Fluorescent dyes undergoing intramolecular proton transfer with improved sensitivity to surface charge in lipid bilayers.

4'-(Dialkylamino)-3-hydroxyflavones are characterized by an excited-state proton transfer reaction between two tautomeric excited states, which results in two emission bands well separated on the wavelength scale. Due to the high sensitivity of the relative intensities of the two emission bands to solvent polarity, hydrogen bonding and local electric fields, these dyes found numerous applications in biomembrane studies. In order to further improve their fluorescence characteristics, we have synthesized new dyes where the 2-phenyl group is substituted with a 2-thienyl group. In organic solvents, the new dyes exhibit red shifted absorption and dual fluorescence. Although they show lower sensitivity to solvent polarity and H-bond donor ability (acidicity) than their parent 3-hydroxyflavone dyes, they exhibit a much higher sensitivity to solvent H-bond acceptor ability (basicity). Moreover, when tested in lipid vesicles of different surface charge, the new dyes show much better resolved dual emission and higher sensitivity to the surface charge of lipid bilayers than the parent dyes. The response of the new dyes to surface charge is probably connected with the H-bond basicity of the membrane surface, which is the highest for negatively charged surfaces. As a consequence, the new dyes appear as prospective fluorophores for the development of new fluorescent probes for biomembranes.     

Liquid ordered and gel phases of lipid bilayers: fluorescent probes reveal close fluidity but different hydration

Hydration and fluidity of lipid bilayers in different phase states were studied using fluorescent probes selectively located at the interface. The probe of hydration was a recently developed 3-hydroxyflavone derivative, which is highly sensitive to the environment, whereas the probe of fluidity was the diphenylhexatriene derivative, 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. By variation of the cholesterol content and temperature in large unilamellar vesicles composed of sphingomyelin or dipalmitoylphosphatidlycholine, we generated different phases: gel, liquid ordered (raft), liquid crystalline, and liquid disordered (considered as liquid crystalline phase with cholesterol). For these four phases, the hydration increases in the following order: liquid ordered < gel approximately liquid disordered < liquid crystalline. The membrane fluidity shows a somewhat different trend, namely liquid ordered approximately gel < liquid disordered < liquid crystalline. Thus, gel and liquid ordered phases exhibit similar fluidity, whereas the last phase is significantly less hydrated. We expect that cholesterol due to its specific H-bonding interactions with lipids and its ability to fill the voids in lipid bilayers expels efficiently water molecules from the highly ordered gel phase to form the liquid ordered phase. In this study, the liquid ordered (raft) and gel phases are for the first time clearly distinguished by their strong difference in hydration.     

Influence d'une infection aBeauveria bassiana sur les survivants et la descendance deChilo suppressalis [Lep.: Pyralidae]

Les répercussions secondaires d'une infection subléthale àBeauveria bassiana des chenilles et chrysalides deChilo suppressalis sont étudiées du point de vue de la réduction de la fécondité des femelles et de la fertilité des œufs. Ces phénomènes semblent pouvoir être considérés comme assez généraux en raison des divers exemples connus aussi bien chez les lépidoptères que les coléoptères. Compte tenu de cette propriété, l'interprétation des expérimentations de lutte microbiologique à l'aide de champignons entomopathogènes est discutée. Summary Chilo suppressalis mature larvae and pupae reared on a semi-synthetic medium were infected by spraying titrated spore suspensions ofBeauveria bassiana. Potentialities of the imagos having survived to the mycosis were evaluated by measuring the fecundity of female adults and the egg fertility. The mature larvae contaminated with a spore suspension titrated at 3×10⁷ conidia/ml, as well as the pupae and imagos, were attacked by the muscardine disease. The fertility of the eggs laid by the surviving insects (25% of the total) was very low compared with the control insects. Some pupae, contaminated under the same conditions but with concentrations of inocula varying between 3,3×10⁵ et 3,3×10⁶ conidia/ml also showed mycosis in the pupal and adult stages (53 to 87% of the insects were contaminated, according to the spore concentration). The number of eggs laid by the surviving insects seemed in inverse ratio to the spore dosage, and they showed no fertility, apart from the pupae infected with the weaker suspension.

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Ce travail fait partie d'une thèse de docteur ingénieur soutenue le 28 mai 1975 devant la Commission de l'Université Paul-Sabatier de Toulouse, France.