Biofilm and Rivers: The Natural Association to Reduce Metals in Waters.

This article focuses on a very peculiar habitat, the thin biofilm that covers the surface of rocks, cobbles, sediment grains, leaf litter, and vegetation on a riverbed. Species composition changes over time and depends on environmental conditions and perturbation of water quality. It provides several ecosystem services, contributing to the biogeochemical fluxes and reducing contamination by absorbing the pollutants. Biofilm into the Toce River (Ossola Valley, Piedmont, Italy) was investigated to assess its capacity to accumulate the metals and macroions from the water column. In this preliminary work, we investigated three sample points, in two different seasons. The community composition of biofilm was determined via morphological analysis (diatoms and non-diatoms algal community). We characterize the biofilm, a community of different organisms, from different perspectives. In the biofilm, Hg was analyzed with an automated mercury analyzer, other metals and macroions with inductively coupled plasma mass spectrometry (ICP-MS) (Al, As, Ba, Ca, Cr, Cu, Fe, K, Mg, Mn, Ni, P, Pb, and Zn), and the carotenoid and chlorophyll composition of the photosynthetic organism with HPLC analysis for the primary producers. The results evidence a seasonal pattern in metals and macroions levels in the biofilm, and a significant difference in the biofilm community and in carotenoid composition, suggesting the utility of using the biofilm as an additional bioindicator to monitor the water quality of the river.

In Streptococcus thermophilus, Ammonia from Urea Hydrolysis Paradoxically Boosts Acidification and Reveals a New Regulatory Mechanism of Glycolysis.

Streptococcus thermophilus is widely used in the dairy industry for the manufacturing of fermented milk and cheeses and probiotic formulations. S. thermophilus evolved from closely phylogenetically related pathogenic streptococci through loss-of-function events counterbalanced by the acquisition of relevant traits, such as lactose and urea utilization for the adaptation to the milk environment. In the context of regressive evolution, the urease gene cluster accounts for 0.9% of the total coding sequence belonging to known functional categories. The fate of ammonia and carbon dioxide derived by urea hydrolysis in several biosynthetic pathways have been depicted, and the positive effect of urease activity on S. thermophilus growth fitness and lactic acid fermentation in milk has been already addressed by several authors. However, the mechanistic effect of urea hydrolysis on the energetic metabolisms of S. thermophilus is still unclear. This study aimed to assess the effect of urease activity on the growth and energy metabolism of Streptococcus thermophilus in milk. In milk, 13C-urea was completely hydrolyzed in the first 150 min of S. thermophilus growth, and urea hydrolysis was accompanied by an increase in cell density and a reduction in the generation time. By using energetically discharged cells with gene transcription and translation blocked, we showed that in the presence of fermentable carbon sources, urease activity, specifically the production of ammonia, could dramatically boost glycolysis and, in cascade, homolactic fermentation. Furthermore, we showed that ammonia, specifically ammonium ions, were potent effectors of phosphofructokinase, a key glycolytic enzyme. IMPORTANCE Finding that ammonia-generating enzymes, such as urease, and exogenous ammonia act on phosphofructokinase activity shed new light on the regulatory mechanisms that govern glycolysis. Phosphofructokinase is the key enzyme known to exert a regulatory role on glycolytic flux and, therefore, ammonia as an effector of phosphofructokinase acts, in cascade, modulating the glycolytic pathway. Apart from S. thermophilus, due to the high conservation of glycolytic enzymes in all branches of the tree of life and being aware of the role of ammonia as an effector of phosphofructokinase, we propose to reevaluate the physiological role of the ammonia production pathways in all organisms whose energy metabolism is supported by glycolysis.

G-quadruplex binding properties of a potent PARP-1 inhibitor derived from 7-azaindole-1-carboxamide.

Poly ADP-ribose polymerases (PARP) are key proteins involved in DNA repair, maintenance as well as regulation of programmed cell death. For this reason they are important therapeutic targets for cancer treatment. Recent studies have revealed a close interplay between PARP1 recruitment and G-quadruplex stabilization, showing that PARP enzymes are activated upon treatment with a G4 ligand. In this work the DNA binding properties of a PARP-1 inhibitor derived from 7-azaindole-1-carboxamide, (2-[6-(4-pyrrolidin-1-ylmethyl-phenyl)-pyrrolo[2,3-b]pyridin-1-yl]-acetamide, compound 1) with model duplex and quadruplex DNA oligomers were studied by NMR, CD, fluorescence and molecular modelling. We provide evidence that compound 1 is a strong G-quadruplex binder. In addition we provide molecular details of the interaction of compound 1 with two model G-quadruplex structures: the single repeat of human telomeres, d(TTAGGGT)4, and the c-MYC promoter Pu22 sequence. The formation of defined and strong complexes with G-quadruplex models suggests a dual G4 stabilization/PARP inhibition mechanism of action for compound 1 and provides the molecular bases of its therapeutic potential.

