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At least 1,081 records · Page 60Linked to original sources
PubMed1994

Dynamics of merocyanine 540 in model biomembranes: photoisomerization studies in small unilamellar vesicles.

The fluorescence lifetime, tau f, of merocyanine 540 (MC540) in small unilamellar vesicles was measured as a function of temperature and cholesterol content by using phase modulation fluorometry. These vesicles were formed by probe sonication of aqueous suspensions of egg phosphatidylcholine (PC) and cholesterol. The fluorescence lifetime of MC540 in these vesicles decreased with increasing temperature, but was independent of cholesterol. The decrease in tau f with temperature is attributed to trans-cis photoisomerization. At low temperatures, the inverse of tau f, or the fluorescence rate constant, kf, approaches a constant value of 0.45 +/- 0.02 ns-1, which corresponds to the value of the radiative rate constant, kr, of the dye. The photoisomerization rate constant, kiso, was obtained by subtracting kr from kf. The temperature dependence of kiso is well described by an Arrhenius equation, with an activation energy of 31.5 +/- 0.9 kJ mol-1. This Arrhenius behavior is rationalized in terms of the Smoluchowski limit for the Kramers theory for activated barrier crossing. The electronic spectra and kiso for MC540 in these vesicles are consistent with the dye being located in the polar headgroup region of the lipid bilayer.

Cholesterol↗
PubMed1975

Biosynthesis of retinol in bovine corpus luteum tissue.

The bovine corpus luteum was found to contain retinol, as analyzed by fluorometry and the formation of anhydroretinol. After the incubation of beta-[15,15 foot-3H] carotene with slices of corpus luteum tissue the biosynthesis of radioactive retinol was demonstrated. Alumina column chromatography, thin layer chromatography, and high speed liquid chromatography were used to isolate and identify radioactive vitamin A. Retinol was also found to form a polar complex, from which could be released by acid hydrolysis.

Animals↗
PubMed1985

Enzyme immunoassay of 6-ketoprostaglandin F1 alpha in a solid phase.

A solid-phase enzyme immunoassay of 6-ketoprostaglandin F1 alpha (a stable degradation product of prostaglandin I2) was developed in which the hapten molecule was labeled with beta-galactosidase. The antiserum was bound to a polystyrene tube, and the enzyme-labeled and unlabeled 6-ketoprostaglandin F1 alpha were allowed to react in a competitive manner with the immobilized antibody. The activity of the immunologically bound beta-galactosidase was assayed by fluorometry, and correlated with the amount of unlabeled 6-ketoprostaglandin F1 alpha. According to a calibration curve 6-ketoprostaglandin F1 alpha was detectable in a range of 30 fmol-10 pmol. When 6-ketoprostaglandin F1 alpha was extracted from human serum by using an octadecylsilyl silica column (Sep-Pak C18) and the crude extract was subjected to the enzyme immunoassay, the content of 6-ketoprostaglandin F1 alpha was 50-60 pmol/ml of human serum. An endogenous substance(s) which disturbed the immunoreaction and gave such an apparently high concentration of 6-ketoprostaglandin F1 alpha separated from the endogenous 6-ketoprostaglandin F1 alpha by HPLC. With the purified 6-ketoprostaglandin F1 alpha fraction there was a good correlation (r = 0.994) between enzyme immunoassay and radioimmunoassay. The validity of the enzyme immunoassay was confirmed by gas chromatography-selected ion monitoring.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Effect of cholesterol on membrane microheterogeneity: a study using 1,6-diphenyl-1,3,5-hexatriene fluorescence lifetime distributions.

