Expression of Size-Selected RNA Encoding Brain Serotonin Transporter in Xenopus laevis Oocytes

The mRNA that encodes a serotonin transporter was expressed using the Xenopus laevis oocyte expression system. Poly(A)+ RNA isolated from mouse brainstem was injected into Xenopus laevis oocytes, and the ability of oocytes to take up serotonin was measured 3 days postinjection. RNA-dependent serotonin uptake was sensitive to citalopram, a specific inhibitor of serotonin uptake, whereas background levels of serotonin uptake were not citalopram sensitive. Two RNA size fractions, 4.0 and 4.5 kb, were most efficient in stimulating uptake. Injection into Xenopus laevis oocytes of the 4.5-kb size fraction of mouse brainstem RNA resulted in threefold more serotonin uptake than did injection of unfractionated poly(A)+ RNA.     

Functional expression of basolateral Cl−/HCO3− exchange from rat jejunum in Xenopus laevis oocytes

Poly(A)⁺ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of Cl⁻/HCO₃⁻ antiport was investigated by means of ³⁶Cl⁻ uptake. Two days after injection of 50 ng of poly(A)⁺ RNA, Cl⁻ uptake was significantly increased with respect to water-injected oocytes. The expressed transport was inhibited by 0·2 mM DIDS, whereas endogenous Cl⁻ uptake was unaffected by this disulphonic stilbene. After sucrose density gradient fractionation, the highest expression of DIDS-sensitive Cl⁻ uptake was detected with mRNA size fraction of about 2–4 kb in length. The expressed Cl⁻ uptake can occur against a Cl⁻ concentration gradient and is unaffected by the known Cl⁻ channel blocker anthracene-9-carboxylic acid. Cl⁻ transport mechanism has properties similar to jejunal basolateral Cl⁻/HCO₃⁻ exchange with regard to Na⁺ dependence. © 1998 John Wiley & Sons, Ltd.     

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near‐native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4‐M23) was expressed in the X. laevis oocytes following their injection with AQP4‐M23 cRNA. AQP4‐M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4‐M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over‐expressed AQP4‐M23, the membranes from AQP4‐M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher‐order arrays of AQP4‐M23. In addition, but only infrequently, AQP4‐M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

ANP and CNP activate CFTR expressed in Xenopus laevis oocytes by direct activation of PKA

Abstract

Context: Acting through different receptors, natriuretic peptides (atrial natriuretic peptide [ANP], brain type natriuretic peptide [BNP] and C-type natriuretic peptide [CNP]) increase intracellular cGMP, which then stimulates different pathways that activate fluid secretion. Objective: We used two-electrode voltage clamping to define the dominant pathway that is employed when natriuretic peptides activate cystic fibrosis transmembrane conductance regulator (CFTR) in the Xenopus oocyte expression system. Natriuretic peptides could activate CFTR by 1) cGMP cross-activation of protein kinase A (PKA), 2) cGMP activation of cGMP-dependent protein kinase II, 3) cGMP inhibition of phosphodiesterase type III (PDE3), or 4) direct activation of CFTR. Materials and Methods: cRNA-microinjected Xenopus laevis oocytes were perfused with diverse compounds that examined these pathways of natriuretic peptide signaling. Results and Discussion: ANP stimulated the shark CFTR (sCFTR)-mediated chloride conductance and this activation was inhibited by H-89, a specific inhibitor of PKA. After co-expression of the CNP receptor (NPR-B), sCFTR became stimulatable by CNP and was similarly inhibited by H-89, pointing to cross-activation of PKA. 8-pCPT-cGMP, a relatively cGKII-selective cGMP, failed to stimulate sCFTR. Another membrane-permeable and non-hydrolyzable analog of cGMP, 8-Br-cGMP, stimulated CFTR only at millimolar concentrations, consistent with cross-activation of PKA. The PDE inhibitors EHNA, rolipram, cilostamide, and amrinone did not significantly increase chloride conductance, arguing against a significant role for PDE2, PDE3 and PDE4 signaling in the oocyte. Sildenafil, a PDE5 inhibitor, caused a partial activation of sCFTR channels and this effect was again inhibited by H-89. Conclusion: From these experiments we conclude that in the Xenopus oocyte system, natriuretic peptides, 8-Br-cGMP, and PDE5 inhibitors activate CFTR by cross-activation of PKA.     

