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Because of their importance as chemical mediators, the presence of a rich and varied family of lipoxygenase (LOX) products, collectively named oxylipins, has been investigated thoroughly in diatoms, and the involvement of these products in important processes such as bloom regulation has been postulated. Nevertheless, little information is available on the enzymes and pathways operating in these protists. Exploiting transcriptome data, we identified and characterized a LOX gene, PaLOX, in Pseudo-nitzschia arenysensis, a marine diatom known to produce different species of oxylipins by stereo- and regio-selective oxidation of eicosapentaenoic acid (EPA) at C12 and C15. PaLOX RNA interference correlated with a decrease of the lipid-peroxidizing activity and oxylipin synthesis, as well as with a reduction of growth of P. arenysensis. In addition, sequence analysis and structure models of the C-terminal part of the predicted protein closely fitted with the data for established LOXs from other organisms. The presence in the genome of a single LOX gene, whose downregulation impairs both 12- and 15-oxylipins synthesis, together with the in silico 3D protein modelling suggest that PaLOX encodes for a 12/15S-LOX with a dual specificity, and provides additional support to the correlation between cell growth and oxylipin biosynthesis in diatoms.

Abstract Bacteria belonging to Rhodococcus genus represent ideal candidates for microbial biotechnology applications because of their metabolic versatility, ability to degrade a wide range of organic compounds, and resistance to various stress conditions, such as metal toxicity, desiccation, and high concentration of organic solvents. Rhodococcus spp. strains have also peculiar biosynthetic activities that contribute to their strong persistence in harsh and contaminated environments and provide them a competitive advantage over other microorganisms. This review is focused on the metabolic features of Rhodococcus genus and their potential use in biotechnology strategies for the production of compounds with environmental, industrial, and medical relevance such as biosurfactants, bioflocculants, carotenoids, triacylglycerols, polyhydroxyalkanoate, siderophores, antimicrobials, and metal-based nanostructures. These biosynthetic capacities can also be exploited to obtain high value-added products from low-cost substrates (industrial wastes and contaminants), offering the possibility to efficiently recover valuable resources and providing possible waste disposal solutions. Rhodococcus spp. strains have also recently been pointed out as a source of novel bioactive molecules highlighting the need to extend the knowledge on biosynthetic capacities of members of this genus and their potential utilization in the framework of bioeconomy. Key points • Rhodococcus possesses promising biosynthetic and bioconversion capacities. • Rhodococcus bioconversion capacities can provide waste disposal solutions. • Rhodococcus bioproducts have environmental, industrial, and medical relevance.

Diterpenes represent a wider class of isoprenoids, with more than 18,000 isolated compounds, and are present in plants, fungi, bacteria, and animals in both terrestrial and marine environments. Here, we report on the fully characterised structures of 251 new diterpenes, isolated from higher plants and published from 2017, which are shown to have antitumoral activity. An overview on the most active compounds, showing IC50 < 20 μM, is provided for diterpenes of different classes. The most active compounds were extracted from 29 different plant families; particularly, Euphorbiaceae (69 compounds) and Lamiaceae (54 compounds) were the richest sources of active compounds. A better activity than the positive control was obtained with 33 compounds against the A549 cell line, 28 compounds against the MCF-7 cell line, 9 compounds against the HepG2 cell line, 8 compounds against the Hep3B cell line, 19 compounds against the SMMC-7721 cell line, 9 compounds against the HL-60 cell line, 24 compounds against the SW480 cell line, and 19 compounds against HeLa.

Nature offers interesting structures and chemicals for photochemistry and photocatalysis. Therefore, highly performing photocatalysts have been derived with improved morphology, photoadsorption, energy transfer, and photostability. Different green synthesis methods have been developed employing phytochemicals, biotemplates, and bioreactors. Smart natural hierarchical structures and enzyme-like materials have been faithfully reproduced providing unique physico-chemical proprieties and constructing artificial photosynthesis. Hybridization with natural pigments and polymers has been investigated, obtaining a new class of heterogenous nano-photocatalysts with improved dispersibility and efficacy. A biology-inspired approach has expanded the spectrum of photocatalysis applications carrying out increasingly biocompatible and effective systems for bioremediation, active food packaging, and nanomedicine.

Rhodococcus members excrete secondary metabolites, especially compounds which act as biosurfactants. In this work, we demonstrated the ability of Rhodococcus opacus R7 to produce a novel bioactive compound belonging to the class of biosurfactants with antimicrobial properties during the growth on naphthalene. Chemical and biochemical analyses of the isolated compound demonstrated that the biosurfactant could be classified as a hydrophobic peptide. The ESI-full mass spectrometry revealed that the isolated biosurfactant showed a molecular weight of 1292 Da and NMR spectra evidenced the composition of the following amino acid residues: Ala, Thr, Asp, Gly, Ser. Surfactant activity of the R. opacus R7 compound was quantified by the critical micelle dilution (CMD) method and the critical micelle concentration (CMC) was estimated around 20 mg L−1 with a corresponding surface tension of 48 mN m−1. Moreover, biological assays demonstrated that R. opacus R7 biosurfactant peptide exhibited antimicrobial activity against Escherichia coli ATCC 29522 and Staphylococcus aureus ATCC 6538 with the minimum inhibition growth concentration (MIC) values of 2.6 mg mL−1 and 1.7 mg mL−1, respectively. In this study for the first time, a hydrophobic peptide with both biosurfactant and antimicrobial activity was isolated from a bacterium belonging to Rhodococcus genus.

