14C-labeled Acetate Research Articles

Toward Cleavable Metabolic/pH Sensing "Double Agents" Hyperpolarized by NMR Signal Amplification by Reversible Exchange.

We show the simultaneous generation of hyperpolarized 13 C-labeled acetate and 15 N-labeled imidazole following spin-relay of hyperpolarization and hydrolysis of the acetyl moiety on 1-13 C-15 N2 -acetylimidazole. Using SABRE-SHEATH (Signal Amplification by Reversible Exchange in SHield Enables Alignment Transfer to Heteronuclei), transfer of spin order occurs from parahydrogen to acetylimidazole 15 N atoms and the acetyl 13 C site (≈263-fold enhancement), giving rise to relatively long hyperpolarization lifetimes at 0.3 T (T1 ≈52 s and ≈149 s for 13 C and 15 N, respectively). Immediately following polarization transfer, the 13 C-labeled acetyl group is hydrolytically cleaved to produce hyperpolarized 13 C-acetate/acetic acid (≈140-fold enhancement) and 15 N-imidazole (≈180-fold enhancement), the former with a 13 C T1 of ≈14 s at 0.3 T. Straightforward synthetic routes, efficient spin-relay of SABRE hyperpolarization, and facile bond cleavage open a door to the cheap and rapid generation of long-lived hyperpolarized states within a wide range of molecular targets, including biologically relevant carboxylic acid derivatives, for metabolic and pH imaging.

Specificity of exogenous acetate and glutamate as astrocyte substrates examined in acute brain slices from female mice using methionine sulfoximine (MSO) to inhibit glutamine synthesis.

Removal of endogenously released glutamate is mediated primarily by astrocytes and exogenous 13 C-labeled glutamate has been applied to study glutamate metabolism in astrocytes. Likewise, studies have clearly established the relevance of 13 C-labeled acetate as an astrocyte specific metabolic substrate. Recent studies have, however, challenged the arguments used to anchor this astrocyte specificity of acetate and glutamate. The aim of the current study was to evaluate the specificity of acetate and glutamate as astrocyte substrates in brain slices. Acutely isolated hippocampal and cerebral cortical slices from female NMRI mice were incubated in media containing [1,2-13 C]acetate or [U-13 C]glutamate, with or without methionine sulfoximine (MSO) to inhibit glutamine synthetase (GS). Tissue extracts were analyzed by gas chromatography-mass spectrometry. Blocking GS abolished the majority of glutamine 13 C-labeling from [1,2-13 C]acetate as intended. However, 13 C-labeling of GABA was only 40-50% reduced by MSO, suggesting considerable neuronal uptake of acetate. Moreover, labeling of glutamate from [1,2-13 C]acetate in the presence of MSO exceeded the level probable from exclusive labeling of the astrocytic pool, which likewise suggests neuronal acetate metabolism. Approximately 50% of glutamate was uniformly labeled in slices incubated with [U-13 C]glutamate in the presence of MSO, suggesting that neurons exhibit substantial uptake of exogenously provided glutamate. © 2017 Wiley Periodicals, Inc.

Systemic availability and metabolism of colonic-derived short-chain fatty acids in healthy subjects: a stable isotope study.

The short-chain fatty acids (SCFAs) are bacterial metabolites produced during the colonic fermentation of undigested carbohydrates, such as dietary fibre and prebiotics, and can mediate the interaction between the diet, the microbiota and the host. We quantified the fraction of colonic administered SCFAs that could be recovered in the systemic circulation, the fraction that was excreted via the breath and urine, and the fraction that was used as a precursor for glucose, cholesterol and fatty acids. This information is essential for understanding the molecular mechanisms by which SCFAs beneficially affect physiological functions such as glucose and lipid metabolism and immune function. The short-chain fatty acids (SCFAs), acetate, propionate and butyrate, are bacterial metabolites that mediate the interaction between the diet, the microbiota and the host. In the present study, the systemic availability of SCFAs and their incorporation into biologically relevant molecules was quantified. Known amounts of 13 C-labelled acetate, propionate and butyrate were introduced in the colon of 12 healthy subjects using colon delivery capsules and plasma levels of 13 C-SCFAs 13 C-glucose, 13 C-cholesterol and 13 C-fatty acids were measured. The butyrate-producing capacity of the intestinal microbiota was also quantified. Systemic availability of colonic-administered acetate, propionate and butyrate was 36%, 9% and 2%, respectively. Conversion of acetate into butyrate (24%) was the most prevalent interconversion by the colonic microbiota and was not related to the butyrate-producing capacity in the faecal samples. Less than 1% of administered acetate was incorporated into cholesterol and <15% in fatty acids. On average, 6% of colonic propionate was incorporated into glucose. The SCFAs were mainly excreted via the lungs after oxidation to 13 CO2 , whereas less than 0.05% of the SCFAs were excreted into urine. These results will allow future evaluation and quantification of SCFA production from 13 C-labelled fibres in the human colon by measurement of 13 C-labelled SCFA concentrations in blood.

