RESUMO
In non-viscous aqueous solutions, the cyanine fluorescent dyes Cy3 and Cy5 have rather low fluorescence efficiency (the fluorescence quantum yields of Cy3 and Cy5 are 0.04 and 0.3, respectively [1, 2]) and short excited state lifetimes due to their structural features. In this work, we investigated the effect of solubility and rotational degrees of freedom on the fluorescence efficiency of Cy3 and Cy5 in several ways. We compared the fluorescence efficiencies of two cyanine dyes sCy3 and sCy5 with the introduction of a sulfonyl substituent in the aromatic ring as well as covalently bound to T10 oligonucleotides. The results show that because of the different lengths of the polymethine chains between the aromatic rings of the dyes, cis-trans-isomerization has a much greater effect on the Cy3 molecule than on the Cy5 molecule, while the effect of aggregation is also significant.
RESUMO
Arachidonic acid (ARA) is a major component of lipid bilayers as well as the key substrate for the eicosanoid cascades. ARA is readily oxidized, and its non-enzymatic and enzymatic oxidation products induce inflammatory responses in nearly all tissues, including lung tissues. Deuteration at bis-allylic positions substantially decreases the overall rate of ARA oxidation when hydrogen abstraction is an initiating event. To compare the effects of dosing of arachidonic acid (H-ARA) and its bis-allylic hexadeuterated form (D-ARA) on lungs in conventionally healthy mice and in an acute lung injury model, mice were dosed with H-ARA or D-ARA for six weeks through dietary supplementation and then challenged with intranasal lipopolysaccharide (LPS) for subsequent analysis of bronchoalveolar lavage fluid and lung tissue. Dosing on D-ARA resulted in successful incorporation of D-ARA into various tissues. D-ARA significantly reduced LPS-induced adverse effects on alveolar septal thickness and the bronchoalveolar area. Oral deuterated ARA is taken up efficiently and protects against adverse LPS-induced pathology. This suggests novel therapeutic avenues for reducing lung damage during severe infections and other pathological conditions with inflammation in the pulmonary system and other inflammatory diseases.
RESUMO
Bright fluorescent probes with enhanced intensities in the fluorescein channel are of great value for plenty of biological applications. To design effective probes one should introduce as many as possible fluorophores to the biomolecule while leaving its native structure as intact as possible. To reach this compromise, we designed and synthesized fluorescein bifluorophores on the 3,5-diaminobenzoic acid scaffold, which allows for insertion of two fluorophores at one modification site of a biomolecule. Rigid structure of the branching linker group allows to minimize self-quenching the fluorophores. However, despite the structure similarities of fluorescein isomers (5-FAM and 6-FAM), different photophysical behavior was observed for the corresponding bifluorophores. Here we made efforts to get insight into these effects with the focus on the media viscosity impact.
RESUMO
Autoxidation of polyunsaturated fatty acids (PUFAs) damages lipid membranes and generates numerous toxic by-products implicated in neurodegeneration, aging, and other pathologies. Abstraction of bis-allylic hydrogen atoms is the rate-limiting step of PUFA autoxidation, which is inhibited by replacing bis-allylic hydrogens with deuterium atoms (D-PUFAs). In cells, the presence of a relatively small fraction of D-PUFAs among natural PUFAs is sufficient to effectively inhibit lipid peroxidation (LPO). Here, we investigate the effect of various D-PUFAs on the stability of liposomes under oxidative stress conditions. The permeability of vesicle membranes to fluorescent dyes was measured as a proxy for bilayer integrity, and the formation of conjugated dienes was monitored as a proxy for LPO. Remarkably, both approaches reveal a similar threshold for the protective effect of D-PUFAs in liposomes. We show that protection rendered by D-PUFAs depends on the structure of the deuterated fatty acid. Our findings suggest that protection of PUFAs against autoxidation depends on the total level of deuterated bi-sallylic (CD2 ) groups present in the lipid bilayer. However, the phospholipid containing 6,6,9,9,12,12,15,15,18,18-d10 -docosahexaenoic acid exerts a stronger protective effect than should be expected from its deuteration level. These findings further support the application of D-PUFAs as preventive/therapeutic agents in numerous pathologies that involve LPO.