Oxygenated derivatives of fatty acids, collectively called oxylipins, are a highly diverse family of lipoxygenase (LOX) products well described in planktonic diatoms. Here we report the first investigation of these molecules in four benthic diatoms, Cylindrotheca closterium, Nanofrustulum shiloi, Cocconeis scutellum, and Diploneis sp. isolated from the leaves of the seagrass Posidonia oceanica from the Gulf of Naples. Analysis by hyphenated MS techniques revealed that C. closterium, N. shiloi, and C. scutellum produce several polyunsaturated aldehydes (PUAs) and linear oxygenated fatty acids (LOFAs) related to the products of LOX pathways in planktonic species. Diploneis sp. also produced other unidentified fatty acid derivatives that are not related to LOX metabolism. The levels and composition of oxylipins in the benthic species match their negative effects on the reproductive success in the sea urchin Paracentrotus lividus. In agreement with this correlation, the most toxic species N. shiloi revealed the same LOX pathways of Skeletonema marinoi and Thalassiosira rotula, two bloom-forming planktonic diatoms that affect copepod reproduction. Overall, our data highlight for the first time a major role of oxylipins, namely LOFAs, as info-chemicals for benthic diatoms, and open new perspectives in the study of the structuring of benthic communities.

Melleins are 3,4-dihydroisocoumarins mainly produced by fungi, but also by plants, insects and bacteria. These specialized metabolites play important roles in the life cycles of the producers and they are involved in many biochemical and ecological processes. This review outlines the isolation and chemical and biological characterizations of natural-occurring melleins from the first report of (R)-mellein in 1933 to the most recent advances in their characterization in 2019. In addition, the pathways that could be involved in mellein biosynthesis are discussed, along with the enzymes and genes involved.

Resistance to antibiotics is one of the main current threats to human health and every year multi-drug resistant bacteria are infecting millions of people worldwide, with many dying as a result. Ever since their discovery, some 40 years ago, the antimicrobial peptides (AMPs) of innate defense have been hailed as a potential alternative to conventional antibiotics due to their relatively low potential to elicit resistance. Despite continued effort by both academia and start-ups, currently there are still no antibiotics based on AMPs in use. In this study, we discuss what we know and what we do not know about these agents, and what we need to know to successfully translate discovery to application. Understanding the complex mechanics of action of these peptides is the main prerequisite for identifying and/or designing or redesigning novel molecules with potent biological activity. However, other aspects also need to be well elucidated, i.e., the (bio)synthetic processes, physiological and pathological contexts of their activity, and a quantitative understanding of how physico-chemical properties affect activity. Research groups worldwide are using biological, biophysical, and algorithmic techniques to develop models aimed at designing molecules with the necessary blend of antimicrobial potency and low toxicity. Shedding light on some open questions may contribute toward improving this process.

International audience; Natural products from marine animals show high potential for the development of new medicines, but drug development based on these compounds is commonly hampered by their low natural abundance. Since many of these metabolites are suspected or known to be produced by uncultivated bacterial symbionts, the rapidly growing diversity of sequenced prokaryotic genomes offers the opportunity to identify alternative, culturable sources of natural products computationally. In this work, we investigated the potential of using this sequenced resource to facilitate the production of meroterpenoid-like compounds related to those from marine sources. This genome-mining strategy revealed a biosynthetic gene cluster for highly modified cytotoxic meroterpenoids related to pelorol and other compounds isolated from sponges. Functional characterization of the terpene cyclase MstE showed that it generates an ent-sterol-like skeleton fused to an aryl moiety from an open-chain precursor and is therefore a promising tool for the chemoenzymatic preparation of synthetically challenging chemical scaffolds.

Anthocyanins are water-soluble pigments providing certain color for various plant parts, especially in edible berries. Earlier these compounds were only known as natural food colorants, the stability of which depended on pH, light, storage temperature and chemical structure. However, due to the increase of the in vitro, in vivo experimental data, as well as of the epidemiological studies, today anthocyanins and their metabolites are also regarded as potential pharmaceutical compounds providing various beneficial health effects on either human or animal cardiovascular system, brain, liver, pancreas and kidney. Many of these effects are shown to be related to the free-radical scavenging and antioxidant properties of anthocyanins, or to their ability to modulate the intracellular antioxidant systems. However, it is generally overlooked that instead of acting exclusively as antioxidants certain anthocyanins affect the activity of mitochondria that are the main source of energy in cells. Therefore, the aim of the present review is to summarize the major knowledge about the chemistry and regulation of biosynthesis of anthocyanins in plants, to overview the facts on bioavailability, and to discuss the most recent experimental findings related to the beneficial health effects emphasizing mitochondria.

