An expedient synthesis of fluorescent labeled ceramide-1-phosphate analogues

A synthesis for fluorescent analogs of ceramide-1-phosphate bearing 9-anthrylvinyl or 4,4-difluoro-3a,4a-diaza-s-indacene-8-yl (Me₄-BODIPY) fluorophore at co-position of fatty acid residue was carried out. The key stage of the synthesis is hydrolysis of corresponding sphingomyelins catalyzed by phospholipase D from Streptomyces chromofuscus; the enzymatic yield has been raised to 50–70% by appliance of organic solvent in the incubation medium.     

Mitochondrial H+-ATPase: Identification of Subunits of the F0 Subcomplex that Contact Membrane Lipids

The subunits of the F₀ membrane sector of bovine heart mitochondrial H⁺-ATPase that contact the lipids of the mitochondrial inner membrane were identified with the use of specially synthesized proteoliposomes that contained active mitochondrial H⁺-ATPase and a photoreactive lipid, which was 1-acyl-2-[12-[di-azocyclopentadiene-2-carbonylamino)-[12-¹⁴C]dodecanoyl]-sn-glycero-3-phosphocholine, 1-acyl-2-[11-([¹²⁵I]diazoiodocyclopentadiene-2-carbonyloxy)undecanoyl]-sn-glycero-3-phosphocholine, or 1-acyl-2-[12-(diazocyclopentadiene-2-carbonylamino)dodecanoyl]-sn-glycero-3-phosphocholine, where acyl is a mixture of the residues of palmitic (70%) and stearic (30%) acids. An analysis of the cross-linked products obtained upon the UV-irradiation of these proteoliposomes indicated that subunits c and a of the F₀ membrane sector contact the lipids. The crosslinked products were identified by SDS-PAGE and MALDI mass spectrometry.     

Cholesterol modulates interaction between an amphipathic class A peptide, Ac-18A-NH2, and phosphatidylcholine bilayers

Cholesterol (Chol) in phosphatidylcholine large unilamellar vesicles (PC LUV) modulated interaction of the bilayers with a class A amphipathic peptide, Ac-18A-NH2: Chol increased the peptide binding capacity and reduced the affinity together with the peptide-induced leakage of calcein from LUV. Similar effects of Chol have been observed on the interaction of LUV with apoA-I [Saito, H., Miyako, Y., Handa, T., and Miyajima, K. (1997) J. Lipid Res. 38, 287-294]. Circular dichroism (CD) spectra of the peptide indicated a similar helical structure formation in LUV with and without Chol. The fluorescence spectral shift, quantum yield, anisotropy, and acrylamide-quenching of the peptide Trp indicated that in PC:Chol (3:2) LUV, Ac-18A-NH2 was located in a more polar membrane environment with increased motional freedom and greater accessibility to the aqueous medium. Fluorescence energy transfer from the Trp indole ring to acceptors situated at different depths in the bilayers revealed that the amphipathic peptide penetrated the hydrophobic interior of PC bilayers, while the peptide was located at the polar zwitterionic surface in PC:Chol LUV. The inclusion of Chol causes the headgroup separation of PC at the surface of LUV and increases the binding maximum of the wedge-shaped amphipathic peptide without disrupting the membrane structure. In addition, the rigidifying effect of Chol on PC acyl chains prevents the penetration of the peptide into the bilayer interior. These findings imply that Chol in membranes affects the binding and motional freedom of exchangeable plasma apolipoproteins containing class A amphipathic sequences, e.g., apoA-I and apoCs.     

Lipid-specific fluorescent probes in studies of biological membranes

Lipid-specific fluorescent probes are natural lipids carrying an apolar fluorophore in one of the hydrocarbon chains. Since such probes retain the head groups and resemble the molecular shape of native membrane lipids, they largely mimic the behaviour of their natural prototypes in biological membranes. Information provided by the lipid-specific probes is more differentiated and easier to interpret than that obtained from non-lipid probes. The principles of design of lipid-specific probes are formulated and the relative advantages and disadvantages of various fluorophores are discussed. In order to reduce ambiguities caused by perturbation of the probe environment, it is proposed to use, in a comparative manner, two or more lipid-specific probes resembling each other in all aspects except the polar head groups (the 'two probes' concept). Two types of fluorophores, the anthrylvinyl group and the perylenoyl group, were found to be well suited for the synthesis of lipid-specific probes. Use of both types of probes 'in tandem' opens new possibilities for studying lipid-protein and lipid-lipid interactions in biological membranes. The anthrylvinyl- and perylenoyl-labeled lipids were applied in studies of serum lipoproteins and erythrocyte membranes. A new highly sensitive ligand-receptor binding assay and a new approach to biological signal amplifying based on the use of lipid-specific probes are described.     

