Genome Sequences of Four Cluster P Mycobacteriophages.

Four bacteriophages infecting Mycobacterium smegmatis mc2155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages.

Evidence of evolutionary conservation of function between the thyroxine transporter Oatp1c1 and major facilitator superfamily members.

Organic anion transporting polypeptide 1c1 (Oatp1c1) is a high-affinity T(4) transporter expressed in brain barrier cells. To identify Oatp1c1 amino acid residues critical for T(4) transport, consensus membrane topology was predicted and a three-dimensional Oatp1c1 structure was generated using the known structures of major facilitator superfamily (MFS) transporters, glycerol 3-phosphate transporter, lactose permease, and the multidrug transporter Escherichia coli multidrug resistance protein D as templates. A total of nine amino acid mutations were generated based on amino acid conservation, localization to putative transmembrane domains, and side chain functionality. Mutant constructs were transiently transfected into human embryonic kidney 293 cells and assessed for plasma membrane localization and the capacity to transport substrate (125)I-T(4). Wild-type Oatp1c1, R601S, P609A, W277A/W278A, W277F/W278F, G399A/G409A, and G399L/G409L were all expressed at the plasma membrane. Wild-type Oatp1c1 and W277F/W278F displayed biphasic T(4) transport kinetics, albeit the mutant did so with an approximately 10-fold increase in high-affinity Michaelis constant. The W277A/W278A mutation abolished Oatp1c1 T(4) transport. G399A/G409A and G399V/G409V mutants displayed near wild-type activity in an uptake screen but exhibited diminished T(4) transport activity at high-substrate concentrations, suggesting a substrate binding site collapse or inability to convert between input and output states. Finally, transmembrane domain 11 mutants R601S and P609A displayed partial T(4) transport activity with significantly reduced maximum velocities and higher Michaelis constant. Arg601 is functionally strongly conserved with members of the MFS whose structures and function have been extensively studied. These data provide the experimental foundation for mapping Oatp1c1 substrate binding sites and reveal evolutionary conservation with bacterial MFS transporter members.

The blood-brain barrier thyroxine transporter organic anion-transporting polypeptide 1c1 displays atypical transport kinetics.

Organic anion-transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter expressed in brain barrier cells. Oatp1c1 transports a variety of additional ligands including the conjugated sterol estradiol 17beta-glucuronide (E(2)17betaG). Intriguingly, published data suggest that E(2)17betaG inhibition of Oatp1c1-mediated T(4) transport exhibits characteristics suggestive of atypical transport kinetics. To determine whether Oatp1c1 exhibits atypical transport kinetics, we first performed detailed T(4) and E(2)17betaG uptake assays using Oatp1c1 stably transfected HEK293 cells and a wide range of T(4) and E(2)17betaG concentrations (100 pm to 300 nm and 27 nm to 200 mum, respectively). Eadie-Hofstee plots derived from these detailed T(4) and E(2)17betaG uptake experiments display a biphasic profile consistent with atypical transport kinetics. These data along with T(4) and E(2)17betaG cis-inhibition dose-response measurements revealed shared high- and low-affinity Oatp1c1 binding sites for T(4) and E(2)17betaG. T(4) and E(2)17betaG recognized these Oatp1c1 binding sites with opposite preferences. In addition, sterols glucuronidated in the 17 or 21 position, exhibited preferential substrate-dependent inhibition of Oatp1c1 transport, inhibiting Oatp1c1-mediated E(2)17betaG transport more strongly than T(4) transport. Together these data reveal that Oatp1c1-dependent substrate transport is a complex process involving substrate interaction with multiple binding sites and competition for binding with a variety of other substrates. A thorough understanding of atypical Oatp1c1 transport processes and substrate-dependent inhibition will allow better prediction of endo- and xenobiotic interactions with the Oatp transporter.

Competitive inhibition of organic anion transporting polypeptide 1c1-mediated thyroxine transport by the fenamate class of nonsteroidal antiinflammatory drugs.

Organic anion transporting polypeptide (Oatp) 1c1 is a high-affinity T(4) transporter with narrow substrate specificity expressed at the blood-brain barrier. A transport model using cells overexpressing Oatp1c1 was created to identify novel Oatp1c1 substrates and inhibitors. Rat Oatp1c1 was cloned and stably expressed in human embryonic kidney 293 cells. Oatp1c1-transfected human embryonic kidney 293 cells transported (125)I-labeled T(4) in a time-dependent manner that was completely abolished in the presence of excess unlabeled T(4). Next, various compounds, including inhibitors of thyroid hormone uptake, were screened for inhibitory effects on Oatp1c1-mediated T(4) uptake. Phenytoin (64%), indocyanine green (17%), fenamic acid (68%), diclofenac (51%), and meclofenamic acid (33%) all reduced T(4) uptake by Oatp1c1 when assayed at concentrations of 10 microM. Dose-response assays for the fenamic acids, iopanoic acid, indocyanine green, and phenytoin revealed IC(50) values for Oatp1c1 T(4) uptake below or near the blood plasma levels after therapeutic doses. Further kinetic assays and reciprocal plot analyses demonstrated that the fenamic acid diclofenac inhibited in a competitive manner. Finally, microvessels were isolated from adult rat brain and assessed for T(4) uptake. Ten micromolar of fenamate concentrations inhibited T(4) microvessel uptake with a similar hierarchical inhibition profile [fenamic acid (43%), diclofenac (78%), and meclofenamic acid (85%)], as observed for Oatp1c1 transfected cells. Oatp1c1 is expressed luminally and abluminally in the blood-brain barrier endothelial cell, and exhibits bidirectional transport capabilities. Together, these data suggest that Oatp1c1 transports fenamates into, and perhaps across, brain barrier cells.

Organic anion-transporting polypeptides at the blood-brain and blood-cerebrospinal fluid barriers.

Organic anion-transporting polypeptides (Oatps) are solute carrier family members that exhibit marked evolutionary conservation. Mammalian Oatps exhibit wide tissue expression with an emphasis on expression in barrier cells. In the brain, Oatps are expressed in the blood-brain barrier endothelial cells and blood-cerebrospinal fluid barrier epithelial cells. This expression profile serves to illustrate a central role for Oatps in transporting endo- and xenobiotics across brain barrier cells. This chapter will detail the expression patterns and substrate specificities of Oatps expressed in the brain, and will place special emphases on the role of Oatps in prostaglandin synthesis and in the transport of conjugated endobiotics.