Inhibited Leucine Incorporation Research Articles

Overexpression of 12-lipoxygenase causes cardiac fibroblast cell growth.

Evidence suggests that leukocyte type 12-lipoxygenase (12-LO) plays an important role in cell growth. However, the role of 12-LO in cardiac cell growth has not been tested. We have now stably overexpressed 12-LO cDNA in rat fetal cardiac fibroblasts to evaluate the role of the 12-LO pathway in cardiac cell growth. Overexpression of 12-LO increased cell [(3)H]leucine incorporation by 2.1+/-0.1-fold (P<0.01) and cell protein content by 2.2+/-0. 3-fold (P<0.01) over mock-transfected cells. These findings were confirmed in additional clones. Baicalein, a 12-LO enzyme inhibitor, dose-dependently inhibited serum-induced leucine incorporation in cardiac fibroblast cells as well as partially inhibited leucine incorporation in cells overexpressing 12-LO. 12-LO overexpression also caused cell [(3)H]thymidine incorporation to increase by 3.4+/-0.3-fold (P<0.01). Cell flow cytometry analysis showed that the size of 12-LO-overexpressing cells was markedly enlarged compared with that of mock-transfected cells. The fibronectin content of the 12-LO-overexpressing cardiac fibroblasts was also significantly increased. We next evaluated the effects of 12-LO RNA overexpression on kinase pathways linked to cellular growth. The overexpression of 12-LO enhanced extracellular signal-regulated kinase activity (4. 1+/-0.5-fold), c-Jun NH(2)-terminal kinase activity (2.9+/-0.5-fold), and p38 mitogen-activated protein kinase activity (2.2+/-0.3-fold). Pretreatment with SB202190 (100 nmol/L), a specific inhibitor of p38, prevented the increases in protein content of 12-LO-overexpressing cardiac fibroblast cells. These data clearly demonstrate that the overexpression of 12-LO causes cell growth of cardiac fibroblasts, thus supporting the role of 12-LO as a novel growth-promoting pathway in the heart.

An Examination of the Role of Increased Cytosolic Free Ca2+Concentrations in the Inhibition of mRNA Translation

Mobilization of Ca2+sequestered by the endoplasmic reticulum (ER) produces the phosphorylation of initiation factor (eIF) 2, whereas an increase in cytosolic free Ca2+([Ca2+]i) due to plasmalemmal Ca2+influx increases the phosphorylation of elongation factor (eEF) 2. In nucleated mammalian cells, depletion of ER Ca2+stores has been demonstrated to inhibit translational initiation, but evidence that increased [Ca2+]iper se causes slowing of peptide chain elongation is lacking. L-type Ca2+channel activity of GH3pituitary cells, which are enriched in calmodulin-dependent eEF-2 kinase, was manipulated such that the impact of [Ca2+]ion eEF-2 phosphorylation and translational rate could be examined for up to 10 min without inhibiting initiation. At 1 mM extracellular Ca2+, resting [Ca2+]ivalues were high (154–255 nM) and eEF-2 was phosphorylated. The Ca2+channel antagonist, nisoldipine, lowered [Ca2+]iand reduced eEF-2 phosphorylation by half but had no effect on amino acid incorporation. The Ca2+channel agonist, Bay K 8644, produced sustained elevations of [Ca2+]ithat were associated with 25–50% increases in eEF-2 phosphorylation, but no changes in protein synthetic rates occurred. Larger Ca2+influxes were achievable with either 25 mM KCl or KCl plus Bay K 8644. These treatments further increased eEF-2 phosphorylation (50–100% above control) and inhibited leucine incorporation by 20–70% but ATP content was reduced by 25–50% and total cell-associated Ca2+contents rose by 3- to 13-fold. eIF-2α was not phosphorylated during these treatments. Addition of low concentrations of ionomycin, which do not lower ATP content, was associated with complex changes in [Ca2+]ithat resembled alterations in eEF-2 phosphorylation. The inhibition of leucine incorporation in response to ionomycin, however, coincided only with the phosphorylation of eIF-2α, not eEF-2. It is concluded that changes in [Ca2+]ioccurring in the absence of ATP depletion alter the phosphorylation state of eEF-2 but are not regulatory for mRNA translation.

