Guanosine inhibits CD40 receptor expression and function induced by cytokines and beta amyloid in mouse microglia cells

Growing evidence implicates CD40, a member of the TNFR superfamily, as contributing to the pathogenesis of many neurodegenerative diseases. Thus, strategies to suppress its expression may be of benefit in those disorders. To this aim, we investigated the effect of guanosine, a purine nucleoside that exerts neurotrophic and neuroprotective effects. CD40 expression and function are increased by exposure of mouse microglia cultures or the N9 microglia cell line to IFN-gamma (10 ng/ml) plus TNF-alpha (50 ng/ml) or beta amyloid (Abeta) peptide (Abeta(1-42); 500 nM). Culture pretreatment with guanosine (10-300 microM), starting 1 h before cytokine or Abeta addition, dose-dependently inhibited the CD40-induced expression as well as functional CD40 signaling by suppressing IL-6 production promoted by IFN-gamma/TNF-alpha challenge in the presence of CD40 cross-linking. Moreover, guanosine abrogated IFN-gamma-induced phosphorylation on Ser(727) and translocation of STAT-1alpha to the nucleus as well as TNF-alpha-/Abeta-induced IkappaBalpha and NF-kappaB p65/RelA subunit phosphorylation, thus inhibiting NF-kappaB-induced nuclear translocation. Guanosine effects were mediated by an increased phosphorylation of Akt, a PI3K downstream effector, as well as of ERK1/2 and p38 in the MAPK system, because culture pretreatment with selective ERK1/2, p38 MAPK, and PI3K antagonists (U0126, SB203580, or LY294002, respectively) counteracted guanosine inhibition on IFN-gamma/TNF-alpha-induced CD40 expression and function as well as on STAT-1alpha or NF-kappaB nuclear translocation. These findings suggest a role for guanosine as a potential drug in the experimental therapy of neuroinflammatory/neurodegenerative diseases, particularly Alzheimer's disease.     

The required role of endogenously produced lipoxin A4 and annexin-1 for the production of IL-10 and inflammatory hyporesponsiveness in mice

The appropriate development of an inflammatory response is central for the ability of a host to deal with any infectious insult. However, excessive, misplaced, or uncontrolled inflammation may lead to acute or chronic diseases. The microbiota plays an important role in the control of inflammatory responsiveness. In this study, we investigated the role of lipoxin A4 and annexin-1 for the IL-10-dependent inflammatory hyporesponsiveness observed in germfree mice. Administration of a 15-epi-lipoxin A4 analog or an annexin-1-derived peptide to conventional mice prevented tissue injury, TNF-alpha production, and lethality after intestinal ischemia/reperfusion. This was associated with enhanced IL-10 production. Lipoxin A4 and annexin-1 failed to prevent reperfusion injury in IL-10-deficient mice. In germfree mice, there was enhanced expression of both lipoxin A4 and annexin-1. Blockade of lipoxin A4 synthesis with a 5-lipoxygenase inhibitor or Abs against annexin-1 partially prevented IL-10 production and this was accompanied by partial reversion of inflammatory hyporesponsiveness in germfree mice. Administration of BOC-1, an antagonist of ALX receptors (at which both lipoxin A4 and annexin-1 act), or simultaneous administration of 5-lipoxygenase inhibitor and anti-annexin-1 Abs, was associated with tissue injury, TNF-alpha production, and lethality similar to that found in conventional mice. Thus, our data demonstrate that inflammatory responsiveness is tightly controlled by the presence of the microbiota and that the innate capacity of germfree mice to produce IL-10 is secondary to their endogenous greater ability to produce lipoxin A4 and annexin-1.     

Feijoa sellowiana derived natural Flavone exerts anti-cancer action displaying HDAC inhibitory activities

Curative properties of some medicinal plants such as the Feijoa sellowiana Bert. (Myrtaceae), have been often claimed, although the corresponding molecular mechanism(s) remain elusive. We report here that the Feijoa acetonic extract exerts anti-cancer activities on solid and hematological cancer cells. Feijoa extract did not show toxic effects on normal myeloid progenitors thus displaying a tumor-selective activity. In the Feijoa acetonic extract, fractionation and subsequent purification and analyses identified Flavone as the active component. Flavone induces apoptosis which is accompanied by caspase activation and p16, p21 and TRAIL over-expression in human myeloid leukemia cells. Use of ex vivo myeloid leukemia patients blasts confirms that both the full acetonic Feijoa extract and its derived Flavone are able to induce apoptosis. In both cell lines and myeloid leukemia patients blasts the apoptotic activity of Feijoa extract and Flavone is accompanied by increase of histone and non-histone acetylation levels and by HDAC inhibition. Our findings show for the first time that the Feijoa apoptotic active principle is the Flavone and that this activity correlates with the induction of HDAC inhibition, supporting the hypothesis of its epigenetic pro-apoptotic regulation in cancer systems.     

