Persistent pollutants exceed toxic thresholds in a freshwater top predator decades after legislative control.

Declining emissions of persistent organic pollutants (POPs), subject to international control under the Stockholm convention, are not consistently reflected in biotic samples. To assess spatial and temporal variation in organochlorine pesticides and PCBs in UK freshwaters, we analysed tissues of a sentinel predator, the Eurasian otter, Lutra lutra between 1992 and 2009. Past declines in otter populations have been linked to POPs and it is unclear whether otter recovery is hampered in any areas by their persistence. PCBs, DDT (and derivatives), dieldrin and HCB were detected in over 80% of 755 otter livers sampled. Concentrations of ∑PCB, ∑DDT and dieldrin in otter livers declined across the UK, but there was no significant time trend for ∑PCB-TEQ (WHO toxic equivalency, Van den Berg et al., 2006) or HCB. In general, higher concentrations were found in the midlands and eastern regions, and lowest concentrations in western regions. Concentrations of PCBs and HCB in otters increased near the coast, potentially reflecting higher pollutant levels in estuarine systems. Decades after legislative controls, concentrations of these legacy pollutants still pose a risk to otters and other freshwater predators, with spatially widespread exceedance of thresholds above which reproduction or survival has been reduced in related species.

Effect of inhibiting dolichol phosphate-mannose formation on the biosynthesis of the N-acetylglucosaminylpyrophosphoryl polyprenols by the retina.

Previously (Kean, E.L. (1982) J. Biol. Chem. 257, 7952-7954), it was shown that dolichol phosphate-mannose stimulated the formation of the N-acetylglucosamine-containing mono-, di- and trisaccharide intermediates of the dolichol pathway. Consistent with this activating role, the present report demonstrates that inhibition of the formation of dolichol phosphate-mannose by the antibiotics, showdomycin and diumycin, also blocked the stimulatory phenomenon.

Influence of metal ions on the biosynthesis of N-acetylglucosaminyl polyprenols by the retina.

The following enzymatic process was investigated, catalyzed by an enzyme preparation from the retina of the embryonic chick: UDP-GlcNAc + dolichol phosphate GDPmannose leads to metal ions GlcNAc-P-P-polyprenol + (GlcNAc)2-P-P-polyprenol + Man-(GlcNAc)2-P-P-polyprenol. These reactions were carried out in the presence of a dolichol phosphate mannose-synthesizing system, shown previously to be an activator of GlcNAc-lipid synthesis. The process was also strongly influenced by the choice of the divalent cation used during the reactions. In the presence of Mg2+, not only was the extent of incorporation of radioactivity from UDP-[3H]GlcNAc increased 4-fold into the GlcNAc lipids, as compared to Mn2+, but the relative distribution of the products was affected as well. In the presence of Mg2+ the reaction was driven mainly in the direction of the formation of the first intermediate of the dolichol pathway, GlcNAc-P-P-polyprenol. Many of the other characteristics of the GlcNAc-transferases, such as pH optimum, requirement for dolichol phosphate and specificity for stimulation by sugar nucleotides, were similar for either the Mn2+ or Mg2+ systems. Retinol phosphate could not replace the requirement for dolichol phosphate. The influence of metal ions, in addition to the stimulation by dolichol phosphate mannose, on GlcNAc-lipid synthesis may be aspects of metabolic regulation of the dolichol pathway.

Selective inhibition of acyl-CoA dehydrogenases by a metabolite of hypoglycin.

Extracts of liver mitochondria from donor rats given hypoglycin, the toxic amino acid from the ackee plant (Blighia sapida) showed drastically reduced levels of acyl-CoA dehydrogenase activity with butyryl-CoA as substrate. Activity with octanoyl- and palmitoyl-CoA was unaffected. Evidence that the active agent is methylenecyclopropylacetyl-CoA, a hypoglycin metabolite, was obtained by observing effects of the compound on a partially purified enzyme mixture prepared from rabbit liver. At 13 muM concentration, it strongly inhibited butyryl-CoA dehydrogenase (EC 1.3.99.2) with butyryl-CoA as substrate; it was far less effective with palmitoyl-CoA as substrate for the other similar enzymes present in the preparation. Unlike normal substrates of the acyl-CoA dehydrogenases, the compound itself, and not a reaction product, is inhibitory. The observed effect is consistent with quite general inhibition of fatty acid beta-oxidation by hypoglycin.

