Analysis of the biosynthetic gene cluster for the polyether antibiotic monensin in Streptomyces cinnamonensis and evidence for the role of monB and monC genes in oxidative cyclization

The analysis of a candidate biosynthetic gene cluster (97 kbp) for the polyether ionophore monensin from Streptomyces cinnamonensis has revealed a modular polyketide synthase composed of eight separate multienzyme subunits housing a total of 12 extension modules, and flanked by numerous other genes for which a plausible function in monensin biosynthesis can be ascribed. Deletion of essentially all these clustered genes specifically abolished monensin production, while overexpression in S. cinnamonensis of the putative pathway-specific regulatory gene monR led to a fivefold increase in monensin production. Experimental support is presented for a recently-proposed mechanism, for oxidative cyclization of a linear polyketide intermediate, involving four enzymes, the products of monBI, monBII, monCI and monCII. In frame deletion of either of the individual genes monCII (encoding a putative cyclase) or monBII (encoding a putative novel isomerase) specifically abolished monensin production. Also, heterologous expression of monCI, encoding a flavin-linked epoxidase, in S. coelicolor was shown to significantly increase the ability of S. coelicolor to epoxidize linalool, a model substrate for the presumed linear polyketide intermediate in monensin biosynthesis.     

Mitochondrial DNA damage triggers mitochondrial dysfunction and apoptosis in oxidant-challenged lung endothelial cells

Oxidant-induced death and dysfunction of pulmonary vascular cells play important roles in the evolution of acute lung injury. In pulmonary artery endothelial cells (PAECs), oxidant-mediated damage to mitochondrial DNA (mtDNA) seems to be critical in initiating cytotoxicity inasmuch as overexpression of the mitochondrially targeted human DNA repair enzyme, human Ogg1 (hOgg1), prevents both mtDNA damage and cell death (Dobson AW, Grishko V, LeDoux SP, Kelley MR, Wilson GL, and Gillespie MN. Am J Physiol Lung Cell Mol Physiol 283: L205-L210, 2002). The mechanism by which mtDNA damage leads to PAEC death is unknown, and the present study tested the specific hypothesis that enhanced mtDNA repair suppresses PAEC mitochondrial dysfunction and apoptosis evoked by xanthine oxidase (XO). PAECs transfected either with an adenoviral vector encoding hOgg1 linked to a mitochondrial targeting sequence or with empty vector were challenged with ascending doses of XO plus hypoxanthine. Quantitative Southern blot analyses revealed that, as expected, hOgg1 overexpression suppressed XO-induced mtDNA damage. Mitochondrial overexpression of hOgg1 also suppressed the XO-mediated loss of mitochondrial membrane potential. Importantly, hOgg1 overexpression attenuated XO-induced apoptosis as detected by suppression of caspase-3 activation, by reduced DNA fragmentation, and by a blunted appearance of condensed, fragmented nuclei. These observations suggest that mtDNA damage serves as a trigger for mitochondrial dysfunction and apoptosis in XO-treated PAECs.     

Engineering of beta-propeller protein scaffolds by multiple gene duplication and fusion of an idealized WD repeat

The ability to design specific amino acid sequences that fold into desired structures is central to engineering novel proteins. Protein design is also a good method to assess our understanding of sequence-structure and structure-function relationships. While beta-sheet structures are important elements of protein architecture, it has traditionally been more difficult to design beta-proteins than alpha-helical proteins. Taking advantage of the tandem repeated sequences that form the structural building blocks in a group of beta-propeller proteins; we have used a consensus design approach to engineer modular and relatively large scaffolds. An idealized WD repeat was designed from a structure-based sequence alignment with a set of structural guidelines. Using a plasmid sequential ligation strategy, artificial concatemeric genes with up to 10 copies of this idealized repeat were then constructed. Corresponding proteins with 4 through to 10 WD repeats were soluble when over-expressed in Escherichia coli. Notably, they were sufficiently stable in vivo surviving attack from endogenous proteases, and maintained a homogeneous, non-aggregated form in vitro. The results show that the beta-propeller scaffold is an attractive platform for future engineering work, particularly in experiments in which directed evolution techniques might improve the stability of the molecules and/or tailor them for a specific function.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

