Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Effect of Calmodulin Antagonists and Chlorinated Phenoxy Compounds

Recognized calmodulin antagonists and chlorinated phenoxyalkylamines were tested as inhibitors of mevalonate incorporation into triterpenols and their fatty acid esters in a centrifuged pellet from the latex of Euphorbia lathyris. The calmodulin antagonists, chlorpromazine (II), fluphenzine, and trifluoperazine were good inhibitors; I₅₀ values for II and trifluoperazine were 150 and 55 micromolar, respectively. Inhibition by the phenoxyalkylamines increased with increasing chlorine substitution, and I₅₀ for 2-(pentachlorophenoxy)ethyl N,N-diethylamine (IX) was 35 micromolar. The calmodulin-stimulated phosphodiesterase catalyzed hydrolysis of cAMP was used as an assay to quantitate the calmodulin antagonism of the tested compounds. Compounds II and IX were calmodulin antagonists over a concentration range similar to their effective range in the biosynthesis of triterpenes. The antagonism of the chlorinated phenoxy compounds increased in parallel to their inhibitory effect upon triterpene biosynthesis.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

Sesquiterpene biosynthesis in maritime pine needles

The biosynthesis of sesquiterpene hydrocarbons was studied in maritime pine (Pinus pinaster) needles by incorporation of ¹⁴CO₂, [1-¹⁴C] acetate and [2-¹⁴C] mevalonate. It was shown that the mechanisms of sesquiterpene biosynthesis are different according to the applied tracer. The important role of the acyclic compound, trans-β-farnesene, before cyclisation processes is discussed.     

Calcium Dependence of Rapid Auxin Action in Maize Roots

We investigated the interaction of Ca²⁺ and auxin on root elongation in seedlings of Zea mays L. The seedlings were raised either in the presence of Ca²⁺ (high calcium; HC = imbibed and raised in 10 millimolar CaCl₂), in the absence of additional Ca²⁺ (intermediate calcium; IC = imbibed and raised in distilled H₂O, calcium supply from seed only), or without additional Ca²⁺ and subsequently depleting them of Ca²⁺ (low calcium; LC = imbibed and raised in distilled H₂O and subsequently treated with 1 millimolar ethyleneglycol-bis-[β-aminoethylether]-N,N,N′,N′ -tetraacetic acid [EGTA]). Exposure of roots of either HC or IC seedlings to auxin concentrations from 0.1 to 10 micromolar resulted in strong inhibition of elongation. In roots of LC seedlings, on the other hand, auxin concentrations as high as 10 micromolar caused only slight inhibition of elongation. Adding 0.5 millimolar Ca²⁺ to LC roots in the presence of IAA allowed normal expression of the inhibitory action of the hormone. Inhibition of elongation in IC roots by indoleacetic acid was reversible upon treatment of the roots with 1 millimolar EGTA. The inhibitory action of auxin could then be re-established by supplying 0.5 millimolar Ca²⁺. The data indicate that Ca²⁺ may be necessary to the growth-regulating action of auxin. The significance of this finding is discussed with respect to the potential role of Ca²⁺ as a second messenger of auxin action and the relevance of this model to recent evidence for gravi-induced redistribution of Ca²⁺ and its role in establishing gravitropic curvature.     

Acid Metallophosphatase from the Ameba Amoeba proteus

Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl₂ or CaCl₂ in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl₂, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl₂, CaCl₂ or ZnCl₂ (10 and 100 mM) before gel staining. However, 1 M ZnCl₂, unlike 1 M CaCl₂ or 1 M MgCl₂, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H₂O₂ (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg²⁺, Ca²⁺, Fe²⁺, Fe³⁺, and Zn²⁺), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.     

