Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Demonstration of transthyretin mRNA in the brain and other extrahepatic tissues in the rat

Studies were conducted to ascertain if transthyretin mRNA was present in extrahepatic tissues of the rat. A trnasthyretin cDNA clone was isolated from a lambda gt11 human liver cDNA library by antibody screening and its identity was confirmed by nucleotide sequence analysis. This transthyretin cDNA clone was used to survey poly(A+) RNA isolated from 12 different rat tissues for transthyretin mRNA by Northern blot analysis. The liver contained the highest level of transthyretin mRNA and this level was not altered by the vitamin A status of the rat. A significant amount of transthyretin mRNA was found in the brain (30% of the level of the liver) which was localized in specific regions of the brain. In addition, detectable levels of transthyretin mRNA (1% to 2% of that of the liver) were observed in the stomach, heart, skeletal muscle, and spleen. Translation of brain poly(A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-transthyretin antiserum resulted in a protein band of the same size as liver pre-transthyretin. Liver pre-transthyretin was processed by the cotranslational addition of dog pancreas microsomal membranes to a protein that migrated coincidentally with monomeric serum transthyretin. These data suggest that transthyretin in the brain and the cerebrospinal fluid results from de novo synthesis and that transthyretin may play a significant physiological function, as yet unknown, within the nervous system.     

Comparison of populations of mRNA coding fumarase in rat brain and liver

The populations of mRNA encoding mitochondrial and cytosolic fumarases in the poly A+ RNA fractions purified from polysomes of rat brain and liver were examined. When the in vitro translation products programmed by the poly A+ RNA fraction obtained from rat brain were purified by immunoprecipitation with anti-fumarase antibody and analyzed by SDS polyacrylamide gel electrophoresis and fluorography, only one polypeptide of 50 KD was detected as a precursor of fumarase. In contrast, by the same method, two polypeptides of 50 KD and 45 KD, which is the same size as mature fumarase, were detected as precursors of rat liver fumarase. These results suggest that rat brain polysomes contain only one population of mRNA coding a 50 KD precursor of mitochondrial fumarase with little or no mRNA of the cytosolic fumarase, whereas rat liver polysomes contain two types of mRNA for mitochondrial and cytosolic fumarases, respectively. These findings are consistent with the fact that the brain is the only organ in rats known not to contain cytosolic fumarase.     

Studies on protein kinases involved in regulation of nucleocytoplasmic mRNA transport.

The rate of energy-dependent nucleoside triphosphatase (NTPase)-mediated nucleocytoplasmic translocation of poly(A)-containing mRNA [poly(A)+mRNA] across the nuclear envelope is thought to be regulated by poly(A)-sensitive phosphorylation and dephosphorylation of nuclear-envelope protein. Studying the phosphorylation-related inhibition of the NTPase, we found that phosphorylation of one polypeptide of rat liver envelopes by endogenous NI- and NII-like protein kinase was particularly sensitive to poly(A). This polypeptide (106 kDa) was also phosphorylated by nuclear-envelope-bound Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C). Activation of kinase C by tumour-promoting phorbol esters resulted in inhibition of nuclear-envelope NTPase activity and in a concomitant decrease of mRNA (actin) efflux rate from isolated rat liver nuclei. Protein kinase C, but not nuclear envelope NI-like or NII-like protein kinase, was found to be solubilized from the envelope by Triton X-100, whereas the presumable poly(A)-binding site [the 106 kDa polypeptide, representing the putative carrier for poly(A)+mRNA transport] remained bound to this structure. RNA efflux from detergent-treated nuclei lost its susceptibility to phorbol esters. Addition of purified protein kinase C to these nuclei restored the effect of the tumour promoters. Protein kinase C was found to bind also to isolated rat liver nuclear matrices in the absence but not in the presence of ATP. The NII-like nuclear-envelope protein kinase co-purified together with the 106 kDa polypeptide which specifically binds to poly(A) in an ATP-labile linkage.     

Functional dissection of nuclear envelope mRNA translocation system: effects of phorbol ester and a monoclonal antibody recognizing cytoskeletal structures

