Incorporation Of Precursors Research Articles

Studies on paraherquamide biosynthesis: synthesis of deuterium-labeled 7-hydroxy-pre-paraherquamide, a putative precursor of paraherquamides A, E, and F

The stereocontrolled, asymmetric synthesis of triply deuterium-labeled 7-hydroxy-pre-paraherquamide ( 27) was accomplished, employing a diastereoselective intramolecular S N2′ cyclization strategy. The deuterium-labeled substrate was interrogated in a precursor incorporation experiment in the paraherquamide-producing organism Penicillium fellutanum. The isolated sample of paraherquamide A revealed incorporation of one of the two geminal deuterons of the CD 2-group at C-12 exclusively. The lack of signals for the second deuteron of the CD 2-group at C-12 and for the CH 2D-group (C-22/C-23) suggests that this substrate suffered an unexpectedly selective catabolic degradation and metabolic re-incorporation of deuterium thus casting doubt on the proposed biosynthetic intermediacy of 27. Consideration of alternative biosynthetic pathways, including oxidation of the indole C-6 position prior to hydroxylation at C-7 or oxidative spiro-contraction of pre-paraherquamide prior to construction of the dioxepin is discussed. The synthesis of 27 also provides for a concise, asymmetric stereocontrolled synthesis of an advanced intermediate that will be potentially useful in the synthesis of paraherquamides E and F.

Durable and rechargeable biocidal polypropylene polymers and fibers prepared by using reactive extrusion

Incorporation of N-halamine precursor onto polypropylene was explored by using a reactive extrusion process. Several cyclic and acyclic halamine precursors were grafted onto polypropylene backbone through a melt free radical graft copolymerization. The structures and morphology of the grafted polymer were characterized with FTIR, and scanning electron microscope. Thermal properties of the polymers were evaluated by differential scanning calorimetry. The halogenated products of the corresponding grafted samples exhibited potent antimicrobial properties against Escherichia coli, and the antimicrobial properties were durable and regenerable. The relationship between effective surface contact and biocidal efficacy are further discussed.

Biosynthetic studies of Nocathiacin-I

Nocathiacin-I is one of the newest members of thiazolyl peptide class of antibiotics. It is a potent inhibitor of bacterial protein synthesis and showed potent in vitro and in vivo Gram-positive antibacterial activity. Understanding of the biosynthesis of natural products is important for improvement of titer and precursor directed biosynthesis for new compounds. Biosynthesis of nocathiacin-I in Amycolatopsis fastidiosa using stable isotope precursor incorporation is described.

Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus

The proposed lethal action of daptomycin on Staphylococcus aureus results from the loss of K(+) and membrane depolarization. However, whether these events alone cause cell death has been questioned. We sought to determine whether other consequences of daptomycin-mediated membrane damage may contribute to cell death. Previously established assays were used to evaluate the membrane damaging activity of daptomycin at a single time-point of 10 min. More detailed time-course experiments were also performed to determine the kinetics of membrane depolarization and leakage of K(+), Mg(2+) and ATP. The kinetics of inhibition of macromolecular synthesis following exposure to daptomycin were also determined by assaying the incorporation of radioactive precursors into macromolecules. Daptomycin exhibited no membrane damaging activity in single time-point assays following exposure to the antibiotic for 10 min. Kinetic analysis confirmed these results as leakage of intracellular components did not occur until 20-30 min, membrane depolarization was gradual and cells remained biosynthetically active for at least 30 min after exposure to daptomycin. Viability declined rapidly after exposure to daptomycin and appeared to precede other detectable changes. These data show that daptomycin-induced loss of Mg(2+) and ATP occurs in conjunction with the previously reported leakage of K(+) and membrane depolarization. We propose that the lethal activity of daptomycin is not simply due to loss of K(+) and probably involves more general damage to the membrane.

