New 4,4-difluoro-3a,4a-diaza-<Emphasis Type="Italic">s</Emphasis>-indacene (BODIPY)-labeled sphingolipids for membrane studies

The synthesis of a series of new fluorescently labeled sphingolipids containing a 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY-8) group at the ω-position of a fatty acyl residue is described. The obtained probes were used in studies of biological and model membrane systems.     

Identification of a new carbohydrate-binding site of influenza virus

It has recently been shown that the influenza virus can specifically bind the residue of a nonsialylated sulfated oligosaccharide Gal(6SO₃H)β1-4GlcNAcβ (6’SLacNAc). To identify by photoaffinity labeling the virion component that binds 6’SLacNAc, we synthesized a carbohydrate probe containing a ¹²⁵I labeled diazocyclopentadien-2-yl carbonyl group as an aglycone. According to the electrophoretic data, the labeled areas corresponded to a large hemagglutinin subunit, a nucleocapsid protein, and neuraminidase (NA). Probing in the presence of an excess of 6’SLacNAcβ-OCH₂CH₂NHAc glycoside resulted in redistribution of the labeling intensity, with the maximum inhibition being observed for NA. The data obtained indicate that NA is a viral 6’SLacNAc-binding protein.     

Synthesis of New Photoaffine Probes on the Basis of Ganglioside GM1

New photoaffine probes, photoreactive derivatives of ganglioside GM1 bearing a carbene-generating diazocyclopentadien-2-ylcarbonyl group at various distances from the carbohydrate moiety in their molecules were synthesized.     

Synthesis of a Lipid Derivative of the Antitumor Agent Methotrexate

A lipophilic methotrexate prodrug capable of incorporation into membranes of carrier liposomes was synthesized. The conjugate consists of a lipophilic rac-1,2-dioleoylglycerol anchor connected to methotrexate through a Ala–N-carbonylmethylene linker, which should be located in the polar region of the lipid bilayer. The ester bond between the hydrophilic linker and the antitumor agent can be hydrolyzed by intracellular esterases. The liposomal formulation of the prodrug exhibited a cytotoxic activity in vitro.     

Depth-dependent investigation of the apolar zone of lipid membranes using a series of fluorescent probes,Me<Subscript>4</Subscript>-BODIPY-8-labeled phosphatidylcholines

A series of lipid probes, phosphatidylcholines labeled with Me₄-BODIPY-8 (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl) fluorophore attached to the end of acyl residue at different distances from the polar head, were used as depth-dependent probes for the apolar zone of the model membrane systems, large unilamellar vesicles (LUV). Data on the anisotropy of probe fluorescence demonstrated a different mobility profiles for the fluorophore microenvironment in LUVs of different composition at various temperatures, which indicates a high sensitivity of these probes as tools for studying membrane systems. An interesting anomaly was observed for LUVs from dimiristoylphosphatidylcholine (DMPC) or from a DMPC-cholesterol mixture: the anisotropy of the fluorophore located near the bilayer center is larger than that of the fluorophore located further from the center; i.e., the mobility of the microenvironment is lower in the first case. This anomaly is supposed to result from the penetration of unlabeled long chain of the probes to the opposite bilayer leaflet. Such a possibility should be taken into account when constructing the fluorescent probes and interpreting the results.     

The capsule polysaccharide structure and biogenesis for non-O1 Vibrio cholerae NRT36S: genes are embedded in the LPS region

Background  

In V. cholerae, the biogenesis of capsule polysaccharide is poorly understood. The elucidation of capsule structure and biogenesis is critical to understanding the evolution of surface polysaccharide and the internal relationship between the capsule and LPS in this species. V. cholerae serogroup O31 NRT36S, a human pathogen that produces a heat-stable enterotoxin (NAG-ST), is encapsulated. Here, we report the covalent structure and studies of the biogenesis of the capsule in V. cholerae NRT36S.     

