Effect of 3-methyl-branching on the myocardial retention of radioiodinated 19-iodo-18-nonadecenoic acid analogues

The effect of methyl-branching at the 3(β-)-position on myocardial uptake and retention of fatty acids where radioiodide has been stabilized as a terminal trans-(E)-vinyl iodide has been evaluated in fasted rats. The syntheses of two new dimethyl-branched fatty acids, 17-iodo-3,3-dimethylheptadecanoic acid (14) and (E)-19-iodo-3,3-dimethyl-18-nonadecenoic acid (19), are described. Tissue distribution studies in fasted rats with [¹²⁵I]-19 showed significant heart uptake (2 min, 4.56% dose/g), and prolonged retention (60 min 4.10% dose-g). These results suggest that [¹²³I]-19 is a good candidate for further studies of regional myocardial fatty acid uptake patterns by SPECT.     

A simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate

The 5-position of tetrahydrofolate was found to be unusually reactive with low concentrations of formic acid in the presence of a water-soluble carbodiimide. The product of this reaction has neutral and acid ultraviolet spectra and chromatographic behavior consistent with its identity as 5-formyltetrahydrofolate (leucovorin). When enzymatically synthesized (6S)-tetrahydrofolate was used as starting material, the product supported the growth of folatedepleted L1210 cells at one-half the concentration required for authentic (6R,S)-leucovorin. This reaction has been used to produce high specific activity (44 Ci/mmol) [³H](6S)-5-formyltetrahydrofolate in high yield. Experiments with [¹⁴C]formic acid indicate that 1 mol of formate reacted per mol of tetrahydrofolate but that no reaction occurred with a variety of other folate compounds. (6S)-5-Formyltetrahydrofolate, labeled in the formyl group with ¹⁴C, has also been synthesized using this reaction. These easily produced, labeled folates should allow close examination of the transport and utillization of leucovorin and of the mechanism of reversal of methotrexate toxicity by reduced folate cofactors.     

Cyclization and hydroxylation stereochemistry in the biosynthesis of gibberellic acid

In Gibberella fujikuroi cultures, ent-[3β-³H,17-¹⁴C]kaurene is converted to gibberellic acid with retention of the tritium label at the 3α-position. This evidence for the stereochemistry of 3-hydroxylation also permits the stereochemistry of the ‘proton-initiated’ cyclization step in gibberellic acid biosynthesis to be deduced.     

7′-hydroxy (−)-R-abscisic acid: A metabolite of feeding (−)-R-abscisic acid to Xanthium strumarium

Feeding of(±)-abscisic acid to leaves of Xanthium strumarium resulted in formation of a new metabolite. The compound was identified as 7′-hydroxy (−)-R-abscisic acid by high resolution mass spectrometry of its methyl ester and monoacetate, and by optical rotary dispersion. The numbering system for abscisic acid has been extended to include the exocyclic methyl groups. Feeding racemic [2-¹⁴C]abscisic acid to Xanthium leaves resulted in ca 20% conversion of the radiolabelled compound into the new metabolite. Evidence is presented that, in Xanthium, only the synthetic (−)-R-enantiomer of abscisic acid is hydroxylated at the 7′-position.     

Hormone selective lipase activation in the isolated rabbit heart

The synthesis and release of PGs by the isolated perfused rabbit heart upon bradykinin stimulation results from lipase stimulation which liberates arachidonic acid for PG biosynthesis. The [¹⁴C]-labelled fatty acids, arachidonate, linoleate, and oleate, when infused into the heart preparation, were efficiently incorporated into the phospholipid pool in the heart, mostly in the 2-position of phosphatidylcholine. On the other hand, [¹⁴C]-palmitate was esterified into both the 1- and the 2-position. Bradykinin released bioassayable PG when injected into the rabbit hearts regardless of which fatty acid label was incorporated into the phospholipid pool. However, only [¹⁴C]-arachidonic acid (but not [¹⁴C]-linoleate, oleate or palmitate) was liberated from the variously labelled hearts upon hormone stimulation. This selective bradykinin effect on fatty acid release suggests that hormone stimulation either activates a specific lipase that distinguishes different fatty acids in the 2-position or activates lipase which is selectively compartmented with arachidonate-containing phospholipids. Ischemia, on the other hand, appeared to non-specifically stimulate tissue lipases, resulting in a non-selective release of oleic as well as arachidonic acid. A disproportionally large release of arachidonic acid was observed accompanying a relatively small PG (10:1 arachidonate: PG ratio) production during ischemia, as compared to bradykinin (3:1 ratio), suggesting distinct mechanisms for PG biosynthesis induced by bradykinin and ischemia.This work was supported by NIH grants: SCOR-HL-17646, HE-14397, HL-20787, and Experimental Pathology training grant (WH) 5 TO1 GM00897-16. Address correspondence to Dr. Philip Needleman, Department of Pharmacology, Washington University Medical School, St. Louis, Missouri 63110.     