The effect of cholesterol on microheterogeneity of liposomes obtained from saturated and unsaturated phospholipids was studied by measuring the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Data obtained by frequency domain fluorometry have been analyzed either by discrete exponential or continuous lifetime distribution approaches. In egg phosphatidylcholine liposomes, the addition of cholesterol increases the lifetime value or the centre of the lifetime distribution. At high cholesterol concentration, good fits are obtained using a monomodal distribution analysis or single exponential component. At low cholesterol concentration an additional short component of low fractional intensity must be included to obtain a good fit. In dipalmitoylphosphatidylcholine, the addition of cholesterol decreases the long lifetime component centre value both in the gel and in the liquid-crystalline state. The DPH lifetime value is sensitive to the dielectric constant of the probe microenvironment, and cholesterol has been shown to modify water penetration in the bilayer. Using this information our data indicate that cholesterol affects the polarity of the microenvironment in liposomes of unsaturated phosphatidylcholine and saturated phosphatidylcholine in different ways. Although the major conclusions of this paper are obtained using changes of the distribution centre upon cholesterol addition, there are also preliminary indications that the lifetime distribution width decreases as cholesterol is added. We have interpreted this observation as being due to the homogenizing effect of cholesterol.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1976

Studies on the binding of FMN by apoflavodoxin from Peptostreptococcus elsdenii, pH and NaCl concentration dependence.

1. The pH and ionic strength dependence of the interaction of FMN with apoflavodoxin has been studied by fluorometry in the pH region 2-5, at 22 degrees C. 2. The rate constant of dissociation and the dissociation constant were experimentally determined; the rate constants of association were claculated at a given pH value. These constants depend on the ionic strength. The plots of these constants against the square root of the ionic strength are straight. 3. Our data have been interpreted in terms of the Brönsted theory, which relates chemical reaction rates to ionic strength. The data indicate that the apoenzyme reaches its maximum net positive charge at pH 2.0-2.6. The calculated net charge in this pH region is between 11 and 12 and is in agreement with the theoretical value of 12 as deduced from the primary structure of the protein. The isoelectric point of the holoenzyme is about 4. 4. The rate constant of association extrapolated to zero ionic strength is 3.2-10(5)M-1-s-1 and is pH-independent. 5. The rate constant of dissociation and the dissociation constant extrapolated to zero ionic strength depend on the pH. The results are explained by assuming that there are two protein ionizations with a pK value of 3.4; these ionizing groups are possibly close to the FMN binding site.

Apoproteins↗
PubMed1978

Fluorescence energy transfer between epsilon-ATP at the nucleotide binding site and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide at Cys-373 of G-actin.

The method of fluorescence energy transfer is used to measure the distance from the nucleotide binding site to Cys-373 of G-actin. The fluorescent ATP analogue 1-N6-ethenoadenosine 5'-triphosphate was used as donor and N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide was used as acceptor. From the measurements of the efficiency of fluorescence energy transfer by both static and time resolved fluorometries, the distance between nucleotide binding site and Cys-373 residue of G-actin was calculated to be about 30 A.

Actins↗
PubMed1983

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P.

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.

Amino Acid Sequence↗
PubMed1984

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver.

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.

Animals↗
PubMed1987

Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions.

Chimeric genes composed of immunoglobulin (Ig)-derived variable (V) regions and T-cell receptor (TCR)-derived constant (C) regions were constructed. The VL and VH genes showing anti-phosphorylcholine (PC) activity were used in this study. Two pairs of chimeric genes, VL-C beta and VH-C alpha genes, and VL-C alpha and VH-C beta genes, were inserted into an expression vector containing both Ecogpt and neo genes, and transfected into EL4 cells. Cells which express both chimeric receptor molecules were established. The activity of the transformants to the antigen was examined by using stopped-flow fluorometry. An increase in the concentration of cytoplasmic calcium ion was observed after addition of Staphylococcus pneumoniae R36A bacteria grown in the choline-containing medium which express PC molecules, but not after the PC-negative bacteria grown in the ethanolamine-containing medium.

Amino Acid Sequence↗
PubMed1989

DPH lifetime distributions in vesicles containing phospholipid hydroperoxides.

Multifrequency phase fluorometry was used to determine the lifetime distributions of 1,6 diphenyl-1,3,5-hexatriene in 1-palmitoyl-2 linoleoyl phosphatidylcholine small unilamellar vesicles containing 2% incorporation of phospholipid hydroperoxides. A biexponential decay was observed in both vesicle preparations over a temperature range of 5 to 35 degrees C. Vesicles containing phospholipid hydroperoxides showed an overall longer lifetime as well as a greater distribution in width. These findings suggest that phospholipid hydroperoxides create structural heterogeneity in membrane structure.