Skin wound healing in different aged Xenopus laevis

Xenopus froglets can perfectly heal skin wounds without scarring. To explore whether this capacity is maintained as development proceeds, we examined the cellular responses during the repair of skin injury in 8‐ and 15‐month‐old Xenopus laevis. The morphology and sequence of healing phases (i.e., inflammation, new tissue formation, and remodeling) were independent of age, while the timing was delayed in older frogs. At the beginning of postinjury, wound re‐epithelialization occurred in form of a thin epithelium followed by a multilayered epidermis containing cells with apoptotic patterns and keratinocytes stained by anti‐inducible nitric oxide synthase (iNOS) antibody. The inflammatory response, early activated by recruitment of blood cells immunoreactive to anti‐tumor necrosis factor (TNF)‐α, iNOS, transforming growth factor (TGF)‐β1, and matrix metalloproteinase (MMP)‐9, persisted over time. The dermis repaired by a granulation tissue with extensive angiogenesis, inflammatory cells, fibroblasts, and anti‐α‐SMA positive myofibroblasts. As the healing progressed, wounded areas displayed vascular regression, decrease in cellularity, and rearrangement of provisional matrix. The epidermis restored to a prewound morphology while granulation tissue was replaced by a fibrous tissue in a scar‐like pattern. The quantitative PCR analysis demonstrated an up‐regulated expression of Xenopus suppressor of cytokine signaling 3 (XSOCS-3) and Xenopus transforming growth factor-β2 (XTGF-β2) soon after wounding and peak levels were detected when granulation tissue was well developed with a large number of inflammatory cells. The findings indicate that X. laevis skin wound healing occurred by a combination of regeneration (in epidermis) and repair (in dermis) and, in contrast to froglet scarless wound healing, the growth to a more mature adult stage is associated with a decrease in regenerative capacity with scar‐like tissue formation. J. Morphol. 274:956–964, 2013. © 2013 Wiley Periodicals, Inc.     

Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes

Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I. After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase. Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions. How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase. By using lethal toxin from Clostridium sordellii (LT), a glucosyl-transferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner. The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here.     

Glucose transporter GLUT12-functional characterization in Xenopus laevis oocytes

We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.     

Antioxidant metabolism in Xenopus laevis embryos is affected by stratospheric balloon flight

To test the effects of low levels of radiation from space on living organisms, we flew Xenopus laevis embryos at different stages of development on a stratospheric balloon (BI.R.BA mission). After recovery, different parameters were analyzed to assess the effects of flight, with particular regard to oxidative stress damage. Because of failed temperature control during flight, the flight shielded embryos (FC) could not be used for biochemical or morphological comparisons. In contrast, the incubation conditions (i.e. temperature, containers, volumes) for the flight embryos (F) were parallel to those for the ground controls. Mortality data show that younger embryos (16 h) flown on the balloon (F) are more sensitive to radiation exposure than older ones (40 h and 6 days). Exposure during flight lowered the antioxidant potential in all embryos, particularly older ones. These preliminary data demonstrate that flight on a stratospheric balloon might affect antioxidant metabolism, though it is not yet possible to correlate these results with low radiation exposure during flight.     

Novel short antimicrobial peptide isolated from Xenopus laevis skin

A rich source of bioactive peptides, including a large number of antimicrobial peptides, has been found in amphibian skin. In this study, a novel short antimicrobial peptide was purified from Xenopus laevis skin and characterised through reversed‐phase high‐performance liquid chromatography, Edman degradation and matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry. The peptide was composed of six amino acids with a sequence of DEDLDE and thus named X. laevis antibacterial peptide‐P2 (XLAsp‐P2). Transmission electron microscopy revealed that this peptide showed potential antimicrobial abilities against bacteria by damaging the bacterial cell membrane. XLAsp‐P2 maybe inhibit bacterial growth by binding to the microbial genomic DNA. The peptide also exhibited a weak haemolytic activity against rabbit red blood cells. Therefore, XLAsp‐P2 is a novel short anionic antibacterial peptide with broad activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

非洲爪蟾卵母细胞P2X受体存在的证明

目的：D1中除有P2Y受体介导的成分外,可能还有P2X受体介导的电流参与。本研究的目的在于进一步证实D1电流中是否有P2X受体介导电流的存在。方法：采用双电极电压钳记录方法。结果：Zn^2＋对不依赖于G蛋白之IATP（D1）具有增强作用;重复施加ATP Dl电流逐步减小,而D2电流则逐步增大。在仅有D12之IATP（D2）情况下,重复加ATP后D2电流不但不增大反而有所下降。结论：非洲爪蟾卵母细胞上存在P2X受体。     

Analysis of aspartic acid and asparagine metabolism in Xenopus laevis oocytes using a simple and sensitive HPLC method

The amino acids in methanol-soluble extracts of Xenopus oocytes were measured using a method involving precolumn derivatization with phenylisothiocyanate and reverse phase HPLC of the derivatized amino acids. This technique allows the estimation of asparagine and glutamine pools in oocytes, estimated as 70 and 283 pmoles per oocyte, respectively. The pool sizes of the other amino acids were similar to previously reported results obtained using conventional ion exchange chromatography and postcolumn derivatization with ninhydrin. The advantages of the method developed here include picomolar sensitivity and the enhanced resolution of asparagine and glutamine from other amino acids. The kinetics of aspartic acid and asparagine utilization were monitored following microinjection of oocytes with [³H]aspartic acid and [¹⁴C]asparagine. The aspartic acid pool turned over rapidly with a half-time of <30 min. The asparagine pool was metabolized much more slowly and appeared to be utilized almost completely for protein synthesis. The absolute rate of protein synthesis in oocytes was calculated from the incorporation data and chemical pool measurements as ～25 ng/hr-oocyte. The methodology developed here may be useful in experimental situations involving limited amounts of biological material. © 1994 Wiley-Liss, Inc.     

