Circular dichroism studies of the self-association of amphotericin B

Circular dichroism (CD) spectroscopy has been used to evaluate the ability of 21 different solvents to influence the aggregation state of amphotericin B. Using the relative donor/acceptor tendencies known for each solvent system, it was possible to deduce information as to the factors which goven the self-association of amphotericin B. It was concluded that in the absence of strong solvent interaction, amphotericin B prefers to self-associate into oligomeric species. This intrinsic driving force can be overcome through the use of solvents which function as strong electron pair donors, probably forming specific solvent—solute species. © 1994 Wiley-Liss, Inc.     

Structural changes of low density lipoproteins with Cu2+ and glucose induced oxidation

The compositional and structural changes of lipids and apolipoproteins during in vitro oxidation of low density lipoprotein (LDL) are investigated in this study by IR spectroscopy. For comparison, LDL samples containing either copper or glucose at physiological or pathological concentrations are considered in order to know the separate affects of these chemical factors on LDL oxidation. The results show that the initial steps of lipid oxidation proceed through hydrogen atom loss from methylene groups, as well as loss of cholesteryl ester molecules, and later a recovering of carbonyl compounds resulting from aldehyde formation that generally occurs in autooxidation processes. Lipid oxidation is induced by copper ions, and glucose enhances metal ion induced LDL oxidation as determined by conjugated diene formation. As to the protein conformational changes, IR spectroscopy reveals for the first time that LDL oxidation involves formation of beta-sheet structures, these being more abundant in LDL samples with pathological concentrations of glucose or copper. Consequently, the LDL structural changes may contribute to the recognition of oxidized LDL particles by scavenger receptors.     

Monoacylglycerol accumulation in low and high density lipoproteins during the hydrolysis of very low density lipoprotein triacylglycerol by lipoprotein lipase.

We have demonstrated that low and high density lipoproteins from monkey plasma are capable of accepting and accumulating monoacylglycerol that is formed by the action of lipoprotein lipase on monkey lymph very low density lipoproteins. Furthermore, the monoacylglycerol that accumulates in both low and high density lipoproteins is not susceptible to further hydrolysis by lipoprotein lipase but is readily degraded by the monoacylglycerol acyltransferase of monkey liver plasma membranes. These observations suggest a new mechanism for monoacylglycerol transfer from triacylglycerol rich lipoproteins to other lipoproteins. In addition, the finding that monoacylglycerol bound to low and high density lipoprotein is degraded by the liver enzyme but not lipoprotein lipase lends support to the hypothesis that there are distinct and consecutive extrahepatic and hepatic stages in the metabolism of triacylglycerol in plasma lipoproteins.     

Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

[²H]Chondroitin sulfate was prepared by partial $\text{N}$-deacetylation of chondroitin sulfate ($\text{via}$ hydrazinolysis) followed by treatment with [²H₆]acetic anhydride. ²H NMR spectra of [²H]chondroitin sulfate in the presence of human plasma low density lipoprotein provide evidence for a soluble complex stoichiometry of 3 (and possibly 2) lipoproteins per polysaccharide molecule, and allow a rough estimation of the dissociation constant K_d.     

Effect of homocysteinylation of low density lipoproteins on lipid peroxidation of human endothelial cells

Homocysteine-thiolactone (HcyT) is a toxic product whose synthesis is directly proportional to plasma homocysteine (Hcy) levels. Previous studies demonstrated that the interaction between HcyT and low density lipoproteins (LDL) induces the formation of homocystamide-LDL adducts (Hcy-LDL). Structural and functional alterations of Hcy-LDL have been described and it has been suggested that homocysteinylation could increase atherogenicity of LDL. Oxidative damage of endothelial cells (EC) is considered to be a critical aspect of the atherosclerotic process. To further investigate the molecular mechanisms involved in the atherogenicity of homocysteinylated LDL, we studied the effect of interaction between Hcy-LDL and EC on cell oxidative damage, using human aortic endothelial cells (HAEC) as experimental model. Homocysteinylation of LDL was carried out by incubation of LDL, isolated from plasma of healthy normolipemic subjects, with HcyT (10-100 microM). In our experimental conditions, homocysteinylation treatment was not accompanied by oxidative damage of LDL. No modifications of apoprotein structure and physico-chemical properties were observed in Hcy-LDL with respect to control LDL (c-LDL), as evaluated using the intrinsic fluorescence of tryptophan and the probe Laurdan incorporated in lipoproteins. Our results demonstrated that Hcy-LDL incubated at 37 degrees C for 3 h with HAEC, induced an oxidative damage on human EC with a significant increase of lipid hydroperoxides in cells incubated with Hcy-LDL with respect to cell incubated with c-LDL. The compositional changes were associated with a significant decrease viability in cells treated with Hcy-LDL. The relationship between the levels of -SH groups of LDL and the oxidative damage of HAEC has been demonstrated. These results suggest that Hcy-LDL exert a cytotoxic effect that is likely related to an increase in lipid peroxidation and oxidative damage of EC.     

