Evaluation of infection course in mice induced by L. major in presence of positively charged liposomes containing CpG ODN

An inoculation of virulent Leishmania major is known as leishmanization [LZ] which is proven to be the most effective control measure against Cutaneous Leishmaniasis [CL]. However, using LZ is restricted due to various side effects such as uncontrolled lesion development. In the present research, the efficacy of cationic nanoliposomes containing CpG oligodeoxynucleotides [CpG ODN] as an improved adjuvant delivery system was studied to diminish the lesion development and infection course of L. major after inoculation into the mice. BALB/c mice were inoculated subcutaneously [SC] with L. major plus empty DSPC, DSPC [CpG ODN], DSPC [Non CpG ODN], empty DMPC, DMPC [CpG ODN], DMPC [Non CpG ODN] or HEPES buffer. The results showed that group of mice received DMPC [CpG ODN] nanoliposomes developed a significantly smaller lesion and showed minimum number of L. major in the spleen and draining lymph nodes. In addition, using DMPC [CpG ODN] liposomes resulted in a Th1 type of immune response with a preponderance of IgG2a isotype which is concurrent with the production of DMPC [CpG] induced IFN-gamma in the spleen of the mice. Taken together, the results suggested that immune modulation using DMPC [CpG ODN] nanoliposomes might be a practical approach to improve the safety of LZ

Tumor Necrosis Factor-Alpha Induced VCAM-l Expression is Inhibited by High Density Lipoprotein in Human Astroglioma Cells / 체질인류학회지

Astrocytes, the major glial cells in the central nervous system (CNS), can express vascular cell adhesion molecule-1(VCAM-1) in response to cytokines, such as TNF-alpha. In CNS, an increased VCAM-1 expression may contribute to inflammatory processes. We, in the present study, have examined the effect of human plasma High Density Lipoproteins (HDL) and other lipoproteins on VCAM-1 expression in astroglioma cells since astrocytes secrete HDL-like lipoprotein particles which contain apo E and cholesterol, phospholipid. The exposure of astroglioma cells to the major plasma lipoprotein fractions (VLDL, LDL and HDL) had no effect on the VCAM-1 expression. However, TNF-alpha-induced VCAM-l was inhibited by HDL in a dose-dependent manner, but not by VLDL or LDL. The inhibitory effect of HDL on TNF-alpha-induced VCAM-l was reversed by the inclusion of Apo A-I antibody, the major apolipoprotein of HDL, demonstrating the specificity of this response. Reconstituted HDL (discoidal complex of apo HDL and DMPC), but not apo HDL or DMPC, was effective in suppressing the VCAM-1 expression. RNase protection assay (RPA) revealed that TNF-alpha- induced VCAM-l mRNA expression was markedly inhibited by HDL (500 microgram cholesterol/mL). These results indicate that HDL-like particles in the CNS may function as an immunosuppressive molecule in pathologic conditions of CNS.

Photophysical studies of zinc phthalocyanine and chloroaluminum phthalocyanine incorporated into liposomes in the presence of additives

The photophysical properties of zinc phthalocyanine (ZnPC) and chloroaluminum phthalocyanine (AlPHCl) incorporated into liposomes of dimyristoyl phosphatidylcholine in the presence and absence of additives such as cholesterol or cardiolipin were studied by time-resolved fluorescence, laser flash photolysis and steady-state techniques. The absorbance of the drugs changed linearly with drug concentration, at least up to 5.0 æM in homogeneous and heterogeneous media, indicating that aggregation did not occur in these media within this concentration range. The incorporation of the drugs into liposomes increases the dimerization constant by one order of magnitude (for ZnPC, 3.6 x 10(4) to 1.0 x 10(5) M-1 and for AlPHCl, 3.7 x 10(4) to 1.5 x 10(5) M-1), but this feature dose does not rule out the use of this carrier, since the incorporation of these hydrophobic drugs into liposomes permits their systemic administration. Probe location in biological membranes and predominant positions of the phthalocyanines in liposomes were inferred on the basis of their fluorescence and triplet state properties. Both phthalocyanines are preferentially distributed in the internal regions of the liposome bilayer. The additives affect the distribution of these drugs within the liposomes, a fact that controls their delivery when both are used in a biological medium, retarding their release. The addition of the additives to the liposomes increases the internalization of phthalocyanines. The interaction of the drugs with a plasma protein, bovine serum albumin, was examined quantitatively by the fluorescence technique. The results show that when the drugs were incorporated into small unilamellar liposomes, the association with albumin was enhanced when compared with organic media, a fact that should increase the selectivity of tumor targeting by these phthalocyanines (for ZnPC, 0.71 x 10(6) to 1.30 x 10(7) M-1 and for AlPHCl, 4.86 x 10(7) to 3.10 x 10(8) M-1).

Conformational flexibility of voltage gated dihydropyridine sensitive calcium channel in hydrated DMPC bilayer.