Tyrosine phosphorylation of phosphoinositide-dependent kinase 1 by the insulin receptor is necessary for insulin metabolic signaling

In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IR(Delta43)) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IR(Delta43) (L6(IRDelta43)) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr(308). Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IR(WT)), but not IR(Delta43). Based on overlay blotting, PDK-1 directly bound IR(WT), but not IR(Delta43). Insulin-activated IR(WT), and not IR(Delta43), phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr-->Phe substitution prevented this inhibitory action. In the L6(hIR) cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IR(WT), on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr(308), and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6(IRDelta43) compared to control cells. In L6 cells expressing IR(WT), the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.     

Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation

In the present study we evaluated the role of neurokinins in the modulation of inducible nitric oxide synthase (iNOS) inflammatory cell expression in guinea pigs with chronic allergic airway inflammation. In addition, we studied the acute effects of nitric oxide inhibition on this response. Animals were anesthetized and pretreated with capsaicin (50 mg/kg sc) or vehicle 10 days before receiving aerosolized ovalbumin or normal saline twice weekly for 4 wk. Animals were then anesthetized, mechanically ventilated, given normal saline or N(G)-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg ic), and challenged with ovalbumin. Prechallenge exhaled NO increased in ovalbumin-exposed guinea pigs (P < 0.05 compared with controls), and capsaicin reduced this response (P < 0.001). Compared with animals inhaled with normal saline, ovalbumin-exposed animals presented increases in respiratory system resistance and elastance and numbers of total mononuclear cells and eosinophils, including those expressing iNOS (P < 0.001). Capsaicin reduced all these responses (P < 0.05) except for iNOS expression in eosinophils. Treatment with l-NAME increased postantigen challenge elastance and restored both resistance and elastance previously attenuated by capsaicin treatment. Isolated l-NAME administration also reduced total eosinophils and mononuclear cells, as well as those cells expressing iNOS (P < 0.05 compared with ovalbumin alone). Because l-NAME treatment restored lung mechanical alterations previously attenuated by capsaicin, NO and neurokinins may interact in controlling airway tone. In this experimental model, NO and neurokinins modulate eosinophil and lymphocyte infiltration in the airways.     

Angiogenesis and lipoxins

Angiogenesis, the growth of new capillaries from pre-existing ones, occurs through dynamic functions of the endothelial cells (EC), including migration, proliferation and maturation, which are essential to achieve an organized formation of the vessel sprout. Aspirin-triggered lipoxins (ATL), the 15R enantiomeric counterparts of native lipoxins, are endogenous lipid mediators generated within the vascular lumen during multicellular responses, which display potent and well-described immunomodulatory actions. Here we present some of the findings regarding the inhibition of EC responses in vitro and in vivo by these novel compounds and the modulation of fundamental steps of the angiogenic process, identifying previously unappreciated vascular actions of locally generated ATL and their longer acting synthetic analogs.     

Comparison of early and late responses to antigen of sensitized guinea pig parenchymal lung strips.

The peripheral lung parenchyma has been studied as a component of the asthmatic inflammatory response. During induced constriction, tissue resistance increases in different asthma models. Approximately 60% of the asthmatic patients show early and late responses. The late response is characterized by more severe airway obstruction. In the present study, we evaluated lung parenchymal strips mechanics in ovalbumin-sensitized guinea pigs, trying to reproduce both early and late inflammatory responses. Oscillatory mechanics of lung strips were performed in a control group (C), in an early response group (ER), and in two late response groups: 17 h (L1) and 72 h (L2) after the last ovalbumin challenge. Measurements of resistance and elastance were obtained before and after ovalbumin challenge in C and ER groups and before and after acetylcholine challenge in all groups. Using morphometry, we assessed the density of eosinophils and smooth muscle cells, as well as collagen and elastin content in lung strips. The baseline and postagonist values of resistance and elastance were increased in ER, L1, and L2 groups compared with C (P < or = 0.001). The morphometric analysis showed an increase in alveolar eosinophil density in ER and L2 groups compared with C (P < 0.05). There was a significant correlation between eosinophil density in parenchymal strips of C, L1, and L2 groups and values of resistance and elastance postacetylcholine (r = 0.71, P = 0.001 and r = 0.74, P < 0.001, respectively). The results show that the lung parenchyma is involved in the late response, and the constriction response in this phase is related to the eosinophilic inflammation.     