Abstract 1164: Semaphorin 3C promotes de novo steroidogenesis in androgen-deprived prostate cancer cells

Abstract Introduction: This project aims to study the biological role of Semaphorin 3C (SEMA3C) in castration resistant prostate cancer (CRPC) progression. We have found that SEMA3C is a key driver of prostate cancer (PCa) growth through activation of epidermal growth factor receptor (EGFR) and cMet receptor tyrosine kinase (RTK) pathways. RTK signalling activation has been shown to induce de novo steroidogenesis, therefore we hypothesize that SEMA3C-mediated RTK signalling may contribute to CRPC progression through promotion of androgen biosynthesis in androgen-deprived PCa cells. Methods: LNCaP and 22Rv1 cells stably overexpressing SEMA3C were compared to mock transfected cells with respect to their in vitro steroidogenesis, secreted hormone levels were measured by Liquid chromatography-mass spectrometry (LC-MS) technique. Also the expression of steroidogenic enzymes and regulatory proteins were tested by quantitative PCR and western blotting. De novo steroidogenesis was detected by pulsing cells with C14-labeled acetate as a steroid precursor; production of intermediates was measured by scintillation. Results: Steroid analysis showed that SEMA3C overexpression can significantly elevate testosterone and dihydrotestosterone secretion by prostate cancer cells. SEMA3C is also able to upregulate STAR, CYP17A1, HSD17β3, AKR1C3 and SRD5A1 expression. Steroid analysis of SEMA3C-overexpressing LNCaP cells incubated with C14-labeled acetate resulted in the increase of the radioisotope-labeled testosterone and pregnenolone. Conclusions: These findings indicate that SEMA3C can promote steroid biosynthesis in androgen deprived prostate cancer cells, thereby SEMA3C signaling pathway can contribute to maintain active AR signaling in CRPC cells. This research line will be followed to measure intratumoral androgen level by inducing subcutaneously xenografted tumors in athymic nude mice. Tumor homogenates’ steroids will then be extracted and run on LC-MS system. Citation Format: Parvin Yenki, Hans Adomat, Chi Wing Cheng, Christopher Ong. Semaphorin 3C promotes de novo steroidogenesis in androgen-deprived prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1164.

Biosynthetic Study of Amphidinin A and Amphidinolide P.

The biogenetic origins of amphidinin A (1) and amphidinolide P (2) were investigated by feeding experiments with (13)C-labeled acetates. (13)C-NMR data of (13)C-enriched samples revealed that the all carbons of 1 and 2 were derived from acetates. The polyketide chain of 1 was formed from one triketide chain, two diketide chains, and three unusual isolated C1 units derived from C-2 of cleaved acetates, while the polyketide chain of 2 was formed from one pentaketide chain, two acetate units, and three unusual isolated C1 units derived from C-2 of cleaved acetates. The all branched C1 units of 1 and 2 were derived from C-2 of cleaved acetates.

Uncultured Desulfobacteraceae and Crenarchaeotal group C3 incorporate 13C-acetate in coastal marine sediment.

