Background: Members of the eukaryotic Hsp90 family function as important molecular chaperones in the assembly, folding and activation of cellular signaling in development. Twohsp90 genes, hsp90 a and hsp90 b, have been identified in fish and homeothermic vertebrates but not in poikilothermic vertebrates. In the present study, the expression ofhsp90 a and hsp90 b genes in Xenopus laevis, which is phylogenetically positioned between zebrafish and mammals, has been addressed.Methods: Partial Xenopus hsp90 a and hsp90 b cDNA were identified and isolated using RT-PCR, and a full-lengthXenopus hsp90 b cDNA was isolated from an embryonic cDNA library. Northern-blot analysis was used to study the expression ofhsp90 a and hsp90 bgenes in total RNA of the embryos andin situ hybridization was used to compare the expression of these genes with that ofhsp70 and MyoD genes in Xenopus embryogenesis.Results: Northern-blot analysis revealed that thehsp90 b gene was strongly expressed constitutively at all stages of embryogenesis, but weakly induced following the heat shock. In contrast, thehsp90 a gene was weakly expressed in embryos at control temperature, but strongly up-regulated following heat shock.In situ hybridization results showed that hsp90 agene was observed predominantly in cells of the developing somite. Microscopic sections showed thathsp90 aandMyoD mRNA are expressed in similar regions in somite and this pattern was distinct from that of hsp70 andhsp90 b.Conclusion: These data support the hypothesis that the presence of hsp90 a and hsp90 b genes is conserved among vertebrates, and these genes are differentially regulated in a tissue, stress, and development stage-specific manner.

Introduction: ROMK channel is localized on the apical membrane of nephrons. Recent studies suggest that endocytosis of ROMK channels is important for regulation of K+ secretion in cortical collecting ducts. In this study, the effect of S362A mutation is examined on the membrane turnover and stability of ROMK2 channel when expressed in Xenopus laevis oocytes.Methods: oocytes were isolated by standard protocols using collagenase (Type 1A). Mutations of the cytoplasmic termini of ROMK2 were performed using the quik-change approach for site-directed mutagenesis. Xenopus oocytes were injected with cRNA encoding ROMK2 or S362A mutant 3 days prior to treatment with Brefeldin A added to the OR3 medium (+BFA) at the concentration of 25 μM or ethanol as BFA vehicle (-BFA). BFA inhibits the insertion of new proteins into the cell membrane. Two-electrode voltage clamp (TEVC) was used to measure oocyte ROMK-dependent currents and membrane potential. Data was analyzed using Student’s t-tests or ANOVA as appropriate.Results: Incubation of oocytes expressing ROMK2 channels in 25 mM BFA caused a reduction in the currents and membrane voltage. In oocytes expressing the S362A mutant, there was no decay in current and membrane voltage after 48 hours incubation with BFA at 25 mM. The fractional current for ROMK2 at 48h following treatment of oocytes with BFA was 0.24 ± 0.05 (n=24), which was significantly different from S362A mutant (0.96±0.05, n=24). Conclusion: These results show that the S362A mutation increases the general stability of ROMK and renders the protein resistance to endocytosis. This is consistent with the idea that there is an interaction between the C-terminal of ROMK2 and components of the endocytotic pathway. A functional PDZ domain (the S-E-V) plays a key role in determining the stability of ROMK.

