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Convergent extension of the chordamesoderm is the best-examined gastrulation movement in Xenopus. Here we study general features of cell-cell contacts in this tissue by combining depletion of adhesion factors C-cadherin, Syndecan-4, fibronectin, and hyaluronic acid, the analysis of respective contact width spectra and contact angles, and La3+ staining of the pericellular matrix. We provide evidence that like in other gastrula tissues, cell-cell adhesion in the chordamesoderm is largely mediated by different types of pericellular matrix. Specific glycocalyx structures previously identified in Xenopus gastrula tissues are absent in chordamesoderm but other contact types like 10–20 nm wide La3+ stained structures are present instead. Knockdown of any of the adhesion factors reduces the abundance of cell contacts but not the average relative adhesiveness of the remaining ones: a decrease of adhesiveness at low contact widths is compensated by an increase of contact widths and an increase of adhesiveness proportional to width. From the adhesiveness-width relationship, we derive a model of chordamesoderm cell adhesion that involves the interdigitation of distinct pericellular matrix units. Quantitative description of pericellular matrix deployment suggests that reduced contact abundance upon adhesion factor depletion is correlated with excessive accumulation of matrix material in non-adhesive gaps and the loss of some contact types. [ABSTRACT FROM AUTHOR]

Activation of Map kinase/Erk signalling downstream of fibroblast growth factor (Fgf) tyrosine kinase receptors regulates gene expression required for mesoderm induction and patterning of the anteroposterior axis during Xenopus development. We have proposed that a subset of Fgf target genes are activated in the embyo in response to inhibition of a transcriptional repressor. Here we investigate the hypothesis that Cic (Capicua), which was originally identified as a transcriptional repressor negatively regulated by receptor tyrosine kinase/Erk signalling in Drosophila, is involved in regulating Fgf target gene expression in Xenopus. We characterise Xenopus Cic and show that it is widely expressed in the embryo. Fgf overexpression or ectodermal wounding, both of which potently activate Erk, reduce Cic protein levels in embryonic cells. In keeping with our hypothesis, we show that Cic knockdown and Fgf overexpression have overlapping effects on embryo development and gene expression. Transcriptomic analysis identifies a cohort of genes that are up-regulated by Fgf overexpression and Cic knockdown. We investigate two of these genes as putative targets of the proposed Fgf/Erk/Cic axis: fos and rasl11b, which encode a leucine zipper transcription factor and a ras family GTPase, respectively. We identify Cic consensus binding sites in a highly conserved region of intron 1 in the fos gene and Cic sites in the upstream regions of several other Fgf/Cic co-regulated genes, including rasl11b. We show that expression of fos and rasl11b is blocked in the early mesoderm when Fgf and Erk signalling is inhibited. In addition, we show that fos and rasl11b expression is associated with the Fgf independent activation of Erk at the site of ectodermal wounding. Our data support a role for a Fgf/Erk/Cic axis in regulating a subset of Fgf target genes during gastrulation and is suggestive that Erk signalling is involved in regulating Cic target genes at the site of ectodermal wounding. [ABSTRACT FROM AUTHOR]

Amphibian metamorphosis is controlled by thyroid hormone (TH), which binds TH receptors (TRs) to regulate gene expression programs that underlie morphogenesis. Gene expression screens using tissues from premetamorphic tadpoles treated with TH identified some TH target genes, but few studies have analyzed genome-wide changes in gene regulation during spontaneous metamorphosis. We analyzed RNA sequencing data at four developmental stages from the beginning to the end of spontaneous metamorphosis, conducted on the neuroendocrine centers of Xenopus tropicalis tadpole brain. We also conducted chromatin immunoprecipitation sequencing (ChIP-seq) for TRs, and we compared gene expression changes during metamorphosis with those induced by exogenous TH. The mRNA levels of 26% of protein coding genes changed during metamorphosis; about half were upregulated and half downregulated. Twenty four percent of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks. Genes involved with neural cell differentiation, cell physiology, synaptogenesis and cell-cell signaling were upregulated, while genes involved with cell cycle, protein synthesis, and neural stem/progenitor cell homeostasis were downregulated. There is a shift from building neural structures early in the metamorphic process, to the differentiation and maturation of neural cells and neural signaling pathways characteristic of the adult frog brain. Only half of the genes modulated by treatment of premetamorphic tadpoles with TH for 16 h changed expression during metamorphosis; these represented 33% of the genes whose mRNA levels changed during metamorphosis. Taken together, our results provide a foundation for understanding the molecular basis for metamorphosis of tadpole brain, and they highlight potential caveats for interpreting gene regulation changes in premetamorphic tadpoles induced by exogenous TH. [ABSTRACT FROM AUTHOR]

As the first and sometimes only skeletal tissue to appear, cartilage plays a fundamental role in the development and evolution of vertebrate body shapes. This is especially true for amphibians whose largely cartilaginous feeding skeleton exhibits unparalleled ontogenetic and phylogenetic diversification as a consequence of metamorphosis. Fully understanding the evolutionary history, evolvability and regenerative potential of cartilage requires in-depth analysis of how chondrocytes drive growth and shape change. This study is a cell-level description of the larval growth and postembryonic shape change of major cartilages of the feeding skeleton of a metamorphosing amphibian. Histology and immunohistochemistry are used to describe and quantify patterns and trends in chondrocyte size, shape, division, death, and arrangement, and in percent matrix from hatchling to froglet for the lower jaw, hyoid and branchial arch cartilages of Xenopus laevis. The results are interpreted and integrated into programs of cell behaviors that account for the larval growth and histology, and metamorphic remodeling of each element. These programs provide a baseline for investigating hormone-mediated remodeling, cartilage regeneration, and intrinsic shape regulating mechanisms. These programs also contain four features not previously described in vertebrates: hypertrophied chondrocytes being rejuvenated by rapid cell cycling to a prechondrogenic size and shape; chondrocytes dividing and rearranging to reshape a cartilage; cartilage that lacks a perichondrium and grows at single-cell dimensions; and an adult cartilage forming de novo in the center of a resorbing larval one. Also, the unexpected superimposition of cell behaviors for shape change onto ones for larval growth and the unprecedented exploitation of very large and small cell sizes provide new directions for investigating the development and evolution of skeletal shape and metamorphic ontogenies. [ABSTRACT FROM AUTHOR]

Immersion in tricaine methanesulfonate (i.e. TMS) has been used for euthanasia of Xenopus laevis (African Clawed frogs). However, the time for preparation and potential human health hazards may pose as a barrier for large group culls. Here, we aimed to investigate whether immersion in bupivacaine is an effective means to euthanize this species. In experiment one, frogs (n = 10/group) were randomly assigned to 1-h immersion in 1 of 3 treatment groups: 1) TMS-5 (MS-222, 5g/L); 2) TMS-10 (MS-222, 10 g/L); or 3) Bupi-1.5 (0.5% Bupivacaine, 1.5 g/L). Frogs were then removed from solutions, rinsed with system water, and placed into a recovery cage. Heart rate was evaluated audibly via doppler ultrasound flow over 1 min at immediate removal (T1h), at 2 (T2h), and 3 (T3h) h in the recovery cage. In experiment two, frogs (n = 7/group) underwent 5-h & 19-h immersion in either TMS-5 or Bupi-1.5, with heart rate assessment at 5 and 19 hrs. Righting reflex and withdrawal reflex of the hindlimb were tested during the experiments. Experiment one—after the 1-h immersion, Bupi-1.5 treated animals had decreased heart rates compared to TMS-5 and TMS-10 treated animals by T2h. Neither TMS-5, TMS-10, nor Bupi-1.5 ceased heart rate after the 1-h immersion. Experiment two—after the 5-h immersion, Bupi-1.5 and TMS-5 treated animals were comparable in heart rates. 43% of TMS-5 animals and 14% of the Bupi-1.5 animals had completely ceased heart rates at T5h. At 19 h all remaining animals exhibited rigor mortis and had ceased heart rate. We recommend 19-h of immersion using either TMS-5 or Bupi-1.5 for cessation of heart rate in African Clawed frogs. These data are strong support for the use of secondary physical methods for euthanasia in African Clawed frogs when euthanasia by immersion is performed. [ABSTRACT FROM AUTHOR]