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: Difference in specificity between human and sea urchin NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure–activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel “caged” 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca²⁺ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.     

Biosynthesis of nimbolide from [2-14C,(4R)4-3H1] mevalonic acid lactone in the leaves of Azadirachta indica

Nimbolide was biosynthesized from [2-¹⁴C, (4R)4-³H₁]mevalonic acid lactone in the leaves of Azadirachta indica. The nimbolide had a ³H:¹⁴C ratio of 3:5 which gives support to the suggestion of the involvement of a triterpenoid intermediate with a double bond at the Δ⁸⁽⁹)-position in the biosynthesis of nimbolide.     

Docking and molecular dynamics studies on triclosan derivatives binding to FabI

FabI, enoyl-ACP reductase (ENR), is the rate-limiting enzyme in the last step for fatty acids biosynthesis in many bacteria. Triclosan (TCL) is a commercial bactericide, and as a FabI inhibitor, it can depress the substrate (trans-2-enoyl-ACP) binding with FabI to hinder the fatty acid synthesis. The structure-activity relationship between TCL derivatives and FabI protein has already been acknowledged, however, their combination at the molecular level has never been investigated. This paper uses the computer-aided approaches, such as molecular docking, molecular dynamics simulation, and binding free energy calculation based on the molecular mechanics/Poisson-Bolzmann surface area (MM/PBSA) method to illustrate the interaction rules of TCL derivatives with FabI and guide the development of new derivatives. The consistent data of the experiment and corresponding activity demonstrates that electron-withdrawing groups on side chain are better than electron-donating groups. 2-Hydroxyl group on A ring, promoting the formation of hydrogen bond, is vital for bactericidal effect; and the substituents at 4-position of A ring, 2′-position and 4′-position of B ring benefit antibacterial activity due to forming a hydrogen bond or stabilizing the conformation of active pocket residues of receptor. While the substituents at 3′-position and 5′-position of B ring destroy the π-π stacking interaction of A ring and NAD⁺ which depresses the antibacterial activity. This study provides a new sight for designing novel TCL derivatives with superior antibacterial activity.     

Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins

A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of ¹⁴C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the ¹⁴C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the ¹⁴C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the ¹⁴C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.     

Biosynthesis of α-spinasterol from (2- 14C (4R)-4-3H1) mevalonic acid by Spinacea oleracea and Medicago sativa

Leaves of Spinacea oleracea and Medicago sativa were incubated with (2-¹⁴C, (4R)-4³H₁ mevalonic acid and the sterols isolated. Cycloartenol had a ³H: ¹⁴C atomic ratio of 6:6 whilst oxidation to cycloartenone resulted in a ratio of 5:6 showing that tritium was present in the 3α-position and that the cycloartenol was symmetrically labelled. Separation of the 4-demethyl sterols gave α-spinasterol and a mixture of stigmast-7-enol and 24-methylcholest-7-enol, which had ³H: ¹⁴C atomic ratios of 3:5. Ozonolysis of α-spinastery] acetate gave the terminal side chain fragment as 2-ethyl-3-methyl butanoic acid. The acid contained ¹⁴C but no tritium thus showing that the C-24 hydrogen of cycloartenol is lost during the alkylation reactions leading to the C-24 ethyl group of α-spinasterol.     

Liquid chromatography of pyridine nucleotides and associated compounds and isolation of several analogs of nicotinamide adenine dinucleotide phosphate.

