Search Results (401)

Scorpion neurotoxins are small peptides that target ion channels and offer opportunities for novel therapeutic discovery. This study analyzed the functional effects of the venom and toxins from the Costa Rican endemic scorpion, Tityus championi. Initially, crude venom was tested on different isoforms of voltage-gated sodium channels. Our findings revealed that the venom contains toxins that affect mammalian Na_V1.6 and Na_V1.7, as well as the cockroach BgNa_V1 channel. Increased currents through Na_V1.6 and BgNa_V1 channels were associated with bigger window currents and inhibition of inactivation. Decreased Na_V1.7 currents were associated with smaller conductance. Crude venom and TCh3 toxin inhibited action potential generation in invertebrate neurons expressing Na_V1.7-like channels. In these neurons, Tch2 and Tch4 toxins shifted voltage sensitivity to more negative potentials, ultimately widening the window current but decreasing channel availability. Conversely, Tch3 behaved as an inhibitory toxin, closing window currents and decreasing channel availability. Structural modeling showed that Tch3 adopts an αββ fold and binds the S3–S4 loop of Domain II in human Na_V1.7. These data show the diverse effects of scorpion venoms on channels and neurons, characterize its principal toxins, and show that Tch3 has therapeutic potential for pain relief. Full article

Rare earth elements (REE), such as gadolinium (Gd) and erbium (Er), are increasingly recognised as emerging environmental contaminants due to their widespread use in industrial processes, electronics, and medical imaging applications. Despite their extensive presence in aquatic ecosystems, little is known about their developmental toxicity. In this study, Xenopus laevis embryos were exposed to environmentally relevant concentrations of Gd and Er during critical early developmental stages. The assessed endpoints included survival, malformations, growth (body length), and heart rate. Both Gd and Er caused significant sublethal effects, including increased axial malformations, reduced growth, and altered cardiac activity. To explore potential mechanisms of toxicity, the expression patterns of key developmental genes (fgf8, bmp4, sox9, egr2, rax1, pax6) and pro-inflammatory cytokines (tnfα, il1β, p65) were analysed using Real-Time PCR. The results showed dysregulation of gene expression, indicating disruption to pathways involved in morphogenesis and neurodevelopment. Elevated reactive oxygen species levels suggested that oxidative stress was a contributing factor. Raman spectroscopy confirmed biochemical changes affecting proteins, lipids, and nucleic acids, providing evidence of cellular stress and metabolic imbalance. Overall, our findings demonstrate that even low-level exposure to Gd and Er can impair amphibian embryonic development and disturb molecular homeostasis. These results emphasise the ecological risks of REE pollution and highlight the importance of ongoing environmental monitoring and long-term toxicological research. Full article

VESTIGIAL-LIKE proteins constitute a family of evolutionarily conserved proteins that act as cofactors in regulating gene expression through their binding to TEAD transcription factors. Among the four members of this family in vertebrates, VESTIGIAL-LIKE 4 has emerged as a tumor suppressor that competes with YAP in binding TEADs, thus inhibiting the HIPPO pathway downstream of YAP. Nevertheless, very few studies have addressed its function during early vertebrate development. Here, we used gain- and loss-of-function strategies to investigate the role of vestigial-like 4 during Xenopus laevis development. Our data show that vestigial-like 4 is a key regulator of neurogenesis and neural crest formation. In embryos depleted of vestigial-like 4, neurogenesis is severely impaired, and neither neurog1 nor neurod1 is able to stimulate neurogenesis. Vestigial-like 4 is also required for neural crest formation through pax3 and sox9 regulation, and this property does not necessarily require its interaction with tead. Collectively, our findings demonstrate that vestigial-like 4 is an important regulator of neurogenesis and neural crest formation. Although vestigial-like 4 can bind to tead proteins in the embryo, its function does not depend solely on this interaction, suggesting a complex level of regulation with which vestigial-like 4 regulates early steps in development and differentiation. Full article

