Water-soluble Hexasulfobutyl[60]fullerene inhibit low-density lipoprotein oxidation in aqueous and lipophilic phases

Oxidative modification of low-density lipoprotein (LDL) plays a pivotal role in the pathogenesis of atherosclerosis. Increasing the resistance of LDL to oxidation may therefore mitigate, or even prevent, atherosclerosis. A new water-soluble C60 derivative, hexasulfobutyl[60]fullerene [C60 - (CH2CH2CH2CH2-SO3Na)6; FC4S], consisting of 6 sulfobutyl moieties covalently bound onto the C60 cage is a potent free radical scavenger. This study explored the antioxidative effect of sulfobutylated fullerene derivatives (FC4S) on LDL oxidation. FC4S was found to be effective in protecting LDL against oxidation induced by either Cu2+ or azo peroxyl radicals generated initially in the aqueous or lipophilic phase, respectively. Levels of the oxidative products, conjugated diene and thiobarbituric acid-reactive substances, and the relative electrophoresis mobility of the LDL were decreased. The addition of 20 microM FC4S at the early stage of oxidation increased the kinetic lag time from 69 +/- 11 to 14 +/- 10 min (P < 0.05) and decreased the propagation rate from 17.1 +/- 2.6 to 6.3 +/- 1.0 mOD/min (P < 0. 005). Persistent suppression of peroxidation reaction was observed upon further addition of FC4S after full consumption of all endogenous antioxidants during the propagation period. Intravenous injection of hypercholesterolemic rabbits with FC4S (1 mg/kg/day) efficiently decreased atheroma formation. Data substantiate the use of FC4S as an excellent hydrophilic antioxidant in protecting atheroma formation, via removing free radicals, in either aqueous or lipophilic phase.     

Extraction of rifamycin B from native and aqueous solutions]

Optimal conditions for extraction of rifamycin B from aqueous solutions and fermentation broth filtrates at pH values within 2.0-7.0 were determined. When the antibiotic was extracted from the aqueous solutions, the highest yield was obtained at pH 2.0. When the antibiotic was extracted from the fermentation broth filtrates, it was found that chloroform was the most selective solvent with respect to rifamycin B, the chloroform selectivity being increased at pH 3.5-4.0. It was shown that rifamicin B passed from the buffer solutions with a concentration of 3-20 mg/ml to chloroform in amounts of 6-7 mg/ml and to ethylacetate and butanol in amounts of 20 mg/ml. Such conditions of chloroform and butanol (9 : 1) increased the rifamycin B contents in the extract up to 40 mg/ml.     

Bioanalytical liquid chromatography tandem mass spectrometry methods on underivatized silica columns with aqueous/organic mobile phases

This review article summarizes the recent progress on bioanalytical LC-MS/MS methods using underivatized silica columns and aqueous/organic mobile phases. Various types of polar analytes were extracted by using protein precipitation (PP), liquid/liquid extraction (LLE) or solid-phase extraction (SPE) and were then analyzed using LC-MS/MS on the silica columns. Use of silica columns and aqueous/organic mobile phases could significantly enhance LC-MS/MS method sensitivity, due to the high organic content in the mobile phase. Thanks to the very low backpressure generated from the silica column with low aqueous/high organic mobile phases, LC-MS/MS methods at high flow rates are feasible, resulting in significant timesaving. Because organic solvents have weaker eluting strength than water, direct injection of the organic solvent extracts from the reversed-phase solid-phase extraction onto the silica column was possible. Gradient elution on the silica columns using aqueous/organic mobile phases was also demonstrated. Contrary to what is commonly perceived, the silica column demonstrated superior column stability. This technology can be a valuable supplement to the reversed-phase LC-MS/MS.     

Rifamycin B production by Nocardia mediterranei immobilized in a dual hollow fibre bioreactor

Continuous production of rifamycin B was studied using Nocardia mediterranei (ATCC 21789) immobilized in a dual hollow fibre bioreactor designed for cultivating aerobic cells. In the reactor operation the volumetric productivity based on the volume occupied by the immobilized cells was 108 mg l⁻¹ h⁻¹ when air was used for aeration and was 143 mg l⁻¹ h⁻¹ with pure oxygen. These corresponded to 22 and 30-fold increases over the productivity of the comparable batch system. These high productivities were due to the high cell mass density of 550 g l⁻¹. However, the specific productivity of the cell was 30–40% of that in the shake flask culture. As the residence time of medium in the reactor increased, pH of effluent rose to an alkaline region that was outside its optimum condition (pH 6.5–7.0) and the yield and productivity decreased.     

