Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

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.

Lack of Galectin-3 Drives Response to Paracoccidioides brasiliensis toward a Th2-Biased Immunity

There is recent evidence that galectin-3 participates in immunity to infections, mostly by tuning cytokine production. We studied the balance of Th1/Th2 responses to P. brasiliensis experimental infection in the absence of galectin-3. The intermediate resistance to the fungal infection presented by C57BL/6 mice, associated with the development of a mixed type of immunity, was replaced with susceptibility to infection and a Th2-polarized immune response, in galectin-3-deficient (gal3(-/-)) mice. Such a response was associated with defective inflammatory and delayed type hypersensitivity (DTH) reactions, high IL-4 and GATA-3 expression and low nitric oxide production in the organs of infected animals. Gal3(-/-) macrophages exhibited higher TLR2 transcript levels and IL-10 production compared to wild-type macrophages after stimulation with P. brasiliensis antigens. We hypothesize that, during an in vivo P. brasiliensis infection, galectin-3 exerts its tuning role on immunity by interfering with the generation of regulatory macrophages, thus hindering the consequent Th2-polarized type of response.

Transmission potential, skin inflammatory response, and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background: Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector. Results: The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs). Conclusion: Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.

Contralateral cortical response of a subpopulation of interneurons and the glial glutamate transporter GLT1 to an ischemic core

Introduction: Cerebral ischemia is an important cause of brain lesion in humans. The target in research has been the ischemic core or the penumbra zones; little attention has been given to areas outside the core or the penumbra but connected with the primary site of injury. Objective: Evaluate the laminar response of a subpopulation of gabaergic cells, those that are parvalbumin (PV) positive and the astrocytes through the expression of the glial transporter GLT1 on the contralateral cortex to an ischemic core. Methodology: For this purpose we used the medial cerebral artery occlusion model in rats. The artery was occluded for 90 minutes and the animals were sacrificed at 24 and 72 hours post-ischemia. The brains were removed, cut in a vibratome at 50 microns and incubated with the primary antibodies against PV or GLT1. Sections were developed using the vectastain Kit. In control tissue the primary antibody was omitted. Results: When compared with control animals, treated ones show a decrease in the expression of GLT1, especially in layers III and IV of the contralateral cortex to the ischemic core. PV positive cells increases in layers II and V. Conclusion: Increases in the expression of PV cells could correspond to an adaptation associated with glutamate increases in the synaptic compartment. These increases may be due to decreases in the expression of GLT1 transporter, that could not remove the glutamate present in the synaptic cleft, generating hyperactivity in the contralateral cortex. These changes could represent an example of neuronal and glial plasticity in remote areas to an ischemic core but connected to the primary site of injury.

Description and Phylogenetic Relationships of a New Genus and Species of Lizard (Squamata, Gymnophthalmidae) from the Amazonian Rainforest of Northern Brazil

Marinussaurus curupira, a new genus and species of Gymnophthalmidae lizard is described from Iranduba, state of Amazonas, Brazil. The genus is characterized by an elongate body; short and stout pentadactyl limbs; all digits clawed; single frontonasal; two prefrontals; absence of frontoparietals; interparietal and parietals forming a straight posterior margin, with interparietal shorter than parietals; distinctive ear opening and eyelid; few temporals; three pairs of chin shields; nasal divided; a distinct collar; smooth, mainly hexagonal, dorsal scales; smooth quadrangular ventral scales; two precloacal and three femoral pores on each side in males; pores between three or four scales. Parsimony (PAR) and partitioned Bayesian (BA) phylogenetic analyses with morphological and molecular data recovered the new genus as a member of the Ecpleopodini radiation of the Cercosaurinae. A close relationship of the new genus with Arthrosaura is postulated.

A New Genus of Microteiid Lizard from the Caparao Mountains, Southeastern Brazil, with a Discussion of Relationships among Gymnophthalminae (Squamata)

