Effects of Glutamic Acid on C-type Inactivation of Kvl.4△N Channel

Objectives Acidosis has an inhibitory effect on the inactivation of Kv1.4 ΔN channel through the position H508. So in order to show the effects of glutamic acid on the mutant Kv 1.4 channel that lacks N-type inactivation （Kv1.4 Δ2-146）, we studied in the expression system of the Xenopus oocytes. Methods The two-electrode voltage-clamp technique （TEV） was used to record the currents. Results Acidosis increased fKv1.4 Δ2-146 C-type inactivation. After application of glutamic acid （1 mmol/L） to Kv1.4 Δ2-146 increased C-type inactivation further, changed inactivation time constants from （2.02 ± 0.39 s ） to （1.71 ± 0.23 s） （P〈 0.05） at ＋50mv, and shifted the steady-state inactivation curves of Kv1.4 ΔN to positive potential, which was from （-44.30 ± 0.59 mV） to （-39.88 ± 0.29 mV）（P〈0.05）. and slowed the rate of recovery from inactivation, which was from （1.64 ± 0.19 s） to （1.91 ± 0.23 s）（P〈 0.05）. Conclusions Together, these results suggest that 1 mmol/L glutamic acid accelerates the C-type inactivation of Kv1.4 ΔN in pH 6.8.     

Regulation of early Xenopus development by the PIAS genes

Originally identified as cytokine inhibitors, protein inhibitors of activated STAT (PIAS) are shown to regulate activities of a plethora of proteins and influence diverse processes such as immune response, cancer formation, and cell cycle progression. However, the roles of PIAS during vertebrate embryogenesis are less understood. In this study, we report isolation and initial characterization of all four PIAS genes from Xenopus laevis. The Xenopus PIAS genes are expressed throughout early development and have overlapping and distinct expression patterns, with, for example, high levels of PIAS2 in the notochord and strong expression of PIAS4 in the neural and neural crest derivatives. Overexpression of PIAS disrupts mesoderm induction and impairs body axis formation. PIAS proteins have differential ability to regulate signals from the growth factors activin, bone morphogenetic protein 4 (BMP4), and Wnt8. Our data suggest that Xenopus PIAS play important roles in mesodermal induction and patterning during early frog development. Developmental Dynamics 240:2120–2126, 2011. © 2011 Wiley‐Liss, Inc.     

BMP inhibition initiates neural induction via FGF signaling and Zic genes

Neural induction is the process that initiates nervous system development in vertebrates. Two distinct models have been put forward to describe this phenomenon in molecular terms. The default model states that ectoderm cells are fated to become neural in absence of instruction, and do so when bone morphogenetic protein (BMP) signals are abolished. A more recent view implicates a conserved role for FGF signaling that collaborates with BMP inhibition to allow neural fate specification. Using the Xenopus embryo, we obtained evidence that may unite the 2 views. We show that a dominant-negative R-Smad, Smad5-somitabun—unlike the other BMP inhibitors used previously—can trigger conversion of Xenopus epidermis into neural tissue in vivo. However, it does so only if FGF activity is uncompromised. We report that this activity may be encoded by FGF4, as its expression is activated upon BMP inhibition, and its knockdown suppresses endogenous, as well as ectopic, neural induction by Smad5-somitabun. Supporting the importance of FGF instructive activity, we report the isolation of 2 immediate early neural targets, zic3 and foxD5a. Conversely, we found that zic1 can be activated by BMP inhibition in the absence of translation. Finally, Zic1 and Zic3 are required together for definitive neural fate acquisition, both in ectopic and endogenous situations. We propose to merge the previous models into a unique one whereby neural induction is controlled by BMP inhibition, which activates directly, and, via FGF instructive activity, early neural regulators such as Zic genes.     