Stable isotope probing (SIP) of deoxyribonucleic acid (DNA) was used to identify microbes incorporating (13) C-labeled acetate in sulfate-reducing sediment from Aarhus Bay, Denmark. Sediment was incubated in medium containing 10 mM sulfate and different (13) C-acetate (10, 1, 0.1 mM) concentrations. The resultant changes in microbial community composition were monitored in total and SIP-fractionated DNA during long-term incubations. Chemical analyses demonstrated metabolic activity in all sediment slurries, with sulfate-reducing activity largely determined by initial acetate concentrations. Sequencing of 16S rRNA gene PCR amplicons showed that the incubations shifted the bacterial but not the archaeal community composition. After 3 months of incubation, only sediment slurries incubated with 10 mM (13) C-acetate showed detectable (13) C-DNA labeling. Based on 16S rRNA and dsrB gene PCR amplicon sequencing, the (13) C-labeled DNA pool was dominated by a single type of sulfate reducer representing a novel genus in the family Desulfobacteraceae. In addition, members of the uncultivated Crenarchaeotal group C3 were enriched in the (13) C-labeled DNA. Our results were reproducible across biological replicate experiments and provide new information about the identities of uncultured acetate-consuming bacteria and archaea in marine sediments.

The ratio of acetate-to-glucose oxidation in astrocytes from a single 13C NMR spectrum of cerebral cortex.

The (13) C-labeling patterns in glutamate and glutamine from brain tissue are quite different after infusion of a mixture of (13) C-enriched glucose and acetate. Two processes contribute to this observation, oxidation of acetate by astrocytes but not neurons, and preferential incorporation of α-ketoglutarate into glutamate in neurons, and incorporation of α-ketoglutarate into glutamine in astrocytes. The acetate:glucose ratio, introduced previously for analysis of a single (13) C NMR spectrum, provides a useful index of acetate and glucose oxidation in the brain tissue. However, quantitation of relative substrate oxidation at the cell compartment level has not been reported. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes, based on the standard assumption that neurons do not oxidize acetate. Mice were infused with [1,2-(13) C]acetate and [1,6-(13) C]glucose, and proton decoupled (13) C NMR spectra of cortex extracts were acquired. A fit of those spectra to the model indicated that (13) C-labeled acetate and glucose contributed approximately equally to acetyl-CoA (0.96) in astrocytes. As this method relies on a single (13) C NMR spectrum, it can be readily applied to multiple physiologic and pathologic conditions. Differences in (13) C labeling of brain glutamate and glutamine have been attributed to metabolic compartmentation. The acetate:glucose ratio, introduced for description of a (13) C NMR (nuclear magnetic resonance) spectrum, is an index of glucose and acetate oxidation in brain tissue. A simple mathematical method is presented to quantify the ratio of acetate-to-glucose oxidation in astrocytes from a single NMR spectrum. As kinetic analysis is not required, the method is readily applicable to analysis of tissue extracts. α-KG=alpha-ketoglutarate; CAC=citric acid cycle; GLN=glutamine; GLU = glutamate.

Identification and activity of acetate-assimilating bacteria in diffuse fluids venting from two deep-sea hydrothermal systems.

Diffuse hydrothermal fluids often contain organic compounds such as hydrocarbons, lipids, and organic acids. Microorganisms consuming these compounds at hydrothermal sites are so far only known from cultivation-dependent studies. To identify potential heterotrophs without prior cultivation, we combined microbial community analysis with short-term incubations using (13)C-labeled acetate at two distinct hydrothermal systems. We followed cell growth and assimilation of (13)C into single cells by nanoSIMS combined with fluorescence in situ hybridization (FISH). In 55 °C-fluids from the Menez Gwen hydrothermal system/Mid-Atlantic Ridge, a novel epsilonproteobacterial group accounted for nearly all assimilation of acetate, representing the first aerobic acetate-consuming member of the Nautiliales. In contrast, Gammaproteobacteria dominated the (13) C-acetate assimilation in incubations of 37 °C-fluids from the back-arc hydrothermal system in the Manus Basin/Papua New Guinea. Here, 16S rRNA gene sequences were mostly related to mesophilic Marinobacter, reflecting the high content of seawater in these fluids. The rapid growth of microorganisms upon acetate addition suggests that acetate consumers in diffuse fluids are copiotrophic opportunists, which quickly exploit their energy sources, whenever available under the spatially and temporally highly fluctuating conditions. Our data provide first insights into the heterotrophic microbial community, catalyzing an under-investigated part of microbial carbon cycling at hydrothermal vents.

