• Journal of the Korean Society of Food Science and Nutrition
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• v.26 no.4
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• pp.743-750
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• 1997

Direct measurement of the kinetics of free fatty acid transfer between phospholipid model membrane is technically limited by the rapid nature of the transfer process. Separation of membrane-bound fatty acid by centrifugation has shown that although the equilibrium distribution of free fatty acid is determined by this method, fatty acid transfer occurs too rapidly for accurate kinetic measurements. Recently fluorescence resonance energy transfer(FRET) assay has been developed to examine transfer of fatty acids between membranes. Donor membranes which has fluorescent fatty acid, anthroyloxy fatty acid(AOFA), is mixed with acceptor membranes which has non-interchangeable fluorescent quencher, nitrobenzo-xadiazol(NBD), using stopped flow apparatus. As the fluorescent fatty acids transfer from donor membrane to acceptor membrane, fluorescence intensity would be decreased and the rate and degree of fatty acid transfer can be analyzed. Fatty acid transfer between micelles is more complicated because of bile salt. Therefore in experiments with micelles, fluorescence self quenching assay is used. At high concentrations, a fluorophore tends to quench its own fluorescence causing a reduction in fluorescence intensity. Donor micelles contained self quenching concentrations of fluorophore and acceptor micelles had no fluorophore. Upon mixing of donor and acceptor micelles, the rate of transfer of the fluorophore from the donor to the acceptor was measured by monitoring the release in self quenching when its concentration in donor decreased over time.

• YAKHAK HOEJI
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• v.38 no.6
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• pp.637-645
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• 1994

The effect of various drugs on the stability of the liposomal membrane of phosphatidylcholine and cholesterol was studied, employing the fluorescence self-quenching method. Calcein was entrapped into the phospholipid small unilamellar vesicles and the leakage of the fluorescence probe was monitored on adding the drug to the system. The results of the experiments showed that phenothiazine derivatives, some potent local anesthetics and surface active agents were very effective in inducing the leakage of calcein from the liposome. The leakage-inducing activity of these drug substances has been ascribed to their surface activity and the perturbation of the liposomal membrane by these substances. On the other hand drug substance with low surface activity or without amphiphilic moieties did not show any effect or only small effect on the leakage of calcein from the liposomes. The effect of lipid concentration on the stability of the liposomes was also investigated to show that the higher concentrations of lipid more drug was required to induce the leakage. The effect of surface charges of vesicles was also studied, and the results showed that the charge on the liposomes enhanced the stability of the liposomes against the leakage-inducing activity of these drug substances.

• YAKHAK HOEJI
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• v.38 no.2
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• pp.131-139
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• 1994

Calcein-encapsulated small unilamellar vesicles of various lipid composition were prepared using the sonication technique, and their stabilities at $20^{\circ}C$ were examined by measuring calcein leakage from the liposomes. The fluidity of these liposomal bilayers was also investigated by measuring the fluorescence polarization of DPH labelled into the liposomes. The results showed that liposomes made of PC mixtures with different acyl chain length were very stable, which may be due to the formation of interdigitated bilayer structure. The addition of cholesterol further stabilized these PC liposomes. However, addition of cholesterol reduced the encapsulation efficiences of liposomes. The fluidity of the liposomes was significantly decreased by cholesterol in the liquid crystalline state, but not changed in the gel state. These results suggest that the enhanced stability of PC mixture liposomes may be ascribed to the formation of stable interdigitated bilayer structure. In membrane-mimetic and drug-delivery studies, vesicles made of mixtures of various phospholipids are recommended instead of addition of cholesterol to the phospholipid.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.91-100
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• 2008

Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism(SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer(FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci(QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 $F_2$ plants from the cross of PI 96354$\times$Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot(FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.