Interaction between uric acid and HMGB1 translocation and release from endothelial cells

We aimed to investigate the potential relationship between alarmins [acting via Toll-like receptor-4 (TLR4)], uric acid (UA), and high-mobility group box-1 protein (HMGB1) during acute kidney injury. UA, which is significantly increased in the circulation following renal ischemia-reperfusion injury (IRI), was used both in vitro and in vivo as an early response-signaling molecule to determine its ability to induce the secretion of HMGB1 from endothelial cells. Treatment of human umbilical vein endothelial cells (HUVEC) with UA resulted in increased HMGB1 mRNA expression, acetylation of nuclear HMGB1, and its subsequent nuclear-cytoplasmic translocation and release into the circulation, as determined by Western blotting and immunofluorescence. Treatment of HUVEC with UA and a calcium mobilization inhibitor (TMB-8) or a MEK/Erk pathway inhibitor (U0126) prevented translocation of HMGB1 from the nucleus, resulting in reduced cytoplasmic and circulating levels of HMGB1. Once released, HMGB1 in autocrine fashion promoted further HMGB1 release while also stimulating NF-κB activity and increased angiopoietin-2 expression and protein release. Transfection of HUVEC with TLR4 small interfering (si) RNA reduced HMGB1 levels during UA and HMGB1 treatment. In summary, UA after IRI mediates the acetylation and release of HMGB1 from endothelial cells by mechanisms that involve calcium mobilization, the MEK/Erk pathway, and activation of TLR4. Once released, HMGB1 promotes its own further cellular release while acting as an autocrine and paracrine to activate both proinflammatory and proreparative mediators.     

Design, synthesis, and biological evaluation of hydroquinone derivatives as novel inhibitors of the sarco/endoplasmic reticulum calcium ATPase

Analogues of the compound 2,5-di-tert-butylhydroquinone (BHQ) are capable of inhibiting the enzyme sarco/endoplasmic reticulum ATPase (SERCA) in the low micromolar and submicromolar concentration ranges. Not only are SERCA inhibitors valuable research tools, but they also have potential medicinal value as agents against prostate cancer. This study describes the synthesis of 13 compounds representing several classes of BHQ analogues, such as hydroquinones with a single aromatic substituent, symmetrically and unsymmetrically disubstituted hydroquinones, and hydroquinones with ω-amino acid tethers attached to their hydroxyl groups. Structure–activity relationships were established by measuring the inhibitory potencies of all synthesized compounds in bioassays. The assays were complemented by computational ligand docking for an analysis of the relevant ligand/receptor interactions.     

Sildenafil attenuates pulmonary inflammation and fibrin deposition, mortality and right ventricular hypertrophy in neonatal hyperoxic lung injury

Background

Phosphodiesterase-5 inhibition with sildenafil has been used to treat severe pulmonary hypertension and bronchopulmonary dysplasia (BPD), a chronic lung disease in very preterm infants who were mechanically ventilated for respiratory distress syndrome.

Methods

Sildenafil treatment was investigated in 2 models of experimental BPD: a lethal neonatal model, in which rat pups were continuously exposed to hyperoxia and treated daily with sildenafil (50–150 mg/kg body weight/day; injected subcutaneously) and a neonatal lung injury-recovery model in which rat pups were exposed to hyperoxia for 9 days, followed by 9 days of recovery in room air and started sildenafil treatment on day 6 of hyperoxia exposure. Parameters investigated include survival, histopathology, fibrin deposition, alveolar vascular leakage, right ventricular hypertrophy, and differential mRNA expression in lung and heart tissue.

Results

Prophylactic treatment with an optimal dose of sildenafil (2 × 50 mg/kg/day) significantly increased lung cGMP levels, prolonged median survival, reduced fibrin deposition, total protein content in bronchoalveolar lavage fluid, inflammation and septum thickness. Treatment with sildenafil partially corrected the differential mRNA expression of amphiregulin, plasminogen activator inhibitor-1, fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2 in the lung and of brain and c-type natriuretic peptides and the natriuretic peptide receptors NPR-A, -B, and -C in the right ventricle. In the lethal and injury-recovery model we demonstrated improved alveolarization and angiogenesis by attenuating mean linear intercept and arteriolar wall thickness and increasing pulmonary blood vessel density, and right ventricular hypertrophy (RVH).

Conclusion

Sildenafil treatment, started simultaneously with exposure to hyperoxia after birth, prolongs survival, increases pulmonary cGMP levels, reduces the pulmonary inflammatory response, fibrin deposition and RVH, and stimulates alveolarization. Initiation of sildenafil treatment after hyperoxic lung injury and continued during room air recovery improves alveolarization and restores pulmonary angiogenesis and RVH in experimental BPD.     

Caste differentiation responses of two sympatric <Emphasis Type="Italic">Reticulitermes</Emphasis> termite species to juvenile hormone homologs and synthetic juvenoids in two laboratory assays

Summary We report on our investigations comparing three juvenile hormone (JH) homologs and two synthetic juvenoids to induce caste differentiation in laboratory colonies of Reticulitermes flavipes and R. tibialis. Two laboratory assays were evaluated as model systems for inducing caste differentiation: (1) shorter-term dish assays on groups of 20 individuals and (2) longer-term feeding assays on groups of 500 individuals. Each assay possessed attributes that can be considered advantageous under certain conditions. Specifically, dish assays were most suitable for presoldier and soldier induction, while jar assays provided for the induction of nymphs, presoldiers, soldiers, neotenic reproductives, and intercastes. Differences in response to the JH homologs and synthetic juvenoids were noted between species, suggesting differences in JH physiology may exist between R. flavipes and R. tibialis. Substantial morpholo-gical impacts were noted in association with some treatments, including (1) juvenoid-induced mandibular mal-formation in presoldiers, (2) JH II-induced abdominal elongation in R. flavipes soldiers and workers (associated with a presence of internal reproductive anatomy that is consistent with what would be expected to occur in pseudergates), and (3) JH II-induced soldier-nymph intercastes in R. tibialis that were able to further molt into soldier-alate intercastes. Findings are discussed in relation to the potential differences in JH-related physiology between R. flavipes and R. tibialis, and the use of model systems to induce rare castes and intercastes for molecular investigations of caste differentiation.     

