Membrane-protective properties of isobornylphenols-a new class of antioxidants

The membrane protective and membrane active properties and the antioxidative activity of new semisynthetic antioxidants—isobornylphenols were studied. The presence of oxidant and cytotoxic properties of the compounds were evaluated considering the degree of hemolysis of erythrocytes, either spontaneous or induced by hydrogen peroxide. All the studied compounds were found to have significant antioxidative activity in certain conditions. But their capacity to protect membrane erythrocytes from oxidative stress substantially depended on the structure and concentration of the compound. The highest membrane protective activity was observed for 2,6-diisobornyl-4-methylphenol, which has isobornyl in both of its ortho-positions. Scanning electron microscopy of blood erythrocyte surface architectonics confirmed the ability of the studied compounds to interact with the cell membrane and to change its structure. A relationship between erythrocyte morphological transformation according to bilayer-couple hypothesis depending on isobornylphenols membrane behavior and the cytotoxic effect of certain compound high concentrations reflected in low membrane protective activity in the model cell system was shown. The data obtained allow us to conclude that the biological activity of isobornylphenols is due to both their antioxidative properties and their ability to interact with the cell membranes.     

Gemini ester quat surfactants and their biological activity

Cationic gemini surfactants are an important class of surface-active compounds that exhibit much higher surface activity than their monomeric counterparts. This type of compound architecture lends itself to the compound being easily adsorbed at interfaces and interacting with the cellular membranes of microorganisms. Conventional cationic surfactants have high chemical stability but poor chemical and biological degradability. One of the main approaches to the design of readily biodegradable and environmentally friendly surfactants involves inserting a bond with limited stability into the surfactant molecule to give a cleavable surfactant. The best-known example of such a compound is the family of ester quats, which are cationic surfactants with a labile ester bond inserted into the molecule. As part of this study, a series of gemini ester quat surfactants were synthesized and assayed for their biological activity. Their hemolytic activity and changes in the fluidity and packing order of the lipid polar heads were used as the measures of their biological activity. A clear correlation between the hemolytic activity of the tested compounds and their alkyl chain length was established. It was found that the compounds with a long hydrocarbon chain showed higher activity. Moreover, the compounds with greater spacing between their alkyl chains were more active. This proves that they incorporate more easily into the lipid bilayer of the erythrocyte membrane and affect its properties to a greater extent. A better understanding of the process of cell lysis by surfactants and of their biological activity may assist in developing surfactants with enhanced selectivity and in widening their range of application.     

The actions of low-molecular fragments of substrate and their analogs on the activity of isoenzymes of phospholipase C from Clostridium perfringens]

The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl. perfringens was studied by gel-diffusion in agarose-lecithin gels. It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme. The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups. However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2.     

Cytotoxic effect of unilamellar detergent dialysis liposomes on Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro

Liposomes from egg yolk lecithin and egg yolk lecithin/ganglioside are cytotoxic for Trypanosoma brucei and Trypanosoma congolense bloodstream forms in vitro. The trypanocidal effect is influenced by the liposome age and concentration. This effect is diminished in the presence of whole blood in vitro and could not be observed in vivo. Freeze-fractured parasite membrane showed an intramembranous particle aggregation after incubation with liposomes. Liposomes from egg yolk lecithin kill trypanosomes more rapidly than do liposomes from egg yolk lecithin/cholesterol.     

The influence of LP-X and other lipoproteins associated with hepatic dysfunction on the activity of lecithin:cholesterol acyltransferase.

