scholarly journals Expression of rat liver Na+/l-alanine co-transport in Xenopus laevis oocytes. Effect of glucagon in vivo

1990 ◽  
Vol 270 (1) ◽  
pp. 189-195 ◽  
Author(s):  
M Palacin ◽  
A Werner ◽  
J Dittmer ◽  
H Murer ◽  
J Biber
Keyword(s):  
Xenopus Laevis ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Transport Systems ◽  
Xenopus Laevis Oocytes ◽  
Alanine Transport ◽  
Dependent Component ◽  
Gradient Fractionation

Poly(A)+ RNA (mRNA) isolated from rat liver was injected into Xenopus laevis oocytes, and expression of Na+/L-alanine transport was assayed by measuring Na(+)-dependent uptake of L-[3H]alanine. Expression of Na+/L-alanine transport was detected 3-7 days after mRNA injection, and was due to an increment of the Na(+)-dependent component. After injection of 40 ng of total mRNA, Na(+)-dependent uptake of L-alanine was 2.5-fold higher than in water-injected oocytes. In contrast with Na+/L-alanine transport by water-injected oocytes, expressed Na+/L-alanine transport was inhibited by N-methylaminoisobutyric acid, was inhibited by an extracellular pH of 6.5 and was saturated at approx. 1 mM-L-alanine. After sucrose-density-gradient fractionation, highest expression of Na+/L-alanine uptake was observed with mRNA of 1.9-2.5 kb in length. Compared with mRNA isolated from control rats, mRNA isolated from glucagon-treated rats showed a approx. 2-fold higher expression of Na+/L-alanine transport. The results demonstrate that both liver Na+/L-alanine transport systems (A and ASC) can be expressed in X. laevis oocytes. Furthermore, the data obtained with mRNA isolated from glucagon-treated rats suggest that glucagon regulates liver Na+/L-alanine transport (at least in part) via the availability of the corresponding mRNA.

Characterization and Partial Purification of Heterodisperse Polysomal RNAs in Normal and Neoplastic Cells

1972 ◽  
Vol 58 (2) ◽  
pp. 71-94
Author(s):  
Ada Sacchi ◽  
Gianni Chinali ◽  
Susetta Pons ◽  
Michela Galdieri ◽  
Piero Cammarano
Keyword(s):  
Size Distribution ◽  
Density Gradient ◽  
Rat Liver ◽  
Sucrose Density Gradient ◽  
Partial Purification ◽  
Messenger Rnas ◽  
Alkaline Ph ◽  
Sedimentation Profile ◽  
Molecular Weights ◽  
Phenol Extraction

The size distribution of cytoplasmic messenger RNAs (m-RNA) has been studied in rat liver and in monodifferentiated cells (mouse reticulocytes and myelomas). It has been found that the RNA which exhibits a « rapid turnover » and a polydisperse profile of radioactivity is refractory to phenol extraction. This property has been exploited to selectively isolate m–RNA from the phenol residue by means of an extraction at an alkaline pH. The sucrose density gradient profiles of m–RNA isolated from monodifferentiated cells show monodisperse peaks having the sedimentation coefficients expected on the basis of the molecular weights of monocistronic messages for α and β chains of hemoglobin (reticulocytes) and L and H chains of immunoglobulin (myelomas). The sedimentation profile of cytoplasmic m–RNA associated with rat liver polysomes shows a much broader distribution, with sedimentation coefficients ranging from 8 S to 28 S.

trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes

1989 ◽  
Vol 9 (11) ◽  
pp. 5244-5247
Author(s):  
N Benvenisty ◽  
T Shoshani ◽  
Y Farkash ◽  
H Soreq ◽  
L Reshef
Keyword(s):  
Gene Expression ◽  
Xenopus Laevis ◽  
Reporter Gene ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Xenopus Laevis Oocytes ◽  
Activation Process ◽  
Tissue Specific ◽  
Chimeric Construct

To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.

scholarly journals Expression of Na+-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA

1992 ◽  
Vol 281 (3) ◽  
pp. 717-723 ◽  
Author(s):  
J Bertran ◽  
A Werner ◽  
G Stange ◽  
D Markovich ◽  
J Biber ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
Transport System ◽  
Sucrose Density Gradient ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Xenopus Laevis Oocytes ◽  
Transport Pathways ◽  
Arginine Uptake ◽  
Dose Dependent Increase

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

scholarly journals Actions of Agonists, Fipronil and Ivermectin on the Predominant In Vivo Splice and Edit Variant (RDLbd, I/V) of the Drosophila GABA Receptor Expressed in Xenopus laevis Oocytes

PLoS ONE ◽  
2014 ◽  
Vol 9 (5) ◽  
pp. e97468 ◽  
Author(s):  
Kristin Lees ◽  
Maria Musgaard ◽  
Siros Suwanmanee ◽  
Steven David Buckingham ◽  
Philip Biggin ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Gaba Receptor ◽  
Xenopus Laevis Oocytes

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.

scholarly journals The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1970 ◽  
Vol 117 (2) ◽  
pp. 319-324 ◽  
Author(s):  
G. J. Mulder
Keyword(s):  
Enzyme Activity ◽  
Density Gradient ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Male Rats ◽  
Uridine Diphosphate ◽  
Triton X

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The Km of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.

Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate

FEBS Letters ◽  
1989 ◽  
Vol 251 (1-2) ◽  
pp. 219-224 ◽  
Author(s):  
Odile Mulner-Lorillon ◽  
Robert Poulhe ◽  
Patrick Cormier ◽  
Jean-Claude Labbe ◽  
Marcel Doree ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Xenopus Laevis Oocytes
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