Differential inhibition of AE1 and AE2 anion exchangers by oxonol dyes and by novel polyaminosterol analogs of the shark antibiotic squalamine

1998 ◽  
Vol 76 (5) ◽  
pp. 799-806 ◽  
Author(s):  
Seth L Alper ◽  
Marina N Chernova ◽  
Jon Williams ◽  
Michael Zasloff ◽  
Foon-Yee Law ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Anion Exchange ◽  
Xenopus Oocytes ◽  
Red Cells ◽  
Xenopus Laevis Oocytes ◽  
Red Cell ◽  
Cell Type ◽  
Anion Exchangers ◽  
Oxonol Dyes ◽  
Assay Conditions

Oxonol and polyaminosterol drugs were examined as inhibitors of recombinant mouse AE1 and AE2 anion exchangers expressed in Xenopus laevis oocytes and were compared as inhibitors of AE1-mediated anion flux in red cells and in HL-60 cells that express AE2. The oxonols WW-781, diBA(5)C4, and diBA(3)C4 inhibited HL-60 cell Cl-/Cl- exchange with IC50 values from 1 to 7 µM, 100-1000 times less potent than their IC50 values for red cell Cl-/anion exchange. In Xenopus oocytes, diBA(5)C4 inhibited AE1-mediated Cl- efflux several hundred times more potently than that mediated by AE2. Several novel squalamine-related polyaminosterols were also evaluated as anion exchange inhibitors. In contrast to diBA(5)C4, polyaminosterol 1361 inhibited oocyte-expressed AE2 8-fold more potently than AE1 (IC50 0.6 versus 5.2 µM). The 3-fold less potent desulfo-analog, 1360, showed similar preference for AE2. It was found that 1361 also partially inhibited Cl- efflux from red cells, whereas neither polyaminosterol inhibited Cl efflux from HL60 cells. Thus, the oxonol diBA(5)C4 is >100-fold more potent as an inhibitor of AE1 than of AE2, whereas the polyaminosterols 1360 and 1361 are 8-fold more potent as inhibitors of AE2 than of AE1. Assay conditions and cell type influenced IC50 values for both classes of compounds.Key words: band 3, oxonols, squalamine, Xenopus laevis oocytes, HL-60 cells.

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 Xenopus laevis lectin is localized at several sites in Xenopus oocytes, eggs, and embryos.

1983 ◽  
Vol 97 (6) ◽  
pp. 1875-1881 ◽  
Author(s):  
M M Roberson ◽  
S H Barondes
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Immunoperoxidase Staining ◽  
Xenopus Laevis Oocytes ◽  
Embryonic Cells ◽  
Cortical Granules ◽  
Glutaraldehyde Fixation ◽  
Blastula Stage ◽  
Vitelline Envelope ◽  
Developing System

The endogenous lectin of Xenopus laevis oocytes, unfertilized eggs, and blastula-stage embryos was immunohistochemically localized using a highly specific antiserum. Each tissue was examined with several techniques, including paraformaldehyde or glutaraldehyde fixation, frozen or plastic sections, and immunofluorescence or immunoperoxidase staining. In oocytes and unfertilized eggs, lectin was detected in association with yolk platelets, cortical granules, and the vitelline envelope. In embryos, cortical granules had disappeared and lectin was found in the cleavage furrows between the embryonic cells. The distribution of the lectin suggests that it plays more than one role in this developing system.

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.

Protooncogene product, c-mos kinase, is involved in upregulating Na+/H+ antiporter in Xenopus oocytes

1994 ◽  
Vol 267 (6) ◽  
pp. C1717-C1722 ◽  
Author(s):  
K. Rezai ◽  
A. Kulisz ◽  
W. J. Wasserman
Keyword(s):  
Plasma Membrane ◽  
Xenopus Laevis ◽  
Intracellular Ph ◽  
Xenopus Oocytes ◽  
Stage Iv ◽  
Meiotic Maturation ◽  
Xenopus Laevis Oocytes ◽  
Cell Systems ◽  
Oocyte Meiotic Maturation

Progesterone-stimulated Xenopus laevis oocytes undergo an increase in their intracellular pH from 7.3 to 7.7 because of the activation of Na+/H+ antiporters in their plasma membrane. Activation of Na+/H+ exchangers (NHE) in other cell systems appears to be regulated by phosphorylation of the NHE protein. In the current study we demonstrated that cytoplasm taken from steroid-stimulated oocytes rapidly induced an increase in intracellular pH when microinjected into full-grown stage VI recipient oocytes. The protein within the cytoplasm that appears to be responsible for this activity is c-mos kinase. Microinjected pure mosxe kinase protein rapidly activated the Na+/H+ exchangers in full-grown recipient oocytes. Furthermore, injected mosxe protein rapidly activated the Na+/H+ exchangers in smaller progesterone-insensitive stage IV oocytes. Therefore, it appears that the protooncogene product, p39 c-mos kinase, which is normally synthesized in full-grown stage VI oocytes in response to progesterone stimulation, is involved in the upregulation of the Na+/H+ antiporters during oocyte meiotic maturation.

