TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Nuclear gene dosage effects on mitochondrial mass and DNA.

In order to assess the effect of nuclear gene dosage on the regulation of mitochondria we have studied serial sections of a set of isogenic haploid and diploid cells of Saccharomyces cerevisiae, growing exponentially in the absence of catabolite repression, and determined the amount of mitochondrial DNA per cell. Mitochondria accounted for 14% of the cytoplasmic and 12% of the total cellular volume in all cells examined regardless of their ploidy or their apparent stage in the cell cycle. The mean number of mitochondria per cell was 22 in the diploid and 10 in the haploids. The volume distribution appeared unimodal and identical in haploids and diploids. The mitochondrial DNA accounted for 12.6 +/- 1.2% and 13.5 +/- 1.3% of the total cellular DNA in the diploid and haploid populations, respectively. These values correspond to 3.6 x 10(-15) g, 2.2 x 10(9) daltons, or 44 genomes (50 x 10(6) daltons each) per haploid and twice that per diploid cell. On this basis, the average mitochondrion in these cells contains four mitochondrial genomes in both the haploid and the diploid.

Dimethyl sulfoxide: an inhibitor of liver alcohol dehydrogenase.

Dimethyl sulfoxide inhibits horse liver alcohol dehydrogenase. In the direction of aldehyde reduction, this inhibition is competitive with aldehyde, with an inhibition constant of 5 x 10(-3)M. Dimethyl sulfoxide reacts with the binary complex consisting of enzyme and the reduced form of nicotinamide -adenine dinucleotide to form a highly fluorescent ternary complex, with a dissociation constant similar to the inhibition constant. The inhibition of aldehyde reduction can be interpreted as due to competition between aldehyde and dimethyl sulfoxide for the carbonyl binding site of the above-mentioned binary complex.

Cyclic adenosine monophosphate in bacteria.

Both cyclic AMP and a specific inducer acting in concert are required for the synthesis of many inducible enzymes in E. coli. Little enzyme is made in the absence of either. In contrast to the specific inducers which stimulate the synthesis only of the proteins required for their metabolism, cyclic AMP controls the synthesis of many proteins. Glucose and certain other carbohydrates decrease the differential rate of synthesis of inducible enzymes by lowering cyclic AMP concentrations. In the lac operon, cyclic AMP acts at the promoter site to facilitate initiation of transcription. This action requires another protein, the cyclic AMP receptor protein. The nucleotide stimulates tryptophanase synthesis at a translational level. The action of cyclic AMP in E. coli may serve as a model to understand its action on transcriptional and translational processes in eukaryotes.

Omega-conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.

Previous studies have identified two components of whole-cell Ca2+ current in bovine chromaffin cells. The "standard" component was activated by single depolarizations, while "facilitation" could be activated by large prepulses or repetitive depolarizations. Neither current component was sensitive to changes in holding potential between -100 and -50 mV; thus neither appeared to be carried by N-type Ca2+ channels. We now report that the facilitation Ca2+ current is insensitive to omega-conotoxin GVIA (omega-CgTx), but that the toxin blocks approximately 50% of the standard Ca2+ current. In some cells the toxin blocks all of the standard Ca2+ current, in others about half of the current, while in others it has no effect. Kinetic differences in current activation are observed after toxin application. These results suggest that the standard component of chromaffin cell Ca2+ current is composed of two pharmacologically distinct channels-one is omega-CgTx sensitive and the other is not. Two kinetically distinct types of 14 pS Ca2+ channels that may correspond to the omega-CgTx-sensitive and -insensitive components were observed in single-channel experiments. Because omega-CgTx blocked Ca2+ channels that were not inactivated by a depolarized holding potential, the commonly used Ca2+ channel categorization scheme may be inadequate to describe the Ca2+ channels found in chromaffin cells.

A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Catecholamine secretion by isolated adrenal cells.

Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. Acetylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents (atropine and hexamethonium) and by local anaesthetics (tetracaine), and is dependent upon the concentration of Ca2+ in the incubation medium. In the presence of Ca2+, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca2+ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.

Purification and characterization of a phosphoprotein phosphatase from bovine adrenal cortex.

