The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.

Transcriptome analysis of nicotine-exposed cells from the brainstem of neonate spontaneously hypertensive and Wistar Kyoto rats

In this study, the effects of nicotine on global gene expression of cultured cells from the brainstem of spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats were evaluated using whole-genome oligoarrays. We found that nicotine may act differentially on the gene expression profiles of SHR and WKY. The influence of strain was present in 321 genes that were differentially expressed in SHR as compared with WKY brainstem cells independently of the nicotine treatment. A total of 146 genes had their expression altered in both strains after nicotine exposure. Interaction between nicotine treatment and the strain was observed to affect the expression of 229 genes that participate in cellular pathways related to neurotransmitter secretion, intracellular trafficking and cell communication, and are possibly involved in the phenotypic differentiation between SHR and WKY rats, including hypertension. Further characterization of their function in hypertension development is warranted. The Pharmacogenomics Journal (2010) 10, 134-160; doi:10.1038/tpj.2009.42; published online 15 September 2009

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

The Evolution of Modularity in the Mammalian Skull I: Morphological Integration Patterns and Magnitudes

Morphological integration refers to the modular structuring of inter-trait relationships in an organism, which could bias the direction and rate of morphological change, either constraining or facilitating evolution along certain dimensions of the morphospace. Therefore, the description of patterns and magnitudes of morphological integration and the analysis of their evolutionary consequences are central to understand the evolution of complex traits. Here we analyze morphological integration in the skull of several mammalian orders, addressing the following questions: are there common patterns of inter-trait relationships? Are these patterns compatible with hypotheses based on shared development and function? Do morphological integration patterns and magnitudes vary in the same way across groups? We digitized more than 3,500 specimens spanning 15 mammalian orders, estimated the correspondent pooled within-group correlation and variance/covariance matrices for 35 skull traits and compared those matrices among the orders. We also compared observed patterns of integration to theoretical expectations based on common development and function. Our results point to a largely shared pattern of inter-trait correlations, implying that mammalian skull diversity has been produced upon a common covariance structure that remained similar for at least 65 million years. Comparisons with a rodent genetic variance/covariance matrix suggest that this broad similarity extends also to the genetic factors underlying phenotypic variation. In contrast to the relative constancy of inter-trait correlation/covariance patterns, magnitudes varied markedly across groups. Several morphological modules hypothesized from shared development and function were detected in the mammalian taxa studied. Our data provide evidence that mammalian skull evolution can be viewed as a history of inter-module parcellation, with the modules themselves being more clearly marked in those lineages with lower overall magnitude of integration. The implication of these findings is that the main evolutionary trend in the mammalian skull was one of decreasing the constraints to evolution by promoting a more modular architecture.

The Evolution of Modularity in the Mammalian Skull II: Evolutionary Consequences

Changes in patterns and magnitudes of integration may influence the ability of a species to respond to selection. Consequently, modularity has often been linked to the concept of evolvability, but their relationship has rarely been tested empirically. One possible explanation is the lack of analytical tools to compare patterns and magnitudes of integration among diverse groups that explicitly relate these aspects to the quantitative genetics framework. We apply such framework here using the multivariate response to selection equation to simulate the evolutionary behavior of several mammalian orders in terms of their flexibility, evolvability and constraints in the skull. We interpreted these simulation results in light of the integration patterns and magnitudes of the same mammalian groups, described in a companion paper. We found that larger magnitudes of integration were associated with a blur of the modules in the skull and to larger portions of the total variation explained by size variation, which in turn can exert a strong evolutionary constraint, thus decreasing the evolutionary flexibility. Conversely, lower overall magnitudes of integration were associated with distinct modules in the skull, to smaller fraction of the total variation associated with size and, consequently, to weaker constraints and more evolutionary flexibility. Flexibility and constraints are, therefore, two sides of the same coin and we found them to be quite variable among mammals. Neither the overall magnitude of morphological integration, the modularity itself, nor its consequences in terms of constraints and flexibility, were associated with absolute size of the organisms, but were strongly associated with the proportion of the total variation in skull morphology captured by size. Therefore, the history of the mammalian skull is marked by a trade-off between modularity and evolvability. Our data provide evidence that, despite the stasis in integration patterns, the plasticity in the magnitude of integration in the skull had important consequences in terms of evolutionary flexibility of the mammalian lineages.

