ABSTRACT
Stable isotope probing with [(13)C]biphenyl was used to explore the genetic properties of indigenous bacteria able to grow on biphenyl in PCB-contaminated River Raisin sediment. A bacterial 16S rRNA gene clone library generated from [(13)C]DNA after a 14-day incubation with [(13)C]biphenyl revealed the dominant organisms to be members of the genera Achromobacter and Pseudomonas. A library built from PCR amplification of genes for aromatic-ring-hydroxylating dioxygenases from the [(13)C]DNA fraction revealed two sequence groups similar to bphA (encoding biphenyl dioxygenase) of Comamonas testosteroni strain B-356 and of Rhodococcus sp. RHA1. A library of 1,568 cosmid clones was produced from the [(13)C]DNA fraction. A 31.8-kb cosmid clone, detected by aromatic dioxygenase primers, contained genes of biphenyl dioxygenase subunits bphAE, while the rest of the clone's sequence was similar to that of an unknown member of the Gammaproteobacteria. A discrepancy in G+C content near the bphAE genes implies their recent acquisition, possibly by horizontal transfer. The biphenyl dioxygenase from the cosmid clone oxidized biphenyl and unsubstituted and para-only-substituted rings of polychlorinated biphenyl (PCB) congeners. A DNA-stable isotope probing-based cosmid library enabled the retrieval of functional genes from an uncultivated organism capable of PCB metabolism and suggest dispersed dioxygenase gene organization in nature.
Subject(s)
Bacteria/classification , Carbon Isotopes/metabolism , DNA/genetics , Dioxygenases/genetics , Geologic Sediments , Polychlorinated Biphenyls/analysis , Water Pollutants, Chemical/analysis , Bacteria/genetics , Bacteria/isolation & purification , Biphenyl Compounds/metabolism , Cluster Analysis , DNA/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Dioxygenases/isolation & purification , Gene Library , Gene Order , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Rivers , Sequence Analysis, DNA , Water/chemistryABSTRACT
In a continuing effort to discover novel chemotypes as potent and selective PDE5 inhibitors for the treatment of male erectile dysfunction (ED), we have found that 4-benzylaminoquinoline derivatives are very potent and selective PDE5 inhibitors. Some compounds in this series had PDE5 IC(50)'s as low as 50 pM. While an electron withdrawing group at the C6-position of the quinoline substantially improved PDE5 potency, an ethyl group at the C8-position not only improved the PDE5 potency but also the isozyme selectivity. Substitutents at the C3-position can incorporate a variety of different groups. The synthesis and primary structure-activity relationship of this new series of potent PDE5 inhibitors are described.
Subject(s)
3',5'-Cyclic-GMP Phosphodiesterases/antagonists & inhibitors , Erectile Dysfunction/drug therapy , Phosphodiesterase Inhibitors/pharmacology , Quinolines/pharmacology , 3',5'-Cyclic-GMP Phosphodiesterases/metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5 , Erectile Dysfunction/enzymology , Humans , Male , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/therapeutic use , Quinolines/chemistry , Quinolines/therapeutic useABSTRACT
Novel pyrazolopyridopyridazine derivatives have been prepared as potent and selective PDE5 inhibitors. Compound 6 has been identified as a more potent and selective PDE5 inhibitor than sildenafil (1). It is as efficacious as sildenafil in in vitro and in vivo PDE5 inhibition models, and it is orally bioavailable in rats and dogs. The superior isozyme selectivity of 6 is expected to exert less adverse effects in humans when used for erectile dysfunction treatment.