Antibacterial activity of the secondary metabolites from in vitro cultures of Drosera aliciae

Preparations from carnivorous plants belonging to genus Drosera (Droseraceae) were shown to possess antimicrobial properties [1]. The objective of this research was to evaluate antibacterial properties of secondary metabolites obtained from Drosera aliciae Raym.-Hamet plants grown in vitro and to examine mechanism of their antimicrobial action. Bactericidal activity of chloroform and methanol extracts from D. aliciae as well as pure ramentaceone as the major naphthoquinone present in this plant were examined against resistant and susceptible to antibiotics strains from the following species of human bacterial pathogens (hospital isolates): Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Chloroform extract proved to be more effective than methanol preparation against all of the tested strains except for P. aeruginosa isolates with the lowest minimal bactericidal concentration (MBC) values in case of S. aureus (25–50mg FW ml-1). Methanol extract from D. aliciae caused 3 and 8-fold increase (for 0.1 and 0.25 MBC respectively) in generation time of E. faecalis in relation to control, chloroform preparation elevated generation time 1.2 and 2-fold, while both tested concentrations of ramentaceone caused no statistically significant growth retardation. The influence of D. aliciae extracts and ramentaceone on the synthesis of DNA, RNA and proteins in cultures of E. faecalis was estimated by measurement of incorporation of radioactive precursors: [3H]thymidine, [3H]uridine or [3H]leucine, respectively. Methanol extract from D. aliciae except for a moderate effect on DNA synthesis had no influence on neither RNA nor protein synthesis. Chloroform preparation caused about 75% decrease in [3H]uridine incorporation into RNA in comparison to control after 60min and a significant reduction of DNA and protein synthesis (44% and 30% respectively). Ramentaceone also reduced DNA and RNA synthesis but less efficiently than chloroform extract and caused no changes in protein synthesis.

Characterization of carbon nitride deposition from CH4∕N2 glow discharge plasma beams using optical emission spectroscopy

The properties of plasmas in a CH4∕N2 dc abnormal glow discharge with the percentage of methane from 1% to 20% have been studied in order to understand the effect of precursor incorporation into the carbon nitride (CNx) films. The appearance of CN radicals as well as C2, CH, and NH has been revealed by optical emission spectroscopy (OES). The evolution of CN, N2, N2+, and C2 emission lines from mixed CH4∕N2 and pure N2 plasma on changing mixture ratio and polarity of discharge-field has been studied. The possible mechanisms behind their variations have been discussed. Besides, a CH4∕N2 ratio of 1∕50 and a top-needle anode is considered to be the best conditions for synthesis of β-C3N4, which has been confirmed in the as-deposited carbon nitride films with quite good crystalline features by XRD analyses.

Differential control of mesangial cell proliferation by interferon-gamma

Rat mesangial cells were shown to be sensitive to recombinant interferon-gamma (IFN-gamma). IFN-gamma reduced thymidine uptake by these cells and inhibited cell proliferation. Incubation of the cells with 1000 U/ml IFN-gamma decreased thymidine uptake by up to 64% and cell numbers were decreased by 17%. The effects of IFN-gamma were dose and time dependent and were partially reversible by the anti-IFN-gamma monoclonal antibody DB-1. This lymphokine did not reduce incorporation of RNA and protein precursors however. Measurements of 3H-uridine and 3H-leucine incorporation indicated significant increases in RNA and protein synthesis (37% and 45%, respectively) on a per cell basis. The mitogenic effects of IL-1 and platelet-derived growth factor (PDGF) were also susceptible to IFN-gamma-mediated inhibition but the mitogenic response to epidermal growth factor (EGF) was much less sensitive. We conclude that while IFN-gamma may act to modulate the mitogenic signals provided by some factors such as IL-1 and PDGF, the response to EGF appears to be unaffected.

Molecular structure of organic components in cephalopods: Evidence for oxidative cross linking in fossil marine invertebrates

Analysis of the jaws of the Humboldt squid ( Dosidicus gigas) and Nautilus belauensis using mass spectrometric techniques revealed a chitin–protein composition, confirmed by analysis of chitin and amino acid model compounds. Analysis of comparable fossil material from several cephalopod taxa ( Placenticeras, Nanaimoteuthis jeletzkyi, Anagaudryceras limatum and Hoploscaphites) from Upper Cretaceous (Campanian and Maastrichtian) localities from North America and Japan revealed no compounds from the chitin–protein biopolymer complex but showed the presence of aromatic compounds, including alkyl benzene derivatives, alkyl phenols, naphthalenes, and phenanthrene. An n-alkyl component, ranging in chain length up to C 24, was also detected . There was no evidence of the presence of these compounds in the host sediment. Cleavage of ester bonds in the fossil macromolecules (including fossils with both predominantly aromatic and aliphatic compounds) using thermally assisted methylation (THM) revealed a carboxylic acid distribution from C 6 to C 18. Additionally, methyl ketones were detected in the fossils, in which the n-alkyl (aliphatic) component was significant. Methyl ketones (alkan-2-ones) have been reported in fossil algae and kerogen and are indicative of ether functional groups in macromolecules, which are probably crosslinked via oxidative reticulation. Thus, the aliphatic component is likely derived from in situ incorporation and polymerisation of labile lipid precursors, in part crosslinked via ether and ester bonds. This is the first demonstration of ether linkages and of oxidative crosslinking in animal fossils, suggesting that such a process may be important in the long term preservation of both animal- and plant-derived organic matter.

