Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.
The objective of this study was to develop an acylation agent for the radioiodination of monoclonal antibodies that would maximize retention of the label in tumor cells following receptor- or antigen-mediated internalization. The strategy taken was to add a polar substituent to the labeled aromatic ring to impede transport of labeled catabolites across lysosomal and cell membranes after antibody degradation. Preparation of unlabeled N-succinimidyl 4-guanidinomethyl-3-iodobenzoate (SGMIB) was achieved in six steps from 3-iodo-4-methylbenzoic acid. Preparation of 4-guanidinomethyl-3-[131I]iodobenzoic acid from the silicon precursor, 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylsilylbenzoic acid proceeded in less than 5% radiochemical yield. A more successful approach was to prepare [131I]SGMIB directly from the tin precursor, N-succinimidyl 4-(N1,N2-bis-tert-butyloxycarbonyl)guanidinomethyl-3-trimethylstannylbenzoate, which was achieved in 60-65% radiochemical yield. A rapidly internalizing anti-epidermal growth factor receptor variant III antibody L8A4 was labeled using [131I]SGMIB in 65% conjugation efficiency and with preservation of immunoreactivity. Paired-label in vitro internalization assays demonstrated that the amount of radioactivity retained in cells after internalization for L8A4 labeled with [131I]SGMIB was 3-4-fold higher than that for L8A4 labeled with 125I using either Iodogen or [125I]SIPC. Catabolite assays documented that the increased retention of radioiodine in tumor cells for antibody labeled using [131I]SGMIB was due to positively charged, low molecular weight species. These results suggest that [131I]SGMIB warrants further evaluation as a reagent for labeling internalizing antibodies.
Highly ionic conductive solid polymer electrolytes have been prepared by blending polyethylene oxide) (MW 600 000) andpoly(2-vinylpyridine) (MW 200 000) orpoly(4-vinylpyridine) (MW 50 000) and LiC104. All blends were prepared by the solution blending process. Several different blend compositions have been studied and optimum compositions required for preparing solid polymer electrolytes have been determined. The polyethylene oxide) (85% by weight)/poly(2-vinylpyridine) (15% by weightVLiClCh blend at an ethylene oxide/Li+ mole ratio of 10 exhibits an ionic conductivity value of 1.0 X 10-® S cm-' at 25 °C and is an elastomeric material with dimensional stability. Furthermore, this blend exhibits ionic conductivities >3.0 X 10-® S cm-1 at 25 °C over a wide salt concentration range. Several other blends prepared are also elastomeric materials with ionic conductivities ~5.0 X10-8 S cm-1, e.g. polyethylene oxide) (85 % by weight)/ poly(2-vinylpyridine) (15% by weight)/LiC104 at an ethylene oxide to Li+ mole ratio of 6 exhibits a value of 7.0 X 10-8 S cm-1 at 30 °C. Studies indicate that the LiC104 salt compatibilizes the polyethylene oxide) and the poly(2-vinylpyridine) by the simultaneous interaction of the Li+ with the oxygens of the PEO and nitrogen of the pyridyl units. Scanning electron microscopy (SEM) on the internal structure of the blends shows the presence of a two phase microstructure, most likely, stabilized by the emulsifying effect of LiCICh.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.