4,5-Seco-Guaiane and a Nine-Membered Sesquiterpene Lactone from Holostylis reniformis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Helivypolide G. A novel dimeric bioactive sesquiterpene lactone

Helivypolide G was isolated from leaves of Helianthus annuus L. cv. Stella. In the course of our ongoing research for new allelochemicals from Helianthus annuus, a novel dimeric bioactive sesquiterpene lactone, helivypolide G has been isolated and characterized from the medium polar active fractions of the leaves of cultivar variety Stella. The monomers are connected through carbons C-15 of each unit and an oxygen bridge, forming an enolic oxane ring. © 2004 Elsevier Ltd. All rights reserved.

Microbial transformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus

The biotransformation of the sesquiterpene lactone tagitinin C by the fungus Aspergillus terreus MT 5.3 yielded a rare derivative that was elucidated by spectrometric methods. The fungus led to the formation of a different product through an unusual epoxidation reaction between C4 and C5, formation of a C3,C10 ether bridge, and a methoxylation of the C1 of tagitinin C. The chemical structure of the product, namely 1 beta-methoxy-3 alpha-hydroxy-3,10 beta-4,5 alpha-diepoxy-8 beta-isobutyroyloxygermacr-11(13)-en-6 alpha,12-olide, is the same as that of a derivative that was recently isolated from the flowers of a Brazilian population of Mexican sunflower (Tithonia diversifolia), which is the source of the substrate tagitinin C. The in vitro cytotoxic activity of the substrate and the biotransformed product were evaluated in HL-60 cells using an MTT assay, and both compounds were found to be cytotoxic. We show that soil fungi may be useful in the biotransformation of sesquiterpene lactones, thereby leading to unusual changes in their chemical structures that may preserve or alter their biological activities, and may also mimic plant biosynthetic pathways for production of secondary metabolites.

Hyperverzweigte Lacton-Copolymere: enzymatische Synthese, Charakterisierung und Darstellung strukturierter Polymernetzwerke = (Hyperbranched lactone copolymers by enzyme catalysis: synthesis, characterization and preparation of structured polymer networks)

Hyperverzweigte Polymere erfuhren in den letzten Jahren immer mehr Beachtung, da sie im Vergleich zu ihren linearen Analoga besondere Eigenschaften besitzen. Im Jahre 2002 wurde die erste enzymkatalysierte Darstellung hyperverzweigter Poly(epsilon-caprolacton)e (hb-PCL) beschrieben. Hier ermöglichte das Konzept der konkurrierenden ringöffnenden Polymerisation und Polykondensation die Kontrolle der Eigenschaften des dargestellten Polymers. Detaillierte Untersuchungen in Hinblick auf Grenzen und Möglichkeiten, aber auch die Synthese im Technikumsmaßstab sind wesentliche Aspekte dieser Arbeit. Außerdem wird ein neues Konzept eingeführt, das Reknitting genannt wurde. Ziel desselben ist das Recycling kommerziellen, linearen PCLs mittels Umesterung zu hb-PCL durch Enzymkatalyse. Diese hb-PCLs zeigen vergleichbare Eigenschaften zu den aus den Comonomeren dargestellten. Ausgehend von hb-PCL sollte eine geeignete Route zu methacrylierten Vernetzerverbindungen entwickelt werden. Aus Mischungen derselben mit 2-Hydroxyethylmethacrylat wurden komplexe Netzwerkarchitekturen durch Copolymerisation erhalten. Diese Netzwerke wurden in Hinblick auf ihre mechanisch physikalischen Eigenschaften untersucht. Zuletzt wurden Screeningexperimente an anderen zyklischen Estern durchgeführt, da ein Transfer des oben vorgestellten Konzepts angestrebt wurde. Zwei neue hyperverzweigte Polymerklassen, hb-Poly(delta-valerolacton) und hb-Polytrimethylencarbonat wurden detaillierter untersucht und in Ihren Eigenschaften mit hb-PCL verglichen.

Discovery of a highly potent anti-inflammatory epoxyisoprostane-derived lactone.

Epoxyisoprostanes EI (1) and EC (2) are effective inhibitors of the secretion of proinflammatory cytokines IL-6 and IL-12. In detailed studies toward the investigation of the molecular mode of action of these structures, a highly potent lactone (3) derived from 1 was identified. The known isoprostanoids 1 and 2 are most likely precursors of 3, the product of facile intramolecular reaction between the epoxide with the carboxylic acid in 2.

Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

Two enzymes of diacylglyceryl-O-4′-(N,N,N,-trimethyl)homoserine biosynthesis are encoded by btaA and btaB in the purple bacterium Rhodobacter sphaeroides

Betaine lipids are ether-linked, nonphosphorous glycerolipids that resemble the more commonly known phosphatidylcholine in overall structure. Betaine lipids are abundant in many eukaryotes such as nonseed plants, algae, fungi, and amoeba. Some of these organisms are entirely devoid of phosphatidylcholine and, instead, contain a betaine lipid such as diacylglyceryl-O-4′-(N,N,N,-trimethyl)homoserine. Recently, this lipid also was discovered in the photosynthetic purple bacterium Rhodobacter sphaeroides where it seems to replace phosphatidylcholine under phosphate-limiting growth conditions. This discovery provided the opportunity to study the biosynthesis of betaine lipids in a bacterial model system. Mutants of R. sphaeroides deficient in the biosynthesis of the betaine lipid were isolated, and two genes essential for this process, btaA and btaB, were identified. It is proposed that btaA encodes an S-adenosylmethionine:diacylglycerol 3-amino-3-carboxypropyl transferase and btaB an S-adenosylmethionine-dependent N-methyltransferase. Both enzymatic activities can account for all reactions of betaine lipid head group biosynthesis. Because the equivalent reactions have been proposed for different eukaryotes, it seems likely that orthologs of btaA/btaB may be present in other betaine lipid-containing organisms.

Expression of an Arabidopsis Aspartate Kinase/Homoserine Dehydrogenase Gene Is Metabolically Regulated by Photosynthesis-Related Signals but Not by Nitrogenous Compounds1

Although the control of carbon fixation and nitrogen assimilation has been studied in detail, relatively little is known about the regulation of carbon and nitrogen flow into amino acids. In this paper we report our study of the metabolic regulation of expression of an Arabidopsis aspartate kinase/homoserine dehydrogenase (AK/HSD) gene, which encodes two linked key enzymes in the biosynthetic pathway of aspartate family amino acids. Northern blot analyses, as well as expression of chimeric AK/HSD-β-glucuronidase constructs, have shown that the expression of this gene is regulated by the photosynthesis-related metabolites sucrose and phosphate but not by nitrogenous compounds. In addition, analysis of AK/HSD promoter deletions suggested that a CTTGACTCTA sequence, resembling the binding site for the yeast GCN4 transcription factor, is likely to play a functional role in the expression of this gene. Nevertheless, longer promoter fragments, lacking the GCN4-like element, were still able to confer sugar inducibility, implying that the metabolic regulation of this gene is apparently obtained by multiple and redundant promoter sequences. The present and previous studies suggest that the conversion of aspartate into either the storage amino acid asparagine or aspartate family amino acids is subject to a coordinated, reciprocal metabolic control, and this biochemical branch point is a part of a larger, coordinated regulatory mechanism of nitrogen and carbon storage and utilization.

Transport, Compartmentation, and Metabolism of Homoserine in Higher Plant Cells : Carbon-13- and Phosphorus-31-Nuclear Magnetic Resonance Studies

The transport, compartmentation, and metabolism of homoserine was characterized in two strains of meristematic higher plant cells, the dicotyledonous sycamore (Acer pseudoplatanus) and the monocotyledonous weed Echinochloa colonum. Homoserine is an intermediate in the synthesis of the aspartate-derived amino acids methionine, threonine (Thr), and isoleucine. Using 13C-nuclear magnetic resonance, we showed that homoserine actively entered the cells via a high-affinity proton-symport carrier (Km approximately 50–60 μm) at the maximum rate of 8 ± 0.5 μmol h−1 g−1 cell wet weight, and in competition with serine or Thr. We could visualize the compartmentation of homoserine, and observed that it accumulated at a concentration 4 to 5 times higher in the cytoplasm than in the large vacuolar compartment. 31P-nuclear magnetic resonance permitted us to analyze the phosphorylation of homoserine. When sycamore cells were incubated with 100 μm homoserine, phosphohomoserine steadily accumulated in the cytoplasmic compartment over 24 h at the constant rate of 0.7 μmol h−1 g−1 cell wet weight, indicating that homoserine kinase was not inhibited in vivo by its product, phosphohomoserine. The rate of metabolism of phosphohomoserine was much lower (0.06 μmol h−1 g−1 cell wet weight) and essentially sustained Thr accumulation. Similarly, homoserine was actively incorporated by E. colonum cells. However, in contrast to what was seen in sycamore cells, large accumulations of Thr were observed, whereas the intracellular concentration of homoserine remained low, and phosphohomoserine did not accumulate. These differences with sycamore cells were attributed to the presence of a higher Thr synthase activity in this strain of monocot cells.