Sisymbrium Officinale (the Singers' Plant) as an Ingredient: Analysis of Somatosensory Active Volatile Isothiocyanates in Model Food and Drinks.

Sisymbrium officinale (L.) Scop. (hedge mustard) is a wild common plant of the Brassicaceae family. It is known as "the singers' plant" for its traditional use in treating aphonia and vocal disability. The plant is rich in glucosinolates and isothiocyanates; the latter has been demonstrated to be a strong agonist in vitro of the Transient Receptor Potential Ankirine 1 (TRPA1) channel, which is involved in the somatosensory perception of pungency as well as in the nociception pathway of inflammatory pain. Volatile ITCs are released by the enzymatic or chemical hydrolysis of GLSs (glucosinolates) during sample crushing and/or by the mastication of fresh plant tissues when the plant is used as an ingredient. Some functional food and drink model preparations have been realised: honey enriched with seeds and flowers, infusions, cold drink (voice drink), artisanal beer, and a fermented tea (kombucha). Using SPME-GCMS chromatography, we analysed samples of the plant and of the food preparations adopting conditions that simulate the release of isothiocyanates (ITCs) during oral assumption. Two active compounds, iso-propylisothiocyanate and 2-butylisothiocyanate, have been assayed. The concentration of ITCs varies according to temperature, pH, grinding conditions, and different plant organs used. Kombucha-type fermentation seems to eliminate the ITCs, whereas they are retained in beer. The ITCs' concentration is higher when entire seeds and flowers are used.

Phosphorus speciation during anaerobic digestion and subsequent solid/liquid separation.

This study aims to investigate the effect of anaerobic digestion (AD) on P species and how the different species are distributed in the digestate and digestate fractions, i.e. liquid and solid fractions. To do so, six full scale AD plants were used in this work and representative biomass samples were collected for investigation. P fractionation proceeded by adopting fractionation protocols consisting in step-by-step extraction with different solvents, (i.e. NaHCO3, HCl and NaOH-EDTA). Subsequently P species in the different fractions were identified by using 31PNMR. On average, AD did not substantially affect P speciation that depended on the P-fraction content of feeds. A high NaHCO3 fraction content in the ingestate determined, also, a high content of this fraction in the digestate, with consequently lower contents of both P-HCl and P-NaOH-EDTA, i.e. digestate P-fraction contents represented an inheritance of P speciation in the ingestate. A feed effect was observed in single plants. Highest pig/cow slurry content in the feeds seemed to decrease readily soluble P (extracted with NaHCO3) content and increased P associated with both organic matter and amorphous Fe/Al in the digestate. Again, using a large amount of digestate in the feed increased P-soluble content in the digestate. 31P NMR analyses revealed that inorganic P compounds dominated the spectra of all biomasses and fractions, with orthophosphate as the predominant species. When present, organic phosphorus compounds were typically represented by monophosphate esters, DNA and phospholipids, with a predominance of monophosphate esters.

Stabilization of c-KIT G-Quadruplex DNA Structures by the RNA Polymerase I Inhibitors BMH-21 and BA-41.

The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3'-end, stabilized by an extensive network of π-π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M-1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.

Synthesis of a leopolic acid-inspired tetramic acid with antimicrobial activity against multidrug-resistant bacteria.

