Multifunctional polypeptides for purine de novo synthesis

The pathway leading to the synthesis of purines for ATP, RNA, DNA and other cellular molecules involves the same enzymatic steps for all groups of organisms. However, the organization of the polypeptides catalyzing some of these steps differs strikingly from organism to organism.     

Calmodulin antagonist W-7 inhibits de novo synthesis of cholesterol and suppresses secretion of de novo synthesized and preformed lipids from cultured hepatocytes

The effects of a calmodulin antagonist W-7 were studied on the synthesis and secretion of lipids in primary rat hepatocytes and McArdle-RH7777 cells. In time course experiments, W-7 (20 microM) inhibited secretion of newly synthesized triacyl[(3)H]glycerol by 35%. When the cells were pre-treated overnight with W-7 (20 microM), followed by incubation with [(3)H]oleate, a significant decrease in the secretion of triacylglycerol (TG) and cholesteryl ester (CE) was observed. De novo synthesis of cholesterol from acetate or mevalonolactone was inhibited by W-7, but not glycerolipid synthesis from glycerol and oleic acid precursors. Concentration-response curves for the effects of overnight pre-incubation with W-7 followed labeling with [(3)H]glycerol and [(14)C]mevalonolactone revealed that: (1). the inhibitory effect of W-7 was concentration-dependent and appeared even at the lowest concentration examined (1 microM). W-7 at a concentration of 20 microM suppressed secretion of TG by 60% (P相似文献   

Stimulation of de novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol ester

The present study was undertaken to determine the mechanism by which phorbol ester stimulates eicosanoid synthesis in endothelial cells. We observed that phorbol 12-myristate 13-acetate (PMA) actively stimulated eicosanoid synthesis over a prolonged period of time, and the stimulatory effect was abolished by cycloheximide and actinomycin D. Western blot was employed to test the hypothesis that PMA elicited sustained eicosanoid synthesis via the stimulation of de novo synthesis of prostaglandin G/H synthase (cyclooxygenase, EC 1.14.99.1). Treatment of cultured human umbilical vein endothelial cells resulted in an enhancement of the 70-kDa immunoreactive prostaglandin G/H synthase band over the control cells treated with medium alone. The enhancement was abolished by cycloheximide. Human umbilical vein endothelial cells were then metabolically labeled with L-[35S]methionine, and the effect of PMA on methionine incorporation was evaluated by immunoblotting. PMA increased the synthetic rate of prostaglandin G/H synthase over the control cells. By pulse-chase experiments, we further showed that prostaglandin G/H synthase has a rapid turnover rate (t1/2 less than 10 min) in control cells, and PMA had no effect on the enzyme turnover. Our data indicate that PMA increases the synthesis of prostaglandin G/H synthase which is required for circumventing the autoinactivation of prostaglandin G/H synthase and hence permit sustained conversion of arachidonic acid into eicosanoids.     

Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors

The effect of various herbicides on glutathione levels in barley (Hordeum vulgare L.), tobacco (Nicotiana tabacum L.), soybean (Glycine max [L.] Merr.), and corn (Zea mays L.) was examined. Illumination of excised barley, tobacco, and soybean plants for 8 hours in solution containing 2 millimolar aminotriazole (a catalase inhibitor) resulted in an increase in leaf glutathione from 250 to 400 nanomoles per gram fresh weight to 600 to 1800 nanomoles per gram fresh weight, depending on the species tested. All of this increase could be accounted for as oxidized glutathione. Between 25 and 50% of this oxidized glutathione was reduced when plants were darkened for 16 hours, but there was no significant decline in total glutathione. Another catalase inhibitor, thiosemicarbazide, was as effective as aminotriazole in elevating glutathione in soybean but was less effective in barley and tobacco. Glyphosate, an inhibitor of aromatic amino acid biosynthesis, had no significant effect on glutathione levels in any of the plants examined. Whereas methyl viologen (paraquat), which is a sink for photosystem I electrons, caused oxidation of leaf glutathione in all of the plants but did not increase the total amount of glutathione present.     