Effect of cocaine on macromolecular syntheses and cell proliferation in cultured glial cells

In the present study the effect of cocaine on thymidine, uridine and leucine incorporation was assessed in primary cortical glial and C6 glioma cells. Cocaine exposure for 24 h inhibited thymidine and uridine incorporation in cortical glial and C6 glioma cells. However, the effect of cocaine on uridine incorporation was less prominent compared to thymidine incorporation. High concentrations of cocaine inhibited leucine incorporation in C6 glioma cells but not in cortical glia. Cocaine exposure for four days decreased cell proliferation of cortical glial and C6 glioma cells. Cocaine-induced attenuation of macromolecular syntheses was not due to cell death since cocaine-treated cells were not stained with Trypan Blue and did not release lactate dehydrogenase into culture supernatants. Furthermore, cocaine had no effect on glutamate uptake either in cortical glia or in C6 glioma cells. These results indicate that cocaine inhibits macromolecular syntheses in glial cells. The inhibition of macromolecular syntheses in glial cells may be the mechanism involved in cocaine-induced fetal brain growth retardation.

Induction of Na(+)-K(+)-ATPase subunit mRNAs by cycloheximide in a rat liver cell line

Exposure of confluent Clone 9 cells to 40 microM cycloheximide (CHX), a concentration sufficient to inhibit leucine incorporation by 95% within 5 min, coordinately increased the abundances of Na(+)-K(+)-ATPase subunit mRNAs, mRNA alpha 1 and mRNA beta 1. The CHX-induced increases in mRNA alpha 1 and mRNA beta 1 abundances were, respectively, 1.8- and 1.9-fold at 40 min and 3.0- and 3.3-fold at 6 h. Augmented subunit mRNA contents were also observed after exposure to other protein synthesis inhibitors including 100 microM anisomycin and 100 microM emetine. Upon removal of CHX, the rate of leucine incorporation returned to control values within 1 h, but mRNA alpha 1 and mRNA beta 1 content decreased only slowly and were still elevated at 24 h at 1.7- and 1.8-fold the respective control values. Despite the persistence of increased levels of the subunit mRNAs and normalization of the rate of leucine incorporation, Na(+)-K(+)-ATPase activity was unchanged at 3, 6, 24, and 48 h after removal of CHX. In cells "depleted" of protein kinase C (PKC) activity after a 24-h preincubation in the presence of 160 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), mRNA alpha 1 and mRNA beta 1 abundances were still inducible by CHX. It is concluded that exposure of Clone 9 cells to CHX and other inhibitors of protein synthesis results in increased abundances of Na(+)-K(+)-ATPase subunit mRNAs independently of PKC activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Selenium metabolism in isolated hepatocytes: inhibition of incorporation in proteins by mono(2-ethylhexyl)phthalate, a metabolite of the peroxisome proliferator di(2-ethylhexyl)phthalate

Di(2-ethylhexyl)phthalate (DEHP), a rat liver carcinogen, induces peroxisomal proliferation and a concomitant oxidative stress, but decreases liver glutathione peroxidase (GSH-Px) activity. This enzyme is a selenoprotein and we have investigated the influence of mono(2-ethylhexyl)phthalate (MEHP), a major metabolite of DEHP, on selenium incorporation in hepatocellular proteins. [75Se]Selenious acid (6 nM) was added to primary cultures of rat hepatocytes and protein incorporation was assessed by SDS-PAGE and autoradiography. High concentrations of MEHP (1.0-3.0 mM) inhibited selenium labeling of all major selenoproteins in 3-24 h experiments, but also inhibited protein synthesis as assessed by leucine incorporation. The protein synthesis inhibition was reversible. Lower concentrations of MEHP (0.3-0.5 mM) did not decrease the 75Se-labeling in 24 h experiments and did not inhibit leucine incorporation. However, conditions that significantly induced peroxisomal proliferation also affected the 75Se-labeling. Thus in 72 h experiments, 0.05-0.25 mM MEHP increased the labeling of a 58 kd protein, decreased the labeling of a 23 kd protein (with the same mol. wt as GSH-Px), had no effect on a 20 kd protein and decreased the labeling of a 15 kd protein (as compared to MEHP-free control plates). The pattern of changes associated with peroxisomal proliferation mimicked that seen in livers from selenium-deficient animals, as reported by others. These data indicate that the bioavailability of selenium is decreased by DEHP. This effect may relate to a transient inhibition of protein synthesis, but also to the DEHP-induced peroxisomal proliferation.

Effects of herbicides and herbicide analogs on [ 14C]leucine incorporation by suspension-cultured Solanum nigrum cells

Assays of [ 14C]leucine incorporation were used to measure effects of herbicides on suspensioncultured heterotrophic Solanum nigrum cells. Most herbicidal vs. nonherbicidal chemicals in a set of 47 compounds could be distinguished from each other based on their extent of inhibition of leucine incorporation by S. nigrum cells. Herbicides which failed to inhibit leucine incorporation were photosynthetic inhibitors. Both phytotoxic and nonphytotoxic thiocarbamate analogs (as determined by whole-plant studies) tended to inhibit leucine incorporation. It was concluded that the leucine incorporation screen could detect a majority of compounds tested which are herbicidal, and that it may also be useful to detect compounds which have cellular toxicity which is not observed in the whole plant.