The increasing emergence of multidrug-resistant pathogens is one of the biggest threats to human health and food security. The discovery of new antibacterials, and in particular the finding of new scaffolds, is an imperative goal to stay ahead of the evolution of antibiotic resistance. Herein we report the synthesis of a 3-decyltetramic acid analogue of the ureido dipeptide natural antibiotic leopolic acid A. The key step in the synthetic strategy is an intramolecular Lacey-Dieckmann cyclization reaction of a linear precursor to obtain the desired 3-alkyl-substituted tetramic acid core. The synthesized analogue is more effective than the parent leopolic acid A against Gram-positive (Staphylococcus pseudintermedius) and Gram-negative (E. coli) bacteria (MIC 8 µg/mL and 64 µg/mL, respectively). Interestingly, the compound shows a significant activity against Staphylococcus pseudintermedius strains expressing a multidrug-resistant phenotype (average MIC 32 µg/mL on 30 strains tested). These results suggest that this molecule can be considered a promising starting point for the development of a novel class of antibacterial agents active also against resistant strains.

c-MYC G-quadruplex binding by the RNA polymerase I inhibitor BMH-21 and analogues revealed by a combined NMR and biochemical Approach.

BACKGROUND: Pyridoquinazolinecarboxamides have been reported as RNA polymerase I inhibitors and represent a novel class of potential antitumor agents. BMH-21, was reported to intercalate with GC-rich rDNA, resulting in nucleolar stress as a primary mechanism of cytotoxicity. METHODS: The interaction of BMH-21 and analogues with DNA G-quadruplex structures was studied by NMR and molecular modelling. The cellular response was investigated in a panel of human tumor cell lines and protein expression was examined by Western Blot analysis. RESULTS AND CONCLUSIONS: We explored the ability of BMH-21 and its analogue 2 to bind to G-quadruplex present in the c-MYC promoter, by NMR and molecular modelling studies. We provide evidence that both compounds are not typical DNA intercalators but are effective binders of the tested G-quadruplex. The interaction with c-MYC G-quadruplex was reflected in down-regulation of c-Myc expression in human tumor cells. The inhibitory effect was almost complete in lymphoma cells SUDHL4 characterized by overexpression of c-Myc protein. This downregulation reflected an early and persistent modulation of cMyc mRNA. Given the relevance of c-MYC in regulation of ribosome biogenesis, it is conceivable that the inhibition of c-MYC contributes to the perturbation of nuclear functions and RNA polymerase I activity. Similar experiments with CX-5461, another RNA polymerase I transcription inhibitor, indicate the same behaviour in G-quadruplex stabilization. GENERAL SIGNIFICANCE: Our results support the hypothesis that BMH-21 and analogue compounds share the same mechanism, i.e. G-quadruplex binding as a primary event of a cascade leading to inhibition of RNA polymerase I and apoptosis.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7.

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.

Total synthesis of leopolic acid A, a natural 2,3-pyrrolidinedione with antimicrobial activity.

The first total synthesis of leopolic acid A, a fungal metabolite with a rare 2,3-pyrrolidinedione nucleus linked to an ureido dipeptide, was designed and carried out. Crucial steps for the strategy include a Dieckmann cyclization to obtain the 2,3-pyrrolidinedione ring and a Wittig olefination to install the polymethylene chain. An oxazolidinone-containing leopolic acid A analogue was also synthesized. The antibacterial activity showed by both compounds suggests that they could be considered as promising candidates for future developments.

Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22.

BACKGROUND: Intra-molecular G-quadruplex structures are present in the guanine rich regions of human telomeres and were found to be prevalent in gene promoters. More recently, the targeting of c-MYC transcriptional control has been suggested, because the over expression of the c-MYC oncogene is one of the most common aberration found in a wide range of human tumors. METHODS: The interaction of nemorubicin and doxorubicin with DNA G-quadruplex structures has been studied by NMR, ESI-MS and molecular modelling, in order to obtain further information about the complex and the multiple mechanisms of action of these drugs. RESULTS AND CONCLUSIONS: Nemorubicin intercalates between A3 and G4 of d(TTAGGGT)4 and form cap-complex at the G6pT7 site. The presence of the adenine in this sequence is important for the stabilization of the complex, as was shown by the interaction with d(TTGGGTT)4 and d(TTTGGGT)4, which form only a 1:1 complex. The interaction of doxorubicin with d(TTAGGGT)4 is similar, but the complex appears less stable. Nemorubicin also binds with high efficiency the c-MYC G-quadruplex sequence Pu22, to form a very well defined complex. Two nemorubicin molecules bind to the 3'-end and to the 5'-end, forming an additional plane of stacking over each external G-tetrad. The wild type c-MYCPu22 sequence forms with nemorubicin the same complex. GENERAL SIGNIFICANCE: Nemorubicin and doxorubicin, not only intercalate into the duplex DNA, but also result in significant ligands for G-quadruplex DNA segments, stabilizing their structure; this may in part explain the multiple mechanisms of action of their antitumor activity.