Identification and Functional Characterization of a Novel Mitochondrial Carrier for Citrate and Oxoglutarate in Saccharomyces cerevisiae

Mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and coenzymes across the mitochondrial membrane. The function of only a few of the 35 Saccharomyces cerevisiae mitochondrial carriers still remains to be uncovered. In this study, we have functionally defined and characterized the S. cerevisiae mitochondrial carrier Yhm2p. The YHM2 gene was overexpressed in S. cerevisiae, and its product was purified and reconstituted into liposomes. Its transport properties, kinetic parameters, and targeting to mitochondria show that Yhm2p is a mitochondrial transporter for citrate and oxoglutarate. Reconstituted Yhm2p also transported oxaloacetate, succinate, and fumarate to a lesser extent, but virtually not malate and isocitrate. Yhm2p catalyzed only a counter-exchange transport that was saturable and inhibited by sulfhydryl-blocking reagents but not by 1,2,3-benzenetricarboxylate (a powerful inhibitor of the citrate/malate carrier). The physiological role of Yhm2p is to increase the NADPH reducing power in the cytosol (required for biosynthetic and antioxidant reactions) and probably to act as a key component of the citrate-oxoglutarate NADPH redox shuttle between mitochondria and cytosol. This protein function is based on observations documenting a decrease in the NADPH/NADP⁺ and GSH/GSSG ratios in the cytosol of ΔYHM2 cells as well as an increase in the NADPH/NADP⁺ ratio in their mitochondria compared with wild-type cells. Our proposal is also supported by the growth defect displayed by the ΔYHM2 strain and more so by the ΔYHM2ΔZWF1 strain upon H₂O₂ exposure, implying that Yhm2p has an antioxidant function.     

Aqueous dispersions of DMPG in low salt contain leaky vesicles

Aqueous dispersions of dimyristoyl phosphatidylglycerol (DMPG), at low ionic strength, display uncommon thermal behavior. Models for such behavior need to assign a form to the lipid aggregate. Although most studies accept the presence of lipid vesicles in the lipid gel and fluid phases, this is still controversial. With electron spin resonance (ESR) spectra of spin labels incorporated into DMPG aggregates, quantification of [(14)C]sucrose entrapped by the aggregates, and viscosity measurements, we demonstrate the existence of leaky vesicles in dispersions of DMPG at low ionic strength, in both gel and fluid phases of the lipid. As a control system, the ubiquitous lipid dimyristoyl phosphatidylcholine (DMPC) was used. For DMPG in the gel phase, spin labeling only indicated the presence of lipid bilayers, strongly suggesting that DMPG molecules are organized as vesicles and not micelles or bilayer fragments (bicelles), as the latter has a non-bilayer structure at the edges. Quantification of [(14)C]sucrose entrapping by DMPG aggregates revealed the presence of highly leaky vesicles. Due to the short hydrocarbon chains ((14)C atoms), DMPC vesicles were also found to be partially permeable to sucrose, but not as much as DMPG vesicles. Viscosity measurements, with the calculation of the intrinsic viscosity of the lipid aggregate, showed that DMPG vesicles are rather similar in the gel and fluid phases, and quite different from aggregates observed along the gel-fluid transition. Taken together, our data strongly supports that DMPG forms leaky vesicles at both gel and fluid phases.     

Estimation of population density of European pine marten in central Italy using camera trapping

Evaluating presence and abundance of small carnivores is essential for their conservation. In Italy, there is scarce information on European pine marten distribution, and no data are published on its abundance. Camera traps have been widely used to estimate population density applying capture–recapture models for species in which individual recognition is possible. Here we estimate the abundance of European pine martens in central Italy using camera trapping and a model that allows the estimation of population density without the need for individual recognition Rowcliffe et al. (Anim Conserv 11:185–186, 2008). Camera trapping was also used to evaluate habitat use patterns by martens. Fifteen camera traps were deployed in 90 placements for 15 days each, for a total of 1,334 camera days. Pine martens were captured in 24% of camera trap placements with a mean trap success rate of 0.33 photographs per camera placement. Estimated pine marten population density in the study area was 0.34 individuals km⁻². Marten trap rate was not strongly associated with any habitat type, although there were trends towards lower probability of records at locations with high coverage of cultivated fields and higher probability of records at locations with high coverage of human-made woodland. The results suggest that pine martens in this area are not confined to wooded habitat. To our knowledge, this study is the first application of the Rowcliffe et al. (Anim Conserv 11:185–186, 2008) method to a wild carnivore population and, furthermore, the first estimation of population density of pine martens in Italy.