A study was made of the in vitro effects of the abnormal serum lipoproteins associated with liver disease on the activity of the enzyme lecithin:cholesterol acyltransferase. At lipoprotein concentrations equivalent to those found in hepatic disease sera, the results indicate that: (1) LP-X levels greater than 2.5 mg/ml produced total inhibition of enzyme activity. (2) LP-X levels remained constant even up to 36 h incubation, despite active cholesterol esterification in the presence of LP-X concentrations less than 2.5 mg/ml. In addition, the specific activity of radiolabelled LP-X, and its electrophoretic properties remained unchanged after incubation showing that the molecule remained intact. (3) Low density lipoproteins other than LP-X stimulated the enzymes activity, but this effect was overcome by LP-X. (4) Additional concentrations of high density lipoproteins also produced enhancement of enzyme activity, but at higher levels inhibition was seen. LP-X prevented the enhancement of lecithin:cholesterol acyltransferase activity. (5) Small samples of pure LP-X, obtained with the minimum of physical manipulation, showed a complete absence of cholesterol ester and triacylglycerol from the molecule. The implications of these results are discussed, particularly in relation to other reports which have presented evidence that LP-X is a substrate for lecithin:cholesterol acyltransferase.     

Chemical Identification and Ethological Function of Soldier-Specific Secretion in Japanese Subterranean Termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70–80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier’s frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

Chemical identification and ethological function of soldier-specific secretion in Japanese subterranean termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70-80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier's frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

Interaction of apolipoprotein A-I with lecithin-cholesterol vesicles in the presence of phospholipase C

Here we study the anti-nucleating mechanism of apolipoprotein A-I (apo A-I) on model biliary vesicles in the presence of phospholipase C (PLC) utilizing dynamic light scattering (DLS), steady-state fluorescence spectroscopy, cryogenic transmission electron microscopy (cryo-TEM), and UV/Vis spectroscopy. PLC induces aggregation of cholesterol-free lecithin vesicles from an initial, average size of 100 nm to a maximal size of 600 nm. The presence of apo A-I likely inhibits vesicle aggregation by shielding the PLC-generated hydrophobic moieties, which results in vesicles of an average size of 200 nm. A similar phenomenon is observed in cholesterol-enriched lecithin vesicles. Whereas PLC alone produces aggregates of 300 nm, no aggregation is observed when apo A-I is present along with PLC. However, the ability of apo A-I to inhibit aggregation is temporary, and after 8 h, a broad particle size distribution with sizes as high as 800 nm is observed. Apo A-I possibly induces the formation of small apo A-I/lecithin/cholesterol complexes of about 5-20 nm similar to the discoidal pre-HDL complexes found in blood when it can no longer effectively shield all the DAG molecules. Concomitant with formation of complexes, DAG molecules coalesce into large oil droplets, which account for the large particles observed by light scattering. Thus, apo A-I acts as an anti-nucleating agent by two mechanisms, anti-aggregation and microstructural transition. The mode of protection is dependent on the cholesterol content and the relative amounts of DAG and apo A-I present. This study supports the possibility of apo A-I solubilizing lipids in bile in a similar fashion as it does in blood and also delineates the mechanism of formation of the complexes.     

Activity of Zn and Mg phthalocyanines and porphyrazines in amyloid aggregation of insulin

Formation of the deposits of protein aggregates—amyloid fibrils in an intracellular and intercellular space—is common to a large group of amyloid‐associated disorders. Among the approaches to develop of therapy of such disorders is the use of agents preventing protein fibrillization. Polyaromatic complexes—porphyrins and phthalocyanines—are known as compounds possessing anti‐fibrillogenic activity. Here, we explore the impact of related macrocyclic complexes—phthalocyanines (Pc) and octaphenyl porphyrazines (Pz) of Mg and Zn—on aggregation of amyloidogenic protein insulin. Pz complexes are firstly reported as compounds able to affect protein fibrillization. The effect of Pc and Pz complexes on the kinetics and intensity of insulin aggregation was studied by the fluorescent assay using amyloid sensitive cyanine dye. This has shown the impact of metal ion on the anti‐fibrillogenic properties of macrocyclic complexes—the effect on the fibrillization kinetics of Mg‐containing compounds is much more pronounced comparing to that of Zn analogues. Scanning electron microscopy experiments have demonstrated that filamentous fibrils are the main product of aggregation both for free insulin and in the presence of macrocyclic complexes. However, those fibrils are distinct by their length and proneness to lateral aggregation. The Pc complexes cause the increase in variation of fibrils length 0.9 to 2.7 nm in opposite to 1.4 to 2.0 nm for free insulin, whereas Pz complexes cause certain shortening of the fibrils to 0.8 to 1.6 nm. The averaged size of the fibrils population was estimated by dynamic light scattering; it correlates with the size of single fibrils detected by scanning electron microscopy.     

