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Purpose: Stereotactic body radiation therapy (SBRT) in the management of adrenal metastases is emerging as a well-tolerated, effective method of treatment for patients with limited metastatic disease. SBRT planning and treatment utilization are widely variable, and publications report heterogeneous radiation dose fractionation schemes and treatment outcomes. The objective of this analysis was to review the current literature on SBRT for adrenal metastases and to develop treatment guidelines and a model for tumor control probability of SBRT for adrenal metastases based on these publications., Methods and Materials: A literature search of all studies on SBRT for adrenal metastases published from 2008 to 2017 was performed, and outcomes in these studies were reviewed. Local control (LC) rates were fit to a statistically significant Poisson model using maximum likelihood estimation techniques., Results: One-year LC greater than 95% was achieved at an approximated biological equivalent dose with α/β = 10 Gy of 116.4 Gy., Conclusions: While respecting normal tissue tolerances, tumor doses greater than or equal to a biological equivalent dose with α/β = 10 Gy of 116.4 Gy are recommended to achieve high LC. Further studies following unified reporting standards are needed for more robust prediction., (Published by Elsevier Inc.)

Purpose: Patients with human EGFR2-positive (HER2 + ) breast cancer have a high incidence of brain metastases, and trastuzumab emtansine (T-DM1) is often employed. Stereotactic radiosurgery (SRS) is frequently utilized, and case series report increased toxicity with combination SRS and T-DM1. We provide an update of our experience of T-DM1 and SRS evaluating risk of clinically significant radionecrosis (CSRN) and propose a mechanism for this toxicity., Experimental Design: Patients with breast cancer who were ≤45 years regardless of HER2 status or had HER2 + disease regardless of age and underwent SRS for brain metastases were included. Rates of CSRN, SRS data, and details of T-DM1 administration were recorded. Proliferation and astrocytic swelling studies were performed to elucidate mechanisms of toxicity., Results: A total of 45 patients were identified; 66.7% were HER2 + , and 60.0% were ≤ 45 years old. Of the entire cohort, 10 patients (22.2%) developed CSRN, 9 of whom received T-DM1. CSRN was observed in 39.1% of patients who received T-DM1 versus 4.5% of patients who did not. Receipt of T-DM1 was associated with a 13.5-fold ( P = 0.02) increase in CSRN. Mechanistically, T-DM1 targeted reactive astrocytes and increased radiation-induced cytotoxicity and astrocytic swelling via upregulation of Aquaporin-4 (Aqp4)., Conclusions: The strong correlation between development of CSRN after SRS and T-DM1 warrants prospective studies controlling for variations in timing of T-DM1 and radiation dosing to further stratify risk of CSRN and mitigate toxicity. Until such studies are completed, we advise caution in the combination of SRS and T-DM1., (©2019 American Association for Cancer Research.)

Objective: In this study, we analyzed patterns of care for patients with locally advanced cervical cancer to identify predictors for upfront surgery compared with definitive chemoradiation (CRT)., Methods: The National Cancer Database was queried for patients aged 18 years or older with Federation of Gynecology and Obstetrics IB2-IIB cervical cancer. All patients underwent either upfront hysterectomy with or without postoperative radiation therapy versus definitive CRT. Logistic regression was used to assess variables associated with modality of treatment (surgery vs CRT)., Results: Of the 9494 patients included, 2151 (22.7%) underwent upfront surgery. Of those undergoing surgery, 380 (17.7%) had positive margins, 478 (22.2%) had positive nodes, and 458 (21.3%) had pathologic involvement of the parametrium. Under multiple logistic regression, rates of surgery significantly increased from 2004 (12.2%) to 2012 (31.2%) (odds ratio [OR] per year increase, 1.15; confidence interval [CI], 1.12-1.17; P < 0.001). Upfront surgery was more commonly performed in urban (OR, 1.21; 95% CI, 1.03-1.41; P = 0.018) and rural counties (OR, 1.79; 95% CI, 1.24-2.58; P = 0.002), for adenocarcinoma (OR, 2.14; 1.88-2.44; P < 0.001) and adenosquamous (OR, 2.69; 2.11-3.43; P < 0.001) histologies, and in patients from higher median income communities (ORs, 1.19-1.37). Upfront surgery was less common at academic centers (OR, 0.73; 95% CI, 0.58-0.93; P = 0.011)., Conclusions: Rates of upfront surgery relative to definitive CRT have increased significantly over the past decade. In the setting of level 1 evidence supporting the use of definitive CRT alone for these women, the rising rates of upfront surgery raises concern for both unnecessary surgical procedures with higher rates of treatment-related morbidity and greater health care costs.

