Progesterone reduces erectile dysfunction in sleep-deprived spontaneously hypertensive rats

Background  

Paradoxical sleep deprivation (PSD) associated with cocaine has been shown to enhance genital reflexes (penile erection-PE and ejaculation-EJ) in Wistar rats. Since hypertension predisposes males to erectile dysfunction, the aim of the present study was to investigate the effects of PSD on genital reflexes in the spontaneously hypertensive rat (SHR) compared to the Wistar strain. We also extended our study to examine how PSD affect steroid hormone concentrations involved in genital events in both experimental models.     

Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation

Eosinophils are effector cells that have an important role in the pathogenesis of allergic disease. Defective removal of these cells likely leads to chronic inflammatory diseases such as asthma. Thus, there is great interest in understanding the mechanisms responsible for the elimination of eosinophils from inflammatory sites. Previous studies have demonstrated a role for certain mediators and molecular pathways responsible for the survival and death of leukocytes at sites of inflammation. Reactive oxygen species have been described as proinflammatory mediators but their role in the resolution phase of inflammation is poorly understood. The aim of this study was to investigate the effect of reactive oxygen species in the resolution of allergic inflammatory responses. An eosinophilic cell line (Eol-1) was treated with hydrogen peroxide and apoptosis was measured. Allergic inflammation was induced in ovalbumin sensitized and challenged mouse models and reactive oxygen species were administered at the peak of inflammatory cell infiltrate. Inflammatory cell numbers, cytokine and chemokine levels, mucus production, inflammatory cell apoptosis and peribronchiolar matrix deposition was quantified in the lungs. Resistance and elastance were measured at baseline and after aerosolized methacholine. Hydrogen peroxide accelerates resolution of airway inflammation by induction of caspase-dependent apoptosis of eosinophils and decrease remodeling, mucus deposition, inflammatory cytokine production and airway hyperreactivity. Moreover, the inhibition of reactive oxygen species production by apocynin or in gp91^phox−/− mice prolonged the inflammatory response. Hydrogen peroxide induces Eol-1 apoptosis in vitro and enhances the resolution of inflammation and improves lung function in vivo by inducing caspase-dependent apoptosis of eosinophils.Eosinophils express numerous receptors and secrete a wide variety of inflammatory mediators that influence many innate and adaptive immune responses. These multifunctional cells are important in the defense against helminth infection and are involved in the pathogenesis of many eosinophilic dominant allergic diseases.¹ High levels of eosinophil granule proteins (such as major basic protein (MBP)) have been found in bronchoalveolar lavage fluid from patients with asthma and evidence indicates that high-concentration granule products have contributed to the development of airway hyperreactivity (AHR), a cardinal feature of asthma.² Asthma is an inflammatory disease of the airways with participation of many cell types including leukocytes especially eosinophils and lymphocytes.^{3, 4} Activation of these cells (mainly lymphocytes) leads to the release of proinflammatory mediators and cytokines such as leukotriene B₄, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-13 (IL-13) and colony-stimulating factor granulocyte-macrophage (GM-CSF).^{3, 5, 6, 7} Investigations using preclinical animal models of asthma and clinical studies in patients with asthma have demonstrated that the presence of eosinophils in the lungs are associated with epithelial damage, goblet cell hyperplasia, smooth muscle hypertrophy and airway hyperresponsiveness resulting in airflow limitation which can be fatal.^{3, 8, 9, 10} Recently, anti-IL-5 treatment has been shown to ameliorate lung function in patients with eosinophilic asthma.¹¹Apoptosis of leukocytes is regarded as an important process for the successful resolution of inflammatory responses. Reduced eosinophil apoptosis in bronchoalveolar lavage (BAL) fluid has been shown to correlate positively with severity of asthma.^{3, 12, 13, 14} Indeed, defective leukocyte apoptosis and subsequent removal of apoptotic cells by phagocytes is thought to be important for the initiation and propagation of chronic inflammatory diseases such as asthma.¹⁵ Therefore, a balance in the tissue microenvironment between pro- and antiapoptotic signals is likely to greatly influence the load of eosinophils in the asthmatic lung.¹⁶ Thus, there is a great interest in understanding the mechanisms responsible for the elimination of eosinophils and other leukocytes and inactivation of proinflammatory mediators in inflammatory sites.¹⁷Several molecular pathways have been shown to modulate the survival and death of leukocytes at sites of inflammation, including reactive oxygen species (ROS).¹⁸ ROS are a family of molecules containing oxygen and includes hydrogen peroxide (H₂O₂), superoxide O₂⁻, hydroxyl radical (OH) and nitric oxide (NO).¹⁹ In inflammatory conditions, ROS are increased as they help in neutralizing invading organisms during infection either directly or indirectly by formation of extracellular traps (ETs).²⁰ ROS have traditionally been regarded as quintessentially proinflammatory. However, evidence for ROS-mediated anti-inflammatory actions has been described.²¹ The importance for ROS production in the context of infection can be exemplified in patients with chronic granulomatous disease (CGD) where defective production in ROS results in multiple infections and often early death.^{22, 23} Furthermore, studies in mouse models have shown that NADPH oxidase is key for regulating lung inflammation and injury as well as NF-κB activation and downstream cytokine production in response to LPS.²⁴ More recently, our group has demonstrated that NADPH oxidase-derived H₂O₂ is directly linked to induction of apoptosis of neutrophils and resolution of inflammation in a model of antigen-induced arthritis.¹⁸ However, the role of ROS in the context of the resolution of allergic inflammation is still unknown.Here, we evaluated whether H₂O₂ drives apoptosis of eosinophils and thereby influences the resolution of established eosinophilic inflammation and reduction of airflow obstruction. Our study provides evidence that H₂O₂ is released during allergic inflammation in a gp91^phox−/−-dependent manner and induces a caspase-dependent proapoptotic effect in eosinophils, thus having a crucial role in the resolution of allergic inflammation.     