Effect of blood components on the stability of liposomes

The human blood plasma is studied for its effect on the outlet of different substances from lecithin liposomes. It is shown that in the presence of the whole blood the permeability of liposomal membrane for the low-molecular weight compounds increases sharply. Blood plasma proteins play a key role in this process but the influence of albumin, gamma-globulins, fibrinogen as well as of serum lipases is rather insignificant. An increase in the rate of outlet of substances in the presence of blood plasma depends on their molecular weight and may be explained by the formation of dynamic defects or pores of definite size in the liposomal membrane. The formation of these defects (pores) is supposed to proceed due to insertion of blood plasma proteins into the phospholipid matrix of liposomes.     

Interaction of model discoidal complexes of phosphatidylcholine and apolipoprotein A-I with plasma components. Physical and chemical properties of the transformed complexes

Conversion of model discoidal complexes of egg yolk phosphatidylcholine and apolipoprotein A-I, upon interaction with a source of lecithin:cholesterol acyltransferase (plasma d greater than or equal to 1.21 g/ml fraction or partially purified enzyme) and with different sources of substrate unesterified cholesterol (LDL, VLDL or cholesterol incorporated into complexes), was investigated by gradient gel electrophoresis, gel filtration, equilibrium density gradient ultracentrifugation, electron microscopy and chemical analysis. When the incubation mixture contained an inhibitor of lecithin:cholesterol acyltransferase, discoidal complexes with mean long dimension of approximately 10.5 +/- 1.9 nm were converted (within 1 h) predominantly to small round particles and were partially depleted of their phospholipid content. Upon electrophoresis the small particles showed peak maxima within the migration intervals of the human plasma ( HDL3b ) gge and ( HDL3c ) gge subpopulations with associated particle size ranges of 7.8-8.2 and 7.2-7.8 nm, respectively. Within 1 h, in the presence of activated enzyme, the complexes were again converted in major part to the small particles. However, further incubation resulted in an apparent single-step conversion to a larger major product with peak maximum occurring within the migration intervals of the ( HDL2a ) gge and the ( HDL3a ) gge subpopulations (particle size ranges 8.8-9.8 and 8.2-8.8 nm, respectively). Formation of an apolar core was indicated by detection of cholesteryl esters in the conversion product. The form in which the substrate unesterified cholesterol was introduced did not markedly influence the size properties of the final conversion product. With VLDL as source of substrate, considerable incorporation of triacylglycerol occurred in company with a lower level of cholesteryl esters, suggesting transfer of these lipids during formation of the apolar core. Incubation of complexes with a partially purified (3000-fold) preparation of lecithin:cholesterol acyltransferase yielded a product similar in properties to that when the d greater than or equal to 1.21 g/ml fraction was used. Our model discoidal complexes and their conversion products exhibit properties very similar to those of potential precursors to HDL as well as of mature HDL particles. Their further investigation shows promise of providing detailed insight into the possible origin and heterogeneity of human plasma HDL.     

Aggregated human gamma-globulin-induced proliferation and polyclonal activation of murine B lymphocytes

Murine splenic B lymphocytes are stimulated to proliferate and undergo polyclonal activation in the presence of heat-aggregated human gamma-globulin (AHGG). Splenic macrophages are required to generate the proliferative and polyclonal antibody responses. The polyclonal response as opposed to the proliferative response is dependent upon the presence of T lymphocytes. The stimulatory molecule responsible for the AHGG-induced proliferative and polyclonal activation is derived from incubation of AHGG with splenic macrophages. The biologically active moiety is derived from the Fc portion of the HGG molecule and is approximately 14,000 m.w. The method of aggregation is critical for the generation of stimulatory HGG preparations. Heat aggregation is the only method produced a biologically active preparation     

Characterization of bioactive glycolipids from Scytonema julianum (cyanobacteria)