Topography of basal and stimulated biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol: variation in effect of proteolysis.

Evidence has recently appeared indicating the cytoplasmic orientation in microsomal vesicles of the GlcNAc-transferases that participate in the biosynthesis of GlcNAc-P-P-dolichol and (GlcNAc)2-P-P-dolichol. The topography of the stimulation of these activities brought about by dolichol-P-mannose and phosphatidylglycerol, however, is not known. The present report continues studies of the topography of these early reactions of the dolichol pathway, examined under basal and stimulatory conditions after proteolysis of intact microsomes isolated from livers of the embryonic chick. Under all conditions which were examined, the GlcNAc-transferase concerned with the biosynthesis of the chitobiosyl derivative was completely inhibited, consistent with its cytoplasmic orientation. The effect of proteolysis on the formation of GlcNAc-P-P-dolichol, however, varied with different proteolytic enzymes, ranging from no inhibition to over 90% inhibition. This variation in response suggests that the GlcNAc-transferase may be partially buried within the microsomal bilayer which only some preparations of proteolytic enzymes can penetrate. Evidence was obtained indicating the cytoplasmic orientation of the stimulation of GlcNAc-P-P-dolichol biosynthesis. These studies support the conclusion that both the catalytic and allosteric activating sites of the GlcNAc-transferase have a similar topography.

The biogenesis of dicarboxylic acid in rats given hypoglycin.

The metabolic origin of dicarboxylic acids which are produced as a result of hypoglycin poisoning (Jamaican vomiting sickness) was investigated. 14C- and 3H-labelled palmitic acid was administered with hypoglycin to rats, and radioactivity was measured in urinary dicarboxylic acids that were isolated by gas-liquid chromatography. Both isotopes were incorporated into adipic and sebacic acids, indicating a precursor-product relationship. Glutaric acid was, essentially, unlabelled. Preferential incorporation of C-16, relative to C-1 of palmitate, while not evident from data for fraction of isotopic dose incorporated, could be deduced by comparing ratios of 14C:3H in precursor with those ratios in products. It thus appears that omega-oxidation of the fatty acid intervenes predominantly at an intermediate stage of chain-shortening, when inhibition of beta-oxidation by hypoglycin becomes more pronounced.

Isoelectric forms of bacteriorhodopsin from Halobacterium halobium.

Isoelectric focusing was performed on bacteiorhodopsin isolated from Halobacterium halobium, R1 strain, solubilized in Nonidet P-40, on sucrose density gradient stabilized columns. When 560 nm absorbance was monitored, four forms of bacteriorhodopsin were observed, having isoelectric points (pI) of: A. 3.93; B, 4.43; C, 5.03; D, 5.49. Instability of some of the isoelectric forms during the very process of electrofocusing was observed. When focused over a period of 6 days, the relative abundance of the forms changed although their pI values remains constant. Refocusing of the isolated forms A or B led to the production f form C. The latter species was stable to refocusing. Form B was unstable to storage either as an aqueous suspension of the purple membrane or as a detergent extract. Each of the forms had the absorption spectrum typical for bacteriorhodopsin and each showed identical patterns after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Immunoreactivity of rhodopsin and opsin.

An examination by a radioimmunoassay of the relative affinity of opsin and rhodopsin for rabbit antibody raised against bovine rhodopsin revealed that opsin was the preferred antigen. About 10-fold greater amounts of rhodopsin than opsin were required to achieve 50% inhibition of binding of 125I-labeled ligand in the RIA. Opsin was more reactive when examined in the light or dark, compared to rhodopsin incubated in the dark. Mixtures of opsin and rhodopsin (prepared by partial bleaching of rhodopsin or synthetic mixtures) exhibited increased reactivity with increasing mole fraction of opsin. This response was nonlinear, with small increases in opsin producing relatively large increases in reactivity. A partial fractionation of the antibody into two groups showing differential reactivities toward opsin and rhodopsin was achieved by affinity chromatography on opsin-Sepharose. However, with both groups, opsin was still the preferred antigen. Scatchard analysis of 125I-labeled rhodopsin and opsin produced nonlinear plots, indicating the presence of multiple species of antibody. The affinities and binding capacities were similar for both labeled antigens. In competitive binding studies, the antibody showed a strong preference for either labeled ligand (rhodopsin or opsin) as compared to the unlabeled material. These latter observations indicate that altering rhodopsin either by bleaching or iodination produced changes in the relative immunoreactivity of the molecule.

