Inhibitors of sterol synthesis. Studies of the metabolism of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one in Chinese hamster ovary cells and its effects on activities of early enzymes in cholesterol biosynthesis

The metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I) has been studied in Chinese hamster ovary (CHO-K1) cells which were maintained in a lipid-deficient medium. The incorporation of I into the cells was linear with respect to sterol concentration in the medium over the ranges of concentrations studied and was more than 3.5 times that of the uptake of cholesterol. The results of detailed chromatographic analyses of the lipids recovered from the cells after 6 h of incubation with [2,4-3H]I (0.5 microM or 6.0 microM) indicated that most of the 3H was associated with free I. Considerably lesser amounts of the 3H was associated with esters of I. No formation of [3H]cholesterol or [3H]cholesteryl esters (or other C27 monohydroxysterols) from labeled I was observed. The labeled material with the chromatographic behavior of the esters of I gave, after mild alkaline hydrolysis, the free 15-ketosterol which was characterized by the results of chromatographic and cocrystallization studies. Upon transfer of the CHO-K1 cells from a culture medium containing 8% newborn calf serum to the same medium containing 8% lipid-deficient newborn calf serum, increases in the levels of activity of cytosolic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and of HMG-CoA reductase were observed. These increases were blocked by the addition of I at a concentration of 1.0 microM. I (1.0 microM) also caused a decrease in the levels of activity of the three enzymes in cells previously grown in medium containing lipid-deficient serum. These results demonstrate that I not only affects the enzymatic reduction of HMG-CoA but also the enzymatic formation of this key intermediate in cholesterol biosynthesis.     

Inhibitors of sterol synthesis. Exogenous oleate reduces the inhibitory effect of 5 alpha-cholest-8(14)-en-3 beta-ol-15-one on the growth of CHO-K1 cells

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one, a hypocholesterolemic agent and a potent inhibitor of sterol biosynthesis, inhibited the growth of CHO-K1 cells incubated in medium containing fetal calf serum. The concentration of the oxysterol required to inhibit growth by 50% was 13 microM. Sodium oleate (82 microM) reduced the inhibitory effects of the sterol, and increased the concentration of the 15-ketosterol required to cause a 50% inhibition of growth to 25 microM. The ACAT inhibitor N'-(2,4-difluoro-phenyl)-N-[4-(2,2-dimethylpropy)-phenyl]-methyl)- N- heptylurea (5 microM) abolished the effect of sodium oleate, and reduced the concentration of the 15-ketosterol required to inhibit growth by 50% to 5 microM.     

Meiosis-activating sterol and the maturation of isolated mouse oocytes

This study was carried out to examine the effects of the meiosis-activating C(29) sterol, 4,4-dimethyl-5 alpha-cholesta-8,14, 24-trien-3 beta-ol (FF-MAS), on mouse oocyte maturation in vitro. Cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) from hormonally primed, immature mice were cultured 17-18 h in minimum essential medium (MEM) containing 4 mM hypoxanthine plus increasing concentrations of FF-MAS. The sterol induced maturation in DO with an optimal concentration of 3 microg/ml but was without effect in CEO, even at concentrations as high as 10 microg/ml. Some stimulation of maturation in hypoxanthine-arrested CEO was observed when MEM was replaced by MEMalpha. Interestingly, the sterol suppressed the maturation of hypoxanthine-arrested CEO in MEM upon removal of glucose from the medium. FF-MAS also failed to induce maturation in DO when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP). The rate of maturation in FF-MAS-stimulated, hypoxanthine-arrested DO was slow, as more than 6 h of culture elapsed before significant meiotic induction was observed, and this response required the continued presence of the sterol. Although the oocyte took up radiolabeled lanosterol, such accumulation was restricted by the presence of cumulus cells. In addition, lanosterol failed to augment FSH-induced maturation and was even inhibitory at a high concentration. Moreover, the downstream metabolite, cholesterol, augmented the inhibitory action of dbcAMP on maturation in both CEO and DO. Two inhibitors of 14 alpha-demethylase, ketoconazole, and 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol that can suppress FF-MAS production from lanosterol failed to block consistently FSH-induced maturation. These results confirm the stimulatory action of FF-MAS on hypoxanthine-arrested DO but do not support a universal meiosis-inducing function for this sterol.     