A new genus and species of microteiid lizard is described based on a series of specimens obtained at Parque Nacional do Caparao (20 degrees 28'S, 41 degrees 49'W), southeastern Brazil, along the division line between the States of Minas Gerais and Espirito Santo. The new lizard occurs in isolated high-altitude, open, rocky habitats above the altitudinal lit-nits of the Atlantic forest. It is characterized by the presence of prefrontals, frontoparietals, parietals, interparietal, and occipital scales; ear opening and eyelid distinct; three pairs of genials; absence of collar; lanceolate and mucronate dorsal scales; six regular transverse and longitudinal series of smooth ventrals that are longer than wide, with the lateral ones narrower. Maximum parsimony (MP) and partitioned Bayesian (PBA) phylogenetic analyses based on morphological and molecular characters with all known genera of Gymnophthalminae (except for Scriptosaura) Plus Rhachisaurus recovered this new lizard in a clade having Colobodactylus and Heterodactylus as its closest relatives. Both analyses recovered the monophyly of Gymnophthalminae and Gymnophthalmini. The monophyly of the Heterodactylini received moderate support in MP analyses but was not recovered in PBA. To eliminate classification controversy between these results, the present concept of Heterodactylini is restricted to accommodate the new genus, Colobodactylus and Heterodactylus, and a new tribe Iphisiini is proposed to allocate Alexandresaurus, Iphisa, Colobosaura, Acratosaura, and Stenolepis. Current phylogenetic knowledge of Gymnophthalminae suggests that fossoriality and increase of body elongation arose as adaptive responses to avoid extreme surface temperatures, either cold or hot, depending on circumstances.

Cellular Renewal and Improvement of Local Cell Effector Activity in Peritoneal Cavity in Response to Infectious Stimuli

The peritoneal cavity (PerC) is a singular compartment where many cell populations reside and interact. Despite the widely adopted experimental approach of intraperitoneal (i.p.) inoculation, little is known about the behavior of the different cell populations within the PerC. To evaluate the dynamics of peritoneal macrophage (Mempty set) subsets, namely small peritoneal Mempty set (SPM) and large peritoneal Mempty set (LPM), in response to infectious stimuli, C57BL/6 mice were injected i.p. with zymosan or Trypanosoma cruzi. These conditions resulted in the marked modification of the PerC myelo-monocytic compartment characterized by the disappearance of LPM and the accumulation of SPM and monocytes. In parallel, adherent cells isolated from stimulated PerC displayed reduced staining for beta-galactosidase, a biomarker for senescence. Further, the adherent cells showed increased nitric oxide (NO) and higher frequency of IL-12-producing cells in response to subsequent LPS and IFN-gamma stimulation. Among myelo-monocytic cells, SPM rather than LPM or monocytes, appear to be the central effectors of the activated PerC; they display higher phagocytic activity and are the main source of IL-12. Thus, our data provide a first demonstration of the consequences of the dynamics between peritoneal Mempty set subpopulations by showing that substitution of LPM by a robust SPM and monocytes in response to infectious stimuli greatly improves PerC effector activity.

The Spleen CD4(+) T Cell Response to Blood-Stage Plasmodium chabaudi Malaria Develops in Two Phases Characterized by Different Properties

The pivotal role of spleen CD4(+) T cells in the development of both malaria pathogenesis and protective immunity makes necessary a profound comprehension of the mechanisms involved in their activation and regulation during Plasmodium infection. Herein, we examined in detail the behaviour of non-conventional and conventional splenic CD4(+) T cells during P. chabaudi malaria. We took advantage of the fact that a great proportion of CD4(+) T cells generated in CD1d(-/-) mice are I-A(b)-restricted (conventional cells), while their counterparts in I-Ab(-/-) mice are restricted by CD1d and other class IB major histocompatibility complex (MHC) molecules (non-conventional cells). We found that conventional CD4(+) T cells are the main protagonists of the immune response to infection, which develops in two consecutive phases concomitant with acute and chronic parasitaemias. The early phase of the conventional CD4(+) T cell response is intense and short lasting, rapidly providing large amounts of proinflammatory cytokines and helping follicular and marginal zone B cells to secrete polyclonal immunoglobulin. Both TNF-alpha and IFN-gamma production depend mostly on conventional CD4(+) T cells. IFN-gamma is produced simultaneously by non-conventional and conventional CD4(+) T cells. The early phase of the response finishes after a week of infection, with the elimination of a large proportion of CD4(+) T cells, which then gives opportunity to the development of acquired immunity. Unexpectedly, the major contribution of CD1d-restricted CD4(+) T cells occurs at the beginning of the second phase of the response, but not earlier, helping both IFN-gamma and parasite-specific antibody production. We concluded that conventional CD4(+) T cells have a central role from the onset of P. chabaudi malaria, acting in parallel with non-conventional CD4(+) T cells as a link between innate and acquired immunity. This study contributes to the understanding of malaria immunology and opens a perspective for future studies designed to decipher the molecular mechanisms behind immune responses to Plasmodium infection.