Impacts of residual insecticide barriers on perimeter-invading ants, with particular reference to the odorous house ant, Tapinoma sessile

Three liquid insecticide formulations were evaluated as barrier treatments against perimeter-invading ants at a multifamily housing complex in West Lafayette, IN. Several ant species were present at the study site, including (in order of abundance) pavement ant, Tetramorium caespitum (L.); honey ant, Prenolepis imparis (Say); odorous house ant, Tapinoma sessile (Say); thief ant, Solenopsis molesta (Say); acrobat ant, Crematogaster ashmeadi (Mayr); crazy ant, Paratrechina longicornis (Latrielle), field ants, Formica spp.; and carpenter ant Camponotus pennsylvanicus (DeGeer). Studies began in May 2001 and concluded 8 wk later in July. Individual replicate treatments were placed 0.61 in (2 feet) up and 0.92 m (3 feet) out from the ends of 46.1 by 10.1-m (151 by 33-foot) apartment buildings. Ant sampling was performed with 10 placements of moist cat food for 1 h within treatment zones, followed by capture and removal of recruited ants for later counting. All treatments led to substantial reductions in ant numbers relative to untreated controls. The most effective treatment was fipronil, where 2% of before-treatment ant numbers were present at 8 wk after treatment. Both imidacloprid and cyfluthrin barrier treatments had efficacy comparative with fipronil, but to 4 and 2 wk, respectively. Odorous house ants were not sampled before treatment. Comparisons of ant species composition between treatments and controls revealed an increase in odorous house ant frequencies at 1-8 wk after treatment in treated locations only. These results demonstrate efficacy for both nonrepellent and repellent liquid insecticides as perimeter treatments for pest ants. In addition, our findings with odorous house ant highlight an apparent invasive-like characteristic of this species that may contribute to its dramatic increase in structural infestation rates in many areas of the United States.     

An unstructured initiation site is required for efficient proteasome-mediated degradation

The proteasome is the main ATP-dependent protease in eukaryotic cells and controls the concentration of many regulatory proteins in the cytosol and nucleus. Proteins are targeted to the proteasome by the covalent attachment of polyubiquitin chains. The ubiquitin modification serves as the proteasome recognition element but by itself is not sufficient for efficient degradation of folded proteins. We report that proteolysis of tightly folded proteins is accelerated greatly when an unstructured region is attached to the substrate. The unstructured region serves as the initiation site for degradation and is hydrolyzed first, after which the rest of the protein is digested sequentially. These results identify the initiation site as a novel component of the targeting signal, which is required to engage the proteasome unfolding machinery efficiently. The proteasome degrades a substrate by first binding to its ubiquitin modification and then initiating unfolding at an unstructured region.     

APC stimulated by CpG oligodeoxynucleotide enhance activation of MHC class I-restricted T cells

Oligonucleotides containing unmethylated CpG motifs (cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG ODN)) are potent immunostimulatory agents capable of enhancing the Ag-specific Th1 response when used as immune adjuvants. We evaluated the cellular mechanisms responsible for this effect. Development of a CTL response was enhanced when mice were immunized with peptide-pulsed dendritic cells (DCs) treated with CpG ODN. However, in vitro, CpG ODN had no direct effect on highly purified T cells. In vitro, CpG ODN treatment of peptide- or protein-pulsed DCs enhanced the ability of the DCs to activate class I-restricted T cells. The presence of helper T cells enhanced this effect, indicating that treatment with CpG ODN does not obviate the role of T cell help. The enhanced ability of CpG ODN-treated DCs to activate T cells was present but blunted when DCs derived from IL-12 knockout mice were used. Fixation of Ag-pulsed, CpG ODN-treated DCs limited their ability to activate T cells. In contrast, fixation had little effect on DC activation of T cells when DCs were not exposed to CpG ODN. This indicates that production of soluble factors by DCs stimulated with CpG ODN plays a particularly important role in their ability to activate class I-restricted T cells. We conclude that CpG ODN enhances the development of a cellular immune response by stimulating APCs such as DCs, to produce IL-12 and other soluble factors.     

Mitochondrial gene divergence of Colombian Drosophila pseudoobscura

Isolated populations of drosophila pseudoobscura, separated from North American populations by about 2,400 km, were found in Colombia in 1960. We compared for sequences of the small ribosomal RNA (srRNA) gene on the mitochondria between North American and Colombian D. pseudoobscura in order to clarify the age of the Colombian isolates. The North American populations were not genetically different from each other but were genetically different from the Colombian populations. The Mexican strains represent the area from which the Colombian founders might have come. The estimated net nucleotide divergence between Mexican and Colombian D. pseudoobscura indicates that the Colombian population is not an ancient lineage. Phylogenies using both distance and parsimony methodologies reinforced this conclusion. The Colombian samples group together with both methods but, according to the bootstrap analysis, not significantly. It appears that the populations have not been separated long enough for their DNA sequences to show much divergence.