Left-right symmetry breaking in most studied vertebrates makes use of so-called leftward flow, a mechanism which was studied in detail especially in mouse and Xenopus laevis embryos and is based on rotation of monocilia on specialized epithelial surface designated as left-right organizer or laterality coordinator. However, it has been argued that prior to emergence of leftward flow an additional mechanism operates during early cleavage stages in Xenopus embryo which is based on cytoskeletal processes. Evidence in favour of this early mechanism was supported by left-right abnormalities after chemical inhibition of cytoskeletal protein formin. Here we analyzed temporal dimension of this effect in detail and found that reported abnormalities arise only after treatment at gastrula-neurula stages, i.e. just prior to and during the operation of left-right organizer. Moreover, molecular and morphological analysis of the left-right organizer reveals its abnormal development. Our results strongly indicate that left-right abnormalities reported after formin inhibition cannot serve as support of models based on early symmetry breaking event in Xenopus embryo. [ABSTRACT FROM AUTHOR]

The fully aquatic African clawed frog, Xenopus laevis, has an unusual and highly adapted nose that allows it to separately sample both airborne and waterborne stimuli. The function of the adult water nose has received little study, despite the fact that it is quite likely to receive information about conspecifics through secretions released into the water and could aid the frog in making decisions about social and reproductive behaviors. To assess the potential for chemical communication in this species, we developed an in situ electroolfactogram preparation and tested the olfactory responses of adult males to cloacal fluids and skin secretions from male and female conspecifics. We found robust olfactory responses to all conspecific stimuli, with greatest sensitivity to female cloacal fluids. These results open the door to further testing to identify compounds within cloacal fluids and skin secretions that are driving these responses and examine behavioral responses to those compounds. Understanding the role of chemical communication in social and reproductive behaviors may add to our rich understanding of vocal communication to create a more complete picture of social behavior in this species. [ABSTRACT FROM AUTHOR]

Block of proliferation 1 (Bop1) is a nucleolar protein known to be necessary for the assembly of the 60S subunit of ribosomes. Here, we show a specific bop1 expression in the developing anterior tissue of the South African clawed frog Xenopus laevis. Morpholino oligonucleotide-mediated knockdown approaches demonstrated that Bop1 is required for proper development of the cranial cartilage, brain, and the eyes. Furthermore, we show that bop1 knockdown leads to impaired retinal lamination with disorganized cell layers. Expression of neural crest-, brain-, and eye-specific marker genes was disturbed. Apoptotic and proliferative processes, which are known to be affected during ribosomal biogenesis defects, are not hindered upon bop1 knockdown. Because early Xenopus embryos contain a large store of maternal ribosomes, we considered if Bop1 might have a role independent of de novo ribosomal biogenesis. At early embryonic stages, pax6 expression was strongly reduced in bop1 morphants and synergy experiments indicate a common signaling pathway of the two molecules, Bop1 and Pax6. Our studies imply a novel function of Bop1 independent of ribosomal biogenesis. [ABSTRACT FROM AUTHOR]

We wanted to clone the glucocorticoid receptor (GR) from slender African lungfish (Protopterus dolloi) for comparison to the P. dolloi mineralocorticoid receptor (MR), which we had cloned and were characterizing, as well as for comparison to the GRs from humans, elephant shark and zebrafish. However, although sequencing of the genome of the Australian lungfish (Neoceratodus forsteri), as well as, that of the West African lungfish (Protopterus annectens) were reported in the first three months of 2021, we could not retrieve a GR sequence with a BLAST search of GenBank, when we submitted our research for publication in July 2021. Moreover, we were unsuccessful in cloning the GR from slender African lungfish using a cDNA from the ovary of P. dolloi and PCR primers that had successfully cloned a GR from elephant shark, Xenopus and gar GRs. On October 21, 2021 the nucleotide sequence of West African lungfish (P. annectens) GR was deposited in GenBank. We used this GR sequence to construct PCR primers that successfully cloned the GR from the slender spotted lungfish. Here, we report the sequences of nine P. dolloi GR isoforms and explain the basis for the previous failure to clone a GR from slender African lungfish using PCR primers that cloned the GR from elephant shark, Xenopus and gar. Studies are underway to determine corticosteroid activation of these slender African lungfish GRs. [ABSTRACT FROM AUTHOR]

Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, we've used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development. [ABSTRACT FROM AUTHOR]

Combating the spread of antimicrobial resistance (AMR) among bacteria requires a new class of antimicrobials, which desirably have a narrow spectrum because of their low propensity for the spread of AMR. Antimicrobial peptides (AMPs), which target the bacterial cell membrane, are promising seeds for novel antimicrobials because the cell membrane is essential for all cells. Previously, we reported the antimicrobial and haemolytic effects of a natural AMP, magainin 2 (Mag2), isolated from the skin of Xenopus laevis (the African clawed frog), four types of synthesised Mag2 derivatives, and three types of rationally designed AMPs on gram-positive and gram-negative bacteria. To identify novel antimicrobial seeds, we evaluated the effect of AMPs on Mycoplasma pneumoniae, which also exhibits AMR. We also evaluated the antimicrobial effects of an AMP, NK2A, which has been reported to have antimicrobial effects on Mycoplasma bovis, in addition to Mag2 and previously synthesised seven AMPs, on four strains of M. pneumoniae using colorimetric, biofilm, and killing assays. We found that three synthesised AMPs, namely 17base-Ac6c, 17base-Hybrid, and Block, had anti-M. pneumoniae (anti-Mp) effect at 8–30 μM, whereas others, including NK2A, did not have any such effect. For the further analysis, the membrane disruption activities of AMPs were measured by propidium iodide (PI) uptake assays, which suggested the direct interaction of AMPs to the cell membrane basically following the colorimetric, biofilm, and killing assay results. PI uptake assay, however, also showed the NK2A strong interaction to cell membrane, indicating unknown anti-Mp determinant factors related to the peptide sequences. Finally, we conclude that anti-Mp effect was not simply determined by the membrane disruption activities of AMPs, but also that the sequence of AMPs were important for killing of M. pneumoniae. These findings would be helpful for the development of AMPs for M. pneumoniae. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) oligomers associated with Alzheimer's disease (AD) form Ca2+-permeable plasma membrane pores, leading to a disruption of the otherwise well-controlled intracellular calcium (Ca2+) homeostasis. The resultant up-regulation of intracellular Ca2+ concentration has detrimental implications for memory formation and cell survival. The gating kinetics and Ca2+ permeability of Aβ pores are not well understood. We have used computational modeling in conjunction with the ability of optical patch-clamping for massively parallel imaging of Ca2+ flux through thousands of pores in the cell membrane of Xenopus oocytes to elucidate the kinetic properties of Aβ pores. The fluorescence time-series data from individual pores were idealized and used to develop data-driven Markov chain models for the kinetics of the Aβ pore at different stages of its evolution. Our study provides the first demonstration of developing Markov chain models for ion channel gating that are driven by optical-patch clamp data with the advantage of experiments being performed under close to physiological conditions. Towards the end, we demonstrate the up-regulation of gating of various Ca2+ release channels due to Aβ pores and show that the extent and spatial range of such up-regulation increases as Aβ pores with low open probability and Ca2+ permeability transition into those with high open probability and Ca2+ permeability.