Background: For patients with resectable gastric adenocarcinoma, perioperative chemotherapy and adjuvant chemoradiotherapy (CRT) are considered standard options. In the current study, the authors used the National Cancer Data Base to compare overall survival (OS) between these regimens., Methods: Patients who underwent gastrectomy for nonmetastatic gastric adenocarcinoma from 2004 through 2012 were divided into those treated with perioperative chemotherapy without RT versus those treated with adjuvant CRT. Survival was estimated and compared using univariate and multivariate models adjusted for patient and tumor characteristics, surgical margin status, and the number of lymph nodes examined. Subset analyses were performed for factors chosen a priori, and potential interactions between treatment and covariates were assessed., Results: A total of 3656 eligible patients were identified, 52% of whom underwent perioperative chemotherapy and 48% of whom received postoperative CRT. The median follow-up was 47 months, and the median age of the patients was 62 years. Analysis of the entire cohort demonstrated improved OS with adjuvant RT on both univariate (median of 51 months vs 42 months; P = .013) and multivariate (hazard ratio, 0.874; 95% confidence interval, 0.790-0.967 [P = .009]) analyses. Propensity score-matched analysis also demonstrated improved OS with adjuvant RT (median of 49 months vs 39 months; P = .033). On subset analysis, a significant interaction was observed between the survival impact of adjuvant RT and surgical margins, with a greater benefit of RT noted among patients with surgical margin-positive disease (hazard ratio with RT: 0.650 vs 0.952; P for interaction <.001)., Conclusions: In this National Cancer Data Base analysis, the use of adjuvant RT in addition to chemotherapy was associated with a significant OS advantage for patients with resected gastric cancer. The survival advantage observed with adjuvant CRT was most pronounced among patients with positive surgical margins. Cancer 2017;123:3402-9. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Objectives: Among patients with T2N0M0 glottic larynx cancer undergoing definitive radiotherapy, recent retrospective and prospective data have suggested improved outcomes with altered fractionation over conventional fractionation (CFxn). We sought to characterize national fractionation patterns and to compare outcomes among them., Materials and Methods: We queried the National Cancer Database for T2N0M0 squamous cell carcinomas of the glottis diagnosed from 2004-2014 and managed with definitive radiotherapy. Dose-per-fraction and duration of radiotherapy were used to define cohorts undergoing CFxn, hypofractionation (HypoFxn), and hyperfractionation (HyperFxn). Logistic regression was performed to identify predictors of receiving altered fractionation. Cox regression and propensity-score matching (PSM) analyses were used to compare survival between schedules., Results: We abstracted 2 006 CFxn patients, 1 166 HypoFxn patients, and 161 HyperFxn patients. Fractionation patterns changed significantly from 2004 to 2014, with use of HyperFxn decreasing from 6.3% to 1.8% and use of HypoFxn increasing from 23.9% to 54.1% (p<0.001). Receipt of altered fractionation was independently associated with later year of diagnosis and higher facility volume. On Cox regression, both HypoFxn (hazard ratio [HR] for mortality 0.84, 95% confidence interval [95%CI] 0.73-0.97) and HyperFxn (HR 0.74, 95%CI 0.56-0.99) were associated with improved survival over CFxn. The survival advantage of each altered fractionation schedule over CFxn was redemonstrated on comparison of PSM groups., Conclusion: Increasing utilization of HypoFxn for T2N0M0 glottic cancer is driving national practice patterns away from CFxn. Our findings support the use of altered fractionation, particularly HypoFxn, for patients undergoing definitive radiotherapy, although HyperFxn remains understudied in a prospective fashion., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The enzymic hydrolysis of acyl-coenzyme A occurs in intact and purified chloroplasts. The different components of spinach chloroplasts were separated after a slight osmotic shock and the purified envelope membranes were shown to be the site of very active acyl-CoA thioesterase activity (EC 3.1.2.2.). The enzyme, which had a pH optimum of 9.0, was not affected by sulfhydryl reagents or by serine esterase inhibitors. However, the acyl-CoA thioesterase was strongly inhibited by unsaturated fatty acids, especially oleic acid, at concentrations above 100 micromolar. In marked contrast, saturated fatty acids had only a slight effect on the thioesterase activity. Substrate specificities showed that the velocity of the reaction increased with the chain length of the substrate from decanoyl-CoA to myristoyl-CoA and then decreased with the chain length from myristoyl-CoA to stearoyl-CoA. Interestingly, oleoyl-CoA was only slowly hydrolyzed. These results suggest that the envelope acyl-CoA thioesterase coupled with an envelope acyl-CoA synthetase may be involved in a switching system which indirectly allows acyl transfer from acyl carrier protein derivatives to unsaturated acyl-CoA derivatives and ensures the predominance of unsaturated 18 carbon fatty acids in plants. Furthermore, the position of both acyl-CoA thioesterase and synthetase in the envelope membranes suggest that these two enzymes may be involved in the transport of oleic acid from the stroma phase to the cytosol compartment of the leaf cell.

