The evaluation of the oxidative state of native-LDL: three methods compared

LDL-oxidation is considered a contributing factor to the development of atherosclerotic lesions. However, to utilise the oxidative state of LDL as a marker of cardiovascular risk, reliable analytical methods for its detection must be defined. We have compared three methods for their capacity to evaluate the difference in the oxidation state of isolated LDL subjected to either dialysis (D-LDL) or gel filtration (F-LDL) to remove EDTA. Their susceptibility to oxidation promoted by Cu(2+) was monitored by following the time course of conjugated diene (CD) and lipid hydroperoxide (ROOH) accumulation. The relative electrophoretic mobility (REM) of the same LDL samples was evaluated by capillary electrophoresis. As measured by all three methods, F-LDL are less prone to oxidation than D-LDL when added with CuSO(4). REM of F-LDL and D-LDL significantly differs already before the addition of the metal catalyst, whereas CD and ROOH contents become significantly different only after it. Besides confirming that a rapid centrifugation followed by gel filtration is a more convenient procedure than dialysis to remove EDTA during LDL isolation, our study suggests the REM of isolated-LDL as the biochemical marker of choice in the evaluation of its oxidative state.     

The surface roughness of lactose particles can be modulated by wet-smoothing using a high-shear mixer

The purpose of this research was to encapsulate superoxide dismutase (SOD) and catalase (CAT) in biodegradable microspheres (MS) to obtain suitable sustained protein delivery. A modified water/oil/water double emulsion method was used for poly(D,L-lactide-co-glycolide) (PLGA) and poly(D,L-lactide) PLA MS preparation co-encapsulating mannitol, trehalose, and PEG400 for protein stabilization. Size, morphology, porosity, mass loss, mass balance, in vitro release and in vitro activity were assessed by using BCA protein assay, scanning electron microscopy, BET surface area, and particle-sizing techniques. In vitro activity retention within MS was evaluated by nicotinammide adenine dinucleotide oxidation and H₂O₂ consumption assays. SOD encapsulation efficiency resulted in 30% to 34% for PLAMS and up to 51% for PLGA MS, whereas CAT encapsulation was 34% and 45% for PLGA and PLAMS, respectively. All MS were spherical with a smooth surface and low porosity. Particle mean diameters ranged from 10 to 17 μm. CAT release was prolonged, but the results were incomplete for both PLA and PLGA MS, whereas SOD was completely released from PLGA MS in a sustained manner after 2 months. CAT results were less stable and showed a stronger interaction than SOD with the polymers. Mass loss and mass balance correlated well with the release profiles. SOD and CAT in vitro activity was preserved in all the preparations, and SOD was better stabilized in PLGA MS. PLGA MS can be useful for SOD delivery in its native form and is promising as a new depot system.     

Methylenetetrahydrofolate reductase gene C677T polymorphism,homocysteine, vitamin B12, and DNA damage in coronary artery disease

Elevated levels of plasma homocysteine (Hcy), a risk factor for coronary artery disease (CAD), can result from genetic errors, e.g., the methylenetetrahydrofolate reductase (MTHFR) polymorphism, or nutritional deficiencies, e.g., in vitamin B12 and folate. The mechanism by which Hcy induces atherosclerosis is not fully understood. Recently, Hcy has also been observed to induce DNA damage. In this study, we have investigated whether DNA damage is related to the C677T variant in the MTHFR gene and to plasma levels of Hcy, B12, and folate in patients with CAD. Patients ( n=46) with angiographically proven CAD were studied by using the micronucleus (MN) test, an accepted method for evaluating genetic instability. TT patients had plasma Hcy levels higher than those with the CT or CC genotypes (27.8+/-5.2 vs 13.7+/-2.2 and 12.9+/-1.9 micro mol/l, respectively; P=0.02). Patients with multi-vessel disease had higher plasma Hcy levels (11.6+/-1.2, 22.0+/-4.7, 19.3+/-3.9 micromol/l for one-, two- and three-vessel disease, respectively; P=0.05). The MN index increased with the number of affected vessels (8.4+/-0.7, 11.1+/-2.0, 14.2+/-1.7 for one-, two-, and three-vessels disease, respectively; P=0.02) and was significantly higher in subjects with the TT genotype compared with the CC or CT genotypes (15.7+/-2.4 vs 8.9+/-1.7 and 9.9+/-0.8; P=0.02). The MN index was also correlated negatively with plasma B12 concentration ( r=-0.343; P=0.019) and positively with plasma Hcy ( r=0.429, P=0.005). These data indicate that the MN index is associated with the severity of CAD and is related to the MTHFR polymorphism, suggesting an interesting link between coronary atherosclerosis and genetic instability in humans.     