Synthesis and biological properties of the fluorescent ether lipid precursor 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol

The synthesis of an omega-pyrene-labeled 1-O-alkyl-sn-glycerol was performed using a chirospecific method starting from R-(-)-2,3-O-isopropylidene-sn-glycerol. The product, 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol (pAG), is a fluorescent ether lipid that has a pyrene moiety covalently attached at the alkyl chain terminus. pAG was taken into CHO-K1 cells and a plasmalogen-deficient variant of CHO-K1, NRel-4. This variant is defective in dihydroxyacetonephosphate acyltransferase, which catalyzes the first step in plasmenylethanolamine (PlsEtn) biosynthesis. pAG was incorporated primarily into ethanolamine and choline phospholipids as well as a neutral lipid fraction tentatively identified as alkyldiacylglycerol. NRel-4 accumulated more fluorescence in the phospholipid fraction than CHO-K1, specifically in the ethanolamine phospholipids. Analysis of the fluorescent lipids showed that 93% of the pAG was incorporated into glycerolipids with the ether bond intact. Although the addition of 20 microM 1-O-hexadecyl-sn-glycerol to the medium fully restored PlsEtn biosynthesis in NRel-4 cells, pAG only partially restored PlsEtn synthesis. Incubation of cells with pAG followed by irradiation with long-wavelength (>300 nm) ultraviolet light resulted in cytotoxicity. NRel-4 cells displayed an increased sensitivity to this treatment compared with CHO-K1 cells. This photodynamic cytotoxicity approach could be used to select for mutants that are defective in downstream steps in ether lipid biosynthesis.     

Inhibition of cholesterol synthesis and cell growth by 24(R,S),25-iminolanosterol and triparanol in cultured rat hepatoma cells

24(R,S),25-Iminolanosterol (IL) and triparanol added to cultures of rat hepatoma cells, H4-II-C3 (H4), interrupt the conversion of lanosterol to cholesterol and, depending on their concentrations, cause the accumulation in the cells of intermediates in the lanosterol to cholesterol conversion. At 45 microM, both substances cause the accumulation of 5 alpha-cholesta-8(9),24-dien-3 beta-ol (zymosterol), and at the low concentration of 4.5 microM, they cause the accumulation of cholesta-5.24-dien-3 beta-ol (desmosterol). The effect of intermediate concentrations of 9 or 22.5 microM of either substance is to cause the accumulation in the cells of three sterols: cholesta-5,7,24-trien-3 beta-ol, zymosterol, and desmosterol. The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol. The synthesis of cholesterol from [14C]acetate or [2-14C]mevalonate is completely blocked by either inhibitor even at 4.5 microM. IL or triparanol inhibits the growth of H4 cells. Cells seeded into either full growth or lipid-depleted medium containing 22.5 microM IL will not grow unless the media are supplemented with low density lipoproteins (60 micrograms/ml). Supplementation of the media with 4.6 mM mevalonate does not counteract the inhibitory effect of IL on cell growth.     

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.     

Regulation of hepatic cholesterol biosynthesis. Effects of a cytochrome P-450 inhibitor on the formation and metabolism of oxygenated sterol products of lanosterol.

The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.     