The metabolism of (14)C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C(16), C(18:1), C(18:2)) but the kernel contained about 68% C(12) and C(14) fatty acids. Haustorium fatty acids were intermediate between the two. [(14)C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO(2), and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C(16), C(18), and C(18:1) fatty acids but in the kernel about 18% to 32% of the (14)C-fatty acids were C(12) fatty acids.[(14)C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[(14)C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C(16) and C(18)) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[(14)C]laurin.When tri-[(14)C]laurin or [(14)C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[(14)C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

Incubation of stroma preparations from spinach chloroplasts with low concentrations of cerulenin (10 muM) resulted in severe inhibition of fatty acid synthesis but stimulated the release of medium-chain acids in very high proportions (60-70%). Preincubation of these preparations with cerulenin in the absence of substrate exerted no additional effect on subsequent fatty acid synthesis (as measured by incorporation of [14C]acetate into fatty acids) or the pattern of radioactive acids obtained. Acyl-protein, acyl-CoA, free fatty acids and lipids were resolved from each other and analysed for their distribution of 14C-labelled fatty acids. Acyl-protein derived from cerulenin-treated preparations was the only fraction which contained short- and medium-chain acids (C6--C12). The other fractions from both control and cerulenin-treated groups consisted exclusively of C16 and C18 acids. Acyl-protein was purified by gel filtration chromatography and was characterized as acyl-acyl carrier protein.

The fatty-acid composition of C. roseus and N. tabacum cell suspension cultures was unaffected by subculture on Wood and Braun, Murashige and Skoog, or Gamborg B5C media. However, placing the cultures - which were normally grown at 25° C - at 15° C reduced growth but resulted in enhanced formation of oleic and linolenic acids in C. roseus cultures and increased levels of linoleic and linolenic acids in cultures of G. max and N. tabacum, respectively. The incorporation of [(14)C]acetate into [(14)C]linoleic acid was more rapid in N. tabacum cells than in G. max cells, but was very poor in C. roseus where the [(14)C] label was distributed mainly between palmitic and oleic acids.

