Inactivation of G-protein genes: double knockout in cell lines.
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Animals↗
Biomedical subjects
R M Mortensen
Publications and source records attributed to R M Mortensen.
Thyroid hormone receptor-alpha inhibits retinoic acid-responsive gene expression and modulates retinoic acid-stimulated neural differentiation in mouse embryonic stem cells.
Thyroid hormone (T3) and retinoic acid (RA) are essential for normal vertebrate development and are known to coregulate several genes. Early development is predominantly retinoic acid sensitive, yet thyroid hormone receptor-alpha (T3R alpha) is expressed along with retinoic acid receptors (RAR)-alpha, -beta, and -gamma. To determine the role of unliganded T3R alpha in early development and on RA-stimulated neural development, we used homologous recombination techniques to inactivate both T3R alpha gene alleles in mouse embryonic stem (ES) cells. Loss of both T3R alpha alleles resulted in an increase in basal and RA-induced expression of the endogenous RA-responsive genes, RAR beta and alkaline phosphatase, which demonstrates that T3R alpha has an inhibitory effect on the RA response. A similar magnitude of T3R inhibition of the RA response was seen in transient transfection assays of RA response elements in both ES and assays of RA response elements in both ES and JEG cells. Cotransfection experiments were used to demonstrate that inhibition of the RA response could be mediated by T3R alpha 1. The addition of T3R alpha 1, but not the T3R alpha variant c-erbA alpha 2, to T3R alpha-null ES cells restored the inhibitory effect on RA-induced gene expression. RA-stimulated neural differentiation was seen in the wild-type, but not in T3R alpha-null ES, cells, consistent with reports of abnormal neural development as a consequence of premature RA stimulation. Our results demonstrate that the early expression of unliganded T3R alpha functions to modulate the RA response and RA-stimulated neural differentiation.
Alkaline Phosphatase↗
Double knockouts. Production of mutant cell lines in cardiovascular research.
Double knockouts by homologous recombination is a method for producing cell lines with an inactivating mutation in any desired gene. The biochemical analysis of genetically altered cell lines has been important in determining the function of specific proteins. Until recently, mutant cell lines have been produced by random mutagenesis and then selection for a particular phenotypic change. Recent technological advances in gene targeting by homologous recombination now enable the production of mutants in any desired gene. Diploid cells contain two copies or alleles of each gene encoded on an autosome (nonsex) chromosome. In most cases, both alleles must be inactivated to produce a phenotypic change in a mutant cell line, hence the term "double knockout." We and others have described the production of mutationally altered cell lines by inactivating both alleles by the production of two targeting vectors, two separate homologous recombination events, and selection. A simpler procedure, involving considerably less effort and time, has been used to inactivate several alpha-subunits of G proteins and other genes. This method facilitates the inactivation of more than one gene in a single cell line.
Alleles↗
Growth and differentiation of embryonic stem cells that lack an intact c-fos gene.
The c-fos protooncogene encodes a transcription factor that is thought to play a critical role in proliferation and differentiation as well as in the physiological response of mature cells to their environment. To test directly the role of c-fos in growth and differentiation, we generated mouse embryonic stem cell lines in which both copies of the c-fos gene were specifically disrupted by homologous recombination. Remarkably, the disruption of both copies of c-fos in these cells has no detectable effect on embryonic stem cell viability, growth rate, or differentiation potential. Embryonic stem cells lacking c-fos can differentiate into a wide range of cell types in tissue culture and also in chimeric mice. We conclude that despite a large body of literature suggesting an important role for c-fos in cell growth and differentiation, in at least some cell types this gene is not essential for these processes.
Alleles↗
Production of homozygous mutant ES cells with a single targeting construct.
We have developed a simple method for producing embryonic stem (ES) cell lines whereby both alleles have been inactivated by homologous recombination and which requires a single targeting construct. Four different ES cell lines were created that were heterozygous for genes encoding two guanine nucleotide-binding protein subunits, alpha i2 and alpha i3, T-cell receptor alpha, and beta-cardiac myosin heavy chain. When these heterozygous cells were grown in high concentrations of G418, many of the surviving cells were homozygous for the targeted allele and contained two copies of the G418 resistance gene. This scheme provides an easy method for obtaining homozygous mutationally altered cells, i.e., double knockouts, and should be generally applicable to other genes and to cell lines other than ES cells. This method should also enable the production of cell lines in which more than one gene have had both alleles disrupted. These mutant cells should provide useful tools for defining the role of particular genes in cell culture.
Animals↗
Dose effect of adrenocorticotropin on aldosterone and cortisol biosynthesis in cultured bovine adrenal glomerulosa cells: in vitro correlate of hyperreninemic hypoaldosteronism.