Iodination of insulin in aqueous and organic solvents

1. The iodination of insulin was studied under various experimental conditions in aqueous media and in some organic solvents, by measuring separately the uptake of iodine by the four tyrosyl groups and the relative amounts of monoiodotyrosine and di-iodotyrosine that are formed. In aqueous media from pH1 to pH9 the iodination occurs predominantly on the tyrosyl groups of the A chain. Some organic solvents increase the iodine uptake of the B-chain tyrosyl groups. Their efficacy in promoting iodination of Tyr-B-16 and Tyr-B-26 is in the order: ethylene glycol and propylene glycol approximately methanol and ethanol>dioxan>8m-urea. 2. It is suggested that each of the four tyrosyl groups in insulin has a different environment: Tyr-A-14 is fully exposed to the solvent; Tyr-A-19 is sterically influenced by the environmental structure, possibly by the vicinity of a disulphide interchain bond; Tyr-B-16 is embedded into a non-polar area whose stability is virtually independent of the molecular conformation; Tyr-B-26 is probably in a situation similar to Tyr-B-16 with the difference that its non-polar environment depends on the preservation of the native structure.     

Lipase-catalyzed cellulose acetylation in aqueous and organic media

Screening for lipases capable of catalyzing acetylation of cellulosic substrates was conducted in aqueous buffer solution using water-soluble carboxymethyl cellulose (CMC) as substrate. Lipase A12 from Aspergillus niger (A. niger) showed the most promising acetylation activity among 11 tested commercial microbial lipases and was further applied to catalyzing acetylation of solid cellulose in aqueous solution. This reaction was shown to be feasible with an acetylation extent of 0.16 wt % achieved compared with no detectable acetylation in the absence of enzyme. Pretreatments on cellulose substrate by ultrasonic irradiation and surfactant solution only slightly improved the acetylation extent by 44 and 27%, respectively. Alternatively, this lipase-catalyzed acetylation was remarkably improved with solubilized cellulose as substrate in the dimethyl sulfoxide/paraformaldehyde solvent system, with an acetylation extent (7.87 wt %) nearly 50 times higher than that achieved in aqueous solution. This improvement was attributed to (1) the absence of bulk water and the increase in substrate solubility by the transition of reaction media from aqueous solution to organic solvents and (2) the ability of lipase A12 to remain catalytically active in highly polar DMSO. This discovery that the A. niger lipase was capable of surviving its contact with polar solvents was further confirmed by its considerably preserved catalytic activity on CMC acetylation in aqueous media after enzyme pretreatments with organic solvents of various polarities and in mixture media with the aqueous phase partially replaced by organic solvents.     

Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases

Human peripheral blood monocyte-enriched fractions (identified by staining for peroxidase and by sizing) were obtained by velocity sedimentation at unit gravity of peripheral blood mononuclear cells. They were then fractionated by countercurrent distribution (a multiple-extraction procedure) in a charged Dextran/poly(ethylene glycol) aqueous phase system. The monocytes remained viable after the separation (order of 90%). Cells obtained from different cavities along the extraction train were tested for their ability to phagocytize latex particles. With increasing partition coefficient (presumably higher charge-associated membrane properties) the ratio of monocytes that phagocytized to monocytes that did not phagocytize increased appreciably. When, however, monocytes were permitted to phagocytize particles prior to countercurrent distribution, an increase in partition coefficient was associated with an appreciable decrease in the above-specified ratio. Control experiments indicate that the observed change in partitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitiartitioning behavior cannot be ascribed to an alteration in size and/or density of the monocytes as a function of phagocytosis. It may be due to the internalization of charged surface groups during phagocytosis. We conclude that there is a correlation between the surface properties of monocytes (as reflected by partitioning) and their ability to ingest particles. Furthermore, an alteration in the surface charge-associated properties of monocytes as a consequence of phagocytosis is indicated by the cells' reduced partition coefficient.     

The distribution of phosphorus between soluble and particulate phases for seven Scottish East Coast rivers

Phosphorus (P) fractions were quantified in water samples collected on four occasions from sites at the lower tidal limit of seven Scottish East Coast rivers. Individual catchment characteristics ranged from those dominated by semi-natural land use to those where agriculture predominated. Together the rivers displayed attributes ranging from nearly pristine to those impacted by point and diffuse sources of pollutants. Sampling times were chosen to coincide with periods of low river flows where conditions should result in low concentrations of suspended particulate matter (SPM) but favourable for phytoplankton growth. Total phosphorus (TP) concentrations were < 0.004 mg l^–1, 0.005–0.048 mg l^–1 and 0.28–2.2 mg l^–1 for pristine, agricultural and point source impacted rivers respectively. Soluble reactive phosphorus (SRP) represented from < 5% to > 90% of TP and dissolved P dominated all samples. The total phosphorus content (TPC) of SMP ranged from 0.1 to 1.1% and was significantly related to SRP. Organic matter was a significant component of SPM and organically bound phosphorus was the dominant form of particulate P. The C/P ratio of organic matter was wide, between 500–1200 for the more pristine systems which narrowed to < 400 for heavily impacted rivers. Exchangeable P increased during the summer but was generally a minor component of TP and therefore likely to be a significant source of SRP only in pristine rivers. Phytoplankton constituted between 5 and 46% of organic matter and concentrations of chlorophyll-a were significantly correlated with both TP and SRP.     

Photolysis of formylmethylflavin in aqueous and organic solvents.

