Detection of in vivo lipid peroxidation using the thiobarbituric acid assay for lipid hydroperoxides

Thiobarbituric acid (TBA) assays which have been modified for detection of lipid hydroperoxides appear to be useful for demonstration of in vivo lipid peroxidation. Since these methods require heating tissue membranes with the buffered TBA, there is a possibility of interference from the detection of autoxidation that occurs during heating. These studies were undertaken to investigate conditions which favor TBA color production from hydroperoxide while limiting autoxidation during the assay. An acetic acid-sodium acetate buffered (pH 3.6) TBA assay was used. Heating linoleic acid hydroperoxide with 50 microM ferric iron or under nitrogen nearly doubled color production compared to heating it with no added iron or under air. The lipid antioxidant butylated hydroxytoluene inhibited color production from fatty acid hydroperoxides. When tissue fractions, including liver and lung microsomes and lung whole membranes, were heated in the assay, color production was greater under air than under nitrogen and was much greater under oxygen. When liver microsomes from carbon tetrachloride-exposed rats were used, color was increased only when oxygen was present in the heating atmosphere. The results with tissue fractions appear to demonstrate autoxidation during color development rather than the presence of preformed hydroperoxides. Finally, it was found that color production from membrane fractions was dependent on the vitamin E content of the membranes. It appears that autoxidation during heating should be limited by heating under nitrogen and not by adding antioxidants, which inhibit color production from hydroperoxides. As the vitamin E effect demonstrates, antioxidant status must be considered, since a change in color production could result from a change in antioxidant content without the accumulation of lipid hydroperoxides.     

The problems of eukaryotic and prokaryotic DNA packaging and in vivo conformation posed by superhelix density heterogeneity

Systems for gel electrophoresis in the presence of one of the intercalative unwinding ligands, ethidium or chloroquine, have been developed which permit the resolution of highly supercoiled closed circular DNA molecules differing by unit values of the topological winding number, alpha. All native closed circular DNAs examined, including the viral and intracellular forms of SV40 and polyoma DNA, bacterial plasmid DNAs, and the double stranded closed circular DNA genome of the marine bacteriophage, PM2, are more heterogeneous with respect to the number of superhelical turns present than are the thermal distributions observed in the limit products of the action of nicking-closing (N-C) enzyme on the respective DNAs. In the cases of SV40 and polyoma, where it has been shown that the supercoiling is a combined consequence of the binding of the four nucleosomal histones, H2a, H2b, H3 and H4, and the action of N-C enzyme, the breadth of the distributions within the form I DNAs poses specific problems since the work of other laboratories indicates that the number of nucleosomes on the respective minichromosomes falls within a narrow distribution of 21. If it is assumed that all nucleosomes have identical structures, and that the DNA within a nucleosome is not free to rotate, the native DNA would be anticipated to be less heterogeneous than the thermal equilibrium mixtures present in N-C enzyme relaxed SV40 and polyoma DNAs.The absolute number of superhelical turns (at 37 degrees C in 0.2 M NaCl) in virion polyoma DNA has been determined to be 26 +/- 1, which is the same value obtained for virion SV40 DNA. This is consistent with the observations that polyoma DNA has a higher molecular weight, a lower superhelix density, but the same number of nucleosomes as SV40 DNA. In addition, the distributions within the virion and intracellular form I DNAs of both SV40 and polyoma were found to be indistinguishable.Images     

The governance of human genetics: policy discourse and constructions of public trust

The collection of practices now commonly understood as 'biotechnology' poses a challenge to traditional mechanisms of regulating science and technology, just as it challenges traditional practices of science. The task of regulation is to reconcile the often conflicting political demands of protecting science, economy and the public interest. Public trust is the key measure of political success or failure. The purpose of this paper is to use policy discourse analysis as a vehicle for exploring the politics of the relationship between human genetics governance and public trust. An analysis of 30 policy documents produced six identifiable discourse streams relevant to public trust. These findings will be discussed, and an analysis of their impact on effective governance presented in conclusion.     

Combinations of two capsid regions controlling canine host range determine canine transferrin receptor binding by canine and feline parvoviruses

Feline panleukopenia virus (FPV) and its host range variant, canine parvovirus (CPV), can bind the feline transferrin receptor (TfR), while only CPV binds to the canine TfR. Introducing two CPV-specific changes into FPV (at VP2 residues 93 and 323) endowed that virus with the canine TfR binding property and allowed canine cell infection, although neither change alone altered either property. In CPV the reciprocal changes of VP2 residue 93 or 323 to the FPV sequences individually resulted in modest reductions in infectivity for canine cells. Changing both residues in CPV to the FPV amino acids blocked the canine cell infection, but that virus was still able to bind the canine TfR at low levels. This shows that both CPV-specific changes control canine TfR binding but that binding is not always sufficient to mediate infection.     

The effect of estrogens and phytoestrogenic lignans on macrophage uptake of atherogenic lipoproteins

The present study examined the effect of estrogens and compounds with estrogenic activity on the uptake of atherogenic lipoproteins into macrophages, thought to be the initiating step in the development of atherosclerotic lesions. Isolated low density lipoprotein (LDL) and lipoprotein(a) (Lp(a)) were radiolabelled with (3)H-cholesterol linoleate, and incubated with J774 macrophages for 24 hours in the presence of pharmacological doses of estrogens and phytoestrogens. At a concentration of 0.1 microM, the estrogen 17beta-estradiol significantly reduced LDL uptake by macrophages by 14% (p < 0.05), but estrone did not have any effect. At 10 microM, both estrogens significantly reduced macrophage LDL uptake, but the phytoestrogenic-lignans enterodiol and enterolactone had no effect on LDL uptake. Lp(a) uptake into cells was significantly reduced by both estrone and estradiol, and by enterolactone and enterodiol at concentrations of 10 microM (p < 0.01), with enterodiol being most effective. The results of this study suggest that the uptake of these structurally similar lipoproteins is regulated differently. Macrophage Lp(a) uptake appears more phytoestrogen sensitive than does LDL uptake.     