Alzheimer's disease (AD) is a devastating illness affecting over 40 million people worldwide. Intraneuronal rise of amyloid beta in its oligomeric forms (iAβOs), has been linked to the pathogenesis of AD by disrupting cytosolic Ca2+ homeostasis. However, the specific mechanisms of action are still under debate and intense effort is ongoing to improve our understanding of the crucial steps involved in the mechanisms of AβOs toxicity. We report the development of a mathematical model describing a proposed mechanism by which stimulation of Phospholipase C (PLC) by iAβO, triggers production of IP3 with consequent abnormal release of Ca2+ from the endoplasmic reticulum (ER) through activation of IP3 receptor (IP3R) Ca2+ channels. After validating the model using experimental data, we quantify the effects of intracellular rise in iAβOs on model solutions. Our model validates a dose-dependent influence of iAβOs on IP3-mediated Ca2+ signaling. We investigate Ca2+ signaling patterns for small and large iAβOs doses and study the role of various parameters on Ca2+ signals. Uncertainty quantification and partial rank correlation coefficients are used to better understand how the model behaves under various parameter regimes. Our model predicts that iAβO alter IP3R sensitivity to IP3 for large doses. Our analysis also shows that the upstream production of IP3 can influence Aβ-driven solution patterns in a dose-dependent manner. Model results illustrate and confirm the detrimental impact of iAβOs on IP3 signaling. [ABSTRACT FROM AUTHOR]

Uracil is removed from DNA by the conserved enzyme uracil DNA N-glycosylase (UNG). Previously, we observed that inhibiting UNG in Xenopus egg extracts blocked assembly of CENP-A, a histone H3 variant. CENP-A is an essential protein in all species, since it is required for chromosome segregation during mitosis. Thus, the implication of UNG in CENP-A assembly implies that UNG would also be essential, but UNG mutants lacking catalytic activity are viable in all species. In this paper, we present evidence that UNG2 colocalizes with CENP-A and H2AX phosphorylation at centromeres in normally cycling cells. Reduction of UNG2 in human cells blocks CENP-A assembly, and results in reduced cell proliferation, associated with increased frequencies of mitotic abnormalities and rapid cell death. Overexpression of UNG2 induces high levels of CENP-A assembly in human cells. Using a multiphoton laser approach, we demonstrate that UNG2 is rapidly recruited to sites of DNA damage. Taken together, our data are consistent with a model in which the N-terminus of UNG2 interacts with the active site of the enzyme and with chromatin.

Depolarization induced charge movement associated currents, analogous to gating currents in channels, were recently demonstrated in G-protein coupled receptors (GPCRs), and were found to affect the receptor's Agonist binding Affinity, hence denoted AA-currents. Here we study, employing a combined theoretical-experimental approach, the properties of the AA-currents using the m2-muscarinic receptor (m2R) as a case study. We found that the AA-currents are characterized by a "bump", a distinct rise followed by a slow decline, which appears both in the On and the Off responses. The cumulative features implied a directional behavior of the AA-currents. This forced us to abandon the classical chemical reaction type of models and develop instead a model that includes anisotropic processes, thus producing directionality. This model fitted well the experimental data. Our main findings are that the AA-currents include two components. One is extremely fast, approximately 0.2 ms, at all voltages. The other is slow, 2-3 ms at all voltages. Surprisingly, the slow component includes a process which strongly depends on voltage and can be as fast as 0.3 ms at + 40 mV. The reason that it does not affect the overall time constant of the slow component is that it carries very little charge. The two fast processes are suitable candidates to link between charge movement and agonist binding affinity under physiological conditions.

With the increasing availability of high quality genomic data, there is opportunity to deeply explore the genealogical relationships of different gene loci between closely related species. In this study, we utilized genomes of Xenopus laevis (XLA, a tetraploid species with (L) and (S) sub-genomes) and X. tropicalis (XTR, a diploid species) to investigate whether synonymous substitution rates among orthologous or homoeologous genes displayed any heterogeneity. From over 1500 orthologous/homoeologous genes collected, we calculated proportion of synonymous substitutions between genomes/sub-genomes (k) and found variation within and between chromosomes. Within most chromosomes, we identified higher k with distance from the centromere, likely attributed to higher substitution rates and recombination in these regions. Using maximum likelihood methods, we identified further evidence supporting rate heterogeneity, and estimated species divergence times and ancestral population sizes. Estimated species divergence times (XLA.L-XLA.S: ~25.5 mya; XLA-XTR: ~33.0 mya) were slightly younger compared to a past study, attributed to consideration of population size in our study. Meanwhile, we found very large estimated population size in the ancestral populations of the two species (NA = 2.55 x 106). Local hybridization and population structure, which have not yet been well elucidated in frogs, may be a contributing factor to these possible large population sizes. [ABSTRACT FROM AUTHOR]

The FLRT family of transmembrane proteins has been implicated in the regulation of FGF signalling, neurite outgrowth, homotypic cell sorting and cadherin-mediated adhesion. In an expression screen we identified the Netrin receptors Unc5B and Unc5D as high-affinity FLRT3 interactors. Upon overexpression, Unc5B phenocopies FLRT3 and both proteins synergize in inducing cell deadhesion in Xenopus embryos. Morpholino knock-downs of Unc5B and FLRT3 synergistically affect Xenopus development and induce morphogenetic defects. The small GTPase Rnd1, which transmits FLRT3 deadhesion activity, physically and functionally interacts with Unc5B, and mediates its effect on cell adhesion. The results suggest that FLRT3, Unc5B and Rnd1 proteins interact to modulate cell adhesion in early Xenopus development.

The nicotinic acetylcholine receptor (nAChR) is an excitatory pentameric ligand-gated ion channel (pLGIC), homologous to the inhibitory γ-aminobutyric acid (GABA) type A receptor targeted by pharmaceuticals and endogenous sedatives. Activation of the GABAA receptor by the neurosteroid allopregnanolone can be inhibited competitively by thyroid hormone (L-3,3’,5-triiodothyronine, or T3), but modulation of nAChR by T3 or neurosteroids has not been investigated. Here we show that allopregnanolone inhibits the nAChR from Torpedo californica at micromolar concentrations, as do T3 and the anionic neurosteroid pregnenolone sulfate (PS). We test for the role of protein and ligand charge in mediated receptor inhibition by varying pH in a narrow range around physiological pH. We find that both T3 and PS become less potent with increasing pH, with remarkably similar trends in IC50 when T3 is neutral at pH < 7.3. After deprotonation of T3 (but no additional deprotonation of PS) at pH 7.3, T3 loses potency more slowly with increasing pH than PS. We interpret this result as indicating the negative charge is not required for inhibition but does increase activity. Finally, we show that both T3 and PS affect nAChR channel desensitization, which may implicate a binding site homologous to one that was recently indicated for accelerated desensitization of the GABAA receptor by PS. [ABSTRACT FROM AUTHOR]

