Size distribution of plasma lipoproteins

Particle diameters of very low density lipoprotein (VLDL)--29.5, 36.3, 22.0; low density lipoprotein (LDL)--18.4, 19.0, 22.0; high density lipoprotein subclass 2 (HDL2)--8.5, 9.7, 15.0; and high density lipoprotein subclass 3 (HDL3)--7.2, 7.6, 14.4 nm were evaluated by means of flotation velocity (FV), optical mixing (OM), and fluorescent probe (FP) respectively. On the basis of the calculated frictional ratio f/f0 from FV and OM data for VLDL, LDL, HDL2, and HDL3--1.51, 1.07, 1.31, and 1.10, assuming the sphericity lipoprotein particles as an index for structural peculiarity a conclusion is made that in contrast to LDL and HDL3 HDL2 have asymmetrical weight distribution per particle volume. A model is suggested suitable for the structural peculiarity of VLDL established on the data of three independent methods which outlines VLDL as particles consisting of several associated subunits with the mean diameter of about 20 nm.     

Quorum-sensing inhibitory compounds from extremophilic microorganisms isolated from a hypersaline cyanobacterial mat

In this study, extremely halophilic and moderately thermophilic microorganisms from a hypersaline microbial mat were screened for their ability to produce antibacterial, antidiatom, antialgal, and quorum-sensing (QS) inhibitory compounds. Five bacterial strains belonging to the genera Marinobacter and Halomonas and one archaeal strain belonging to the genus Haloterrigena were isolated from a microbial mat. The strains were able to grow at a maximum salinity of 22–25 % and a maximum temperature of 45–60 °C. Hexanes, dichloromethane, and butanol extracts from the strains inhibited the growth of at least one out of nine human pathogens. Only butanol extracts of supernatants of Halomonas sp. SK-1 inhibited growth of the microalga Dunaliella salina. Most extracts from isolates inhibited QS of the acyl homoserine lactone producer and reporter Chromobacterium violaceum CV017. Purification of QS inhibitory dichloromethane extracts of Marinobacter sp. SK-3 resulted in isolation of four related diketopiperazines (DKPs): cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), cyclo(l-Pro-l-isoLeu), and cyclo(l-Pro-d-Phe). QS inhibitory properties of these DKPs were tested using C. violaceum CV017 and Escherichia coli-based QS reporters (pSB401 and pSB1075) deficient in AHL production. Cyclo(l-Pro-l-Phe) and cyclo(l-Pro-l-isoLeu) inhibited QS-dependent production of violacein by C. violaceum CV017. Cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Leu), and cyclo(l-Pro-l-isoLeu) reduced QS-dependent luminescence of the reporter E. coli pSB401 induced by 3-oxo-C6-HSL. Our study demonstrated the ability of halophilic and moderately thermophilic strains from a hypersaline microbial mat to produce biotechnologically relevant compounds that could be used as antifouling agents.     

Dipolar relaxation in a lipid bilayer detected by a fluorescent probe, 4'-dimethylaminochalcone

The dynamic behavior of polar molecules in egg phosphatidylcholine (PC) bilayers has been studied using a membrane fluorescent probe, 4'-dimethylaminochalcone (DMAC). Time and spectrally resolved fluorescence spectroscopy of DMAC incorporated in PC liposomes, as compared to studies of the probe in organic solvents, shows the existence of two independent populations, associated with different extent and speed of dipolar solvent relaxation. The first DMAC population represents approximately 69% of the fluorescence-emitting molecules, has a short fluorescence decay time (0.32 ns) and undergoes Stokes shift of 80 nm. The remaining 31% fraction of DMAC molecules has a decay time of 0.74 ns and undergoes a high (106 nm) Stokes shift. A fraction of the shift, ca. 24 nm for the first and 46 nm for the second population, is attributed to the fast (<0.1 ns) rotational relaxation of nearby dipolar molecules, which might be water. This two-state model accounts well for the detailed fluorescence properties of DMAC in egg PC, i.e. its broadened steady-state spectrum, its average fluorescence quantum yield and its complex wavelength-dependent fluorescence decays.     

Porcine kidney extract contains a specific inhibitor of the ouabain-sensitive alpha2-isoform of Na, K-ATPase present in rat diaphragm fibres

In experiments on isolated rat diaphragm muscle, acetylcholine (100 nmol/l) hyperpolarized muscle fibres due to activation of the alpha 2 isoform of Na,K-ATPase. This hyperpolarization was blocked in a dose-dependent manner by ouabain (K0.5 = 8 +/- 4 nmol/l) as well as by a solution of porcine kidney extract (10 kDa cut-off filtration), with the K0.5 approximately equal to a 1:20,000-fold dilution. The inhibitory activity of the developed slowly over a period of 3 hours and, in contrast to ouabain, was still present after 1 hour of washing. Ouabain, but not the extract, inhibits Rb+ uptake in human erythrocytes that only express the alpha = 1 isoform of Na, K-ATPase. Our data suggest that in rat skeletal muscle the alpha 1 isoform of Na,K-ATPase is primarily responsible for ionic homeostasis, while the alpha 2 isoform provides a "regulatable" function and may be controlled by cholinergic stimulation and/or endogenous digitalis-like factors (EDLFs). Porcine kidney extract contains a factor (M. W. < 10 kDa) that selectively inhibits the rat alpha 2 isoform and differs from ouabain. Our experimental protocol can be used as a highly sensitive physiological assay for factors that selectively inhibit the alpha 2 isoform of Na,K-ATPase.     

