Assessment of natural variation in the first pore domain of the tomato HKT1;2 transporter and characterization of mutated versions of SlHKT1;2 expressed in Xenopus laevis oocytes and via complementation of the salt sensitive athkt1;1 mutant

The human whipworm, Trichuris trichiura, is estimated to infect 289.6 million people globally. Control of human trichuriasis is a particular challenge, as most anthelmintics have a limited single-dose efficacy, with the striking exception of the narrow-spectrum anthelmintic, oxantel. We recently identified a novel ACR-16-like subunit from the pig whipworm, T. suis which gave rise to a functional acetylcholine receptor (nAChR) preferentially activated by oxantel. However, there is no ion channel described in the mouse model parasite T. muris so far. Here, we have identified the ACR-16-like and ACR-19 subunits from T. muris, and performed the functional characterization of the receptors in Xenopus laevis oocytes using two-electrode voltage-clamp electrophysiology. We found that the ACR-16-like subunit from T. muris formed a homomeric receptor gated by acetylcholine whereas the ACR-19 failed to create a functional channel. The subsequent pharmacological analysis of the Tmu-ACR-16-like receptor revealed that acetylcholine and oxantel were equally potent. The Tmu-ACR-16-like was more responsive to the toxic agonist epibatidine, but insensitive to pyrantel, in contrast to the Tsu-ACR-16-like receptor. These findings confirm that the ACR-16-like nAChR from Trichuris spp. is a preferential drug target for oxantel, and highlights the pharmacological difference between Trichuris species.

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Functional characterization of leucine transport induced in Xenopus laevis oocytes injected with mRNA isolated from midguts of lepidopteran larvae (Philosamia cynthia).

Journal of Experimental Biology ◽

10.1242/jeb.198.4.961 ◽

1995 ◽

Vol 198 (4) ◽

pp. 961-966

Author(s):

V F Sacchi ◽

C Perego ◽

S Magagnin

Keyword(s):

Xenopus Laevis ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Fourfold Increase ◽

Leucine Uptake ◽

Lepidopteran Larvae ◽

Nacl Concentration ◽

Leucine Transport ◽

The Difference

The injection of poly(A)+ mRNA prepared from Philosamia cynthia midgut caused time- and dose-dependent increases of leucine transport in Xenopus laevis oocytes, with an increase in leucine uptake 1.5-3 times that of oocytes injected with water. When the NaCl concentration was reduced from 100 to 5 mmol l-1, the difference between mRNA- and water-injected oocytes was greater and a fourfold increase of L-leucine uptake was measured. D-Leucine (10 mmol l-1) completely inhibited the induced uptake of 0.1 mmol l-1 L-leucine. The newly expressed component of L-leucine uptake increased at alkaline pH and was abolished by incubation for 15 min with 15 mmol l-1 phenylglyoxal. The mean Km values, calculated using Na+ activation curves of leucine uptake, were 23.3 +/- 6.1 mmol l-1 in water-injected oocytes and 0.4 +/- 0.2 mmol l-1 for the newly expressed component of leucine uptake in mRNA-injected oocytes. On the basis of these results, we conclude that the increase of L-leucine uptake in mRNA-injected oocytes was due to the expression of a new transport system, which differs from the endogenous ones and shares many features with that found previously in Philosamia cynthia midgut.

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Characterization of the monocarboxylate transporter 1 expressed in Xenopus laevis oocytes by changes in cytosolic pH

Biochemical Journal ◽

10.1042/bj3330167 ◽

1998 ◽

Vol 333 (1) ◽

pp. 167-174 ◽

Cited By ~ 211

Author(s):

Stefan BRÖER ◽

Hans-Peter SCHNEIDER ◽

Angelika BRÖER ◽

Basim RAHMAN ◽

Bernd HAMPRECHT ◽

...

Keyword(s):

Xenopus Laevis ◽

Intracellular Ph ◽

Expression System ◽

Lactate Concentration ◽

Monocarboxylate Transporter ◽

Kinetic Properties ◽

Xenopus Laevis Oocytes ◽

Lactate Transport ◽

Monocarboxylate Transporter 1

Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)-expressing Xenopus laevis oocytes by using pH-sensitive microelectrodes and [14C]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, d,l-3-hydroxybutyrate, acetoacetate, α-oxoisohexanoate and α-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor α-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.

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Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes

Biology of the Cell ◽

10.1016/s0248-4900(03)00058-3 ◽

2003 ◽

Vol 95 (5) ◽

pp. 311-320 ◽

Cited By ~ 1

Author(s):

Franck Chesnel ◽

Christophe Heligon ◽

Laurent Richard-Parpaillon ◽

Daniel Boujard

Keyword(s):

Xenopus Laevis ◽

Molecular Cloning ◽

Adaptor Protein ◽

Xenopus Laevis Oocytes

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The Use of Xenopus laevis Oocytes for the Functional Characterization of Heterologously Expressed Membrane Proteins

Cellular Physiology and Biochemistry ◽

10.1159/000016341 ◽

2000 ◽

Vol 10 (1-2) ◽

pp. 1-12 ◽

Cited By ~ 116

Author(s):

Carsten A. Wagner ◽

Björn Friedrich ◽

Iwan Setiawan ◽

Florian Lang ◽

Stefan Bröer

Keyword(s):

Membrane Proteins ◽

Xenopus Laevis ◽

Functional Characterization ◽

Xenopus Laevis Oocytes

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Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4257-4269.1988 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4257-4269

Author(s):

A Shimamura ◽

D Tremethick ◽

A Worcel

Keyword(s):

Xenopus Laevis ◽

Transcriptional Activation ◽

High Speed ◽

Histone H1 ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

5S Rna ◽

Rna Transcription

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

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