Src family tyrosine kinases regulate oocyte maturation and fertilization in many species, yet their physiological roles in Xenopus laevis (X. laevis) remain incompletely defined. Here, we generated three X. laevis Src (xSrc) constructs with defined point mutations allowing for selective immunochemical detection and controlled modulation of kinase activity: wild type (xSrcWT, Arg121His), constitutively active (xSrcKA, Arg121His/Tyr526Phe), and kinase-negative (xSrcKN, Arg121His/Lys294Met). Capped mRNAs were microinjected into immature oocytes, and effects on meiotic maturation and egg activation were analyzed. All constructs produced detectable Src protein within 4–5 h after injection without inducing progesterone-independent maturation. Following progesterone treatment, MAP kinase phosphorylation, CDK1 activation, and germinal vesicle breakdown (GVBD) occurred normally in all groups, although xSrcKA-expressing oocytes showed a modest but reproducible acceleration of MAPK activation and GVBD. Global tyrosine phosphorylation analysis revealed increased phosphorylation of several proteins, including a prominent ~50 kDa substrate, specifically in xSrcKA oocytes. After maturation, oocytes were subjected to artificial activation. xSrcKN-expressing oocytes responded normally to Ca²⁺ ionophore (A23187), indicating that Src activity is not required for direct Ca²⁺-mediated activation. In contrast, xSrcKN oocytes exhibited markedly reduced activation in response to hydrogen peroxide or Cathepsin B, which stimulate membrane-associated signaling pathways. These findings demonstrate that Src kinase activity is required for membrane signal-mediated egg activation but is dispensable for activation driven by direct intracellular Ca²⁺ elevation. Collectively, our results identify Src kinase as a positive regulator of progesterone-induced meiotic maturation and a critical mediator of specific fertilization-like activation pathways in X. laevis. Full article

The transport of metabolites across biological membranes is vital for normal cellular functions, including nutrient uptake, homeostasis, and toxin efflux. In eukaryotes, mitochondrial transporters in the inner mitochondrial membrane (IMM) play a pivotal role in energy production, metabolism, and the biosynthesis of a wide range of compounds. While functional assignments exist for over half of the mitochondrial transporters, emerging high-throughput methodologies underscore the need for reassessment and expansion of the current knowledge, particularly as evidence suggesting functional redundancy and substrate promiscuity has emerged. In this study, we investigated the substrate specificity of five yeast mitochondrial transporters—Crc1 (YOR100c), Ctp1 (YBR291c), Oac1 (YKL120w), Pet9 (YBL030c), and Yhm2 (YMR241w)—via heterologous gene expression in Xenopus laevis oocytes and liquid chromatography-mass spectrometry (LC-MS)-based transport assays. We used two substrate mixtures: a 17-compound organic acid mix and a ¹³C-labeled yeast metabolite extract. Our results revealed broader substrate specificities than previously reported, as partially supported by substrate docking simulations. Pet9 transported several organic acids and amino acids, while Yhm2 showed uptake of nine amino acids and fumaric acid. Additional promiscuous transport activity was observed for Crc1, indicating that these proteins may have more extensive metabolic roles than previously known. This study advances the understanding of yeast mitochondrial transporter function, demonstrating redundancy and broad substrate specificity among mitochondrial carriers. It highlights the importance of utilizing in vivo heterologous systems and physiologically relevant substrate mixtures to elucidate transporter functionality. Full article

Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution. Full article

The availability of biobanked tissues represents an important resource for translational research; however, functional investigations are generally limited to freshly collected samples. To address this limitation, we developed an innovative strategy to restore functional properties of frozen biopsies by microtransplanting patient-derived membrane proteins into Xenopus laevis oocytes. This study aimed to recover and characterize the physiological properties of human colon cancer cell membranes and to investigate the role of neurotransmitter-related signaling and ion currents in cancer. Membrane incorporation was assessed by immunohistochemical detection of tumor-specific markers, including carcinoembryonic antigen, together with confocal microscopy and ultrastructural analyses. Functional viability was evaluated using two-electrode voltage clamp recordings to assess endogenous calcium-activated chloride currents and responses to selected neurotransmitters. The successful incorporation of colon cancer membranes was confirmed by specific immunoreactivity and ultrastructural features consistent with cancer cell architecture. Although no functional responses to the tested neurotransmitters were detected, oocytes microinjected with cancer membranes showed a marked reduction or complete suppression in endogenous calcium-activated chloride currents. These findings demonstrate that membrane microtransplantation into Xenopus oocytes is a reliable translational approach to functionally investigate cancer cell membranes from frozen biopsies, and suggest that altered chloride channel activity may represent a baseline for new studies to investigate new potential therapeutic targets for colon cancer. Full article

In this study, the biocatalytic activity of four steroid-transforming strains isolated from the African frog Xenopus laevis and identified as Streptomyces rochei towards pregnane steroids has been investigated. All the isolated strains facilitated the reduction of the C20-carbonyl group and the structures of the metabolites were confirmed by mass spectrometric (MS) and ¹H NMR spectroscopic analyses. Hydrocortisone and progesterone were poorly transformed by the streptomycete strains, whereas cortexolone (Reichstein’s substance S) was effectively biotransformed, yielding more than 90% of 17α,20β,21α-trihydroxy-4-pregnen-3-one (20β-S). Primarily, 20α-reduction was detected when the microbial isolates were incubated with 17α-hydroxyprogesterone with the yield of 17α,20α-dihydroxy-4-pregnen-3-one (17,20α-P) reaching 70%. The biological activity of 20β-S was evaluated in Danio rerio. The results demonstrated that 20β-S modulated stress- and anxiety-related behavioral responses and activated Pgr-dependent transcriptional pathways in the brain and ovarian tissues. These observations support the potential relevance of the synthesized progestin as a functional regulator in teleost physiology. The findings enhance our understanding of the biodiversity of steroid-transforming actinomycetes inhabiting amphibians and can be successfully employed for the effective microbiological synthesis of biologically active 20-hydroxylated progestins that serve as bioregulators in teleosts. Full article