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthesis of polyamines, organic cations that are implicated in many cellular processes. The enzyme is regulated at the post-translational level by an unusual system that includes antizymes (AZs) and antizyme inhibitors (AZINs). Most studies on this complex regulatory mechanism have been focused on human and rodent cells, showing that AZINs (AZIN1 and AZIN2) are homologues of ODC but devoid of enzymatic activity. Little is known about Xenopus ODC and its paralogues, in spite of the relevance of Xenopus as a model organism for biomedical research. We have used the information existing in different genomic databases to compare the functional properties of the amphibian ODC1, AZIN1 and AZIN2/ODC2, by means of transient transfection experiments of HEK293T cells. Whereas the properties of xlODC1 and xlAZIN1 were similar to those reported for their mammalian orthologues, the former catalyzing the decarboxylation of L-ornithine preferentially to that of L-lysine, xlAZIN2/xlODC2 showed important differences with respect to human and mouse AZIN2. xlAZIN2 did not behave as an antizyme inhibitor, but it rather acts as an authentic decarboxylase forming cadaverine, due to its higher affinity to L-lysine than to L-ornithine as substrate; so, in accordance with this, it should be named as lysine decarboxylase (LDC) or lysine/ornithine decarboxylase (LODC). In addition, AZ1 stimulated the degradation of xlAZIN2 by the proteasome, but the removal of the 21 amino acid C-terminal tail, with a sequence quite different to that of mouse or human ODC, made the protein resistant to degradation. Collectively, our results indicate that in Xenopus there is only one antizyme inhibitor (xlAZIN1) and two decarboxylases, xlODC1 and xlLDC, with clear preferences for L-ornithine and L-lysine, respectively. [ABSTRACT FROM AUTHOR]

A comprehensive, accurate, and revisable alpha taxonomy is crucial for biodiversity studies, but is challenging when data from reference specimens are difficult to collect or observe. However, recent technological advances can overcome some of these challenges. To illustrate this, we used modern approaches to tackle a centuries-old taxonomic enigma presented by Fraser’s Clawed Frog, Xenopus fraseri, including whether X. fraseri is different from other species, and if so, where it is situated geographically and phylogenetically. To facilitate these inferences, we used high-resolution techniques to examine morphological variation, and we generated and analyzed complete mitochondrial genome sequences from all Xenopus species, including >150-year-old type specimens. Our results demonstrate that X. fraseri is indeed distinct from other species, firmly place this species within a phylogenetic context, and identify its minimal geographic distribution in northern Ghana and northern Cameroon. These data also permit novel phylogenetic resolution into this intensively studied and biomedically important group. Xenopus fraseri was formerly thought to be a rainforest endemic placed alongside species in the amieti species group; in fact this species occurs in arid habitat on the borderlands of the Sahel, and is the smallest member of the muelleri species group. This study illustrates that the taxonomic enigma of Fraser’s frog was a combined consequence of sparse collection records, interspecies conservation and intraspecific polymorphism in external anatomy, and type specimens with unusual morphology. [ABSTRACT FROM AUTHOR]

uman ther-à-go-go-elated ene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine’s relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens. [ABSTRACT FROM AUTHOR]

Kidneys are composed of numerous ciliated epithelial tubules called nephrons. Each nephron functions to reabsorb nutrients and concentrate waste products into urine. Defects in primary cilia are associated with abnormal formation of nephrons and cyst formation in a wide range of kidney disorders. Previous work in Xenopus laevis and zebrafish embryos established that loss of components that make up the Wnt/PCP pathway, Daam1 and ArhGEF19 (wGEF) perturb kidney tubulogenesis. Dishevelled, which activates both the canonical and non-canonical Wnt/PCP pathway, affect cilia formation in multiciliated cells. In this study, we investigated the role of the noncanoncial Wnt/PCP components Daam1 and ArhGEF19 (wGEF) in renal ciliogenesis utilizing polarized mammalian kidney epithelia cells (MDCKII and IMCD3) and Xenopus laevis embryonic kidney. We demonstrate that knockdown of Daam1 and ArhGEF19 in MDCKII and IMCD3 cells leads to loss of cilia, and Daam1’s effect on ciliogenesis is mediated by the formin-activity of Daam1. Moreover, Daam1 co-localizes with the ciliary transport protein Ift88 and is present in cilia. Interestingly, knocking down Daam1 in Xenopus kidney does not lead to loss of cilia. These data suggests a new role for Daam1 in the formation of primary cilia. [ABSTRACT FROM AUTHOR]

It has been suggested that Jmjd6 plays an important role in gene regulation through its demethylation or hydroxylation activity on histone and transcription factors. In addition, Jmjd6 has been shown to regulate RNA splicing by interaction with splicing factors. In this study, we demonstrated that Jmjd6a is expressed in developing Xenopus laevis eye during optic vesicle formation and retinal layer differentiation stages. Knockdown of Jmjd6a by an antisense morpholino resulted in eye malformation including a deformed retinal layer and no lens formation. We further found down-regulation of gene expression related to eye development such as Rx1, Otx2, and Pax6 in Jmjd6a morpholino injected embryos. Jmjd6 interacts with splicing factor U2AF25 and GSK3β RNA in the anterior region of Xenopus embryos. Knockdown of Jmjd6a led to deletion of GSK3β RNA exon 1 and 2, which resulted in generation of N’-terminal truncated GSK3β protein. This event further caused decreased phosphorylation of β-catenin and subsequently increased β-catenin stability. Therefore, our result may suggest that Jmjd6a plays an important role in Xenopus eye development through regulation of GSK3β RNA splicing and canonical Wnt/β-catenin signaling. [ABSTRACT FROM AUTHOR]

Advanced imaging techniques generate large datasets capable of describing the structure and kinematics of tissue spreading in embryonic development, wound healing, and the progression of many diseases. These datasets can be integrated with mathematical models to infer biomechanical properties of the system, typically identifying an optimal set of parameters for an individual experiment. However, these methods offer little information on the robustness of the fit and are generally ill-suited for statistical tests of multiple experiments. To overcome this limitation and enable efficient use of large datasets in a rigorous experimental design, we use the approximate Bayesian computation rejection algorithm to construct probability density distributions that estimate model parameters for a defined theoretical model and set of experimental data. Here, we demonstrate this method with a 2D Eulerian continuum mechanical model of spreading embryonic tissue. The model is tightly integrated with quantitative image analysis of different sized embryonic tissue explants spreading on extracellular matrix (ECM) and is regulated by a small set of parameters including forces on the free edge, tissue stiffness, strength of cell-ECM adhesions, and active cell shape changes. We find statistically significant trends in key parameters that vary with initial size of the explant, e.g., for larger explants cell-ECM adhesion forces are weaker and free edge forces are stronger. Furthermore, we demonstrate that estimated parameters for one explant can be used to predict the behavior of other similarly sized explants. These predictive methods can be used to guide further experiments to better understand how collective cell migration is regulated during development. [ABSTRACT FROM AUTHOR]

In insects, the gustatory system has a critical function not only in selecting food and feeding behaviours but also in growth and metabolism. Gustatory receptors play an irreplaceable role in insect gustatory signalling. Trichogramma chilonis is an effective biocontrol agent against agricultural insect pests. However, the molecular mechanism of gustation in T. chilonis remains elusive. In this study, we found that T. chilonis adults had a preference for D-fructose and that D-fructose contributed to prolong longevity and improve fecundity. Then, We also isolated the full-length cDNA encoding candidate gustatory receptor (TchiGR43a) based on the transcriptome data of T. chilonis, and observed that the candidate gustatory receptor gene was expressed from the larval to adult stages. The expression levels of TchiGR43a were similar between female and male. A Xenopus oocyte expression system and two-electrode voltage-clamp recording further verified the function analysis of TchiGR43a. Electrophysiological results showed that TchiGR43a was exclusively tuned to D-fructose. By the studies of behaviour, molecular biology and electrophysiology in T. chilonis, our results lay a basic fundation of further study on the molecular mechanisms of gustatory reception and provide theoretical basis for the nutritional requirement of T. chilonis in biocontrol. [ABSTRACT FROM AUTHOR]