A phosphoprotein phosphatase which is active against chemically phosphorylated protamine has been purified about 500-fold from bovine adrenal cortex. The enzyme has a pH optimum between 7.5 and 8.0, and has an apparent Km for phosphoprotamine of about 50 muM. The hydrolysis of phosphoprotamine is stimulated by salt, and by Mn2+. Hydrolysis of phosphoprotamine is inhibited by ATP, ADP, AMP, and Pi, but is not affected by AMP or cyclic GMP. The purified phosphoprotein phosphatase preparation also dephosphorylates p-nitrophenyl phosphate and phosphohistone, and catalyzes the inactivation of liver phosphorylase, the inactivation of muscle phosphorylase a (and its conversion to phosphorylase b), and the inactivation of muscle phosphorylase b kinase. Phosphatase activities against phosphoprotamine and muscle phosphorylase a copurify over the last three stages of purification. Phosphoprotamine inhibits phosphorylase phosphatase activity, and muscle phosphorylase a inhibits the dephosphorylation of phosphoprotamine. These results suggest that one enzyme possesses both phosphoprotamine phosphatase and phosphorylase phosphatase activities. The stimulation of phosphorylase phosphatase activity, but not of phosphoprotamine phosphatase activity, by caffeine and by glucose, suggests that the different activities of this phosphoprotein phosphatase may be regulated separately.

Stimulation of sugar transport in rat diaphragm by 8-bromoguanosine 3', 5'-monophosphate.

The cyclic GMP derivative, 8-bromo cyclic GMP, increases the uptake of D-xylose and of 2-deoxy D-glucose into intact rat diaphragm incubated in vitro. 8-Bromo cyclic GMP does not stimulate the incorporation of [14C]glucose into glycogen in the diaphragm, or the uptake of alphalpha-amino isobutyric acid into this tissue. The effect of 8-bromo cyclic GMP on the diaphragm is consistent with the hypothesis that cyclic GMP plays a role in the regulation of sugar transport in muscle.

Permeant anions are not required for norepinephrine secretion from pheochromocytoma cells.

The 'chemiosmotic' model for secretion proposed by Pollard and his colleagues (Int. Rev. Cytol. 58, 159-197, 1979) was tested with pheochromocytoma cells. Contrary to the prediction of this model, norepinephrine secretion did not require the presence of a permeant anion in the medium. Secretion was not blocked by replacing much of the Cl- of the medium with isethionate or by replacing all of the Cl- salts of the medium with isotonic sucrose. Biochemical evidence is presented to indicate that the cells secreted by the normal exocytotic mechanism in the sucrose medium. Making the normal bathing medium hypertonic with 300 mM sucrose increased the basal level of norepinephrine release, but also suppressed secretion in response to a strong secretagogue (1 mM Ba2+). The data indicate that the Pollard model does not apply to pheochromocytoma cells, but suggest the possible involvement of osmotic pressure in exocytosis.

Preganglionic stimulation increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion by both cAMP-dependent and Ca2+-dependent protein kinases.

Electrical stimulation of the preganglionic cervical sympathetic trunk increases the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat by a nicotinic mechanism and by a noncholinergic mechanism. We have measured the incorporation of [32P]Pi into specific tryptic phosphopeptides in tyrosine hydroxylase in order to identify the protein kinases that phosphorylate this enzyme in electrically stimulated ganglia. 32P-labeled tyrosine hydroxylase was isolated from the ganglion by immunoprecipitation and polyacrylamide gel electrophoresis and was subjected to tryptic hydrolysis. Seven tryptic peptides were resolved from these hydrolysates by two-dimensional thin-layer electrophoresis and chromatography. Preganglionic stimulation (20 Hz, 5 min) increased the incorporation of 32P into four of these peptides. In the presence of cholinergic antagonists, however, electrical stimulation increased the labeling of only one phosphopeptide. From a comparison of the effects of preganglionic stimulation with the effects of agonists that activate specific protein kinases, we conclude that electrical stimulation increases the phosphorylation of tyrosine hydroxylase by both a cAMP-dependent protein kinase and a Ca2+/calmodulin-dependent protein kinase. The nicotinic component of preganglionic stimulation appears to be mediated by a Ca2+/calmodulin-dependent protein kinase, while the noncholinergic component appears to be mediated by cAMP-dependent protein kinase. Although protein kinase C can phosphorylate tyrosine hydroxylase, this kinase does not appear to participate in the stimulation-induced phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.

Myosin and myosin phosphorylation in pheochromocytoma (PC12) cells.

Myosin was isolated from extracts of a clonal cell line of pheochromocytoma (PC12) cells by ammonium sulfate fractionation and gel filtration. This myosin consisted of heavy chains and two light chains (20 and 17 kDa). The 20 kDa light chain could be phosphorylated by a protein kinase which was also present in the extracts and which eluted after myosin from the gel filtration column. Myosin phosphorylation was partly inhibited by EGTA and by the calmodulin-inhibiting drug trifluoperazine. The Mg2+-ATPase of phosphorylated myosin, but not of unphosphorylated myosin, was activated by skeletal muscle actin. Ca2+ did not affect the Mg2+-ATPase activity of either myosin preparation at low ionic strength. The phosphorylation of myosin may activate a contractile mechanism controlling the Ca2+-dependent secretion of norepinephrine from the cells.

Phosphorylation of tyrosine hydroxylase in the superior cervical ganglion.