Correlation between viral production and carbon mineralization under nitrate-reducing conditions in aquifer sediment.

A variety of microbially mediated metabolic pathways impact biogeochemical cycling in terrestrial subsurface environments. However, the role that viruses have in influencing microbial mortality and microbial community structure is poorly understood. Here we investigated the production of viruses and change in microbial community structure within shallow alluvial aquifer sediment slurries amended with (13)C-labeled acetate and nitrate. Biostimulation resulted in production of viruses concurrent with acetate oxidation, (13)CO2 production and nitrate reduction. Interestingly, change in viral abundance was positively correlated to acetate consumption (r(2)=0.6252, P<0.05) and (13)CO2 production (r(2)=0.6572, P<0.05); whereas change in cell abundance was not correlated to acetate consumption or (13)CO2 production. Viral-mediated cell lysis has implications for microbial community structure. Betaproteobacteria predominated microbial community composition (62% of paired-end reads) upon inoculation but decreased in relative abundance and was negatively correlated to changes in viral abundance (r(2)=0.5036, P<0.05). As members of the Betaproteobacteria decreased, Gammaproteobacteria, specifically Pseudomonas spp., increased in relative abundance (82% of paired-end reads) and was positively correlated with the change in viral abundance (r(2)=0.5368, P<0.05). A nitrate-reducing bacterium, Pseudomonas sp. strain Alda10, was isolated from these sediments and produced viral-like particles with a filamentous morphology that did not result in cell lysis. Together, these results indicate that viruses are linked to carbon biogeochemistry and community structure in terrestrial subsurface sediments. The subsequent cell lysis has the potential to alter available carbon pools in subsurface environments, additionally controlling microbial community structure from the bottom-up.

Non-growing Rhodopseudomonas palustris Increases the Hydrogen Gas Yield from Acetate by Shifting from the Glyoxylate Shunt to the Tricarboxylic Acid Cycle

When starved for nitrogen, non-growing cells of the photosynthetic bacterium Rhodopseudomonas palustris continue to metabolize acetate and produce H2, an important industrial chemical and potential biofuel. The enzyme nitrogenase catalyzes H2 formation. The highest H2 yields are obtained when cells are deprived of N2 and thus use available electrons to synthesize H2 as the exclusive product of nitrogenase. To understand how R. palustris responds metabolically to increase H2 yields when it is starved for N2, and thus not growing, we tracked changes in biomass composition and global transcript levels. In addition to a 3.5-fold higher H2 yield by non-growing cells we also observed an accumulation of polyhydroxybutyrate to over 30% of the dry cell weight. The transcriptome of R. palustris showed down-regulation of biosynthetic processes and up-regulation of nitrogen scavenging mechanisms in response to N2 starvation but gene expression changes did not point to metabolic activities that could generate the reductant necessary to explain the high H2 yield. We therefore tracked (13)C-labeled acetate through central metabolic pathways. We found that non-growing cells shifted their metabolism to use the tricarboxylic acid cycle to metabolize acetate in contrast to growing cells, which used the glyoxylate cycle exclusively. This shift enabled cells to more fully oxidize acetate, providing the necessary reducing power to explain the high H2 yield.

Electrolyte-free milk protein solution influences sodium and fluid retention in rats.