Concerning the role of 24,25-dihydrolanosterol and lanostanol in sterol biosynthesis by cultured cells

Rat hepatoma cells (H4-II-E-C3) efficiently converted a dietary supplement of [2-3H]24,25-dihydrolanosterol (1) to [3H]cholesterol while [2-3H]lanostanol (4,4,14 alpha-trimethylcholestanol (2) was recovered from the cells without apparent transformation, although it was esterified and induced an accumulation of lanosterol. A comparison of the chromatographic (TLC, GLC and HPLC), spectral (MS and 1H-NMR) and physical properties of 1 and 2 is given for the first time. The inability to detect 2 in nature coupled with our findings that 1 but not 2 is metabolized to cholesterol by H4 cells is interpreted to imply that the biosynthetic inclusion of the delta 8(9)-bond during the cyclization process of squalene-oxide to a tetracyclic product is an evolutionary adaptation selected for because the olefinic linkage is structually important in the subsequent conversion of lanosterol and its stereoisomers, e.g., cycloartenol, to delta 5-sterols.     

Molecular cloning of Chinese hamster ceramide glucosyltransferase and its enhanced expression in peroxisome-defective mutant Z65 cells

To clarify the metabolic bases of characteristic increases in the concentrations of glucosylceramide (CMH) and GM3 in peroxisome-defective mutant Chinese hamster ovary (CHO) cells (Z65), we measured the ceramide glucosyltransferase (CGT) and beta-glucosidase activities in Z65 and CHO-K1 cells, and found that the former enzyme was responsible for the accumulation of CMH in Z65 cells. Inhibition of CGT by D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) caused a marked reduction in a incorporation of [3-14C]serine to CMH in both CHO-K1 and Z65 cells, but resulted in the accumulation of ceramide in Z65 cells in a concentration higher than that in CHO-K1 cells. Then, we cloned the cDNA encoding CGT from CHO-K1 cells, which exhibited sequence homology with the human gene product (98.7%). Northern blot analysis of CGT revealed increased expression of it in Z65 cells compared with that in CHO-K1 cells, which probably caused the simultaneous increase in GM3. With an immunohistochemical procedure, GM3 was found to be more strongly expressed in the cell membrane of Z65 cells than in CHO-K1 cells.     

The role of ubiquinones in the regulation of lipid metabolism in rat thymocytes

The effect of ubiquinones Q-1, Q-2, Q-8 and Q-9 on lipid metabolism in rat thymocytes in vitro was studied. The cells were incubated in a medium containing ubiquinones within the concentration range from 1 to 100 microM. A 2-fold decreased cholesterol synthesis was observed in thymocytes incubated with ubiquinone Q-9 at a concentration of exogenous ubiquinone of no less than 40 microM. Incubation of thymocytes with ubiquinones UQ-1 and UQ-2 that are characteristic of rats (40 microM and 100 microM) resulted in a decrease of cholesterol synthesis. Ubiquinone-8 had a tendency to inhibit the cholesterogenesis in rat thymocytes.     

Cholesterol starvation decreases p34(cdc2) kinase activity and arrests the cell cycle at G2.

As a major component of mammalian cell plasma membranes, cholesterol is essential for cell growth. Accordingly, the restriction of cholesterol provision has been shown to result in cell proliferation inhibition. We explored the potential regulatory role of cholesterol on cell cycle progression. MOLT-4 and HL-60 cell lines were cultured in a cholesterol-deficient medium and simultaneously exposed to SKF 104976, which is a specific inhibitor of lanosterol 14-alpha demethylase. Through HPLC analyses with on-line radioactivity detection, we found that SKF 104976 efficiently blocked the [(14)C]-acetate incorporation into cholesterol, resulting in an accumulation of lanosterol and dihydrolanosterol, without affecting the synthesis of mevalonic acid. The inhibitor also produced a rapid and intense inhibition of cell proliferation (IC(50) = 0.1 microM), as assessed by both [(3)H]-thymidine incorporation into DNA and cell counting. Flow cytometry and morphological examination showed that treatment with SKF 104976 for 48 h or longer resulted in the accumulation of cells specifically at G2 phase, whereas both the G1 traversal and the transition through S were unaffected. The G2 arrest was accompanied by an increase in the hyperphosphorylated form of p34(cdc2) and a reduction of its activity, as determined by assaying the H1 histone phosphorylating activity of p34(cdc2) immunoprecipitates. The persistent deficiency of cholesterol induced apoptosis. However, supplementing the medium with cholesterol, either in the form of LDL or free cholesterol dissolved in ethanol, completely abolished these effects, whereas mevalonate was ineffective. Caffeine, which abrogates the G2 checkpoint by preventing p34(cdc2) phosphorylation, reduced the accumulation in G2 when added to cultures containing cells on transit to G2, but was ineffective in cells arrested at G2 by sustained cholesterol starvation. Cells arrested in G2, however, were still viable and responded to cholesterol provision by activating p34(cdc2) and resuming the cell cycle. We conclude that in both lymphoblastoid and promyelocytic cells, cholesterol availability governs the G2 traversal, probably by affecting p34(cdc2) activity.     