Telmisartan induces apoptosis and regulates Bcl-2 in human renal cancer cells

It has been well-characterized that the renin-angiotensin system (RAS) physiologically regulates systemic arterial pressure. However, RAS signaling has also been shown to increase cell proliferation during malignancy, and angiotensin receptor blockers (ARBs) are able to decrease pro-survival signaling by inhibiting anti-apoptotic molecules and suppressing caspase activity. In this study, the apoptotic effects of telmisartan, a type of ARB, was evaluated using a non-cancerous human renal cell line (HEK) and a human renal cell carcinoma (RCC) cell line (786). Both types of cells were treated with telmisartan for 4 h, 24 h, and 48 h, and then were assayed for levels of apoptosis, caspase-3, and Bcl-2 using MTT assays, flow cytometry, and immunostaining studies. Analysis of variance was used to identify significant differences between these data (P < 0.05). Following the treatment of 786 cells with 100 µM and 200 µM telmisartan, a marked inhibition of cell proliferation was observed. 50 µM cisplatin also caused high inhibition of these cells. Moreover, these inhibitions were both concentration- and time-dependent (P < 0.05). Various apoptotic effects were also observed compared with control cells at the 24 h and 48 h timepoints assayed (P < 0.001). Furthermore, positive caspase-3 staining and down-regulation of Bcl-2 were detected, consistent with induction of cell death. In contrast, treatment of HEK cells with telmisartan did not produce an apoptotic effect compared with control cells at the 24 h timepoint (P > 0.05). Treatment with cisplatin promoted in HEK cells high index of apoptosis (P < 0.001). Taken together, these results suggest that telmisartan induces apoptosis via down-regulation of Bcl-2 and involvement of caspase-3 in human RCC cells.     