Novel OTOF mutations in Brazilian patients with auditory neuropathy

The OTOF gene encoding otoferlin is associated with auditory neuropathy (AN), a type of non-syndromic deafness. We investigated the contribution of OTOF mutations to AN and to non-syndromic recessive deafness in Brazil. A test for the Q829X mutation was carried out on a sample of 342 unrelated individuals with non-syndromic hearing loss, but none presented this mutation. We selected 48 cases suggestive of autosomal recessive inheritance, plus four familial and seven isolated cases of AN, for genotyping of five microsatellite markers linked to the OTOF gene. The haplotype analysis showed compatibility with linkage in 11 families (including the four families with AN). Samples of the 11 probands from these families and from seven isolated cases of AN were selected for an exon-by-exon screening for mutations in the OTOF gene. Ten different pathogenic variants were detected, among which six are novel. Among the 52 pedigrees with autosomal recessive inheritance (including four familial cases of AN), mutations were identified in 4 (7.7%). Among the 11 probands with AN, seven had at least one pathogenic mutation in the OTOF gene. Mutations in the OTOF gene are frequent causes of AN in Brazil and our results confirm that they are spread worldwide. Journal of Human Genetics (2009) 54, 382-385; doi: 10.1038/jhg.2009.45; published online 22 May 2009

Forebrain projections to brainstem nuclei involved in the control of mandibular movements in rats

Mandibular movements occur through the triggering of trigeminal motoneurons. Aberrant movements by orofacial muscles are characteristic of orofacial motor disorders, such as nocturnal bruxism (clenching or grinding of the dentition during sleep). Previous studies have suggested that autonomic changes occur during bruxism episodes. Although it is known that emotional responses increase jaw movement, the brain pathways linking forebrain limbic nuclei and the trigeminal motor nucleus remain unclear. Here we show that neurons in the lateral hypothalamic area, in the central nucleus of the amygdala, and in the parasubthalamic nucleus, project to the trigeminal motor nucleus or to reticular regions around the motor nucleus (Regio h) and in the mesencephalic trigeminal nucleus. We observed orexin co-expression in neurons projecting from the lateral hypothalamic area to the trigeminal motor nucleus. In the central nucleus of the amygdala, neurons projecting to the trigeminal motor nucleus are innervated by corticotrophin-releasing factor immunoreactive fibers. We also observed that the mesencephalic trigeminal nucleus receives dense innervation from orexin and corticotrophin-releasing factor immunoreactive fibers. Therefore, forebrain nuclei related to autonomic control and stress responses might influence the activity of trigeminal motor neurons and consequently play a role in the physiopathology of nocturnal bruxism.

Augmentation of insulin secretion by leucine supplementation in malnourished rats: possible involvement of the phosphatidylinositol 3-phosphate kinase/mammalian target protein of rapamycin pathway

A regimen of low-protein diet induces a reduction of pancreatic islet function that is associated with development of metabolic disorders including diabetes and obesity afterward. In the present study, the influence of leucine supplementation on metabolic parameters, insulin secretion to glucose and to amino acids, as well as the levels of proteins that participate in the phosphatidylinositol 3-phosphate kinase (PI3K) pathway was investigated in malnourished rats. Four groups were fed with different diets for 12 weeks: a normal protein diet (17%) without (NP) or with leucine supplementation (NPL) or a low (6%)-protein diet without (LP) or with leucine supplementation (LPL). Leucine was given in the drinking water during the last 4 weeks. As indicated by the intraperitoneal glucose tolerance test, LPL rats exhibited increased glucose tolerance as compared with NPL group. Both NPL and LPL rats had higher circulating insulin levels than controls. The LPL rats also showed increased insulin secretion by pancreatic islets in response to glucose or arginine compared with those observed in islets from LP animals. Glucose oxidation was significantly reduced in NPL, LP, and LPL isolated islets as compared with NP; but no alteration was observed for leucine and glutamate oxidation among the 4 groups. Western blotting analysis demonstrated increased PI3K and mammalian target protein of rapamycin protein contents in LPL compared with LP islets. A significant increase in insulin-induced insulin receptor substrate I associated PI3K activation was also observed in LPL compared with LP islets. These findings indicate that leucine supplementation can augment islet function in malnourished rats and that activation of the PI3K/maminalian target protein of rapamycin pathway may play a role in this process. (C) 2010 Elsevier Inc. All rights reserved.