We studied the phosphorylation of tyrosine hydroxylase in the superior cervical ganglion of the rat. Ganglia were preincubated with [32P]Pi and were then incubated in non-radioactive medium containing a variety of agents that are known to activate tyrosine hydroxylase in this tissue. Tyrosine hydroxylase was isolated from homogenates of the ganglia by immunoprecipitation followed by polyacrylamide gel electrophoresis. 32P-labelled tyrosine hydroxylase was visualized by radioautography, and the incorporation of 32P into the enzyme was quantitated by densitometry of the autoradiograms. Veratridine produced a concentration-dependent increase in the incorporation of 32P into tyrosine hydroxylase, with 50 microM veratridine producing a 5-fold increase in 32P incorporation. The nicotinic agonist, dimethylphenylpiperazinium (100 microM), caused a 7-fold increase in the phosphorylation of tyrosine hydroxylase. The effect of dimethylphenylpiperazinium was maximal within 1 min and decreased upon continued exposure of the ganglia to this agent. The actions of dimethylphenylpiperazinium and of veratridine were dependent on extracellular Ca2+. Muscarine, 8-Br-cAMP, forskolin, vasoactive intestinal peptide, isoproterenol, deoxycholate and phospholipase C also stimulated the incorporation of 32P into tyrosine hydroxylase. These data support the hypothesis that phosphorylation plays a role in activation of tyrosine hydroxylase produced by all of these agents.

Estimation of the cytoplasmic catecholamine concentrations in pheochromocytoma cells.

Pheochromocytoma cells contain amine oxidase (flavin-containing), and convert dopamine and norepinephrine to deaminated metabolites. Dihydroxyphenylacetic acid is the major dopamine metabolite produced by the cells, whereas dihydroxyphenylglycol is the predominant metabolite of norepinephrine. Cells incubated under control conditions produce deaminated dopamine metabolites at a rate of about 30 pmol/min per mg protein, and dihydroxyphenylglycol at a rate of approx. 10 pmol/min per mg protein. Activation of tyrosine 3-monooxygenase increases the formation of dihydroxyphenylacetic acid, but does not greatly affect the production of dihydroxyphenylglycol. Inhibition of aromatic-L-amino-acid decarboxylase decreases the production of dihydroxyphenylacetic acid, but does not alter the production of dihydroxyphenylglycol. These results are consistent with the idea that newly synthesized dopamine represents the major source of cytoplasmic dopamine, whereas cytoplasmic norepinephrine is derived largely from catecholamine stores in secretory vesicles. The concentrations of dopamine and of norepinephrine in the cytoplasm of pheochromocytoma cells were estimated by measuring the substrate dependence of amine oxidase activity in extracts of these cells. By this method, the cytoplasmic concentrations of dopamine and of norepinephrine were estimated to be in the range of 0.5 to 1 microM. Incubation of the cells with extracellular norepinephrine or with reserpine results in an increase in the production of dihydroxyphenylglycol, and in inhibition of tyrosine 3-monoxygenase activity. Both of these effects are presumably mediated by a rise in the cytoplasmic norepinephrine concentration. Analysis of the relationship between norepinephrine metabolism and tyrosine 3-monooxygenase activity indicates that the apparent Ki of this enzyme for norepinephrine in intact cells is 10-15-times the basal cytoplasmic concentration of norepinephrine, or approx. 10 microM.

Transmembrane signalling in Saccharomyces cerevisiae.

Baker's yeast, a unicellular eukaryote, has been a model organism for biochemists, geneticists and most recently for molecular biologists. Pioneering biochemical studies were conducted on yeast, such as the study of glucose fermentation and amino acid metabolism. The powerful tools of yeast genetics have allowed a comprehensive study of important issues such as the cell cycle and meiosis. In recent years, it has been established that Saccharomyces cerevisiae, the most extensively characterized of the yeasts, shares key molecules and biochemical pathways with higher eukaryotes. For example, actin, tubulin, ubiquitin, calmodulin, GTP regulatory proteins, different protein kinases including protein tyrosine kinases, were all found to play central roles in yeast. Furthermore, structurally homologous proteins, as well as transcription regulating elements, of yeast and higher eukaryotes, including mammals, were shown to be structurally and functionally interchangeable. It has also been found that yeast can express human genes. Technically, yeasts are simple to handle, inexpensive to grow, complete a cell cycle within 90 min, and therefore can yield relatively quick results. These qualities are useful in biotechnological applications. Saccharomyces cerevisiae, can be genetically manipulated fairly easily, and has been tinkered with more than any other system. A cloned, in vitro mutated gene, can be transformed into wild type yeast and by homologous recombination, can replace the native gene and generate the desired mutant. Such manipulations, not possible yet in other eukaryotic cells, allow the precise definition of the role played by different genes and their domains. These unique features of Saccharomyces cerevisiae, together with rapidly evolving techniques of molecular biology, have made it a successful model organism for the study of numerous questions.(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid intracellular alkalinization of Saccharomyces cerevisiae MATa cells in response to alpha-factor requires the CDC25 gene product.

