Potential Contribution of Translational Factors to Triiodo-L-Thyronine-Induced Insulin Synthesis by Pancreatic Beta Cells

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect. Methods: INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K. Results: It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment. Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.

Collybistin and gephyrin are novel components of the eukaryotic translation initiation factor 3 complex

Abstract Background Collybistin (CB), a neuron-specific guanine nucleotide exchange factor, has been implicated in targeting gephyrin-GABAA receptors clusters to inhibitory postsynaptic sites. However, little is known about additional CB partners and functions. Findings Here, we identified the p40 subunit of the eukaryotic translation initiation factor 3 (eIF3H) as a novel binding partner of CB, documenting the interaction in yeast, non-neuronal cell lines, and the brain. In addition, we demonstrated that gephyrin also interacts with eIF3H in non-neuronal cells and forms a complex with eIF3 in the brain. Conclusions Together, our results suggest, for the first time, that CB and gephyrin associate with the translation initiation machinery, and lend further support to the previous evidence that gephyrin may act as a regulator of synaptic protein synthesis.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal beta-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized beta-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)

Suppression of Hypoxia-Inducible Factor-1 alpha Contributes to the Antiangiogenic Activity of Red Propolis Polyphenols in Human Endothelial Cells

Polyphenol-enriched fractions from natural sources have been proposed to interfere with angiogenesis in pathological conditions. We recently reported that red propolis polyphenols (RPP) exert antiangiogenic activity. However, molecular mechanisms of this activity remain unclear. Here, we aimed at characterizing molecular mechanisms to explain the impact of RPP on endothelial cells' (EC) physiology. We used in vitro and ex and in vivo models to test the hypothesis that RPP inhibit angiogenesis by affecting hypoxia-inducible factor-1 alpha (HIF1 alpha) stabilization in EC. RPP (10 mg/L) affected angiogenesis by reducing migration and sprouting of EC, attenuated the formation of new blood vessels, and decreased the differentiation of embryonic stem cells into CD31-positive cells. Moreover, RPP (10 mg/L) inhibited hypoxia- or dimethyloxallylglycine-induced mRNA and protein expression of the crucial angiogenesis promoter vascular endothelial growth factor (VEGF) in a time-dependent mariner. Under hypoxic conditions, RPP at 10 mg/L, supplied for 1-4 h, decreased HIF1 alpha protein accumulation, which in turn attenuated VEGF gene expression. In addition, RPP reduced the HIF1 alpha protein half-life from similar to 58 min to 38 min under hypoxic conditions. The reduced HIF1 alpha protein half-life was associated with an increase in the von Hippel-Lindau (pVHL)-dependent proteasomal degradation of HIF1 alpha. RPP (10 mg/L, 4 h) downregulated Cdc42 protein expression. This caused a corresponding increase in pVHL protein levels and a subsequent degradation of HIF1 alpha. In summary, we have elucidated the underlying mechanism for the antiangiogenic action of RPP, which attenuates HIF1 alpha protein accumulation and signaling. J. Nutr. 142: 441-447, 2012.

Is there a therapeutic window for pentoxifylline after the onset of acute pancreatitis?

PURPOSE: To investigate the effects of pentoxifylline (PTX) in experimental acute pancreatitis (AP) starting drug administration after the induction of the disease. METHODS: One hundred male Wistar rats were submitted to taurocholate-induced AP and divided into three groups: Group Sham: sham-operated rats, Group Saline: AP plus saline solution, and Group PTX: AP plus PTX. Saline solution and PTX were administered 1 hour after induction of AP. At 3 hours after AP induction, peritoneal levels of tumor necrosis factor (TNF)-alpha, and serum levels of interleukin (IL)-6 and IL-10 levels were assayed by Enzyme-Linked Immunosorbent Assay (ELISA). Determinations of lung myeloperoxidase activity (MPO), histological analysis of lung and pancreas, and mortality study were performed. RESULTS: PTX administration 1 hour after induction of AP caused a significant decrease in peritoneal levels of TNF-alpha and in serum levels of IL-6 and IL-10 when compared to the saline group. There were no differences in lung MPO activity between the two groups with AP. A decrease in mortality was observed in the PTX treatment compared to the saline group. CONCLUSIONS: Administration of PTX after the onset of AP decreased the systemic levels of proinflammatory cytokines, raising the possibility that there is an early therapeutic window for PTX after the initiation of AP.