Genomics and the renaissance of generalism

A meeting report of the sessions on human, eukaryotic and bacterial genome sequencing at the American Society for Microbiology and Institut Pasteur joint conference: Genomes 2000 International Conference on Microbial and Model Genomes, Paris, April 11-15, 2000     

New fluorescent cholesterol analogs as membrane probes.

New fluorescent cholesterol analogs, (22E, 20R)-3beta-hydroxy-23-(9-anthryl)-24-norchola-5,22-die ne (R-AV-Ch), and the 20S-isomer (S-AV-Ch) were synthesized, their spectral and membrane properties were characterized. The probes bear a 9-anthrylvinyl (AV) group instead of C22-C27 segment of the cholesterol alkyl chain. Computer simulations show that both of the probes have bulkier tail regions than cholesterol and predict some perturbation in the packing of membranes, particularly for R-AV-Ch. In monolayer experiments, the force-area behavior of the probes was compared with that of cholesterol, pure and in mixtures with palmitoyloleoyl phosphatidylcholine (POPC) and N-stearoyl sphingomyelin (SSM). The results show that pure R-AV-Ch occupies 35-40% more cross-sectional area than cholesterol at surface pressures below film collapse (0-22 mN/m); whereas S-AV-Ch occupies nearly the same molecular area as cholesterol. Isotherms of POPC or SSM mixed with 0.1 mol fraction of either probe are similar to isotherms of the corresponding mixtures of POPC or SSM with cholesterol. The probes show typical AV absorption (lambda 386, 368, 350 and 256 nm) and fluorescence (lambda 412-435 nm) spectra. Steady-state anisotropies of R-AV-Ch and S-AV-Ch in isotropic medium or liquid-crystalline bilayers are higher than the values obtained for other AV probes reflecting hindered intramolecular mobility of the fluorophore and decreased overall rotational rate of the rigid cholesterol derivatives. This suggestion is confirmed by time-resolved fluorescence experiments which show also, in accordance with monolayer data, that S-AV-Ch is better accommodated in POPC-cholesterol bilayers than R-AV-Ch. Model and natural membranes can be labeled by either injecting the probes via a water-soluble organic solvent or by co-lyophilizing probe and phospholipid prior to vesicle production. Detergent-solubilization studies involving 'raft' lipids showed that S-AV-Ch almost identically mimicked the behavior of cholesterol and that of R-AV-Ch was only slightly inferior. Overall, the data suggest that the AV-labeled cholesterol analogs mimic cholesterol behavior in membrane systems and will be useful in related studies.     

Gangliosides induce cell apoptosis in the cytotoxic line CTLL-2, but not in the promyelocyte leukemia cell line HL-60

Gangliosides induce apoptosis in the cells of the IL-2-dependent cytotoxic mouse line CTLL-2. Upon incubation with gangliosides for 24 h, their effect resulting in appearance of apoptotic cells, falls in a series GM2 > GM3 > GM1 > GD1a > GD1b > GT1b. In the presence of rIL-2, apoptosis induced by GM1 is suppressed, whereas that induced by GM2 is enhanced (the effect of intracellular agent C2-Cer is independent of this cytokine). The GM1-induced apoptosis is cancelled by the caspase I inhibitor. The gangliosides under study are not able to induce apoptosis in the promyelocyte leukemia cell line HL-60. Physiological aspects of the phenomenon found are discussed.     

Unusual Fluorescent Properties of N-(9-Anthroyl) Derivatives of Aromatic Amines

9-Anthroyl derivatives of some aromatic amines exhibit unusual fluorescence characteristics. In solvents of low and medium polarity (hexane, chloroform, DMF, and tert-butanol), their emission maxima are shifted to longer wavelengths as compared to the spectra recorded in polar solvents (ethanol and methanol); the red shift is accompanied by an increase in the fluorescence quantum yield. Possible reasons of such an anomalous spectral shift are discussed.