Characterization of Fatty Acid biosynthesis in isolated pea root plastids

Fatty acid biosynthesis from Na[1-¹⁴C]acetate was characterized in plastids isolated from primary roots of 7-day-old germinating pea (Pisum sativum L.) seeds. Fatty acid synthesis was maximum at 82 nanomoles per hour per milligram protein in the presence of 200 micromolar acetate, 0.5 millimolar each of NADH, NADPH, and coenzyme A, 6 millimolar each of ATP and MgCl₂, 1 millimolar each of MnCl₂ and glycerol-3-phosphate, 15 millimolar KHCO₃, 0.31 molar sucrose, and 0.1 molar Bis-Tris-propane, pH 8.0, incubated at 35°C. At the standard incubation temperature of 25°C, fatty acid synthesis was essentially linear for up to 6 hours with 80 to 120 micrograms per milliliter plastid protein. ATP and coenzyme A were absolute requirements, whereas divalent cations, potassium bicarbonate, and reduced nucleotides all variously improved activity two- to 10-fold. Mg²⁺ and NADH were the preferred cation and nucleotide, respectively. Glycerol-3-phosphate had little effect, whereas dithiothreitol and detergents generally inhibited the incorporation of [¹⁴C]acetate into fatty acids. On the average, the principal radioactive products of fatty acid biosynthesis were approximately 39% palmitic, 9% stearic, and 52% oleic acid. The proportions of these fatty acids synthesized depended on the experimental conditions.     

Photosynthetic formation of the aspartate family of amino acids in isolated chloroplasts

The metabolism of ¹⁴C-labeled aspartic acid, diaminopimelic acid, malic acid and threonine by isolated pea (Pisum sativum L.) chloroplasts was examined. Light enhanced the incorporation of [¹⁴C] aspartic acid into soluble homoserine, isoleucine, lysine, methionine and threonine and protein-bound aspartic acid plus asparagine, isoleucine, lysine, and threonine. Lysine (2 millimolar) inhibited its own formation as well as that of homoserine, isoleucine and threonine. Threonine (2 millimolar) inhibited its own synthesis and that of homoserine but had only a small effect on isoleucine and lysine formation. Lysine and threonine (2 millimolar each) in combination strongly inhibited their own synthesis as well as that of homoserine. Radioactive [1,7-¹⁴C]diaminopimelic acid was readily converted into [¹⁴C]threonine in the light and its labeling was reduced by exogenous isoleucine (2 millimolar) or a combination of leucine and valine (2 millimolar each). The strong light stimulation of amino acid formation illustrates the point that photosynthetic energy is used in situ for amino acid and protein biosynthesis, not solely for CO₂ fixation.     

Orotic acid biosynthesis in rat liver: studies on the source of carbamoylphosphate.

Measurements of the incorporation of radiolabeled precursors into orotic acid in tissue slices and minces provided evidence of the participation of the intramitochondrial carbamoylphosphate synthetase (CPSase-I) in the de novo biosynthesis of pyrimidines in rat liver. Ammonia, the only nitrogen source utilized by CPSase-I, markedly stimulated the incorporation of NaH¹⁴CO₃ into orotic acid in liver slices, and ornithine, which enhances the intramitochondrial consumption of carbamoylphosphate (CP) in citrulline synthesis, antagonized the stimulation by ammonia. Sensitivity of the incorporation of NaH¹⁴CO₃ into orotic acid to stimulation by ammonia was found to increase with age in concert with the emergence of CPSase-I in the liver during late fetal and neonatal development. Tissues lacking in CPSase-I activity did not exhibit the responses to ammonia and ornithine observed with the adult rat liver. While the occurrence of CPSase-I in the liver contributes extensively toward the exceptionally high capacity of that tissue for the de novo biosynthesis of orotic acid, our results also indicate that the physiological rate of orotic acid biosynthesis in rat liver is approximately one-third of capacity; the incorporation of NaH¹⁴CO₃ into orotic acid averaged 488 nmol/g of tissue in 3 h in the presence of toxic levels of ammonia, but declined to 160 nmol/g of tissue in 3 h when physiological levels of both ammonia and ornithine were provided. However, the rate of orotic acid biosynthesis observed with physiological concentrations of ammonia and ornithine could be reduced further, to about one-quarter of the physiological rate, by providing additional ornithine; thus, physiological levels of ornithine do not prevent the escape of intramitochondrial CP into the cytoplasm. Finally, over 80% of the incorporation of NaH¹⁴CO₃ into orotic acid at physiological levels of ammonia and ornithine was found to be ammonia dependent, and all but a small fraction of the ammonia-dependent incorporation could be blocked by providing ornithine in amounts in excess of physiological. These results indicate that CPSase-I is the major source of CP in the biosynthesis of hepatic pyrimidines under normal (physiological) conditions as well as in ammonia toxicity.     