Unidirectional transport of poly(A)-containing mRNA [poly(A)+ mRNA] through the nuclear envelope pore complex is thought to be an energy (ATP or GTP)-dependent process which involves a nuclear envelope nucleoside triphosphatase (NTPase). In the intact envelope, this enzyme is regulatable by poly(A) binding and by poly(A)-dependent phosphorylation/dephosphorylation of other components of the mRNA translocation system, which are as yet unidentified. Monoclonal antibodies (mAbs) were elicited against the poly(A) binding nuclear envelope fraction isolated from rat liver. The mAbs were screened for their modulatory effects on mRNA transport in vitro. One stable clone decreased the efflux of rapidly labeled RNA and of one specific mRNA (ovalbumin) from isolated nuclei. It increased the binding of poly(A) to the envelope and increased the maximal catalytic rate of the NTPase, but it did not alter the apparent Km of the enzyme or the extent of its stimulation by poly(A). The nuclear envelope-associated protein kinase that down-regulates the NTPase was inhibited by the antibody, while other protein kinases were not affected. Because both the NTPase and mRNA efflux were inhibited by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, the sensitive kinase is probably protein kinase C. Protein kinase C was found to be associated with the isolated nuclear envelope. The antibody reacted with both a Mr 83,000 and a Mr 65,000 nuclear envelope polypeptide from rat liver and other tissues. By immunofluorescence microscopy in CV-1 cells, the antibody localized to the nuclear envelope and, in addition, to cytoplasmic filaments which show some superposition with the microfilament network.     

Modification of the half-life of poly (A+)mRNA in liver and brain cells of the rat during embryogenesis and postnatal development

The poly(A+)/poly(A-)mRNA ratio and the half-life time of poly(A+)mRNA for mRNA metabolism in the liver and brain of rat in the course of ontogensis, late embryogenesis, postnatal development and upon ageing were determined. It was shown that in the course of ontogenesis both the ratio of poly(A+)/poly(A-)mRNA of free and membrane-bound polyribosomes and the half-life time of poly(A+)mRNA determined from the degradation kinetics in the presence of actinomycin D are changed. A possible role of poly(A) sequences in the regulation of mRNA life-time is discussed.     

Somatomedin-C/insulin-like growth factor-I and insulin-like growth factor-II mRNAs in rat fetal and adult tissues

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyse rat Sm-C/IGF-I and IGF-II mRNAs in poly(A+) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobases (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. The abundance of a 7.5-kb Sm-C/IGF-I mRNA in poly(A+) RNAs from adult rat liver was 10-50-fold higher than in other adult rat tissues which provides further evidence that in the adult rat the liver is a major site of Sm-C/IGF-I synthesis and source of circulating Sm-C/IGF-I. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A+) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. Some samples of adult rat intestine contained the 4.7- and 3.9-kb IGF-II mRNAs and some samples of adult liver and lung contained the 4.7-kb mRNA. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded.     

Retinol-binding protein messenger RNA levels in the liver and in extrahepatic tissues of the rat

A retinol-binding protein (RBP) cDNA clone was used to examine the effect of retinol status on the level of RBP mRNA in the liver, and to explore whether extrahepatic tissues contain RBP mRNA. In the first series of experiments, poly(A+) RNA was isolated from the livers of normal, retinol-depleted, and retinol-repleted rats and the levels of RBP mRNA in these samples were determined by both Northern blot and RNA Dot blot analyses. The levels of RBP mRNA in liver were similar in all three groups of rats. These findings confirm and extend previous studies which showed that retinol did not alter the in vivo rate of RBP synthesis or the translatable levels of RBP mRNA. In a second series of experiments, the RBP cDNA clone was used to survey poly (A+) RNA isolated from 12 different rat tissues for RBP mRNA by Northern blot analysis. We found that, along with the liver, many extrahepatic tissues contained RBP mRNA. Kidney contained RBP mRNA at a level of 5-10% of that of the liver, and the lungs, spleen, brain, stomach, heart, and skeletal muscle contained 1-3% of that of the liver. Translation of kidney poly (A+) RNA in rabbit reticulocyte lysates and immunoprecipitation of the translation products with anti-RBP antiserum resulted in a protein band of the same size as liver preRBP. These data suggest that RBP is synthesized in many extrahepatic tissues.It is possible that this extra-hepatically synthesized RBP may function in the recycling of retinol from these tissues back to the liver or to other target organs.     

An analysis of the rate of metallothionein mRNA poly(A)-shortening using RNA blot hybridization.

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.     

Expression of the mammalian system A neutral amino acid transporter in Xenopus oocytes

In this report, we demonstrate the expression of the mammalian System A neutral amino acid transporter in Xenopus laevis oocytes following microinjection of mRNA from rat liver, Chinese hamster ovary (CHO) cells, and human placenta. Stage 6 oocytes were injected with poly(A+) mRNA from one of these three sources and incubated for 24 h prior to assaying Na(+)-dependent 2-aminoisobutyric acid transport to monitor the increase in System A activity. The endogenous 2-aminoisobutyric acid uptake rates in oocytes were sufficiently slow so as to provide a low background value that was subtracted to obtain transport rates for the mammalian carrier alone. The degree of expression of the mammalian System A activity in Xenopus oocytes corresponded to the known transport rates in the tissue from which the mRNA was prepared. For example, hepatic mRNA from glucagon-treated rats produced greater System A activity than mRNA from control animals, and the mRNA from the CHO transport mutant cell line alar4-H3.9, which overproduces System A, resulted in higher transport rates than mRNA from the parental cell line (CHO-K1). Fractionation of total mRNA poly(A+) by nondenaturing agarose gel electrophoresis revealed transport activity associated with a 2.0-2.5-kilobase mRNA fraction common to each of the three tissues tested.     