Uptake and kinetic properties of choline and ethanolamine in <i>Plasmodium falciparum</i>

Purpose: The asexual proliferation of Plasmodium, inside the erythrocyte, is accompanied by the synthesis of huge quantities of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). These needful phospholipids for the cytoplasmic membrane of the merozoites are provided by the precursors choline and ethanolamine. PtdCho and PtdEtn are synthesized by the parasite because the erythrocyte is unable to do it. In order to assess the dynamism of the phospholipid pathways, we aimed to investigate the respective shape of the uptake of choline and ethanolamine by Plasmodium falciparum. Method: Time-course experiments and kinetic assays were performed respectively with fixed and ranged concentrations of radioactively-labelled choline and ethanolamine. The labelled-precursors were added in the culture of P. falciparum infected-erythrocytes and the incorporated molecules in phospholipids were measured with a scintigraph counter. Result: The results showed that the incorporation of precursors in the infected-erythrocyte occurred with a Michaelis-Menten\'s kinetic shape. According to the maximum rate (Vmax), the pathway of ethanolamine incorporation was faster than that of choline. Similarly, affinity for ethanolamine was greater than that of choline. Conclusion: Although PtdCho is the major phospholipid in the membrane, this study rules out that the influx of ethanolamine in the infected-erythrocyte, in vivo conditions, is more dynamic than choline. Keywords: Plasmodium, phospholipids, kinetics, metabolism, choline, ethanolamine Tropical Journal of Pharmaceutical Research Vol. 7 (2) 2008: pp. 953-959

Procarbazine – A Traditional Drug in the Treatment of Malignant Gliomas

The methylhydrazine derivative Procarbazine (PCZ) as monotherapy or in combination with CCNU and vincristine (PCV) was evaluated in a vast number of clinical trials and is still used in patients with high-grade and low-grade gliomas. The compound is an antineoplastic agent with multiple sites of action. It inhibits incorporation of small DNA precursors, as well as RNA and protein synthesis. PCZ can also directly damage DNA through an alkylation reaction. The drug is not cross-resistant with other mustard-type alkylating agents. As PCZ was in almost all trials used in a combination with CCNU and Vincristin, the efficacy can only be evaluated in the view of the PCV regimen. The published data suggest a role of PCV as a salvage regimen, especially in oligodendroglial tumors; however, well designed studies with high evidence are rare in all entities. This article summarizes the existing data with the goal to define the role of PCZ/PCV in modern neurooncology.

Investigations of valanimycin biosynthesis: Elucidation of the role of seryl-tRNA

The antibiotic valanimycin is a naturally occurring azoxy compound produced by Streptomyces viridifaciens MG456-hF10. Precursor incorporation experiments showed that valanimycin is derived from l-valine and l-serine via the intermediacy of isobutylamine and isobutylhydroxylamine. Enzymatic and genetic investigations led to the cloning and sequencing of the valanimycin biosynthetic gene cluster, which was found to contain 14 genes. A novel feature of the valanimycin biosynthetic gene cluster is the presence of a gene (vlmL) that encodes a class II seryl-tRNA synthetase. Previous studies suggested that the role of this enzyme is to provide seryl-tRNA for the valanimycin biosynthetic pathway. Here, we report the results of investigations to elucidate the role of seryl-tRNA in valanimycin biosynthesis. A combination of enzymatic and chemical studies has revealed that the VlmA protein encoded by the valanimycin biosynthetic gene cluster catalyzes the transfer of the seryl residue from seryl-tRNA to the hydroxyl group of isobutylhydroxylamine to produce the ester O-seryl-isobutylhydroxylamine. These findings provide an example of the involvement of an aminoacyl-tRNA in an antibiotic biosynthetic pathway.