Regulation of dolichol and of cholesterol biosynthesis in cholesterol-fed rabbits

The feeding of rabbits with a diet supplemented with 2% cholesterol caused a significant increase in the concentration of serum and hepatic microsomal cholesterol while not affecting serum high-density lipoprotein cholesterol concentration. The concentration of cytochrome b₅ was also increased in the cholesterol-fed rabbits but no change in the concentration of cytochrome P-450 was apparent. The increase in microsomal cholesterol was accompanied by an inhibition of hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and a marked stimulation of acyl-coenzyme A:cholesterol acyltransferase activity. The incorporation of [1-¹⁴C]acetate into cholesterol and dolichol was strongly inhibited in liver slices of cholesterol-fed animals. In contrast, while incorporation of [2-¹⁴C]mevalonate into cholesterol was also inhibited by approximately 90%, incorporation of this precursor into dolichol was stimulated fourfold. The increased incorporation of mevalonate into dolichol was consistent with a threefold increase in the activity of the dolichol phosphate-dependent mannosyl transferase. The possible significance of these differences is discussed.     

Regulation of Apoplastic pH in Source Leaves of Vicia faba by Gibberellic Acid

Leaf discs of broad bean (Vicia faba L.), peeled on the spongy mesophyll side, rapidly altered the pH of the surrounding medium (apoplast). Using pH indicator paper appressed against the leaf, immediately after peeling, initial apoplastic pH was estimated to be 4.5. Changes in the apoplastic pH were measured with a microelectrode placed into a 100-microliter drop of an unbuffered solution (2 millimolar KCl, 0.5 millimolar CaCl₂, and 200 millimolar mannitol) on the peeled surface. Discs acidified the medium until the pH stabilized at about 5.0 (about 10 minutes). Acidification was inhibited by 50 micromolar sodium vanadate, an inhibitor of the plasmalemma H⁺-ATPase and attenuated by omitting the osmoticum or potassium ions from the medium. Fusicoccin (10 micromolar) greatly enhanced the rate of acidification. The presence of 0.1 to 1 micromolar gibberellic acid resulted in a slower rate of medium acidification. Gibberellic acid appeared to modulate the activity of the H⁺-translocating ATPase located at the plasma membrane of the mesophyll cells.     

Control of alpha-amylase mRNA accumulation by gibberellic Acid and calcium in barley aleurone layers

Pulse-labeling of barley (Hordeum vulgare L. cv Himalaya) aleurone layers incubated for 13 hours in 2.5 micromolar gibberellic acid (GA₃) with or without 5 millimolar CaCl₂ shows that α-amylase isozymes 3 and 4 are not synthesized in vivo in the absence of Ca²⁺. A cDNA clone for α-amylase was isolated and used to measure α-amylase mRNA levels in aleurone layers incubated in the presence and absence of Ca²⁺. No difference was observed in α-amylase mRNA levels between layers incubated for 12 hours in 2.5 micromolar GA₃ with 5 millimolar CaCl₂ and layers incubated in GA₃ alone. RNA isolated from layers incubated for 12 hours in GA₃ with and without Ca²⁺ was translated in vitro and was found to produce the same complement of translation products regardless of the presence of Ca²⁺ in the incubation medium. Immunoprecipitation of translation products showed that the RNA for α-amylase synthesized in Ca²⁺-deprived aleurone layers was translatable. Ca²⁺ is required for the synthesis of α-amylase isozymes 3 and 4 at a step after mRNA accumulation and processing.     

The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH¹⁴CO₃ into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH₄Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH¹⁴CO₃ into orotic acid was increased 3–4-fold in liver homogenate with NH₄Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH₄Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH₄Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH₄Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.     