The use of Xenopus laevis as a model for vertebrate developmental biology is limited by a lack of antibodies specific for embryonic antigens. This study evaluated the use of immune and non-immune phage display libraries for the isolation of single domain antibodies, or nanobodies, with specificities for Xenopus embryonic antigens. The immune nanobody library was derived from peripheral blood lymphocyte RNA obtained from a llama immunized with Xenopus gastrula homogenates. Screening this library by immunostaining of embryonic tissues with pooled periplasmic material and sib-selection led to the isolation of several monoclonal phages reactive with the cytoplasm and nuclei of gastrula cells. One antigen recognized by a group of nanobodies was identified using a reverse proteomics approach as nucleoplasmin, an abundant histone chaperone. As an alternative strategy, a semi-synthetic non-immune llama nanobody phage display library was panned on highly purified Xenopus proteins. This proof-of-principle approach isolated monoclonal nanobodies that specifically bind uclear istribution lement-ike 1 (Ndel1) in multiple immunoassays. Our results suggest that immune and non-immune phage display screens on crude and purified embryonic antigens can efficiently identify nanobodies useful to the Xenopus developmental biology community. [ABSTRACT FROM AUTHOR]

Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSPs (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32–36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function. [ABSTRACT FROM AUTHOR]

Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4β2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4β2)2β2- and LS-(α4β2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)β2 subunit binding interfaces. However LS-(α4β2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4β2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4β2)2β2-nAChR exhibit a single conductance state, whereas LS-(α4β2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4β2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site’s role in mediating LS-(α4β2)2α4-nAChR responses. Small large amplitude openings are highly-correlated within individual LS-(α4β2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)β2 subunit interfaces, rather than independent induction of high conductance channel openings. [ABSTRACT FROM AUTHOR]

Digestion of food and metabolism of frogs are little studied at the moment, and such processes could be very particular in the case of amphibians, given their ectothermic and carnivorous nature which may lead them to use nutrients through specific biochemical pathways. In the present study, 24 adult Xenopus laevis (six replicates with two frogs per treatment) were randomly assigned to two diets with different carbohydrate:fat ratio (4.5:1 and 2.1:1), changing the dietary glucogenic and lipogenic proportions. Food intake (FI) per unit metabolic body weight (MBW) as well as macronutrient digestibility were calculated, and circulating blood acylcarnitines and amino acids measured, in order to evaluate the effect of the diet treatments. Results demonstrated that food intake regulated most of the changes in the parameters evaluated; significant differences were obtained in crude protein and fat digestibilities through the effect of FI/MBW (p<0.05), whereas diet treatment had a significant effect on the levels of malonyl-CoA. Food intake also significantly impacted malonyl, isovaleryl, hydroxyisovaleryl and long chain fatty acid concentrations; significant (p<0.05) interactions between those metabolites were detected owing to diet. The findings obtained suggest that food intake was the main factor controlling digestion and metabolism in X. laevis, with frogs preferring to utilise protein and fat as primary sources for energy production in the citric acid cycle, reflecting characteristics of a strict carnivore physiological model. [ABSTRACT FROM AUTHOR]

The cross-section of a cell in a monolayer epithelial tissue can be modeled mathematically as a k-sided polygon. Empirically studied distributions of the proportions of k-sided cells in epithelia show remarkable similarities in a wide range of evolutionarily distant organisms. A variety of mathematical models have been proposed for explaining this phenomenon. The highly parsimonious simulation model of (Patel et al., PLoS Comput. Biol., 2009) that takes into account only the number of sides of a given cell and cell division already achieves a remarkably good fit with empirical distributions from Drosophila, Hydra, Xenopus, Cucumber, and Anagallis. Within the same modeling framework as in that paper, we introduce additional options for the choice of the endpoints of the cleavage plane that appear to be biologically more realistic. By taking the same data sets as our benchmarks, we found that combinations of some of our new options consistently gave better fits with each of these data sets than previously studied ones. Both our algorithm and simulation data are made available as research tools for future investigations. [ABSTRACT FROM AUTHOR]

Sodium-glucose transporters (SGLT) belong to the solute carrier 5 family, which is characterized by sodium dependent transport of sugars and other solutes. In contrast, the human SGLT3 (hSGLT3) isoform, encoded by SLC5A4, acts as a glucose sensor that does not transport sugar but induces membrane depolarization by Na+ currents upon ligand binding. Whole-exome sequencing (WES) of several extended pedigrees with high density of attention-deficit/hyperactivity disorder (ADHD) identified a triplet ATG deletion in SLC5A4 leading to a single amino acid loss (ΔM500) in the hSGLT3 protein imperfectly co-segregating with the clinical phenotype of ADHD. Since mutations in homologous domains of hSGLT1 and hSGLT2 were found to affect intestinal and renal function, respectively, we analyzed the functional properties of hSGLT3[wt] and [ΔM500] by voltage clamp and current clamp recordings from cRNA-injected Xenopus laevis oocytes. The cation conductance of hSGLT3[wt] was activated by application of glucose or the specific agonist 1-desoxynojirimycin (DNJ) as revealed by inward currents in the voltage clamp configuration and cell depolarization in the current clamp mode. Almost no currents and changes in membrane potential were observed when glucose or DNJ were applied to hSGLT3[ΔM500]-injected oocytes, demonstrating a loss of function by this amino acid deletion in hSGLT3. To monitor membrane targeting of wt and mutant hSGLT3, fusion constructs with YFP were generated, heterologously expressed in Xenopus laevis oocytes and analyzed for membrane fluorescence by confocal microscopy. In comparison to hSGLT3[wt] the fluorescent signal of mutant [ΔM500] was reduced by 43% indicating that the mutant phenotype might mainly result from inaccurate membrane targeting. As revealed by homology modeling, residue M500 is located in TM11 suggesting that in addition to the core structure (TM1-TM10) of the transporter, the surrounding TMs are equally crucial for transport/sensor function. In conclusion, our findings indicate that the deletion [ΔM500] in hSGLT3 inhibits membrane targeting and thus largely disrupts glucose-induced sodium conductance, which may, in interaction with other ADHD risk-related gene variants, influence the risk for ADHD in deletion carriers. [ABSTRACT FROM AUTHOR]

Mammalian oocytes and zygotes have nucleoli that are transcriptionally inactive and structurally distinct from nucleoli in somatic cells. These nucleoli have been termed nucleolus precursor bodies (NPBs). Recent research has shown that NPBs are important for embryonic development, but they are only required during pronuclear formation. After fertilization, multiple small NPBs are transiently formed in male and female pronuclei and then fuse into a single large NPB in zygotes. In cloned embryos produced by somatic cell nuclear transfer (SCNT), multiple NPBs are formed and maintained in the pseudo-pronucleus, and this is considered an abnormality of the cloned embryos. Despite this difference between SCNT and normal embryos, it is unclear how the size and number of NPBs in pronuclei is determined. Here, we show that in mouse embryos, the volume of NPB materials plays a major role in the NPB scaling through a limiting component mechanism and determines whether a single or multiple NPBs will form in the pronucleus. Extra NPB- and extra MII spindle-injection experiments demonstrated that the total volume of NPBs was maintained regardless of the pronucleus number and the ratio of pronucleus/NPB is important for fusion into a single NPB. Based on these results, we examined whether extra-NPB injection rescued multiple NPB maintenance in SCNT embryos. When extra-NPBs were injected into enucleated-MII oocytes before SCNT, the number of NPBs in pseudo-pronuclei of SCNT embryos was reduced. These results indicate that multiple NPB maintenance in SCNT embryos is caused by insufficient volume of NPB. [ABSTRACT FROM AUTHOR]