In some critically ill patients, aldosterone secretion is diminished despite hyperreninemia. These same patients demonstrate appropriately elevated plasma ACTH and cortisol levels. In addition, infusion of ACTH or angiotensin-II (AII) fails to elicit the normal aldosterone response, implying that the defect is at the level of the zona glomerulosa (ZG) cell. To test the hypothesis that elevated ACTH levels induce this defect, Percoll-purified bovine ZG cells were plated in serum-free defined medium and cultured for 5 days. On days 1-4, cells were exposed to various concentrations of ACTH for 1 h. On the fifth day of culture, half of the wells pretreated with ACTH were treated for 1 h with AII (10(-7) M); the other half of the wells received another dose of ACTH for 1 h. Additionally, cells were exposed to daily 1-h pulses of AII (10(-7) M) alone or in combination with ACTH (10(-8) M) for 5 days. Acutely dispersed bovine ZG cells showed dose-dependent increases in aldosterone when incubated with ACTH, potassium, or AII, with minimal cortisol production. Acutely dispersed bovine fasciculata cells produced no aldosterone, but demonstrated a dose-dependent cortisol response to ACTH and AII, but not potassium. On day 1 of culture, the ZG cells demonstrated a significant (P less than 0.001 in all cases) dose-related increase in aldosterone secretion in response to ACTH. However, continued daily pulsation with ACTH resulted in a dose-dependent decrease in aldosterone secretion, with a concomitant dose- and time-related rise in cortisol production. Indeed, ACTH-induced cortisol production in ZG became similar to ACTH-induced cortisol production in zona fasciculata cells. The addition of AII to the daily ACTH pulse did not significantly alter the aldosterone or cortisol response patterns to ACTH alone. In contrast, ZG cells treated with AII alone for 5 days showed a minimal change in cortisol production and no reduction in aldosterone production until day 4. Northern blot analysis of total RNA isolated from ZG cells pulsed with ACTH for 5 days demonstrated a parallel dose-dependent increase in 17 alpha-hydroxylase mRNA, which did not occur in cells pulsed with AII alone. These in vitro results suggest that elevated ACTH levels over time induce 17 alpha-hydroxylase activity in ZG cells, thereby shifting steroid biosynthesis from an aldosterone-producing to a cortisol-producing pathway. It is likely that the chronically elevated ACTH levels in critically ill patients induce a similar change in ZG cell biosynthesis, resulting in their hyperreninemic hypoaldosterone state.
Adrenocorticotropic Hormone↗
Embryonic stem cells lacking a functional inhibitory G-protein subunit (alpha i2) produced by gene targeting of both alleles.
The alpha i2 subunit of the inhibitory heterotrimeric guanine nucleotide-binding proteins is highly conserved in mammals and is expressed in all cell types, but its exact function is not yet defined. We have investigated the role of this protein by producing embryonic stem (ES) cells lacking a functional alpha i2 gene. These alpha i2-null cell lines regulate adenylyl cyclase and grow and differentiate in vitro the same as wild-type ES cells. Homologous recombination was used to sequentially inactivate both copies of the alpha i2 gene. The first allele was inactivated by insertion of a neomycin-resistance gene. We modified the hygromycin B-resistance gene for improved expression in ES cells and used this gene to inactivate the remaining normal allele. The techniques used should be generally applicable to other genes whether or not they are expressed in ES cells.
Adenylyl Cyclases↗
Hysteresis in the relationship between serum ionized calcium and intact parathyroid hormone during recovery from induced hyper- and hypocalcemia in normal humans.
We have used an immunoradiometric assay for intact PTH in conjunction with calcium and citrate infusions to study whether levels of intact PTH are responsive to reversal of the direction of change in ionized calcium in normal humans. Eleven normal subjects received graded infusions of citrate or calcium on separate days to produce linear rates of decrease or increase in calcium. After discontinuation of the infusions, the return of calcium toward baseline was followed. Six subjects were given an infusion of citrate after the calcium infusion to speed the recovery of calcium toward baseline. Citrate-induced hypocalcemia produced a rise in serum PTH levels from 28.1 +/- 3.6 to 69.4 +/- 4.8 ng/L as calcium fell from 1.26 +/- 0.01 to 1.06 +/- 0.02 mmol/L. As calcium returned toward baseline, PTH levels fell dramatically, reaching levels indistinguishable from baseline despite persistent hypocalcemia. Slopes of regression lines defining the PTH-calcium relationships during decreasing and increasing calcium levels were significantly different. Those subjects receiving a calcium infusion alone showed a prompt suppression of PTH levels. As calcium returned toward baseline after the infusion, a modest decline in calcium produced no significant change in the PTH-calcium relationship. When citrate was used to return calcium to baseline, PTH levels rose from 7.8 +/- 2.0 to 55.0 +/- 6.8 ng/L as calcium fell from 1.42 +/- 0.02 to 1.26 +/- 0.02 mmol/L and defined a regression relationship significantly different from the period of increasing calcium. Thus, a hysteretic relationship between ionized calcium and levels of intact PTH can be induced in normal humans by reversing the direction of change in calcium. Therefore, the role played by calcium concentration per se in controlling PTH secretion may be part of more complex and dynamic regulatory mechanisms.