Stimulation of glutathione synthesis in iron-loaded mice

We have previously reported that the iron-loading of mice, by feeding them carbonyl iron, caused an elevation of hepatic glutathione concentration and an increase in glutathione excretion from the liver (Kawabata, T., Ogino, T. and Awai, M. (1989) Biochim. Biophys. Acta 1004, 89-94). To elucidate the mechanism of glutathione elevation, hepatic cysteine concentration and gamma-glutamylcysteine synthetase (L-glutamate: L-cysteine gamma-ligase (ADP-forming), EC 6.3.2.2) activity were measured and possible changes in cysteine metabolism were also compared between iron-loaded and control mice. Hepatic cysteine concentration was higher in iron-loaded mice (185 +/- 12 nmol/g wet wt.) than in the controls (164 +/- 8 nmol/g wet wt.), and gamma-glutamylcysteine synthetase activity was also elevated in iron-loaded mice (34.3 +/- 3.2 nmol/mg protein per min) compared with the controls (28.6 +/- 3.8 nmol/mg protein per min). A comparison of the metabolic pathways with intravenously injected [35S]cysteine showed that organ distribution of the isotope was not significantly different, and also the rate of [35S]cysteine uptake into the hepatic glutathione fraction exhibited no difference between the two groups of mice. This shows that hepatic cysteine turnover may not be different between the two groups of mice. Since hepatic cysteine concentration was higher in iron-loaded mice, the apparently equal turnover of hepatic cysteine suggests that GSH synthesis may be elevated in iron-loaded mice. The high gamma-glutamylcysteine synthetase activity is suggested to stimulate GSH synthesis in iron-loaded mice.     

Regulation of de novo phosphatidylinositol synthesis

Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition. Potential sites of enzymatic regulation are discussed.     

Protein-mediated error correction for de novo DNA synthesis

The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.     

More chores for TOR: de novo ceramide synthesis

In this issue of Cell Metabolism, Aronova et al. (2008) show that target of rapamycin complex 2 (TORC2) controls de novo ceramide synthesis in yeast by regulating the activity of ceramide synthase. This work may provide insight into how chemotherapeutic drugs increase de novo ceramide synthesis and promote cell death in humans.     

Stimulation of fibrinogen synthesis in cultured rat hepatocytes by fibrinogen fragment E

Because of the inherent difficulties of experimentation in intact animals, we used primary monolayer cultures of non-proliferating adult rat hepatocytes to study the effects of fibrinogen degradation products on fibrinogen biosynthesis. The freshly isolated hepatocytes obtained by collagenase perfusion of the liver in situ were cultured in a chemically defined serum-free medium. The rate of fibrinogen synthesis in control cultures was $\text{40–50}\text{pmol}\text{2.5·10}{}^6$ cells per 24 h. Additions of 20, 60 or 100 μg of homologous stage I fibrinogen degradation products had no effect on fibrinogen synthesis. In contrast, addition of the same amounts of homologous or heterologous (human) stage III fibrinogen degradation products resulted in a concentration-dependent increase in fibrinogen biosynthesis without affecting the rate of synthesis of albumin. When purified stage III fibrinogen degradation products D and E (human) were tested in 10, 30 or 50 μg/3 ml medium only fragment E showed a significant increase in fibrinogen biosynthesis (1.9-, 2.8- and 5.6-fold, respectively, over the control cultures). The presence of excess fibrinogen had no effect. These results suggest that fibrinogen fragment E may be a specific stimulator of fibrinogen biosynthesis which may play an important role in maintaining normal levels of plasma fibrinogen.     