Multiple isoelectric forms of detergent-solubilized bovine rhodopsin. I. Identity, composition and properties.

Isoelectric focusing was performed on crude bovine rhodopsin (a detergent extract of rod outer segments) and on rhodopsin after purification by adsorption chromatography on calcium phosphate-Celite and affinity chromatography on Con A-Sepharose. The purified unbleached (native) material resolved into three forms of nearly equal amounts having isoelectric points (pI) of 5.19, 5.58 and 6.14. Each form was stable to refocusing. The composition of the three forms as determined by amino acid analysis, phosphate analysis and polyacrylamide gel electrophoresis was the same as that found for unfocused rhodopsin. Each isoelectric form cross-reacted with rabbit antibody to bovine rhodopsin. After photobleaching, one form of opsin was observed, having an isoelectric point of 5.58. While crude rhodopsin showed a single species in the native state (pI of 6.20), multiple forms were revealed after treatment with phospholipase. Purified rhodopsin, after reduction, showed a single major isoelectric point of 6.09 rather than the multiple forms. Exposure of rod outer segments or rhodopsin to antioxidants or rcrude rhodopsin showed a single species in the native state (pI of 6.20), multiple forms were revealed after treatment with phospholipase. Purified rhodopsin, after reduction, showed a single major isoelectric point of 6.09 rather than the multiple forms. Exposure of rod outer segments or rhodopsin to antioxidants or removal of phospholipids led to an alteration in the relative abundance of the multiple isoelectric forms, but not to a change in their isoelectric point.

Isolation of a branched-chain alpha-keto acid decarboxylase from rat liver.

An enzyme which catalyses oxidative decarboxylation of branched-chain alpha-keto acids was extracted from rat liver mitochondria with the aid of NaClO4. Purification yielded a product which appeared homogenous upon electrophoresis. Some kinetic data are reported; however, the enzyme is inactive with alpha-ketoisovalerate. The tenacity of binding to mitochondria, specificity, and other features, suggest that the decarboxylase may be a component of an enzyme complex named alpha-ketoisocaproate: alpha-keto-beta-methylvalerate dehydrogenase.

Reactivity with lectins of the saccharide components of rhodopsin in reconstituted membranes. Orientation of the carbohydrates.

Rhodopsin-containing liposomes may provide a model for investigating the interaction of intrinsic membrane glycoproteins in biological systems. As part of the characterization of this preparation, the surface orientation of the carbohydrates of rhodopsin, assembled from purified bovine rhodopsin and egg phosphatidylcholine was examined, and is the topic of this report. The major tool used in these studies was the interaction with the carbohydrate-specific reagents, plant lectins. Two techniques were used: lectin-mediated aggregation of the liposomes, as measured by light scattering; the binding of 125I-labeled succinylated concanavalin A, and Scatchard analysis as a measure of affinity. The preparation most extensively examined had a mole ratio of rhodopsin:phospholipid of 1:100. Among a variety of lectins which were examined, only concanavalin A, succinylated concanavalin A, and wheat germ agglutinin were able to mediate the aggregation of rhodopsin-containing liposomes, as expected. The aggregation with concanavalin A was prevented by the presence of sugars having the alpha-D-glucopyranosyl configuration, and that brought about with wheat germ agglutinin, by N-acetylglucosamine (GlcNAc). In addition, the aggregation with concanavalin A was reversed with methyl alpha-D-mannoside, and with wheat germ agglutinin, by GlcNAc, suggesting that membrane fusion did not take place. On a molar basis, wheat germ agglutinin brought about a greatly reduced extent of aggregation as compared to concanavalin A, suggesting the relative inaccessibility of GlcNAc residues in the liposomes as compared to mannose. The initial rate of the aggregation, however, were similar with either lectin. The first-order rate constants were unaffected by wide variation in the concentrations of concanavalin A and wheat germ agglutinin, and by variation in the mole ratios of rhodopsin in the liposomes from 0.2 to 19 moles per 100 moles of egg lecithin. Rhodopsin-liposomes were also prepared from a total lipid extract of rod outer segments instead of egg lecithin. Similar kinetic properties were exhibited by this preparation as were obtained with the liposome prepared with the purified phospholipid. Scatchard analysis of the binding of 125I-labeled succinylated concanavalin A by rhodopsin liposomes indicated the presence of a single class of binding site as the preferred fit, with an apparent Kd of 2.8 X 10(-7) M. The binding was destroyed or extensively interfered with by trypsinization and by periodate treatment.