The phylogenetic relationships between Dryocosmus, Chilaspis and allied genera of oak gallwasps (Hymenoptera, Cynipidae: Cynipini)

Abstract Oak gallwasps (Hymenoptera; Cynipidae, tribe Cynipini) are cyclically parthenogenetic insects that induce galls on specific plant hosts in the family Fagaceae. Understanding the processes underlying the evolution of specific oak associations requires knowledge of the phylogenetic relationships among oak gallwasp genera. Although three major lineages of oak gallwasps have been identified, the status and relationships of several species‐poor but biologically significant genera remain unresolved. Two such genera are Chilaspis and Dryocosmus, whose western palaearctic species all gall oaks in the section Cerris. Dryocosmus is particularly significant biologically because it includes: (a) the only palaearctic gallwasp to gall chestnuts, Castanea, and (b) nearctic species. The oak section Cerris is wholly absent from the nearctic, and the relationship between palaearctic and nearctic Dryocosmus is significant for patterns of host plant evolution in the tribe as a whole. We examined the relationships between Chilaspis, Dryocosmus and other oak cynipid genera using cladograms from sequence data for two mitochondrial loci (cytochrome c oxidase subunit I and cytochrome b) and two nuclear loci (the 28S ribosomal gene regions D2 and D3–5). Our analyses support the following conclusions: (1) palaearctic Chilaspis and Dryocosmus species form an intermingled monophyletic group. (2) We propose that Chilaspis Mayr, 1881 is a syn.n. of Dryocosmus Giraud, 1859 and propose the name D. mayri as a comb.rev. for the species previously named C. mayri, and D. nitidus and D. israeli as comb.n. of C. nitida and C. israeli, respectively. (3) We reassess the utility of morphological characters previously regarded as diagnostic for these genera. (4) Two species previously known only from a single generation represent two halves of a single species lifecycle. Dryocosmus nervosus is here designated a syn.n. of D. cerriphilus. (5) The nearctic species D. favus lies outside the palaearctic Chilaspis/Dryocosmus clade, and Dryocosmus as currently recognized is not a monophyletic group. (6) Dryocosmus/Chilaspis is closely related to the other oak gallwasp taxa (Aphelonyx, Plagiotrochus, Pseudoneuroterus, Trichagalma, and some Neuroterus species) galling section Cerris oaks. This implies an early branching evolution of this oak association within this group, and supports previous work showing the rarity of oak gallwasp host shifts.     

Synthesis and SAR of 2-phenoxypyridines as novel c-Jun N-terminal kinase inhibitors

The design and synthesis of a novel series of c-jun N-terminal kinase (JNK3) inhibitors is described. The development and optimization of the 2-phenoxypyridine series was carried out from an earlier pyrimidine series of JNK1 inhibitors. Through the optimization of the scaffold 2, several potent compounds with good in vivo profiles were discovered.     

Aldose reductase deficiency in mice protects from ragweed pollen extract (RWE)-induced allergic asthma

Background

Childhood hospitalization related to asthma remains at historically high levels, and its incidence is on the rise world-wide. Previously, we have demonstrated that aldose reductase (AR), a regulatory enzyme of polyol pathway, is a major mediator of allergen-induced asthma pathogenesis in mouse models. Here, using AR null (AR^-/-) mice we have investigated the effect of AR deficiency on the pathogenesis of ragweed pollen extract (RWE)-induced allergic asthma in mice and also examined the efficacy of enteral administration of highly specific AR inhibitor, fidarestat.

Methods

The wild type (WT) and AR^-/- mice were sensitized and challenged with RWE to induce allergic asthma. AR inhibitor, fidarestat was administered orally. Airway hyper-responsiveness was measured in unrestrained animals using whole body plethysmography. Mucin levels and Th2 cytokine in broncho-alveolar lavage (BAL) were determined using mouse anti-Muc5A/C ELISA kit and multiplex cytokine array, respectively. Eosinophils infiltration and goblet cells were assessed by H&E and periodic acid Schiff (PAS)-staining of formalin-fixed, paraffin-embedded lung sections. T regulatory cells were assessed in spleen derived CD4⁺CD25⁺ T cells population.