Milk is an effective post-exercise rehydration drink that maintains the net positive fluid balance. However, it is unclear which components are responsible for this effect. We assessed the effect of milk protein solution (MPS) obtained by dialysis on body fluid retention. Milk, MPS, milk electrolyte solution (MES), sports drink and water were administered to male Wistar rats at a dose of 6ml/rat after treadmill exercise. Total body fluid retention was assessed by urine volume 4h after administration of hydrating liquids. The rate of gastric emptying was evaluated by a tracer method using (13)C-labelled acetate. Plasma osmolality, Na and K levels, and urinary Na and K were measured by HPLC and osmometry, respectively. The gastric emptying rate was not delayed by MPS. During 4h of rehydration, cumulative urine volumes differed significantly between treatment groups (P<0·05) with 4·9, 2·2 and 3·4ml from water-, milk- and MPS-fed rats, respectively. Thus, MPS elicited 50% of the total body fluid retention of milk. Plasma aldosterone levels were significantly higher in MPS- and milk-fed rats compared with water-fed rats. Plasma osmolality was maintained at higher levels in MPS-fed rats than in water- and MES-fed rats (P<0·05). Cumulative urine Na excretion was also suppressed in the milk- and MPS-fed groups compared with the MES-fed group. Our results demonstrate that MPS obtained by dialysis clearly affects net body water balance without affecting gastric emptying after exercise. This effect was attributed to retention of Na and water, and maintenance of plasma osmolality.

Methanogenic pathway and community structure in a thermophilic anaerobic digestion process of organic solid waste

The methanogenic pathway and microbial community in a thermophilic anaerobic digestion process of organic solid waste were investigated in a continuous-flow stirred-tank reactor using artificial garbage slurry as a feedstock. The decomposition pathway of acetate, a significant precursor of CH(4) and a key intermediate metabolite in the anaerobic digestion process, was analyzed by using stable isotopes. A tracer experiment using (13)C-labeled acetate revealed that approximately 80% of the acetate was decomposed via a non-aceticlastic oxidative pathway, whereas the remainder was converted to methane via an aceticlastic pathway. Archaeal 16S rRNA analyses demonstrated that the hydrogenotrophic methanogens Methanoculleus spp. accounted for >90% of detected methanogens, and the aceticlastic methanogens Methanosarcina spp. were the minor constituents. The clone library targeting bacterial 16S rRNA indicated the predominance of the novel Thermotogales bacterium (relative abundance: ~53%), which is related to anaerobic acetate oxidizer Thermotoga lettingae TMO, although the sequence similarity was low. Uncultured bacteria that phylogenetically belong to municipal solid waste cluster I were also predominant in the microflora (~30%). These results imply that the microbial community in the thermophilic degrading process of organic solid waste consists exclusively of unidentified bacteria, which efficiently remove acetate through a non-aceticlastic oxidative pathway.

ChemInform Abstract: Biosynthesis of Dynemicin A, a 3-Ene-1,5-diyne Antitumor Antibiotic

Biosynthetic studies of dynemicin A (DNM-A) were carried out by examining the incorporation of singly and doubly 13 C-labeled acetates, [methyl- 13 C]methionine and [ 15 N,2- 13 C]glycine, into DNM-A by cultures of Micromonospora chersina M956-1. The results show that the compound is biosynthesized from two heptaketide chains, which form the bicyclic enediyne core and the anthraquinone moiety, respectively; both are derived from seven head-to-tail coupled acetate units, while the carboxyl group is derived from one carbon of an acetate unit and the O-methyl group from methionine

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.

A Biosynthetic Pathway for BE-7585A, a 2-Thiosugar-Containing Angucycline-Type Natural Product

Sulfur is an essential element found ubiquitously in living systems. However, there exist only a few sulfur-containing sugars in nature and their biosyntheses have not been studied. BE-7585A produced by Amycolatopsis orientalis subsp. vinearia BA-07585 has a 2-thiosugar and is a member of the angucycline class of compounds. We report herein the results of our initial efforts to study the biosynthesis of BE-7585A. Spectroscopic analyses verified the structure of BE-7585A, which is closely related to rhodonocardin A. Feeding experiments using (13)C-labeled acetate were carried out to confirm that the angucycline core is indeed polyketide-derived. The results indicated an unusual manner of angular tetracyclic ring construction, perhaps via a Baeyer-Villiger type rearrangement. Subsequent cloning and sequencing led to the identification of the bex gene cluster spanning approximately 30 kbp. A total of 28 open reading frames, which are likely involved in BE-7585A formation, were identified in the cluster. In view of the presence of a homologue of a thiazole synthase gene (thiG), bexX, in the bex cluster, the mechanism of sulfur incorporation into the 2-thiosugar moiety could resemble that found in thiamin biosynthesis. A glycosyltransferase homologue, BexG2, was heterologously expressed in Escherichia coli. The purified enzyme successfully catalyzed the coupling of 2-thioglucose 6-phosphate and UDP-glucose to produce 2-thiotrehalose 6-phosphate, which is the precursor of the disaccharide unit in BE-7585A. On the basis of these genetic and biochemical experiments, a biosynthetic pathway for BE-7585A can now be proposed. The combined results set the stage for future biochemical studies of 2-thiosugar biosynthesis and BE-7585A assembly.