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. (14)C-Fatty acids synthesized by the extract from [2-(14)C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-(14)C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg(-1) protein·h(-1) occurred at a substrate level of 73 μM malonyl CoA, cofactor levels of 500 μM NADPH, 30 μg·ml(-1) E. coli ACP, and 1.0 mg·ml(-1) extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual (14)C-fatty acids were regulated by concentrations of [(14)C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [(14)C]palmitic acid were rapidly saturated at a low substrate level (0.3 μM malonyl CoA). Increasing the level of [2-(14)C]malonyl CoA permitted further synthesis of [(14)C]stearate and [(14)C]oleate. Desaturation of [(14)C]stearate to [(14)C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.

When individual enzyme activities of the fatty acid synthetase (FAS) system were assayed in extracts from five different plant tissues, acetyl-CoA:acyl carrier protein (ACP) transacylase and beta-ketoacyl-ACP synthetases I and II had consistently low specific activities in comparison with the other enzymes of the system. However, two of these extracts synthesized significant levels of medium chain fatty acids (rather than C16 and C18 acid) from [14C]malonyl-CoA; these extracts had elevated levels of acetyl-CoA:ACP transacylase. To explore the role of the acetyl transacylase more carefully, this enzyme was purified some 180-fold from spinach leaf extracts. Varying concentrations of the transacylase were then added either to spinach leaf extracts or to a completely reconstituted FAS system consisting of highly purified enzymes. The results suggested that: (a) acetyl-CoA:ACP transacylase was the enzyme catalyzing the rate-limiting step in the plant FAS system; (b) increasing concentration of this enzyme markedly increased the levels of the medium chain fatty acids, whereas increase of the other enzymes of the FAS system led to increased levels of stearic acid synthesis; and (c) beta-ketoacyl-ACP synthetase I was not involved in the rate-limiting step. It is suggested that modulation of the activity of acetyl-CoA:ACP transacylase may have important implications in the type of fatty acid synthesized, as well as the amount of fatty acids formed.

Two enzymes involved in oleic acid biosynthesis have been purified from immature safflower seed. The stearoyl-acyl carrier protein (ACP) desaturase which catalyzes the formation of the double bond of oleate has been purified 200-fold and is a dimer with a molecular weight of 68,000. The enzyme shows strong preference for stearoyl-ACP as substrate; by comparison of its activity with stearoyl-CoA and palmitoyl-ACP as substrates, it appears that the ACP moiety is primarily important for binding of substrate and the chain length is important for catalytic activity. The desaturase requires 56 microM oxygen for half-maximal activity, 400 microM oxygen for maximal activity, and is stimulated severalfold by catalase. The acyl-ACP thioesterase has been purified 700-fold and is also a dimer of molecular weight 74,000. It shows a 5-fold preference for oleoyl-ACP versus stearoyl-ACP and is relatively inactive with corresponding acyl-CoAs.

The chloroplast envelope is the site of a very active long-chain acylcoenzyme A (CoA) synthetase. Furthermore, we have recently shown that an acyl CoA thioesterase is also associated with envelope membrane (Joyard J, PK Stumpf 1980 Plant Physiol 65: 1039-1043). To clarify the interacting roles of both the acyl-CoA thioesterase and the acyl-CoA synthetase, the formation of acyl-CoA in envelope membranes was examined with different techniques which permitted the measurement of the actual rates of acyl-CoA formation. Using [(14)C]ATP or [(14)C]oleic acid as labeled substrates, it can be shown that the envelope acyl-CoA synthetase required both Mg(2+) and dithiothreitol. Triton X-100 slightly stimulated the activity. The specificity of the acyl-CoA synthetase was determined either with [(14)C]ATP or with [(3)H]CoA as substrates. The results obtained in both cases were similar, that is, as substrates, the unsaturated fatty acids were more effective than saturated fatty acids, the velocity of the reaction increased from lauric acid to palmitic acid, and the maximum velocity was obtained with unsaturated C(18) fatty acids.The results obtained suggest that the acyl-CoA thioesterase associated with envelope membranes could be an ultimate control to prevent the transport (outside of the chloroplast) or the insertion (into chloroplast lipids) of fatty acids with chains shorter than C(16).