Multiple isoelectric forms of detergent-solubilized bovine rhodopsin. II. Spectral analysis.

The absorption and circular dichroic spectra of three stable isoelectric forms of purified rhodopsin (in Emulphogene BC 720) with isoelectric points of 5.19, 5.58 and 6.14 were analysed over the accessible wavelength region of 190-800 nm. It was found from the spectral analysis of the 5.58 and 6.14 forms that the tertiary structure of these isoelectric forms was different, without any change being observed in the secondary structure of these proteins. The difference in structure was removed by denaturation with SDS. Specifically, the aromatic amino acid residues of the 6.14 isoelectric form were suggested to be in a more polar environment as compared to those for the 5.58 isoelectric form. The changes of structure in the microenvironment of retinal in these proteins were minor in comparison to the tertiary changes observed in the proteins. This indicated that the site of charge perturbation is not localized in the retinylidene microenvironment. Furthermore, the spectral features of these isoelectric forms were features common to the spectra of both purified unfocused rhodopsin and crude rhodopsin, suggesting that the isoelectric forms are not a consequence of purification. Spectral analysis of the 5.19 isoelectric form indicated that this form of rhodopsin had an increased tendency to aggregate after focusing. Based on the denaturation properties of these isoelectric forms, it was shown that the 5.19 and 5.58 isoelectric forms had different conformations. It was concluded from this study that preparations of 'purified' rhodopsin are a heterogeneous mixture of stable proteins with different tertiary structures and different isoelectric points.

Glycosidases of the retinal pigment epithelium.

The pH optima and apparent Km and Vmax values were determined for nine glycosidases of the retinal pigment epithelium (RPE) of the calf. In terms of micromoles of substrate cleaved per milligram protein per hour, the following relative order of enzymatic activities was observed: beta-N-acetylglucosaminidase greater than alpha-glucosidase = beta-N-acetylgalactosaminidase greater than alpha-mannosidase greater than beta-galactosidase greater than beta-glucosidase greater than alpha-fucosidase greater than alpha-galactosidase greater than beta-glucuronidase. The pH optimum of each of these enzymes was in the acidic range (below pH 6). All these findings refer to enzymatic activities of bovine RPE preparations obtained by the brushing procedure of Glocklin and Potts and washing as described by Berman and Feeney. Thus they may relate to those activities associated with particulate components of the RPE cell and not to the more soluble glycosidases. The distribution of the glycosidases between the washes of the cells and the final pellet of bovine RPE cells was examined. The activities of 10 glycosidases in the RPE of the embryonic chick were also examined. Neither beta-mannosidase nor beta-fucosidase activities could be detected in washed bovine RPE cells, although beta-mannosidase was detected in RPE of the embryonic chick. The presence of isoenzymes of beta-glucuronidase in bovine RPE was indicated. Specificity by beta-glucuronidase of bovine RPE for synthetic substrates was observed.

Are sugars involved in the binding of rhodopsin-membranes by the retinal pigment epithelium?

The binding by the retinal pigment epithelium (RPE) cells of the embryonic chick, in culture, of recombinant membranes composed of phosphatidylcholine and bovine rhodopsin (rhodopsin-liposomes [RL]) was investigated to explore the influence that the carbohydrate groups of rhodopsin might have on this process. The reaction was quantitated by measuring the amount of RL associated with the cells using a radioimmunoassay for rhodopsin. The process was saturated at about 8 microM RL (calculated as rhodopsin-equivalents), and a two- to threefold greater rate and extent of binding was observed at 37 degrees C as compared with 4 degrees C. The presence of up to 30 X 10(3)-fold molar excess over RL of mannose and N-acetylglucosamine, the sugars present on rhodopsin, as well as other saccharides, had little or no effect on the binding. These observation indicate that lectin-like recognition involving the sugar groups of rhodopsin is not involved in the binding of rhodopsin-containing membranes by the RPE cells. In contrast, exogenously added concanavalin A and wheat germ agglutinin enhanced the binding, an effect which was blocked by appropriate sugars.

The lipid intermediate pathway in the retina for the activation of carbohydrates involved in the glycosylation of rhodopsin.

