Nitric Oxide Produced in Response to Engagement of beta 2 Integrins on Human Neutrophils Activates the Monomeric GTPases Rap1 and Rap2 and Promotes Adhesion

We found that engagement of beta 2 integrins on human neutrophils increased the levels of GTP-bound Rap1 and Rap2. Also, the activation of Rap1 was blocked by PP1, SU6656, LY294002, GF109203X, or BAPTA-AM, which indicates that the downstream signaling events in Rap1 activation involve Src tyrosine kinases, phosphoinositide 3-kinase, protein kinase C, and release of calcium. Surprisingly, the integrin-induced activation of Rap2 was not regulated by any of the signaling pathways mentioned above. However, we identified nitric oxide as the signaling molecule involved in beta 2 integrin-induced activation of Rap1 and Rap2. This was illustrated by the fact that engagement of beta 2 integrins increased the production of nitrite, a stable end-product of nitric oxide. Furthermore, pretreatment of neutrophils with N-monomethyl-L-arginine, or 1400W, which are inhibitors of inducible nitric-oxide synthase, blocked integrin-induced activation of Rap1 and Rap2. Similarly, Rp-8pCPT-cGMPS, an inhibitor of cGMP-dependent serine/threonine kinases, also blunted the integrin-induced activation of Rap GTPases. Also nitric oxide production and its downstream activation of cGMP-dependent serine/threonine kinases were essential for proper neutrophil adhesion by beta 2 integrins. Thus, we made the novel findings that beta 2 integrin engagement on human neutrophils triggers production of nitric oxide and its downstream signaling is essential for activation of Rap GTPases and neutrophil adhesion.

A family with erythrocytosis establishes a role for prolyl hydroxylase domain protein 2 in oxygen homeostasis

The number of red blood cells is normally tightly regulated by a classic homeostatic mechanism based on oxygen sensing in the kidney. Decreased oxygen delivery resulting from anemia induces the production of erythropoietin, which increases red cell production and hence oxygen delivery. Investigations of erythropoietin regulation identified the transcription factor hypoxia-inducible factor (HIF). HIF is now recognized as being a key regulator of genes that function in a comprehensive range of processes besides erythropoiesis, including energy metabolism and angiogenesis. HIF itself is regulated through the -subunit, which is hydroxylated in the presence of oxygen by a family of three prolyl hydroxylase domain proteins (PHDs)/HIF prolyl hydroxylases/egg-laying-defective nine enzymes. Hydroxylation allows capture by the von Hippel–Lindau tumor suppressor gene product, ubiquitination, and destruction by the proteasome. Here we describe an inherited mutation in a mammalian PHD enzyme. We show that this mutation in PHD2 results in a marked decrease in enzyme activity and is associated with familial erythrocytosis, identifying a previously unrecognized cause of this condition. Our findings indicate that PHD2 is critical for normal regulation of HIF in humans.

Assembly intermediates in polyketide biosynthesis; enantioselective syntheses of beta-hydroxycarbonyl compounds

A versatile approach for the enantioselective synthesis of functionalised beta-hydroxy N-acetylcysteamine thiol esters has been developed which allows the facile incorporation of isotopic labels. It has been shown that a remarkable reversal of selectivity occurs in the titanium mediated aldol reaction of the acyloxazolidone intermediate using either (S)- or (R)-tert-butyldimethylsilyloxybutanal. The aldol products are valuable intermediates in the synthesis of 4-hydroxy-6-substituted gamma-lactones.

Inactivation of Human beta-Defensins 2 and 3 by Elastolytic Cathepsins

beta-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human beta-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that beta-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of beta-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization.

Development of a rapid screening test for veterinary sedatives and the beta-blocker carazolol in porcine kidney by ELISA

Sedatives and tranquillisers are frequently used to reduce stress during the transportation of food producing animals. The most widely used classes of sedatives include the butyrophenone azaperone, the phenothiazines acepromazine, propionylpromazine, chlorpromazine and the beta-blocker, carazolol. For regulatory control purposes, tolerances for azaperone and carazolol have been set by the European Union as 100 and 25 mug kg(-1), respectively. Furthermore, the use of the phenothiazines is prohibited and therefore has a zero tolerance. A method for the detection of residues of five tranquillisers and one beta-blocker using a single ELISA plate has been developed. Kidney samples (2.5 g) were extracted with dichloromethane and applied to a competitive enzyme immunoassay using three polyclonal antibodies raised in rabbits against azaperol, propionylpromazine and carazolol conjugates. In sample matrix, the azaperol antibody cross-reacted 28.0% with azaperone and the propionylpromazine antibody cross-reacted 24.9% with acepromazine and 11.7% with chlorpromazine. In the ELISA, the detection capabilities of the six sedatives, azaperol, azaperone, carazolol, acepromazine, chlorpromazine, and propionylpromazine are 5, 15, 5, 5, 20 and 5 mug kg(-1), respectively. The proposed method is a sensitive and rapid multi-residue technique that offers a cost effective alternative to current published procedures, without any concession on the ability to detect sedative misuse.