scholarly journals Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider Cupiennius salei to Potentiate Toxicity on Insects

Toxins ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 250 ◽  
Author(s):  
Benjamin Clémençon ◽  
Lucia Kuhn-Nentwig ◽  
Nicolas Langenegger ◽  
Lukas Kopp ◽  
Steve Peigneur ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Direct Interaction ◽  
Structural Type ◽  
Structure Type ◽  
Open Loop ◽  
Cytolytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Cupiennius Salei

The venom of Cupiennius salei is composed of dozens of neurotoxins, with most of them supposed to act on ion channels. Some insecticidal monomeric neurotoxins contain an α-helical part besides their inhibitor cystine knot (ICK) motif (type 1). Other neurotoxins have, besides the ICK motif, an α-helical part of an open loop, resulting in a heterodimeric structure (type 2). Due to their low toxicity, it is difficult to understand the existence of type 2 peptides. Here, we show with the voltage clamp technique in oocytes of Xenopus laevis that a combined application of structural type 1 and type 2 neurotoxins has a much more pronounced cytolytic effect than each of the toxins alone. In biotests with Drosophila melanogaster, the combined effect of both neurotoxins was enhanced by 2 to 3 log units when compared to the components alone. Electrophysiological measurements of a type 2 peptide at 18 ion channel types, expressed in Xenopus laevis oocytes, showed no effect. Microscale thermophoresis data indicate a monomeric/heterodimeric peptide complex formation, thus a direct interaction between type 1 and type 2 peptides, leading to cell death. In conclusion, peptide mergers between both neurotoxins are the main cause for the high cytolytic activity of Cupiennius salei venom.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

The acquisition of L3 variation among early bilinguals

2019 ◽  
Vol 10 (5) ◽  
pp. 657-689
Author(s):  
Mihi Park ◽  
Rebecca Lurie Starr
Keyword(s):  
Learning Experience ◽  
Prior Experience ◽  
Structure Type ◽  
Home Language ◽  
Bilingual Learners ◽  
Language Background ◽  
Argument Realization ◽  
Korean Learners

Abstract The present study investigates whether prior experience with formal study of an L2 influences L3 Korean learners’ Type 1 variation (i.e., use of obligatory forms) and Type 2 variation (i.e., variation between alternative acceptable variants). The patterns of variation in Korean argument realization of early bilingual learners (English-Chinese/Malay/Indonesian/Tamil) of L3 Korean were assessed in light of the distribution of variants present in classroom input, learners’ prior L2 learning experience and home language background, argument animacy and number, and familiarity of verb structure type. Our findings demonstrate that prior experience with a typologically-similar L2 facilitates acquisition of grammatical patterns as well as acquisition of native-like patterns of variation between grammatical forms that are constrained by a range of internal linguistic factors. Any L2 experience, regardless of typological proximity, is found to facilitate acquisition of internal linguistic constraints, but not acquisition of grammatical patterns.

Effects of Ethanol and Anesthetics on Type 1 and 5 Metabotropic Glutamate Receptors Expressed in Xenopus laevis Oocytes

1998 ◽  
Vol 53 (1) ◽  
pp. 148-156 ◽  
Author(s):  
Kouichiro Minami ◽  
Robert W. Gereau ◽  
Makiko Minami ◽  
Stephen F. Heinemann ◽  
R. Adron Harris
Keyword(s):  
Xenopus Laevis ◽  
Glutamate Receptors ◽  
Metabotropic Glutamate Receptors ◽  
Xenopus Laevis Oocytes ◽  
Metabotropic Glutamate

Cloning and functional expression of an SGLT-1-like protein from the Xenopus laevisintestine

1999 ◽  
Vol 276 (5) ◽  
pp. G1251-G1259 ◽  
Author(s):  
Katsumi Nagata ◽  
Naohiro Hori ◽  
Kenzo Sato ◽  
Kunimasa Ohta ◽  
Hideaki Tanaka ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Functional Expression ◽  
Hill Coefficient ◽  
Xenopus Laevis Oocytes ◽  
Membrane Hyperpolarization ◽  
Apparent Concentration ◽  
Inward Currents ◽  
Rapid Amplification ◽  
The Hill

A cDNA encoding an Na+-glucose cotransporter type 1 (SGLT-1)-like protein was cloned from the Xenopus laevis intestine by the 5′- and 3′-rapid amplification of cDNA ends method. The deduced amino acid sequence was 673 residues long, with a predicted mass of 74.1 kDa and 52–53% identity to mammalian SGLT-1s. This gene was expressed in the small intestine and kidney, reflecting a tissue distribution similar to that of SGLT-1. The function of the protein was studied using the two-microelectrode voltage-clamp technique after injection of cRNA into Xenopus laevis oocytes. Perfusion with myo-inositol elicited about twofold larger inward currents than perfusion withd-glucose. The order of the substrate specificity was myo-inositol > d-glucose >d-galactose ≥ α-methyl-d-glucoside. The current induced by myo-inositol increased with membrane hyperpolarization and depended on external myo-inositol and Na+: the apparent Michaelis-Menten constant was 0.25 ± 0.07 (SD) mM with myo-inositol, whereas the apparent concentration for half-maximal activation was 12.5 ± 1.0 mM and the Hill coefficient was 1.6 ± 0.1 with Na+. In conclusion, the cloned protein shares features with both SGLT-1 and the Na+- myo-inositol cotransporter.

scholarly journals Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion

1996 ◽  
Vol 319 (3) ◽  
pp. 755-760 ◽  
Author(s):  
Roland B GREGORY ◽  
Greg J BARRITT
Keyword(s):  
Endoplasmic Reticulum ◽  
Xenopus Laevis ◽  
Membrane Fusion ◽  
Direct Interaction ◽  
Protein S ◽  
Detectable Effect ◽  
Xenopus Laevis Oocytes ◽  
Endoplasmic Reticulum Membrane ◽  
Rate Of Increase

The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro-Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+o-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trpl cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2–17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5´-[γ-thio]triphosphate (GTP[S]), guanosine 5´-[β,γ-imido]triphosphate (p[NH]ppG) and AlF4-, but not guanosine 5´-[β-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] or AlF4-. Staurosporine largely prevented the inhibition of store-activated Ca2+ inflow by GTP[S]. It is concluded that membrane fusion processes are unlikely to be involved in the link between the release of Ca2+ from the endoplasmic reticulum and activation of SACCs. The idea that this link is achieved by direct interaction of a protein(s) in the endoplasmic reticulum membrane with the SACC protein is briefly discussed.

scholarly journals Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product.

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414 ◽  
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

Volume sensitivity of cation-Cl− cotransporters is modulated by the interaction of two kinases: Ste20-related proline-alanine-rich kinase and WNK4

2006 ◽  
Vol 290 (1) ◽  
pp. C134-C142 ◽  
Author(s):  
Kenneth B. E. Gagnon ◽  
Roger England ◽  
Eric Delpire
Keyword(s):  
Catalytic Activity ◽  
Xenopus Laevis Oocytes ◽  
Transport Function ◽  
Yeast Two Hybrid ◽  
Functional Interaction ◽  
Catalytic Domains ◽  
Volume Sensitivity ◽  
Two Hybrid

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl− cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl− cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.

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