The presence of and many of the properties of enzymes in the retina involved in the biosynthesis of key intermediates in the dolichol pathway of carbohydrate activation has been demonstrated. The formation of dolichol phosphate-mannose and of mannose-containing oligosaccharide-polyprenols, and their use as substrates in further biosynthetic reactions was shown. The biosynthesis of the core-region glucosamine-containing mono, di and tri-saccharide pyrophosphoryl polyprenols, and the novel function of GDP-mannose as a regulator in their biosynthesis, was described. The participation of the dolichol pathway in the glycosylation of rhodopsin was demonstrated by the inhibition of core-region glycosylation of this glycoprotein by the antibiotic, tunicamycin. Preliminary evidence was obtained that glycosylation of rhodopsin was not a requirement for its insertion into disc membranes.

Jamaican vomiting sickness: a study of two adult cases.

An acute illness (Jamaican vomiting sickness) which affected two adults after eating unripe ackee fruit was investigated. Analyses of serum and urine samples were performed to compare the patterns of organic acidaemia and aciduria with those reported from childhood cases. The main conclusion from the comparison is that the toxic ackee constitutent, hypoglycin, produces essentially the same metabolic effects in adults as in children.

Identification of ethylmalonic acid in urine of two patients with the vomitting sickness of Jamaica.

Large amounts of ethylmalonic acid have been identified in urines from two patients with the vomitting sickness of Jamaica. The amounts were 178 and 882 mug per mg creatinine which are 70 and 350 times, respectively, over control values. Other short and medium chain dicarboxylic acids including glutaric and adipic acids and those with eight and ten carbon chain, saturated and cis-unsaturated, were also detected in large quantities as in the case of hypoglycin treated rats; urine. However, the large increase of urinary ethylmalonic acid in these two human cases is in a sharp contrast to the findings in hypoglycin treated rats in which urinary ethylmalonic acid increased only 3 times over control. It appears that ethylmalonic acid is produced in the cases with the vomiting sickness of Jamaica by carboxylation of n-butyryl-CoA which is not oxidized further due to the inhibition by hypoglycin A. In case of hypoglycin-treated rats, n-butyryl-CoA is mainly conjugated with glycine or deacylated to free butyric acid.

Galactosylation of rhodopsin by the human retina.

The major oligosaccharide chains of bovine, frog and human rhodopsins are abridged, hybrid, asparagine-linked structures that contain only mannose and N-acetylglucosamine as the constituent sugars. Isomers that also contain galactose have been detected in varying amounts in rhodopsins from different species. As part of studies to examine the mechanisms used by the human retina to control the glycosylation of rhodopsin, the kinetics of the retinal galactosyltransferase was examined using Golgi-enriched fractions of human retina as the enzyme source and rhodopsin, opsin, and the oligosaccharide isolated from rhodopsin as acceptors. These reactions were compared to those using bovine retina and rat liver Golgi. A comparison of the Vmax/Km ratios revealed that the efficiency of the human retinal galactosyltransferase was some 20-fold lower than that of the rat liver. In addition, consistent with the higher state of galactosylation of human as compared to bovine rhodopsin, greater efficiency was observed with the human preparation. While the conformation of the visual pigment exerted an effect, its low galactosylation was not due to a major directing influence on glycosylation by the polypeptide matrix as indicated by the even lower activity toward the isolated oligosaccharide. The galactosylated oligosaccharides obtained by these procedures were identified by chromatographic methods. The relatively low activity of the retinal galactosyltransferases observed in this study may help explain the limited glycosylation that is typical of rhodopsin.

Biogenesis and content of rhodopsin in the retina of the chick during development.

Developmental aspects of the formation of rhodopsin in the chick were investigated. The content of rhodopsin was measured in the retina of the developing chick embryo, newly hatched and adult chickens by both spectral and immunological procedures. Rhodopsin was first detected spectrally at day 18 (stage 43) with the level increasing about 6 fold through hatching. The concentration in the adult retina was about one half that present in the 1 day old chick. By the more sensitive procedure of radioimmunoassay (RIA), rhodopsin was detected as early as day 12 (stage 37), but was undetectable in the 8 day old embryo. The rhodopsin concentration remained low and relatively constant until day 17 (stage 42), at which point it increased rapidly up to hatching. Retina from the newly-hatched chick had a rhodopsin concentration some 30 fold higher than the 16 day old embryo (stage 41). The results of RIA indicate that the biosynthesis of rhodopsin may significantly precede the morphological appearance of outer segments.