Local Differences in GABA Release Induced by Excitatory Amino Acids During Retina Development: Selective Activation of NMDA Receptors by Aspartate in the Inner Retina

Glutamate and GABA are the major excitatory and inhibitory neurotransmitters in the CNS. In the retina, it has been shown that glutamate and aspartate and their agonists kainate and NMDA promote the release of GABA. In the chick retina, at embryonic day 14 (E14), glutamate and kainate were able to induce the release of GABA from amacrine and horizontal cells as detected by GABA-immunoreactivity. NMDA also induced GABA release restricted to amacrine cell population and its projections to the inner plexiform layer (E14 and E18). Although aspartate reduced GABA immunoreactivity, specifically in amacrine cells of E18 retinas, it was not efficient to promote GABA release from retinas at E14. As observed in differentiated retinas, dopamine inhibited the GABA release promoted by NMDA and aspartate but not by kainate. Our data show that different retinal sites respond to distinct EAAs via different receptor systems.     

Glutamate 59 is critical for transport function of the amino acid cotransporter KAAT1

KAAT1 is a neutral amino acid transporter activated by K⁺ or by Na⁺ (9). The protein shows significant homology with members of the Na⁺/Cl^–-dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20–30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of V_max and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of Na⁺ or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta     

Protein kinase B activation by reactive oxygen species is independent of tyrosine kinase receptor phosphorylation and requires SRC activity

Reactive oxygen species (ROS) participate as second messengers in the mitogenic signal transduction. Most of the experimental data supporting the role of ROS as signaling molecules have been obtained by using H2O2. Exposure of cells to H2O2 rapidly increases tyrosine phosphorylation of tyrosine kinase receptors (TKRs) in the absence of growth factor binding, thus inducing the activation of downstream signaling cascades, like that of protein kinase B (AKT). Another molecule able to induce an increase of intracellular ROS levels is diethylmaleate (DEM), which acts by depleting the ROS scavenger reduced glutathione (GSH). A comparison of the effects exerted by H2O2 and DEM shows that the latter induces redox modifications milder than those generated by H2O2. We also demonstrated that DEM-induced redox modifications are not accompanied by platelet-derived growth factor-receptor (PDGF-R) and epidermal growth factor-receptor Tyr phosphorylation, although they are able to activate ERKs and AKT, with kinetics different from those observed following H2O2 treatment. The activation of these two pathways is not blocked by AG1296, a selective inhibitor of PDGF-R Tyr kinase, thus confirming that the effects of DEM are not mediated by the TKR phosphorylation. On the contrary, PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazole[3,4-d]pyrimidine), an inhibitor of Src kinase, completely prevents DEM- and H2O2-induced AKT activation but has no effect on the pathway of ERKs. Finally, nitration of Tyr residues in PDGF-R is observed in DEM-treated cells, thus suggesting that ROS-induced modifications different from Tyr phosphorylation can occur at the growth factor-receptor level and can be involved in the regulation of signaling pathways.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

Hydrogen-bonding propensities of sphingomyelin in solution and in a bilayer assembly: a molecular dynamics study

Sphingomyelin is enriched within lipid microdomains of the cell membrane termed lipid rafts. These microdomains play a part in regulating a variety of cellular events. Computer simulations of the hydrogen-bonding properties of sphingolipids, believed to be central to the organization of these domains, can delineate the possible molecular interactions that underlie this lipid structure. We have therefore used molecular dynamics simulations to unravel the hydrogen-bonding behavior of palmitoylsphingomyelin (PSM). A series of eight simulations of 3 ns each of a single PSM molecule in water showed that the sphingosine OH and NH groups can form hydrogen bonds with the phosphate oxygens of their own polar head, in agreement with NMR data. Simulations of PSM in a bilayer assembly were carried out for 8 ns with three different force field parameterizations. The major physico-chemical parameters of the simulated bilayer agree with those established experimentally. The sphingosine OH group was mainly involved in intramolecular hydrogen bonds, in contrast to the almost exclusive intermolecular hydrogen bonds formed by the amide NH moiety. During the bilayer simulations the intermolecular hydrogen bonds among lipids formed a dynamic network characterized by the presence of hydrogen-bonded lipid clusters of up to nine PSM molecules.     

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.     

Cytogenetic mapping of five YAC clones to human chromosome region 2q31 --> q32.1 in relation to the FRA2G common fragile site

In this work, five YAC clones have been mapped by fluorescent in situ hybridization (FISH) to human chromosome region 2q31 q32.1 and ordered in relation to each other and to the FRA2G common fragile site. YAC clones that span the fragile site have been identified. Moreover a deleted HOXD 13 gene has been identified on the 942D2 YAC.