Selection of the N-Acylhomoserine Lactone-Degrading Bacterium Alteromonas stellipolaris PQQ-42 and of Its Potential for Biocontrol in Aquaculture

The production of virulence factors by many pathogenic microorganisms depends on the intercellular communication system called quorum sensing, which involves the production and release of signal molecules known as autoinducers. Based on this, new-therapeutic strategies have emerged for the treatment of a variety of infections, such as the enzymatic degradation of signaling molecules, known as quorum quenching (QQ). In this study, we present the screening of QQ activity amongst 450 strains isolated from a bivalve hatchery in Granada (Spain), and the selection of the strain PQQ-42, which degrades a wide range of N-acylhomoserine lactones (AHLs). The selected strain, identified as Alteromonas stellipolaris, degraded the accumulation of AHLs and reduced the production of protease and chitinase and swimming motility of a Vibrio species in co-cultivation experiments in vitro. In the bio-control experiment, strain PQQ-42 significantly reduced the pathogenicity of Vibrio mediterranei VibC-Oc-097 upon the coral Oculina patagonica showing a lower degree of tissue damage (29.25 ± 14.63%) in its presence, compared to when the coral was infected with V. mediterranei VibC-Oc-097 alone (77.53 ± 13.22%). Our results suggest that this AHL-degrading bacterium may have biotechnological applications in aquaculture.

The Sesquiterpene Lactone Dehydroleucodine Triggers Senescence and Apoptosis in Association with Accumulation of DNA Damage Markers

Sesquiterpene lactones (SLs) are plant-derived compounds that display anti-cancer effects. Some SLs derivatives have a marked killing effect on cancer cells and have therefore reached clinical trials. Little is known regarding the mechanism of action of SLs. We studied the responses of human cancer cells exposed to various concentrations of dehydroleucodine (DhL), a SL of the guaianolide group isolated and purified from Artemisia douglasiana (Besser), a medicinal herb that is commonly used in Argentina. We demonstrate for the first time that treatment of cancer cells with DhL, promotes the accumulation of DNA damage markers such as phosphorylation of ATM and focal organization of γH2AX and 53BP1. This accumulation triggers cell senescence or apoptosis depending on the concentration of the DhL delivered to cells. Transient DhL treatment also induces marked accumulation of senescent cells. Our findings help elucidate the mechanism whereby DhL triggers cell cycle arrest and cell death and provide a basis for further exploration of the effects of DhL in in vivo cancer treatment models.

Sequential alcohol oxidation/putative homo Claisen-Tishchenko-type reaction to give esters: a key process in accessing novel biologically active lactone macrocycles

We report an efficient methodology for the direct oxidative esterification of primary alcohols to diether-esters using pyridinium chlorochromate (PCC). Numerous studies were carried out to probe the reaction mechanism and at the same time optimize the reaction conditions. The reaction could be conducted with 1 equivalent of PCC and 1 equivalent of BF3 center dot OEt2. Indications based on literature precedent were that the reaction may proceed via a sequential alcohol oxidation to the aldehyde followed by a putative Cr or boron catalyzed Claisen-Tishchenko-type reaction. Using this efficient methodology, we synthesized a family of novel diether-esters in very good yields; some of these molecules were subsequently tested against both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In addition, we also disclose a new synthetic strategy for the synthesis of lactam macrocycles with potential biological activity. This methodology included the regioselective borylation of the ester substrate and a subsequent Suzuki-Miyaura coupling to obtain the desired lactam macrocycle.

Anti-Inflammatory and antiosteoclastogenic activities of parthenolide on human periodontal ligament cellsIn vitro

Periodontitis is an inflammatory disease that causes osteolysis and tooth loss. It is known that the nuclear factor kappa B (NF-κB) signalling pathway plays a key role in the progression of inflammation and osteoclastogenesis in periodontitis. Parthenolide (PTL), a sesquiterpene lactone extracted from the shoots of Tanacetum parthenium, has been shown to possess anti-inflammatory properties in various diseases. In the study reported herein, we investigated the effects of PTL on the inflammatory and osteoclastogenic response of human periodontal ligament-derived cells (hPDLCs) and revealed the signalling pathways in this process. Our results showed that PTL decreased NF-κB activation, I-κB degradation, and ERK activation in hPDLCs. PTL significantly reduced the expression of inflammatory (IL-1β, IL-6, and TNF-α) and osteoclastogenic (RANKL, OPG, and M-CSF) genes in LPS-stimulated hPDLCs. In addition, PTL attenuated hPDLC-induced osteoclastogenic differentiation of macrophages (RAW264.7 cells), as well as reducing gene expression of osteoclast-related markers in RAW264.7 cells in an hPDLC-macrophage coculture model. Taken together, these results demonstrate the anti-inflammatory and antiosteoclastogenic activities of PTL in hPDLCs in vitro. These data offer fundamental evidence supporting the potential use of PTL in periodontitis treatment.