Effects of cycloheximide on inverse regulation of phenolic and nonphenolic ring deiodinases in monkey hepatocarcinoma cells

Both phenolic and nonphenolic ring deiodinase activities in monkey hepatocarcinoma cells (NCLP-6E) were increased by addition of serum in a concentration-dependent manner: the stimulatory effect of serum was evident at a concentration as low as 1.5%, and was maximal at 5%. Lineweaver-Burk analysis showed that the increases in the deiodinase activities are due to the increase in Vmax, but not in Km. The addition of cycloheximide at concentrations ranging from 0.1 to 50 micrograms/ml inhibited the stimulatory effect of serum on phenolic ring deiodinase activity progressively. On the other hand, nonphenolic ring deiodinase activity was increased as much as 4-fold by the addition of 0.5-5 micrograms/ml cycloheximide together with 0.5% serum; a high concentration of the drug, 50 micrograms/ml, however, did not elicit such an increase. Actinomycin D at 5 micrograms/ml completely abolished the increase in nonphenolic ring deiodinase activity by serum or cycloheximide. In addition, actinomycin D inhibited the increase in phenolic ring deiodinase activity by serum in a dose-dependent manner at concentrations ranging from 0.05 to 5 micrograms/ml. It is concluded that phenolic and nonphenolic ring deiodinases are regulated by different mechanisms in monkey hepatocarcinoma cells (NCLP-6E).     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Effects of propylthiouracil and methylmercaptoimidazol on metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells.

Monkey hepatocarcinoma cell monolayer cultures (NCLP-6E) metabolized thyroxine, 3,5,3'-triiodothyronine, 3,3',5'-triiodothyronine and 3,3'-diiodothyronine by phenolic and nonphenolic ring deiodinations and sulfation of the deiodinated products, as shown in previous work with this system. The effects of the antithyroid drugs, propylthiouracil (PTU) and methylmercaptoimidazole (MMI), on these processes was investigated. PTU, at 0.1 and 1 mM, inhibited only phenolic ring deiodination. MMI at 1 mM had no effect, but 32 mM inhibited deiodination of both rings as well as sulfation. The findings suggest that the increased serum rT3 level caused by PTU in vivo is the result of decreased rT3 deiodination, in contrast to the increased rT3 production which is caused by starvation.     

Increases in phenolic and nonphenolic ring deiodinase activity due to serum in monolayer liver cell cultures

When monkey hepatocarcinoma cells (NCLP-6E) were treated with 10% of various serum preparations for 24 h at 37 degrees C, nonphenolic ring deiodinase activity increased 2.0- to 2.3-fold. Phenolic ring deiodinase activity also increased 0.9- to 2.1-fold. Dialysis of the sera enhanced the effect on deiodinase activities in some preparations, but reduced activity in other serum preparations. Similarly, a 1.3- to 3.1-fold increase in phenolic ring deiodinase activity was observed in rat hepatoma cells (R-Y121B). In this case, dialysis usually reduced the effect of the sera. It is concluded that both large molecule(s) (undialyzable) and small molecule(s) (dialyzable) in serum contribute to the regulation of phenolic and nonphenolic ring deiodinase activity in NCLP-6E and R-Y121B cells.     

Uptake and metabolism of thyroid hormones by cultured monkey hepatocarcinoma cells. Effects of potassium cyanide and dinitrophenol

Cultured monkey hepatocarcinoma cell (NCLP-6E) were used to investigate the uptake and metabolism of thyroid hormones. Intracellular accumulation was shown by the failure to acutely release hormone from cells subsequently exposed to serum proteins, and by the metabolic trnasformation of the hormones to deiodinated products and their sulfates. When hepatocarcinoma cell monolayers were studied at hormone concentrations below 10(-10) M, neither KCN nor dinitrophenol inhibited uptake. Taken together with previous findings that uptake was neither saturable nor reduced at low temperature, these results indicate that this process was not active transport. Deiodination of both the phenolic and non-phenolic rings, however, was partially inhibited by KCN but not by dinitrophenol. Sulfation of 3,3'-diiodothyronine and 3'-monoiodothyronine was strongly inhibited by both KCN and dinitrophenol. Uptake of the hormones and their metabolites was also measured in suspended hepatocarcinoma cells and compared with the uptake by normal rat hepatocytes, human fibroblasts and human lymphocytes. In these experiments 1 micrometer triidothyronine and 0.47 mM dinitrophenol were used to inhibit deiodination and sulfation, respectively. Uptake was similar in all cell types. Accumulation was highest with 3,5,3'-triiodothyronine, intermediate with other compounds having iodines in both rings, lowest with compounds iodinated in only one ring, and absent with iodothronine sulfates. These findings help to explain the relative rates of metabolism of the iodothyronines and their release from the cells.     