Identification of iron-reducing microorganisms in anoxic rice paddy soil by 13C-acetate probing

In anoxic rice field soil, ferric iron reduction is one of the most important terminal electron accepting processes, yet little is known about the identity of iron-reducing microorganisms. Here, we identified acetate-metabolizing bacteria by RNA-based stable isotope probing in the presence of iron(III) oxides as electron acceptors. After reduction of endogenous iron(III) for 21 days, isotope probing with (13)C-labeled acetate (2 mM) and added ferric iron oxides (ferrihydrite or goethite) was performed in rice field soil slurries for 48 and 72 h. Ferrihydrite reduction coincided with a strong suppression of methanogenesis (77%). Extracted RNA from each treatment was density resolved by isopycnic centrifugation, and analyzed by terminal restriction fragment length polymorphism, followed by cloning and sequencing of 16S rRNA of bacterial and archaeal populations. In heavy, isotopically labeled RNAs of the ferrihydrite treatment, predominant (13)C-assimilating populations were identified as Geobacter spp. (approximately 85% of all clones). In the goethite treatment, iron(II) formation was not detectable. However, Geobacter spp. (approximately 30%), the delta-proteobacterial Anaeromyxobacter spp. (approximately 30%), and novel beta-Proteobacteria were predominant in heavy rRNA fractions indicating that (13)C-acetate had been assimilated in the presence of goethite, whereas none were detected in the control heavy RNA. For the first time, active acetate-oxidizing iron(III)-reducing bacteria, including novel hitherto unrecognized populations, were identified as a functional guild in anoxic paddy soil.

Biosynthesis of Resorcylic Acid Lactone Lasiodiplodin inLasiodiplodia theobromae

The biosynthesis of lasiodiplodin (1) and its (5S)-5-hydroxylated derivative (2) were investigated by the administration of (13)C-labeled acetates to Lasiodiplodia theobromae. The labeling patterns of biosynthetically (13)C-labeled 1 and 2 were determined by (13)C-NMR and INADEQUATE spectra, demonstrating the octaketide origins of 1 and 2. Taking into account the biosynthetic study of resorcylic acid lactones, the involvement of highly reduced acyl intermediates in the biosynthesis of lasiodiplodins was presumed; thus, we synthesized (2)H-labeled hypothetical acyl intermediates of 1, 9-hydroxydecanoic acid (4) and its N-acetylcysteamine thioester (SNAC, 5). When L. theobromae was incubated with 5 mM of a (2)H-labeled intermediate, the (2)H-label from the intermediate was incorporated at the expected position of 1. These incorporation studies revealed that 1 was produced via a pathway which closely resembles that of resorcylic acid lactone biosynthesis.

Carbon isotope-labelling experiments indicate that ladderane lipids of anammox bacteria are synthesized by a previously undescribed, novel pathway

Ladderane lipids are unusual membrane lipids of bacteria that anaerobically oxidize ammonium to dinitrogen gas (anammox). Ladderane lipids contain linearly concatenated cyclobutane rings for which the pathway of biosynthesis is currently unknown. To investigate the possible biosynthetic routes of these lipids, 2-(13)C-labelled acetate was added to a culture of the anammox bacterium Candidatus Brocadia fulgida. Labelling patterns obtained by high-field (13)C nuclear magnetic resonance spectroscopy of isolated lipids indicated that C. Brocadia fulgida synthesizes C(16:0) and isoC(16:0) fatty acids according to the known pathway of type II fatty acid biosynthesis. The (13)C-labelling pattern of the C(8) alkyl chain of the C(20) [3] ladderane monoether also indicated the use of this route. However, carbon atoms in the cyclobutane rings and the cyclohexane ring were nonspecifically labelled and did not correspond to known patterns of fatty acid synthesis. Taken together, our results indicate that it is unlikely that ladderane lipids are formed from the cyclization of polyunsaturated fatty acids as hypothesized previously and suggest an alternative, although as yet unknown, pathway of biosynthesis.