During fatty acids biosynthesis the elongating acyl chain is sequestered within the core of the highly conserved acyl carrier protein (ACP). At each catalytic step, the acyl intermediates are transiently delivered from ACP to the active site of the enzymatic counterparts and, at the same time, are protected from the solvent to prevent nonselective reactivity. Yet, the molecular determinants of such a universal transition-termed chain flipping-remain poorly understood. Here we capture the atomic-level details of the chain-flipping mechanism by using metadynamics simulations. We observe the fatty-acid chain gliding through the protein-protein interface with barely 30% of its surface exposed to water molecules. The small ACP's helix III acts as gatekeeper of the process, and we find its conformational plasticity critical for a successful substrate transfer. The results are in agreement with a wide range of experimental observations and provide unprecedented insight on the molecular determinants and driving forces of the chain-flipping process.

Several epidemiological studies have demonstrated that high vitamin E intakes are related to a reduced risk of non-communicable diseases, while other dietary antioxidants are not, suggesting that vitamin E exerts specific healthy functions in addition to its antioxidant role. In this regard, tomato (Solanum lycopersicum), one of the most consumed vegetables of the whole world population, is an important source of both tocopherols and tocotrienols. However, vitamin E content may strongly depend on several biotic and abiotic factors. In this review we will debate the elements affecting the synthesis of tocopherols and tocotrienols in tomato fruit, such as environmental conditions, genotype, fruit maturity level, and the impact of classical processing methods, such as pasteurization and lyophilization on the amount of these compounds. In addition we will analyze the specific vitamin E mechanisms of action in humans and the consequent functional effects derived from its dietary intake. Finally, we will examine the currently available molecular techniques used to increase the content of vitamin E in tomato fruit, starting from the identification of genetic determinants and quantitative trait loci that control the accumulation of these metabolites.

Lipopolysaccharide, the outer cell-wall component of Gram-negative bacteria, has been shown to be important for symbiotic associations. We recently reported that the lipopolysaccharide O-antigen of Burkholderia enhances the initial colonization of the midgut of the bean bug, Riptortus pedestris However, the midgut-colonizing Burkholderia symbionts lack the O-antigen but display the core oligosaccharide on the cell surface. In this study, we investigated the role of the core oligosaccharide, which directly interacts with the host midgut, in the Riptortus-Burkholderia symbiosis. To this end, we generated the core oligosaccharide mutant strains, ΔwabS, ΔwabO, ΔwaaF, and ΔwaaC, and determined the chemical structures of their oligosaccharides, which exhibited different compositions. The symbiotic properties of these mutant strains were compared with those of the wild-type and O-antigen-deficient ΔwbiG strains. Upon introduction into Riptortus via the oral route, the core oligosaccharide mutant strains exhibited different rates of colonization of the insect midgut. The symbiont titers in fifth-instar insects revealed significantly reduced population sizes of the inner core oligosaccharide mutant strains ΔwaaF and ΔwaaC These two strains also negatively affected host growth rate and fitness. Furthermore, R. pedestris individuals colonized with the ΔwaaF and ΔwaaC strains were vulnerable to septic bacterial challenge, similar to insects without a Burkholderia symbiont. Taken together, these results suggest that the core oligosaccharide from Burkholderia symbionts plays a critical role in maintaining a proper symbiont population and in supporting the beneficial effects of the symbiont on its host in the Riptortus-Burkholderia symbiosis.

Objectives: Lung ischemia-reperfusion injury is the main cause of primary graft dysfunction after lung transplantation and results in increased morbidity and mortality. Fas-mediated apoptosis is one of the pathologic mechanisms involved in the development of ischemia-reperfusion injury. We hypothesized that the inhibition of Fas gene expression in lungs by intratracheal administration of small interfering RNA could reduce lung ischemia-reperfusion injury in an ex vivo model reproducing the procedural sequence of lung transplantation. Design: Prospective, randomized, controlled experimental study. Setting: University research laboratory. Subjects: C57/BL6 mice weighing 28-30 g. Interventions: Ischemia-reperfusion injury was induced in lungs isolated from mice, 48 hours after treatment with intratracheal small interfering RNA targeting Fas, control small interfering RNA, or vehicle. Isolated lungs were exposed to 6 hours of cold ischemia (4°C), followed by 2 hours of warm (37°C) reperfusion with a solution containing 10% of fresh whole blood and mechanical ventilation with constant low driving pressure. Measurements and Main Results: Fas gene expression was significantly silenced at the level of messenger RNA and protein after ischemia-reperfusion in lungs treated with small interfering RNA targeting Fas compared with lungs treated with control small interfering RNA or vehicle. Silencing of Fas gene expression resulted in reduced edema formation (bronchoalveolar lavage protein concentration and lung histology) and improvement in lung compliance. These effects were associated with a significant reduction of pulmonary cell apoptosis of lungs treated with small interfering RNA targeting Fas, which did not affect cytokine release and neutrophil infiltration. Conclusions: Fas expression silencing in the lung by small interfering RNA is effective against ischemia-reperfusion injury. This approach represents a potential innovative strategy of organ preservation before lung transplantation. © 2016 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc.