Profiling of Glycan Receptors for Minute Virus of Mice in Permissive Cell Lines Towards Understanding the Mechanism of Cell Recognition

The recognition of sialic acids by two strains of minute virus of mice (MVM), MVMp (prototype) and MVMi (immunosuppressive), is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA) capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM). Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3′SIA-LN) and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3′SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3′SIA-Le^X identified in a previous glycan microarray screen.     

Identification of the sialic acid structures recognized by minute virus of mice and the role of binding affinity in virulence adaptation

Sialic acid binding is required for infectious cell surface receptor recognition by parvovirus minute virus of mice (MVM). We have utilized a glycan array consisting of approximately 180 different carbohydrate structures to identify the specific sialosides recognized by the prototype (MVMp) and immunosuppressive (MVMi) strains of MVM plus three virulent mutants of MVMp, MVMp-I362S, MVMp-K368R, and MVMp-I362S/K368R. All of the MVM capsids specifically bound to three structures with a terminal sialic acid-linked alpha2-3 to a common Galbeta1-4GlcNAc motif: Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-4Galbeta1-4GlcNAc (3'SiaLN-LN), Neu5Acalpha2-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc (3'SiaLN-LN-LN), and Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)-GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4(Fucalpha1-3)GlcNAc (sLe(x)-Le(x)-Le(x)). In addition, MVMi also recognized four multisialylated glycans with terminal alpha2-8 linkages: Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha ((Sia)(3)), Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GD3), Neu5Acalpha2-8Neu5Acalpha2-8Neu5Acalpha2-3Galbeta1-4Glc (GT3), and Neu5Acalpha2-8Neu5Acalpha2-3(GalNAcbeta1-4)Galbeta1-4Glc (GD2). Interestingly, the virulent MVMp-K368R mutant also recognized GT3. Analysis of the relative binding affinities using a surface plasmon resonance biospecific interaction (BIAcore) assay showed the wild-type MVMp and MVMi capsids binding with higher affinity to selected glycans compared with the virulent MVMp mutants. The reduced affinity of the virulent MVMp mutants are consistent with previous in vitro cell binding assays that had shown weaker binding to permissive cells compared with wild-type MVMp. This study identifies the sialic acid structures recognized by MVM. It also provides rationale for the tropism of MVM for malignant transformed cells that contain sLe(x) motifs and the neurotropism of MVMi, which is likely mediated via interactions with multisialylated glycans known to be tumor cell markers. Finally, the observations further implicate a decreased binding affinity for sialic acid in the in vivo adaptation of MVMp to a virulent phenotype.     

Serum oxidized low density lipoprotein, paraoxonase 1 and lipid peroxidation levels during oral glucose tolerance test.

Increasing evidence suggests that the postprandial state is a contributing factor to the development of atherosclerosis. To evaluate the effects of acute hyperglycemia on the oxidative stress, concentrations of serum-oxidized low density lipoprotein (oxLDL), paraoxonase 1 (PON1), and thiobarbituric acid reactive substances (TBARS) were measured in subjects with normal glucose tolerance (NGT) (n=35), impaired glucose tolerance (IGT) (n=25), and diabetic glucose tolerance (DGT) (n=20). In NGT group, the 2 hours' TBARS and oxLDL levels were not statistically different when compared to baseline, and 2 hours' PON1 activities were higher when compared to baseline (p<0.01). Subjects with IGT and DGT have higher 2 hours' serum TBARS and oxLDL levels than their baseline levels (p<0.01, for each). Baseline oxLDL levels of both IGT and DGT groups were higher than NGT group (p<0.01 and p<0.01, respectively). While there were not any significant differences in 2 hours' versus baseline PON1 activities in the IGT group, the 2 hours' versus baseline PON1 activities in the DGT group were significantly lower (p<0.01). The postchallenge 2 hours' PON1 activities of both IGT and DGT groups were lower than NGT group (p<0.01 and p<0.01, respectively). Baseline oxLDL was positively correlated with 2 hours' glucose (r=0.613, p<0.01) in IGT and DGT groups. PON1 activities were correlated with HDL-cholesterol, total cholesterol, and fasting glucose (r=0.680, r=0.698 and r=0.431, respectively, for each p<0.01) in NGT. In conclusion, oxidative stress occurs at an early stage in diabetes, and protective effects of HDL against atherosclerosis may be dependent on the PON1 activities.     

The age of the British upper coal measures with reference to their miospore content

Details are given of miospore distributions and zonation in the British Upper Coal Measures. These are compared with miospore distributions in strata of comparable age in France, Belgium, The Netherlands, Germany, Spain, and parts of Russia and the United States. Various published correlations of these strata are discussed and an amended correlation between the British and French sequences suggested. It is concluded that there is as yet little palynological evidence for the presence in Britain of Carboniferous strata of an age younger than that of the Westphalian D stage.