Expression of the rabbit intestinal N2 Na+/nucleoside transporter in Xenopus laevis oocytes.
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ABSTRACT:
SUBMITTER: Jarvis SM
PROVIDER: S-EPMC1151387 | biostudies-other | 1991 Sep
REPOSITORIES: biostudies-other
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Project description:The expression of the organic cation transport system of rat renal proximal tubules has been studied in Xenopus laevis oocytes injected with poly(A)+ RNA from the rat renal cortex. The effectiveness of the technique was confirmed by examining expression of the Na+/D-glucose co-transporter. Compared with water-injected and non-injected oocytes, the injection of total poly(A)+ RNA resulted in about a 3-fold increase in tetraethylammonium (TEA) uptake activity. TEA uptake by poly(A)(+)-RNA-injected oocytes was time-dependent and was inhibited by cimetidine and HgCl2, but not by p-aminohippurate. After size-fractionation on a sucrose density gradient, a 1.4-2.4 kb poly(A)+ RNA fragment was identified that expressed the organic cation transport system in oocytes. These results demonstrate that the renal organic cation transporter was expressed in oocytes and that this expression system can provide an effective assay procedure for cloning of the organic cation transporter.
| S-EPMC1131049 | biostudies-other
Project description: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.
| S-EPMC1138087 | biostudies-other
Project description:Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.
| S-EPMC1130750 | biostudies-other
Project description:BackgroundRapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.ResultsWe used the generally available software (http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/) to predict a DNA sequence for the plant gene that is better suited for Xenopus laevis. Rice OsNAR2.1 and OsNRT2.3a DNA optimized sequences were commercially synthesized for Xenopus expression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and original OsNAR2.1 and OsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3- than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.ConclusionThe functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in the Xenopus oocyte system.
| S-EPMC3878178 | biostudies-literature
Project description:The urate transporter, GLUT9, is responsible for the basolateral transport of urate in the proximal tubule of human kidneys and in the placenta, playing a central role in uric acid homeostasis. GLUT9 shares the least homology with other members of the glucose transporter family, especially with the glucose transporting members GLUT1-4 and is the only member of the GLUT family to transport urate. The recently published high-resolution structure of XylE, a bacterial D-xylose transporting homologue, yields new insights into the structural foundation of this GLUT family of proteins. While this represents a huge milestone, it is unclear if human GLUT9 can benefit from this advancement through subsequent structural based targeting and mutagenesis. Little progress has been made toward understanding the mechanism of GLUT9 since its discovery in 2000. Before work can begin on resolving the mechanisms of urate transport we must determine methods to express, purify and analyze hGLUT9 using a model system adept in expressing human membrane proteins. Here, we describe the surface expression, purification and isolation of monomeric protein, and functional analysis of recombinant hGLUT9 using the Xenopus laevis oocyte system. In addition, we generated a new homology-based high-resolution model of hGLUT9 from the XylE crystal structure and utilized our purified protein to generate a low-resolution single particle reconstruction. Interestingly, we demonstrate that the functional protein extracted from the Xenopus system fits well with the homology-based model allowing us to generate the predicted urate-binding pocket and pave a path for subsequent mutagenesis and structure-function studies.
| S-EPMC4186817 | biostudies-literature
Project description:The Na+/bile salt co-transporter of the pig ileal brush border membrane has been expressed in Xenopus laevis oocytes. Injection of pig ileal poly (A)+ RNA into oocytes resulted in the functional expression of an Na(+)-gradient-stimulated taurocholate uptake within 2-5 days. The expressed Na(+)-dependent taurocholate uptake exhibited saturation kinetics (apparent Km of 48 microM), and displayed similar competitive substrate inhibition by taurodeoxycholate as the native brush border Na+/bile salt co-transporter studied in pig ileal brush border membrane vesicles. Interestingly, injection of pig proximal and mid intestinal poly (A)+ RNA into oocytes also resulted in the expression of the Na+/bile salt co-transporter, though the Na(+)-dependent transport of bile salts does not occur in brush border membrane vesicles (BBMV) isolated from pig proximal and mid intestine. This suggests that the mRNA coding for the co-transporter is present in the enterocytes lining the whole length of the small intestine, but that the function is only expressed in the brush border of the distal small intestine. The transport of D-glucose into BBMV, and the transport of methyl-alpha-D-glucopyranoside (a non-metabolizable hexose derivative) into oocytes were used throughout the study as methods of confirming the integrity of vesicles and oocytes.