Bioactive pyridine-N-oxide disulfides from Allium stipitatum.

From Allium stipitatum, three pyridine-N-oxide alkaloids (1-3) possessing disulfide functional groups were isolated. The structures of these natural products were elucidated by spectroscopic means as 2-(methyldithio)pyridine-N-oxide (1), 2-[(methylthiomethyl)dithio]pyridine-N-oxide (2), and 2,2'-dithio-bis-pyridine-N-oxide (3). The proposed structure of 1 was confirmed by synthetic S-methylthiolation of commercial 2-thiopyridine-N-oxide. Compounds 1 and 2 are new natural products, and 3 is reported for the first time from an Allium species. All compounds were evaluated for activity against fast-growing species of Mycobacterium, methicillin-resistant Staphylococcus aureus, and a multidrug-resistant (MDR) variants of S. aureus. Compounds 1 and 2 exhibited minimum inhibitory concentrations (MICs) of 0.5-8 microg/mL against these strains. A small series of analogues of 1 were synthesized in an attempt to optimize antibacterial activity, although the natural product had the most potent in vitro activity. In a whole-cell assay at 30 microg/mL, 1 was shown to give complete inhibition of the incorporation of (14)C-labeled acetate into soluble fatty acids, indicating that it is potentially an inhibitor of fatty acid biosynthesis. In a human cancer cell line antiproliferative assay, 1 and 2 displayed IC(50) values ranging from 0.3 to 1.8 microM with a selectivity index of 2.3 when compared to a human somatic cell line. Compound 1 was evaluated in a microarray analysis that indicated a similar mode of action to menadione and 8-quinolinol by interfering with the thioredoxin system and up-regulating the production of various heat shock proteins. This compound was also assessed in a mouse model for in vivo toxicity.

Use of benzoate as an electron acceptor by Syntrophus aciditrophicus grown in pure culture with crotonate

In methanogenic environments, the main fate of benzoate is its oxidization to acetate, H(2) and CO(2) by syntrophic associations of hydrogen-producing benzoate degraders and hydrogen-using methanogens. Here, we report the use of benzoate as an electron acceptor. Pure cultures of S. aciditrophicus simultaneously degraded crotonate and benzoate when both substrates were present. The growth rate was 0.007 h(-1) with crotonate and benzoate present compared with 0.025 h(-1) with crotonate alone. After 8 days of incubation, 4.12 +/- 0.50 mM of cyclohexane carboxylate and 8.40 +/- 0.61 mM of acetate were formed and 4.0 +/- 0.04 mM of benzoate and 4.8 +/- 0.5 mM of crotonate were consumed. The molar growth yield was 22.7 +/- 2.1 g (dry wt) of cells per mol of crotonate compared with about 14.0 +/- 0.1 g (dry wt) of cells per mol of crotonate when S. aciditrophicus was grown with crotonate alone. Cultures grown with [ring-(13)C]-benzoate and unlabelled crotonate initially formed [ring-(13)C]-labelled cyclohexane carboxylate. No (13)C-labelled acetate was detected. In addition to cyclohexane carboxylate, (13)C-labelled cyclohex-1-ene carboxylate was detected as an intermediate. Once almost all of the benzoate was gone, carbon isotopic analyses showed that cyclohexane carboxylate was formed from both labelled and non-labelled metabolites. Glutarate and pimelate were also detected at this time and carbon isotopic analyses showed that each was made from a mixture labelled and non-labelled metabolites. The increase in molar growth yield with crotonate and benzoate and the formation of [ring-(13)C]-cyclohexane carboxylate from [ring-(13)C]-benzoate in the presence of crotonate are consistent with benzoate serving as an electron acceptor.