Cyanobacteria serve as a rich source of novel bioactive metabolites. Few studies on glycolipids reported them as having specific biological activities. In this study, total lipids of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral and phospho- and glycolipids, and the latter were further fractionated by high-performance liquid chromatography (HPLC). Each glycolipid fraction was tested in vitro for its ability to inhibit platelet-activating factor (PAF)- and thrombin-induced washed rabbit platelet aggregation and/or to cause platelet aggregation. The structures of the most active fractions were elucidated by biological assays, by chemical determinations and identified by electrospray mass spectrometry. One fraction was a potent inhibitor of PAF-induced platelet aggregation. Structural studies of this fraction indicated the existence of a phosphoglyco-analog of acyl-sphingosine. Two fractions causing platelet aggregation were detected and identified as phosphoglycolipids. The first one was identified as a phosphoglyco-analog of acyl-acetylated sphingosine and the second one as a glyco-analog of phosphatidylglycerol. The presence of the above bioactive compounds demonstrates new types of lipids in cyanobacteria in regard to the structure and biological activity. In addition, the identified bioactive lipids may contribute to the allergic character of cyanobacteria.     

Enzymatic synthesis and 3-D structure of anti-proliferative acidic (MeGlcUA) xylotetrasaccharide

Anomerically free acidic xylo-oligosaccharides have shown interesting biological properties when tested against Gram-positive and Gram-negative aerobically grown bacteria, as well as against Helicobacter pylori, sarcoma-180 and other tumors. We report here a structure–activity relationship study on the role of 4-O-methyl glucuronic acid (MeGlcUA) in regulating aggregation of β-polyxylosides of (9H-fluoren-9-yl)- methanol obtained via the action of Thermotoga neapolitana xylanase. Neutral compounds from mono- to penta-β-1,4 xylosides were obtained from this biocatalyzed reaction. In addition, acidic components among products, carrying an α-1,2 4-O-methyl glucuronic acid (MeGlcUA) were also isolated. An anti-proliferative test of these compounds on human epithelial EFO 27 ovarian cancer cells indicated that the presence of MeGlcUA modulates their biological activity, while its absence induces molecular aggregation. The three-dimensional structure of the most active MeGlcUA β-polyxyloside was investigated by resorting to NOESY experiments supported by dynamic force-field calculations with/without constraints. The 3D structure is characterized by all sugars possessing a ⁴C₁ chair conformation. The MeGlcUA moiety, and the external and middle xyloses adopt a hairpin-shaped conformation, generating a non-planar arrangement of the molecule with the aromatic ring folding back toward the carbohydrate chain. Such a non-planar conformation may justify the lack of aggregation.     

Cl- transport in apical plasma membrane vesicles isolated from bovine tracheal epithelium

Gradually altered synthetic entities were employed as molecular probes, and arachidonic acid, ADP, human alpha-thrombin and the Ca2+ ionophore A23187 as aggregation-inducing agents, in a comprehensive study on the response profile of human blood platelets with an emphasis on the effects of exogenous and increased intracellular Ca2+. Corroborating further previous conclusions, some representative carbamoylpiperidine derivatives, at concentrations effecting substantial inhibition of ADP-induced aggregation, failed to retain that effect when 5.0 mM Ca2+ was introduced into the otherwise identical test medium; reference compounds chlorpromazine and propranolol registered corresponding inhibitory patterns. At increased concentrations the compounds' inhibitory potency was regenerated even in the presence of 5 mM Ca2+. In fact, in sufficiently high concentrations, the compounds were even capable of inhibiting aggregation elicited by 15 microM of the ionophore A23187; so did chlorpromazine and propranolol. Another set of congeners revealed the striking sensitivity of ionophore A23187-induced human blood platelet aggregation to the surface active potencies of inhibitor molecules. The loss in inhibitory potency was directly related to the lesser hydrophobic character of the molecule.     

