Determination of the nucleoside triphosphate contents of eggs and oocytes of Xenopus laevis.
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ABSTRACT:
SUBMITTER: Woodland HR
PROVIDER: S-EPMC1178701 | biostudies-other | 1972 Apr
REPOSITORIES: biostudies-other
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Project description:Polyadenylated [poly(A)+] mRNA isolated from rabbit small-intestinal mucosa was injected into Xenopus laevis oocytes, and expression of the N2 Na+/nucleoside co-transporter was assayed by measuring Na(+)-dependent thymidine uptake. Expression of Na(+)-dependent thymidine uptake steadily increased after mRNA injection and was on average increased 11-fold by day 6 over background. Na(+)-dependent thymidine uptake was saturable (apparent Km approximately 30 microM at 22 degrees C) and inhibited by uridine and cytidine, but not by guanosine and inosine. These properties of the expressed thymidine transport strongly suggest that the epithelial N2 Na+/nucleoside co-transporter can be expressed in X. laevis oocytes.
| S-EPMC1151387 | biostudies-other
Project description:We determine the calcium fluxes through inositol 1,4,5-trisphosphate receptor/channels underlying calcium puffs of Xenopus laevis oocytes using a simplified version of the algorithm of Ventura et al. An analysis of 130 puffs obtained with Fluo-4 indicates that Ca2+ release comes from a region of width approximately 450 nm, that the release duration is peaked around 18 s and that the underlying Ca2+ currents range between 0.12 and 0.95 pA. All these parameters are independent of IP(3) concentration. We explore what distributions of channels that open during a puff, N(p), and what relations between current and number of open channels, I(N(p)), are compatible with our findings and with the distribution of puff-to-trigger amplitude ratio reported in Rose et al. To this end, we use simple "mean field" models in which all channels open and close simultaneously. We find that the variability among clusters plays an important role in shaping the observed puff amplitude distribution and that a model for which I(N(p)) approximately N(p) for small N(p) and I(N(p)) approximately N(p)(1/alpha) (alpha > 1) for large N(p), provides the best agreement. Simulations of more detailed models in which channels open and close stochastically show that this nonlinear behavior can be attributed to the limited time resolution of the observations and to the averaging procedure that is implicit in the mean-field models. These conclusions are also compatible with observations of approximately 400 puffs obtained using the dye Oregon green.
| S-EPMC2839043 | biostudies-literature
Project description:Using a combination of deep sequencing and bioinformatics approach, we for the first time identify miRNAs and their relative abundance in mature, metaphase II arrested eggs in Xenopus laevis. We characterize 115 miRNAs that have been described either in Xenopus tropicalis (85), X. laevis (9), or other vertebrate species (21) that also map to known Xenopus pre-miRNAs and to the X. tropicalis genome. In addition, 72 new X. laevis putative candidate miRNAs are identified based on mapping to X. tropicalis genome within regions that have the propensity to form hairpin loops. These data expand on the availability of genetic information in X. laevis and identify target miRNAs for future functional studies.
| S-EPMC3309558 | biostudies-literature
Project description:The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein post-translational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 2636 phosphosites. We used structural information and phosphoproteomic data for 13 other species in order to predict functionally important phospho-regulatory events. We found that the degree of conservation of phosphosites across species is predictive of sites with known molecular function. In addition, we predicted kinase-protein interactions for a set of cell-cycle kinases across all species. The degree of conservation of kinase-protein interactions was found to be predictive of functionally relevant regulatory interactions. Finally, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. Our analysis suggests that a small class of phosphosites occurs in positions that have the potential to regulate protein conformation.
| S-EPMC4552029 | biostudies-literature
Project description:Xenopus laevis oocytes are a widely used model system because of their capacity to translate exogenous mRNA, but their high intrinsic background fluorescence is a disadvantage for fluorescence recordings. Here, we developed two distinct methods for improving fluorescence recordings from oocytes. One was a pharmacological method in which a small-molecule salt-inducible kinase inhibitor was co-injected with the mRNA of interest to stimulate melanin production. We interrogated the oocytes using cut-open voltage clamp with simultaneous fluorescence recording and found that by increasing the amount of light-absorbing melanin in these oocytes, we decreased their intrinsic background fluorescence. The treated oocytes produced fluorescence signals that were approximately four times larger. The second method consisted of direct injection of synthetic melanin. This method also significantly improved (doubled) fluorescence signals and allowed any oocyte to be used for fluorescence recording. These two methods provide significant improvements of the signal quality for fluorescent oocyte recordings and allow all healthy oocytes to be used for high-sensitivity recordings.
