Uptake of biotin by native Xenopus laevis oocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C397-C401 ◽  
Author(s):  
H. M. Said ◽  
L. Polenzani ◽  
S. Khorchid ◽  
D. Hollander ◽  
R. Miledi
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Significant Inhibition ◽  
Metabolic Inhibitors ◽  
Xenopus Laevis Oocytes ◽  
Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Repair of heteroduplex DNA in Xenopus laevis oocytes.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 459-470 ◽  
Author(s):  
C W Lehman ◽  
S Jeong-Yu ◽  
J K Trautman ◽  
D Carroll
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Xenopus Laevis Oocytes ◽  
Heteroduplex Dna ◽  
Strand Breaks ◽  
Single Base ◽  
Mismatch Correction

Abstract We have hypothesized that the inheritance of heteroallelic markers during recombination of homologous DNAs in Xenopus oocytes is determined by resolution of a heteroduplex intermediate containing multiple single-base mismatches. To test this idea, we prepared synthetic heteroduplexes carrying 8 separate mispairs in vitro and injected them into oocyte nuclei. DNA was recovered and analyzed directly, by Southern blot-hybridization, and indirectly, by cloning individual repair products in bacteria. Mismatch correction was quite efficient in the oocytes; markers on the same strand were commonly co-corrected, indicating a long-patch mechanism; and the distribution of markers was very similar to that obtained by recombination. This supports our interpretation of the recombination outcome in terms of a resection-annealing mechanism. The injected heteroduplexes carried strand breaks (nicks) as a result of their method of preparation. We tested the idea that mismatch correction might be nick-directed by ligating the strands of the heteroduplex substrate to form covalently closed circles. Repair in oocytes was still efficient, and long patches predominated; but the pattern of recovered markers was quite different than with the nicked substrate. This suggests that nicks, when present, do indeed direct repair, but that, in their absence, recognition of specific mismatches governs repair of the ligated heteroduplexes.

An endogenous ATP-sensitive glutathione S-conjugate efflux mechanism in Xenopus laevis oocytes

1996 ◽  
Vol 270 (5) ◽  
pp. R1156-R1162 ◽  
Author(s):  
N. Ballatori ◽  
W. Wang ◽  
L. Li ◽  
A. T. Truong
Keyword(s):  
Xenopus Laevis ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Organic Anion ◽  
Atp Depletion ◽  
Transport Systems ◽  
Disulfonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Temperature Sensitive ◽  
Comparable Rate

Constitutive efflux mechanisms for reduced glutathione (GSH) and the glutathione S-conjugates S-ethylglutathione (ethyl-SG) and S-(2,4-dinitrophenol)-glutathione (DNP-SG) were examined in Xenopus laevis oocytes. Oocytes were loaded by either microinjection with 50 nl of the 3H-labeled compounds or were exposed to unlabeled 1-chloro-2,4-dinitrobenzene and efflux of DNP-SG synthesized within the oocytes measured spectrophotometrically. Efflux of unlabeled DNP-SG (approximately 1.2 mM intracellular concentration) and microinjected 0.5 mM [3H]DNP-SG was a linear function of time, with approximately 20% released in 3 h at 25 degrees C. [3H] ethyl-SG, 0.5 mM, was released at a comparable rate, whereas only 4% of a tracer dose of [3H]GSH (2.5 mM intracellular GSH) was released in 3 h. Efflux of all three compounds was temperature sensitive and inhibited after ATP depletion but unaffected when Na+ in the culture medium was replaced with K+ or when the pH was changed from 7.5 to either 6.8 or 8.0. Efflux was saturable, with apparent Michaelis constant values of 0.91 +/- 0.19, 0.44 +/- 0.25, and 5.3 +/- 2.2 mM for DNP-SG, ethyl-SG, and GSH, respectively. Bilirubin ditaurate, 0.5 mM, cis-inhibited efflux of 0.5 mM [3H]DNP-SG, 0.5 mM [3H]ethyl-SG, and 2.5 mM [3H]GSH. DNP-SG and ethyl-SG efflux was also cis-inhibited by other glutathione S-conjugates, 0.25 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, 0.5 mM sulfobromophthalein, and 0.5 mM dibromosulfophthalin, but not by 0.25 mM taurocholate. [3H]GSH release (2.5 mM) was unaffected by these compounds or by 10 mM extracellular GSH or methionine. These findings indicate that Xenopus oocytes have an endogenous ATP-sensitive mechanism for extruding glutathione S-conjugates, with properties comparable to ATP-dependent glutathione S-conjugate/organic anion transport systems described in a variety of cell types. However, in contrast to mammalian cells, GSH and ethyl-SG release from Xenopus oocytes was also inactivated after cellular ATP depletion but was not sensitive to membrane depolarization in high-K+ medium or trans-stimulated by extracellular GSH, indicating that efflux of these organic anions from Xenopus laevis oocytes is also mediated by an ATP-sensitive mechanism.