Articular chondrocytes derived from distinct tissue zones differentially respond to in vitro oscillatory tensile loading

The cell morphology, gene expression, and matrix synthesis of articular chondrocytes are known to vary with depth from the tissue surface. The objective of this study was to investigate if chondrocytes from different zones respond to in vitro oscillatory tensile loading in distinct ways and whether tensile strain, which is most prevalent near the articular surface, would preferentially stimulate superficial zone chondrocytes. Chondrocytes were separately isolated from the superficial, middle, and deep zones of articular cartilage and seeded into three-dimensional fibrin hydrogel constructs. An intermittent protocol of oscillatory tensile loading was applied for 3 days, and the effects on extracellular matrix (ECM) synthesis were assessed by measuring the incorporation of radiolabed precursors, size exclusion gel chromatography, and western blotting. Tensile loading was found to be a potent stimulus for proteoglycan synthesis only in superficial zone chondrocytes. Although overall biosynthesis rates by deep zone chondrocytes were unaffected by tensile loading, the molecular characteristics of proteins and proteoglycans released to the culture medium were significantly altered so as to resemble those of superficial zone chondrocytes. Oscillatory tensile loading differentially affected subpopulations of articular chondrocytes in three-dimensional fibrin hydrogel constructs. Cells isolated from deeper regions of the tissue developed some characteristics of superficial zone chondrocytes after exposure to tensile loading, which may indicate an adaptive response to the new mechanical environment. Understanding how exogenous mechanical stimuli can differentially influence chondrocytes from distinct tissue zones will yield important insights into mechanobiological processes involved in cartilage tissue development, maintenance, disease, and repair.

10.1007/s11827-008-1008-5

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-14C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-14C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-14C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.

Following Fungal Melanin Biosynthesis with Solid-State NMR: Biopolymer Molecular Structures and Possible Connections to Cell-Wall Polysaccharides

Melanins serve a variety of protective functions in plants and animals, but in fungi such as Cryptococcus neoformans they are also associated with virulence. A recently developed solid-state nuclear magnetic resonance (NMR) strategy, based on the incorporation of site-specific (13)C-enriched precursors into melanin, followed by spectroscopy of both powdered and solvent-swelled melanin ghosts, was used to provide new molecular-level insights into fungal melanin biosynthesis. The side chain of an l-dopa precursor was shown to cyclize and form a proposed indole structure in C. neoformans melanin, and modification of the aromatic rings revealed possible patterns of polymer chain elongation and cross-linking within the biopolymer. Mannose supplied in the growth medium was retained as a beta-pyranose moiety in the melanin ghosts even after exhaustive degradative and dialysis treatments, suggesting the possibility of tight binding or covalent incorporation of the pigment into the polysaccharide fungal cell walls. In contrast, glucose was scrambled metabolically and incorporated into both polysaccharide cell walls and aliphatic chains present in the melanin ghosts, consistent with metabolic use as a cellular nutrient as well as covalent attachment to the pigment. The prominent aliphatic groups reported previously in several fungal melanins were identified as triglyceride structures that may have one or more sites of chain unsaturation. These results establish that fungal melanin contains chemical components derived from sources other than l-dopa polymerization and suggest that covalent linkages between l-dopa-derived products and polysaccharide components may serve to attach this pigment to cell wall structures.

Vascular thickness and cytostatic-resistant bronchial carcinoma

A new way of treatment of malignant tumours was suggested by blocking angiogenesis and thus creating subpopulations of cells which, due to their decreased oxygen content, react differently to treatment. The relationship between angiogenesis (vessel density) and resistance to cytostatic drugs was investigated. Results were analysed retrospectively for 83 untreated patients (73 men, 10 women; mean age 60 [37-75] years) with non-small cell bronchial carcinoma. Vessel density and resistance proteins were determined immunohistochemically. Radioactive incorporation of nucleic acid precursors with and without doxorubicin was measured with the in vitro short-term test. There were significant differences between vessel density and various resistance proteins. With low vessel density (less than mean value) glutathione-S-transferase pi (P < 0.01), thymidylate synthase (P < 0.05) and metallothioneine (P < 0.05) were significantly increased. P-glycoprotein showed a corresponding relationship, but it was not statistically significant, while there was no relationship with topoisomerase II. Bronchial carcinoma with low vessel density was more frequently resistant in vitro to doxorubicin than carcinomas with higher vessel density (P < 0.05). Non-small cell bronchial carcinoma with low vessel density more frequently expresses resistance proteins and is more commonly resistant to doxorubicin. Blocking angiogenesis to improve the results of treatment is therefore problematic.