The photolysis of formylmethylflavin (FMF), a major intermediate in the photodegradation sequence of riboflavin, has been carried out in water (pH 7.0) and in several organic solvents. FMF produces lumichrome (LC) in organic solvents and LC and lumiflavin (LF) in aqueous solution. FMF and its photoproducts have been analysed using a specific multicomponent spectrophotometric method. FMF undergoes a bimolecular redox reaction on photolysis. The second-order rate constants for the reaction range from 0.66 (chloroform) to 2.44 M(-1) s(-1) (water) and are a linear function of the solvent dielectric constant. A plot of ln k against 1/epsilon is linear for the reactions in 1-butanol, 1-propanol, ethanol, methanol, acetonitrile and water (epsilon approximately 17-79) and non-linear in chloroform and dichloroethane (epsilon approximately 5-10) suggesting a change in reaction mechanism in the two regions. This may be explained on the basis of the existence of a dipolar intermediate along the reaction pathway. The rate of photolysis is governed by the solvation of the intermediate and is thus influenced by the dielectric constant of the medium. The solvent effect on the rate of photolysis of FMF has been expressed in terms of the solvent acceptor number. A linear relationship has been found between ln k and the solvent acceptor number.     

New ordered metastable phases between the gel and subgel phases in hydrated phospholipids

Formation of low-temperature ordered gel phases in several fully hydrated phosphatidylethanolamines (PEs) and phosphatidylcholines (PCs) with saturated chains as well as in dipalmitoylphosphatidylglycerol (DPPG) was observed by synchrotron x-ray diffraction, microcalorimetry, and densitometry. The diffraction patterns recorded during slow cooling show that the gel-phase chain reflection cooperatively splits into two reflections, signaling a transformation of the usual gel phase into a more ordered phase, with an orthorhombic chain packing (the Y-transition). This transition is associated with a small decrease (2-4 microl/g) or inflection of the partial specific volume. It is fully reversible with the temperature and displays in heating direction as a small (0.1-0.7 kcal/mol) endothermic event. We recorded a Y-transition in distearoyl PE, dipalmitoyl PE (DPPE), mono and dimethylated DPPE, distearoyl PC, dipalmitoyl PC, diC(15)PC, and DPPG. No such transition exists in dimyristoyl PE and dilauroyl PE where the gel L(beta) phase transforms directly into subgel L(c) phase, as well as in the unsaturated dielaidoyl PE. The PE and PC low-temperature phases denoted L(R1) and SGII, respectively, have different hydrocarbon chain packing. The SGII phase is with tilted chains, arranged in an orthorhombic lattice of two-nearest-neighbor type. Except for the PCs, it was also registered in ionized DPPG. In the L(R1) phase, the chains are perpendicular to the bilayer plane and arranged in an orthorhombic lattice of four-nearest-neighbor type. It was observed in PEs and in protonated DPPG. The L(R1) and SGII phases are metastable phases, which may only be formed by cooling the respective gel L(beta) and L(beta') phases, and not by heating the subgel L(c) phase. Whenever present, they appear to represent an indispensable intermediate step in the formation of the latter phase.     

The vital activities of enterobacteria in an aqueous medium in the absence of sources of organic nutrition]

Heterotrophic bacteria were found to be capable of proliferation in physiological saline and distilled water. In 1988-1989 experimental studies were made with a view to establish the role of the gaseous phase of atmospheric air and the products of the autolysis of dead bacteria as the sources of organic nutrition. The studies revealed that the complete removal of atmospheric air from vials with bacterial suspension completely stopped the stimulation of reproduction. In vials with a higher concentration of dead bacterial bodies the proliferation rate was 2- to 400-fold (on the average, 118-fold) higher. The products of the autolysis of bacterial bodies proved to be of no importance as an independent source of organic nutrition for heterotrophic bacteria. The mechanism of the assimilation of autolysis products is "switched on" by biologically active geomagnetic disturbances. The mechanisms of the increase of bacterial biomass remain unclear.     

The activity of PEG-modified chymotrypsin in aqueous and organic media

Summary Polyethylene glycol-modified chymotrypsin was prepared by activation of monomethoxypolyethylene glycols with phenylchloroformates and subsequent coupling with free enzyme. The modification was correlated with enzyme activity in aqueous solution and in organic solvents. The activity in aqueous solution was high (80% of that of non-modified enzyme), even at high degrees of modification. The modified protease was soluble in benzene and DMF and catalyzed transesterification in cyclohexane.     

Modeling pore size distribution in cellulose rolled stationary phases

Rolled stationary phases are fabrics (i.e., nonparticulate phases) that rapidly separate proteins from salts on the basis of size exclusion. Pore size and pore size distributions in the stationary phase determine how different size molecules distribute between the stationary and mobile phases in liquid chromatography columns. The potential for size exclusion chromatography by fabrics is not initially obvious because their interlaced structures are atypical for size exclusion supports. A simple logistic model fits the pore size distribution of a rolled stationary phase when pore sizes were measured using PEG, Dextran, D2O, glucose, and NaCl probes. When the fabric is treated with cellulase enzymes, the water-accessible pores uniformly decrease and peak retention is lower. The logistic function model captures this result and enables comparison of pore size distribution curves between enzyme-treated and untreated fabrics in rolled stationary phase columns.