Chromatography, High Pressure Liquid↗
PubMed1989

Free and bound sodium in pancreatic beta-cells exposed to glucose and tolbutamide.

The effects of glucose and tolbutamide on the sodium handling of the pancreatic beta-cells were evaluated by measuring the total sodium content in intact islets from ob/ob-mice by integrating flame photometry and the free ion in individual beta-cells by dual wavelength fluorometry. Whereas increasing the glucose concentration from 3 to 20 mM resulted in a lowering of sodium, the addition of 100 microM tolbutamide caused a rise. The above-mentioned effects were most marked (about 50%) for the physiologically significant free sodium. The data indicate a more important role for Na+ in the regulation of insulin release than so far acknowledged. Increase of Na+ may contribute to the secretory response to hypoglycemic sulfonylureas by providing an additional rise of cytoplasmic Ca2+.

Animals↗
PubMed1989

Permeability of oxidized phosphatidylcholine liposomes.

Permeability of liposomes made from mixtures of unoxidized and singlet oxygen oxidized phosphatidylcholine has been related to the degree of lipid oxidation expressed as hydroperoxide moiety content in the lipids. The effect of oxidation on the liposomes permeability has been studied by fluorometry using calcein as a fluorescent probe that undergoes self quenching when highly concentrated inside liposomes. The liposomes containing 73% and 5% of hydroperoxides retain respectively 64.5 and 96.3% of calcein with respect to that retained by the liposomes made from unoxidized phosphatidylcholine. The fluorescence data show a linear relationship between the liposome permeability and the oxidation degree of lipids.

Chromatography, High Pressure Liquid↗
PubMed1990

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides.

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.

Animals↗
PubMed1991

Endothelin induces the Ca(2+)-transient in endothelial cells in situ.

Using front-surface fluorometry of fura-2 and valvular strips of the pig aorta, we recorded changes in the cytosolic Ca2+ concentration, [Ca2+]i, of endothelial cells in situ, quantitatively, and investigated the effects of endothelin-1 and -3 on these endothelial cells. Both endothelin-1 and -3 elevated [Ca2+]i of a peak (the first phase) and sustained type. This first phase is considered to be due to a release of Ca2+ from intracellular storage sites. The sustained phase depended on extracellular Ca2+ and is considered to be due to an influx of Ca2+ through the plasma membrane. At equimolar concentrations, the peak elevations of [Ca2+]i induced by endothelin-1 were much higher than those induced by endothelin-3. We suggest that, in endothelial cells in situ, endothelin-1 mobilizes stored Ca2+ and may activate Ca(2+)-sensitive pathways, including the release of prostacyclin and endothelium-derived relaxing factors, more potently than does endothelin-3.

Animals↗
PubMed1992

Endothelin-1 inhibits and enhances contraction of porcine coronary arterial strips with an intact endothelium.

Using front-surface fluorometry and fura-2-loaded porcine coronary arterial strips with an intact endothelium, changes in cytosolic Ca2+ concentrations ([Ca2+]i) and tension of smooth muscle were simultaneously monitored in an attempt to determine the vasoactive properties of endothelin-1 (ET-1). ET-1 in low concentrations (0.1-1nM) caused a significant transient decrease in [Ca2+]i and tension of the strips precontracted with 10(-7) M U-46619. The maximal decreases in [Ca2+]i and tension were obtained with 0.6nM ET-1. In higher concentrations (1nM-100nM), there was no reduction in [Ca2+]i or tension; the contraction induced by U-46619 was potentiated. The decreases in [Ca2+]i and tension induced by ET-1 were inhibited by the mechanical removal of the endothelium or by pretreatment with NG-nitro-L-arginine and were slightly attenuated by indomethacin. Thus, ET-1 in low concentrations can induce endothelium-dependent transient relaxations accompanied by transient reductions of [Ca2+]i in isolated porcine coronary arteries. This effect is mainly mediated by the release of endothelium-derived relaxing factor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1992

Creation of amyloid fibrils from mutant Asn187 gelsolin peptides.

