Two new fluorescent labels are presented that are optimized for excitation with He/Ne laser and red diode lasers. Application in FCS and labeling of proteins and oligomers are demonstrated. A strong rise of quantum yield and emission life time upon binding to biomolecules are characteristic features of the dyes.
Oligomers of (R)-3-hydroxybutanoic acid (OHBs) consisting of 8, 16, and 32 HB units have
been investigated by CD spectroscopy in solution and in a racemic phospholipid bilayer. A Freudenberg
plot (ϑ/n vs (n − 2)/n) of the data obtained in solution can be interpreted as an indication for the presence
of a chiral secondary structure of the oligoester chainon the very short time scale of UV spectroscopy!
The OHBs were also combined with fluorescent groups, including donor/acceptor pairs (coumarin,
rhodamine, cyanine, oxazine). This enabled their detection by fluorescent microscopy and fluorescence-resonance-energy-transfer (FRET) measurements. With these techniques it was possible to calculate the
distance between the fluorescent groups, thus providing information about the conformation of the OHB
chain. With a calculated Förster distance R
0 of 60 Å the distance (in 10-6 M CHCl3) between the termini
of HB 8-, 16-, and 32-mer were calculated to be 41, 38, and 49 Å, respectively. This result is compatible
with conclusions drawn from other observations, indicating that longer HB chains fold in a hairpin-type
fashion. Incorporation of fluorescent 16- and 32-mers in liposomes shows that the latter tend to aggregate
while the former do not: The investigation described here did not lead to a decision as to whether an
OHB chain, and thus also the poly(hydroxybutanoate) (PHB) chain, attains a 21- or a 31-helical
arrangement in solution.
Our results demonstrate that an activation of the renal urine concentrating mechanism by desmopressin causes renal medullary hypoxia and an upregulation of hypoxia-inducible gene expression.
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