A single-column technique is described for separating the naturally occurring nicotinic acid- and nicotinamide-containing nucleosides and nucleotides. A unique feature of the system is the use of conductivity measurements as a reproducible means of peak identification. The chromatographic system has been used to isolate a number of NADP⁺ analogs and to characterize their enzymatic hydrolysis products. These analogs include the α-anomer of NADP⁺, the nicotinic acid analog of NADP⁺, and a series of NADP⁺ derivatives that contain phosphate in the 3′-position.     

Synthesis of the 15N-Gln11 labeled peptaibol antibiotic zervamicin-IIB

Summary Synthesis of zervamicin IIB, specifically labeled at the α-position of glutamine-11 with¹⁵N, was achieved by the Fmoc/tert.-butyl strategy in solution using a fragment condensation approach. Three fragments of zervamicin IIB were obtained by stepwise elongation with Fmoc amino acids using BOP as a coupling reagent. For the introduction of the highly sterically hindered α-aminoisobutyric acid residues, BOP/DMAP activation was applied. Peptide fragments were coupled by means of the coupling reagent, CF₃-PyBOP. Using the strategy developed, zervamicin IIB specifically¹⁵N labeled has been synthesized in 30% overall yield based on the isotopically labeled amino acid. From 600 MHz NMR spectroscopy the position of the¹⁵N-label was clearly detected. The isotope enrichment (98 ±2%) was determined by FAB-mass spectrometry.     

Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. : III. Conversion of 4-Methoxycinnamic Acids into 4-Hydroxybenzoic Acids

Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamic acid and 3,4,5-trimethoxycinnamic acid to cell suspension cultures of Vanilla planifolia resulted in the formation of 4-hydroxybenzoic acid, vanillic acid, and syringic acid, respectively. The homologous 4-methoxybenzoic acids were demethylated to the same products. It is concluded that the side chain degrading enzyme system accepts the 4-methoxylated substrates while the demethylation occurs at the benzoic acid level. The demethylating enzyme is specific for the 4-position. Feeding of [O-¹⁴C-methyl]-3,4-dimethoxycinnamic acid revealed that the first step in the conversion is the glycosylation of the cinnamic acid to its glucose ester. A partial purification of a UDP-glucose: trans-cinnamic acid glucosyltransferase is reported. 4-Methoxy substituted cinnamic acids are better substrates for this enzyme than 4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxy substituted cinnamic acids are intermediates in the biosynthetic conversion of cinnamic acids to benzoic acids in cells of V. planifolia.     

Incorporation of 14C-Formaldehyde into Hexose Phosphate by Cell-free Extract of a Methanolutilizing Yeast,Candida sp.

The persistence and degradation of isoxathion ¹⁴C-labeled at the 5-position of the isoxazole ring were studied in three soil types under laboratory conditions. Persistence was influenced by soil type and moisture content; approx. half life at 30 ppmw dose level varied from 15 to 40 days in nonflooded models. In a flooded model isoxathion disappeared much faster. Isoxathion underwent biodegradation to a number of products with concomitant release of ¹⁴CO₂. 3-Hydroxy-5-phenylisoxazole, 5-phenyl-4-oxazolin-2-one, benzoylacetamide and benzoic acid were the unequivocally identified metabolites; oxon derivative of isoxathion, 3-methoxy-5-phenylisoxazole, 2-methyl-5-phenyl-4-isoxazolin-3-one, 2-acetyl-5-phenyl-4-isoxazolin-3-one, 2, 5-diphenylpyrazine and acetophenone were tentatively identified as the minor products. None of these major products was persistent in soils. 3-Hydroxy-5-phenylisoxazole, the initial metabolite or hydrolyzate of isoxathion, was adsorbed to soil to a much greater extent than isoxathion, which explains the rapid disappearance of its fungicidal activity in soil.     

Spin label saturation transfer ESR studies of protein-lipid interactions in Photosystem II-enriched membranes

Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 10⁵ range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.     

Studies on plant growth-regulating substances

Nitrophenols structurally similar to 4-hydroxy-3-nitrobenzoic acid and 4-hydroxy-3-nitrobenzaldehyde have been examined for their ability to inhibit chloroplast development in linseed and oat seedlings, and their activity has been estimated quantitatively in specifically designed clover and Lemna bioassays. Twelve compounds were found to be active and activity is considered in relation to chemical structure. A nitro group in the 3-position and a hydroxyl group or ether linkage in the 4-position were found to be essential for activity whereas the nature of the functional group in the 1-position could vary considerably. Possibilities in which activity might arise from metabolism of the applied compound were investigated using excised wheat and pea tissue, and compounds with various groupings in the 1-position were shown to be converted to the corresponding active benzoic acid derivative.     