Febrile response induced by cecal ligation and puncture (CLP) in rats: involvement of prostaglandin E-2 and cytokines

The purpose of the present study was to better understand the events involved in the febrile response induced by cecal ligation and puncture (CLP), a complex infectious process. To this end, we conducted in vivo experiments in rats examining (1) fever development, (2) bacterial number in the infection focus and in blood, (3) peripheral and hypothalamic synthesis of cytokines, (4) hypothalamic and cerebrospinal fluid (CSF) synthesis of prostaglandin E-2 (PGE(2)), (5) the effect of anti-IL-6 antibody on fever, and (6) the effect of celecoxib on fever and hypothalamic synthesis of PGE(2) after CLP induction. We found that CLP promotes fever and animal death depending on the number of punctures. The peak of CLP-induced fever overlapped with the maximal increase in the number of bacteria in the infectious focus and blood, which occurred at 6 and 12 h. The peak of the febrile response also coincided with increased amounts of interleukin (IL)-1 beta, IL-6 and IL-10 in the peritoneal exudate and serum; IL-6 in the hypothalamus and PGE(2) in the CSF and predominantly in the hypothalamus. Moreover, intracerebroventricularly injected anti-IL-6 antibody reduced the febrile response while celecoxib reduced the fever and PGE(2) amount in the hypothalamus induced by CLP. Tumor necrosis factor (TNF)-alpha peaked at 3 h at all sites studied. Conversely, IL-10 concentration decreased in the hypothalamus. These findings show that the peak of CLP-induced fever is accompanied by an increase of bacteria in peritoneal fluid (local infection) and blood; local synthesis of pyrogenic (IL-1 beta, IL-6) and antipyretic (IL-10) cytokines and central production of IL-6 and PGE(2), suggesting that these last are the central mediators of this response.

Direct effect of PGF2 alpha pulses on PRL pulses, based on inhibition of PRL or PGF2 alpha secretion in heifers

The relationships between PRL and PGF(2 alpha) and their effect on luteolysis were studied. Heifers were treated with a dopamine-receptor agonist (bromocriptine; Bc) and a Cox-1 and -2 inhibitor (flunixin meglumine [FM]) to inhibit PRL and PGF(2 alpha), respectively. The Bc was given (Hour 0) when ongoing luteolysis was indicated by a 12.5% reduction in CL area (cm(2)) from the area on Day 14 postovulation, and FM was given at Hours 0, 4, and 8. Blood samples were collected every 8-h beginning on Day 14 until Hour 48 and hourly for Hours 0 to 12. Three groups of heifers in ongoing luteolysis were used: control (n = 7), Bc (n = 7), and FM (n = 4). Treatment with Bc decreased (P < 0.003) the PRL concentrations averaged over Hours 1 to 12. During the greatest decrease in PRL (Hours 2-6), LH concentrations were increased. Progesterone concentrations averaged over hours were greater (P < 0.05) in the Bc group than in the controls. In the FM group, no PGFM pulses were detected, and PRL concentrations were reduced. Concentrations of PGFM were not reduced in the Bc group, despite the reduction in PRL. Results supported the hypothesis that a decrease (12.5%) in CL area (cm(2)) is more efficient in targeting ongoing luteolysis (63%) than using any day from Days 14 to >= 19 (efficiency/day, 10-24%). The hypothesis that PRL has a role in luteolysis was supported but was confounded by the known positive effect of LH on progesterone. The hypothesis was supported that the synchrony of PGFM and PRL pulses represents a positive effect of PGF(2 alpha), on PRL, rather than an effect of PRL on PGF(2 alpha). (C) 2012 Elsevier Inc. All rights reserved.

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Cytokine Balance in Human Malaria: Does Plasmodium vivax Elicit More Inflammatory Responses than Plasmodium falciparum?

Background: The mechanisms by which humans regulate pro-and anti-inflammatory responses on exposure to different malaria parasites remains unclear. Although Plasmodium vivax usually causes a relatively benign disease, this parasite has been suggested to elicit more host inflammation per parasitized red blood cell than P. falciparum. Methodology/Principal Findings: We measured plasma concentrations of seven cytokines and two soluble tumor necrosis factor (TNF)-alpha receptors, and evaluated clinical and laboratory outcomes, in Brazilians with acute uncomplicated infections with P. vivax (n = 85), P. falciparum (n = 30), or both species (n = 12), and in 45 asymptomatic carriers of low-density P. vivax infection. Symptomatic vivax malaria patients, compared to those infected with P. falciparum or both species, had more intense paroxysms, but they had no clear association with a pro-inflammatory imbalance. To the contrary, these patients had higher levels of the regulatory cytokine interleukin (IL)-10, which correlated positively with parasite density, and elevated IL-10/TNF-alpha, IL-10/interferon (IFN)-gamma, IL-10/IL-6 and sTNFRII/TNF-alpha ratios, compared to falciparum or mixed-species malaria patient groups. Vivax malaria patients had the highest levels of circulating soluble TNF-alpha receptor sTNFRII. Levels of regulatory cytokines returned to normal values 28 days after P. vivax clearance following chemotherapy. Finally, asymptomatic carriers of low P. vivax parasitemias had substantially lower levels of both inflammatory and regulatory cytokines than did patients with clinical malaria due to either species. Conclusions: Controlling fast-multiplying P. falciparum blood stages requires a strong inflammatory response to prevent fulminant infections, while reducing inflammation-related tissue damage with early regulatory cytokine responses may be a more cost-effective strategy in infections with the less virulent P. vivax parasite. The early induction of regulatory cytokines may be a critical mechanism protecting vivax malaria patients from severe clinical complications.