The alpha-factor mating pheromone induces a transient intracellular alkalinization of MATa cells within minutes after exposure to the pheromone, and is the earliest biochemical event that can be identified subsequent to the exposure. Dissipation of the pheromone induced pH gradient, using 2,4-dinitrophenol or sodium orthovanadate, does not inhibit the biological response of the yeast to the pheromone such as mating and 'schmoo' formation. These findings suggest that the pheromone mediated pH change per se is not a part of the transmembrane signalling but rather the consequence of a biochemical reaction triggered by the alpha-pheromone interaction with its receptor and may have a permissive effect on the pheromonal response. The cdc25ts mutation causes MATa cells to become nonresponsive to alpha-factor subsequent to a shift to the restrictive temperature, suggesting that the CDC25 gene product participates in the pheromone response pathway.

Insulin and insulin-like growth factors stimulate deoxyribonucleic acid synthesis in PC12 pheochromocytoma cells.

The effects of insulin and insulin-like growth factors (IGFs) on the replication of PC12 pheochromocytoma cells were investigated. Incubation of PC12 cells for 2-3 days in low (0.3%) serum medium decreased [3H]thymidine incorporation into PC12 cell DNA to approximately 30% of that in control (15% serum) medium. Incubation of the cells in low serum medium also slowed the growth of the cultures and increased the percentage of cells in the G0/G1 phase of the cell cycle. Addition of insulin to cells in low serum medium increased [3H]thymidine incorporation into the cells, increased the number of cells in PC12 cultures, and decreased the percentage of cells in the G0/G1 phase of the cell cycle. IGF-I and IGF-II also increased [3H]thymidine incorporation into PC12 cells incubated in low serum medium. IGF-I (EC50, approximately 0.3 nM) was a more potent stimulus of [3H]thymidine incorporation than was insulin (EC50, approximately 3.5 nM). These data suggest that insulin and IGFs are growth factors for PC12 cells, and that the growth-promoting effects of these agents may be mediated by a type I IGF receptor on PC12 cells.

Insulin-degrading enzyme does not require peroxisomal localization for insulin degradation.

Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal location and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site-directed mutagenesis was used to destroy the peroxisomal targeting signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (approximately 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions.

Insulin regulates placental lactogen and estradiol secretion by cultured human term trophoblast.

We have described a human term trophoblast cell culture system which synthesizes hormones de novo from their precursors. In the present report we utilized this monolayer system to show that insulin produced a dose-dependent stimulation of hPL secretion, with a significant effect at 10(-10) M insulin. At 10(-9) M concentration, insulin also inhibited estradiol secretion by the cultured trophoblast. We conclude that insulin regulates secretion of estradiol and placental lactogen by cultured human term trophoblast.

Both nicotinic and muscarinic receptors mediate catecholamine secretion by isolated guinea-pig chromaffin cells.

We have studied the roles of nicotinic and muscarinic receptors in the acetylcholine-evoked secretion of catecholamine from guinea-pig chromaffin cells. Isolated guinea-pig chromaffin cells secrete catecholamine in response to acetylcholine, nicotine, and a variety of muscarinic agonists. Optimal concentrations of acetylcholine (50-200 microM) induce the release of 10-25% of the catecholamine content of the cells in 10 min. Maximal secretion evoked by nicotine or by muscarinic agonists is 5-12% of the catecholamine content of the cells. Secretion evoked by optimal concentrations of nicotine (50 microM) and muscarine (200 microM) are additive, and together these agonists cause catecholamine release equivalent to that produced by optimal concentrations of acetylcholine. Atropine causes a biphasic inhibition of acetylcholine-induced catecholamine secretion; low concentrations of atropine (0.02-0.01 microM) inhibit by 35-45% the catecholamine secretion evoked by 100 microM acetylcholine. Increasing the atropine concentration from 0.1 to 5 microM causes no further decrease in acetylcholine-evoked release, but at concentrations above 5 microM, a second distinct phase of inhibition appears. At 100 microM, atropine reduces acetylcholine-evoked secretion by 85%. At 0.1 microM, atropine significantly inhibits secretion induced by muscarinic, but not nicotinic, agonists. Tubocurarine (50 microM) does not block muscarinic stimulation of release, but inhibits acetylcholine- and nicotine-evoked release by 70 and 80%, respectively. Our experiments indicate that nicotinic and muscarinic stimulation represent distinct mechanisms for the activation of catecholamine release from guinea-pig chromaffin cells.(ABSTRACT TRUNCATED AT 250 WORDS)