The lipid A from Vibrio fischeri lipopolysaccharide: a unique structure bearing a phosphoglycerol moiety

Vibrio fischeri, a bioluminescent marine bacterium, exists in an exclusive symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, whose light organ it colonizes. Previously, it has been shown that the lipopolysaccharide (LPS) or free lipid A of V. fischeri can trigger morphological changes in the juvenile squid's light organ that occur upon colonization. To investigate the structural features that might be responsible for this phenomenon, the lipid A from V. fischeri ES114 LPS was isolated and characterized by multistage mass spectrometry (MS(n)). A microheterogeneous mixture of mono- and diphosphorylated diglucosamine disaccharides was observed with variable states of acylation ranging from tetra- to octaacylated forms. All lipid A species, however, contained a set of conserved primary acyl chains consisting of an N-linked C14:0(3-OH) at the 2-position, an unusual N-linked C14:1(3-OH) at the 2'-position, and two O-linked C12:0(3-OH) fatty acids at the 3- and 3'-positions. The fatty acids found in secondary acylation were considerably more variable, with either a C12:0 or C16:1 at the 2-position, C14:0 or C14:0(3-OH) at the 2'-position, and C12:0 or no substituent at the 3'-position. Most surprising was the presence of an unusual set of modifications at the secondary acylation site of the 3-position consisting of phosphoglycerol (GroP), lysophosphatidic acid (GroP bearing C12:0, C16:0, or C16:1), or phosphatidic acid (GroP bearing either C16:0 + C12:0 or C16:0 + C16:1). Given their unusual nature, it is possible that these features of the V. fischeri lipid A may underlie the ability of E. scolopes to recognize its symbiotic partner.     

Studies on the biosynthesis of N-(γ-L-glutamyl)-4-hydroxyaniline] in Agaricus bisporus: Identification of the position in shikimic acid at which the amination occurs

Labelled shikimic acid was efficiently incorporated into the aniline moiety of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$, a characteristic aromatic compound of the common mushroom, Agaricus bisporus. Incubations with [3-³H]- and [1,6-¹⁴C]shikimic acid clearly proved that the amination of shikimic acid occurs at its 4-position during the biosynthesis of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

The temperature-mediated metabolism of 1-acyl-lysophosphatidyl glycerol in cerulenin-treated cultures of Bacillus megaterium.

When [U-¹⁴C]palmitate was added to a culture of $\text{B. megaterium}$ that had been grown at 35°, transferred to 20° and treated with cerulenin, label was initially incorporated into lysophosphatidyl glycerol. The labeled lyso derivative, in turn, was converted to phosphatidyl glycerol, apparently by esterification of the 2-position with endogenous acyl groups. Labeled lysophosphatidyl glycerol synthesis at 20° was observed only when a culture was treated with cerulenin prior to the addition of [U-¹⁴C]palmitate. When [U-¹⁴C]palmitate was added before cerulenin, labeled lysophosphatidyl glycerol formation was not detected. When chloramphenicol was added with cerulenin at the time of culture transfer from 35° to 20°, the synthesis of lysophosphatidyl glycerol was unaffected but the rate of its esterification to phosphatidyl glycerol was significantly retarded. Transfer of such a culture back to 35° resulted in a marked acceleration in the rate of conversion of lysophosphatidyl glycerol to phosphatidyl glycerol.     

Biogenesis of nonacosan-15-one in brassica oleracea

The incorporation of [4-¹⁴C]4-oxostearic acid, [4-¹⁴C]stearic acid, and [1-¹⁴C]eicosanoic acid, respectively, into the nonacosan-15-one of Brassica oleracea has been investigated. While the oxo-acid was found to be a poor precursor of nonacosan-15-one, the two n-fatty acids were efficiently incorporated as intact units into n-nonacosane and the related 15-ketone. In accordance with expectation, the label in the 4-position of stearic acid appeared in the carbonyl carbon of the ketone. These results suggest that the C₂₉ hydrocarbon is first formed and subsequently oxidized to nonacosan-15-one.