Glycosylation and Deglycosylation of Storage Proteins in Developing Agrostemma githago L. Seeds

Summary A 23 kD protein is the major glycoprotein in embryos of Agrostemma githago seeds. This glycoprotein is synthesized by cleavage of a 88 kD protein which is also glycosylated. Because cycloheximide inhibited leucine incorporation considerably more than glucosamine incorporation, glycosylation occurs to some extent post-translationally. Tunicamycin inhibited glycosylation only to a small extent. Since after tunicamycin treatment a small amount of the nonglycosylated form of the 23 kD glycoprotein was present, glycosylation is not necessary for processing and transport of this protein. A small decrease in the relative molecular weight which occurs as a final processing step in the synthesis of two storage proteins of 17 kD and 38 kD, very probably represents a deglycosylation event as was shown by the disappearance of [ 3 H]mannose and [ 3 H]glucosamine label at the appropriate positions on the gel during a chase. By analogy, a small decrease of the relative molecular weight in the synthesis of the 23 kD glycoprotein may be attributed to a partial deglycosylation.

Protein synthesis inhibitors enhance secretagogue sensitivity of in vitro rat pancreatic acini.

Isolated rat pancreatic acini were treated with cycloheximide and amylase release was measured. This agent increased the sensitivity to both synthetic octapeptide of cholecystokinin (CCK8) and carbamylcholine, the major secretagogues known to utilize Ca2+ as a second messenger. The mechanism of the cycloheximide effect was via inhibition of protein synthesis, as indicated by the following: 1) the concentration of cycloheximide used inhibited leucine incorporation by greater than 90%; 2) this effect was not instantaneous but increased up to a 2-h pretreatment; and 3) a similar effect was obtained with puromycin, a chemically different inhibitor of protein synthesis. Cycloheximide acted on the steps by which secretagogues mobilize cellular Ca2+ because the dose-response curve for 45Ca2+ efflux was shifted to the same extent as that for amylase release, whereas the dose-response curve for amylase release induced by the Ca2+ ionophore A23187 was not altered. The results suggest, therefore, that a rapidly turning-over protein present in pancreatic acinar cells exerts an inhibitory influence on Ca2+ mobilization by secretagogues.

Inhibition by Polyamines of Macromolecular Synthesis and Its Implication for Ethylene Production and Senescence Processes

Applied diamines and polyamines inhibited the incorporation of radioactively labeled leucine and uridine into trichloroacetic acid-insoluble material in apple (Malus domestica Borkh, cv Golden Delicious) fruit tissue. The inhibitory effect was in general more pronounced with the higher molecular weight amines. Putrescine at 5 millimolar inhibited leucine incorporation by 37% and uridine by 44%. Spermidine and spermine at the same concentration inhibited uridine incorporation by 60%. The polyamines at concentrations between 0.1 and 1.0 millimolar inhibited leucine incorporation by 55 to 90%. The inhibitory effect of 0.1 to 10 millimolar polyamines on dark- and wound-induced senescence or ethylene production, is discussed in the light of interference with macromolecular synthesis.

Modulation of bone marrow heme and protein synthesis by trace elements

We examined the effect of heavy metals and trace elements, such as Cu, Au, Cd, Zn, and Pb, on heme synthetic and degradatory enzymes in rat bone marrow suspensions. Cu, Cd, and Au, at a low concentration of 5 × 10 −6 m, inhibited δ-aminolevulinic acid (δALA) dehydratase by 61, 47, and 47% respectively. A similar concentration of Zn did not inhibit the enzyme activity and, in fact, was able to protect against Au and Pb inhibition in the normal marrow. Cd was unable to recover any of the enzyme activity and, in fact, caused further inhibition. We determined the rate of cellular protein synthesis by measuring the rate of incorporation of radiolabeled precursor [ 14C]leucine. Cu and Au, at a concentration of 10 −5 m, inhibited leucine incorporation into marrow globin and nonglobin protein by 26 and 34%, respectively. Substantially higher concentrations of Zn, as well as Fe, were required to inhibit leucine incorporation into protein. These studies led us to postulate that the inhibition of δALA dehydratase by heavy metals was directly related to the inhibition of heme and protein synthesis in the bone marrow cell suspensions. No evidence of a significant change in heme oxygenase activity in vitro was observed after treatment with any of the heavy metals or trace elements used. These results suggest that trace elements by themselves or in a combination reduce the capacity of the erythroid cell to synthesize heme, the prosthetic group of many hemoproteins. In addition, we have shown that in an iron-overloaded rat the bone marrow contains approximately 45% more heme oxygenase activity than that of the control. Administration of Au alone did not increase heme degradation, but the combination of Fe and Au resulted in a marked induction of heme oxygenase. Finally, for the first time we were able to demonstrate that excess iron and/or decreased Zn concentration may predispose δALA dehydratase to inhibition by an interaction with other metals, such as Pb, by an undefined mechanism(s).