| S-EPMC1132864 | biostudies-other
Project description:The ribonucleotide and deoxyribonucleotide contents of eggs and oocytes of Xenopus laevis were measured. Eggs contained most deoxyribonucleotide in the form of triphosphates. dCTP, dTTP, dATP and dGTP were present in similar amounts. The egg contained sufficient deoxynucleotide triphosphate to make approximately 2500 nuclei. Oocytes contained less pyrimidine deoxyribonucleoside triphosphates than did eggs, and purine deoxyribonucleoside triphosphates were not detected. These differences may be correlated with the ability of eggs to induce nuclear DNA synthesis, a property not shown by oocytes. Both oocytes and eggs seem to contain non-phosphorylated, alpha-unsubstituted aldehydes, which may be deoxyribose derivatives. Eggs and oocytes contain similar amounts of ribonucleoside triphosphates. The low rate of RNA synthesis found in eggs, but not in oocytes, is therefore not caused by simple precursor control.
| S-EPMC1178701 | biostudies-other
Project description:Observations on lactate transport in brain cells and cardiac myocytes indicate the presence of a high-affinity monocarboxylate transporter. The rat monocarboxylate transporter isoform MCT2 was analysed by expression in Xenopus laevis oocytes and the results were compared with the known characteristics of lactate transport in heart and brain. Monocarboxylate transport via MCT2 was driven by the H(+) gradient over the plasma membrane. Uptake of lactate strongly increased with decreasing pH, showing half-maximal stimulation at pH 7.2. A wide variety of monocarboxylates and ketone bodies, including lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, 2-oxoisovalerate and 2-oxoisohexanoate, were substrates of MCT2. All substrates had a high affinity for MCT2. For lactate a K(m) value of 0.74+/-0.07 mM was determined at pH 7.0. For the other substrates, K(i) values between 100 microM and 1 mM were measured for inhibition of lactate transport, which is about one-tenth of the corresponding values for the ubiquitously expressed monocarboxylate transporter isoform MCT1. Monocarboxylate transport via MCT2 could be inhibited by alpha-cyano-4-hydroxycinnamate, anion-channel inhibitors and flavonoids. It is suggested that cells which express MCT2 preferentially use lactate and ketone bodies as energy sources.
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Project description:MicroRNA-protein complexes (microRNPs) can activate translation of target reporters and specific mRNAs in quiescent (i.e., G0) mammalian cell lines. Induced quiescent cells, like folliculated immature oocytes, have high levels of cAMP that activate protein kinase AII (PKAII) to maintain G0 and immature states. We report microRNA-mediated up-regulated expression of reporters in immature Xenopus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that maintenance of cAMP levels and downstream PKAII signaling are required for microRNA-mediated up-regulated expression in oocytes. We identify an important, endogenous cell state regulator, Myt1 kinase, as a natural target of microRNA-mediated up-regulation in response to xlmiR16, ensuring maintenance of oocyte immaturity. Our data reveal the physiological relevance of cAMP/PKAII-controlled posttranscriptional gene expression activation by microRNAs in maintenance of the immature oocyte state.
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Project description:We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca2+ release comes from a region of width approximately 450 nm, that the release duration is peaked around 18 s and that the underlying Ca2+ currents range between 0.12 and 0.95 pA. All these parameters are independent of IP(3) concentration. We explore what distributions of channels that open during a puff, N(p), and what relations between current and number of open channels, I(N(p)), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al. To this end, we use simple "mean field" models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(N(p)) approximately N(p) for small N(p) and I(N(p)) approximately N(p)(1/alpha) (alpha > 1) for large N(p), provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of approximately 400 puffs obtained using the dye Oregon green.
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