Subcellular-fractionation techniques were used to characterize the endocytic pathway followed by ricin in rat liver in vivo and tentatively identify the site(s) at which the ricin interchain disulphide bridge is split. After injection of 125I-ricin, hepatic uptake of radioactivity was maximum at 30 min (40% of injected dose). At 5 min, about 80% of the radioactivity in the homogenate was recovered in the microsomal (P) fraction, but later on the recovery of the radioactivity in the mitochondrial-lysosomal (ML) fractions progressively increased (50% at 30 min) at the expense of that in the P fraction. Subfractionation of the P and ML fractions on analytical sucrose-density gradients revealed a time-dependent translocation of the radioactivity from low- to high-density endocytic structures, with median relative densities at 5 and 60 min of about 1.15 and 1.16 (P fraction) and 1.19 and 1.22 (ML fraction) respectively. The late distribution of the radioactivity in the ML fraction was similar to that of the lysosomal marker acid phosphatase. Studies with co-injected lactose and mannan showed that ricin was internalized mainly via the mannose receptor. In the presence of mannan, the late recovery of radioactivity in the ML fraction was decreased, and the distribution of the radioactivity associated with the P fraction was shifted toward lower densities (median relative density 1.13), indicating a different pathway of endocytosis. Analysis of the radioactivity associated with the ML and S fractions by SDS/PAGE revealed a time-dependent increase in the amount of intact A- and B-chains and low-molecular-mass products. When ML fractions containing partially processed ricin were incubated at 37 degrees C at pH 5 or at pH 7.2 in the presence of ATP, only low-molecular-mass products were generated. We conclude that internalized ricin associates with endocytic structures whose size and density of equilibration increase with time, and that, although detectable in these structures, reduction of the ricin interchain disulphide bridge occurs to a large extent in the cytosol.
SummaryMature human milk samples from young healthy women on an equilibrated diet contained a mean of 81 ng/ml total iodine. Iodide represented a mean of 77% of the total iodine. Of the 22 ng/ml organic iodine, there was about 1 ng thyroxine and triiodothyronine and,,after pepsin hydrolysis, up to 40% of organic iodine in monoiodotyrosine form. By electrophoresis, after incubation with radioactive thyroid hormone, we found an absence of binding on thyroxine-binding globulin, but thyroxine and triiodothyronine were bound to albumin with a maximal capacity 50 times higher than in human serum. All these differences did not favor transport of iodinated compounds from maternal serum to milk. In conclusion, milk iodide was taken up by newborn thyroid to make thyroid hormones. Abbreviations T4, thyroxine T3, triiodothyronineFor the past 10 years, thyroid congenital screening has brought a new approach to the neonatal thyroid function, and thyroxine appeared as the most important feature. These considerations have been certainly related to the interest recently developed by breast milk studies. Sack et a/. (19) had found a significant amount of thyroxine in human milk. These last 2 years, using two different sensitive methods, Mallol et a/. (10) and Moller et a/. (13) found that the thyroxine supplied by breast milk was inadequate for normal neonatal development.Recently. we have noticed a very low content of thyroid hormones in human breast milk and determined its total iodine level (4). This study presents the chemical form of the iodine and the proteins contained in the human milk with the aim of comparing milk to serum. MATERIALS AND METHODSPooled and individual mature breast milks were obtained from the Lactarium of the lnstitut de Pukriculture of Paris for the neonates in the Hbpital des Enfants-Malades. The mothers were young, healthy, living in and around the city, and had an equilibrated diet. Some samples of commercial cow milks were used for comparison.Fresh milk samples were used immediately or kept frozen at -18°C. For total iodine determination, the catalytic effect of iodine on the reduction of ceric ions by arsenious acid is used in a Technicon Autoanalyzer. The automatic micromethod was applicable in the range of 0.5 to 6 ng/O.l-ml sample. Milk was free of substance interfering with the reaction; the recovery of a stable amount of iodine added to the milk was complete. Iodide was determined either after 24-h dialysis against water and/or after shaking with an anion exchange resin (Bio-Rad AG 1-X2 type). Radioimmunoassays used were: T4 (6), T3 (18), both with curve sensitivity of 20 to 600 pglassay, and thyroxine-binding globulin with a commercial kit (Behringwerke, Marburg, Germany). Radioactive T4 and Tg used for radioimmunoassays and for the different incubations had high specific activity (>I200 pCi/pg, Amersham, UK). Peroxidase was determined according to Fragu and Nataf (5). Protein determinations were performed after trichloracetic acid precipitation, albumin after ammonium sulfate separation by t...
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