Estimation of surface area in very low density human serum lipoproteins

The surface area of very low density lipoproteins (VLDL) from the serum of 15 healthy donors and the surface area of artificial lipid particles have been estimated. The artificial particles were prepared as a mixture of egg phosphatidylcholine and triolein. Two fluorescent probes - energy donor and acceptor - were placed on the surface, and Forster's nonradiative energy transfer was measured; the transfer efficiency is a function of surface area. The fluorescent probe K-68 (4-[5-(phenyloxazolyl-2)-1-pentadecyl)pyridinium) was used as a donor, and DSP-12 (dimethylamino)styryl-N-dodecylpyridinium) was used as an acceptor. The specific surface area of the artificial lipid particles was estimated to be 0.585 +/- 0.015 nm2 per phosphatidylcholine molecule, which is 15% less than in lipid bilayers. The specific area of VLDL particles was 259 +/- 65 m2 per g of total VLDL. This value is close to the specific area of low density lipoproteins (LDL), and corresponds to the area of a spherical particle 10-12 nm in radius. However, VLDL are assumed to be much larger particles as compared with LDL. Therefore, the new data of the VLDL surface area raise a problem of revision of the existing VLDL models.     

Apolipoprotein A-I localization and dipalmitoylphosphatidylcholine dynamics in reconstituted high density lipoproteins

The structure and molecular dynamics of recombinant high density lipoproteins (rHDL) were studied by non-radiative energy transfer (NRET), fluorescence anisotropy and intensity measurements. The rHDL particles contained human plasma apolipoprotein (apo) A-I and dipalmitoylphosphatidylcholine (DPPC). Fluorescent cis- and trans-parinaric acids were used both as probes of molecular motion in the particle lipid phase and as acceptors in the Forster's energy transfer from apo A-I tryptophan residues to determine particle dimensions, apolipoprotein localization and lipid dynamics. The probes are sensitive to thermal wobbling (macromobility) and conformational deformations (micromobility) of phospholipid acyl chains. The experimental data fitted to various models of the particle structure are compatible with the following: (a) at T < Tt the particles appeared as lens-like discs with a radius of the lipid phase of 5 nm and a mean thickness of 4 nm, the value being more by 20% in the particle centre, the alpha-helices of about 1 nm thickness were located around the edge of the lipid core. Compared to liposomes, both macro- and micromobility of DPPC molecules in rHDL were more rapid due to a significant disorder of the boundary lipid molecules close to the apo A-I molecule. This disorder led to the increase of the specific surface area per one lipid molecule, S(o). The lipid phase can be divided into three regions: (i) zone I of the most tightly packed lipid (0-1.7 nm from the disc axis) with a S(o) value small as 0.5 nm2; (ii) intermediate zone II (from 1.7 to 4.0 nm); and (iii) boundary lipid zone III (4-5 nm) of significantly disordered lipid with a S(o) value large as 0.65 nm2. (b) at T> Tt the S(o) heterogeneity disappeared, the radius of the lipid phase did not increase significantly, not exceeding 5.2-5.4 nm, but protein-induced immobilization of lipid molecules which affected about half or more of the total lipid, became remarkable. The overall effect was the suppression of the transition amplitude in rHDL compared to liposomes. The structural inhomogeneity might underlie the function of the native plasma HDL as the key component of the transport and metabolism of plasma lipids.     

Adrenergic Stimulation of Na/K Pump Current in Adult Rat Cardiac Myocytes in Short-term Culture

Passive membrane properties, steady-state Na/K pump current (I _p) and modulation of I _p by adrenergic agonists were studied with patch-clamp techniques in adult rat ventricular myocytes that were freshly isolated or maintained in culture for 1–4 days. Freshly isolated (day 0) myocytes had a 1.7–1.8 times smaller specific membrane resistance compared with that of cells on any day in culture. From day 0 to 4 there was a progressive decrease in cell capacitance (−17.6 ± 0.8 pF/day) without a parallel decline in cell dimensions. The pump current density (1.55 pA/pF) was stable over the 0–4 days in culture. In rod-shaped myocytes norepinephrine (NE) and isoproterenol (ISO) stimulated I _p in a dose-dependent manner, with an apparent affinity of 36 ± 8 and 1.5 ± 0.4 nm, and maximum stimulation of 0.65 ± 0.02 and 0.57 ± 0.02 pA/pF, respectively. Nadolol suppressed this effect, suggesting that it was mediated by β-adrenergic receptor activation. An inverse relationship was found between steady-state I _p and the stimulation of I _p by NE. In contrast to what was shown in guinea pig cardiac myocytes, in rat myocytes isoproterenol stimulation of I _p was not increased by intracellular [Ca] and it did not change the I _p -membrane potential relation. These results show that in adult rat cardiac myocytes NE and ISO are potent stimulators of Na/K pump activity, and this effect may be studied using rat myocytes maintained in short-term culture. Received: 12 November 1997/Revised: 5 March 1998     

On mechanisms of fluorescence quenching by water

Mechanisms of fluorescence quenching of aromatic chromophores by water are reviewed. The mechanisms include polarity of chromophore environment, proton or electron transfer between the excited chromophore and water. A hypothesis is proposed that the quenching can be a result of chromophore-solvent hydrogen bond breaking in the excited state.     

Low-density lipoproteins with different capacity for aggregation

Preparations of low-density lipoproteins from healthy donor blood contain lipoprotein particles with different capacity for aggregation: upon stirring, some particles form aggregates significantly more quick than others. After stirring, lipoprotein particles are separated by ultracentrifugation into two fractions: a fraction of large aggregates and a fraction of small particles without intermediate forms. It is known that lipoprotein aggregates can accelerate intracellular accumulation of lipids. Therefore, it is supposed that particles of high aggregation ability are more atherogenic.