Brainstem oxytocinergic modulation of heart rate control in rats: effects of hypertension and exercise training

Oxytocinergic brainstem projections participate in the autonomic control of the circulation. We investigated the effects of hypertension and training on cardiovascular parameters after oxytocin (OT) receptor blockade within the nucleus tractus solitarii (NTS) and NTS OT and OT receptor expression. Male spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats were trained (55% of maximal exercise capacity) or kept sedentary for 3 months and chronically instrumented (NTS and arterial cannulae). Mean arterial blood pressure (MAP) and heart rate (HR) were measured at rest and during an acute bout of exercise after NTS pretreatment with vehicle or OT antagonist (20 pmol of OT antagonist (200 nl of vehicle)-1). Oxytocin and OT receptor were quantified (35S-oligonucleotide probes, in situ hybridization) in other groups of rats. The SHR exhibited high MAP and HR (P < 0.05). Exercise training improved treadmill performance and reduced basal HR (on average -11%) in both groups, but did not change basal MAP. Blockade of NTS OT receptor increased exercise tachycardia only in trained groups, with a larger effect on trained WKY rats (+31 +/- 9 versus +12 +/- 3 beats min-1 in the trained SHR). Hypertension specifically reduced NTS OT receptor mRNA density (-46% versus sedentary WKY rats, P < 0.05); training did not change OT receptor density, but significantly increased OT mRNA expression (+2.5-fold in trained WKY rats and +15% in trained SHR). Concurrent hypertension- and training-induced plastic (peptide/receptor changes) and functional adjustments (HR changes) of oxytocinergic control support both the elevated basal HR in the SHR group and the slowing of the heart rate (rest and exercise) observed in trained WKY rats and SHR.

16-Bromoepiandrosterone, an activator of the mammalian immune system, inhibits glucose 6-phosphate dehydrogenase from Trypanosoma cruzi and is toxic to these parasites grown in culture

Glucose 6-phosphate dehydrogenase (G6PDH) catalyzes the first step of the pentose-phosphate pathway which supplies cells with ribose 5-phosphate (R5P) and NADPH. R5P is the precursor for the biosynthesis of nucleotides while NADPH is the cofactor of several dehydrogenases acting in a broad range of biosynthetic processes and in the maintenance of the cellular redox state. RNA interference-mediated reduction of G6PDH levels in bloodstream-form Trypanosoma brucei validated this enzyme as a drug target against Human African Trypanosomiasis. Dehydroepiandrosterone (DHEA), a human steroidal pro-hormone and its derivative 16 alpha-bromoepiandrosterone (16BrEA) are uncompetitive inhibitors of mammalian G6PDH. Such steroids are also known to enhance the immune response in a broad range of animal infection models. It is noteworthy that the administration of DHEA to rats infected by Trypanosoma cruzi, the causative agent of Human American Trypanosomiasis (also known as Chagas` disease), reduces blood parasite levels at both acute and chronic infection stages. In the present work, we investigated the in vitro effect of DHEA derivatives on the proliferation of T. cruzi epimastigotes and their inhibitory effect on a recombinant form of the parasite`s G6PDH (TcG6PDH). Our results show that DHEA and its derivative epiandrosterone (EA) are uncompetitive inhibitors of TcG6PDH, with K(i) values of 21.5 +/- 0.5 and 4.8 +/- 0.3 mu M, respectively. Results from quantitative inhibition assays indicate 16BrEA as a potent inhibitor of TcG6PDH with an IC(50) of 86 +/- 8 nM and those from in vitro cell viability assays confirm its toxicity for T. cruzi epimastigotes, with a LD(50) of 12 +/- 8 mu M. In summary, we demonstrated that, in addition to host immune response enhancement, 16BrEA has a direct effect on parasite viability, most likely as a consequence of TcG6PDH inhibition. Crown Copyright (C) 2010 Published by Elsevier Ltd. All rights reserved.

Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas` disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T cruzi. The infective trypomastigotes showed the highest transport activity (V(max) = 5.34 +/- 0.54 nmol/min per mg of protein: K(m) = 0.38 +/- 0.01 mM) when compared to intracellular epimastigotes (V(max) = 2.18 +/- 0.20 nmol/min per mg of protein; K(m) = 0.39 +/- 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T cruzi. (C) 2009 Elsevier B.V. All rights reserved.

The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.