A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

1985 ◽  
Vol 5 (10) ◽  
pp. 2697-2704
Author(s):  
L J Lorenz ◽  
J D Richter
Keyword(s):  
Xenopus Laevis ◽  
Cdna Clone ◽  
Rna Binding ◽  
Binding Protein ◽  
Single Gene ◽  
In Vitro Translation ◽  
Xenopus Laevis Oocytes ◽  
Cdna Insert ◽  
Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

Selective enhancement of bovine papillomavirus type 1 DNA replication in Xenopus laevis eggs by the E6 gene product

1989 ◽  
Vol 9 (2) ◽  
pp. 406-414
Author(s):  
H Romanczuk ◽  
W M Wormington
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
Gene Product ◽  
Mammalian Cells ◽  
Xenopus Laevis Oocytes ◽  
Bovine Papillomavirus ◽  
In Vitro Synthesis ◽  
E6 Gene ◽  
E6 Protein

Genetic analyses of bovine papillomavirus type 1 (BPV-1) DNA in transformed mammalian cells have indicated that the E6 gene product is essential for the establishment and maintenance of a high plasmid copy number. In order to analyze the direct effect of the E6 protein on the replication of a BPV-1-derived plasmid, a cDNA containing the BPV-1 E6 open reading frame was subcloned into an SP6 vector for the in vitro synthesis of the corresponding mRNA. The SP6 E6 mRNA was injected into Xenopus laevis oocytes to determine the subcellular localization of the E6 gene product and to analyze the effect of the protein on BPV-1 DNA replication. SP6 E6 mRNA microinjected into stage VI oocytes was translated into a 15.5-kilodalton protein that was specifically immunoprecipitated by antibodies directed against the E6 gene product. The E6 protein preferentially accumulated in oocyte nuclei, a localization which is consistent with the replicative functions in which it has been implicated. The expression of E6 in replication-competent mature oocytes selectively enhanced the replication of a BPV-derived plasmid, indicating a direct role for this gene product in the control of BPV-1 DNA replication.

The secretion of proteins in vitro from Xenopus oocytes and their accessory cells: a biochemical and morphological study

Development ◽  
1981 ◽  
Vol 61 (1) ◽  
pp. 367-383
Author(s):  
T. J. Mohun ◽  
C. D. Lane ◽  
A. Colman ◽  
C. C. Wylie
Keyword(s):  
Gel Electrophoresis ◽  
Messenger Rna ◽  
Morphological Study ◽  
Xenopus Oocytes ◽  
Secretory Proteins ◽  
Xenopus Laevis Oocytes ◽  
Follicular Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Accessory Cells

Protein secretion by Xenopus laevis oocytes and their surrounding follicular cells in vitro has been investigated using two-dimensional gel electrophoresis. Viable oocytes, devoid of follicle layers, were prepared by treatment with collagenase; they retain in full their capacity to synthesize, sequester and export secretory proteins following microinjection with heterologous messenger RNA. Both RNA-injected and normal cells export a large number of endogenous oocyte proteins and, as with heterologous secretory translation products, these proteins are found within the oocyte in a vesicle fraction. Electron microscopy indicates that secretion involves exocytotic release of cortical vesicle contents. The follicular cells themselves also seem to contribute a number of proteins to the incubation medium surrounding isolated oocytes, but the presence of follicle layers is not required for the export of endogenous oocyte proteins.