The GABA-gated chloride channels of the Cys-loop receptor family, known as GABAA receptors, function as the primary gatekeepers of fast inhibitory neurotransmission in the central nervous system. Formed by the pentameric arrangement of five identical or homologous subunits, GABAA receptor subtypes are defined by the subunit composition that shape ion channel properties. An understanding of the structural basis of distinct receptor properties has been hindered by the absence of high resolution structural information for heteromeric assemblies. Robust heterologous expression and purification protocols of high expressing receptor constructs are vital for structural studies. Here, we describe a unique approach to screen for well-behaving and functional GABAA receptor subunit assemblies by using the Xenopus oocyte as an expression host in combination with fluorescence detection size exclusion chromatography (FSEC). To detect receptor expression, GFP fusions were introduced into the α1 subunit isoform. In contrast to expression of α1 alone, co-expression with the β subunit promoted formation of monodisperse assemblies. Mutagenesis experiments suggest that the α and β subunits can tolerate large truncations in the non-conserved M3/M4 cytoplasmic loop without compromising oligomeric assembly or GABA-gated channel activity, although removal of N-linked glycosylation sites is negatively correlated with expression level. Additionally, we report methods to improve GABAA receptor expression in mammalian cell culture that employ recombinant baculovirus transduction. From these methods we have identified a well-behaving minimal functional construct for the α1/β1 GABAA receptor subtype that can be purified in milligram quantities while retaining high affinity agonist binding activity. [ABSTRACT FROM AUTHOR]

Background: A new type of influenza virus, known as type D, has recently been identified in cattle and pigs. Influenza D virus infection in cattle is typically asymptomatic; however, its infection in swine can result in clinical disease. Swine can also be infected with all other types of influenza viruses, namely A, B, and C. Consequently, swine can serve as a “mixing vessel” for highly pathogenic influenza viruses, including those with zoonotic potential. Currently, the only antiviral drug available targets influenza M2 protein ion channel is not completely effective. Thus, it is necessary to develop an M2 ion channel blocker capable of suppressing the induction of resistance to the genetic shift. To provide a basis for developing novel ion channel-blocking compounds, we investigated the properties of influenza D virus M2 protein (DM2) as a drug target. Results: To test the ion channel activity of DM2, the DNA corresponding to DM2 with cMyc-tag conjugated to its carboxyl end was cloned into the shuttle vector pNCB1. The mRNA of the DM2–cMyc gene was synthesized and injected into Xenopus oocytes. The translation products of DM2–cMyc mRNA were confirmed by immunofluorescence and mass spectrometry analyses. The DM2–cMyc mRNA-injected oocytes were subjected to the two-electrode voltage-clamp (TEVC) method, and the induced inward current was observed. The midpoint (Vmid) values in Boltzmann modeling for oocytes injected with DM2–cMyc RNA or a buffer were −152 and −200 mV, respectively. Assuming the same expression level in the Xenopus oocytes, DM2 without tag and influenza C virus M2 protein (CM2) were subjected to the TEVC method. DM2 exhibited ion channel activity under the condition that CM2 ion channel activity was reproduced. The gating voltages represented by Vmid for CM2 and DM2 were –141 and –146 mV, respectively. The reversal potentials observed in ND96 for CM2 and DM2 were −21 and −22 mV, respectively. Compared with intact DM2, DM2 variants with mutation in the YxxxK motif, namely Y72A and K76A DM2, showed lower Vmid values while showing no change in reversal potential. Conclusion: The M2 protein from newly isolated influenza D virus showed ion channel activity similar to that of CM2. The gating voltage was shown to be affected by the YxxxK motif and by the hydrophobicity and bulkiness of the carboxyl end of the molecule. [ABSTRACT FROM AUTHOR]

Insect repellents are widely used to fend off nuisance mosquitoes and, more importantly, to reduce or eliminate mosquito bites in areas where viruses and other vector-borne diseases are circulating. Synthesized more than six decades ago, DEET is the most widely used insect repellent. Plant-derived compounds are used in a plethora of commercial formulations and natural recipes to repel mosquitoes. They are also used as fragrances. We analysed Bombshell® to identify the constituent(s) eliciting a previously reported “off- label” repellence activity. The two major fragrance ingredients in Bombshell, i.e., methyl dihydrojasmonate and lilial, demonstrated strong repellence against the southern house mosquito, Culex quinquefasciatus, in laboratory assays. Both compounds activated a previously identified DEET-sensitive odorant receptor, CquiOR136. These compounds were also major constituents of Ivanka Trump eau de parfum. The methyl dihydrojasmonate content was higher in the Ivanka Trump perfume than in Bombshell, the reverse being true for lilial. Both Bombshell and Ivanka Trump eaux de parfums retained activity for as long as 6 hours in laboratory assays. Although wearing these perfumes may repel nuisance mosquitoes, their use as “off-label” repellents against infected mosquitoes is not recommended. [ABSTRACT FROM AUTHOR]

Acid-sensing ion channels (ASICs) are trimeric proton-gated cation permeable ion channels expressed primarily in neurons. Here we employed site-directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASIC1a by diminazene. This compound inhibits mouse ASIC1a with a half-maximal inhibitory concentration (IC50) of 2.4 μM. At first, we examined whether neutralizing mutations of Glu79 and Glu416 alter diminazene block. These residues form a hexagonal array in the lower palm domain that was previously shown to contribute to pore opening in response to extracellular acidification. Significantly, single Gln substitutions at positions 79 and 416 in ASIC1a reduced diminazene apparent affinity by 6–7 fold. This result suggests that diminazene inhibits ASIC1a in part by limiting conformational rearrangement in the lower palm domain. Because diminazene is charged at physiological pHs, we assessed whether it inhibits ASIC1a by blocking the ion channel pore. Consistent with the notion that diminazene binds to a site within the membrane electric field, diminazene block showed a strong dependence with the membrane potential. Moreover, a Gly to Ala mutation at position 438, in the ion conduction pathway of ASIC1a, increased diminazene IC50 by one order of magnitude and eliminated the voltage dependence of block. Taken together, our results indicate that the inhibition of ASIC1a by diminazene involves both allosteric modulation and blocking of ion flow through the conduction pathway. Our findings provide a foundation for the development of more selective and potent ASIC pore blockers. [ABSTRACT FROM AUTHOR]

Sulphonylurea drugs stimulate insulin secretion from pancreatic β-cells primarily by inhibiting ATP sensitive potassium (KATP) channels in the β-cell membrane. The effective sulphonylurea concentration at its site of action is significantly attenuated by binding to serum albumin, which makes it difficult to compare in vitro and in vivo data. We therefore measured the ability of gliclazide and glibenclamide to inhibit KATP channels and stimulate insulin secretion in the presence of serum albumin. We used this data, together with estimates of free drug concentrations from binding studies, to predict the extent of sulphonylurea inhibition of KATP channels at therapeutic concentrations in vivo. KATP currents from mouse pancreatic β-cells and Xenopus oocytes were measured using the patch-clamp technique. Gliclazide and glibenclamide binding to human plasma were determined in spiked plasma samples using an ultrafiltration-mass spectrometry approach. Bovine serum albumin (60g/l) produced a mild, non-significant reduction of gliclazide block of KATP currents in pancreatic β-cells and Xenopus oocytes. In contrast, glibenclamide inhibition of recombinant KATP channels was dramatically suppressed by albumin (predicted free drug concentration <0.1%). Insulin secretion was also reduced. Free concentrations of gliclazide and glibenclamide in the presence of human plasma measured in binding experiments were 15% and 0.05%, respectively. Our data suggest the free concentration of glibenclamide in plasma is too low to account for the drug’s therapeutic effect. In contrast, the free gliclazide concentration in plasma is high enough to close KATP channels and stimulate insulin secretion. [ABSTRACT FROM AUTHOR]