Inhibition, by trichothecene antibiotics, of brain protein synthesis and fever in rabbits.

1. To test further the hypothesis that brain protein synthesis is necessary for fever, three structurally similar trichothecene antibiotics were injected into the cerebral ventricles of rabbits. They were 3,15-diacetoxy-12-hydroxytrichothec-9-ene (DAHT), 3,15-didesacetyl-calonectrin (DDAC) and T-2 toxin. Their actions on hypothalamic incorporation of [14C]leucine and fever were compared. 2. DDAC (60 micrograms) and T-2 toxin (10 micrograms) strongly inhibited leucine incorporation and fever. DAHT (60 micrograms) did not diminish fever and had a smaller effect upon leucine incorporation. 3. The findings strengthen considerably earlier suggestions that brain protein synthesis is an essential step in pyrogenesis.

Inhibitory effect of cyclic adenosine 2',3'-monophosphate on leucine incorporation by L5178Y cells.

The specificity of the reported stimulation of protein synthesis affected by dibutyryl adenosine cyclic 3',5'-monophosphate in L5178Y cells was tested by assaying the effect of adenosine cyclic 2',3'-monophosphate. Protein synthesis was assayed as total acid precipitable 3H-leucine. Leucine incorporation was inhibited by adenosine cyclic 2',3'-monophosphate. Alone, and in combination with actinomycin, 2',3'-cAMP inhibited leucine incorporation more rapidly and to a greater degree than did actinomycin alone. Since a similar potentiating effect was not observed with cordycepin, the hypothesis has been suggested that adenosine cyclic 2',3'-monophosphate inhibits protein synthesis by interfering with some post transcriptional event, perhaps modification of existing RNA. The results further suggest that 2,3 cyclic AMP may be antagonistic of the effects of 3,5 cyclic AMP, and as such may be useful as an inhibitor of that nucleotide's action.

Differential sensitivity of isolated rat thymocytes to glucocorticoids following concanavalin A stimulation

Concanavalin A (Con A) rapidly stimulates leucine incorporation into trichloroacetic acid (TCA) precipitable, but not TCA soluble material in isolated rat thymocytes. This stimulation of leucine incorporation by Con A is dose dependent, time dependent, and is inhibited by the addition of cycloheximide. In non mitogen stimulated thymocytes, cortisol (10 −6 M) inhibits leucine incorporation into protein by 30%, whereas in Con A stimulated cells, cortisol inhibits leucine incorporation by 50%. The cortisol effect in Con A stimulated cells is present when cortisol and Con A are added simultaneously, is diminished when cortisol is added at intervals after Con A and is dependent on the concentration of cortisol administered. The inhibitory effect of cortisol on leucine incorporation is dose and time dependent in both control and Con A treated cells. The magnitude of the cortisol effect does not differ in control and Con A stimulated thymocytes for the first 3 h of steroid exposure, after which time a significant increase in the glucocorticoid effect occurs only in the Con A stimulated cells. This increased response to cortisol occurs at a time which is coincident with the mean maximal response of these cells to Con A alone. Both progesterone and cortexolone (10 −6 M) inhibit leucine incorporation in control cells only slightly. The magnitude of these effects does not change by stimulation of the thymocytes with Con A. Under the conditions of these experiments, the rate of accumulation and the levels of nuclear glucocorticoid receptors are the same in both control and Con A stimulated thymocytes. These data indicate that thymocyte responsiveness to glucocorticoid is increased following exposure to concanavalin A. This enhanced response to glucocorticoid probably occurs subsequent to nuclear accumulation of steroid receptor complexes.

Effects of cycloheximide on protein synthesis in human lung tumors, regenerating rat liver and hepatomas.