Results

Deficiency of AR in mice led to significantly decreased PENH, a marker of airway hyper-responsiveness, metaplasia of airway epithelial cells and mucus hyper-secretion following RWE-challenge. This was accompanied by a dramatic decrease in infiltration of eosinophils into sub-epithelium of lung as well as in BAL and release of Th2 cytokines in response to RWE-challenge of AR^-/- mice. Further, enteral administration of fidarestat significantly prevented eosinophils infiltration, airway hyper-responsiveness and also markedly increased population of T regulatory (CD4⁺CD25⁺FoxP3⁺) cells as compared to RWE-sensitized and challenged mice not treated with fidarestat.

Conclusion

Our results using AR^-/- mice strongly suggest the role of AR in allergic asthma pathogenesis and effectiveness of oral administration of AR inhibitor in RWE-induced asthma in mice supports the use of AR inhibitors in the treatment of allergic asthma.     

Effects of short-term glucocorticoid treatment on changes in cartilage matrix degradation and chondrocyte gene expression induced by mechanical injury and inflammatory cytokines

Introduction  

Traumatic joint injury damages cartilage and causes adjacent joint tissues to release inflammatory cytokines, increasing the risk of developing osteoarthritis. The main objective of this study was to determine whether the combined catabolic effects of mechanical injury, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R) on cartilage could be abolished by short-term treatment with glucocorticoids such as dexamethasone.     

Enhanced Cytoprotective Effects of the Inhibitor of Apoptosis Protein Cellular IAP1 through Stabilization with TRAF2

Inhibitor of apoptosis (IAP) proteins are key regulators of intracellular signaling that interact with tumor necrosis factor (TNF) receptor superfamily members as well as proapoptotic molecules such as Smac/DIABLO and caspases. Whereas the X-linked IAP is an established caspase inhibitor, the protective mechanisms utilized by the cellular IAP (c-IAP) proteins are less clear because c-IAPs bind to but do not inhibit the enzymatic activities of caspases. In this study, c-IAPs are shown to be highly unstable molecules that undergo autoubiquitination. The autoubiquitination of c-IAP1 is blocked upon coexpression with TNF receptor-associated factor (TRAF) 2, and this is achieved by inhibition of the E3 ubiquitin ligase activity intrinsic to the RING of c-IAP1. Consistent with these observations, loss of TRAF2 results in a decrease in c-IAP1 levels. Stabilized c-IAP1 was found to sequester and prevent Smac/DIABLO from antagonizing X-linked IAP and protect against cell death. Therefore, this study describes an intriguing cytoprotective mechanism utilized by c-IAP1 and provides critical insight into how IAP proteins function to alter the apoptotic threshold.The inhibitors of apoptosis (IAPs)² are an evolutionarily conserved gene family described originally as encoding cell death inhibitors. IAP proteins have subsequently been found to participate in a variety of additional intracellular signaling processes (1), and it has become evident that IAP proteins are versatile molecules playing numerous distinct roles within the cell. Although a more complete understanding of these additional functions for IAP proteins is emerging, the distinct mechanisms utilized by some IAP proteins to function in their originally defined roles as cell death inhibitors remain unclear.Members of the IAP family are characterized by the presence of 1–3 tandem repeats of an ∼70-residue baculovirus IAP repeat domain (2). The baculovirus IAP repeat domains of many IAP proteins have been shown to be the region within IAP proteins that associates with caspases and other proapoptotic molecules (3, 4). IAP proteins have remarkably different apoptotic inhibitory abilities. For example, X-linked IAP (XIAP) is a highly potent cell death inhibitor (5) and is thought to be the only mammalian IAP protein that directly inhibits the enzymatic activities of caspases (2–4, 6). Although cellular IAP1 and -2 (c-IAP1 and c-IAP2) are anti-apoptotic proteins that can bind to caspase-7 and -9, they do not inhibit the enzymatic activities of these caspases (2, 6).Many IAP proteins, including c-IAP1 and c-IAP2, contain a carboxyl-terminal RING domain that can function as an E3 ubiquitin ligase (7). The E3 ubiquitin ligase activity of the RING domain in c-IAP1 and c-IAP2 was previously shown to negatively regulate the apoptotic inhibitory properties of c-IAP proteins and to promote autoubiquitination and degradation of c-IAP1 (8, 9), thus hindering attempts to define the cellular properties of this protein.A specialized property of the c-IAP proteins is their involvement in tumor necrosis family (TNF) signaling (10–12). Both c-IAP1 and c-IAP2 were discovered in a biochemical screen for factors associated with the type 2 TNF receptor. This association was found to be indirect and bridged by interactions with TNF receptor-associated factors (TRAFs), most notably TRAF1 and TRAF2 (11). Though the consequences of the association between TRAF2 and c-IAP1 on TNF-mediated signaling have been investigated (12), less is known about the functional significance of the association between TRAF2 and c-IAP1 on cell death inhibition. Because both c-IAP1 and TRAF2 possess E3 ubiquitin ligase activity in their respective RING domains, it seemed that the association between these molecules might impact the protective properties of c-IAP1 and alter the apoptotic threshold.In this study, the role of TRAF2 in c-IAP1 stability and how the association of TRAF2 with c-IAP1 affects the apoptotic inhibitory properties of c-IAP1 were examined. The presence of TRAF2 greatly enhanced the stability of c-IAP1, and these data suggest that the interaction between TRAF2 and c-IAP1 inhibits the E3 ubiquitin ligase activity intrinsic to the RING domain of c-IAP1. Using stabilized c-IAP1, the anti-apoptotic activity of c-IAP1 was characterized, and it was found that c-IAP1 suppresses apoptosis to a degree comparable with XIAP. Furthermore, we show that c-IAP1 functions to prevent the IAP antagonist, Smac/DIABLO (13, 14), from interfering with XIAP inhibition of caspases. Together, this study demonstrates that although c-IAP1 does not directly inhibit caspase activity, stabilized c-IAP1 can sequester Smac/DIABLO, prevent Smac/DIABLO from antagonizing XIAP, and inhibit cell death.     