Opisthobranchs belong to a subclass of highly evolved and specialised marine gastropods that rely on the use of secondary metabolites for their survival. Here we report the full elucidation of the biosynthesis of aromatic metabolites, lignarenones, in one of these gastropods, the cephalaspidean Scaphander lignarius. Feeding experiments with 2H- and 13C-labelled precursors revealed a mixed acetate/propionate polyketide pathway primed by benzoic acid. Phenylalanine ammonia lyase (PAL), unprecedented in animals, is central to the synthesis of this aromatic precursor by oxidative deamination of L-phenylalanine to cinnamic acid. Lignarenones are synthesised in the cytoplasm of specialised eukaryotic cells named Blochmann's glands, which are distributed in biosynthetic tissue localised in the vulnerable mantle of the mollusc. This result supports the hypothesis that this lineage of gastropods has acquired the genetic information to produce the chemical substances that they use for their survival.

Polypropionates that incorporate pyrones are a family of polyketides featuring the chemistry of a few marine molluscs capable of phototrophic CO2 fixation as a result of storing viable symbiotic chloroplasts in their bodies. The role and origin of these molecules is poorly investigated, although the unusual biological ACHTUNGTRENUNGactivities and chemistry of these natural products have recently received renewed interest. Here, we report the results of in vivo studies on production of g-pyrone-containing polypropionates in the Mediterranean mollusc Elysia viridis. Biosynthesis of the metabolites in the sacoglossan is shown to proceed through condensation of eight intact C3 units by polyketide synthase assembly. LC–MS and NMR spectroscopic studies demonstrate that the process involves a pyrone tetraene (10) as key intermediate, whereas the levels of the final polypropionates (6, 7 and 9) are related to each other and show a significant dependence upon light conditions.

The biosynthesis of lignarenones 1 and 2, the major polyketides of the Mediterranean mollusc Scaphander lignarius is described. The process is primed by benzoic acid and requires acetate and propionate as extender units. The labeling pattern suggests PKS-like synthesis of an unusual E,Z,E-triene chain and origin of the benzoate unit from phenylalanine. 13C-13C NMR COSY has been used to establish the labeling positions due to incorporation of 13C2-acetate.

We investigated the effect of beta-amyloid (A beta) (25-35), a cytotoxic fragment of A beta pepticle, on lipid metabolism and protein ubiquitination in cultured rat hippocampal neurons. After treatment with A beta under conditions leading to apoptotis, as assessed by caspase activity assay, the total cell mass of lipids changed following a biphasic behavior, with an increase that reached a maximum after 16 hr of treatment, followed by a decrease. The increase at 16 hr was 15.3% in the case of phospholipids and 103.0% in the case of gangliosides and was due to enhanced biosynthesis as confirmed by increase of radioactivity incorporation (phospholipids +52.0%, gangliosides +193.1%) in cells fed with tritiated palmitic acid. No change with respect to cholesterol was observed. Strikingly, under these conditions, the ubiquitination state of cell proteins strongly increased. These effects were not observed with the (35-25) reverse sequence pepticle. Similarly to A beta, lactacystin treatment increased lipid synthesis and protein ubiquitination; only lactacystin, and not A beta, induced a strong decrease of proteasome chimotrypsin activity. These results suggest that A beta enhances protein ubiquitination, without inhibiting proteasomal activity, and lipid synthesis. These results may shed new light on the mechanisms of A beta toxicity. (c) 2007 Wiley-Liss, Inc.

The use of stable isotopes has been recently introduced in the biosynthetic studies of metabolites produced by opisthobranch molluscs. This methodology offers numerous advantages since it avoids the complex and tedious manipulations of potentially dangerous radioactive compounds and gives unequivocal evidence for the incorporation. In these studies, high field NMR spectroscopy is a particularly useful tool to localize the labeled atoms in the molecule. This chapter updates the biosynthetic studies on opisthobranch molluscs with particular attention to the recent experiments with precursors labeled with stable isotopes. Opisthobranchs are able to biosynthesize de novo a wide array of chemical skeletons including polyketides, polypropionates, acetogenins, and terpenoids. The studies regarding this latter class is proposed in the light of the recent debate about classical and independent mevalonate pathway.

