8 research outputs found
Vpliv učenja in obsega žoge na spremembo hitrosti rokometnega strela
Thioredoxin, involved in numerous
redox pathways, is maintained
in the dithiol state by the nicotinamide adenine dinucleotide phosphate-dependent
flavoprotein thioredoxin reductase (TrxR). Here, TrxR from <i>Lactococcus lactis</i> is compared with the well-characterized
TrxR from <i>Escherichia coli</i>. The two enzymes belong
to the same class of low-molecular weight thioredoxin reductases and
display similar <i>k</i><sub>cat</sub> values (∼25
s<sup>–1</sup>) with their cognate thioredoxin. Remarkably,
however, the <i>L. lactis</i> enzyme is inactivated by visible
light and furthermore reduces molecular oxygen 10 times faster than <i>E. coli</i> TrxR. The rate of light inactivation under standardized
conditions (λ<sub>max</sub> = 460 nm and 4 °C) was reduced
at lowered oxygen concentrations and in the presence of iodide. Inactivation
was accompanied by a distinct spectral shift of the flavin adenine
dinucleotide (FAD) that remained firmly bound. High-resolution mass
spectrometric analysis of heat-extracted FAD from light-damaged TrxR
revealed a mass increment of 13.979 Da, relative to that of unmodified
FAD, corresponding to the addition of one oxygen atom and the loss
of two hydrogen atoms. Tandem mass spectrometry confined the increase
in mass of the isoalloxazine ring, and the extracted modified cofactor
reacted with dinitrophenyl hydrazine, indicating the presence of an
aldehyde. We hypothesize that a methyl group of FAD is oxidized to
a formyl group. The significance of this not previously reported oxidation
and the exceptionally high rate of oxygen reduction are discussed
in relation to other flavin modifications and the possible occurrence
of enzymes with similar properties
A Novel Ultrasensitive Hybridization-Based ELISA Method for 2-Methoxyphosphorothiolate MicroRNAs and Its In vitro and In vivo Application
MicroRNAs (miRNAs) are endogenous, small non-coding RNAs that bind to target mRNAs and regulate their expression. Recent evidence has indicated the involvement of miRNAs in human malignancies. It has been suggested that aberrantly down-regulated or up-regulated miRNAs may be replaced with synthetic miRNAs or antagomiRNAs, respectively, and restore normal cell functions. As therapeutic development requires analytical support, we developed and validated an ultrasensitive and selective assay for quantification of synthetic 2′-methoxyphosphorothiolate-miRNA in mouse plasma and cell lysate for the first time. The method is based on a hybridization-ligation fluorescence enzyme-linked immunosorbent assay and has provided a linear dynamic range of 10-1,000,000 pM for three synthetic miRNAs both singly and in a mixture. The intra- and inter-day coefficients of variation were <20% and the accuracy values nearly 100%. Using this assay, we performed pharmacokinetic studies of three synthetic miRNAs in mice treated with a single i.v. bolus dose of 7.5 mg kg−1. The 2-methoxyphosphorothiolate-miRNAs reached peak concentrations in the μM and nM ranges in plasma and bone marrow, respectively, and remained measurable at 24 h. These concentrations are in a range that shows biological activities. We conclude that this method provides a general and valuable tool for the pharmacologic study and clinical development of synthetic miRNAs