OsHKT1;1, a member of the high-affinity K⁺ transporter (HKT) family, plays a key role in Na⁺ homeostasis and salinity tolerance in rice. In our previous study, multiple potential OsHKT1;1 splicing variants were identified, as well as the full-length (FL) OsHKT1;1 transcript from the salt-tolerant rice Pokkali. However, most previous studies focused solely on the full-length protein, leaving the transport functions of splice variants largely unexamined. In this study, we focused on the splice variant OsHKT1;1-V2 and compared its function and gene expression with those of OsHKT1;1-FL. Two-electrode voltage clamp experiments using Xenopus laevis oocytes revealed that the 1st start codon of OsHKT1;1-V2 is functional to exhibit bidirectional currents in bath solutions containing NaCl. Unlike the Na⁺-selective feature of OsHKT1;1-FL, OsHKT1;1-V2 primarily mediated Cl⁻ transport with weak Na⁺ selectivity, which was supported by the higher Cl⁻ accumulation in OsHKT1;1-V2–expressing oocytes. Subcellular localization analyses using oocytes and Arabidopsis mesophyll cells indicated plasma membrane localization of OsHKT1;1-V2, similar to OsHKT1;1-FL. Functional assays using a yeast mutant further indicated that OsHKT1;1-FL, but not OsHKT1;1-V2, mediates Na⁺ uptake. The same OsHKT1;1 variants were identified in the japonica cultivar Nipponbare, and OsHKT1;1-V2 of the cultivar showed Cl⁻ transport properties similar to the one from Pokkali. Quantitative PCR analyses revealed higher abundance of OsHKT1;1-FL transcripts in Nipponbare than in Pokkali with markedly lower OsHKT1;1-V2 levels in Pokkali under salt stress. This study provides a new insight into HKT-mediated ion homeostasis under salinity stress. Full article

ATP-dependent chromatin remodeling complexes regulate gene expression by altering chromatin structure through ATP hydrolysis. They are classified into four families—SWI/SNF, ISWI, CHD, and INO80—which remodel chromatin via nucleosome sliding, eviction, assembly, and editing to control transcription. These complexes play critical roles in DNA repair, tumorigenesis, and organogenesis. Recent advances in low-input proteomics have highlighted their importance in vertebrate embryonic development. In mammals, they regulate embryonic genome activation, lineage specification, and stem cell fate determination. In non-mammalian models (e.g., Xenopus laevis), they function from blastocyst formation to pre-organogenesis stages (gastrulation and neurulation)—key windows for chromatin reprogramming and cell fate decisions. This review provides a systematic overview of chromatin remodeling complexes, detailing their classification and conserved mechanisms, and discusses their functions in early embryogenesis and embryonic stem cell maintenance. The collective evidence underscores the implications of these chromatin remodelers for understanding developmental defects and advancing regenerative medicine. Full article

Protein complexes, assembled by scaffold proteins, act as molecular machines driving development. The mechanosensitive adapter protein Zyxin is a key example, integrating actin cytoskeleton dynamics with gene expression. However, the developmental regulation of its interactions and post-translational modifications remains poorly understood. Here, we characterize the dynamic Zyxin interactome across three early developmental stages of Xenopus laevis (from gastrulation to neurulation) using co-immunoprecipitation coupled with quantitative mass spectrometry (DDA and DIA). We identify stage-specific changes in Zyxin’s association with core focal adhesion components, transcriptional regulators and kinases. Furthermore, we uncover developmentally regulated phosphorylation events on isoforms, suggesting dynamic post-translational control of its interactions. Our work provides a comprehensive resource that positions Zyxin as a central orchestrator of cell adhesion, survival, and gene regulatory programs during morphogenesis. These findings underscore the role of Zyxin as a multifaceted regulatory hub, with important implications for understanding tissue homeostasis and related pathologies. Full article

Entomopathogenic nematodes (EPNs), including Steinernema and Heterorhabditis, are obligate insect parasites widely used in biological pest control. However, their efficacy is often limited by susceptibility to environmental stresses like desiccation. Aquaporins (AQPs), channel proteins facilitating water and solute movement across membranes, are hypothesized to play a key role in the osmotic stress response of EPNs. This study identified and cloned three AQP genes (L596_g7661, L596_g18121, and XLOC_007750) from four strains of Steinernema carpocapsae. Bioinformatic analysis confirmed that these AQPs belong to the aquaglyceroporin subfamily and share high sequence homology across strains. Functional characterization in Xenopus oocytes demonstrated that AQP L596_g7661 facilitates glycerol transport. Expression patterns under osmotic dehydration revealed significant upregulation of L596_g7661 and XLOC_007750 in all strains, while L596_g18121 expression remained unchanged. These findings indicate that specific AQPs are involved in the molecular response of S. carpocapsae to osmotic stress, providing crucial insights for breeding resilient EPN strains and enhancing their field application. Full article