Identification of nascent high density lipoproteins containing arginine-rich protein in human plasma.

A high density lipoprotein fraction accumulates in the plasma of patients with alcoholic hepatitis when a severe lecithin:cholesterol acyltransferase (EC 2.3.1.43) deficiency is present. The major apoprotein present in this fraction is arginine-rich protein, the fraction is a preferred substrate for lecithin:cholesterol acyltransferase, and by electron microscopy appears as stacked bilayer discs. It is proposed that the lipoprotein represents the accumulation of nascent high density lipoprotein and is the principal pathway through which arginine-rich protein is secreted by the liver in man. The results also suggest that apoprotein AI is acquired by normal high density lipoprotein during the course of lipoprotein metabolism.     

The carbohydrate content of apolipoprotein E from human very low density lipoproteins.

The carbohydrate content of the E protein of human very low density lipoprotein (VLDL) was evaluated both by colorimetric methods and by gas liquid chromatography of the trifluoroacetylated 0-methyl glycosides. The major unmodified hexose was noted to be galactose with a mole ratio with respect to protein which ranged from 0.81 to 1.54. N-acetyl glucosamine (molar ratios from 0.52 to 1.76) and N-acetyl galactosamine (molar ratios from 0.73 to 1.59) and the respective unacetylated amino sugars were noted for all of the apoproteins evaluated. Sialic acid (molar ratios from 0.79 to 1.69) was a prominent carbohydrate for each of the E protein preparations. When the apoprotein was exposed to neuraminidase with a resultant loss of two-thirds of the sialic acid, the isoelectric focus behavior was found to be unchanged. The E protein isolated from the very low density lipoproteins of Type III patients (dysbetalipoproteinemia) revealed a carbohydrate content similar to the normals or Type IV patients.     

Circular dichroism and the interactions of water soluble porphyrins with DNA

The size, sign, and profile of induced circular dichroism (CD) features in the Soret region are reliable indicators of the binding modes of porphyrins and metalloporphyrins to DNA. Porphyrins shown (using such CD criteria) to be intercalators in monodispersed DNA duplexes prove extremely useful for the detection and characterization of organized, condensed forms of nucleic acids (psi-condensates). In addition, certain select porphyrin derivatives can form extended assemblies on nonaggregated DNA templates. A combination of CD and resonance light scattering (RLS) measurements allows for sensitive detection and characterization of these porphyrin arrays.     

The effects of tunicamycin on the metabolism of acetylated low density lipoproteins

The effect of tunicamycin (TM) on the metabolism of acetylated low-density lipoprotein (AcLDL) was examined to determine whether N-linked glycosylation is required for the proper function of the AcLDL pathway. Proteolytic degradation of [¹²⁵I]-AcLDL was increased twofold in the presence of TM. This did not occur via an increase in total lysosomal enzyme activity or extracellular proteolysis; rather, the rate of uptake of [¹²⁵I]-AcLDL was increased. The enhanced degradation of AcLDL did not lead to a commensurate increase in the rate of synthesis of cholesteryl oleate. Conversely, the rate of cholesterol esterification was reduced in the presence of TM. The uptake of [¹²⁵I]-AcLDL was more sensitive to inhibition by chloroquine in TM-treated cells. However, the presence of TM did not affect the ability of chloroquine to inhibit constitutive recycling of AcLDL binding sites. These results suggest that N-linked glycosylation may be involved in the regulation of AcLDL metabolism in J774 cells.     