Endothelial dysfunction in the pulmonary artery induced by concentrated fine particulate matter exposure is associated with local but not systemic inflammation

Clinical evidence has identified the pulmonary circulation as an important target of air pollution. It was previously demonstrated that in vitro exposure to fine particulate matter (aerodynamic diameter <= 2.5 mu m, PM2.5) induces endothelial dysfunction in isolated pulmonary arteries. We aimed to investigate the effects of in vivo exposure to urban concentrated PM2.5 on rat pulmonary artery reactivity and the mechanisms involved. For this, adult Wistar rats were exposed to 2 weeks of concentrated Sao Paulo city air PM2.5 at an accumulated daily dose of approximately 600 mu g/m(3). Pulmonary arteries isolated from PM2.5-exposed animals exhibited impaired endothelium-dependent relaxation to acetylcholine without significant changes in nitric oxide donor response compared to control rats. PM2.5 caused vascular oxidative stress and enhanced protein expression of Cu/Zn- and Mn-superoxide dismutase in the pulmonary artery. Protein expression of endothelial nitric oxide synthase (eNOS) was reduced, while tumor necrosis factor (TNF)-alpha was enhanced by PM2.5 inhalation in pulmonary artery. There was a significant positive correlation between eNOS expression and maximal relaxation response (E-max) to acetylcholine. A negative correlation was found between vascular TNF-alpha expression and E-max to acetylcholine. Plasma cytokine levels, blood cells count and coagulation parameters were similar between control and PM2.5-exposed rats. The present findings showed that in vivo daily exposure to concentrated urban PM2.5 could decrease endothelium-dependent relaxation and eNOS expression on pulmonary arteries associated with local high TNF-alpha level but not systemic pro-inflammatory factors. Taken together, the present results elucidate the mechanisms underlying the trigger of cardiopulmonary diseases induced by urban ambient levels of PM2.5. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Recognition of enteroinvasive Escherichia coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states

The innate and adaptive immune responses of dendritic cells (DCs) to enteroinvasive Escherichia coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-alpha, whereas S. flexneri induced only the production of TNF-alpha. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4(+) T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL) 20 and TNF-alpha. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.

Oral Administration of Oleic or Linoleic Acids Modulates the Production of Inflammatory Mediators by Rat Macrophages

Oleic (OLA) and linoleic (LNA) acids are commonly consumed fatty acids and they can modulate the inflammatory response, in which macrophages play an important role. The aim of this study was to investigate the effects of these two fatty acids on the production of inflammatory mediators by macrophages. Rats received oral administration of water (control), OLA or LNA (0.22 g/kg body weight) daily for 10 days and peritoneal resident macrophages were then isolated. Subsequently, they were seeded in culture plates and the production of various inflammatory mediators was assessed. Oral administration with OLA decreased the production of IL-1 beta, IL-6 and CINC-2 alpha beta by resident macrophages and LNA decreased the production of IL-1 beta, IL-6 and VEGF in the absence of lipopolysaccharide (LPS), although it accelerated IL-1 beta release and decreased IL-10 synthesis when cells were stimulated with LPS. Neither fatty acid affected the production of superoxide anion, hydrogen peroxide, nitrite, TNF-alpha, PGE(2), LTB4 or 15(S)-HETE. Thus, OLA and LNA influence the production of several inflammatory mediators by macrophages.