The amyloid protein in familial amyloidosis, Finnish type, is a 71 amino acid long fragment of the inner region of mutant Asp187----Asn gelsolin. The mechanism of gelsolin amyloid formation was tested with synthetic 11 and 30 residue peptides corresponding to the normal and mutant sequence of gelsolin. Fibrils meeting the morphologic criteria of amyloid were formed from the mutant Asn187 peptides. Substitution of the normal Asp187 residue with the mutant Asn residue resulted in a 9-fold increase in fibrillogenicity as determined by quantitative fluorometry. The present study demonstrates the first successful in vitro creation of amyloid-like fibrils from Asn187 gelsolin peptides and provides evidence that amyloid formation in Finnish amyloidosis is a direct consequence of the Asp187----Asn substitution in gelsolin.

Amino Acid Sequence↗
PubMed1988

A direct, highly sensitive assay for cytochrome P-450 catalyzed O-deethylation using a novel coumarin analog.

The microsomal O-deethylation of a novel coumarin analog, 7-ethoxy-4-trifluoromethylcoumarin (EFC), to a fluorescent product was characterized. Results indicate that this analog provides a rapid, convenient and highly sensitive means to assay cytochrome P-450-mediated metabolism. Like microsomal 7-ethoxycoumarin (7-EC) O-deethylation, EFC O-deethylation responded to both phenobarbital was greater than that seen with 7-EC (5- to 6-fold over control after 50 mg/kg/day for 4 days in Sprague-Dawley rats compared to approximately 2-fold for 7-EC). Since the reaction was monitored by direct fluorometry of the product, any departures from linearity under a particular set of reaction conditions (e.g. with highly induced samples) were immediately apparent. In the absence of an NADPH-regenerating system, background drift was very low (less than 0.01 fluorescent units), so the sensitivity of the assay was limited primarily by that of the fluorometer employed. This makes the assay particularly useful in situations where test material is limited, e.g. when measuring activity in cultured hepatocytes. Its simplicity, reproducibility, and response to a variety of inducing agents also make it suitable for a rapid screening assay for cytochrome P-450 induction.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1995

Proton release from HeLa cells and alkalization of cytoplasm induced by diferric transferrin or ferricyanide and its inhibition by the diarylsulfonylurea antitumor drug N-(4-methylphenylsulfonyl)-N'-(4-cholorophenyl) urea (LY181984).

Proton release from HeLa cells was stimulated by an external oxidant, potassium ferricyanide, or by the growth factor diferric transferrin. This stimulated proton release was inhibited by the antitumor sulfonylurea LY181984 [N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea] over the concentration range 10 nM to 1 microM. The antitumor-inactive sulfonylurea analog LY181985 [N-(4-methylphenylsulfonyl)-N'-(phenyl)urea] was without effect at 1 microM and required 10-100 microM concentrations to inhibit proton release. Diferric transferrin-induced alkalization of the cytoplasm estimated by BCECF [2',7'-bis(2-carboxyethyl)-5,(and 6)-carboxyfluorescein] fluorescence also was inhibited by 1 microM LY181984 but not by 1 microM LY181985. The inhibited component appeared to be amiloride resistant. The proton release induced by either ferricyanide or diferric transferrin was inhibited by about 35% at a near optimal amiloride concentration of 0.2 mM or at a dimethylamiloride concentration of 0.075 mM. However, the induced proton release was inhibited further by LY181984. Conversely, when proton release was inhibited fully by LY181984 at a near optimal concentration of 10 microM (50% inhibition), increasing concentrations of amiloride or dimethylamiloride resulted in additional inhibitions of 16 and 23%, respectively. However, the inhibitions by LY181984 and the amilorides were additive, suggesting that amiloride and the sulfonylureas may act independently. Evidence for an action of the sulfonylurea in inhibiting proton efflux differently from that of the amilorides came from measurements of sodium uptake either by fluorometry or by direct measurement with 22Na+. Sodium uptake was not inhibited by either LY181984 or LY181985 in HeLa cells at concentrations of LY181984 sufficient to inhibit proton efflux by 80% or more. The results show LY181984 to be a potent inhibitor of diferric transferrin- or ferricyanide-induced proton efflux and cytoplasmic alkalization in HeLa cells and that the inhibition may involve a component of proton transport that is resistant to amiloride.

Amiloride↗