We have built a model for Ca2+ channel using amino acid sequence from S3 helix of the fourth internal repeat of alpha 1 subunit of dihydropyridine sensitive calcium channel from rabbit skeletal muscle, on the basis of X-ray crystallographic data on four helix bundle. The assembling of the geometry of the pore was achieved using a sixteen residues peptide fragment from short SSI/II loop (residues 1010-1025) which had F1013 and E1014 residues, considered to be important for the drug induced activity of the channel. This had hairpin bend between F1013 to W1016. The drug 2,6-dimethyl 3,5-dicarbomethoxy-4 (2-nitrobenzyl) 1,4 dihydropyridine (DHP) (nifedipine), which is a calcium channel inhibitor used in the treatment of cardiovascular diseases, was introduced, interacting with these two residues via Ca2+ ion. Two more Ca2+ ions were introduced in the pore. The model was incorporated in the bilayer of 36 dimyristoyl phosphatidyl choline (DMPC) molecules with 1201 water molecules and simulated for 200 picoseconds (ps) after equilibration for 120 ps. We also simulated the channel model in vacuum and in aqueous environment for comparison. The latter was unstable after 120 ps. The geometric parameters of the pore are analysed by MOLMOL, PCURVE 3.1 and a special program ANHELIX developed by us. Stability of the pore dimensions during simulations is discussed in this paper.

500 picosecond molecular dynamics simulation of amphiphilic polypeptide Ac(LKKL)4 NHEt with 1,2 di-mysristoyl-sn-glycero-3-phosphorylcholine (DMPC) molecules.

Molecular dynamics (MD) simulation of the interaction between amphiphilic polypeptide Ac(LKKL)4NHEt and 4 DMPC (1,2 di-mysristoyl-sn-glycero-3-phosphorylcholine) molecules has been carried out at 310 K for 500 picoseconds (ps) using AMBER 4.0. Interaction energy and a number of conformational parameters are calculated for the subaveraged coordinates, using P-CURVES 3.1 and our MD trajectory analysis program ANALMD. No significant change in DMPC headgroup conformation was observed. However, the mobility of P atoms was found to be restricted. The chains were quite flexible and their flexibility increased towards the ends. They interacted amongst themselves. The polypeptide remained predominantly in alpha-helical conformation. Leu1 and Lys2 at the N terminus and Leu13 to Leu16 at C terminus assumed non helical conformation and were quite flexible. Average interaction energy between the polypeptide and DMPC molecules was found to be -151.828 kcal*mol-1. The main contributory factor was electrostatic interaction of Lys NH3+ groups with the DMPC phosphates. On an average one Lys chain interacted with 1.5 DMPC molecules. Central region of the polypeptide had better contact with DMPC molecules. A model for the fusogenic properties of the polypeptide is presented on the basis of MD results.

Incorporation of synthetic tetramethylene diamine 8,8'-bis(riboflavin tetraacetate) in DMPC vesicles.

The tetramethylene diamine 8,8'-bis(riboflavin tetraacetate) is synthesized by condensation of 8 alpha-bromo riboflavin tetraacetate and 1,4 diaminobutane. The incorporation of bis(riboflavin tetraacetate) in bilayer of DMPC vesicles has been studied by gel permeation chromatography, UV-visible and fluorescence spectroscopy.

200 picosecond molecular dynamics simulation of interaction of nifedipine with 1-2 dimyristoyl phosphatidylcholine membrane.

Interaction of calcium channel antagonist nifedipine (Nif) with 1-2 dimyristoyl phosphatidylcholine (DMPC) membrane has been studied using molecular dynamics approach. The simulations for one molecule of nifedipine with four DMPC molecules were carried out for 200 pico seconds (ps) using AMBER (Assisted Model Building with Energy Refinement) 3.0 adopted to CYBER 180/930 computer and changes in the structural parameters of DMPC were compared with those for DMPC monolayer (a matrix of nine molecules) optimized separately. Dynamics simulations for the latter had been carried for 40 ps. Our results show that the drug molecule (Nif) penetrates a discrete depth within the phospholipid matrix causing hydrocarbon chains of lipid molecules to swing that makes enough room for the receptor adjacent to the drug molecule.

Lectin binding to complex carbohydrate at the interface: a study by resonance energy transfer.

The binding affinity of the oligosaccharide moiety of a neutral glycosphingolipid, asialoGM1, towards Ricinus communis agglutinin (RCAI) was determined for the first time by fluorescence resonance energy transfer (RET). The asialoGM1 was incorporated into a phospholipid (DMPC) vesicle doped with dansylated DPPE and then titrated with an increasing amount of the galactose specific RCAI. The efficiency of RET was determined by a saturable increase in the quenching of 'donor' fluorescence, i.e. the 'trp' residue of RCAI, due to the energy transfer from the 'acceptor' dansyl group on the surface of the vesicle. The apparent binding constant was found to be in the range of 10(5)-10(6) M-1 at 27 degrees C.