Cyclin-dependent kinases (CDKs) are conserved regulators of the eukaryotic cell cycle with different isoforms controlling specific phases of the cell cycle. Mitogenic or growth inhibitory signals are mediated, respectively, by activation or inhibition of CDKs which phosphorylate proteins associated with the cell cycle. The central role of CDKs in cell cycle regulation makes them a potential new target for inhibitory molecules with anti-proliferative and/or anti-neoplastic effects. We describe the crystal structures of the complexes of CDK2 with a weakly specific CDK inhibitor, N6-(delta 2-isopentenyl)adenine, and a strongly specific inhibitor, olomoucine. Both inhibitors are adenine derivatives and bind in the adenine binding pocket of CDK2, but in an unexpected and different orientation from the adenine of the authentic ligand ATP. The N6-benzyl substituent in olomoucine binds outside the conserved binding pocket and is most likely responsible for its specificity. The structural information from the CDK2-olomoucine complex will be useful in directing the search for the next generation inhibitors with improved properties.

To further elucidate the functions of actin in budding yeast and to relate actin structure to specific roles and interactions in vivo, we determined the phenotypes caused by 13 charged-to-alanine mutations isolated previously in the single Saccharomyces cerevisiae actin gene. Defects in actin organization, morphogenesis, budding pattern, chitin deposition, septation, nuclear segregation, and mitochondrial organization were observed. In wild-type cells, mitochondria were found to be aligned along actin cables. Many of the amino acid substitutions that had the most severe effects on mitochondrial organization are located under the myosin "footprint" on the actin monomer, suggesting that actin-myosin interactions might underlie mitochondrial organization in yeast. In addition, one mutant (act1-129; R177A, D179A) produced an actin that assembled into cables and patches that could be visualized by anti-actin immunofluorescence in situ and that assembled into microfilaments of normal appearance in vitro as judged by electron microscopy but which could not be labeled by rhodamine-phalloidin in situ or in vitro. Rhodamine-phalloidin could label actin filaments assembled from all of the other mutant actins, including one (act1-119; R116A, E117A, K118A) that is altered at a residue (E117) that can be chemically cross-linked to phalloidin. The implication of residues R177 and/or D179 in phalloidin binding is in close agreement with a recently reported molecular model in which the phalloidin-binding site is proposed to be at the junction of two or three actin monomers in the filament.

IL-1β is a potent proinflammatory cytokine that is implicated in the pathogenesis of acute respiratory distress syndrome. We hypothesized that LPS and mechanical ventilation (MV) together could lead to IL-1β secretion and the development of acute lung injury (ALI), and that this process would be dependent on caspase-1 and the nucleotide binding domain and leucine-rich repeat (NLR) pyrin domain containing 3 (NLRP3) inflammasome activation. The objectives of this study were to determine the specific role of IL-1β, caspase-1, and the NLRP3 inflammasome in a two-hit model of ALI due to LPS plus MV. We used a two-hit murine model of ALI in which both inhaled LPS and MV were required for the development of hypoxemia, pulmonary neutrophil infiltration, and alveolar leakage. Nlrp3-deficent and Casp1-deficient mice had significantly diminished IL-1β levels in bronchoalveolar lavage fluid, and were specifically protected from hypoxemia, despite similar alveolar neutrophil infiltration and leakage. The IL-1 receptor antagonist, Anakinra, significantly improved the specific development of hypoxemia without significant effects on neutrophil infiltration or alveolar leakage. MV resulted in increased bronchoalveolar lavage extracellular ATP and alveolar macrophage apoptosis as triggers of NLRP3 inflammasome activation. NLRP3 inflammasome activation and IL-1β production play a key role in ALI caused by the combination of LPS and MV, particularly in the hypoxemia associated with acute respiratory distress syndrome. Blocking IL-1 signaling in this model specifically ameliorates hypoxemia, without affecting neutrophil infiltration and alveolar leakage, disassociating these readouts of ALI. MV causes alveolar macrophage apoptosis, a key step in the activation of NLRP3 inflammasome and production of IL-1β.

SUMMARY Acute lung injury (ALI) remains a serious health issue with little improvement in our understanding of the pathophysiology and therapeutic approaches. We investigated the mechanism that lipopolysaccharide (LPS) induces early neutrophil recruitment to lungs and increases pulmonary vascular permeability during ALI. Intratracheal LPS induced release of pro-interleukin-1α (IL-1α) from necrotic alveolar macrophages (AM), which activated endothelial cells (EC) to induce vascular leakage via loss of vascular endothelial (VE)-cadherin. LPS triggered the AM purinergic receptor P2X7(R) to induce Ca2+ influx and ATP depletion, which led to necrosis. P2X7R deficiency significantly reduced necrotic death of AM and release of pro-IL-1α into the lung. CD14 was required for LPS binding to P2X7R, as CD14 neutralization significantly diminished LPS induced necrotic death of AM and pro-IL-1α release. These results demonstrate a key role for pro-IL-1α from necrotic alveolar macrophages in LPS-mediated ALI, as a critical initiator of increased vascular permeability and early neutrophil infiltration.

