| In vitro: |
| Appl Microbiol Biotechnol. 2015 May;99(10):4453-70. | | Isolation of oxygenase genes for indigo-forming activity from an artificially polluted soil metagenome by functional screening using Pseudomonas putida strains as hosts.[Pubmed: 25573469] | Metagenomes contain the DNA from many microorganisms, both culturable and non-culturable, and are a potential resource of novel genes.
METHODS AND RESULTS:
In this study, a 5.2-Gb metagenomic DNA library was constructed from a soil sample (artificially polluted with four aromatic compounds, i.e., biphenyl, phenanthrene, carbazole, and 3-chlorobenzoate) in Escherichia coli by using a broad-host-range cosmid vector. The resultant library was introduced into naphthalene-degrading Pseudomonas putida-derived strains having deficiencies in their naphthalene dioxygenase components, and indigo-forming clones on the indole-containing agar plates were screened. Cosmids isolated from 29 positive clones were classified by their various properties (original screening hosts, hosts showing indigo-forming activity, and digestion patterns with restriction enzymes), and six representative cosmids were chosen.
Sequencing and in vitro transposon mutagenesis of the six cosmids resulted in the identification of genes encoding putative class B and D flavoprotein monooxygenases, a multicomponent hydroxylase, and a reductase that were responsible for the indigo-forming activity in the host cells.
CONCLUSIONS:
Among them, the genes encoding the multicomponent hydroxylase were demonstrated to be involved in phenol degradation. Furthermore, two genes encoding ring-cleavage dioxygenases were also found adjacent to the genes responsible for the indigo formation, and their functions were experimentally confirmed.
| | J. Biol. Chem., 2001, 276(34):31475-8. | | Indirubin and Indigo Are Potent Aryl Hydrocarbon Receptor Ligands Present in Human Urine.[Pubmed: 11425848] | Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes involved in xenobiotic metabolism, cellular proliferation, and differentiation. Numerous xenobiotic and biological compounds are known to interact with AhR, but it remains an orphan receptor, because its physiological ligand is unknown.
METHODS AND RESULTS:
We identified AhR ligands in human urine using a yeast AhR signaling assay and then characterized their properties. Two ligands, indirubin and indigo, were both present at average concentrations of approximately 0.2 nm in the urine of normal donors. Indirubin was also detected in fetal bovine serum and contributed half of the total AhR ligand activity. The activities of indirubin and indigo were comparable with or more potent than that of the archetypal ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, in yeast AhR activation assays.
CONCLUSIONS:
We suggest that the endogenous levels and potencies of indirubin and indigo are such that they activate AhR-mediated signaling mechanisms in vivo. |
|
| In vivo: |
| J Endovasc Ther. 2015 Apr 17. | | Use of Indigo Carmine Angiography to Qualitatively Assess Adequate Distal Perfusion After Endovascular Revascularization in Critical Limb Ischemia.[Pubmed: 25887729] | To report a novel technique to visualize the efficacy of revascularization in critical limb ischemia patients with ischemic foot ulcers.
METHODS AND RESULTS:
An 80-year-old man was admitted with nonhealing ulcers on his left second toe and lateral border of the foot owing to in-stent restenosis of the left popliteal artery. After dilation of the popliteal in-stent restenosis, below-the-knee angiography revealed that the anterior tibial artery (ATA) was occluded, the posterior tibial artery was hypoplastic, and the peroneal artery was enlarged, with 2 plantar arteries. To evaluate the foot circulation before performing additional procedures, a 4-F multipurpose catheter was advanced into the peroneal artery, and 5 mL of indigo carmine was injected. Immediately, the patient's second toe and lateral border ulcers were dyed blue. We concluded that sufficient blood flow had been obtained to the ulcerated area by balloon angioplasty alone, so the procedure was terminated. The ulcers completely healed at 1 month.
CONCLUSIONS:
Indigo carmine angiography provides visual information on foot perfusion, yielding new insights into microcirculation and helping to determine the effectiveness of treatment and procedure endpoint. |
|
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