Showing papers by "James D. Young published in 1994"
TL;DR: The demonstration of transport of AZT by cNT1 expressed in Xenopus oocytes provides the first direct evidence that AZT enters cells by transporter-mediated processes, as well as by passive diffusion.
208 citations
TL;DR: In this paper, the authors investigated the characteristics of glucose transport in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti) Lockington.
Abstract: The characteristics of glucose transport were investigated in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti) Lockington. Transport of glucose by intact hagfish erythrocytes and by phospholipid vesicles reconstituted with n-octylglucoside extract of hagfish erythrocyte membranes was rapid and mediated by a saturable stereospecific mechanism sensitive to inhibition by cytochalasin B. Covalent photoaffinity labelling experiments with [3H]cytochalasin B identified the hagfish glucose transporter on SDS/polyacrylamide gels as a protein with an apparent average Mr of 55 000. Amino acid sequence homology between the hagfish and human erythrocyte glucose transporters (GLUT 1) was investigated in immunoblotting experiments using a panel of 12 different antipeptide antisera and affinity-purified antibodies raised against cytoplasmic extramembranous regions of the human transporter, and with an antibody to the intact purified human protein. The latter antibody labelled a component in the membrane with the same apparent Mr as cytochalasin B. Two affinity-purified antipeptide antibodies, corresponding to residues 240–255 and 450–467 of the human erythrocyte transporter, also labelled a component in the membrane with this relative molecular mass, demonstrating localised sequence similarity between the polypeptides of the two species within the central cytoplasmic loop and within the cytoplasmic C-terminal region. Glucose transport by hagfish erythrocytes was not coupled to the movement of protons.
24 citations
TL;DR: It is established that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that the transporters exhibit N BMPR sensitivity and permeant selectively similar to that of the native transporter.
Abstract: Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.
6 citations
TL;DR: It is concluded that mRNA from rat jejunum encodes novel Na(+)-dependent and Na( +)-independent transport activities unrelated to the D2/4F2hc glycoproteins, as well as a newly discovered zwitterionic/cationic amino acid transporter first described in human erythrocytes.
Abstract: SummaryComplementary DNA clones have been isolated recently from rat (D2) and rabbit kidney (rBAT) which induce increased Na+—independent Leu and Lys transport activities (System b0,+) when expressed in oocytes of Xenopus laevis. These cDNAs encode type II membrane glycoproteins which show significant homology to the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Injection of human 4F2hc cRNA into oocytes also results in induction of Leu/Lys transport activity, but with differing cation requirements for the two amino acids (Na+-dependent for Leu, Na+-independent for Lys: system y+L). System y + L is a newly discovered zwitterionic/cationic amino acid transporter first described in human erythrocytes. Here we have examined the characteristics of Leu transport in Xenopus oocytes microinjected with mRNA from the mucosa of rat jejunum. L-Leu uptake during 10 min (02 mm1, 20d`C) reached 20 pmol/oocyte compared with endogenous uptake by water-injected oocytes of typically 3–4 pmol/oocyte. The e...
5 citations