Balancing de novo synthesis and salvage of lipids by Leishmania amastigotes

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Evidence for stimulated glutathione synthesis by phenobarbital pretreatment during an oxidative challenge in isolated hepatocytes

Hepatocytes isolated from phenobarbital-pretreated and naive male Sprague-Dawley rats were preincubated with 80 microM N, N-bis (2-chloroethyl)-N-nitrosourea and subsequently exposed to varying concentrations of menadione. We observed that the reduced glutathione levels of the hepatocytes isolated from the sodium phenobarbital(PB)-pretreated, but not the naive rats, recovered to near-control levels after exposure to 200 microM menadione. Since this recovery occurred in the presence of N, N-bis (2-chloroethyl)-N-nitrosourea (an inhibitor of glutathione reductase), we hypothesized that this represented a PB-mediated increase in de novo synthesis of glutathione. To test this hypothesis and to further assess the possible contribution of glutathione reductase in the recovery of the glutathione levels, we preincubated hepatocytes isolated from PB-pretreated and naive rats with 2 mM buthionine sulfoximine, with or without N, N-bis (2-chloroethyl)-N-nitrosourea. Following exposure to menadione, samples were periodically removed for glutathione assessment. Consistent with our hypothesis, the addition of buthionine sulfoximine abrogated the ability of the PB-pretreated hepatocytes to restore glutathione levels following a menadione challenge. Buthionine sulfoximine in combination with N, N-bis (2-chloroethyl)-N-nitrosourea completely abolished hepatocellular glutathione homeostasis for all of the concentrations of menadione employed. The findings from this investigation underscore the importance of phenobarbital-mediated increases in glutathione synthesis, as well as the enhanced levels of glutathione reductase, in maintaining the pool of reduced glutathione and ultimately mitigating the consequences of oxidative stress. In addition, these findings suggest that PB pretreatment increases the reserve capacity of the hepatocyte for glutathione synthesis via a hitherto undescribed hormetic mechanism, a reserve expressed fully only on an oxidative stress of sufficient magnitude.     

Requirements for de novo initiation of RNA synthesis by recombinant flaviviral RNA-dependent RNA polymerases

RNA-dependent RNA polymerases (RdRps) that initiate RNA synthesis by a de novo mechanism should specifically recognize the template initiation nucleotide, T1, and the substrate initiation nucleotide, the NTPi. The RdRps from hepatitis C virus (HCV), bovine viral diarrhea virus (BVDV), and GB virus-B all can initiate RNA synthesis by a de novo mechanism. We used RNAs and GTP analogs, respectively, to examine the use of the T1 nucleotide and the initiation nucleotide (NTPi) during de novo initiation of RNA synthesis. The effects of the metal ions Mg(2+) and Mn(2+) on initiation were also analyzed. All three viral RdRps require correct base pairing between the T1 and NTPi for efficient RNA synthesis. However, each RdRp had some distinct tolerances for modifications in the T1 and NTPi. For example, the HCV RdRp preferred an NTPi lacking one or more phosphates regardless of whether Mn(2+) was present or absent, while the BVDV RdRp efficiently used GDP and GMP for initiation of RNA synthesis only in the presence of Mn(2+). These and other results indicate that although the three RdRps share a common mechanism of de novo initiation, each has distinct preferences.     

Lack of inhibition of glutathione reductase by unnitrated derivatives of nitrofurantoin

Nitrofurantoin produced greater than 70% inhibition of glutathione reductase (EC 1.6.4.2) from human blood, rat blood and yeast. In contrast, identical concentrations of unnitrated derivatives produced less than 10% inhibition of the enzyme. Both nitrofurantoin and the unnitrated derivatives are equally effective in causing depletion of erythrocyte ATP and reduced glutathione levels. These data suggest that the drug-induced red cell toxicity may not be mediated solely by inhibition of glutathione reductase.     

Cellular cholesterol metabolism in mitogen-stimulated lymphocytes--requirement for de novo synthesis