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34, reductase) activity and sterol biosynthesis were studied in rat intestinal epithelial cell cultures (IEC-6). Incubation of cells with 0.15-2 microM ketoconazole resulted in a concentration-dependent inhibition of reductase activity. As the drug concentration approached 15 microM, the reductase activity returned to control values, and at 30 microM ketoconazole, a stimulation of enzyme activity was observed. The drug had no effect on reductase activity in homogenates of IEC-6 cells. Ketoconazole (0.15-30 microM) caused a concentration-dependent inhibition of the incorporation of [3H] mevalonolactone into cholesterol with a concomitant accumulation of radioactivity in methyl sterols; e.g. lanosterol and 24,25-epoxylanosterol. Interestingly, the incorporation of radioactivity into polar sterols showed a biphasic response which was inversely proportional to the biphasic response of reductase activity. Thus, incorporation of [3H]mevalonolactone into polar sterols increased at low concentrations of ketoconazole (0.15-2 microM) and decreased to control values at high concentrations of the drug. Treatment of cells with ketoconazole (30 microM) and [3H]mevalonolactone followed by removal of the drug and radiolabel resulted in an inhibition of reductase activity and a redistribution of radioactivity from lanosterol and 24,25-epoxylanosterol to cholesterol and polar sterols. These results suggested that the inhibition of reductase activity at low concentrations of ketoconazole (less than 2 microM) was due to a formation of regulatory polar sterols generated from the methyl sterols. At high concentrations of ketoconazole (30 microM) where no suppression in reductase activity was observed, the conversion of exogenously added [3H]24(S),25-epoxylanosterol to polar sterols was prevented. Exogenously added 24,25-epoxylanosterol inhibited reductase activity in a dose-dependent fashion, and ketoconazole (30 microM) prevented the inhibition caused by low concentrations of epoxylanosterol. The drug, however, was unable to prevent the dose-dependent suppression of reductase activity by 25-hydroxylanosterol, a reduced form of 24,25-epoxylanosterol. These results indicated that 24,25-epoxylanosterol per se was not an inhibitor of reductase activity but could be metabolized to regulatory polar sterols through a cytochrome P-450 dependent reaction which was sensitive to ketoconazole. Treatment of cells with ketoconazole totally abolished the inhibition of reductase activity by low density lipoprotein (LDL).(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of Chinese hamster ovary cell mutants defective in glucose transport