Context: Planarians, zebrafish, and Xenopus laevis are able to mend damage to their spinal cords after they have been damaged. After undergoing metamorphosis, the X. laevis loses the ability to do this. This study examines the genes that are involved in the process of spinal cord regeneration in these animals, investigates the pattern of their expression at various stages after spinal cord injury (SCI), and compares them to animals that do not have the ability to regenerate their spinal cords. This study reviews gene-based studies in regenerative animals, such as zebrafish, X. laevis, and planarians, and compares their expression patterns and fold-change slopes to non-regenerative ones to identify SCI recovery milestones. Evidence Acquisition: A systematic search was carried out with the intention of including all of the studies that had been conducted on the gene expression of X. laevis, zebrafish, and planarians in the context of SCI. Studies have been transferred to Endnote 2019 software. The researchers used the software to remove duplicate studies. Two researchers then assessed the titles and abstracts. A neutral third party resolved the discrepancies and extracted the data to a predesigned Microsoft Excel Worksheet. The genes were retrieved, and the data from the genome-wide studies were also combined to identify genes whose expression patterns in non-regenerative and regenerative species were significantly comparable or disagreeing with one another. Results: This review included 45 original and 2 genome-wide studies. Overall, 112 genes and their pathways were extracted. A total of 238 genes were common in these studies, and 9 significant expression patterns were possible. Among these 238 common genes, genes 23, 9, and 4 followed 4, 5, and 6 of these significant patterns, respectively. Additionally, pooling the genome-wide studies yielded 15 significant genes, with similar patterns in zebrafish and regenerative X. laevis and conflicting patterns between them and non-regenerative X. laevis. Conclusions: The regeneration of the spinal cord involves several processes, including regulation of inflammation, promotion of glial cell proliferation, facilitation of neuroplasticity, and establishment of coordination between newly formed neurons. Genes such as FOXN4, STC1, HSPA5, EGFR, and PRTFDC1 are also important. MCAM and Dkkb genes must be timed to create the right microenvironment for SCI healing in regenerating X. laevis and zebrafish. However, humans and non-regenerative X. laevis share an insufficient microenvironment. In contrast to non-regenerative models of SCI, regenerative animals decrease early-phase agents and increase injury site neuroplasticity in the late phase. As they show parallel expression patterns in regenerative and non-regenerative animals but in competition with one another.

Background: Bartter syndrome is renal tubular disorders that inhibit salt transport and increased renal salt wasting. Type II Bartter syndrome is caused by mutations in the KCNJ1 gene which encodes the inwardly rectifying ATP-sensitive potassium channel Kir1.1 (ROMK). They play a vital role in secretion of potassium into the tubule lumen. The effects of mutation at position 338 of ROMK2 (Kir1.1.b) was investigate.Materials and Methods: Site-directed mutagenesis was used to substitute of threonine for methionine at position 338 of ROMK2 (Kir1.1.b). M338T mutant ROMK2 expressed in oocytes of Xenopus laevis, and in a non-polarized mammalian cell line (MDCK). Two electrode voltage clamp and were used to measure oocyte ROMK-dependent currents. Confocal microscopy of EGFP-tagged ROMK2 determined express and distribution of these channels in MDCK cells.Results: The M338T mutant ROMK2 protein expressed in oocytes was functionally identical to wild type. Its cellular distribution was different in polarized and non-polarized MDCK cells.Conclusion: The M338T mutation is altered residue interactions within the carboxyl terminus of ROMK2 channels. Thus mistargeting of ROMK2 in vivo reduces the driving force for potassium secretion in the TAL and reduces salt reabsorption by this nephron segment.

Introduction: The cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl− ) channel is an essential component of epithelial Cl− transport systems in many organs. CFTR is mainly expressed in the lung and other tissues, such as testis, duodenum, trachea and kidney. The ubiquitin ligase neural precursor cells expressed developmentally down-regulated protein 4-2 (Nedd4-2) has previously been shown to regulate abundance of several channel and carrier proteins in the plasma membrane, an effect reversed by glucocorticoid dependent kinase 1 (SGK1). Methods: The present study was thus performed to elucidate the sensitivity of CFTR to regulation by Nedd4-2 and the serum and SGK1. To this end, the CFTR was heterologously expressed in oocytes alone or together with Nedd4-2 or the SGK1. The cRNAs encoding CFTR, Nedd4-2 and/or the constitutively active S422DSGK1 have been injected into Xenopus oocytes. The activity of CFTR was measured by the two-electrode voltage-clamp technique and CFTR-mediated currents were elicited by the application of forskolin and IBMX (F/I). Results: As a result, forskolin/IBMX treatment triggered cAMP-stimulated ion currents (IcAMP) in Xenopus oocytes expressing CFTR cRNA, but not in oocytes injected with water (control). Co-expression of Nedd4-2 markedly down-regulates the cAMP-stimulated ion current (IcAMP), an effect reversed by Co-expression of the constitutively active S422DSGK1. In Xenopus oocytes co-expressing CFTR with S422DSGK1 the cAMP-stimulated ion current (IcAMP) was similar to that in Xenopus oocytes expressing CFTR alone. Conclusion: The present observations suggest that CFTR is a target for the ubiquitin ligase Nedd4-2, which is inactivated by the SGK1.