d -[U- 14 C]Glucosamine was rapidly taken up by oat coleoptile segments and metabolized to radioactive UDP- N -acetylglucosamine, which acted as specific glycosyl donor for the synthesis of glycolipids and cytosolic, membrane-bound and cell-wall glycoproteins. Cell-wall glycoproteins were solubilized from the walls by either cell-wall-degrading enzymes or chemical extractants. The solubilized cell-wall glycoproteins in the presence of peptide N -glycosidase F released oligosaccharide chains higher than seven glycosidic residues. The combined action of peptide N -glycosidase F and N -acetyl-β- d -glucosaminidase on cell-wall glycoproteins indicated the presence of N -acetylglucosamine residues β-1,2-linked to mannose. Less than 9% of the radioactive oligosaccharide chains was released from the solubilized cell-wall glycoproteins when treated with 0.5 M NaOH at 20°, whereas more than 45% of the radioactivity was released in the presence of 1 M NaOH at 50°. The high hydrolytic sensitivity of cell-wall glycoproteins to peptide N -glycosidase F, N -acetyl-β- d -glucosaminidase NaOH at 50° indicated that most N -acetylglucosamine residues were incorporated into N -linked cell-wall glycoproteins. Further evidence of this was obtained by the use of inhibitors of biosynthesis and processing of N -linked glycoproteins.

The biogenesis of aglajnes, polypropionate allomones of the cephalaspidean mollusc Bulla striata, has been investigated in vivo by feeding experiments. Incorporation of the committed precursor, [1-14C]-propionate, into aglajne-1 (1) and -3 (3) established the de novo origin of these compounds in B. striata. In the letter we also discuss briefly the ecological meaning of the origin of polypropionates in B. striata and in other cephalaspidean molluscs.

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor whose activity is modulated by xenobiotics as well as physiological ligands. These compounds may modulate inflammatory responses and contribute to the rising prevalence of allergic diseases observed in industrialized countries. Mast cells (MCs), located within tissues at the boundary of the external environment, represent a potential target of AhR ligands. In this study, we report that murine and human MCs constitutively express AhR, and its activation by the high-affinity ligand 6-formylindolo[3,2-b]carbazole (FICZ) determines a boost in degranulation. On the contrary, repeated exposure to FICZ inhibits MC degranulation. Accordingly, histamine release, in an in vivo passive systemic anaphylactic model, is exacerbated by a single dose and is attenuated by repetitive stimulation of AhR. FICZ-exposed MCs produce reactive oxygen species and IL-6 in response to cAMP-dependent signals. Moreover, AhR-activated MCs produce IL-17, a critical player in chronic inflammation and autoimmunity, suggesting a novel pathway for MC activation in the pathogenesis of these diseases. Indeed, histological analysis of patients with chronic obstructive pulmonary disease revealed an enrichment in AhR/IL-6 and AhR/IL-17 double-positive MCs within bronchial lamina propria. Thus, tissue-resident MCs could translate external chemical challenges through AhR by modulating allergic responses and contributing to the generation of inflammation-related diseases.

This chapter deals with feeding experiments in biosynthetic studies of secondary metabolites from marine organisms. The evolution of the field is traced through a critical report of both review papers published from the 1980s and selected studies published in the latest years. The progress in the methodologies, from simple modification of advanced precursors, through incorporation of substrates with radioactive or stable isotopes, to recent extensive use of molecular genetic techniques, served as a main guideline for the preparation of this overview. Finally, after a section dedicated to the methodologies, the biosynthesis of oxylipins from diatoms and propionates from Mediterranean sacoglossans, recently conducted in the authors’ institute, will be discussed with more detail.

Structural similarity or even the identity of polyketide compounds does not necessarily imply unique biosynthesis. Feeding experiments with a (13)C labeled precursor establish that the C(3) units in 7-methyl-cyercene-1 (1) are derived from intact propionate in the marine mollusc Ercolania funerea. The same compound in the terrestrial fungus Leptosphaeria maculans/Phoma lingam is synthesized by an acetate/SAM pathway thus proving for the first time metabolic convergence of polyketide biosynthesis in eukaryotes. Traditional (1)H-(13)C NMR correlation spectroscopy has been successfully applied to estimate (13)C incorporation in biosynthetic experiments.

Trans-AT polyketide synthases (PKSs) are a recently discovered group of biosynthetic enzymes present in many bacteria. A strategy for the prediction of natural product structures from trans-AT PKS sequences was demonstrated to be a useful approach for the targeted isolation of bioactive polyketides from chemically poorly studied prokaryotic phyla, as exemplified by the elansolid-type antibiotics.

Structural diversity of polypropionates arises from the amazing combination of acetate and propionate units. Feeding experiments with single and doubly 13 C -labelled precursors prove that the Mediterranean slug Placida dendritica utilizes intact C 3 -units for the biosynthesis of placidenes (e.g., 1 – 5 ), prototypes of a family of pyrone-containing polypropionates largely represented in fungi and marine invertebrates. These results show that fungi and molluscs have elaborated two distinct polyketide pathways for the synthesis of similar or even identical compounds.