Synergistic anticandidal activity of xanthorrhizol in combination with ketoconazole or amphotericin B

Candida species are responsible for the fourth most common nosocominal bloodstream infection. Xanthorrhizol, a sesquiterpene compound isolated from Curcuma xanthorrhiza Roxb. has been reported to have anticandidal activity. The aim of this study is to investigate the synergistic anticandidal effect of xanthorrhizol in combination with ketoconazole or amphotericin B against Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis , and Candida tropicalis . Mostly, xanthorrhizol in combination with ketoconazole or amphotericin B exhibited the synergistic anticandidal effects against all species of Candida tested. In combination with xanthorrhizol, the concentration of ketoconazole or amphotericin B for inhibiting the growth of the tested Candida species could be reduced by ≥50%. Time–kill curves showed that 1/2 minimum inhibitory concentration (MIC) dose of xanthorrhizol, amphotericin B, or ketoconazole alone against each of the six Candida species did not inhibit the growth of all Candida species tested. However, 1/2 MIC dose of xanthorrhizol in combination with 1/2 MIC dose of ketoconazole or 1/2 MIC dose of amphotericin B exhibited growth inhibition of all Candida species tested and reduced viable cells by several logs within 4 h. These results support the potential use of xanthorrhizol as an anticandidal agent, and it can be used complementarily with other conventional antifungal agents.     

Circular dichroism spectroscopic study of non‐covalent interactions of poly‐L‐glutamic acid with a porphyrin derivative in aqueous solutions

The interactions of poly‐L ‐glutamic acid and a cationic porphyrin derivative in aqueous solutions were studied by the combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies. It was found that non‐covalent interactions between both agents influence the structure of the polymeric matrix and the guest porphyrins and vice versa, but the physico‐chemical properties of the solutions, especially the pH and the relative permittivity of the solvent, play a key role in the structure of the polypeptide part of the formed complexes. It was shown that the interaction with porphyrins prevents the precipitation of poly‐L ‐glutamic acid in aqueous solution at acidic pH. In special conditions, the porphyrins attached to the polypeptide probably possess face‐to‐face interaction as demonstrated by the enhancement of the characteristic ECD signal and the appearance of sidebands on its short and long wavelength sides. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

Role of net charge of low density lipoproteins in high affinity binding and uptake by cultured cells.

Selective modification of arginine residues of LDL by cyclohexanedione or acetylation of lysine residues of LDL deminishes their high affinity binding and internalisation by human skin fibroblast up to 50% as compared with native LDL. The enhanced negative charge of the modified LDL particles results in an accelerated electrophoretic mobility towards the anode. Neuraminidase treatment of cyclohexanedione-modified LDL and acetyllysine-LDL normalizes not only their electrophoretic mobility, but also restores more than 80% of the original binding and uptake capacity, the specificity of this effect being indicated by using fibroblasts deficient in LDL receptor and by competitive binding and internalization experiments.     

Oxidative modification of low density lipoprotein (LDL) by activated human monocytes and the cell lines U937 and HL60

Summary Human peripheral blood monocytes, upon activation, have the capacity to oxidize low density lipoprotein (LDL) and render the LDL toxic to cultured cells. Previous studies by our laboratory indicate that this process is mediated by free radicals in that it can be prevented by addition of free radical scavengers and antioxidants during the incubation of monocytes with LDL. Here we report that optimal modification of LDL by monocytes was influenced by media composition. In the absence of added metal ions, oxidation was distinctly dependent on the concentration of monocytes as well as LDL concentration. Exposure of monocytes to lipopolysaccharide or stimulation of phagocytosis by opsonized zymosan resulted in marked enhancement of LDL oxidation compared to other activating agents. After exposure to activated monocytes, lipid oxidation products in the supernatant were found both in a high molecular weight fraction containing LDL (>30 000 Daltons) and in a lipoprotein-free, low molecular weight fraction (<30 000 Daltons), yet only the high molecular weight, LDL-containing fraction was toxic to target cells. In addition, human myelomonocytic cell lines U937 and HL60 were shown to mediate oxidation of LDL. As with monocytes, exposing these cells to opsonized zymosan caused the level of LDL oxidation to be significantly enhanced. These findings offer further insight into the mechanisms of monocyte-mediated oxidation of lipoproteins and will facilitate studies investigating the role of monocyte-modified LDL in tissue injury. This project was funded by grants form the American Heart Association-Northeast Ohio Affiliate and the National Institutes of Health, Bethesda, MD (HL-29582).     

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.     

