Ethanol enhances de novo synthesis of high density lipoprotein cholesterol

Male squirrel monkeys fed ethanol at variable doses were used to assess whether alcohol enhances de novo synthesis of high density lipoprotein (HDL) cholesterol in vivo. Monkeys were divided into three groups: 1) Controls fed isocaloric liquid diet; 2) Low Ethanol monkeys fed liquid diet with vodka substituted isocalorically for carbohydrate at 12% of calories; and 3) High Ethanol animals fed diet plus vodka at 24% of calories. High Ethanol primates had significantly higher levels of HDL nonesterified cholesterol than Control and Low Ethanol animals while serum glutamate oxaloacetate transaminase was similar for the three treatments. There were no significant differences between the groups in HDL cholesteryl ester mass or specific activity following intravenous injection of labeled mevalonolactone. By contrast, High Ethanol monkeys had significantly greater HDL nonesterified cholesterol specific activity with approximately 60% of the radioactivity distributed in the HDL3 subfraction. This report provides the first experimental evidence that ethanol at 24% of calories induces elevations in HDL cholesterol in primates through enhanced de novo synthesis without adverse effects on liver function.     

Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.     

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.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

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.     

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   

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.     

Inhibition of liver lipase in vivo leads to induction of de novo cholesterol synthesis in rat liver

Rats were injected with control-gamma-globulins or anti-liverlipase. The anti-liverlipase treatment resulted in a 69-78% inhibition of the salt-resistant lipase activity (liver lipase) in the liver. De novo cholesterol synthesis was significantly higher in slices of livers from anti-liverlipase treated rats than from control-gamma-globulin injected rats. The anti-liverlipase treatment also affected all plasma lipoprotein fractions. If the rats had been fed a cholestyramine containing diet no effect of anti-liverlipase on cholesterol synthesis was found. In these rats the effects on plasma lipoprotein fractions were more pronounced than in the control fed rats.     

脂肪酸从头合成代谢及其抑制剂在肿瘤中的研究进展

代谢重编程是肿瘤的重要特征之一.肿瘤细胞常会发生过度增殖、局部侵袭、转移、复发和耐药等.这些过程均需要大量的脂肪酸,用于合成肿瘤细胞所必需的生物膜和信号分子等.因此,研究脂肪酸从头合成代谢在肿瘤细胞发生发展过程中所发挥的重要作用有助于深入理解肿瘤的发病机制,为肿瘤的诊断和治疗提供新的思路.本文将对脂肪酸合成代谢在肿瘤中...     

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.     

Evidence for de novo synthesis of isocitratase and malate synthesis in germinating peanut cotyledons

Evidence for de novo synthesis of isocitratase and malate synthetase in cotyledons of germinating peanut (Arachis hypogaea L.) was obtained by the density labeling method. When dry peanut cotyledons were cultured in H₂¹⁸O, a 2.4% increase in the buoyant density of malate synthetase in a cesium chloride gradient was observed. In 100% D₂O the buoyant density shift was 5.5% for isocitratase and 3.5% for malate synthetase in comparison to the water controls. These data suggest that isocitratase and malate synthetase do not pre-exist in some inactive form in the cotyledons, but are completely synthesized after onset of germination from a pool of amino acids which do not derive directly from hydrolysis of storage proteins.     

Activation and de novo synthesis of hydrogenase in chlamydomonas

Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.

Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg²⁺, Ca²⁺, and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.

The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.

     

Localized de novo phospholipid synthesis drives autophagosome biogenesis

ABSTRACT

During (macro)autophagy, cells form transient organelles, termed autophagosomes, to target a broad spectrum of substrates for degradation critical to cellular and organismal health. Driven by rapid membrane assembly, an initially small vesicle (phagophore) elongates into a large cup-shaped structure to engulf substrates within a few minutes in a double-membrane autophagosome. In particular, how autophagic membranes expand has been a longstanding question. Here, we summarize our recent work that delineates a pathway that drives phagophore expansion by localized de novo phospholipid synthesis. Specifically, we found that the conserved acyl-CoA synthetase Faa1 localizes to nucleated phagophores to locally activate fatty acids for de novo phospholipid synthesis in the neighboring ER. These newly synthesized phospholipids are then preferentially incorporated into autophagic membranes and drive the expansion of the phagophore into a functional autophagosome. In summary, our work uncovers molecular principles of how cells coordinate phospholipid synthesis and flux with autophagic membrane formation during autophagy.

Abbreviations: ACS: acyl-CoA synthestases; CoA: coenzyme A; ER: endoplasmic reticulum     

Relationship between the pentose-phosphate pathway and the de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cultures

This study focuses on the activity of the pentose-phosphate pathway and its relationship to de novo synthesis of fatty acids and cholesterol in oligodendrocyte-enriched glial cell cultures derived from 1-week old rat brain. The proportion of glucose that was metabolized along the pentose-phosphate pathway was estimated by measuring ¹⁴CO₂ production from [1-¹⁴C]-, [2-¹⁴C]- and [6-¹⁴C]glucose, the utilization of glucose and the production of lactate. Incorporation of ¹⁴C from [¹⁴C]glucose and from [3-¹⁴C]acetoacetate into lipids was analysed. The pentose- phosphate pathway produced much more CO₂ from glucose than the Krebs cycle, although it accounted for only a small part of the consumption of glucose (< 3%). The higher ¹⁴CO₂ production from [2-¹⁴C]glucose than from [6-¹⁴C]glucose indicated that recycling of the products of the pentose-phosphate pathway takes place in these cells.Gradual inhibition of the pathway with increasing concentrations of 6-aminonicotinamide resulted in a parallel inhibition of the conversion of acetoacetate and of glucose into fatty acids and into cholesterol. Glycolysis was also strongly inhibited in the presence of 6-aminonicotinamide whereas the activity of the Krebs cycle was not affected.These results suggest that de novo synthesis of fatty acids and cholesterol by oligodendrocytes of neonatal rats is closely geared to the activity of the pentose-phosphate pathway in these cells.     

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.     

Methionine metabolism in plants: chloroplasts are autonomous for de novo methionine synthesis and can import S-adenosylmethionine from the cytosol

The subcellular distribution of Met and S-adenosylmethionine (AdoMet) metabolism in plant cells discloses a complex partition between the cytosol and the organelles. In the present work we show that Arabidopsis contains three functional isoforms of vitamin B(12)-independent methionine synthase (MS), the enzyme that catalyzes the methylation of homocysteine to Met with 5-methyltetrahydrofolate as methyl group donor. One MS isoform is present in chloroplasts and is most likely required to methylate homocysteine that is synthesized de novo in this compartment. Thus, chloroplasts are autonomous and are the unique site for de novo Met synthesis in plant cells. The additional MS isoforms are present in the cytosol and are most probably involved in the regeneration of Met from homocysteine produced in the course of the activated methyl cycle. Although Met synthesis can occur in chloroplasts, there is no evidence that AdoMet is synthesized anywhere but the cytosol. In accordance with this proposal, we show that AdoMet is transported into chloroplasts by a carrier-mediated facilitated diffusion process. This carrier is able to catalyze the uniport uptake of AdoMet into chloroplasts as well as the exchange between cytosolic AdoMet and chloroplastic AdoMet or S-adenosylhomocysteine. The obvious function for the carrier is to sustain methylation reactions and other AdoMet-dependent functions in chloroplasts and probably to remove S-adenosylhomocysteine generated in the stroma by methyltransferase activities. Therefore, the chloroplastic AdoMet carrier serves as a link between cytosolic and chloroplastic one-carbon metabolism.