Proteomic Analysis of a Nutritional Shift-up in Saccharomyces cerevisiae Identifies Gvp36 as a BAR-containing Protein Involved in Vesicular Traffic and Nutritional Adaptation

Yeast cells undergoing a nutritional shift-up from a poor to a rich carbon source take several hours to adapt to the novel, richer carbon source. The budding index is a physiologically relevant "global" parameter that reflects the complex links between cell growth and division that are both coordinately and deeply affected by nutritional conditions. We used changes in budding index as a guide to choose appropriate, relevant time points during an ethanol to glucose nutritional shift-up for preparation of samples for the analysis of proteome by two-dimensional electrophoresis/mass spectrometry. About 600 spots were detected. 90 spots, mostly comprising proteins involved in intermediary metabolism, protein synthesis, and response to stress, showed differential expression after glucose addition. Among modulated proteins we identified a protein of previously unknown function, Gvp36, showing a transitory increase corresponding to the drop of the fraction of budded cells. A gvp36Delta strain shares several phenotypes (including general growth defects, heat shock, and high salt sensitivity, defects in polarization of the actin cytoskeleton, in endocytosis and in vacuolar biogenesis, defects in entering stationary phase upon nutrient starvation) with secretory pathway mutants and with mutants in genes encoding the two previously known yeast BAR proteins (RSV161 and RSV167). We thus propose that Gvp36 represents a novel yeast BAR protein involved in vesicular traffic and in nutritional adaptation.

Ultrastructural Studies Of Oogenesis and Sexual Maturation In Female Chlamys (azumapecten) farreri farreri (Jones &amp; Preston, 1904) (Pteriomorphia: Pectinidae) On The Western Coast Of KoreA

Ultastructural studies of the development and degeneration of the oocytes and follicle cells in female Chlamys (Azumapecten) farreri farreri (Jones & Preston, 1904) are described for scallops collected from Daehuksando, Jeollanam-do, Korea. Vitellogenesis occurred by way of endogenous autosynthesis and exogenous heterosynthesis. Auto-synthesis involved the combined activity of the Golgi complex, mitochondria, and rough endoplasmic reticulum, whereas heterosynthesis involved endocytotic incorporation of extraovarian precursors at the basal region of the early vitellogenic oocytes prior to the formation of the vitelline coat. Auxiliary cells were involved in the development of the previtellogenic and early vitellogenic oocytes and appear to play an integral role in vitellogenesis and oocyte degeneration by assimilating products originating from the degenerated oocytes, thus allowed the transfer of yolk precursors needed for vitellogenesis. Auxiliary cells presumably have a lysosomal system for breakdown products of oocyte degeneration. The reproductive cycle in females was classified into five stages: Stage I: early active stage (January to March), Stage II: late active stage (March to April), Stage III: ripe stage (April to August), Stage IV: partially spawned stage (June to August), and Stage V: spent/inactive stage (August to January). The spawning period continued from June to August, with a peak between July and August when the seawater temperature was exceeded 22°C. The percentage of first sexual maturity was 59.3% in individuals of 50.1–60.0 mm in shell height, and 100% in those > 70.1 mm in shell height.Because harvesting clams less than 50.1 mm in shell height could potentially cause a drastic reduction in recruitment, a measure indicating a prohibitory fishing size should be enacted for adequate fisheries management.