Formation and metabolism of 3',5'-diiodothyronine and 3,5-diiodothyronine by cultured monkey hepatocarcinoma cells.

Cultured monkey hepatocarcinoma cells were incubated with [3',5'-125I] diiodo-L--thyronine and with [3,5-125I] diiodo-L-thyronine. In both instances monodeiodination as well as sulfoconjugation took place. [3.-125I] iodothyronine and [3',5'-125I] diiodothyronine were identified as metabolites of [3'-5'-125I]-L-thyroxine in the cells, but neither [3-125I]-iodothyronine nor [3,5-125I] diiodothyronine was detected after incubation of the cells with ]3,5-125I]-L-thyroxine. No products of diphenyl ether splitting were observed in the medium after incubation of the cells with either [3,5-125I] diiodo-L-thyronine or [3,5-125I]-L-thyroxine.     

Phenolic compounds from the cultured mycobionts of Graphis proserpens

Spore-derived mycobionts of the lichen Graphis proserpens were cultivated on a malt-yeast extract medium supplemented with 10% sucrose and their metabolites were investigated. Isocoumarin derivatives 1-3 and 7-oxo-5,7-dihydrooxepino[4,3,2-de]isochromene derivatives, proserins A-C (4-6), were isolated along with three known isocoumarin derivatives and three benzoic acid derivatives. Their structures were determined by spectroscopic methods.     

Glycosphingolipids in cultured lens epithelial cells from dog and rhesus monkey

Vertebrate lens tissues contain several species of acidic andneutral glycosphingolipids in relatively high amounts. However,the epithelia with capsule from dog and rhesus monkey lenseshad a simpler composition and lower content of glycosphingolipidsthan whole lenses. Gangliosides and neutral glycosphingolipidsin monolayer cultures of lens epithelial cells were also differentfrom those in whole lenses. Although -galactosyl (Gal1-3Ga1-R)or Lewis^x (Galß1-4[Fuc1-3]GlcNAc-R) epitopes werefound in glycosphingolipids from whole lenses, they were notdetected in those from monolayer cultures of dog and rhesusmonkey lens cells. In addition, significant changes in ganglio-seriesgangliosides were induced in monolayer cultures of both cells,where GM3 and GD3 were predominant. Immunofluorescence studyrevealed a characteristic distribution of cell surface gangliosidesin confluent monolayers. These findings suggest that glycosphingolipidsynthesis in lens epithelia is intrinsically different fromthat in cortical and nuclear fibres, and that the expressionof Lewis^x and -galactosyl epitopes in glycosphingolipids appearsto be associated with the differentiation of epithelial cellsto fibres. gangliosides glycosphingolipids lens epithelial cells Lewis^x rhesus monkey.     

Thyroxine inactivation by starvation in cultured hepatocarcinoma cells, Formation of reverse triiodothyronine.

The effect of starvation on thyroid hormone metabolism was studied in monkey hepatocarcinoma monolayer cultures. Nonphenolic ring monodeiodination of thyroxine, 3, 5, 3'-triiodothyronine and 3, 3'-diiodothyronine was accelerated. Since phenolic ring deiodination of 3, 3',5'-triiodothyronine (reverse T3) was unaffected, this metabolite accumulated in the medium during thyroxine metabolism. This suggests that increased serum reverse T3 in malnourished humans may be caused by enhanced deiodination of thyroxine rather than decreased rT3 catabolism.     

Evidence for a single enzyme in rat liver catalysing the deiodination of the tyrosyl and the phenolic ring of iodothyronines.

The enzymic 5'-deiodination of 3',5'-di-iodothyronine and 5-deiodination of 3,3',5-tri-iodothyronine by rat liver microsomal fractions were found to be characterized by apparent Km values of 0.77 and 17.4 microM respectively, 3',5'-Di-iodothyronine was a competitive inhibitor of 3,3',5-tri-iodothyronine 5-deiodination (Ki 0.65 microM) and 3,3',5-tri-iodothyronine was a competitive inhibitor of 3',5'-di-iodothyronine 5'-deiodination (Ki 19.6 microM). In addition, several radiographic contrast agents and iodothyronine analogues inhibited both reactions competitively and with equal potencies (r = 0.999). These results strongly suggest the existence of a single hepatic deiodinase acting on both the tyrosyl and phenolic ring of iodothyronines.     