Tanshinone II-A inhibits low density lipoprotein oxidation in vitro

Tanshinone II-A (TSII-A) is a major component of Salvia miltorrhiza Bunge which has long been used for preventing and ameliorating anginal pain in China. However the effect of TSII-A on low density lipoprotein (LDL) oxidation has not been studied. The present study was performed to investigate the effects of TSII-A on LDL oxidation using four oxidizing systems, including copper-, peroxyl radical- and peroxynitriteinitiated and macrophage-mediated LDL oxidation. LDL oxidation was measured in terms of formation of thiobarbituric acid-reactive substances (TBARS), relative electrophoretic mobility (REM) on agarose gel and lag time. In all four systems, TSII-A has apparent antioxidative effects against LDL oxidation, as evidenced by its dose-dependent inhibition of TBARS formation, prolongation of lag time and suppression of increased REM.

Regarding the mechanism underlying its antioxidative effect, TSII-A neither scavenged superoxide nor peroxynitrite. It also did not chelate copper. But it has mild peroxyl radical scavenging activity. The direct binding to LDL particles and conformational change of LDL structure by TSII-A were suggested, because it increased negative charge of LDL which was shown by increased REM on agarose gel. In conclusion, TSII-A is an effective antioxidant against LDL oxidation in vitro. The underlying mechanism appears to be related to its peroxyl radical scavenging and LDL binding activity.     

Release of low density lipoprotein receptors from human fibroblasts or HeLa cells by tryptic digestion.

Trypsin treatment of cultured normal human skin fibroblasts or Hela cells releases material which is retained on a low density lipoprotein (LDL)-Sepharose affinity column, may be eluted from it with 2.5 M KI and, after dialysis, agglutinates LDL or apo-B-coated formocells. Such agglutination is prevented by preincubation of the receptor with LDL in solution or with arginine-rich protamine. Trypsin treatment of “receptor defective” or “receptor negative” mutant fibroblasts releases material which is retained on LDL-Sepharose column but fails to agglutinate LDL-coated formocells. The receptor may be labeled with $\text{6-[}{}^3\text{H]-glucosamine·HCl}$ and [${}^3\text{H}$]-leucine, it is inactivated by heating at 80°C for 10 min and may be obtained from normal fibroblasts or Hela cells, whether they were cultured in presence or in absence of lipoprotein-containing fetal calf serum.     

Vibrational and electronic circular dichroism study of the interactions of cationic porphyrins with (dG-dC)10 and (dA-dT)10

The interactions of two different porphyrins, without axial ligands-5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin-Cu(II) tetrachloride (Cu(II)TMPyP) and with bulky meso substituents-5,10,15,20-tetrakis(N,N,N-trimethylanilinium-4-yl)porphyrin tetrachloride (TMAP), with (dG-dC)10 and (dA-dT)10 were studied by combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopy at different [oligonucleotide]/[porphyrin] ratios, where [oligonucleotide] and [porphyrin] are the concentrations of oligonucleotide per base-pair and porphyrin, respectively. The combination of VCD and ECD spectroscopy enables us to identify the types of interactions, and to specify the sites of interactions: The intercalative binding mode of Cu(II)TMPyP with (dG-dC)(10), which has been well described, was characterized by a new VCD "marker" and it was shown that the interaction of Cu(II)TMPyP with (dA-dT)10 via external binding to the phosphate backbone and major groove binding caused transition from the B to the non-B conformer. TMAP interacted with the major groove of (dG-dC)10, was semi-intercalated into (dA-dT)10, and caused significant variation in the structure of both oligonucleotides at the higher concentration of porphyrin. The spectroscopic techniques used in this study revealed that porphyrin binding with AT sequences caused substantial variation of the DNA structure. It was shown that VCD spectroscopy is an effective tool for the conformational studies of nucleic acid-porphyrin complexes in solution.     

Effect of nystatin,amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 °C or 30 °C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 °C than at 30 °C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K⁺ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K⁺ and before compounds of higher molecular weights.     

Interaction of very low density lipoprotein with chicken oocyte membranes

The interaction of hen 125I-VLDL (very low density lipoprotein) with chicken oocyte membranes was characterized using a rapid sedimentation assay. Equilibrium and kinetic studies showed an apparent dissociation constant (Kd) 8.7-9.1 x 10(-8) M or 43.5-45.5 micrograms VLDL protein/ml. Binding capacity was 2.0 micrograms VLDL protein/mg membrane homogenate protein. The apparent rate constants were k1 = 2.4 x 10(5) M-1 min-1 and k2 = 2.1 x 10(-2) min-1. Specific binding required the presence of divalent cations. Whereas binding was completely restored after treatment with EDTA by the addition of MN++, only 60% of binding was restored using Ca++.