Two-pore domain K+ channels (K2P) are responsible for background K+ currents and regulate the resting membrane potential and cellular excitability. Their activity is controlled by a large variety of physicochemical factors and intracellular signaling pathways. The majority of these effects converge on the intracellular C-terminus of the channels, resulting in the modification of the gating at the selectivity filter. Another gating mechanism, the activation gate at the helix bundle crossing is also well documented in other K+ channel families, however, it remains uncertain whether this type of gating is functional in K2P channels. The regulation of TWIK-related spinal cord K+ channel (TRESK) is different from the other K2P channels. Regulatory factors acting via the C-terminus are not known, instead channel activity is modified by the phosphorylation/dephosphorylation of the unusually long intracellular loop between the 2nd and 3rd transmembrane segments. These unique structural elements of the regulation lead us to examine channel gating at the bundle crossing region. Ba2+ was applied to the intracellular side of excised membrane patches and the characteristics of the channel block were determined. We compared the kinetics of the development of Ba2+ block when the channels were phosphorylated (inhibited) or dephosphorylated (activated) and also in different mutants mimicking the two functional states. Neither the phosphorylation/dephosphorylation nor the point mutations influenced the development of Ba2+ block, suggesting that the conformational changes of the bundle crossing region do not contribute to the phosphorylation-dependent gating of TRESK. [ABSTRACT FROM AUTHOR]

Wnt proteins form a family of highly conserved secreted molecules that are critical mediators of cell-cell signaling during embryogenesis. Partial data on Wnt activity in different tissues and at different stages have been reported in frog embryos. Our objective here is to provide a coherent and detailed description of Wnt activity throughout embryo development. Using a transgenic Xenopus tropicalis line carrying a Wnt-responsive reporter sequence, we depict the spatial and temporal dynamics of canonical Wnt activity during embryogenesis. We provide a comprehensive series of in situ hybridization in whole-mount embryos and in cross-sections, from gastrula to tadpole stages, with special focus on neural tube, retina and neural crest cell development. This collection of patterns will thus constitute a valuable resource for developmental biologists to picture the dynamics of Wnt activity during development. [ABSTRACT FROM AUTHOR]

Thyroid hormone (TH) orchestrates amphibian metamorphosis. Thus, this developmental phase is often used to study TH-dependent responses in specific tissues. However, TH signaling appears early in development raising the question of the control of TH availability in specific cell types prior to metamorphosis. TH availability is under strict temporal and tissue-specific control by deiodinases. We examined the expression of the TH-inactivating enzyme, deiodinase type 3 (D3), during early retinal development. To this end we created a Xenopus laevis transgenic line expressing GFP from the Xenopus dio3 promoter region (pdio3) and followed pdio3–GFP expression in pre-metamorphic tadpoles. To validate retinal GFP expression in the transgenic line as a function of dio3 promoter activity, we used in situ hybridization to compare endogenous dio3 expression to reporter-driven GFP activity. Retinal expression of dio3 increased during pre-metamorphosis through stages NF41, 45 and 48. Both sets of results show dio3 to have cell-specific, dynamic expression in the pre-metamorphic retina. At stage NF48, dio3 expression co-localised with markers for photoreceptors, rods, Opsin-S cones and bipolar neurons. In contrast, in post-metamorphic juveniles dio3 expression was reduced and spatially confined to certain photoreceptors and amacrine cells. We compared dio3 expression at stages NF41 and NF48 with TH-dependent transcriptional responses using another transgenic reporter line: THbZIP-GFP and by analyzing the expression of T3-regulated genes in distinct TH availability contexts. At stage NF48, the majority of retinal cells expressing dio3 were negative for T3 signaling. Notably, most ganglion cells were virtually both dio3-free and T3-responsive. The results show that dio3 can reduce TH availability at the cellular scale. Further, a reduction in dio3 expression can trigger fine-tuned T3 action in cell-type specific maturation at the right time, as exemplified here in photoreceptor survival in the pre-metamorphic retina. [ABSTRACT FROM AUTHOR]

Time delays are known to play a crucial role in generating biological oscillations. The early embryonic cell cycle in the frog Xenopus laevis is one such example. Although various mathematical models of this oscillating system exist, it is not clear how to best model the required time delay. Here, we study a simple cell cycle model that produces oscillations due to the presence of an ultrasensitive, time-delayed negative feedback loop. We implement the time delay in three qualitatively different ways, using a fixed time delay, a distribution of time delays, and a delay that is state-dependent. We analyze the dynamics in all cases, and we use experimental observations to interpret our results and put constraints on unknown parameters. In doing so, we find that different implementations of the time delay can have a large impact on the resulting oscillations. [ABSTRACT FROM AUTHOR]

More than 90% of the cases of Parkinson’s disease have unknown etiology. Gradual loss of dopaminergic neurons of substantia nigra is the main cause of morbidity in this disease. External factors such as environmental toxins are believed to play a role in the cell loss, although the cause of the selective vulnerability of dopaminergic neurons remains unknown. We have previously shown that aquaglyceroporin AQP9 is expressed in dopaminergic neurons and astrocytes of rodent brain. AQP9 is permeable to a broad spectrum of substrates including purines, pyrimidines, and lactate, in addition to water and glycerol. Here we test our hypothesis that AQP9 serves as an influx route for exogenous toxins and, hence, may contribute to the selective vulnerability of nigral dopaminergic (tyrosine hydroxylase-positive) neurons. Using Xenopus oocytes injected with Aqp9 cRNA, we show that AQP9 is permeable to the parkinsonogenic toxin 1-methyl-4-phenylpyridinium (MPP+). Stable expression of AQP9 in HEK cells increases their vulnerability to MPP+ and to arsenite—another parkinsonogenic toxin. Conversely, targeted deletion of Aqp9 in mice protects nigral dopaminergic neurons against MPP+ toxicity. A protective effect of Aqp9 deletion was demonstrated in organotypic slice cultures of mouse midbrain exposed to MPP+ in vitro and in mice subjected to intrastriatal injections of MPP+ in vivo. Seven days after intrastriatal MPP+ injections, the population of tyrosine hydroxylase-positive cells in substantia nigra is reduced by 48% in Aqp9 knockout mice compared with 67% in WT littermates. Our results show that AQP9 –selectively expressed in catecholaminergic neurons—is permeable to MPP+ and suggest that this aquaglyceroporin contributes to the selective vulnerability of nigral dopaminergic neurons by providing an entry route for parkinsonogenic toxins. To our knowledge this is the first evidence implicating a toxin permeable membrane channel in the pathophysiology of Parkinson’s disease. [ABSTRACT FROM AUTHOR]

Melatonin is a neurohormone produced in both animals and plants. It binds at least three G-protein-coupled receptors: MT1 and MT2, and Mel1cGPR. Mammalian GPR50 evolved from the reptilian/avian Mel1c and lost its capacity to bind melatonin in all the therian mammal species that have been tested. In order to determine if binding is lost in the oldest surviving mammalian lineage of monotremes we investigated whether the melatonin receptor has the ability to bind melatonin in the platypus (Ornithorhynchus anatinus), and evaluated its pharmacological profile. Sequence and phylogenetic analysis showed that platypus has in fact retained the ancestral Mel1c and has the capacity to bind melatonin similar to other mammalian melatonin receptors (MT1 and MT2), with an affinity in the 1 nM range. We also investigated the binding of a set of melatoninergic ligands used previously to characterize the molecular pharmacology of the melatonin receptors from sheep, rats, mice, and humans and found that the general profiles of these compounds make Mel1c resemble human MT1 more than MT2. This work shows that the loss of GPR50 binding evolved after the divergence of monotremes less than 190MYA in therian mammals. [ABSTRACT FROM AUTHOR]