10(-4) M cycloheximide (CHM) inhibits leucine incorporation to about the same degree in slices of human lung tumors, rat hepatomas, regenerating livers and normal tissues. At 10(-6) M, CHM has a more pronounced effect on tumor tissue and regenerating liver than on normal tissues. 10(-8) M CHM stimulates protein synthesis in normal rat liver slices.

Phase shifting the circadian rhythm of neuronal activity in the isolated Aplysia eye with puromycin and cycloheximide. Electrophysiological and biochemical studies.

The effects of pulse application of puromycin (PURO) or cycloheximide (CHX) were tested on the circadian rhythm (CR) of spontaneous compound action potential (CAP) activity in the isolated Aplysia eye. CAP activity was recorded from the optic nerve in constant darkness at 15degreesC. PURO pulses (6, 12 h; 12--134 mug/ml) and CHX pulses (12 h, 500--2,000 mug/ml) caused dose-dependent phase delays in the CR when administered during projected night. PURO pulses (6 h, 125 mug/ml) caused phase advances when given during projected day and caused phase delays when given during projected night. In biochemical experiments PURO (12 h, 20 mug/ml) and CHX (12 h, 500 mug/ml) inhibited leucine incorporation into the eye by about 50%. PURO (12 h; 50, 125 mug/ml) also changed the molecular weight distribution of proteins synthesized by the eye during the pulse. The effect of PURO (12 h, 125 mug/ml) on the level of incorporation was almost completely reversible within the next 12 h but the phase-shifted eye showed an latered spectrum of proteins for up to 28 h after the pulse. In electrophysiological experiments spontaneous CAP activity and responses to light were measured before, during, and after drug treatments. In all, eight parameters in three periods were analyzed quantitatively. Of these 24 indices, only 3 showed significant changes. PURO increased spontaneous CAP frequency by 67% 0-7 h after the drug pulse and increased the CAP amplitude of the tonic light response by 23% greater than 7 h after the pulse. CHX increased the intraburst spontaneous CAP frequency by 33% during the pulse and CAP frequency of the tonic light response by 32% 0-7 h after the pulse. The above data indicate that phase-shifting doses of PURO and CHX inhibit protein synthesis in the eye without causing adverse electrophysiological effects, and suggest that protein synthesis is involved in the production of the CR of the isolated Aplysia eye.

The action of cycloheximide on the action potential and protein synthesis in medullated Xenopus axons.

Cycloheximide depresses maximum rate of change in membrane potential observed during the rising phase of the action potential in single medullated axons of Xenopus. To,e course of depression is independent of cycloheximide concentration over a range that almost completely inhibits leucine incorporation into axonal proteins.

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture.

A transient treatment of pregerminated conidia of Neurospora crassa with NaF induced young, submerged cultures to prematurely differentiate conidia. The inductive treatment decreased the rate of respiration (with lower RQ), reduced the relative concentration of nucleoside triphosphates, and inhibited leucine incorporation into protein and adenosine incorporation into RNA.

Separate biochemical actions of inhibitors of short- and long-term memory

In vitro effects of the antibiotics - cycloheximide, puromycin and chloramphenicol, and the sodium pump blockers - ouabain, lithium and copper, on protein synthesis in the crude mitochondrial, postmitochondrial and synaptosomal fractions of chicken forebrain were studied. In the synaptosomal fraction there was a correlation between the inhibitions produced by the sodium pump blockers, of Na +/K + ATPase activity, 14C-leucine uptake and the incorporation of 14C-leucine into protein; whereas the antibiotics only inhibited leucine incorporation into protein. The effects of these drugs on the in vivo incorporation of 14C-leucine into protein in various fractions of chicken forebrain were also studied. The results suggest that there is a biochemical link involving a sodium pump and the transport of amino acids, between nerve impulse activity and neuronal protein synthesis. This link may also interconvert short-term and long-term storage of memory.

Incorporation of Carbohydrate Residues into Peroxidase Isoenzymes in Horseradish Roots

Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-(14)C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-(3)H and mannose-U-(14)C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of (14)C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.

Relationship of protein synthesis and energetics in mitochondria.

Protein synthesis in rat liver mitochondria has been studied by measuring14C-leucine incorporation into mitochondrial proteins. It has been found that addition of uncouplers of oxidative phosphorylation or cyanide inhibits leucine incorporation supported by ATP energy in the presence of oligomycin. Neither cyanide nor oligomycin used separately suppresses the ATP-supported leucine incorporation. Oligomycin arrests respiration-supported protein synthesis. These facts indicate that both ATP and membrane potential or another form of non-phosphorylated energized state are necessary for the use of extramitochondrial amino acids for protein synthesis in mitochondria.