Summary • In recent years, oxylipins (lipoxygenase-derived oxygenated fatty acid products) have been reported in several bloom-forming marine diatoms. Despite increasing attention on the ecophysiological role of these molecules in marine environments, their biosynthesis is largely unknown in these microalgae. • Biochemical methods, including tandem mass spectrometry, nuclear magnetic resonance and radioactive probes were used to identify structures, enzymatic activities and growth-dependent modulation of oxylipin biosynthesis in the pennate diatom Pseudo-nitzschia delicatissima. • Three major compounds, 15S-hydroxy-(5Z,8Z,11Z,13E,17Z)-eicosapentaenoic acid (15S-HEPE), 15-oxo-5Z,9E,11E,13E-pentadecatetraenoic acid and 13,14-threo-13R-hydroxy-14S,15S-trans-epoxyeicosa-5Z,8Z,11Z,17Z-tetraenoic acid (13,14-HEpETE), were produced by three putative biochemical pathways triggered by eicosapentaenoic acid-dependent 15S lipoxygenase. Oxylipin production increases along the growth curve, with remarkable changes that precede the demise of the culture. At least one of the compounds, namely 15-oxoacid, is formed only in the stationary phase immediately before the collapse of the culture. • Synthesis and regulation of phyco-oxylipins seem to correspond to a signaling mechanism that governs adaptation of diatoms along the growth curve until bloom termination. Factors triggering the process are unknown but synthesis of 15-oxoacid, constrained within a time-window of a few days just before the collapse of the culture, implies the involvement of a physiological control not directly dependent on distress or death of diatom cells.

This paper reports the study of terpene biosynthesis in the marine nudibranch Doriopsilla pelseneeri. In ViVo feeding experiments with 13C-glucose proved the de noVo origin of the terpene metabolites via a mevalonate pathway. Characterization of the stereochemical relationship of the new acetyl pelseneeriols (7 and 8) suggests a two-step mechanism for the sesquiterpene cyclization, leading to a slight refinement of terpene tailoring in this family of marine invertebrates.

Oxylipins are important signal transduction molecules widely distributed in animals and plants where they regulate a variety of events associated with physiological and pathological processes. The family embraces several different metabolites that share a common origin from the oxygenase-catalyzed oxidation of polyunsaturated fatty acids. The biological role of these compounds has been especially studied in mammalians and higher plants, although a varied and very high concentration of these products has also been reported from marine macroalgae. This article gives a summary of our results concerning the oxylipin chemistry of marine diatoms, a major class of planktonic microalgae that discourage predation from their natural grazers, zooplanktonic copepods, using chemical warfare. These apparently harmless microscopic cells produce a plethora of oxylipins, including short-chain unsaturated aldehydes, hydroxyl-, keto-, and epoxyhydroxy fatty acid derivatives, that induce reproductive failure in copepods through abortions, congenital malformations, and reduced larval growth. The biochemical process involved in the production of these compounds shows a simple regulation based on decompartmentation and mixing of preexisting enzymes and requires hydrolysis of chloroplast-derived glycolipids to feed the downstream activities of C16 and C20 lipoxygenases.

Marine chemical diversity is generated by a large number of transformations often not noted in terrestrial counterparts. Life in the oceans differs in most respects from life on land and our knowledge of the genetics and biochemistry of marine organisms is still very limited to a small number of species. Biosynthetic studies and biogenetic speculations can therefore be crucial in predicting relevant enzymes and their encoding genes, with a view to setting the stage for rational engineering of marine natural products. A further useful outcome to the identification of biosynthetic pathways is the resulting classification of natural products, which can serve to correlate chemical diversity and biodiversity. This review summarizes the present knowledge on secondary metabolites biogenesis in marine opisthobranchs, a class of organisms that has been emerging as a prolific source of structurally diverse metabolites possessing a broad variety of biological activities.

Immunotherapy may provide valid alternative therapy for patients with hormone-refractory metastatic prostate cancer. However, if the tumor environment exerts a suppressive action on antigen-specific tumor-infiltrating lymphocytes (TIL), immunotherapy will achieve little, if any, success. In this study, we analyzed the modulation of TIL responses by the tumor environment using collagen gel matrix–supported organ cultures of human prostate carcinomas. Our results indicate that human prostatic adenocarcinomas are infiltrated by terminally differentiated cytotoxic T lymphocytes that are, however, in an unresponsive status. We demonstrate the presence of high levels of nitrotyrosines in prostatic TIL, suggesting a local production of peroxynitrites. By inhibiting the activity of arginase and nitric oxide synthase, key enzymes of L-arginine metabolism that are highly expressed in malignant but not in normal prostates, reduced tyrosine nitration and restoration of TIL responsiveness to tumor were achieved. The metabolic control exerted by the tumor on TIL function was confirmed in a transgenic mouse prostate model, which exhibits similarities with human prostate cancer. These results identify a novel and dominant mechanism by which cancers induce immunosuppression in situ and suggest novel strategies for tumor immunotherapy.

marine invertebrates have been emerging as a very promising source of both natural products and enzymatic activities. In this contribution, we have tried to provide a general overview of the present knowledge of the enzymatic transformations and biosynthetic pathways in marine molluscs, suggesting that marine enzymes have great,yet unexplored diversity in both mechanism and structure. We hope that the extraordinary mechanism and catalytic reactivity discussed throughout the manuscript may serve as an inspiration in searching for novel biocatalytic properties.