| S-EPMC6363413 | biostudies-literature
Project description:Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min-1/mg-1) in testes compared to oocytes in Xenopus laevis (X. laevis). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free trans-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F1 (e.g., ATP-α-F1) and Fo (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F1 oxidation at two evolutionary conserved cysteine residues (C244 and C294) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min-1/mg-1 in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in X. laevis oocytes.
| S-EPMC7139892 | biostudies-literature
Project description:Cultured human choriocarcinoma (BeWo) cells have previously been shown to exhibit, in comparison with other cultured cell types, elevated nitrobenzylthioinosine (NBMPR)-sensitive transport activity and large numbers (> 10(7)/cell) of high-affinity NBMPR-binding sites [Boumah, Hogue and Cass (1992) Biochem. J. 288, 987-996]. The present study investigates whether NBMPR-sensitive nucleoside transport activity could be induced in Xenopus laevis oocytes by microinjection of poly(A)+ RNA isolated from proliferating cultures of BeWo cells. Expression of uridine transport activity was assayed by comparing rates of uptake (22 degrees C) of 100 microM [3H]uridine by RNA-injected oocytes with uptake by water-injected or uninjected oocytes. A 4-fold stimulation of uridine uptake (2.0 versus 0.5 pmol/90 min per oocyte) was seen when oocytes were injected with 50 ng of BeWo poly(A)+ RNA, and this stimulation was abolished when the RNA-injected oocytes were assayed in the presence of 10 microM NBMPR. The expressed uridine transport activity in oocytes was highly sensitive to NBMPR, with a 50% reduction seen at 1.1 nM NBMPR (IC50 value). The IC50 value for NBMPR inhibition of uptake of 100 microM [3H]uridine by intact BeWo cells was 1.4 nM. Inward fluxes of [3H]uridine in the RNA-injected oocytes were greatly reduced in the presence of high concentrations (2 mM) of non-radioactive nucleosides (adenosine, thymidine, inosine) that are known permeants of NBMPR-sensitive nucleoside transport processes. These results establish that the abundance of NBMPR-sensitive nucleoside transporter mRNA in poly(A)+ RNA preparations from BeWo cells is sufficient to achieve production of functionally active transporter protein in Xenopus oocytes and that, when expressed in Xenopus oocytes, the transporters exhibit NBMPR sensitivity and permeant selectively similar to that of the native transporters.
| S-EPMC1138087 | biostudies-other
Project description: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.
| S-EPMC6013169 | biostudies-literature
Project description:Epithelial sodium channels (ENaC) are heterotrimeric structures, made up of ?, ?, and ? subunits, and play an important role in maintaining fluid homeostasis. When ?-ENaC subunits are expressed in place of (or in addition to) the ?-ENaC subunit alongside ?- and ?- subunits, fundamental changes in the biophysical properties of ENaC can be observed. Using human ENaC cRNA constructs and the Xenopas laevis oocyte expression system, we show that oxidized glutathione (GSSG) differently effects ???-ENaC and ???-ENaC current. GSSG (400 ?M) significantly decreased normalized whole cell current in oocytes expressing ???-ENaC, and conversely increased whole cell current in ?1??-ENaC and ?2??-ENaC expressing oocytes. GSSG treatment increased current in oocytes expressing all four subunits. Western blot and PCR analysis show that human small airway epithelial cells (hSAEC) express canonical ???-subunits alongside ?-ENaC subunits. Differences in single channel responses to GSSG in hSAECs indicate that airway epithelia redox sensitivity may depend on whether ?- or ?- subunits assemble in the membrane. In silico analysis predict that six Cys amino acids in the ?-ENaC extracellular loop, and a single Cys in the N-terminal domain, are susceptible to post-translational modification by GSSG. Additional studies are needed to better understand the molecular regulation and pathophysiological roles of oxidized glutathione and ?-ENaC in lung disorders.
| S-EPMC7351335 | biostudies-literature
Project description:MicroRNA-protein complexes (microRNPs) can activate translation of target reporters and specific mRNAs in quiescent (i.e., G0) mammalian cell lines. Induced quiescent cells, like folliculated immature oocytes, have high levels of cAMP that activate protein kinase AII (PKAII) to maintain G0 and immature states. We report microRNA-mediated up-regulated expression of reporters in immature Xenopus laevis oocytes, dependent on Xenopus AGO or human AGO2 and on FXR1, as in mammalian cells. Importantly, we find that maintenance of cAMP levels and downstream PKAII signaling are required for microRNA-mediated up-regulated expression in oocytes. We identify an important, endogenous cell state regulator, Myt1 kinase, as a natural target of microRNA-mediated up-regulation in response to xlmiR16, ensuring maintenance of oocyte immaturity. Our data reveal the physiological relevance of cAMP/PKAII-controlled posttranscriptional gene expression activation by microRNAs in maintenance of the immature oocyte state.
| S-EPMC3100953 | biostudies-literature