Compartmental efflux analysis and removal of extracellular cadmium from roots

Profiles of ¹⁰⁹Cd efflux from roots into three solutions were determined for young intact plants of Agrostis gigantea and maize. The solutions were (a) nutrient culture medium containing 3 micromolar Cd at room temperature, (b) ice-cold 5 millimolar CaCl₂, and (c) ice-cold 5 millimolar PbCl₂. Efflux profiles were clearly resolved into three easily discernible components having fast, medium, and slow exchange rates. These results were unexpected for the situation where some intracellular Cd was present both as extractable Cd-binding peptide and in electron-dense granules within the cytoplasm and the vacuoles. Adding a fourth compartment to the curve-fitting model produced a splitting of the fast exchanging component. Use of these efflux kinetics to estimate Cd fluxes through membranes was inappropriate. However, they were useful in determining optimal washing times for the removal of extracellular Cd. A 10 minute wash in ice-cold 5 millimolar CaCl₂ is recommended for this purpose for Agrostis and maize roots.     

Ethylene Biosynthesis and Cadmium Toxicity in Leaf Tissue of Beans (Phaseolus vulgaris L.)

Stress ethylene production in bean (Phaseolus vulgaris L., cv. Taylor's Horticultural) leaf tissue was stimulated by Cd²⁺ at concentrations above 1 micromolar. Cd²⁺-induced ethylene biosynthesis was dependent upon synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) by ACC synthase. Activity of ACC synthase and ethylene production rate peaked at 8 h of treatment. The subsequent decline in enzyme activity was most likely due to inactivation of the enzyme by Cd²⁺, which inhibited ACC synthase activity in vitro at concentrations as low as 0.1 micromolar. Decrease in ethylene production rate was accompanied by leakage of solutes and increasing inhibition of ACC-dependent ethylene production. Ca²⁺, present during a 2-hour preincubation, reduced the effect of Cd²⁺ on leakage and ACC conversion. This suggests that Cd²⁺ exerts its toxicity through membrane damage and inactivation of enzymes. The possibility of an indirect stimulation of ethylene biosynthesis through a wound signal from injured cells is discussed.     

EVIDENCE FOR INVOLVEMENT OF Ca2+ IN THE INCORPORATION OF 32Pi INTO THE PHOSPHATIDYLINOSITOL OF RAT DIAPHRAGM

Abstract— Ethyleneglycol-bis (β-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) inhibited the incorporation of ³²P_i into phosphatidylinositol (PI) in rat diaphragm incubated in Ca²⁺-free Krebs-Ringer medium. Only the labelling of the PI was altered, and no effects on the pool size of PI or on the incorporation of ³²P_i into other phospholipids were observed. The effect of EGTA was concentration-dependent and appeared to be related to its Ca^a+-chelating properties; the inhibition of the incorporation of ³²P_i could be completely reversed by the addition of excess Ca²⁺ but not Mg²⁺. The inhibitory effect of the EGTA was progressively enhanced by lengthening the preincubation of the tissue with EGTA, an observation suggesting that chelation of intracellular or membrane-bound Ca²⁺, rather than extracellular Ca²⁺, was involved in the effect. In contrast to its inhibition of the incorporation of ³²P_i EGTA enhanced the incorporation of [³H]inositol into PI, but this effect was accompanied by an appreciable increase in total uptake of [³Hlinositol by the tissue. Our results suggest that the level of intracellular Ca²⁺ plays a role in the regulation of the incorporation of ³²P_i into PI. Addition of unlabelled α-glycerophosphate to the incubation medium of tissues which had been preincubated with 2-deoxy-d -glucose failed to cause a significant diminution in the inhibition by EGTA of the incorporation of ³²P_i into PI. This experiment suggests, but does not prove, that the effect of EGTA was not at the level of incorporation of ³²P_i into α-glycerophosphate.     