R-limonene has been integrated into various pest control practices as a repellent or an insecticide. However, how limonene induces aversion or mortality remains largely unknown. To elucidate the underlying mechanisms, we conducted behavioral, toxicological, and electrophysiological assays in Aedes aegypti, a primary vector of human diseases. To investigate whether limonene acts on voltage-gated sodium channels and/or the Rdl (Resistance to dieldrin) receptor, two major targets of neuroactive insecticides, we characterized the effect of limonene on Ae. aegypti sodium and Rdl channels expressed in Xenopus oocytes. Limonene significantly potentiated GABA-induced chloride currents through Rdl in a concentration-dependent manner but had no effect on sodium channels. For repellency, limonene evoked spatial repellency in wild-type mosquitoes; however, the spatial repellency by limonene was significantly reduced in knockout mutants of Orco^−/− (odorant receptor co-receptor) and TRPA1^−/− (Transient Receptor Protein, subfamily A and member 1). These results indicate that limonene likely targets the Rdl receptor for insecticidal activity and limonene spatial repellency requires both Orco and TRPA1 channels. Our results reveal the involvement of multiple ion channels and receptors in the mosquito nervous system for limonene’s insecticidal and/or spatial repellency actions, highlighting limonene’s potential as a multi-target neuroactive agent for pest control. Full article

The Xenopus oocyte has long served as a versatile and powerful model for dissecting the molecular underpinnings of reproductive and developmental processes. Its large size, manipulability, and well-characterized cell cycle states have enabled generations of researchers to illuminate key aspects of oocyte maturation, fertilization, and early embryogenesis. This review provides an integrated overview of the cellular and molecular events that define the Xenopus oocyte’s transition from meiotic arrest to embryonic activation—or alternatively, to programmed demise if fertilization fails. We begin by exploring the architectural and biochemical landscape of the oocyte, including polarity, cytoskeletal organization, and nuclear dynamics. The regulatory networks governing meiotic resumption are then examined, with a focus on MPF (Cdk1/Cyclin B), MAPK cascades, and translational control via CPEB-mediated cytoplasmic polyadenylation. Fertilization is highlighted as a calcium-dependent trigger for oocyte activation. During fertilization in vertebrates, sperm-delivered phospholipase C zeta (PLCζ) is a key activator of Ca²⁺ signaling in mammals. In contrast, amphibian species such as Xenopus lack a PLCZ1 ortholog and instead appear to rely on alternative protease-mediated signaling mechanisms, including the uroplakin III–Src tyrosine kinase pathway and matrix metalloproteinase (MMP)-2 activity, to achieve egg activation. The review also addresses the molecular fate of unfertilized eggs, comparing apoptotic and necrotic mechanisms and their relevance to reproductive health. Finally, we discuss recent innovations in Xenopus-based technologies such as mRNA microinjection, genome editing, and in vitro ovulation systems, which are opening new avenues in developmental biology and translational medicine. By integrating classic findings with emerging frontiers, this review underscores the continued value of the Xenopus model in elucidating the fundamental processes of life’s origin. We conclude with perspectives on unresolved questions and future directions in oocyte and early embryonic research. Full article

Variants in CLCN3 and CLCN4, encoding the neuronal endosomal Cl⁻/H⁺ antiporters ClC-3 and ClC-4, are linked to neurodevelopmental disorders with broad phenotypic variability. Over sixty CLCN4 variants have been functionally characterized, showing gain- or loss-of-function (GoF or LoF) effects. While ClC-3 can function as a homodimer, ClC-4 depends on heterodimerization with ClC-3 for efficient endosomal trafficking. CLCN4, located on the X chromosome, exhibits diverse pathogenic outcomes: complete LoF variants often cause non-syndromic presentations in hemizygous males and are asymptomatic in heterozygous females, whereas certain missense variants with partial or complete LoF produce severe syndromic phenotypes in both sexes. Here, we demonstrate dominant effects of three CLCN4 variants within ClC-3/ClC-4 heterodimers using two-electrode voltage-clamp recordings in Xenopus laevis oocytes and whole-cell patch-clamp recordings in mammalian cells co-expressing both proteins via a bicistronic IRES construct. Our findings provide the first evidence of dominant-negative CLCN4 effects within ClC-3/ClC-4 complexes and establish a platform for functional analysis of additional disease-associated variants. Full article