The DNA glycosylase Ogg1 defends against oxidant-induced mtDNA damage and apoptosis in pulmonary artery endothelial cells

Emerging evidence suggests that mitochondrial (mt) DNA damage may be a trigger for apoptosis in oxidant-challenged pulmonary artery endothelial cells (PAECs). Understanding the rate-limiting determinants of mtDNA repair may point to new targets for intervention in acute lung injury. The base excision repair (BER) pathway is the only pathway for oxidative damage repair in mtDNA. One of the key BER enzymes is Ogg1, which excises the base oxidation product 8-oxoguanine. Previously we demonstrated that overexpression of mitochondrially targeted Ogg1 in PAECs attenuated apoptosis induced by xanthine oxidase (XO) treatment. To test the idea that Ogg1 is a potentially rate-limiting BER determinant protecting cells from oxidant-mediated death, PAECs transfected with siRNA to Ogg1 were challenged with XO and the extent of mitochondrial and nuclear DNA damage was determined along with indices of apoptosis. Transfected cells demonstrated significantly reduced Ogg1 activity, which was accompanied by delayed repair of XO-induced mtDNA damage and linked to increased XO-mediated apoptosis. The nuclear genome was undamaged by XO in either control PAECs or cells depleted of Ogg1. These observations suggest that Ogg1 plays a critical and possibly rate-limiting role in defending PAECs from oxidant-induced apoptosis by limiting the persistence of oxidative damage in the mitochondrial genome.     

Fluorescent labeling of proteins with amine-specific 1,3,2-(2H)-dioxaborine polymethine dye

A novel water-soluble amine-reactive dioxaborine trimethine dye was synthesized in a good yield and characterized. The potential of the dye as a specific reagent for protein labeling was demonstrated with bovine serum albumin and lysozyme. Its interaction with proteins was studied by fluorescence spectroscopy and gel electrophoresis. The covalent binding of this almost nonfluorescent dye to proteins results in a 75- to 78-fold increase of its emission intensity accompanied by a red shift of the fluorescence emission maximum by 27 to 45 nm, with fluorescence wavelengths of labeled biomolecules being more than 600 nm. The dye does not require activation for the labeling reaction and can be used in a variety of bioassay applications.     

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.     

Quantifying the complexity of bat wing kinematics

Body motions (kinematics) of animals can be dimensionally complex, especially when flexible parts of the body interact with a surrounding fluid. In these systems, tracking motion completely can be difficult, and result in a large number of correlated measurements, with unclear contributions of each parameter to performance. Workers typically get around this by deciding a priori which variables are important (wing camber, stroke amplitude, etc.), and focusing only on those variables, but this constrains the ability of a study to uncover variables of influence. Here, we describe an application of proper orthogonal decomposition (POD) for assigning importances to kinematic variables, using dimensional complexity as a metric. We apply this method to bat flight kinematics, addressing three questions: (1) Does dimensional complexity of motion change with speed? (2) What body markers are optimal for capturing dimensional complexity? (3) What variables should a simplified reconstruction of bat flight include in order to maximally reconstruct actual dimensional complexity? We measured the motions of 17 kinematic markers (20 joint angles) on a bat (Cynopterus brachyotis) flying in a wind tunnel at nine speeds. Dimensional complexity did not change with flight speed, despite changes in the kinematics themselves, suggesting that the relative efficacy of a given number of dimensions for reconstructing kinematics is conserved across speeds. By looking at subsets of the full 17-marker set, we found that using more markers improved resolution of kinematic dimensional complexity, but that the benefit of adding markers diminished as the total number of markers increased. Dimensional complexity was highest when the hindlimb and several points along digits III and IV were tracked. Also, we uncovered three groups of joints that move together during flight by using POD to quantify correlations of motion. These groups describe 14/20 joint angles, and provide a framework for models of bat flight for experimental and modeling purposes.