scholarly journals Molecular cloning and functional characterization of a high-affinity zinc importer (DrZIP1) from zebrafish (Danio rerio)

2005 ◽  
Vol 388 (3) ◽  
pp. 745-754 ◽  
Author(s):  
Andong QIU ◽  
Majid SHAYEGHI ◽  
Christer HOGSTRAND
Keyword(s):  
Danio Rerio ◽  
Oncorhynchus Tshawytscha ◽  
Functional Characterization ◽  
Cell Types ◽  
Zinc Uptake ◽  
Takifugu Rubripes ◽  
Xenopus Laevis Oocytes ◽  
High Affinity ◽  
Tetraodon Nigroviridis ◽  
Cellular Zinc

Zinc is a vital micronutrient to all organisms and a potential toxicant to aquatic animals. It is therefore of importance to understand the mechanism of zinc regulation. In the present study, we molecularly cloned and functionally characterized a zinc transporter of the SLC39A family [commonly referred to as the ZIP (Zrt- and Irt-related protein) family] from the gill of zebrafish (Danio rerio) (DrZIP1). DrZIP1 protein was found to localize at the plasma membrane and to function as a zinc uptake transporter when being expressed in either chinook salmon (Oncorhynchus tshawytscha) embryonic 214 cells or Xenopus laevis oocytes. In comparison with pufferfish transporter proteins (FrZIP2 and FrECaC) that are known to facilitate cellular zinc uptake, DrZIP1 appears to have high affinity to bind and transport zinc, suggesting that it may be a high-affinity zinc uptake transporter (Km<0.5 μM) in fish. Orthologues of DrZIP1 were also identified in both freshwater and seawater pufferfish (Tetraodon nigroviridis and Takifugu rubripes), indicating that these proteins may be functionally conserved among different fish species. DrZIP1 mRNA is expressed in all the tissues examined in the present study and thus DrZIP1 may be a constitutive zinc uptake transporter in many cell types of zebrafish.

scholarly journals Functional expression of a low-affinity zinc uptake transporter (FrZIP2) from pufferfish (Takifugu rubripes) in MDCK cells

2005 ◽  
Vol 390 (3) ◽  
pp. 777-786 ◽  
Author(s):  
Andong Qiu ◽  
Christer Hogstrand
Keyword(s):  
Oncorhynchus Tshawytscha ◽  
Mdck Cells ◽  
Functional Characterization ◽  
Zinc Uptake ◽  
Takifugu Rubripes ◽  
Xenopus Laevis Oocytes ◽  
Dependent Manner ◽  
Zinc Transport ◽  
Uptake Activity ◽  
Cellular Zinc

Zinc is a vital micronutrient to all organisms and it is therefore very important to determine the mechanisms that regulate cellular zinc uptake. Previously, we reported on zinc uptake transporters from zebrafish (Danio rerio; DrZIP1) and Fugu pufferfish (Takifugu rubripes; FrZIP1) that facilitated cellular zinc uptake of high affinity (Km<0.5 μM) in both CHSE214 [chinook salmon (Oncorhynchus tshawytscha) embryonic 214] cells and Xenopus laevis oocytes. To investigate additional biochemical pathways of zinc uptake in fish, we molecularly cloned the second fish member (FrZIP2) of the SLC39 subfamily II from Fugu pufferfish gill. Functional characterization suggests that FrZIP2 stimulated zinc uptake in a temperature-, time-, concentration- and pH-dependent manner when overexpressed in MDCK cells (Madin–Darby canine kidney cells). In comparison with FrZIP1 and DrZIP1 (<0.5 μM), FrZIP2 appears to represent a low-affinity zinc uptake transporter (Km=13.6 μM) in pufferfish. FrZIP2 protein was selective for zinc, but it might also transport Cu2+, since 20 times excess of Cu2+ completely abolished its zinc uptake activity. The zinc uptake by FrZIP2 was stimulated in a slightly acidic medium (pH 5.5–6.5) and was completely blocked at pH 7.5 and above, suggesting that an inward H+ gradient might provide a driving force for zinc transport by FrZIP2. Furthermore, FrZIP2-mediated zinc uptake activity was slightly inhibited by 0.5 mM HCO3−, indicating that FrZIP2 may employ a different mechanism of zinc translocation from the assumed HCO3−-coupled zinc transport used by human SLC39A2. The FrZIP2 gene was expressed in all the tissues studied herein, with especially high levels in the ovary and intestines. Thus FrZIP2 may be a prominent zinc uptake transporter of low affinity in many cell types of Fugu pufferfish.