Cytoprotective effects of IAPs revealed by a small molecule antagonist

Deregulated expression of members of the IAP (inhibitor of apoptosis) family has been identified in a wide variety of neoplastic cells, and synthetic IAP antagonists represent a promising novel class of chemotherapeutic agents. Early work focused on the ability of these compounds to block the caspase-inhibitory function of XIAP (X-linked IAP). However, recent studies have shown that IAP antagonists, although primarily designed to target XIAP, trigger ubiquitin-mediated degradation of two related proteins, c-IAP (cellular IAP) 1 and c-IAP2, and through this process potentiates the death of tumour cells via autocrine cellular-signalling pathways. In this context, the relative contribution of XIAP as a target of this class of compounds is unclear. In the present study, we examine the involvement of XIAP using a recently described synthetic IAP antagonist, AEG40730, and through comparison of a human XIAP-depleted tumour cell line with its isogenic wild-type control line. Treatment with nanomolar concentrations of AEG40730 resulted in the loss of both XIAP and c-IAP1 proteins, albeit with different kinetics. Although XIAP-deficient HCT116 cells retained some sensitivity to external apoptotic stimuli, the results suggest that IAP antagonists, such as AEG40730, exert their apoptosis-enhancing effects through XIAP in addition to the c-IAPs. These results indicate that IAP antagonists can target multiple IAPs to augment distinct pro-apoptotic signalling pathways, thereby revealing the potential for these compounds in cancer therapy and underscoring the promise of IAP-targeted therapies.     

Benzothiazoles as Rho-associated kinase (ROCK-II) inhibitors

A series of benzothiazole derivatives as ROCK inhibitors have been discovered. Compounds with good biochemical and cellular potency, and sufficient kinase selectivity have been identified.