Diatoms are unicellular plants broadly present in freshwater and marine ecosystems, where they play a primary role in sustaining the marine food chain. In the last 10 years, there has been accumulating evidence that diatoms may have deleterious effects on the hatching success of zooplankton crustaceans such as copepods, thus affecting dynamics of planktonic populations and limiting secondary production. At the molecular level, failure to hatch is ascribed to the presence of a family of inhibitory oxylipins, which we propose to collectively name polyunsaturated short-chain aldehydes (abbreviated here as PUSCAs). Here we describe the origin of PUSCAs produced by the marine diatom Skeletonema costatum via a lipoxygenase-mediated pathways involving non-esterified polyunsaturated fatty acids (PUFA). Experiments with complex lipids proved the pivotal role of chloroplast-derived glycolipids, especially monogalactosyldiacylglycerol (MGDG), in providing hexadecatrienoic acid (C16:3 omega-4), hexadecatetraenoic acid (C16:4 omega-1) and eicosapentaenoic acid (C20:5 omega-3) to the downstream process leading to 2E,4Z-octadienal (C8:2 omega-4), 2E,4Z,7-octatrienal (C8:3 omega-1) and 2E,4Z-heptadienal (C7:2 omega-3), respectively. Under physiological conditions, the hydrolytic process is associated to galactolipid hydrolyzing enzyme capable of removing fatty acids from both sn positions of glycerol.

In comparison with our knowledge of terrestrial plant and microbial systems, little is known about the biogenesis of secondary metabolites in marine organisms, such as sponges, tunicates, algae and molluscs. Although most of these compounds show more than one analogy with terrestrial metabolites, there are a few categories of products that seem to be structurally specific of marine species. One such group of compounds is formed by 3-alkylpyridine alkaloids (also named 3-alkylpiperidine alkaloids), a family of natural products that encompasses a very heterogeneous collection of molecules sharing an hypothetical common origin from a putative 3-alkylpyridine precursor (or a biochemical analogous). Herein, we describe the biosynthesis of haminol-2 (1) in the Mediterranean mollusc Haminoea orbignyana, the first in vivo evidence on the biogenesis of 3-alkylpyridines in marine organisms. The experiments prove the origin of the pyridine ring and C-12 from nicotinic acid, as well as the contribution of acetate to the formation of the alkyl chain.

The paper reports the results of biosynthetic experiments with the marine nudibranch Doriopsilla areolata. Biogensis of the enantiomeric sesquiterpenes 1 and 5 was investigated by feeding of [1-13C]-glucose, [1,2-13C2]-glucose and [1,2-13C2]-acetate. Evidence is presented that supports de novo origin of both compounds via an single metabolic pathway based on mevalonic acid.

Das gemeinsame Motiv der marinen Naturstoffklasse der Alkylpyridin-Alkaloide ist ein Stickstoffheterocyclus mit einem Alkylsubstituenten in 3-Stellung. Diese Zuschrift liefert die ersten experimentellen Hinweise zur Biosynthese dieser Verbindungen

[reaction: see text] Marine diatoms produce alpha,beta,gamma,delta-unsaturated aldehydes that have detrimental effects on the reproduction of their natural predators. The production of these defensive metabolites is suggested to involve enzymatic oxidation of polyunsaturated fatty acids. In this paper, feeding experiments with labeled precursor provide clear evidence in support of the origin of octadienals 1 and 2 from 6,9,12-hexadecatrienoic acid (5), thus proving the involvement of novel lipoxygenase/lyase activity for the oxidation of C(16) fatty acids.

The paper describes the identification of short-chain aldehydes ( 4 – 7 ) from the marine diatom Skeletonema costatum , and their effects on copepod egg viability and sea urchin cell division. Compounds 4 – 7 were isolated as carboethoxyethylidene (CET) derivatives and their characterization was performed by NMR and GCMS analyses. Evidence is presented to support the defensive role and biosynthetic origin of these compounds in S. costatum .

The biosynthesis of drimane esters in marine molluscs Dendrodoris limbata and Dendrodoris grandiflora has been reinvestigated by feeding experiments with D-[14C-(U)]- and D-[6-13C]-glucose. Both experiments proved the incorporation of the precursors in the terpenoid part of drimane esters. Use of [6-13C]-glucose also demonstrated that the labelled material was incorporated via the classical acetate/mevalonate pathway.