Phytochemicals from Ruta graveolens Activate TAS2R Bitter Taste Receptors and TRP Channels Involved in Gustation and Nociception.

Ruta graveolens (rue) is a spontaneous plant in the Mediterranean area with a strong aroma and a very intense bitter taste, used in gastronomy and in folk medicine. From the leaves, stems and fruits of rue, we isolated rutin, rutamarin, three furanocoumarins, two quinolinic alkaloids, a dicoumarin and two long chain ketones. Bitter taste and chemesthetic properties have been evaluated by in vitro assays with twenty receptors of the TAS2R family and four TRP ion channels involved in gustation and nociception. Among the alkaloids, skimmianine was active as a specific agonist of T2R14, whereas kokusaginin did not activate any of the tested receptors. The furanocoumarins activates TAS2R10, 14, and 49 with different degrees of selectivity, as well as the TRPA1 somatosensory ion channel. Rutamarin is an agonist of TRPM5 and TRPV1 and a strong antagonist of TRPM8 ion channels.

Molecular recognition in naphthoquinone derivatives - G-quadruplex complexes by NMR.

BACKGROUND: G-quadruplexes have become important drug-design targets for the treatment of various human disorders such as cancer, diabetes and cardiovascular diseases. Recently, G-quadruplex structures have been visualized in the DNA of human cells and appeared to be dynamically sensitive to the cell cycle and stabilized by small molecule ligands. A small library of isoxazolo naphthoquinones (1a-h), which exhibited a strong antiproliferative activity on different cancer cell lines, was studied as potential ligands of G-quadruplex DNA. METHODS: The DNA binding properties of a series of the selected compounds have been analyzed by fluorescence assays. NMR/modeling studies were performed to describe the complexes between G-quadruplex DNA sequences and two selected compounds 1a and 1b. RESULTS: 1a and 1b in the presence of G-quadruplexes, d(T(2)AG(3)T)(4), d(TAG(3)T(2)A)(4) and d(T(2)G(3)T(2))(4), showed good ability of intercalation and the formation of complexes with 2:1 stoichiometry. 1a showed an important interaction with the sequence Pu22 belonging to the promoter of oncogenes c-myc. CONCLUSIONS: The ligands directly interact with the external G-tetrads of the G-quadruplexes, without alterations in the structure of the G-quadruplex core. The role of the adenine moieties over the G-tetrads in the stabilization of the complexes was discussed. GENERAL SIGNIFICANCE: The results obtained suggested that the strong antiproliferative activity of isoxazolo naphthoquinones is not due to the Hsp90 inhibition, but mainly to the interaction at the level of telomeres and/or at the level of gene promoter. These findings can be used as a basis for the rational drug design of new anticancer agents.

The interaction of nemorubicin metabolite PNU-159682 with DNA fragments d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) shows a strong but reversible binding to G:C base pairs.

The antitumor anthracycline nemorubicin is converted by human liver microsomes to a major metabolite, PNU-159682 (PNU), which was found to be much more potent than its parent drug toward cultured tumor cells and in vivo tumor models. The mechanism of action of nemorubicin appears different from other anthracyclines and until now is the object of studies. In fact PNU is deemed to play a dominant, but still unclear, role in the in vivo antitumor activity of nemorubicin. The interaction of PNU with the oligonucleotides d(CGTACG)(2), d(CGATCG)(2) and d(CGCGCG)(2) was studied with a combined use of (1)H and (31)P NMR spectroscopy and by ESI-mass experiments. The NMR studies allowed to establish that the intercalation between the base pairs of the duplex leads to very stable complexes and at the same time to exclude the formation of covalent bonds. Melting experiments monitored by NMR, allowed to observe with high accuracy the behaviour of the imine protons with temperature, and the results showed that the re-annealing occurs after melting. The formation of reversible complexes was confirmed by HPLC-tandem mass spectra, also combined with endonuclease P1digestion. The MS/MS spectra showed the loss of neutral PNU before breaking the double helix, a behaviour typical of intercalators. After digestion with the enzyme, the spectra did not show any compound with PNU bound to the bases. The evidence of a reversible process appears from both proton and phosphorus NOESY spectra of PNU bound to d(CGTACG)(2) and to d(CGATCG)(2). The dissociation rate constants (k(off)) of the slow step of the intercalation process, measured by (31)P NMR NOE-exchange experiments, showed that the kinetics of the process is slower for PNU than for doxorubicin and nemorubicin, leading to a 10- to 20-fold increase of the residence time of PNU into the intercalation sites, with respect to doxorubicin. A relevant number of NOE interactions allowed to derive a model of the complexes in solution from restrained MD calculations. The conformation of PNU bound to the oligonucleotides was also derived from the coupling constant values.