Biosynthesis of the Brain-specific 14-3-2 Protein in a Cell-free System from Wheat Germ Extract Directed with Poly(A)-containing RNA from Rat Brain

Abstract: 14-3-2 Protein is a neuron-specific protein with a molecular weight of 46,000. Poly(A)-containing RNA was prepared from free polysomes of rat whole brains by means of phenol-chloroform extraction and oligo (dT)-cellulose chromatography. This RNA directed the synthesis of 14-3-2 protein in a cell-free, protein-synthesizing system derived from wheat germ. 14-3-2 Protein was not detected in the products of endogenous incorporation and the products directed with liver poly(A)-containing RNA. These results indicate that mRNA for 14-3-2 protein contains the poly(A) sequence and resides only in the brain.     

Expression of glutathione S-transferases in rat brains

The tissue-specific expression of glutathione S-transferases (GSTs) in rat brains has been studied by protein purification, in vitro translation of brain poly(A) RNAs, and RNA blot hybridization with cDNA clones of the Ya, Yb, and Yc subunit of rat liver GSTs. Four classes of GST subunits are expressed in rat brains at Mr 28,000 (Yc), Mr 27,000 (Yb), Mr 26,300, and Mr 25,000. The Mr 26,3000 species, or Y beta, has an electrophoretic mobility between that of Ya and Yb, similar to the liver Yn subunit(s) reported by Hayes (Hayes, J. D. (1984) Biochem. J. 224, 839-852). RNA blot hybridization of brain poly(A) RNAs with a liver Yb cDNA probe revealed two RNA species of approximately 1300 and approximately 1100 nucleotides. The band at approximately 1300 nucleotides was absent in liver poly(A) RNAs. The Mr 25,000 species, or Y delta, can be immunoprecipitated by antisera against rat heart and rat testis GSTs, but not by antiserum against rat liver GSTs. Therefore, the Y delta subunit may be related to the "Mr 22,000" subunit reported by Tu et al. (Tu, C.-P.D., Weiss, M.J., Li, N., and Reddy, C. C. (1983) J. Biol. Chem. 258, 4659-4662). The abundant liver GST subunits, Ya, are not expressed in rat brains as demonstrated by electrophoresis of purified brain GSTs and a lack of isomerase activity toward the Ya-specific substrate, delta 5-androstene-3,17-dione. This is apparently because of the absence of Ya mRNA expression prior to RNA processing. The data on the preferential expression of Yc subunits in rat brains, together with the differential phenobarbital inducibility of the Ya subunit(s) in rat liver reported by Pickett et al. (Pickett, C. B., Donohue, A. M., Lu, A. Y. H., and Hales, B. F. (1982) Arch. Biochem. Biophys. 215, 539-543), suggest that the Ya and Yc genes for rat GSTs are two functionally distinct gene families even though they share 68% DNA sequence homology. The expression of multiple GSTs in rat brains suggests that GSTs may be involved in physiological processes other than xenobiotics metabolism.     

Differential effect of insulin and epidermal growth factor on the mRNA translocation system and transport of specific poly(A+) mRNA and poly(A-) mRNA in isolated nuclei