In vitro fatty acid synthesis was examined in crude cell extracts, soluble fractions, and 80% (NH(4))(2)SO(4) fractions from Anabaena variabilis M3. Fatty acid synthesis was absolutely dependent upon acyl carrier protein and required NADPH and NADH. Moreover, fatty acid synthesis and elongation occurred in the cytoplasm of the cell. The major fatty acid products were palmitic acid (16:0) and stearic acid (18:0). Of considerable interest, both stearoyl-acyl carrier protein and stearoyl-coenzyme A desaturases were not detected in any of the fractions from A. variabilis. The similarities and differences in fatty acid synthesis between A. variabilis and higher plant tissues are discussed with respect to the endosymbiotic theory of chloroplast evolution.

The effects of the addition of hypolipidemic drugs and 1-acylglycerolipids on the metabolism of oleate in plants have been studied in vivo and in vitro. Using aged potato slices with [14C]oleate as a precursor, it was found that these drugs markedly inhibited both the incorporation into complex lipids and the desaturation of oleate to linoleate. Moreover, in vitro experiments, carried out with microsomes prepared from developing safflower seeds and [14C]oleate or [14C]oleoyl-CoA as precursors, confirmed the inhibitory effect of the drugs on oleate desaturation, and showed that while WY14643 mainly affected oleoyl thiokinase activity, DH990 exerted its strongest effect on the formation of PL, indicating that the mode of action of these two drugs in safflower microsomes is essentially different. Addition of LPC or LPE stimulated the incorporation of radiolabeled precursor into PC and PE, respectively, as well as the desaturation of oleate to linoleate when [14C]oleoyl-CoA was the precursor. The evidence obtained suggests that oleoyl-PE, as well as oleoyl-PC, should be considered as a possible substrate for oleate desaturation in plants.

Stearyl-acyl carrier protein desaturase (EC 1.14.99.6), present in the stroma fraction of spinach (Spinacia oleracea) chloroplasts, rapidly desaturated enzymatically prepared stearyl-acyl carrier protein to oleic acid. No other substrates were desaturated. In addition to stearyl-acyl carrier protein, reduced ferredoxin was an essential component of the system. The electron donor systems were either ascorbate, dichlorophenolindophenol, photosystem I and light, or NADPH and ferredoxin-NADP reductase. The desaturase was more active in extracts prepared from chloroplasts obtained from immature spinach leaves than from mature leaves. Stearyl-acyl carrier protein desaturase also occurs in soluble extracts of avocado (Persea americana Mill.) mesocarp and of developing safflower (Carthamus tinctorius) seeds.

Acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide ligase (ADP forming), EC 6.4.1.2] is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C(3)- and C(4)-plants. The mesophyll tissues of the C(3)-plants Pisum sativum and Allium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using (125)I-streptavidin as a probe, revealed biotinyl proteins of molecular weights 62,000, 51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.In the C(4)-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

Exogenous [1-(14)C]oleic acid and [1-(14)C]linoleic acid were taken up and esterified to complex lipids by greening cucumber (Cucumis sativus L.) cotyledons. Both (14)C-labeled fatty acids were initially esterified to phosphatidylcholine prior to eventual accumulation in triacylglycerols and galactolipids. Kinetic data suggest that esterification occurs prior to desaturation and that phosphatidylcholine is the initial site of both [(14)C]-oleate and [1-(14)C]linoleate esterification and of [1-(14)C]oleate desaturation to [1-(14)C]linoleate. [1-(14)C]Linoleic acid was esterified more rapidly than [(14)C]oleic acid and its desaturation product, [1-(14)C]alpha-linolenate, occurred mainly on monogalactosyl diacylglycerol, although some was also observed on the other major acyl lipids, including phosphatidylcholine.