Respiratory allergy to Blomia tropicalis: Immune response in four syngeneic mouse strains and assessment of a low allergen-dose, short-term experimental model

Background: The dust mite Blomia tropicalis is an important source of aeroallergens in tropical areas. Although a mouse model for B. tropicalis extract (BtE)-induced asthma has been described, no study comparing different mouse strains in this asthma model has been reported. The relevance and reproducibility of experimental animal models of allergy depends on the genetic background of the animal, the molecular composition of the allergen and the experimental protocol. Objectives: This work had two objectives. The first was to study the anti-B. tropicalis allergic responses in different mouse strains using a short-term model of respiratory allergy to BtE. This study included the comparison of the allergic responses elicited by BtE with those elicited by ovalbumin in mice of the strain that responded better to BtE sensitization. The second objective was to investigate whether the best responder mouse strain could be used in an experimental model of allergy employing relatively low BtE doses. Methods: Groups of mice of four different syngeneic strains were sensitized subcutaneously with 100 mu g of BtE on days 0 and 7 and challenged four times intranasally, at days 8, 10, 12, and 14, with 10 mu g of BtE. A/J mice, that were the best responders to BtE sensitization, were used to compare the B. tropicalis-specific asthma experimental model with the conventional experimental model of ovalbumin (OVA)-specific asthma. A/J mice were also sensitized with a lower dose of BtE. Results: Mice of all strains had lung inflammatory-cell infiltration and increased levels of anti-BtE IgE antibodies, but these responses were significantly more intense in A/J mice than in CBA/J, BALB/c or C57BL/6J mice. Immunization of A/J mice with BtE induced a more intense airway eosinophil influx, higher levels of total IgE, similar airway hyperreactivity to methacholine but less intense mucous production, and lower levels of specific IgE, IgG1 and IgG2 antibodies than sensitization with OVA. Finally, immunization with a relatively low BtE dose (10 mu g per subcutaneous injection per mouse) was able to sensitize A/J mice, which were the best responders to high-dose BtE immunization, for the development of allergy-associated immune and lung inflammatory responses. Conclusions: The described short-term model of BtE-induced allergic lung disease is reproducible in different syngeneic mouse strains, and mice of the A/J strain was the most responsive to it. In addition, it was shown that OVA and BtE induce quantitatively different immune responses in A/J mice and that the experimental model can be set up with low amounts of BtE.

The constancy of global regulation across a species: the concentrations of ppGpp and RpoS are strain-specific in Escherichia coli

Background: Sigma factors and the alarmone ppGpp control the allocation of RNA polymerase to promoters under stressful conditions. Both ppGpp and the sigma factor sigma(S) (RpoS) are potentially subject to variability across the species Escherichia coli. To find out the extent of strain variation we measured the level of RpoS and ppGpp using 31 E. coli strains from the ECOR collection and one reference K-12 strain. Results: Nine ECORs had highly deleterious mutations in rpoS, 12 had RpoS protein up to 7-fold above that of the reference strain MG1655 and the remainder had comparable or lower levels. Strain variation was also evident in ppGpp accumulation under carbon starvation and spoT mutations were present in several low-ppGpp strains. Three relationships between RpoS and ppGpp levels were found: isolates with zero RpoS but various ppGpp levels, strains where RpoS levels were proportional to ppGpp and a third unexpected class in which RpoS was present but not proportional to ppGpp concentration. High-RpoS and high-ppGpp strains accumulated rpoS mutations under nutrient limitation, providing a source of polymorphisms. Conclusions: The ppGpp and sigma(S) variance means that the expression of genes involved in translation, stress and other traits affected by ppGpp and/or RpoS are likely to be strain-specific and suggest that influential components of regulatory networks are frequently reset by microevolution. Different strains of E. coli have different relationships between ppGpp and RpoS levels and only some exhibit a proportionality between increasing ppGpp and RpoS levels as demonstrated for E. coli K-12.

Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus)microplus in response to infection with Anaplasma marginale