Synthesis and Properties of a Fluorescent-Labeled Triglyceride Derivative of the Antitumor Drug Merphalan

A new fluorescent probe, a 3-perylenoyl derivative of the lipophilized antitumor drug merphalan (sarcolysine), was synthesized. The probe is suitable for studying intracellular traffic and metabolism of merphalan and its derivatives. The perylenoyl fluorescence is partially quenched by the merphalan chromophore, which broadens the probe potentialities.     

Synthesis and properties of lipophilic derivatives of 5-fluorouracil

A series of N ¹-acyl derivatives of 5-fluorouracil (5-FU) bearing the residues of palmitic, p-myristoylaminobenzoic, p-oleoylaminobenzoic, and adamantane-1-carboxylic acids have been synthesized. The relative hydrolysis rates for the derivatives under physiological conditions (pH 7.2 and 37°C) have been determined, and it has been shown that the resistance of these compounds to hydrolysis increases as the steric accessibility of the amide group at residue N ¹ of 5-FU decreases. The derivatives easily incorporate into the lipid bilayer; their liposomal preparations show a marked cytostatic activity on human breast lymphoma cells (LD50 ～1 μM) and are of interest as potential antitumor preparations. In addition, a fluorescent analogue of the above derivatives, 1-[8-(3-perylenyl)octanoyl]-5-fluorouracil, has been synthesized, which is intended for studying the behavior of 5-FU derivatives in cells and tissues by instrumental methods.     

Immunosuppressive activity of glycosphingolipids. Influence of serum factors on ganglioside inhibition of IL-4-dependent cell proliferation.

Gangliosides have been shown to inhibit proliferation of the interleukin-4 (IL-4) responsive cell line CT.4R. Kinetic analysis has revealed that ganglioside GT1b is a competitive inhibitor of proliferation, while GM and GM3 show a mixed pattern of inhibition, i.e., exhibit more than one inhibition type. Contribution of the competitive cell inhibition for GM1 and GM3 depends on serum factors added: the higher is the percentage of FCS, the larger is the contribution of competitive inhibition. The pattern of proliferation inhibition shown for GT1b does not depend on the FCS content. We have also studied the interaction of the recombinant IL-4 with fluorescent (anthrylvinyl-labelled) gangliosides GM1 and GM3 and lactosylceramide incorporated into liposomes. Dissociation constants of the IL-4-ganglioside complexes have been determined; lactosylceramide does not interact with rIL-4. The K(d) values for the lymphokine complexes with gangliosides support the conclusion based on the kinetic analysis that IL-4 has a higher affinity for GM3 (K(d) = 5 nM) than for GM1 (K(d) = 0.28 microM).     

Nitric oxide-induced membrane tubulovesicular extensions (cytonemes) of human neutrophils catch and hold Salmonella enterica serovar Typhimurium at a distance from the cell surface

Nitric oxide (NO) plays an important role in host defense against bacterial infections such as salmonellosis. NO and 4-bromophenacyl bromide (BPB) induce the formation of long tubulovesicular extensions (TVE, cytonemes, membrane tethers) from human neutrophils. These TVE serve as cellular sensory and adhesive organelles. In the present study, we demonstrated that in the presence of the NO donor, diethylamine NONOate or BPB human neutrophils bound and aggregated Salmonella enterica serovar Typhimurium bacteria extracellularly by TVE. In contrast, inhibition of NO-synthase activity by N ^ω-nitro- l -arginine methyl ester stimulated neutrophil phagocytosis (ingestion) of bacteria. Neutrophil TVE consisted of membrane-covered cytoplasm as was shown by the fluorescent cytoplasmic dye 2',7'-bis(2carboxyethyl)-5,(6)-carboxyfluorescein, and the fluorescent lipid, BODIPY-labeled sulfatide. Disruption and shedding of TVE were accompanied by the appearance of specific invaginations (porosomes) on neutrophil cell bodies. These invaginations corresponded to the variations in diameter of TVE (160–240 nm). We hypothesized that TVE represented protrusions of neutrophil exocytotic trafficking through special structures on the neutrophil surface. In conclusion, we propose a novel mechanism by which NO-induced TVE formation enables neutrophils to bind and aggregate bacteria at a distance.