The relationship between cholesterol synthesis and uptake in proliferating lymphocytes has been examined. [14C]Acetate incorporation into lymphocytes cultured under lipoprotein-deficient conditions increased initially in response to mitogen, decreased after 24 h, and increased rapidly between 72 and 96 h. Addition of LDL (10 micrograms/ml) to the culture during the 'trough' period caused [14C]acetate incorporation to return rapidly to baseline, while at peak periods LDL suppression of cholesterol synthesis was minimal. Lymphocytes cultured in the presence of the HMG-CoA reductase inhibitor, mevinolin, exhibited a time-dependent increase in their capacity to incorporate [14C]acetate into cholesterol, evident when mevinolin was removed by washing prior to assay. PHA enhanced 125I-labelled LDL receptor-mediated binding by lymphocytes cultured in lipoprotein-deficient medium over a 4 day period and mevinolin augmented the effect. [3H]Thymidine incorporation into mitogen-stimulated lipoprotein-deficient cultures was inhibited up to 75% by mevinolin (1 mumol/l). LDL (2.5-10 micrograms/ml) substantially reversed this inhibition in 72 h cultures, but only partially overcame inhibition in cells cultured for 96 h. Results suggest that endogenous cholesterol synthesis may be obligatory for lymphocyte proliferation after the initial round of cell division.     

Design and synthesis of de novo cytochromes c

Natural c-type cytochromes are characterized by the consensus Cys-X-X-Cys-His heme-binding motif (where X is any amino acid) by which the heme is covalently attached to protein by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. In this work, the consensus sequence was used for the heme-binding site of a designed four-helix bundle, and the apoproteins with either a histidine residue or a methionine residue positioned at the sixth coordination site were synthesized and reacted with iron protoporphyrin IX (protoheme) under mild reducing conditions in vitro. These polypeptides bound one heme per helix-loop-helix monomer via a single thioether bond and formed four-helix bundle dimers in the holo forms as designed. They exhibited visible absorption spectra characteristic of c-type cytochromes, in which the absorption bands shifted to lower wavelengths in comparison with the b-type heme binding intermediates of the same proteins. Unexpectedly, the designed cytochromes c with bis-His-coordinated heme iron exhibited oxidation-reduction potentials similar to those of their b-type intermediates, which have no thioether bond. Furthermore, the cytochrome c with His and Met residues as the axial ligands exhibited redox potentials increased by only 15-30 mV in comparison with the cytochrome with the bis-His coordination. These results indicate that highly positive redox potentials of natural cytochromes c are not only due to the heme covalent structure, including the Met ligation, but also due to noncovalent and hydrophobic environments surrounding the heme. The covalent attachment of heme to the polypeptide in natural cytochromes c may contribute to their higher redox potentials by reducing the thermodynamic stability of the oxidized forms relatively against that of the reduced forms without the loss of heme.     

Adenosine blocks hormone-induced meiotic maturation by suppressing purine de novo synthesis

We have examined adenosine (Ado) suppression of FSH-induced germinal vesicle breakdown (GVB) and its relationship to purine de novo synthesis. Oocyte-cumulus cell complexes (OCC) from PMSG-primed, immature mice were cultured 17-18 hr in medium containing 4 mM hypoxanthine (HX) or 300 microM dibutyryl cAMP (dbcAMP) to maintain meiotic arrest, and FSH was added to stimulate meiotic maturation. In the absence of FSH, Ado (1-250 microM) had no effect in dbcAMP-arrested oocytes but dose-dependently suppressed maturation in HX-treated oocytes. FSH-induced maturation was prevented by Ado, though more effectively in dbcAMP-supplemented cultures. Ado affected the magnitude, but not the kinetics pattern, of the response to FSH. Inosine also blocked meiotic induction, but only in dbcAMP-arrested oocytes. Purine de novo synthesis was nearly doubled in OCC by FSH treatment, and this response was completely prevented by Ado. FSH had no effect on HX salvage, although Ado reduced this activity by 98%. Inosine effects on metabolism were intermediate between the control and Ado groups. Experiments with radiolabeled energy substrates showed that Ado suppressed FSH activation of the pentose phosphate pathway but did not prevent significant activation of glycolysis or oxidation of pyruvate. Finally, in cultured follicles from primed mice, hCG-induced maturation was blocked by Ado as effectively as by the purine de novo synthesis inhibitor, azaserine. It is concluded that Ado has an inhibitory action on hormone-induced maturation that is due, at least in part, to suppression of glucose metabolism, leading to compromised purine de novo synthesis.