Structure and expression of the nerve growth factor gene in Xenopus oocytes and embryos

A large part of the coding portion of the Xenopus nerve growth factor (NGF) gene has been identified and cloned by the use of a chicken cDNA probe and its sequence has been determined. Comparison of the derived amino acid sequence of mature Xenopus NGF with that of other species showed a high conservation, whereas comparison of the prepropeptide showed large divergent regions alternated with short conserved regions. Expression of the NGF gene was examined during development of oocytes and embryos. Surprisingly, NGF mRNA was found in the oocyte; it is present in small previtellogenic as well as in fully grown oocytes. NGF mRNA, passed to the embryo at fertilization, is degraded before the gastrula stage and starts accumulating again around the stage of the neurula. The association of NGF mRNA with polysomes is indicative of NGF synthesis during oogenesis. In fact, by using antibodies against mouse NGF it was possible to reveal NGF molecules present as precursors. These molecules accumulate during oogenesis and are maintained in the embryos up to the blastula stage; a very faint band corresponding to a smaller size peptide is sometimes detected. A maternal role for the NGF can be proposed, although a possible activity of NGF in the oocyte cannot be ruled out.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

Ribosomal preparations may induce maturation in Xenopus laevis oocytes

Xenopus laevis oocytes undergo maturation when they are injected with large quantities of crude ribosomes from various origins: X laevis full-grown or matured oocytes, Xenopus ovaries and embryos, Xenopus liver or mouse liver. All have the same efficiency, whatever their origin: they include 50-90% maturation in the injected oocytes at about the same speed as progesterone treatment. The ribosomal preparations are inactive wen injected into recipient oocytes pretreated with cholera toxin or cycloheximide. After dissociation with the high salt extract, but not with the subunits. Hypotheses concernning the mode action of this ribosomal extract are disussed.     

G(alpha)s levels regulate Xenopus laevis oocyte maturation

Progesterone, produced by follicular cells, induces Xenopus laevis oocyte maturation through a very early event that inhibits the activity of the adenylyl cyclase effector system. The participation of a G-protein has been implicated, based on the fact that the inhibitory effect of the steroid is GTP-dependent, and it has been proposed that progesterone acts interfering with G(alpha)s function at the plasma membrane. Here we investigate whether the change in oocyte G(alpha)s levels affects the maturation process induced by progesterone. Overexpression of X. laevis wild type (wt) G(alpha)s and the constitutive activated G(alpha)s(QL) mutant, both blocked progesterone-induced maturation, G(alpha)s(QL) being much more effective than the wt protein. On the other hand, depletion of G(alpha)s, by the use of antisense oligonucleotides, caused spontaneous maturation measured as MAPK activation, indicating clearly that the presence of G(alpha)s is necessary to keep oocytes arrested. Overexpression of three different G-protein coupled receptors (GPCR), the beta2-adrenergic receptor and the m4 and m5 muscarinic receptors, all caused inhibition of MAPK activation induced by progesterone. These receptors, upon their activation with the respective ligands, might be inducing the release of G(beta)gamma from their respective G(alpha), which together with endogenous G(alpha)s-GTP, activate adenylyl cyclase. Our results indicate that G(alpha)s plays an important role in the maturation process and support previous findings of G(beta)gamma participation, suggesting the presence of a mechanism where a constitutively activated G(alpha)s subunit, together with the G(beta)gamma heterodimer, both maintain high levels of intracellular cAMP levels, blocking the G2/M transition.     

Recovery of the Xenopus laevis heart from ROS-induced stress utilizes conserved pathways of cardiac regeneration

Urodele amphibians and some fish are capable of regenerating up to a quarter of their heart tissue after cardiac injury. While many anuran amphibians like Xenopus laevis are not capable of such feats, they are able to repair lesser levels of cardiac damage, such as that caused by oxidative stress, to a far greater degree than mammals. Using an optogenetic stress induction model that utilizes the protein KillerRed, we have investigated the extent to which mechanisms of cardiac regeneration are conserved during the restoration of normal heart morphology post oxidative stress in X. laevis tadpoles. We focused particularly on the processes of cardiomyocyte proliferation and dedifferentiation, as well as the pathways that facilitate the regulation of these processes. The cardiac response to KillerRed-induced injury in X. laevis tadpole hearts consists of a phase dominated by indicators of cardiac stress, followed by a repair-like phase with characteristics similar to mechanisms of cardiac regeneration in urodeles and fish. In the latter phase, we found markers associated with partial dedifferentiation and cardiomyocyte proliferation in the injured tadpole heart, which, unlike in regenerating hearts, are not dependent on Notch or retinoic acid signaling. Ultimately, the X. laevis cardiac response to KillerRed-induced oxidative stress shares characteristics with both mammalian and urodele/fish repair mechanisms, but is nonetheless a unique form of recovery, occupying an intermediate place on the spectrum of cardiac regenerative ability. An understanding of how Xenopus repairs cardiac damage can help bridge the gap between mammals and urodeles and contribute to new methods of treating heart disease.