The effect of PPAR ligands to modulate glucose metabolism alters the incorporation of metabolic precursors into proteoglycans synthesized by human vascular smooth muscle cells

PPAR ligands are important effectors of energy metabolism and can modify proteoglycan synthesis by vascular smooth muscle cells (VSMCs). Describing the cell biology of these important clinical agents is important for understanding their full clinical potential, including toxicity. Troglitazone (10 μM) and fenofibrate (30 μM) treatment of VSMCs reduces (35S)-sulphate incorporation into proteoglycans due to a reduction of glycosaminoglycan (GAG) chain length. Conversely, under physiological glucose conditions (5.5 mM), the same treatment increases (3H)-glucosamine incorporation into GAGs. This apparent paradox is the consequence of an increase in the intracellular (3H)-galactosamine specific activity from 48.2 ± 3.2 μCi/ μmol to 90.7 ± 11.0 μCi/ μmol (P < 0.001) and 57.1 ± 2.6 μCi/ μmol (P < 0.05) when VSMCs were treated with troglitazone and fenofibrate, respectively. The increased specific activity observed with troglitazone (10 μM) treatment correlates with a two-fold increase in glucose consumption, while fenofibrate (50 μM) treatment showed a modest (14.6%) increase in glucose consumption. We conclude that the sole use of glucosamine precursors to assess GAG biosynthesis results in misleading conclusions when assessing the effect of PPAR ligands on VSMC proteoglycan biosynthesis.

Biosynthetic investigations of lactonamycin and lactonamycin z: cloning of the biosynthetic gene clusters and discovery of an unusual starter unit.

The antibiotics lactonamycin and lactonamycin Z provide attractive leads for antibacterial drug development. Both antibiotics contain a novel aglycone core called lactonamycinone. To gain insight into lactonamycinone biosynthesis, cloning and precursor incorporation experiments were undertaken. The lactonamycin gene cluster was initially cloned from Streptomyces rishiriensis. Sequencing of ca. 61 kb of S. rishiriensis DNA revealed the presence of 57 open reading frames. These included genes coding for the biosynthesis of l-rhodinose, the sugar found in lactonamycin, and genes similar to those in the tetracenomycin biosynthetic gene cluster. Since lactonamycin production by S. rishiriensis could not be sustained, additional proof for the identity of the S. rishiriensis cluster was obtained by cloning the lactonamycin Z gene cluster from Streptomyces sanglieri. Partial sequencing of the S. sanglieri cluster revealed 15 genes that exhibited a very high degree of similarity to genes within the lactonamycin cluster, as well as an identical organization. Double-crossover disruption of one gene in the S. sanglieri cluster abolished lactonamycin Z production, and production was restored by complementation. These results confirm the identity of the genetic locus cloned from S. sanglieri and indicate that the highly similar locus in S. rishiriensis encodes lactonamycin biosynthetic genes. Precursor incorporation experiments with S. sanglieri revealed that lactonamycinone is biosynthesized in an unusual manner whereby glycine or a glycine derivative serves as a starter unit that is extended by nine acetate units. Analysis of the gene clusters and of the precursor incorporation data suggested a hypothetical scheme for lactonamycinone biosynthesis.

A Small ATPase Protein of Arabidopsis, TGD3, Involved in Chloroplast Lipid Import

Polar lipid trafficking is essential in eukaryotic cells as membranes of lipid assembly are often distinct from final destination membranes. A striking example is the biogenesis of the photosynthetic membranes (thylakoids) in plastids of plants. Lipid biosynthetic enzymes at the endoplasmic reticulum and the inner and outer plastid envelope membranes are involved. This compartmentalization requires extensive lipid trafficking. Mutants of Arabidopsis are available that are disrupted in the incorporation of endoplasmic reticulum-derived lipid precursors into thylakoid lipids. Two proteins affected in two of these mutants, trigalactosyldiacylglycerol 1 (TGD1) and TGD2, encode the permease and substrate binding component, respectively, of a proposed lipid translocator at the inner chloroplast envelope membrane. Here we describe a third protein of Arabidopsis, TGD3, a small ATPase proposed to be part of this translocator. As in the tgd1 and tgd2 mutants, triacylglycerols and trigalactolipids accumulate in a tgd3 mutant carrying a T-DNA insertion just 5' of the TGD3 coding region. The TGD3 protein shows basal ATPase activity and is localized inside the chloroplast beyond the inner chloroplast envelope membrane. Proteins orthologous to TGD1, -2, and -3 are predicted to be present in Gram- bacteria, and the respective genes are organized in operons suggesting a common biochemical role for the gene products. Based on the current analysis, it is hypothesized that TGD3 is the missing ATPase component of a lipid transporter involving TGD1 and TGD2 required for the biosynthesis of ER-derived thylakoid lipids in Arabidopsis.