The efficiency of efflux of rapidly labeled poly(A)-containing mRNA from isolated rat liver nuclei was found to be modulated by insulin and epidermal growth factor (EGF) in a biphasic but opposite way. At physiological concentrations (10 pM insulin and 1 pM EGF), maximal stimulation of the transport rate by insulin (to 137%) and maximal inhibition by EGF (to 69%) were obtained; at higher concentrations (greater than 100 pM and greater than 10 pM, respectively), the amount of poly(A)-containing mRNA released into the postnuclear supernatant was nearly identical with the level found in untreated nuclei (= 100%). Using mRNA entrapped into closed nuclear envelope (NE) vesicles as a model system, it was found that the modulation of nuclear efflux of mRNA by the two growth factors occurs at the level of translocation through the nuclear pore. The NE nucleoside-triphosphatase (NTPase) activity, which is thought to mediate nucleocytoplasmic transport of at least some mRNAs, responded to insulin and EGF in the same manner as the mRNA transport rate. The increase in NTPase activity caused by insulin and the decrease in NTPase activity caused by EGF were found to be due to changes of the maximal catalytic rate; the Michaelis constant of the enzyme remained almost constant. Investigating the effect of the two growth factors on transport of specific mRNAs, poly(A)-containing actin mRNA was found to display the same alteration in efflux rate as rapidly labeled, total poly(A)-containing mRNA. In contrast, efflux of histone H4 mRNA, which lacks a 3'-poly(A) sequence, decreased in response to insulin and reached minimum levels at the same concentration at which maximum levels of actin mRNA transport rate were obtained. Studying the mechanism of action of insulin and EGF on NE mRNA translocation system, insulin was found to cause an enhancement of NE-associated phosphoprotein phosphatase activity, resulting in a dephosphorylation of the NE poly(A) binding site (= mRNA carrier) and, hence, in a decrease in its affinity to poly(A) [the poly(A) binding affinity of the poly(A)-recognizing mRNA carrier within the envelope is increased after phosphorylation]. EGF, on the other hand, stimulated the protein kinase, which phosphorylates the carrier, and, hence increased the NE poly(A) binding affinity. Because the stage of phosphorylation of the mRNA carrier (which is coupled with the NTPase within the intact NE structure) is inversely correlated with the activity of the NTPase, an enhancement of poly(A)-containing mRNA transport rate by insulin and an inhibition by EGF are observed.     

Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.     

Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells

D-Glucose deprivation of primary rat brain glial cell cultures, by incubation with 25 mM D-fructose for 24 h, resulted in a 4-5-fold induction of D-glucose transport activity. In contrast, 24-h D-glucose starvation of primary rat brain neuronal cultures had only a marginal effect (1.5-2-fold) on D-glucose transport activity. Northern blot analysis of total cellular RNA demonstrated that under these conditions the rat brain glial cells specifically increased the steady-state level of the D-glucose transporter mRNA 4-6-fold, whereas Northern blot analysis of the neuronal cell cultures revealed no significant alteration in the amount of D-glucose transporter mRNA by D-glucose deprivation. These findings demonstrated that the D-glucose-dependent regulation of the D-glucose transporter system occurred in a brain cell type-specific manner. The ED50 for the D-glucose starvation increase in the D-glucose transporter mRNA, in the glial cell cultures, occurred at approximately 3.5 mM D-glucose with maximal effect at 0.5 mM D-glucose. Readdition of D-glucose to the starved cell cultures reversed the increase in the D-glucose transporter mRNA levels and D-glucose transport activity to control values within 24 h. The increase in the D-glucose transporter mRNA was relatively rapid with half-maximal stimulation at approximately 2 h and maximal induction by 6-12 h of D-glucose deprivation. A similar time course was also observed for the starvation-induced increase in D-glucose transport activity and D-glucose transporter protein, as determined by Western blot analysis. These results document that, in rat brain glial cells, D-glucose transport activity, protein, and mRNA are regulated by the extracellular D-glucose concentration. Further, this suggests a potential role for hyperglycemia in the down-regulation of the D-glucose transport system in vivo.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

Regulation of protein disulfide isomerase gene expression in brain and liver during rat development

A 4-fold increase in protein disulfide isomerase (PDI) mRNA is observed in brain of 10 days-old rats and in liver of 20 days-old foetuses when compared with 20 days-old (brain) and 18 days-old (liver) foetuses respectively. During further postnatal development, the mRNA for PDI decreases in both organs to the initial values present in foetuses and remains practically unchanged in brain till the adult. By contrast in liver by 35-40 days after birth, and coincident with sexual maturation, there is a 2.5-fold increase in PDI mRNA that is maintained by 55 days (adult). These results clearly show that protein disulfide isomerase gene expression is differentially regulated in liver and brain during rat development.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Expression of the hepatocyte Na+/bile acid cotransporter in Xenopus laevis oocytes

The expression of the basolateral Na+/bile acid (taurocholate) cotransport system of rat hepatocytes has been studied in Xenopus laevis oocytes. Injection of rat liver poly(A)+ RNA into the oocytes resulted in the functional expression of Na+ gradient stimulated taurocholate uptake within 3-5 days. This Na(+)-dependent portion of taurocholate uptake exhibited saturation kinetics (apparent Km approximately 91 microM) and could be inhibited by 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene. Furthermore, the expressed taurocholate transport activity demonstrated similar substrate inhibition and stimulation by low concentrations of bovine serum albumin as the basolateral Na+/bile acid cotransport system previously characterized in intact liver, isolated hepatocytes, and isolated plasma membrane vesicles. Finally, a 1.5- to 3.0-kilobase size-class of mRNA could be identified that was sufficient to express the basolateral Na+/taurocholate uptake system in oocytes. These results demonstrate that "expression cloning" represents a promising approach to ultimately clone the gene and to further characterize the molecular properties of this important hepatocellular membrane transport system.