Acyl-CoA:O-lysophospholipid acyltransferase activity was measured in extracts of developing safflower (Carthamus tinctorius var. UC-1) seeds. Acyltransferase activity was present in all subcellular fractions. The microsomal acyltransferase activity was solubilized with either 30 mm-n-octylglucoside, 1% deoxycholate, or 100 mum lysophosphatidylcholine. The pH optimum of the lysophosphatidylcholine-dependent acyltransferase activity was at 9.0. Among the various acyl-CoA's tested, oleoyl-CoA was the best substrate and with it the enzyme had a K(m) of 9.5 mum. Among the various acyl acceptors tested, lysophosphatidylcholine (oleoyl) gave the highest activity and the optimal concentration was 30 mum. Acyltransferase activity was stimulated by ethanol and inhibited by bovine serum albumin and divalent cations.

The molecular organization of fatty acid synthetase system in spinach (Spinacia oleracea L. var. Viroflay) leaves was examined by a procedure similar to that employed for the safflower system (Carthamus tinctorius var. UC-1). The crude extract contained all the component activities (acetyl-CoA:ACP transacylase, malonyl-CoA:ACP transacylase, beta-ketoacyl-ACP synthetase, beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase [I]) involved in the synthesis of fatty acids, but enoyl-ACP reductase (II) present in safflower seeds extract could not be detected spectrophotometrically. By polyethylene glycol fractionation followed by several chromatographic procedures, i.e. Sephadex G-200, hydroxyapatite, and blue-agarose, the component enzymes were clearly separated from one another. Properties of beta-ketoacyl-ACP reductase, beta-hydroxyacyl-ACP dehydrase, and enoyl-ACP reductase (I) from spinach were compared with the same enzymes in safflower seeds and Escherichia coli.From these results, it was concluded that the fatty acid synthetase system of spinach leaves, as well as that of safflower seeds, was nonassociated and similar to the Escherichia coli system.

Cell suspension cultures of Catharanthus roseus G. Don, Glycine max (L.) Merr. and Nicotiana tabacum L. were incubated with [(14)C]acetate, [(14)C]oleic acid and [(14)C]linoleic acid at five different temperatures ranging from 15 to 35° C. When the incubation temperature was increased, [(14)C]acetate was incorporated preferentially into [(14)C]palmitate, with a concomitant drop in [(14)C]oleate formation. Between 15 and 20° C, [(14)C]oleic acid accumulated in C. roseus cells. In all cultures, optimum desaturation of [(14)C]oleic acid to [(14)C]linoleic acid occurred between 20 and 25° C, and in G. max this was also the optimal range for desaturation of [(14)C]linoleic acid to [(14)C]linolenic acid. Elongation of [(14)C]palmitic acid was inhibited when cultures grown at 15° C for 25 h were subsequently incubated with [(14)C]acetate at 25° C. [(14)C]oleic acid accumulated in G. max and C. roseus cultures grown at 35° C for 25 h and subsequently incubated at 25° C. Desaturation of [(14)C]oleic acid increased up to 25° C, but then decreased or leveled off depending on the cell line and on the temperature prior to incubation.

beta-Ketoacyl-acyl carrier protein (ACP) synthetase I was purified 180-fold from crude extracts of spinach leaves. The purified preparation was completely free from other component enzymes of the de novo fatty acid synthetase (FAS) system. Its molecular weight was estimated to be 56,000 by gel filtration. The apparent Km value for malonyl-CoA in the presence of ACP and malonyl-CoA:ACP transacylase was 4 microM. Purified synthetase I was highly active with acyl-ACP having chain lengths from C2 to C14, with hexanoyl-ACP being the most effective substrate, but palmitoyl-ACP was far less effective and stearoyl-ACP almost inactive. The antibiotic, cerulenin, strongly inhibited synthetase I activity. The inhibition by cerulenin was protected by prior incubation with hexanoyl-ACP, decanoyl-ACP, and myristoyl-ACP. The synthetase was inhibited by 1 mM p-CMB and 5 mM NEM, but not by 1 mM arsenite.