Remifentanil Directly Activates Human N-methyl-d-aspartate Receptors Expressed in Xenopus laevis Oocytes

2004 ◽  
Vol 100 (6) ◽  
pp. 1531-1537 ◽  
Author(s):  
Klaus Hahnenkamp ◽  
Joke Nollet ◽  
Hugo K. Van Aken ◽  
Hartmut Buerkle ◽  
Tobias Halene ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Postoperative Pain ◽  
Xenopus Laevis ◽  
Nmda Receptors ◽  
Messenger Rna ◽  
Xenopus Laevis Oocytes ◽  
Opiate Tolerance ◽  
Induced Currents ◽  
Pore Blocker

Background Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-D-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva directly stimulates human NMDA receptors. Methods To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. Results Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nM-1 mM each) or Ultiva (0.01 pM-0.1 mM) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 microM/12 microM for glutamate/glycine and 3.5 nM for Ultiva, and NR1A/2B EC50 values were 3.9 microM/1.9 microM for glutamate/glycine and 0.82 microM for Ultiva. Glycine in combination with Ultiva showed no additive effect compared with Ultiva alone. Ultiva-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 microM) did not stimulate NMDA receptors. Conclusion These data indicate that Ultiva but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.

scholarly journals Effect of antisense oligonucleotides on the expression of hepatocellular bile acid and organic anion uptake systems in Xenopus laevis oocytes

1996 ◽  
Vol 316 (3) ◽  
pp. 901-904 ◽  
Author(s):  
Bruno HAGENBUCH ◽  
Bruce F. SCHARSCHMIDT ◽  
Peter J. MEIER
Keyword(s):  
Bile Acid ◽  
Xenopus Laevis ◽  
Rat Liver ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Organic Anion ◽  
Xenopus Laevis Oocytes ◽  
Acid Uptake ◽  
Uptake System ◽  
Anion Uptake

A Na+-dependent bile acid (Na+/taurocholate co-transporting polypeptide; Ntcp) and a Na+-independent bromosulphophthalein (BSP)/bile acid uptake system (organic-anion-transporting polypeptide; oatp) have been cloned from rat liver by using functional expression cloning in Xenopus laevis oocytes. To evaluate the extent to which these cloned transporters could account for overall hepatic bile acid and BSP uptake, we used antisense oligonucleotides to inhibit the expression of Ntcp and oatp in Xenopus laevis oocytes injected with total rat liver mRNA. An Ntcp-specific antisense oligonucleotide co-injected with total rat liver mRNA blocked the expression of Na+-dependent taurocholate uptake by approx. 95%. In contrast, an oatp-specific antisense oligonucleotide when co-injected with total rat liver mRNA had no effect on the expression of Na+-dependent taurocholate uptake, but it blocked Na+-independent uptake of taurocholate by approx. 80% and of BSP by 50%. Assuming similar expression of hepatocellular bile acid and organic anion transporters in Xenopus laevis oocytes, these results indicate that Ntcp and oatp respectively represent the major, if not the only, Na+-dependent and Na+-independent taurocholate uptake systems in rat liver. By contrast, the cloned oatp accounts for only half of BSP transport, suggesting that there must be additional, non-bile acid transporting organic anion uptake systems in rat liver.

Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes

FEBS Letters ◽  
1989 ◽  
Vol 249 (2) ◽  
pp. 367-370 ◽  
Author(s):  
Herbert Steinbeißer ◽  
Ansgar Hofmann ◽  
Pierre Oudet ◽  
Michael F. Trendelenburg
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Laevis Oocytes

scholarly journals The Cleavage and Polyadenylation Specificity Factor in Xenopus laevis Oocytes Is a Cytoplasmic Factor Involved in Regulated Polyadenylation

1999 ◽  
Vol 19 (8) ◽  
pp. 5707-5717 ◽  
Author(s):  
Kirsten S. Dickson ◽  
Andrea Bilger ◽  
Scott Ballantyne ◽  
Marvin P. Wickens
Keyword(s):  
Xenopus Laevis ◽  
Bos Taurus ◽  
Xenopus Laevis Oocytes ◽  
Ns1 Protein ◽  
Amino Acid Sequence Level ◽  
Cytoplasmic Factor ◽  
Cytoplasmic Polyadenylation ◽  
Cleavage And Polyadenylation ◽  
Specificity Factor

ABSTRACT During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurusthymus. This report provides three lines of evidence that implicate theX. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevisoocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit ofX. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevisoocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

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