The airway epithelium in human plays a central role as the first line of defense against environmental contaminants. Most respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and respiratory infections, disturb normal muco-ciliary functions by stimulating the hypersecretion of mucus. Several muco-active agents have been used to treat hypersecretion symptoms in patients. Current muco-active reagents control mucus secretion by modulating either airway inflammation, cholinergic parasympathetic nerve activities or by reducing the viscosity by cleaving crosslinking in mucin and digesting DNAs in mucus. However, none of the current medication regulates mucus secretion by directly targeting airway goblet cells. The major hurdle for screening potential muco-active agents that directly affect the goblet cells, is the unavailability of in vivo model systems suitable for high-throughput screening. In this study, we developed a high-throughput in vivo model system for identifying muco-active reagents using Xenopus laevis embryos. We tested mucus secretion under various conditions and developed a screening strategy to identify potential muco-regulators. Using this novel screening technique, we identified narasin as a potential muco-regulator. Narasin treatment of developing Xenopus embryos significantly reduced mucus secretion. Furthermore, the human lung epithelial cell line, Calu-3, responded similarly to narasin treatment, validating our technique for discovering muco-active reagents. [ABSTRACT FROM AUTHOR]

Amphibian species are experiencing population declines due to infection by the fungal pathogen, Batrachochytrium dendrobatidis (Bd). The African clawed frog (Xenopus laevis), an asymptomatic carrier of Bd, has been implicated in the spread of this pathogen through global trade and established invasive populations on several continents. However, research has not explored the relationships of both life stages of this amphibian with Bd. While the post-metamorphic individuals may act as a reservoir, spreading the infection to susceptible species, the filter-feeding larvae may consume the motile Bd zoospores from the water column, potentially reducing pathogen abundance and thus the likelihood of infection. We explore these contrasting processes by assessing Bd prevalence and infection intensities in field populations of post-metamorphic individuals, and performing laboratory experiments to determine if larval X. laevis preyed upon Bd zoospores. The water flea, Daphnia magna, was included in the Bd consumption trials to compare consumption rates and to explore whether intraguild predation between the larval X. laevis and Daphnia may occur, potentially interfering with control of Bd zoospores by Daphnia. Field surveys of three X. laevis populations in southern California, in which 70 post-metamorphic individuals were tested for Bd, found 10% infection prevalence. All infected individuals had very low infection loads (all Bd loads were below 5 zoospore equivalents). Laboratory experiments found that larval X. laevis consume Bd zoospores and therefore may reduce Bd abundance and transmission between amphibians. However, metamorphic and juvenile X. laevis exhibited intraguild predation by consuming Daphnia, which also prey upon Bd zoospores. The results suggest that X laevis is not a large reservoir for Bd and its larval stage may offer some reduction of Bd transmission through direct predation. [ABSTRACT FROM AUTHOR]

Chondroitin sulfate (CS)/dermatan sulfate (DS) proteoglycans are abundant on the cell surface and in the extracellular matrix and have important functions in matrix structure, cell-matrix interaction and signaling. The DS epimerases 1 and 2, encoded by Dse and Dsel, respectively, convert CS to a CS/DS hybrid chain, which is structurally and conformationally richer than CS, favouring interaction with matrix proteins and growth factors. We recently showed that Xenopus Dse is essential for the migration of neural crest cells by allowing cell surface CS/DS proteoglycans to adhere to fibronectin. Here we investigate the expression of Dse and Dsel in Xenopus embryos. We show that both genes are maternally expressed and exhibit partially overlapping activity in the eyes, brain, trigeminal ganglia, neural crest, adenohypophysis, sclerotome, and dorsal endoderm. Dse is specifically expressed in the epidermis, anterior surface ectoderm, spinal nerves, notochord and dermatome, whereas Dsel mRNA alone is transcribed in the spinal cord, epibranchial ganglia, prechordal mesendoderm and myotome. The expression of the two genes coincides with sites of cell differentiation in the epidermis and neural tissue. Several expression domains can be linked to previously reported phenotypes of knockout mice and clinical manifestations, such as the Musculocontractural Ehlers-Danlos syndrome and psychiatric disorders. [ABSTRACT FROM AUTHOR]

Apolipoprotein CI (ApoCI) belongs to the Apolipoprotein superfamily, members of which are involved in lipid transport, uptake and homeostasis. Excessive ApoCI has been implicated in atherosclerosis and Alzheimer’s disease in humans. In this study we report the isolation of Xenopus laevis apoCI and describe the expression pattern of this gene during early development, using reverse transcription polymerase chain reaction and whole mount in situ hybridization. Xenopus apoCI is enriched in the dorsal ectoderm during gastrulation, and is subsequently expressed in sensory placodes, neural tube and cranial neural crest. These data suggest as yet uncharacterized roles for ApoCI during early vertebrate embryogenesis. [ABSTRACT FROM AUTHOR]

CLC type anion transport proteins are homo-dimeric or hetero-dimeric with an integrated transport function in each subunit. We have identified and partially characterized three members of this family named TbVCL1, TbVCL2 and TbVCL3 in Trypanosoma brucei. Among the human CLC family members, the T. brucei proteins display highest similarity to CLC-6 and CLC-7. TbVCL1, but not TbVCL2 and TbVCL3 is able to complement growth of a CLC-deficient Saccharomyces cerevisiae mutant. All TbVCL-HA fusion proteins localize intracellulary in procyclic form trypanosomes. TbVCL1 localizes close to the Golgi apparatus and TbVCL2 and TbVCL3 to the endoplasmic reticulum. Upon expression in Xenopus oocytes, all three proteins induce similar outward rectifying chloride ion currents. Currents are sensitive to low concentrations of DIDS, insensitive to the pH in the range 5.4 to 8.4 and larger in nitrate than in chloride medium. [ABSTRACT FROM AUTHOR]

In the search for novel anticancer drugs, the potassium channel KV10.1 has emerged as an interesting cancer target. Here, we report a new group of KV10.1 inhibitors, namely the purpurealidin analogs. These alkaloids are produced by the Verongida sponges and are known for their wide variety of bioactivities. In this study, we describe the synthesis and characterization of 27 purpurealidin analogs. Structurally, bromine substituents at the central phenyl ring and a methoxy group at the distal phenyl ring seem to enhance the activity on KV10.1. The mechanism of action of the most potent analog 5 was investigated. A shift of the activation curve to more negative potentials and an apparent inactivation was observed. Since KV10.1 inhibitors can be interesting anticancer drug lead compounds, the effect of 5 was evaluated on cancerous and non-cancerous cell lines. Compound 5 showed to be cytotoxic and appeared to induce apoptosis in all the evaluated cell lines. [ABSTRACT FROM AUTHOR]

During development, a multi-potent group of cells known as the cranial neural crest (CNC) migrate to form craniofacial structures. Proper migration of these cells requires proteolysis of cell adhesion molecules, such as cadherins. In Xenopus laevis, preventing extracellular cleavage of cadherin-11 impairs CNC migration. However, overexpression of the soluble cleavage product (EC1-3) is capable of rescuing this phenotype. The mechanism by which EC1-3 promotes CNC migration has not been investigated until now. Here we show that EC1-3 stimulates phosphorylation of Akt, a target of PI3K, in X.laevis CNC. Through immunoprecipitation experiments, we determined that EC1-3 interacts with all ErbB receptors, PDGFRα, and FGFR1. Of these receptors, only ErbB2 was able to produce an increase in Akt phosphorylation upon treatment with a recombinant EC1-3. This increase was abrogated by mubritinib, an inhibitor of ErbB2. We were able to recapitulate this decrease in Akt phosphorylation in vivo by knocking down ErbB2 in CNC cells. Knockdown of the receptor also significantly reduced CNC migration in vivo. We confirmed the importance of ErbB2 and ErbB receptor signaling in CNC migration using mubritinib and canertinib, respectively. Mubritinib and the PI3K inhibitor LY294002 significantly decreased cell migration while canertinib nearly prevented it altogether. These data show that ErbB2 and Akt are important for CNC migration and implicate other ErbB receptors and Akt-independent signaling pathways. Our findings provide the first example of a functional interaction between the extracellular domain of a type II classical cadherin and growth factor receptors. [ABSTRACT FROM AUTHOR]