Greening cucumber (Cucumis sativus L.) cotyledons exhibited dramatic increases in the ability to desaturate exogenously added [1-(14)C]oleic acid and [1-(14)C]linoleic acid within 2 to 3 hours of illumination. These increases were effectively inhibited by 10 micrograms per milliliter cycloheximide. Oleate desaturation remained at a high level in constant light for 5 to 6 days after induction and then declined by about 50%; when returned to the dark, the tissue showed a sharp decrease in conversion of [(14)C]oleate to [(14)C]linoleate. Linoleate desaturation reached a maximum about 15 hours after induction and declined immediately thereafter while the tissue still was in the light; after induction had peaked return of the tissue to the dark showed a dramatic fall of linoleate desaturation. The changes in desaturation were correlated with the conversion of the principal fatty acid in the etiolated cotyledons, linoleate, to alpha-linolenate, and with the assembly of the chlorophyll-containing photosynthetic membranes. The incorporation of [1-(14)C]acetate into lipids showed no significant light stimulation. The role of light in the regulation of certain aspects of plant metabolism during development is discussed.

The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo.

Soybean suspension cultures very rapidly take up C16 and C18 fatty acids by a nonspecific, nonenzymic binding of exogeneously added fatty acids to cell walls and by a subsequent transfer into the cell where they are rapidly incorporated into triacylglycerols, phosphatidylcholines, and phosphatidylethanolamines. 14C-Palmitic and 14C-stearic acids follow this sequence but are not desaturated, wherease 14C-oleic and 14C-linoleic acids are transferred more rapidly than the saturated fatty acids and are then further modified. All the data fit a sequence of events by which free oleic acid is first activated to a CoA thioester, and then desaturated to linoleyl-CoA; both thioesters are then transferred to triacylglycerols, phosphatidylcholine, and phosphatidylethanolamine.

The storage triacylglycerols of nasturtium (Tropaeolum majus) seeds are composed principally of cis-11-eicosenoate and cis-13-docosenoate. To investigate the biosynthesis of these C(20) and C(22) fatty acids, developing seed tissue was incubated with various (14)C-labeled precursors. Incubation with [1-(14)C]acetate produced primarily cis-11-[1-(14)C]eicosenoate and cis-13-[1,3-(14)C]docosenoate in the triacylglycerol fraction, the odd-carbon [U-(14)C]oleate also formed from [(14)C] acetate was in the polar lipid fraction. Kinetic data showed that this oleate was not channeled into cis-11-eicosenoate nor cis-13-docosenoate over a 24-hour period. Under suitable conditions, nasturtium seed could also produce [(14)C]stearate, [(14)C]eicosenoate, and [(14)C]docosenoate from [1-(14)C]acetate. The results are discussed in terms of the number of pathways producing fatty acids. From pool size and other considerations, the results can be rationalized only in terms of different de novo systems for oleate biosythesis, one supplying oleate for incorporation into phospholipids and the other supplying oleate for chain elongation and subsequent esterification into triacylglycerols. Because of the probable heterogeneous nature of the seed tissue, it is not known if these two systems are operating in different cell types, in the same cell type at different stages of development, or in the same cell type concurrently.

A procedure to detect biotinyl proteins after fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was developed. Proteins were immobilized on nitrocellulose and biotin-containing proteins were detected by probing with 125I-streptavidin. Using this procedure a small survey of biotinyl protein in plants was undertaken. In total four biotin-containing proteins were detected in higher plants of molecular weights 62,000, 50,000, 34,000, and 31,000. These biotinyl proteins were not ubiquitous in the plants surveyed. In the cyanobacterium Anabeana variabilis, a single biotin-containing protein of 21,000 Da was detected. In isolated spinach chloroplasts, the two biotinyl proteins detected were soluble. The results are discussed in relation to acetyl-CoA carboxylase.