Background: Polarized cells are key to the process of differentiation. Xenopus oocyte is a polarized cell that has complete blue-print to differentiate 3 germ layers following fertilization, as key determinant molecules (Proteins and RNAs) are asymmetrically localized. The objective of this work was to localize Na+, K+-ATPase activity along animal-vegetal axis of polarized Xenopus oocyte and following their progesterone-induced maturation (M-phase of the cell cycle) and their role in polarity.Materials and Methods: Enzyme-specific electron microscopy phosphatase histochemistry techniques were used.Results: The Na+, K+-ATPase is the most critical and important protein. The stage-VI Xenopus oocyte has a very distinct animal-vegetal polarity with structural and functional asymmetry. This polarity begins to develop in late stage III with accumulation of pigment granules towards the animal hemisphere. In this study, we have shown the expression and distribution pattern of Na+, K+-ATPase activity in stage-VI oocytes, and following progesterone induced maturation. Using enzyme-specific electron microscopy phosphatase histochemistry, [3H] -ouabain autoradiography, and immunofluorescence cytochemistry, we have shown that Na+, K+-ATPase activity is mainly confined to the entire animal hemisphere as bell (the Nucleus/Germinal Vesicle is present in the animal hemisphere).The polarized distribution of Na+, K+-ATPase activity begins to develop in late stage III and continues through to stage VI. Electron microscopy histochemical results also show this polarized distribution persists following progesterone-induced maturation (leading to M-phase), and it becomes gradually more polarized towards the animal pole. The time course following progesterone-induced maturation suggests that there is gradual down-regulation of Na+, K+-ATPase activity leading to germinal vesicle breakdown (GVBD). By GVBD, the Na+, K+-ATPase activity is completely down-regulated due to endocytotic removal of pump molecules from the plasma membrane into the sub-cortical region of the oocyte.This study provides the first direct evidence for a marked asymmetric localization of Na+, K+-ATPase activity in any vertebrate oocyte. Here, we propose that such asymmetry in Na+, K+-ATPase activity establishes and maintains polarity in Xenopus oocytes due to extracellular and intracellular ionic and electrical gradients. Also such asymmetry in stage VI oocyte and their down regulation following progesterone-induced maturation plays a key role in the active state of the germinal vesicle in stage-VI oocytes and chromosomal condensation after GVBD. This work will entail 1. What are the key Na+dependent genes expressed in cell? 2. What role Na+, K+-ATPase plays in cell cycle? 3. Does down-regulation of Na+, K+-ATPase in MPhase phosphorelate key enzymes involved in cell cycle.4. What is the level of Na+, K+-ATPase activity in uterine cancer and if alpha subunit of the protein acts as receptor for progesterone.Conclusion: At the resolution of electron microscopy and light microscopy, Na+, K+-ATPase activity is found to be localized in the animal hemispheres of polarized Xenopus oocytes and it is completely down regulated following progesterone induced maturation.

Xenopus laevis has been widely used for molecular, cellular, and developmental studies. With development of the sperm-mediated transgenic method, it is now possible to study the gene function during vertebrate development by using this popular model. On the other hand, maintenance of transgenic lines is both expensive and labor-intensive, as for other animal species. In this study, we investigated the possibility of using sperm cryopreservation as a means of preserving the transgenic frog lines. We demonstrated that cryopreserved sperms are viable but not fertilizing under in vitro fertilization conditions. However, by microinjecting the nuclei from cryopreserved sperms, we could successfully regenerate the transgenic line carrying a double promoter transgene construct. One promoter drove the marker gene encoding the green fluorescent protein (GFP), and the second, the heat shock-inducible promoter, drove the expression of GFP fused to matrix metalloproteinase stromelysin-3 (ST3-GFP). We demonstrated the functional transmission of the ST3-GFP transgene by analyzing the phenotype of the F1 animals after heat-shock induction of the transgene expression. Our method thus provides an inexpensive means to preserve transgenic frog lines and a convenient way for distributing the transgenic lines for studies. Furthermore, easy microinjection of nuclei compared to the technically demanding transgenesis procedure with variable outcomes can encourage more laboratories to use transgenic Xenopus laevis for functional studies in vivo.