Adult↗
Case report: chylous ascites and carcinoid tumors: possible association of two rare disorders.
Three cases of chylous ascites are reported in a series of 275 patients with carcinoid tumor. All three patients had diarrhea and elevated serotonin production. The literature is reviewed and the possible causes of the chylous ascites are discussed. It is suggested that a history of clinically significant diarrhea in a patient with chylous ascites should lead to biochemical tests for carcinoid tumors.
Aged↗
Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate.
The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.
Acetyl-CoA C-Acyltransferase↗
Heating RNA before cell-free translation is essential for the efficient and reproducible synthesis of several peroxisomal proteins.
Total RNA, extracted with guanidinium thiocyanate from liver of clofibrate-treated rats, was translated in vitro. Heating the RNA at 60 degrees C for 5 min before translation increased the synthesis of three peroxisomal polypeptides 10-100-fold. Preproalbumin synthesis increased 10-fold. Total incorporation of [35S]methionine into proteins merely doubled. Heating is essential for reproducible and adequate translation of mRNAs coding for peroxisomal and some other proteins.
Animals↗
Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver.
We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate-cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S-translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.
Acyl-CoA Oxidase↗
Acyl-CoA synthetase in rat liver peroxisomes. Computer-assisted analysis of cell fractionation experiments.
The subcellular distribution of the acyl coenzyme A synthetases of rat liver was reinvestigated in order to determine whether part of this activity occurs in peroxisomes. Rat liver was fractionated by differential centrifugation and by equilibrium density centrifugation. Acyl-CoA synthetase was assayed using a new, simple extraction procedure on three substrates: palmitate, laurate, and octanoate. Comparison of the resulting synthetase distributions with the distributions of marker enzymes for peroxisomes, mitochondria, and endoplasmic reticulum demonstrated the presence of some synthetase activity in each of the three organelles. These trimodal synthetase distributions were evaluated quantitatively by means of a computer program that calculated optimal linear combinations of marker enzymes using a least squares criterion. Peroxisomes were found to contain 7% of the liver's palmitoyl-CoA synthetase activity and 6% of its lauroyl-CoA synthetase activity, but no demonstrable octanoyl-CoA synthetase activity. The remainder of these activities are divided between the mitochondria and endoplasmic reticulum, in a manner that agrees with previous studies. The chain length specificity of the synthetase(s) of each organelle appears to be unique. The absolute activity of the peroxisomal palmitoyl-CoA synthetase is sufficient to maintain maximal peroxisomal beta-oxidation. Clofibrate treatment of the rats caused a 2.6- to 3.1-fold increase in the liver's total acyl-CoA synthetase activities. The subcellular distribution was not greatly affected by this drug treatment.
Animals↗
Reactions of cysteamine and other amine metabolites with glyoxylate and oxygen catalyzed by mammalian D-amino acid oxidase.
Pig kidney D-amino acid oxidase [D-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3] catalyzes a rapid uptake of oxygen when high concentrations (50-100 mM) of glyoxylate and the following amines are present under usual assay conditions (pH 8.3): cysteamine, 2-aminoethanol, putrescine, D,L-1-amino-2-propanol, D,L-2-amino-1-propanol, 3-amino-1-propanol, D,L-octopamine, ethylenediamine, and L-cysteine ethyl ester. Notable physiological amines that do not support a rapid O2 reaction under the above conditions include histamine, serotonin, epinephrine, norepinephrine, spermidine, spermine, and cadaverine. A more detailed kinetic investigation of the reactions involving the first four reactive amines listed above indicated that the cysteamine reaction proceeds at a rapid rate even when cysteamine and glyoxylate are present at less than millimolar concentrations, but greater than millimolar concentrations are needed in the other amine reactions in order to observe a reasonable rate. At low concentrations and pH 7.4, the cysteamine-glyoxylate substrate (presumably thiazolidine-2-carboxylic acid) reacts an order of magnitude faster than any other known D-amino acid oxidase substrate. Considerable circumstantial evidence suggests that the reaction involving cysteamine is occurring physiologically, but the reactions of other amines would be occurring in the cell at a very low rate, if at all. It is proposed that the product of the enzymic reaction may be a metabolic effector that can modify the reactivity of proteins or nucleic acids by covalent attachment.
Amines↗