A novel mitochondrial localizing ruthenium(II) peptide conjugate capable of monitoring dynamic changes in local O2 concentrations within living cells is presented. The complex is comprised of luminescent dinuclear ruthenium(II) polypyridyl complex bridged across a single mitochondrial penetrating peptide, FrFKFrFK-CONH2 (r = D-arginine). The membrane permeability and selective uptake of the peptide conjugate at the mitochondria of mammalian cells was demonstrated using confocal microscopy. Dye co-localization studies confirmed very precise localization and preconcentration of the probe at the mitochondria. This precision permitted collection of luminescent lifetime images of the probe, without the need for co-localizing dye and permitted semiquantitative determination of oxygen concentration at the mitochondria using calibration curves collected at 37 °C for the peptide conjugate in PBS buffer. Using Antimycin A the ability of the probe to respond dynamically to changing O2 concentrations within live HeLa cells was demonstrated. Furthermore, based on lifetime data it was evident that the probe also responds to elevated reactive oxygen species (ROS) levels within the mitochondria, where the greater quenching capacity of these species led to luminescent lifetimes of the probe at longer Antimycin A incubation times which lay outside of the O2 concentration range. Although both the dinuclear complex and a mononuclear analogue conjugated to an octaarginine peptide sequence exhibited some cytotoxicity over 24 h, cells were tolerant of the probes over periods of 4 to 6 h which facilitated imaging. These metal-peptide conjugated probes offer a valuable opportunity for following dynamic changes to mitochondrial function which should be of use across domains in which the metabolic activity of live cells are of interest from molecular biology and drug discovery.
The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp. by a differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined to be di-anthracenone (C 44 H 44 Cl 2 O 14 ), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and S. aureus were 0.39 and 0.20 lg ml À1 , and IC 50 measurements against WalK were 1.6 and 5.7 lM, respectively. To clarify the affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation constant, K D1 , of 7.63 lM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK autophosphorylation by binding to the WalK cytoplasmic domain.
A novel Near Infra-Red emitting BODIPY derivative is presented which exhibits the largest Stokes shift thus far reported for a BODIPY compound.
A first investigation into the application of a luminescent osmium(ii) bipyridine complex to live cell imaging is presented. Osmium(ii) (bis-2,2-bipyridyl)-2(4-carboxylphenyl) imidazo[4,5f][1,10]phenanthroline was prepared and conjugated to octaarginine, a cell penetrating peptide. The photophysics, cell uptake and cytotoxicity of this osmium complex conjugate were performed and compared with its ruthenium analogue. Cell uptake and distribution of both ruthenium and osmium conjugates were very similar with rapid transmembrane transport of the osmium probe (complete within approx. 20 min) and dispersion throughout the cytoplasm and organelles. The near-infrared (NIR) emission of the osmium complex (λmax 726 nm) coincides well with the biological optical window and this facilitated luminescent and luminescence lifetime imaging of the cell which was well resolved from cell autofluorescence. The large Stokes shift of the emission also permitted resonance Raman mapping of the dye within CHO cells. Rather surprisingly, the osmium conjugate exhibited very low cytotoxicity when incubated both in the dark and under visible irradiation. This was attributed to the remarkable stability of this complex which was reflected by the complete absence of photo-bleaching of the complex even under extended continuous irradiation. In addition, when compared to its ruthenium analogue its luminescence was short-lived in water therefore rendering it insensitive to O2.
Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os(bpy)2(phen-benz-COOH)]2+ (OsNG, {bpy= 2,2′-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl) benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton’s reagent or by ROS formation during redox cycles involving NADPH, CuII, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of CuII, NADPH, and model metabolites.
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