Interleukin-1β (IL-1β) is a highly inflammatory cytokine that significantly contributes to both acute and chronic inflammatory diseases. The secretion of IL-1β requires a unique protease, caspase-1, which is activated by various protein platforms called inflammasomes. Data suggests a key role for mitochondrial reactive oxygen species for inflammasome activation. Flavonoids constitute a group of naturally occurring polyphenolic molecules with many biological activities, including antioxidant effects. In this study, we investigated the effect of three flavonoids, quercetin (QUC), naringenin, and silymarim on inflammasome activation. We found that QUC inhibits IL-1β secretion by both the NLRP3 and AIM2 inflammasome in a dose dependent manner, but not the NLRC4 inflammasome. QUC inhibition of the inflammasome was still observed in Atg16l1 knockout macrophages, indicating that QUC’s effect was autophagy independent. Since QUC inhibited both NLRP3 and AIM2 inflammasomes but not NLRC4, we assessed ASC speck formation. QUC reduced ASC speck formation and ASC oligomerization compared with controls. Additionally, QUC inhibited IL-1β in Cryopyrin-Associated Periodic Syndromes (CAPS) macrophages, where NLRP3 inflammasome is constitutively activated. In conclusion, QUC inhibits both the NLRP3 and AIM2 inflammasome by preventing ASC oligomerization and may be a potential therapeutic candidate for Kawasaki disease vasculitis and other IL-1 mediated inflammatory diseases.

This large outbreak of P. aeruginosa infections related to bronchoscopy was apparently caused by a loose biopsy-port cap in the bronchoscopes. Instrument safety and surveillance methods for bronchoscopy must be improved, and better recall procedures are needed for medical devices.

BackgroundVentilator-induced lung injury is a form of acute lung injury that develops in critically ill patients on mechanical ventilation and has a high degree of mortality. Nicotinamide phosphoribosyltransferase is an enzyme that is highly upregulated in ventilator-induced lung injury and exacerbates the injury when given exogenously. Nicotinamide (vitamin B3) directly inhibits downstream pathways activated by Nicotinamide phosphoribosyltransferase and is protective in other models of acute lung injury.MethodsWe administered nicotinamide i.p. to mice undergoing mechanical ventilation with high tidal volumes to study the effects of nicotinamide on ventilator-induced lung injury. Measures of injury included oxygen saturations and bronchoalveolar lavage neutrophil counts, protein, and cytokine levels. We also measured expression of nicotinamide phosophoribosyltransferase, and its downstream effectors Sirt1 and Cebpa, Cebpb, Cebpe. We assessed the effect of nicotinamide on the production of nitric oxide during ventilator-induced lung injury. We also studied the effects of ventilator-induced lung injury in mice deficient in C/EBPε.ResultsNicotinamide treatment significantly inhibited neutrophil infiltration into the lungs during ventilator-induced lung injury, but did not affect protein leakage or cytokine production. Surprisingly, mice treated with nicotinamide developed significantly worse hypoxemia during mechanical ventilation. This effect was not linked to increases in nitric oxide production or alterations in expression of Nicotinamide phosphoribosyl transferase, Sirt1, or Cebpa and Cebpb. Cebpe mRNA levels were decreased with either nicotinamide treatment or mechanical ventilation, but mice lacking C/EBPε developed the same degree of hypoxemia and ventilator-induced lung injury as wild-type mice.ConclusionsNicotinamide treatment during VILI inhibits neutrophil infiltration of the lungs consistent with a strong anti-inflammatory effect, but paradoxically also leads to the development of significant hypoxemia. These findings suggest that pulmonary neutrophilia is not linked to hypoxemia in ventilator-induced lung injury, and that nicotinamide exacerbates hypoxemia during VILI.

Aim: This study aimed to assess the association between regional tidal volume (V T), regional functional residual capacity (FRC) and the expression of genes linked with ventilator-induced lung injury. Methods: Two groups of BALB/c mice (n = 8 per group) were ventilated for 2 h using a protective or injurious ventilation strategy, with free-breathing mice as controls. Regional V T and FRC of the ventilated mice was determined from analysis of high-resolution fourdimensional computed tomography (4D-CT) images, taken at baseline and after 2 h of ventilation and corrected for the volume of the region (i.e. specific [s]Vt and specific [s]FRC). RNA levels of 21 genes in ten different lung regions were quantified using qPCR array. Results: sFRC at baseline varied regionally, independent of ventilation strategy, whereas sV T varied regionally depending on ventilation strategy. The expression of IL-6 (P = 0.04), Ccl2 (P < 0.01) and Ang 2 (P < 0.05) were associated with sV T but not sFRC. The expression of seven other genes varied regionally (IL-1β and RAGE) or depended on ventilation strategy (Nfe2l2, c-fos and Wnt1) or both (TNF-α and CxCl2), but were not associated with regional sFRC or sV T. Conclusion: These observations suggest that regional inflammatory responses to mechanical ventilation are primarily driven by tidal stretch.