Background: Bovine anaplasmosis, caused by the rickettsial tick-borne pathogen Anaplasma marginale (Rickettsiales: Anaplasmataceae), is vectored by Rhipicephalus (Boophilus) microplus in many tropical and subtropical regions of the world. A. marginale undergoes a complex developmental cycle in ticks which results in infection of salivary glands from where the pathogen is transmitted to cattle. In previous studies, we reported modification of gene expression in Dermacentor variabilis and cultured Ixodes scapularis tick cells in response to infection with A. marginale. In these studies, we extended these findings by use of a functional genomics approach to identify genes differentially expressed in R. microplus male salivary glands in response to A. marginale infection. Additionally, a R. microplus-derived cell line, BME26, was used for the first time to also study tick cell gene expression in response to A. marginale infection. Results: Suppression subtractive hybridization libraries were constructed from infected and uninfected ticks and used to identify genes differentially expressed in male R. microplus salivary glands infected with A. marginale. A total of 279 ESTs were identified as candidate differentially expressed genes. Of these, five genes encoding for putative histamine-binding protein (22Hbp), von Willebrand factor (94Will), flagelliform silk protein (100Silk), Kunitz-like protease inhibitor precursor (108Kunz) and proline-rich protein BstNI subfamily 3 precursor (7BstNI3) were confirmed by real-time RT-PCR to be down-regulated in tick salivary glands infected with A. marginale. The impact of selected tick genes on A. marginale infections in tick salivary glands and BME26 cells was characterized by RNA interference. Silencing of the gene encoding for putative flagelliform silk protein (100Silk) resulted in reduced A. marginale infection in both tick salivary glands and cultured BME26 cells, while silencing of the gene encoding for subolesin (4D8) significantly reduced infection only in cultured BME26 cells. The knockdown of the gene encoding for putative metallothionein (93 Meth), significantly up-regulated in infected cultured BME26 cells, resulted in higher A. marginale infection levels in tick cells. Conclusions: Characterization of differential gene expression in salivary glands of R. microplus in response to A. marginale infection expands our understanding of the molecular mechanisms at the tick-pathogen interface. Functional studies suggested that differentially expressed genes encoding for subolesin, putative von Willebrand factor and flagelliform silk protein could play a role in A. marginale infection and multiplication in ticks. These tick genes found to be functionally relevant for tick-pathogen interactions will likely be candidates for development of vaccines designed for control of both ticks and tick-borne pathogens.

Characterization of subgraph relationships and distribution in complex networks

A network can be analyzed at different topological scales, ranging from single nodes to motifs, communities, up to the complete structure. We propose a novel approach which extends from single nodes to the whole network level by considering non-overlapping subgraphs (i.e. connected components) and their interrelationships and distribution through the network. Though such subgraphs can be completely general, our methodology focuses on the cases in which the nodes of these subgraphs share some special feature, such as being critical for the proper operation of the network. The methodology of subgraph characterization involves two main aspects: (i) the generation of histograms of subgraph sizes and distances between subgraphs and (ii) a merging algorithm, developed to assess the relevance of nodes outside subgraphs by progressively merging subgraphs until the whole network is covered. The latter procedure complements the histograms by taking into account the nodes lying between subgraphs, as well as the relevance of these nodes to the overall subgraph interconnectivity. Experiments were carried out using four types of network models and five instances of real-world networks, in order to illustrate how subgraph characterization can help complementing complex network-based studies.

Theory of small-signal ac response of a dielectric liquid containing two groups of ions

The analysis of Macdonald for electrolytes is generalized to the case in which two groups of ions are present. We assume that the electrolyte can be considered as a dispersion of ions in a dielectric liquid, and that the ionic recombination can be neglected. We present the differential equations governing the ionic redistribution when the liquid is subjected to an external electric field, describing the simultaneous diffusion of the two groups of ions in the presence of their own space charge fields. We investigate the influence of the ions on the impedance spectroscopy of an electrolytic cell. In the analysis, we assume that each group of ions have equal mobility, the electrodes perfectly block and that the adsorption phenomena can be neglected. In this framework, it is shown that the real part of the electrical impedance of the cell has a frequency dependence presenting two plateaux, related to a type of ambipolar and free diffusion coefficients. The importance of the considered problem on the ionic characterization performed by means of the impedance spectroscopy technique was discussed. (c) 2008 American Institute of Physics.

Transverse momentum and centrality dependence of dihadron correlations in Au plus Au collisions at root s(NN)=200 GeV: Jet quenching and the response of partonic matter

Azimuthal angle (Delta phi) correlations are presented for charged hadrons from dijets for 0.4 < p(T)< 10 GeV/c in Au+Au collisions at root s(NN)=200 GeV. With increasing p(T), the away-side distribution evolves from a broad and relatively flat shape to a concave shape, then to a convex shape. Comparisons to p+p data suggest that the away-side can be divided into a partially suppressed ""head"" region centered at Delta phi similar to pi and an enhanced ""shoulder"" region centered at Delta phi similar to pi +/- 1.1. The p(T) spectrum for the head region softens toward central collisions, consistent with the onset of jet quenching. The spectral slope for the shoulder region is independent of centrality and trigger p(T), which offers constraints on energy transport mechanisms and suggests that it contains the medium response to energetic jets.