Distribution of single wall carbon nanotubes in the Xenopus laevis embryo after microinjection

Single wall carbon nanotubes (SWCNTs) are advanced materials with the potential for a myriad of diverse applications, including biological technologies and large‐scale usage with the potential for environmental impacts. SWCNTs have been exposed to developing organisms to determine their effects on embryogenesis, and results have been inconsistent arising, in part, from differing material quality, dispersion status, material size, impurity from catalysts and stability. For this study, we utilized highly purified SWCNT samples with short, uniform lengths (145 ± 17 nm) well dispersed in solution. To test high exposure doses, we microinjected > 500 µg ml^–1 SWCNT concentrations into the well‐established embryogenesis model, Xenopus laevis, and determined embryo compatibility and subcellular localization during development. SWCNTs localized within cellular progeny of the microinjected cells, but were heterogeneously distributed throughout the target‐injected tissue. Co‐registering unique Raman spectral intensity of SWCNTs with images of fluorescently labeled subcellular compartments demonstrated that even at regions of highest SWCNT concentration, there were no gross alterations to subcellular microstructures, including filamentous actin, endoplasmic reticulum and vesicles. Furthermore, SWCNTs did not aggregate and localized to the perinuclear subcellular region. Combined, these results suggest that purified and dispersed SWCNTs are not toxic to X. laevis animal cap ectoderm and may be suitable candidate materials for biological applications. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of the Larval Amphibian Growth and Development Assay: Effects of benzophenone‐2 exposure in Xenopus laevis from embryo to juvenile

The Larval Amphibian Growth and Development Assay (LAGDA) is a globally harmonized chemical testing guideline developed by the U.S. Environmental Protection Agency in collaboration with Japan's Ministry of Environment to support risk assessment. The assay is employed as a higher tiered approach to evaluate effects of chronic chemical exposure throughout multiple life stages in a model amphibian species, Xenopus laevis. To evaluate the utility of the initial LAGDA design, the assay was performed using a mixed mode of action endocrine disrupting chemical, benzophenone‐2 (BP‐2). X. laevis embryos were exposed in flow‐through conditions to 0, 1.5, 3.0 or 6.0 mg l^–1 BP‐2 until 2 months post‐metamorphosis. Overt toxicity was evident throughout the exposure period in the 6.0 mg l^–1 treatment due to elevated mortality rates and observed liver and kidney pathologies. Concentration‐dependent increases in severity of thyroid follicular cell hypertrophy and hyperplasia occurred in larval tadpoles indicating BP‐2‐induced impacts on the thyroid axis. Additionally, gonads were impacted in all treatments with some genetic males showing both testis and ovary tissues (1.5 mg l^–1) and 100% of the genetic males in the 3.0 and 6.0 mg l^?1 treatments experiencing complete male‐to‐female sex reversal. Concentration‐dependent vitellogenin induction occurred in both genders with associated accumulations of protein in the livers, kidneys and gonads, which was likely vitellogenin and other estrogen‐responsive yolk proteins. This is the first study that demonstrates the endocrine effects of this mixed mode of action chemical in an amphibian species and demonstrates the utility of the LAGDA design for supporting chemical risk assessment. Copyright © 2016 John Wiley & Sons, Ltd.     

An Investigation of the Differential Effects of Ursane Triterpenoids from Centella asiatica,and Their Semisynthetic Analogues,on GABAA Receptors

The ursane triterpenoids, asiatic acid 1 and madecassic acid 2 , are the major pharmacological constituents of Centella asiatica, commonly known as Gotu Kola, which is used traditionally for the treatment of anxiety and for the improvement of cognition and memory. Using the two‐electrode voltage‐clamp technique, these triterpenes, and some semisynthetic derivatives, were found to exhibit selective negative modulation of different subtypes of the GABA_A receptor expressed in Xenopus laevis oocytes. Despite differing by only one hydroxyl group, asiatic acid 1 was found to be a negative modulator of the GABA‐induced current at α₁β₂γ_2L, α₂β₂γ_2L and α₅β₃γ_2L GABA_A receptors, while madecassic acid 2 was not. Asiatic acid 1 exhibited the greatest effect at α₁β₂γ_2L (IC₅₀ 37.05 μm ), followed by α₅β₃γ_2L (IC₅₀ 64.05 μm ) then α₂β₂γ_2L (IC₅₀ 427.2 μm ) receptors. Conversion of the carboxylic acid group of asiatic acid 1 to a carboxamide group (2α,3β,23‐trihydroxy‐urs‐12‐en‐28‐amide 5 ) resulted in enhanced inhibition at both the α₁β₂γ_2L (IC₅₀ 14.07 μm ) and α₂β₂γ_2L receptor subtypes (IC₅₀ 28.41 μm ). The results of this study, and the involvement of α₅‐containing GABA_A receptors in cognition and memory, suggest that asiatic acid 1 may be a lead compound for the enhancement of cognition and memory.