IL-12 activates murine and human B cells, but little information is available as to the expression and function of IL-12R on human B lymphocytes. Here we show that the latter cells, freshly isolated from human tonsils, expressed the transcripts of both β1 and β2 chains of IL-12R and that β2 chain mRNA was selectively increased (4- to 5-fold) by incubation with Staphylococcus aureus Cowan I bacteria or IL-12. B cell stimulation with IL-12 induced de novo expression of the transcripts of the two chains of IL-18R, i.e., IL-1 receptor-related protein and accessory protein-like. Functional studies showed that both IL-12 and IL-18 signaled to B cells through the NF-κB pathway. In the case of IL-12, no involvement of STAT transcription factors, and in particular of STAT-4, was detected. c-rel and p50 were identified as the members of NF-κB family involved in IL-12-mediated signal transduction to B cells. IL-12 and IL-18 synergized in the induction of IFN-γ production by tonsillar B cells, but not in the stimulation of B cell differentiation, although either cytokine promoted IgM secretion in culture supernatants. Finally, naive but not germinal center or memory, tonsillar B cells were identified as the exclusive IL-12 targets in terms of induction of NF-κB activation and of IFN-γ production.

The effect of brefeldin A (BFA) on the synthesis and incorporation of polysaccharides, proteins and glycoproteins into the cell wall of subapical coleoptile segments isolated from etiolated oat seedlings ( Avena sativa L. cv. Angelica) has been investigated. In the presence of D-[U- 14 C]-glucose, the incorporation of radioactive glycosyl residues into buffer-soluble, membrane (matrix polysaccharides) and cell wall polysaccharides was drastically inhibited by increasing concentrations of BFA up to 10 μ·mL −1 . BFA also altered the pattern of these polysaccharides suggesting a different sensitivity of glycosyltransferases toward the action of the drug. The incorporation of [U- 14 C]-glycine or L-[U- 14 C]-leucine into non-covalently- and covalently-bound cell wall proteins as well as the incorporation of radioactive N-acetylglucosamine residues into the newly synthesised oligosaccharidic chains of cytosolic, membrane and cell wall glycoproteins remained unchanged in the presence of 10 μg·mL −1 BFA. The data demonstrate that, in oat coleoptile segments, BFA specifically inhibits the synthesis of cellulose and matrix polysaccharides without altering the synthesis and incorporation of proteins and glycoproteins into the cell wall. In addition, it is demonstrated that BFA does not affect the in vivo activity of glycosyltransferases involved in the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the oligosaccharidic chains of glycoproteins.

Human NK cells cultured in the presence of IL-12 or IL-4 differentiate into cell populations with distinct patterns of cytokine secretion similar to Th1 and Th2 cells. NK cells grown in IL-12 (NK1) produce IL-10 and IFN-gamma, whereas NK cells grown in IL-4 (NK2) produce IL-5 and IL-13. Although these NK cell subsets do not differ in cytotoxic activity, NK1 cells express higher levels of cell surface CD95 (Fas) Ag than NK2 cells and are more sensitive to Ab or chemically induced apoptosis. Like Th1 cells, NK1 cells accumulate much higher levels of the IL-12Rbeta2-chain mRNA and are significantly more responsive to IL-12 than NK2 cells at the level of activation of STAT4 transcription factor. The identification of NK cell subsets that are analogous to T cell subsets suggests a new role for NK cells in innate inflammatory responses and in their effect on adaptive immunity.

Metabolites related to caulerpenyne (1), a toxic sesquiterpene featured by two enol-acetate residues, play a major role in the chemical defence of both algae of the genus Caulerpa and a few molluscs of the order Sacoglossa. Here we report the direct evidence that cell-free preparations of Oxynoe olivacea, a Mediterranean sacoglossan, transform efficiently the algal metabolite 1 to oxytoxin-2 (3), the main defensive metabolite of the mollusc. The process implies two distinct hydrolytic activities, here named LIP-1 and LIP-2, able to operate either hydrolysis of the acetyl residue at C-1 or concerted elimination of the acetyl groups at C-4 and C-13. Incubation experiments with tissue homogenates of O. olivacea or with commercially available lipases suggest a two-step mechanism that involves, in vitro, an unstable metabolite characterized as preoxytoxin-2 (4). The course of the entire process can be easily monitored by reverse phase HPLC/ESI-MS, as well as by NMR measurements, which provides direct evidence of the enzymatic mechanism leading to the formation of this last compound (4). In agreement with the literature, both fresh and frozen tissues of Mediterranean Caulerpa prolifera also have the capability to transform 1 into aldehydic derivatives, namely oxytoxin-1 (2) and oxytoxin-2 (3), through hydrolysis of the acetyl groups. However, differently from experiments with mollusc homogenates, the conversion is not complete and caulerpenyne (1) can be detected in the algal suspension for a few hours. HPLC/ESI-MS monitoring of this transformation suggests that the hydrolytic route involves different activities in the mollusc and seaweed.