scholarly journals pH Dependent Hemolytic Systems. II. Serum Factors Involved in Red Cell Lysis

Blood ◽  
1961 ◽  
Vol 17 (4) ◽  
pp. 474-490 ◽  
Author(s):  
STANLEY YACHNIN ◽  
FRANK H. GARDNER
Keyword(s):  
Human Serum ◽  
Cell Lysis ◽  
Cross Reaction ◽  
Red Cells ◽  
Red Cell ◽  
Cell Type ◽  
Serum Factors ◽  
Specific Serum ◽  
Human Sera ◽  
Ph Dependent

Abstract An analysis of the serum factors involved in the lysis and agglutination of artificially altered red cells in compatible human serum has been presented. Human sera have been found to vary widely in their capacity to hemolyze a panel of artificially altered red cells. These differences have been shown to be the result of a multiplicity of more or less specific serum factors for each altered red cell type. These factors have many of the properties commonly associated with classical antibody. These serum factors are different from complement and properdin. The range of specificity and the degree of cross reaction for these various serum factors have been analyzed. These serum factors are important in the consideration of the use of enzyme treated red cells for the detection of incomplete antibodies, and of certain acquired hemolytic states in man.

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.

scholarly journals Functional expression of the nitrobenzylthioinosine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis

1994 ◽  
Vol 299 (3) ◽  
pp. 769-773 ◽  
Author(s):  
C E Boumah ◽  
C M Harvey ◽  
A R P Paterson ◽  
S A Baldwin ◽  
J D Young ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Oocytes ◽  
Functional Expression ◽  
Transport Processes ◽  
Nucleoside Transport ◽  
Xenopus Laevis Oocytes ◽  
Nucleoside Transporter ◽  
Transport Activity ◽  
Bewo Cells ◽  
Ic50 Value

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.

Voltage-operated channels induced by foreign messenger RNA in Xenopus oocytes

1983 ◽  
Vol 220 (1218) ◽  
pp. 131-140 ◽  
Keyword(s):  
Xenopus Laevis ◽  
Messenger Rna ◽  
Xenopus Oocytes ◽  
Xenopus Laevis Oocytes ◽  
Denervated Muscle ◽  
Synthesis And Processing ◽  
Oocyte Membrane

Poly(A) + messenger RNA (mRNA) extracted from rat brains or from cat muscles was injected into Xenopus laevis oocytes . This led to the incorporation of voltage-operated Na + and K + channels into the oocyte membrane. These channels are not normally present in the oocyte and presumably result from the synthesis and processing of proteins coded by the injected mRNA. Tetrodotoxin blocked the Na + channels induced by mRNA derived from either innervated or denervated muscle.

Characterization of an endogenous Na+/H+ antiporter in Xenopus laevis oocytes

1991 ◽  
Vol 159 (1) ◽  
pp. 359-369
Author(s):  
D. W. Towle ◽  
A. Baksinski ◽  
N. E. Richard ◽  
M. Kordylewski
Keyword(s):  
Xenopus Laevis ◽  
Binding Sites ◽  
Kinetic Data ◽  
Xenopus Oocytes ◽  
Xenopus Laevis Oocytes ◽  
Collagenase Treatment ◽  
Oocyte Aging ◽  
Na Uptake ◽  
Parallel Measurements

The amiloride-sensitive Na+/H+ antiporter in defolliculated oocytes of Xenopus laevis was characterized by measurements of 22Na+ influx and apparent H+ efflux. Uptake of 22Na+ was linear over a 90-min incubation period and was inhibited approximately 80% with 5 × 10(−4) mol l-1 amiloride. Amiloride-sensitive sodium uptake was reduced following collagenase treatment or oocyte aging. K0.5 for amiloride inhibition was 4.13 × 10(−6) +/− 1.33 × 10(−6)mol l-1 and the Km for Na+ was 4.25 × 10(−3) mol l-1. Hill analysis of the kinetic data for Na+ revealed an nH value of 1.14, indicating an absence of interacting binding sites for Na+. Parallel measurements of amiloride-sensitive Na+ uptake and H+ efflux indicated a Na+/H+ exchange ratio of 0.88:1. Our conclusion is that the Na+/H+ antiporter of Xenopus oocytes exhibits a nominal 1:1 Na+/H+ exchange stoichiometry and is similar in its properties to the antiporter of other vertebrate cells.

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