Endogenous hyperphosphorylation in plasma membrane from an ascites hepatocarcinoma cell line

Plasma-membrane-bound kinases of AS-30D ascites from transplantable rat hepatocarcinoma were shown to extensively catalyze the phosphorylation of plasma membrane proteins and membrane lipids, using [gamma-32P]ATP or [gamma-32P]GTP as a phosphate donor. In contrast, plasma membranes from normal adult rat liver or fast-growing regenerating liver (24 h after partial hepatectomy) produce significantly less activity for protein phosphorylation and little phosphorylation of the lipids. However, neonatal (24 h old) rat liver plasma membrane preparations show levels of phosphorylation of proteins and lipids intermediate between those in the tumor cell line and normal adult plasma membrane preparations. Phosphatidic acid was identified as one of the 32P-labelled lipids in the tumor plasma membrane chloroform-methanol (2:1, v/v) extract. Phosphorylation of protein was not affected by cAMP or cGMP. However, calcium ion (in the presence or absence of calmodulin) significantly modifies the 32P labelling of a series of proteins in normal tissue but has little effect with the neoplastic preparations. Some plasma membrane proteins were capable of nucleotide binding, instead or in addition to being phosphorylated. Finally, the presence of membrane-bound phosphoprotein phosphatase(s) was also demonstrated in all the preparations examined by means of chase experiments with nonlabelled ATP or GTP, and (or) by the use of the phosphoprotein phosphatase inhibitor, orthovanadate.     

Sequential events of apoptosis induced by zearalenone in cultured hepatocarcinoma cells

Zearalenone (ZEA) is a fungal metabolite that can contaminate feed and foodstuffs and can cause serious health problems for animals as well as for humans. The present investigation was conducted to determine the chronological succession of the main events that characterise ZEA-induced toxicity in human hepatocarcinoma cells. To this aim, we have monitored the effects of ZEA on (1) cell viability, (2) heat-shock protein expression, (3) oxidative damage, (4) DNA fragmentation, (5) the cell cycle and (6) the cell-death-signalling pathway. Our results demonstrated that ZEA reduced cell viability in a time- and dose-dependent manner. When we exposed HepG2 cells to 100 μM ZEA (80% of cells are viable) for different treatment times (2, 4, 8, 24, 30, 48 and 60 h), we demonstrated an induction of Hsp70 protein, an increase in reactive oxygen species (ROS) generation, DNA fragmentation and cell-cycle arrest. These events begin after only 2 h of mycotoxin exposure and are earlier than those implicated in the execution of apoptosis. However, significant apoptotic cell death was observed after at least 30 h of ZEA exposure as a consequence of increased Bax expression, decreased Bcl-2 expression and mitochondrial membrane potential (Δψm)-released cytochrome c and activated caspase-3 and caspase-9.     

Phenolic components of the primary cell wall. Feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharide.

1. Cell walls from rapidly growing cell suspension cultures of Spinacia oleracea L. contained ferulic acid and p-coumaric acid esterified with a water-insoluble polymer. 2. Prolonged treatment with trypsin did not release may feruloyl esters from dearabinofuranosylated cell walls, and the polymer was also insoluble in phenol/acetic acid/water (2:1:1, w/v/v). 3. Treatment of the cell walls with the fungal hydrolase preparation "Driselase' did liberate low-Mr feruloyl esters. The major esters were 4-O-(6-O-feruloyl-beta-D-galactopyranosyl)-D-galactose and 3?-O-feruloyl-alpha-L-arabinopyranosyl)-L-arabinose. These two esters accounted for about 60% of the cell-wall ferulate. 4. It is concluded that the feruloylation of cell-wall polymers is not a random process, but occurs at very specific sites, probably on the arabinogalactan component of pectin. 5. The possible role of such phenolic substituents in cell-wall architecture and growth is discussed.     

Characterization of Na+-dependent phosphate uptake in cultured kidney cells (JTC-12) from monkey.

