Bovine beta-defensins: identification and characterization of novel bovine beta-defensin genes and their expression in mammary gland tissue.

beta-Defensin genes code for multifunctional peptides with a broad-range antimicrobial activity. In this project we hypothesized that beta-defensin genes may be candidate genes for resistance to mastitis. In this article we describe the identification and genomic characterization of eight bovine beta-defensin genes, including six novel defensin genes and two pseudogenes. Expression in the bovine mammary gland of one of the novel genes, DEFB401, has been demonstrated, as well as the expression of LAP, TAP, DEFB1, BNBD3, BNBD9, and BNBD12. For genomic characterization, 20 BACs from two different bovine BAC libraries (RZPD numbers 750 and 754) were isolated by PCR screening with beta-defensin consensus primers derived from published sequences. PCR products from BACs generated with consensus primers have been subcloned and sequenced, revealing a total of 16 genes and two pseudogenes. Six novel beta-defensin genes share the typical exon-intron structure and are highly homologous to published bovine beta-defensin genes. They are named DEFB401- DEFB405 and LAP-like, and two novel pseudogenes are named EBD-P and EBD-P2. Analysis of mammary gland tissue-derived cDNA from nine cows with different clinical findings demonstrated the expression of several beta-defensin genes mentioned above. First results indicate that the lactational status of the cow presumably has no influence on gene expression. Competent knowledge of antimicrobial activity of beta-defensins from literature, the abundance of beta-defensin mRNA in the bovine mammary gland, and the inducibility of some genes give first evidence that beta-defensins may play a role in local host defense during udder infections.

Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ.

Whereas endothelial responses to shear stress have been studied extensively, the responses to circumferential vascular stretch are yet poorly defined. Circumferential stretch in pulmonary microvessels is largely determined by the transmural pressure gradient, hence by both vascular perfusion and alveolar ventilation pressures. Here, we have studied the production of nitric oxide (NO) by the endothelial nitric oxide synthase (eNOS) in two different models of vascular stretch in the intact lung: In isolated-perfused rat lungs, vascular stretch was induced by elevation of vascular pressure. In situ digital fluorescence microscopy revealed stretch-dependent NO production, which was localized to capillary endothelial cells and inhibited by NOS blockers. In isolated-perfused mouse lungs, vascular stretch was generated by ventilation with elevated negative pressure. Stretch-induced phosphorylation of Akt and eNOS in lung endothelial cells was demonstrated by immunohistochemistry and increased NO production by in situ fluorescence microscopy. Stretch-induced endothelial responses in both models were abrogated by pretreatment with phosphatidylinositol-3-OH kinase inhibitors. These findings demonstrate that circumferential stretch activates NO production in pulmonary endothelial cells by a signaling cascade involving phosphatidylinositol-3-OH kinase, Akt, and eNOS and that this response is independent from the mechanical factors causing vascular distension.