Cultured Chinese hamster ovary (CHO) cells possess an insulin-sensitive facilitated diffusion system for glucose transport. Mutant clones of CHO cells defective in glucose transport were obtained by repeating the selection procedure, which involved mutagenesis with ethyl methanesulfonate, radiation suicide with tritiated 2-deoxy-D-glucose, the polyester replica technique and in situ autoradiographic assaying for glucose accumulation. On the first selection, we obtained mutants exhibiting about half the glucose uptake activity of parental CHO-K1 cells and half the amount of a glucose transporter, the amount of which was determined by immunoblotting with an antibody to the human erythrocyte glucose transporter. The second selection, starting from one of the mutants obtained in the first-step selection, yielded a strain, GTS-31, in which both glucose uptake activity and the quantity of the glucose transporter were 10-20% of the levels in CHO-K1 cells, whereas the responsiveness of glucose transport to insulin, and the activities of leucine uptake and several glycolytic enzymes remained unchanged. GTS-31 cells grew slower than CHO-K1 cells at both 33 and 40 degrees C, and in a medium containing a low concentration of glucose (0.1 mM), the mutant cells lost the ability to form colonies. All the three spontaneous GTS-31 cell revertants, which were isolated by growing the mutant cells in medium containing 0.1 mM glucose, exhibited about half the glucose uptake activity and about half the amount of glucose transporter, as compared to in CHO-K1 cells, these characteristics being similar to those of the first-step mutant. These results indicate that the decrease in glucose uptake activity in strain GTS-31 is due to a mutation which induces a reduction in the amount of the glucose transporter, providing genetic evidence that the glucose transporter functions as a major route for glucose entry into CHO-K1 cells.     

Preparation of 14C-labeled and unlabeled sterol intermediates from yeast using metabolic inhibitors

The method for the preparation of zymosterol was improved (13 mg of zymosterol/g dry cells) by the aerobic adaptation of the cells in the presence of 1 mM DL-ethionine. Lanosterol was also found to accumulate (5.0 mg/g dry cells) when the cells were adapted aerobically in the presence of 10(-4) M buthiobate. Pure lanosterol could be obtained by separation of the unsaponifiable lipids on TLC. Pure [14C]lanosterol with a high specific radioactivity (56 Ci/mol) could be prepared by incubation of the desiccated cells with [14C]isopentenyl pyrophosphate, cofactors such as ATP and NADPH-generating system, and buthiobate in phosphate buffer. The method using desiccated cells may also be applicable to the preparation of other radioactive sterol intermediates.     

Bovine colostrum ultrafiltrate supplemented with adult bovine serum and transferrin: An effective fbs substitute for cultivation of vero and CHO-K1 cells

Summary A mixture containing an ultrafiltrate fraction (UF) of bovine colostrum (6.7%), adult bovine serum (BS) (1%), and human holo-transferrin (hTF) (5 mg/liter) was developed for cultivation of Chinese hamster ovary cells (CHO-K1) and African green monkey kidney cells (Vero). The growth-supporting activity of the mixture (UF/BS/hTF) was comparable to that of 1 to 10% fetal bovine serum (FBS) and considerably better than 1 to 2% BS. Cells could be directly seeded from FBS-supplemented medium to UF/BS/hTF-supplemented medium without any weaning period, even at initial plating density of 1700 cells/ml. Vero and CHO-K1 cells were cultivated in UF/BS/hTF-supplemented media for up to 43 days without any apparent reduction in growth. The UF/BS/hTF mixture could also be used as a freezing medium. Cells were passaged twice in the mixture, frozen, and stored at liquid N₂ for 11 wk. After thawing, the viability of Vero and CHO-K1 cells was reduced 13 and 7%, respectively, and both cell lines started to grow well. Additional hTF could be replaced with bovine holo-transferrin, although a high concentration (150 mg/liter) should be used for CHO-K1 cells. The results suggest that the UF/BS/hTF mixture provides a new economical alternative to FBS in cultivation of Vero and CHO-K1 cells in the presence of reduced protein amounts.     

Protease expression in the supernatant of Chinese Hamster Ovary cells grown in serum-free culture

The protease activity secreted by the Chinese Hamster Ovary (CHO-K1) cell line grown in serum-free medium was examined by substrate gel electrophoresis (zymography). The cell line expressed extracellular proteases that were active on gelatin zymograms but not on casein zymograms. The main protease band visible by gelatin zymography was approx. 92 kDa. Incubation of the conditioned medium with aminophenylmercuric acetate (APMA) resulted in the appearance of gelatinase activity at 82 kDa. Incubation of the conditioned media with EDTA significantly decreased the gelatinolytic activity of both the 92 kDa and 82 kDa forms, indicating the gelatinase responsible was a metalloprotease. Immunoblotting of the conditioned medium showed the gelatinase to be the pro- form of matrix metalloprotease-9 (pro-MMP-9), also known as gelatinase B.     

