Pittman, R.C. et al. A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo. Biochem. J. 212, 791−800

Biochemical Journal (Impact Factor: 4.4). 07/1983; 212(3):791-800.
Source: PubMed


We recently developed a general method for determining tissue sites of degradation of plasma proteins in vivo that made use of covalently attached radioactive sucrose. On degradation of the protein, the sucrose remained trapped in the cells as a cumulative marker of protein degradation. The method described here depends on the same principles, but uses an adduct of cellobiose and tyramine that is radioiodinated to high specific radioactivity and then covalently attached to protein. Use of the radioiodinated ligand increases the sensitivity of the method at least 100-fold and allows simplified tissue analysis. Proteins derivatized with the radioiodinated ligand were recognized as underivatized proteins both in vitro and in vivo. On degradation of derivatized low-density lipoprotein, the rate of leakage from cultured fibroblasts was only 5% during 24 h. Similarly, on injection of labelled proteins into rats and rabbits, urinary excretion of the label was in all cases less than 10% of total labelled catabolic products recovered 24 h after injection. Examination of the tissue contents of label at two times after injection of labelled asialofetuin or apolipoprotein A1 in rats, and asialotransferrin in rabbits showed that the label did not detectably redistribute between tissues after initial uptake and catabolism; a significant leakage from liver was quantitatively accounted for by label appearing in gut contents and faeces. A simple double-label method was devised to provide a correction for intact protein in trapped plasma, the extravascular spaces, and within cells. By using this method it becomes unnecessary to fractionate tissue samples.

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    • "Radio-labelled tyramine cellobiose (125I-TC) was prepared by reacting TC with Na125I (Perkin-Elmer) in Iodogen tubes (Pierce) and was then coupled covalently to heat-denatured collagen (5 mg in 500 μl borate buffer, pH 9.4) after activating the 125I-TC with the cross-linking agent cyanuric chloride, essentially as described by Pittman et al [58]. The labelled protein (125I-TC-collagen) was dialyzed against PBS using 10000 Da dialysis cassettes (Pierce Biotechnology) to remove non-coupled 125I-TC. "
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    ABSTRACT: The urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation. This study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently. These results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.
    BMC Cell Biology 06/2009; 10:39. DOI:10.1186/1471-2121-10-39 · 2.34 Impact Factor
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    • "This technique uses non-hydrolyzable radioactive tracers which become trapped in tissues following uptake. This allows for the measurement of catabolism of HDL by specific tissues without the release of free label into plasma (Glass et al., 1983a; Pittman et al., 1983; Glass et al., 1983b). HDL cholesterol is taken up by tissues either through HDL holo-particle internalization or selective uptake of esterified cholesterol (cholesteryl ester, CE) via scavenger receptor BI (SR-BI) (Acton et al., 1996). "
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    ABSTRACT: The expression of the cholesterol transporter ATP-binding cassette transporter A1 (ABCA1) in the brain and its role in the lipidation of apolipoproteins indicate that ABCA1 may play a critical role in brain cholesterol metabolism. To investigate the role of ABCA1 in brain cholesterol homeostasis and trafficking, we characterized mice that specifically lacked ABCA1 in the CNS, generated using the Cre/loxP recombination system. These mice showed reduced plasma high-density lipoprotein (HDL) cholesterol levels associated with decreased brain cholesterol content and enhanced brain uptake of esterified cholesterol from plasma HDL. Increased levels of HDL receptor SR-BI in brain capillaries and apolipoprotein A-I in brain and CSF of mutant mice were evident. Cholesterol homeostasis changes were mirrored by disturbances in motor activity and sensorimotor function. Changes in synaptic ultrastructure including reduced synapse and synaptic vesicle numbers were observed. These data show that ABCA1 is a key regulator of brain cholesterol metabolism and that disturbances in cholesterol transport in the CNS are associated with structural and functional deficits in neurons. Moreover, our findings also demonstrate that specific changes in brain cholesterol metabolism can lead to alterations in cholesterol uptake from plasma to brain.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 04/2009; 29(11):3579-89. DOI:10.1523/JNEUROSCI.4741-08.2009 · 6.34 Impact Factor
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    • "Albures Ò particles (0.1 mg) were labeled with 125 I-tyramine-cellobiose according to the method originally described by Pittman et al. (1983). A specific activity of around 100 cpm/ng protein was obtained by this method. "
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    ABSTRACT: As the supply of marine fish oil is becoming a limiting factor in the production of Atlantic salmon (Salmo salar), new diets and alternative sources of energy are being tested. Plant oils are natural potential candidates to replace fish oil, but the different levels of essential polyunsaturated fatty acids may influence the health and growth of salmon. In this study, we have investigated the resistance to transport stress and bacterial infection, phagocytic activity in head kidney macrophages and eicosanoid metabolism in salmon fed three different diets. In high-energy fishmeal based diets, 50% and 100% of the supplementary fish oil (FO) was replaced with soybean oil (SO). The three dietary groups were fed for 950day-degrees at 5C (27weeks) and 12C (11weeks) before challenging the fish with Aeromonas salmonicida, analyzing the lipid composition of head kidney and examining macrophage function in vivo and in vitro. Dietary fatty acids affected the lipid composition of the kidney. The level of eicosanoid precursor’s 20:4n-6 and 20:3n-6 were 3 and 7-fold higher in the 100% SO group compared with the FO group. The total fraction of n-3 lipids in kidney was 19% in the SO group, compared to 16% and 12% in the 50% or 100% SO groups, respectively. However, the production of leucotriene B4 (LTB) and prostaglandin E2 (PGE) immunoreactive materiel from exogenously added arachidonic acid in head kidney macrophages was only affected by the composite diet (increased) at 5C. In addition, the phagocytic activity of kidney macrophages in vivo and in vitro was not affected by diet. No effect of diet was observed on transport stress or susceptibility to a bacterial infection with Aeromonas salmonicida. Atlantic salmon therefore seems to tolerate a diet solely based on soybean oil as lipid source, without any detrimental effects on growth, health and immune functions.
    Fish Physiology and Biochemistry 05/2004; 30(2):149-161. DOI:10.1007/s10695-005-4318-7 · 1.62 Impact Factor
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