The p110δ subunit of PI3K regulates bone marrow-derived eosinophil trafficking and airway eosinophilia in allergen-challenged mice

Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.     

Bim inhibits autophagy by recruiting beclin 1 to microtubules

Bim is a proapoptotic BH3-only Bcl-2 family member.?In response to death stimuli, Bim dissociates from the dynein light chain 1 (DYNLL1/LC8), where it is inactive, and can then initiate Bax/Bak-mediated mitochondria-dependent apoptosis. We found that Bim depletion increases autophagosome synthesis in cells and in?vivo, and this effect is inhibited by overexpression of cell death-deficient Bim. Bim inhibits autophagy by interacting with Beclin 1, an autophagy regulator, and this interaction is facilitated by LC8. Bim bridges the Beclin 1-LC8 interaction and thereby inhibits autophagy by mislocalizing Beclin 1 to the dynein motor complex. Starvation, an autophagic stimulus, induces Bim phosphorylation, which abrogates LC8 binding to Bim, leading to dissociation of Bim and Beclin 1. Our data suggest that Bim switches locations between apoptosis-inactive/autophagy-inhibitory and apoptosis-active/autophagy-permissive sites.     

Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.     

A familial deletion 4q syndrome: An outcome of a paracentric inversion

Chromosome inversions are intra-chromosomal rearrangements formed when the chromosome breaks occur at two places, and in the process of repair the intervening segments are joined in an inverted or opposite manner. Inversions themselves do not appear to cause clinical anomalies, if balanced. Abnormal phenotypes can occur due to gene disruption at the point of breakage and reunion or due to duplication/deficiency recombinants formed during crossover at meiosis. We report a case with familial deletion 4q syndrome in a 1-year-old female child with dysmorphism and congenital abnormalities. The deletion was an outcome of a paracentric inversion 4q31.2q35.2. The deletion was confirmed by fluorescence in situ hybridization using telomeric DNA probes for chromosome No. 4. An attempt was made to correlate the genotype with the phenotype. The father had the same rearrangement with a milder phenotype. The recurrence risk in such cases is high.     

The ER-bound RING finger protein 5 (RNF5/RMA1) causes degenerative myopathy in transgenic mice and is deregulated in inclusion body myositis

Growing evidence supports the importance of ubiquitin ligases in the pathogenesis of muscular disorders, although underlying mechanisms remain largely elusive. Here we show that the expression of RNF5 (aka RMA1), an ER-anchored RING finger E3 ligase implicated in muscle organization and in recognition and processing of malfolded proteins, is elevated and mislocalized to cytoplasmic aggregates in biopsies from patients suffering from sporadic-Inclusion Body Myositis (sIBM). Consistent with these findings, an animal model for hereditary IBM (hIBM), but not their control littermates, revealed deregulated expression of RNF5. Further studies for the role of RNF5 in the pathogenesis of s-IBM and more generally in muscle physiology were performed using RNF5 transgenic and KO animals. Transgenic mice carrying inducible expression of RNF5, under control of beta-actin or muscle specific promoter, exhibit an early onset of muscle wasting, muscle degeneration and extensive fiber regeneration. Prolonged expression of RNF5 in the muscle also results in the formation of fibers containing congophilic material, blue-rimmed vacuoles and inclusion bodies. These phenotypes were associated with altered expression and activity of ER chaperones, characteristic of myodegenerative diseases such as s-IBM. Conversely, muscle regeneration and induction of ER stress markers were delayed in RNF5 KO mice subjected to cardiotoxin treatment. While supporting a role for RNF5 Tg mice as model for s-IBM, our study also establishes the importance of RNF5 in muscle physiology and its deregulation in ER stress associated muscular disorders.     

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.     

Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients correlates with the progression of disease

Free radicals are implicated in numerous disease processes including motor neuron degeneration (MND). Antioxidant defense enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the erythrocytes are capable of detoxifying reactive oxygen species produced endogenously or exogenously. In the present study, the extent of lipid peroxidation (LPO) and antioxidant defenses were evaluated in the erythrocytes of 20 sporadic amyotrophic lateral sclerosis (ALS) patients and 20 controls. We observed that lipid peroxidation in the erythrocytes of amyotrophic lateral sclerosis patients significantly increased with respect to controls (P<0.001). On the other hand, catalase activity was found to be significantly lower (P<0.001). The activities of glucose-6-phosphate dehydrogenase, glutathione reductase and glutathione levels were also found to be significantly reduced in ALS patients compared to healthy subjects (P<0.001, P<0.01 and P<0.01, respectively). It was further observed that lipid peroxidation started to increase and catalase, glutathione reductase, glucose-6-phosphate dehydrogenase enzyme activities and glutathione levels started to decrease as amyotrophic lateral sclerosis progressed from 6 to 24 months, suggesting a correlation between these parameters and duration of amyotrophic lateral sclerosis. This study confirms the involvement of oxidative stress during the progression of amyotrophic lateral sclerosis and the need to develop specific peripheral biomarkers.     

Synthesis of 2-[3-(7-Chloro-quinolin-4-ylamino)-alkyl]-1-(substituted phenyl)-2,3,4,9-tetrahydro-1H-beta-carbolines as a new class of antimalarial agents

A series of hybrid molecules 2-[3-(7-Chloro-quinolin-4-ylamino)-alkyl]-1-(substituted phenyl)-2,3,4,9-tetrahydro-1H-beta-carbolines have been synthesized and screened for their in vitro antimalarial activity against chloroquine-sensitive strains of Plasmodium falciparum. Compounds 26, 32, and 34 have shown MIC in the range of 0.05-0.11 microM and are in vitro several folds more active than chloroquine.     

New functionalized 1,2,4-trioxepanes: synthesis and antimalarial activity against multi-drug resistant P. yoelii in mice

A series of new amino functionalized 1,2,4-trioxepanes 8-16 and ester functionalized 1,2,4-trioxepanes 17-19 have been synthesized and evaluated against multi-drug resistant Plasmodium yoelii in Swiss mice. Amino functionalized trioxepanes 14, the most active compound of the series, showed 100% clearance of parasitaemia by oral route on day 4 and 75% protection to the treated mice beyond day 28.