Biosynthesis of squalene and other triterpenes in Mentha piperita from mevalonate-2-14C

Unlike mono- and sesqui-terpenes, squalene and other triterpenes in peppermint readily incorporate mevalonate-2-¹⁴C label (greater than 30% incorporation of R-mevalonate in 4 hr). The labelled squalene produced turns over rapidly. Squalene derived from mevalonate-2-¹⁴C in incorporation times of 1, 4 and 7 hr was degraded chemically and shown to be equivalently labelled, according to theory, in the isopentenyl pyrophosphate-derived and dimethylallyl pyrophosphate-derived portions of the molecule. This contrasts with earlier studies on the biosynthesis of mono- and sesqui-terpenes in peppermint from ¹⁴C-precursors, in which the isopentenyl pyrophosphate-derived portions of the terpene molecules were found to be preferentially labelled, suggesting the presence of endogenous dimethylallyl pyrophosphate pools. The kinetics of squalene biosynthesis, and the labelling pattern of squalene, suggest that sites of triterpene biosynthesis are readily accessible to exogenous mevalonate and that endogenous dimethylallyl pyrophosphate pools do not participate in triterpene biosynthesis to any appreciable extent. The triterpene biosynthetic sites in peppermint thus appear to differ significantly from the monoterpene and sesquiterpene biosynthetic sites.     

Homocysteine Biosynthesis in Green Plants: Physiological Importance of the Transsulfuration Pathway in Lemna paucicostata

To permit an assessment of the relative contributions of the transsulfuration and the direct sulfhydration pathways for homocysteine biosynthesis, the time course of incorporation of ³⁵S from ³⁵SO₄²⁻ into various sulfur-containing compounds in Lemna paucicostata has been determined. Plants were grown with either low (4.5 micromolar) or ample (1,000 micromolar) sulfate in the medium. At the shortest labeling times, ³⁵S-cystathionine was the predominant ³⁵S-containing organic sulfur compound. The flux of sulfur into cystathionine was sufficient to sustain the known rate of methionine biosynthesis. It was calculated that transsulfuration accounted for at least 90 and 85% of the total homocysteine synthesis in low and ample sulfate-grown plants, respectively (and may have accounted for 100%). No marked rise in the ³⁵S-soluble cysteine:³⁵S-homocysteine ratio was observed even at the shortest labeling times, but it is argued that this may be due to (a) the observed compartmentation of soluble cysteine, and (b) the impracticality of using labeling times shorter than 17 seconds. Additional evidence supporting the importance of transsulfuration in Lemna is briefly described.     

Differential effect of Ca+2 on the translation of yeast mitochondrial and some viral RNA'S in an E.coli cell-free system.

Addition of 6mM CaCl₂ to an $\text{E. coli}$ cell-free system resulted in a several-fold enhancement of yeast mt RNA translation and in a severe inhibition of protein synthesis directed by MS2, Qβ and T₅ RNA's. CaCl₂ did not alter the Mg⁺² optimum or the time-course of protein synthesis and had no apparent effect on RNA degradation. Formaldehyde treatment of MS2 RNA markedly diminished the CaCl₂-mediated inhibition of its translation. Addition of equimolar amounts of EGTA, together with CaCl₂, abolished the effect of the latter on cell-free protein synthesis. FMet tRNA binding to ribosomes was enhanced by CaCl₂ in the presence of mt RNA, inhibited in the presence of MS2 RNA, and unaffected in the presence of formaldehyde-treated MS2 RNA. Maximal effect on initiation complex formation was observed with 0.1 mM CaCl₂.     

Biosynthesis of lipids in chloroplasts isolated from jack pine needles

Suspensions of isolated pine needle chloroplasts were shown to incorporate galactose from UDP galactose-[¹⁴C] into galactolipids. The incorporation of the label among galactolipids was always considerably higher in the monogalactosyl diglycerides than in the digalactosyl diglycerides. The galactosyl incorporation into both galactolipid fractions was optimal at pH 8.0 and was inhibited by sulphydryl reagents (p-chloromercuribenzoate, N-ethyl maleimide and CdCl₂). The chloroplast preparations were also able to biosynthesize various phospholipids and galactolipids from palmitoyl-[1-¹⁴C]-CoA; the major portion of the label appeared in phosphatidyl choline. The incorporation of palmitic-[1-¹⁴C] acid into various lipids was very poor compared to that of palmitoyl-[1-¹⁴C]-CoA. However, addition of ATP and CoA markedly stimulated lipid biosynthesis from palmitic-[1-¹⁴C] acid, suggesting the presence of activating enzymes. These chloroplast suspensions did not show any de novo fatty acid synthesis.