleton was also reduced in hypothyroidism, and it is worth mentioning that eEF1A is essential to attach transcripts to the cytoskeleton, which might modulate their stability and rate of translation. Proinsulin poly(A) tail length and cytoskeleton arrangement remained unchanged in hypothyroidism. T3 treatment of control cells for 12 h did not induce any changes in the parameters studied. The data indicate that TH is important for proinsulin mRNA expression and translation, since its total amount and attachment to the cytoskeleton are decreased in hypothyroid beta cells, providing evidence that effects of TH on carbohydrate metabolism also include the control of proinsulin gene expression.

Interstitial and Langerhans' dendritic cells in chronic periodontitis and gingivitis

The aim of the present study was to compare quantitatively the distribution of dendritic cell subpopulations in chronic periodontitis and gingivitis. Fourteen biopsies from patients with chronic periodontitis and fifteen from patients with gingivitis were studied. An immunoperoxidase technique was used to quantify the number of Langerhans' cells (CD1a) and interstitial dendritic cells (factor XIIIa) in the oral and sulcular and junctional/pocket epithelia and in the lamina propria. A greater number of factor XIIIa+ dendritic cells in the lamina propria and CD1a+ dendritic cells in the oral epithelium were observed in gingivitis compared to the periodontitis group (p = 0.05). In the sulcular and junctional/pocket epithelia and in the lamina propria, the number of CD1a+ dendritic cells was similar in the gingivitis and periodontitis groups. In conclusion, the number of Langerhans' cells in the oral epithelium and interstitial dendritic cells in the lamina propria is increased in gingivitis compared to periodontitis, which may contribute to the different pattern of host response in these diseases.

Neural networks and statistical analysis for classification of corneal videokeratography maps based on Zernike coefficients: a quantitative comparison

PURPOSE: The main goal of this study was to develop and compare two different techniques for classification of specific types of corneal shapes when Zernike coefficients are used as inputs. A feed-forward artificial Neural Network (NN) and discriminant analysis (DA) techniques were used. METHODS: The inputs both for the NN and DA were the first 15 standard Zernike coefficients for 80 previously classified corneal elevation data files from an Eyesys System 2000 Videokeratograph (VK), installed at the Departamento de Oftalmologia of the Escola Paulista de Medicina, São Paulo. The NN had 5 output neurons which were associated with 5 typical corneal shapes: keratoconus, with-the-rule astigmatism, against-the-rule astigmatism, "regular" or "normal" shape and post-PRK. RESULTS: The NN and DA responses were statistically analyzed in terms of precision ([true positive+true negative]/total number of cases). Mean overall results for all cases for the NN and DA techniques were, respectively, 94% and 84.8%. CONCLUSION: Although we used a relatively small database, results obtained in the present study indicate that Zernike polynomials as descriptors of corneal shape may be a reliable parameter as input data for diagnostic automation of VK maps, using either NN or DA.

Learning processes and the neural analysis of conditioning

Classical and operant conditioning principles, such as the behavioral discrepancy-derived assumption that reinforcement always selects antecedent stimulus and response relations, have been studied at the neural level, mainly by observing the strengthening of neuronal responses or synaptic connections. A review of the literature on the neural basis of behavior provided extensive scientific data that indicate a synthesis between the two conditioning processes based mainly on stimulus control in learning tasks. The resulting analysis revealed the following aspects. Dopamine acts as a behavioral discrepancy signal in the midbrain pathway of positive reinforcement, leading toward the nucleus accumbens. Dopamine modulates both types of conditioning in the Aplysia mollusk and in mammals. In vivo and in vitro mollusk preparations show convergence of both types of conditioning in the same motor neuron. Frontal cortical neurons are involved in behavioral discrimination in reversal and extinction procedures, and these neurons preferentially deliver glutamate through conditioned stimulus or discriminative stimulus pathways. Discriminative neural responses can reliably precede operant movements and can also be common to stimuli that share complex symbolic relations. The present article discusses convergent and divergent points between conditioning paradigms at the neural level of analysis to advance our knowledge on reinforcement.

A century of research: what have we learned about the interaction of Trypanosoma cruzi with host cells?

Since the discovery of Trypanosoma cruzi and the brilliant description of the then-referred to "new tripanosomiasis" by Carlos Chagas 100 years ago, a great deal of scientific effort and curiosity has been devoted to understanding how this parasite invades and colonises mammalian host cells. This is a key step in the survival of the parasite within the vertebrate host, and although much has been learned over this century, differences in strains or isolates used by different laboratories may have led to conclusions that are not as universal as originally interpreted. Molecular genotyping of the CL-Brener clone confirmed a genetic heterogeneity in the parasite that had been detected previously by other techniques, including zymodeme or schizodeme (kDNA) analysis. T. cruzi can be grouped into at least two major phylogenetic lineages: T. cruzi I, mostly associated with the sylvatic cycle and T. cruzi II, linked to human disease; however, a third lineage, T. cruziIII, has also been proposed. Hybrid isolates, such as the CL-Brener clone, which was chosen for sequencing the genome of the parasite (Elias et al. 2005, El Sayed et al. 2005a), have also been identified. The parasite must be able to invade cells in the mammalian host, and many studies have implicated the flagellated trypomastigotes as the main actor in this process. Several surface components of parasites and some of the host cell receptors with which they interact have been described. Herein, we have attempted to identify milestones in the history of understanding T. cruzi- host cell interactions. Different infective forms of T. cruzi have displayed unexpected requirements for the parasite to attach to the host cell, enter it, and translocate between the parasitophorous vacuole to its final cytoplasmic destination. It is noteworthy that some of the mechanisms originally proposed to be broad in function turned out not to be universal, and multiple interactions involving different repertoires of molecules seem to act in concert to give rise to a rather complex interplay of signalling cascades involving both parasite and cellular components.

Evaluation of follicular lymphoid depletion in the Bursa of Fabricius: an alternative methodology using digital image analysis and artificial neural networks

Fifty Bursa of Fabricius (BF) were examined by conventional optical microscopy and digital images were acquired and processed using Matlab® 6.5 software. The Artificial Neuronal Network (ANN) was generated using Neuroshell® Classifier software and the optical and digital data were compared. The ANN was able to make a comparable classification of digital and optical scores. The use of ANN was able to classify correctly the majority of the follicles, reaching sensibility and specificity of 89% and 96%, respectively. When the follicles were scored and grouped in a binary fashion the sensibility increased to 90% and obtained the maximum value for the specificity of 92%. These results demonstrate that the use of digital image analysis and ANN is a useful tool for the pathological classification of the BF lymphoid depletion. In addition it provides objective results that allow measuring the dimension of the error in the diagnosis and classification therefore making comparison between databases feasible.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.