Cloning, functional characterization, and localization of a rat renal Na+-dicarboxylate transporter

1998 ◽  
Vol 275 (2) ◽  
pp. F298-F305 ◽  
Author(s):  
Takashi Sekine ◽  
Seok Ho Cha ◽  
Makoto Hosoyamada ◽  
Yoshikatsu Kanai ◽  
Nobuaki Watanabe ◽  
...  
Keyword(s):  
Amino Acids ◽  
Positive Charge ◽  
Polyclonal Antibodies ◽  
Functional Characterization ◽  
Xenopus Laevis Oocytes ◽  
Coupling Ratio ◽  
High Affinity ◽  
Luminal Membrane ◽  
Sodium Dependent ◽  
Inward Currents

We report here the isolation, functional characterization, tissue distribution, and membrane localization of rat renal Na+-dicarboxylate transporter (rNaDC-1). rNaDC-1 consists of 2,245 nucleotides, and the deduced amino acid sequence showed 73% and 75% identity to rabbit and human NaDC-1, respectively. When expressed in Xenopus laevis oocytes, rNaDC-1 mediated sodium-dependent uptake of di- and tricarboxylates. Substrates of rNaDC-1 evoked inward currents in oocytes expressed with rNaDC-1; succinate, α-ketoglutarate, and glutarate were relatively high-affinity substrates, and citrate was a low-affinity substrate of rNaDC-1. The coupling ratio of citrate to charge was determined to be 1:1 at pH 7.4; influx of one positive charge per citrate molecule suggests a symport of three Na+with a divalent citrate. Expression of rNaDC-1 mRNA was detected in the kidney and the small and large intestines. Immunohistochemistry using polyclonal antibodies raised against the 14 amino acids at the COOH terminus of rNaDC-1 revealed that rNaDC-1 is localized exclusively in the luminal membrane of S2 and S3.

scholarly journals Functional Characterization of Zebrafish (Danio rerio) Bcl10

PLoS ONE ◽  
2015 ◽  
Vol 10 (4) ◽  
pp. e0122365 ◽  
Author(s):  
Pellegrino Mazzone ◽  
Ivan Scudiero ◽  
Angela Ferravante ◽  
Marina Paolucci ◽  
Luca E. D’Andrea ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Characterization

scholarly journals Functional characterization of a full length pregnane X receptor, expression in vivo, and identification of PXR alleles, in Zebrafish (Danio rerio)

2013 ◽  
Vol 142-143 ◽  
pp. 447-457 ◽  
Author(s):  
Afonso C.D. Bainy ◽  
Akira Kubota ◽  
Jared V. Goldstone ◽  
Roger Lille-Langøy ◽  
Sibel I. Karchner ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Functional Characterization ◽  
Pregnane X Receptor ◽  
Full Length ◽  
Receptor Expression

Subfunctionalization of Duplicate mitf Genes Associated With Differential Degeneration of Alternative Exons in Fish

Genetics ◽  
2002 ◽  
Vol 161 (1) ◽  
pp. 259-267 ◽  
Author(s):  
Joachim Altschmied ◽  
Jacqueline Delfgaauw ◽  
Brigitta Wilde ◽  
Jutta Duschl ◽  
Laurence Bouneau ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Evolutionary History ◽  
Single Gene ◽  
Regulatory Elements ◽  
Fugu Rubripes ◽  
Gene Encoding ◽  
Tetraodon Nigroviridis ◽  
Mammalian Lineage ◽  
History Of ◽  
Fish Proteins

Abstract The microphthalmia-associated transcription factor (MITF) exists in at least four isoforms. These are generated in higher vertebrates using alternative 5′ exons and promoters from a single gene. Two separate genes (mitf-m and mitf-b), however, are present in different teleost fish species including the poeciliid Xiphophorus, the pufferfishes Fugu rubripes and Tetraodon nigroviridis, and the zebrafish Danio rerio. Fish proteins MITF-m and MITF-b correspond at both the structural and the expression levels to one particular bird/mammalian MITF isoform. In the teleost lineage subfunctionalization of mitf genes after duplication at least 100 million years ago is associated with the degeneration of alternative exons and, probably, regulatory elements and promoters. For example, a remnant of the first exon specific for MITF-m is detected within the pufferfish gene encoding MITF-b. Retracing the evolutionary history of mitf genes in vertebrates uncovered the differential recruitment of new introns specific for either the teleost or the bird/mammalian lineage.

scholarly journals Functional characterization of recombinant interleukin (IL)-17A/F1 in the Japanese pufferfish (Takifugu rubripes)

2016 ◽  
Vol 53 ◽  
pp. 79-80 ◽  
Author(s):  
Jun-ichi Hikima ◽  
Koshin Mihara ◽  
Shun Maekawa ◽  
Han-Ching Wang ◽  
Takashi Aoki ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Takifugu Rubripes

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

2021 ◽  
Vol 14 (7) ◽  
pp. 698
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.

Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling

2019 ◽  
Vol 476 (1) ◽  
pp. 39-50 ◽  
Author(s):  
Martin A. Rossotti ◽  
Kevin A. Henry ◽  
Henk van Faassen ◽  
Jamshid Tanha ◽  
Deborah Callaghan ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Growth Factor Receptor ◽  
Single Domain ◽  
Dna Immunization ◽  
Egfr Signaling ◽  
High Affinity ◽  
Single Domain Antibodies ◽  
Human Igg1 ◽  
Protein Immunization

Abstract Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high affinity (KD range: 1–40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the VHHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VHH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC50: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.

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