Activation of AMP-activated protein kinase leads to the phosphorylation of elongation factor 2 and an inhibition of protein synthesis

Protein synthesis, in particular peptide-chain elongation, consumes cellular energy. Anoxia activates AMP-activated protein kinase (AMPK, see ), resulting in the inhibition of biosynthetic pathways to conserve ATP. In anoxic rat hepatocytes or in hepatocytes treated with 5-aminoimidazole-4-carboxamide (AICA) riboside, AMPK was activated and protein synthesis was inhibited. The inhibition of protein synthesis could not be explained by changes in the phosphorylation states of initiation factor 4E binding protein-1 (4E-BP1) or eukaryotic initiation factor 2alpha (eIF2alpha). However, the phosphorylation state of eukaryotic elongation factor 2 (eEF2) was increased in anoxic and AICA riboside-treated hepatocytes and in AICA riboside-treated CHO-K1 cells, and eEF2 phosphorylation is known to inhibit its activity. Incubation of CHO-K1 cells with increasing concentrations of 2-deoxyglucose suggested that the mammalian target of the rapamycin (mTOR) signaling pathway did not play a major role in controlling the level of eEF2 phosphorylation in response to mild ATP depletion. In HEK293 cells, transfection of a dominant-negative AMPK construct abolished the oligomycin-induced inhibition of protein synthesis and eEF2 phosphorylation. Lastly, eEF2 kinase, the kinase that phosphorylates eEF2, was activated in anoxic or AICA riboside-treated hepatocytes. Therefore, the activation of eEF2 kinase by AMPK, resulting in the phosphorylation and inactivation of eEF2, provides a novel mechanism for the inhibition of protein synthesis.     

In vitro mass multiplication of Ophiorrhiza mungo Linn

A protocol for in vitro mass multiplication of plants through seedling (shoot) cultures was established for Ophiorrhiza mungo. Maximum number of adventitious shoots per shoot culture (10.4 +/- 1.72) was initiated on MS solid medium supplemented with BAP (2.22 microM) after 3 weeks. Shoots were further multiplied (12.8 +/- 2.8) through subculture of intact shoots and reculture of nodal segments of aseptic shoots (6.5 +/- 0.94) in MS solid medium containing BAP (0.89 microM). Shoot elongation (1.27 +/- 0.12 cm) was achieved in the medium containing GA3 (1.44 microM) in two weeks. Rooting was favoured in basal agar medium supplemented with IBA (12.3 microM) plus NAA (1.07 microM). The plants were successfully established (100%) in the pots containing sand and top soil (1:1) mixture in a period of two weeks.     

Thyroid hormone stimulation of lipolysis and cyclic adenosine 3', 5'-monophosphate accumulation in human adipose tissue

The role of thyroid hormones on lipolysis in human subcutaneous adipose tissue was investigated. Incubation of subcutaneous fat pads with thyroxine (0.1--10 000 nM) augmented the subsequent isoproterenol stimulation of lipolysis, measured by glycerol release. The basal lipolysis could not by stimulated by thyroxine. The theophylline- and dibutyryl-cyclic AMP stimulated lipolysis also could not be increased by thyroxine at these concentrations. In separate studies, the effect of thyroxine (0.01 pM--1 microM) and triiodothyronine (0.01 pM--1 microM) on cyclic AMP accumulation was examined. No effect of thyroid hormones on cyclic AMP accumulation was seen in non-isoproterenol stimulated tissue. Fat pads stimulated by isoproterenol and then treated with thyroid hormones showed marked increases in accumulation of cyclic AMP as compared to control tissue in the presence of isoproterenol alone.