Cloning and functional characterization of GAD67 upstream regulatory regions in Xenopus laevis

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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Molecular and functional characterization of Xenopus laevis N-methyl-d-aspartate receptors

Molecular and Cellular Neuroscience ◽

10.1016/j.mcn.2009.06.004 ◽

2009 ◽

Vol 42 (2) ◽

pp. 116-127 ◽

Cited By ~ 5

Author(s):

Carsten Schmidt ◽

Michael Hollmann

Keyword(s):

Xenopus Laevis ◽

Functional Characterization

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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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Functional characterization of high-affinity Na+/dicarboxylate cotransporter found in Xenopus laevis kidney and heart

AJP Cell Physiology ◽

10.1152/ajpcell.00295.2004 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1159-C1168 ◽

Cited By ~ 9

Author(s):

Naomi Oshiro ◽

Ana M. Pajor

Keyword(s):

Xenopus Laevis ◽

Cdna Sequence ◽

Citric Acid Cycle ◽

Functional Characterization ◽

Activation Kinetics ◽

Electrophysiological Properties ◽

High Affinity ◽

The Family ◽

Kidney Liver

The SLC13 gene family includes sodium-coupled transporters for citric acid cycle intermediates and sulfate. The present study describes the sequence and functional characterization of a SLC13 family member from Xenopus laevis, the high-affinity Na+/dicarboxylate cotransporter xNaDC-3. The cDNA sequence of xNaDC-3 codes for a protein of 602 amino acids that is ∼70% identical to the sequences of mammalian NaDC-3 orthologs. The message for xNaDC-3 is found in the kidney, liver, intestine, and heart. The xNaDC-3 has a high affinity for substrate, including a Km for succinate of 4 μM, and it is inhibited by the NaDC-3 test substrates 2,3-dimethylsuccinate and adipate. The transport of succinate by xNaDC-3 is dependent on sodium, with sigmoidal activation kinetics, and lithium can partially substitute for sodium. As with other members of the family, xNaDC-3 is electrogenic and exhibits inward substrate-dependent currents in the presence of sodium. However, other electrophysiological properties of xNaDC-3 are unique and involve large leak currents, possibly mediated by anions, that are activated by binding of sodium or lithium to a single site.

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Cloning of the 5′ regulatory regions and functional characterization of the core promoters of ovine PLAU (u-PA) and SERPIN1 (PAI-1)

Gene ◽

10.1016/j.gene.2011.08.020 ◽

2011 ◽

Vol 489 (1) ◽

pp. 11-20 ◽

Cited By ~ 1

Author(s):

A.D. Lampidonis ◽

G. Theodorou ◽

C. Pecorini ◽

R. Rebucci ◽

A. Baldi ◽

...

Keyword(s):

Functional Characterization ◽

Regulatory Regions ◽

Core Promoters ◽

The Core ◽

Pai 1

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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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Molecular and functional characterization of a short-type peptidoglycan recognition protein, PGRP-S in the amphibian Xenopus laevis

Developmental & Comparative Immunology ◽

10.1016/j.dci.2019.04.003 ◽

2019 ◽

Vol 98 ◽

pp. 13-19 ◽

Cited By ~ 5

Author(s):

Jing Hou ◽

Zhen Gan ◽

Shan Nan Chen ◽

Pin Nie

Keyword(s):

Xenopus Laevis ◽

Functional Characterization ◽

Peptidoglycan Recognition Protein ◽

Recognition Protein ◽

Short Type

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Molecular characterization of the murine Slc26a6 anion exchanger: functional comparison with Slc26a1

AJP Renal Physiology ◽

10.1152/ajprenal.00079.2002 ◽

2002 ◽

Vol 283 (4) ◽

pp. F826-F838 ◽

Cited By ~ 144

Author(s):

Qizhi Xie ◽

Rick Welch ◽

Adriana Mercado ◽

Michael F. Romero ◽

David B. Mount

Keyword(s):

Alternative Splicing ◽

Xenopus Laevis ◽

Proximal Tubule ◽

Molecular Characterization ◽

Anion Exchanger ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Membrane Hyperpolarization ◽

Oxalate Transport

We report the molecular and functional characterization of murine Slc26a6, the putative apical chloride-formate exchanger of the proximal tubule. The Slc26a6 transcript is expressed in several tissues, including kidney. Alternative splicing of the second exon generates two distinct isoforms, denoted Slc26a6a and Slc26a6b, which differ in the inclusion of a 23-residue NH2-terminal extension. Functional comparison with murine Slc26a1, the basolateral oxalate exchanger of the proximal tubule, reveals a number of intriguing differences. Whereas Slc26a6 is capable of Cl−, SO[Formula: see text], formate, and oxalate uptake when expressed in Xenopus laevis oocytes, Slc26a1 transports only SO[Formula: see text] and oxalate. Measurement of intracellular pH during the removal of extracellular Cl− in the presence and absence of HCO[Formula: see text] indicates that Slc26a6 functions as both a Cl−/HCO[Formula: see text] and a Cl−/OH− exchanger; simultaneous membrane hyperpolarization during these experimental maneuvers reveals that HCO[Formula: see text] and OH− transport mediated by Slc26a6 is electrogenic. Cis-inhibition and efflux experiments indicate that Slc26a6 can mediate the exchange of both Cl− and SO[Formula: see text]with a number of substrates, including formate and oxalate. In contrast, SO[Formula: see text] and oxalate transport by Slc26a1 are mutually cis-inhibited but activated significantly by extracellular halides, lactate, and formate. The data indicate that Slc26a6 encodes an apical Cl−/formate/oxalate and Cl−/base exchanger and reveal significant mechanistic differences between apical and basolateral oxalate exchangers of the proximal tubule.

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Functional Characterization of the Oxantel-Sensitive Acetylcholine Receptor from Trichuris muris

10.20944/preprints202106.0476.v1 ◽

2021 ◽

Author(s):

Tina V. A. Hansen ◽

Richard K. Grencis ◽

Mohamed Issouf ◽

Cédric Neveu ◽

Claude L. Charvet

Keyword(s):

Single Dose ◽

Mouse Model ◽

Xenopus Laevis ◽

Acetylcholine Receptor ◽

Drug Target ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Trichuris Muris ◽

Electrode Voltage

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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