Structural differences between sensitive and resistant L1210 cells

The main structural differences between sensitive L1210 mouse leukaemic cells and their multidrug resistant counterpart, obtained by adaptation of the parental cell line to vincristine (VCR), concern the size and shape of the cells, their surface properties and changes in organelles involved in proteosynthesis and transport of substances. The resistant cells are larger with higher density of microvilli. In light and electron micrographs containing a group of cells, cells were found to be closer to each other in L1210/VCR cells than in L1210 cells. This difference in cell aggregation suggests different surface properties which could be visualised by decreased staining of L1210/VCR cell surface coat (glycocalyx) with a polycationic dye ruthenium red. A decrease in surface to volume ratio as a consequence of increased cell size in resistant cells is compensated by proliferation of villi and cytoplasmic protrusions of the cell surface. L1210/VCR cells were further distinguished by higher amount of euchromatin, increase in density of rough endoplasmic reticulum, more developed Golgi apparatus and aggregation of free ribosomes into tetrameric and pentameric polyribosomes. These structural changes may be interpreted as a sign of increase in proteosynthesis and transport of substances.     

Isolation and identification of hydroxyl-platelet-activating factor from natural sources

Platelet-activating factor (PAF), a potent inflammatory mediator that has previously been detected in elevated levels in inflamed gingival tissues, in gingival crevicular fluid (GCF) and in saliva, is implicated in periodontal disease. The biologically active phospholipid detected in gingival crevicular fluid is a hydroxyl-PAF analogue. In a preliminary study this bioactive molecule was detected for the first time in human blood derived from volunteers with chronic periodontitis as well as from periodontally healthy volunteers. Compounds isolated from natural sources as well as synthetic ones have been reported as biologically active lipids with physiological importance based on the fact that they induce platelet aggregation with EC50 values ranging from 100 to 0.01 microM through interaction with G-protein-coupled receptors like the PAF receptor, leading to altered signal transduction. In this study, the existence of hydroxyl-PAF analogue in human blood was further studied as well as its distribution in plasma and in blood components. The existence of hydroxyl-PAF analogue was also investigated in samples from rabbit blood hen's egg yolk. The hydroxyl-PAF analogue was purified by high-performance liquid chromatography, detected by biological assays and identified by electrospray MS analysis. Quantitative determination of PAF and hydroxyl-PAF analogue (expressed as PAF-like activity) showed a statistically significant increase in the ratio of plasma hydroxyl-PAF analogue levels to plasma PAF levels in volunteers with periodontitis. Moreover, hydroxyl-PAF analogue was also detected in rabbit blood and hen's egg yolk samples. These data support that this bioactive lipid may play a role in oral inflammation and suggest PAF as a member of a lipid molecule family with different structures and from different sources which share the same or similar biological activities, apparently with different physiological roles in human and animals.     

Resistance of liposomes with different physico-chemical characteristics to the effect of plasma

The chemical composition, liquid content sign and value of charge as well as structure and size of lipid vesicles are studied for the effect they exert on the liposome permeability for 22Na+ in the presence of human blood plasma. The rate of the isotope outlet from the electroneutral lecithin liposomes is determined by the size of vesicles and the quantity of phospholipid bilayers in their membrane. The presence either of a negative or a positive charge on the surface of the liposome membrane has no essential effect on the outlet rate of the radioactive marker. Introduction of different amounts of cholesterol or sphingomyelin into the liposome composition decreases considerably the lipid vesicle permeability and an increase in the liquid content of their membranes due to the temperature elevation is accompanied by a sharp rise in the isotope outlet rate. A conclusion is drawn on the possibility to control the outlet rate of the liposome content in the presence of blood plasma.     

Antigenic properties and molecular weights of murine leukemia virus-binding proteins.

A murine T lymphoma cell line, WEHI-22, has been studied for the presence of murine leukemia virus-binding proteins and for the presence of cell surface molecules that share antigens with mouse immunoglobulins. With surface radioiodination, detergent disruption, and immunoprecipitation, a 60 to 70,000-dalton molecule has been described that is recognized by chicken anti-mouse immunoglobulin serum. In competition experiments this molecule cross-reacts with highly purified mouse IgM myeloma proteins. A cell surface molecule of similar size can be shown to bind to mouse leukemia viruses. Pre-precipitation of the WEHI-22 cell surface material with chicken anti-mouse immunoglobulin removes the material binding to leukemia viruses.