Redescriptions of two species and descriptions of three new species of the louse genus Bizarrifrons Eichler, 1938 (Phthiraptera: Ischnocera: Philopteridae)

Redescriptions of Bizarrifrons magus (Nitzsch [in Giebel], 1866), the type species of Bizarrifrons, and B. picturatus Carriker & Diaz-Ungria, 1961 are given based on material from their type hosts. The nymphal instars of these two species are described and illustrated for the first time. Also, three new species are named and described: B. latifrons, from the russet-backed oropendola, Psarocolius angustifrons alfredi (Des Murs, 1856); B. wecksteini, from the Amazonian oropendola, Psarocolius b. bifasciatus (Spix, 1824); and B. quasisymmetricus, from the solitary cacique, Cacicus solitarius (Vieillot, 1816) (Passeriformes: Icteridae). Two species-groups are proposed, and a checklist and a key for the species of Bizarrifrons are also included. Sequences of a portion of the mitochondrial cytochrome oxidase I (COI) and the nuclear elongation factor 1 alpha (EF-1 alpha) genes for two species are given for the first time in this genus.

Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats

Martins JM, Longhi-Balbinot DT, Soares DM, Figueiredo MJ, Malvar D do C, de Melo MC, Rae GA, Souza GE. Involvement of PGE(2) and RANTES in Staphylococcus aureus-induced fever in rats. J Appl Physiol 113: 1456-1465, 2012. First published August 30, 2012; doi:10.1152/japplphysiol.00936.2011.-This study investigated the involvement of prostaglandins and regulated on activation, normal T cell expressed and secreted (RANTES), in fever induced by live Staphylococcus aureus (no. 25923, American Type Culture Collection) injection in rats. S. aureus was injected intraperitoneally at 10(9), 10(10), and 2 x 10(10) colony-forming units (CFU)/cavity, and body temperature (T-b) was measured by radiotelemetry. The lowest dose of S. aureus induced a modest transient increase in T-b, whereas the two higher doses promoted similar long-lasting and sustained T-b increases. Thus, the 10(10) CFU/cavity dose was chosen for the remaining experiments. The T-b increase induced by S. aureus was accompanied by significant decreases in tail skin temperature and increases in PGE(2) levels in the cerebrospinal fluid (CSF) and hypothalamus but not in the venous plasma. Celecoxib (selective cyclooxygenase-2 inhibitor, 2.5 mg/kg po) inhibited the fever and the increases in PGE(2) concentration in the CSF and hypothalamus induced by S. aureus. Dipyrone (120 mg/kg ip) reduced the fever from 2.5 to 4 h and the PGE(2) increase in the CSF but not in the hypothalamus. S. aureus increased RANTES in the peritoneal exudate but not in the CSF or hypothalamus. Met-RANTES (100 mu g/kg iv), a chemokine (C-C motif) receptor (CCR)1/CCR5 antagonist, reduced the first 6 h of fever induced by S. aureus. This study suggests that peripheral (local) RANTES and central PGE(2) production are key events in the febrile response to live S. aureus injection. As dipyrone does not reduce PGE(2) synthesis in the hypothalamus, it is plausible that S. aureus induces fever, in part, via a dipyrone-sensitive PGE(2)-independent pathway.

Cerebral White Matter Oxidation and Nitrosylation in Young Rodents With Kaolin-Induced Hydrocephalus

Hydrocephalus is associated with reduced blood flow in periventricular white matter. To investigate hypoxic and oxidative damage in the brains of rats with hydrocephalus, kaolin was injected into the cisterna magna of newborn 7- and 21-day-old Sprague-Dawley rats, and ventricle size was assessed by magnetic resonance imaging at 7, 21, and 42 days of age. In-situ evidence of hypoxia in periventricular capillaries and glial cells was shown by pimonidazole hydrochloride binding. Biochemical assay of thiobarbituric acid reaction and immunohistochemical detection of malondialdehyde and 4-hydroxy-2-nonenal indicated the presence of lipid peroxidation in white matter. Biochemical assay of nitrite indicated increased nitric oxide production. Nitrotyrosine immunohistochemistry showed nitrosylated proteins in white matter reactive microglia and astrocytes. Activities of the antioxidant enzymes catalase and glutathione peroxidase were not increased, and altered hypoxia-inducible factor 1 alpha was not detected by quantitative reverse transcription-polymerase chain reaction. Cerebral vascular endothelial growth factor expression determined by quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay was not changed, but vascular endothelial growth factor immunoreactivity was increased in reactive astrocytes of hydrocephalic white matter. To determine if nitric oxide synthase is involved in the pathogenesis, we induced hydrocephalus in 7-day-old wild-type and neuronal nitric oxide synthase-deficient mice. At 7 days, the wild-type and mutant mice exhibited equally severe ventriculomegaly and no behavioral differences, although increased glial fibrillary acidic protein was less in the mutant mice. We conclude that hypoxia, via peroxidation and nitrosylation, contributes to brain changes in young rodents with hydrocephalus and that compensatory mechanisms are negligible.