O-(4-Diazo-3-[125I]iodobenzoyl)sucrose ([125I]DIBS), a novel labelling compound specifically designed to study the catabolic sites of serum proteins [De Jong, Bouma, & Gruber (1981) Biochem. J. 198, 45-51], was applied to study the tissue sites of degradation of serum lipoproteins. [125I]DIBS-labelled apolipoproteins (apo) E and A-I, added in tracer amounts to rat serum, associate with high-density lipoproteins (HDL) just like conventionally iodinated apo E and A-I. No difference is observed between the serum decays of chromatographically isolated [125I]DIBS-labelled and conventionally iodinated HDL labelled specifically in either apo E or apo A-I. When these specifically labelled HDLs are injected into fasted rats, a substantial [125I]DIBS-dependent 125I accumulation occurs in the kidneys and in the liver. No [125I]DIBS-dependent accumulation is observed in the kidneys after injection of labelled asialofetuin or human low-density lipoprotein. It is concluded that the kidneys and the liver are important sites of catabolism of rat HDL apo E and A-I.     

Cleavage of nonphenolic beta-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium.

Purified manganese peroxidase (MnP) from Phanerochaete chrysosporium oxidizes nonphenolic beta-1 diarylpropane lignin model compounds in the presence of Tween 80, and in three- to fourfold lower yield in its absence. In the presence of Tween 80, 1-(3',4'-diethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (I) was oxidized to 3,4-diethoxybenzaldehyde (II), 4-methoxyacetophenone (III) and 1-(3',4'-diethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (IV), while only 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) were detected when the reaction was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the alpha position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4-diethoxybenzaldehyde, 4-methoxyacetophenone remained constant, while the yield of the alpha-keto dimeric product (IV) decreased by approximately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the alpha-keto dimeric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18O2, both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of 18O. Finally, MnP oxidized the substrate 1-(3',5'-dimethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3',5'-dimethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP-catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring.     

Post-secretory deiodination of iodothyronines released from normal human thyroid cells in vitro

The stability of tri-iodothyronine (T3) and thyroxine (T4) in the presence of isolated, cultured normal human thyroid cells has been investigated by radioimmunoassay of the culture medium at intervals to 120 hours.T3 and T4 were both progressively degraded in the presence of cells, and although no significant deiodination was produced by fresh culture medium, the medium withdrawn from confluent cell cultures at 120 hours was capable of degrading subsequently - added iodothyronines.These findings provide evidence for the in vitro release of an iodothyronine deiodinase, and this is discussed in terms of the net decrease in medium iodothyronine levels observed in earlier studies of in vitro T3 and T4 release.     

Characterization of the collagen chains synthesized by cultured smooth muscle cells derived from rhesus monkey thoracic aorta.

Five different collagen chains and one smaller collagenous fragment have been isolated from the collagens found in the combined cell layer and medium of rhesus monkey aortic smooth muscle cell cultures. The collagen chains which can be identified are alpha1 (III), alpha1(I), alpha2, A and B. The smaller collagenous peptide exhibits an apparent molecular weight of 45 000 and has been designated CP45 (Mayne, R., et al. (1977), Arch. Biochem. Biophys. 181, 462). Smooth muscle cells continue to synthesize the collagens from which these components are derived for at least eight passages in culture. At each passage the alpha1 (III) chain consistently represents about one-half of the total collagen which is recovered after initial fractionation by agarose gel chromatography. The results show that smooth muscle cells derived from rhesus monkey thoracic aorta are phenotypically stable for many generations in vitro.     

Pilot-scale extraction of an intracellular recombinant cutinase from E. coli cell homogenate using a thermoseparating aqueous two-phase system

A thermoseparating aqueous two-phase system for extraction of a recombinant cutinase fusion protein from Escherichia coli homogenate has been scaled up to pilot scale. The target protein ZZ-cutinase-(WP)(4) was produced in a fed batch process at 500 l to a concentration of 12% of the total protein and at a cell concentration of 19.7 g l(-1). After harvest and high-pressure homogenisation a first extraction step was performed in an EO(50)PO(50) (50% (w/w) ethylene oxide and 50% (w/w) propylene oxide) thermopolymer/amylopectin rich Waxy barley starch system. The (WP)(4) tag was used for enhanced target protein partitioning to the EO(50)PO(50) phase while the cell debris was collected in the starch phase. A second extraction step followed where the recovered EO(50)PO(50) phase from the first step was supplemented with a non-ionic detergent (C(12-18)EO(5)) and heated to the cloud point (CP) temperature (45 degrees C). One polymer-rich liquid phase and one almost pure aqueous phase were formed. The target protein could be obtained in a water phase after the thermal phase separation at a total recovery over the extraction steps of 71% and a purification factor of 2.5. We were able to demonstrate that a disk-stack centrifugal separator could be adapted for rapid separation of both primary and thermoseparated phase systems.