Alkalizing reactions streamline cellular metabolism in acidogenic microorganisms.

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of ß-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms.

Interaction between double helix DNA fragments and the new antitumor agent sabarubicin, Men10755.

Among the disaccharide derivatives of the antitumor anthracycline doxorubicin, sabarubicin (Men10755) is more active and less cytotoxic than doxorubicin. It showed a strong in vivo antitumor activity in all preclinical models examined, in conjunction with a better tolerability, and is now in phase II clinical trials. The interaction of sabarubicin and Men10749 (a similar disaccharide with a different configuration at C-4' of the proximal sugar) with the hexanucleotides d(CGTACG)(2) and d(CGATCG)(2) was studied by a combined use of 2D-(1)H and (31)P NMR techniques. Both (1)H and (31)P chemical shifts of imino protons and phosphates allowed to established the intercalation sites between the CG base pairs, as it occurs for other anthracyclines of the series. The dissociation rate constants (k(off)) of the slow step of the intercalation process were measured for Men10755 and Men10749, by NMR NOE-exchange experiments. The increase of k(off) , with respect of doxorubicin, showed that the intercalation process is significantly faster for both drugs, leading to an average residence time for sabarubicin into d(CGTACG)(2) sixfold shorter than for doxorubicin. This could give account of both higher cytoplasmic/nuclear ratio and lower cellular uptake of sabarubicin in comparison with doxorubicin and accordingly of the lower cytotoxicity of these disaccharide analogues. A relevant number of NOE interactions allowed the structure of the complexes in solution to be derived through restrained MD calculations. NMR-DOSY experiments were performed with several drug/oligonucleotide mixtures in order to determine the structure and the dimension of the aggregates.

Taste-guided identification of high potency TRPA1 agonists from Perilla frutescens.

Perilla frutescens is a food plant widely used in Asian cuisine. This plant was investigated for its interesting taste and somatosensory properties. Perillaldehyde and perillaketone are among the components of the aromatic extracts from P. Frutescens. These compounds were shown here to activate the cloned TRPA1 channel when expressed in an heterologous cell system and are therefore suggested to be responsible for the chemesthetic properties of this plant.

Interaction between double helix DNA fragments and a new topopyrone acting as human topoisomerase I poison.

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 degrees C. Structural models of the complex with d(CGTACG)(2) were developed on the basis of DOSY, 2D NOESY and (31)P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.

Taste-active compounds in a traditional Italian food: 'lampascioni'.

Nature is a rich source of taste-active compounds, in particular of plant origin, many of which have unusual tastes. Many of these are found in traditional food, where spontaneous plants are used as ingredients. Some taste-active compounds were identified in the bulbs of Muscari comosum, a spontaneous plant belonging to the family of the Liliaceae, very common in the Mediterranean area, and used in traditional gastronomy (called 'lampascioni' in South Italy). The bulbs were extracted with a series of solvents of different polarity. The different fractions were submitted to a preliminary sensory evaluation, and the most interesting ones, characterized by a strong bitter taste and some chemestetic properties, were submitted to further purification and structural analysis. From the ethereal extract, several 3-benzyl-4-chromanones and one stilbene derivative were isolated. Pure compounds were examined for their taste activity by means of sensory evaluation, and proved to be responsible for the characteristic taste of this food. Some of these compounds have been synthesized de novo to confirm their structure.

Synthesis and cytotoxic activity of a new series of topoisomerase I inhibitors.

A series of structurally simple analogues of natural topopyrone C were synthesized and tested for cytotoxic and topoisomerase I inhibitory activities. The removal of the hydroxyl groups at the 5 and 9 positions resulted in an increased cytotoxic potency and ability to stabilize topoisomerase-mediated cleavage. In addition, the results suggest that some structural features, such as the pyrone ring and a polar group in position 11, are fundamental for topoisomerase I inhibitory effect. These structural requirements are also consistent with the cytotoxic activity.