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The red alga Asparagopsis taxiformis (Rhodophyta, Bonnemaisoniaceae) has been shown to produce a large diversity of halogenated volatile organic compounds, with one to four carbons. As the distribution of this alga may expand worldwide, we implemented a research program that aims to understand the functions of its specialized metabolome in marine ecosystems. Phytochemical investigations performed on A. taxiformis gametophyte stages from the Indian Ocean revealed two new highly brominated cyclopentenones named mahorone (1) and 5-bromomahorone (2). They are the first examples of natural 2,3-dibromocyclopentenone derivatives. Their structure elucidation was achieved using spectrometric methods including NMR and MS. A standardized ecotoxicological assay was used as an assessment of their role in the environment, revealing high toxicities for both compounds (EC50 0.16 μM for 1 and 2). Additionally, both compounds were evaluated in antibacterial, antifungal, and cytotoxicity assays. Compounds 1 and 2 exhibit mild antibacterial activities against the human pathogen Acinetobacter baumannii.

Crambe crambe is a Mediterranean marine sponge known to produce original natural substances belonging to two families of guanidine alkaloids, namely crambescins and crambescidins, which exhibit cytotoxic and antiviral activities. These compounds are therefore considered as potential anticancer drugs. The present study focuses on the environmental assessment of a novel in vivo process for the production of pure crambescin and crambescidin using sponge specimens cultured in aquarium. The assessment was performed following the ISO 14040 standard and extended from the production of the different mass and energy flows to the system to the growth of the sponge in indoor aquarium and further periodic extraction and purification of the bioactive compounds. According to the results, the two stages that have a remarkable contribution to all impact categories are the purification of the bioactive molecules followed by the maintenance of the sponge culture in the aquarium. Among the involved activities, the production of the chemicals (particularly methanol) together with the electricity requirements (especially due to the aquarium lighting) are responsible for up to 90% of the impact in most of the assessed categories. However, the contributions of other stages to the environmental burdens, such as the collection of sponges, considerably depend on the assumptions made during the inventory stage. The simulation of alternative scenarios has led to propose improvement alternatives that may allow significant reductions ranging from 20% to 70%, mainly thanks to the reduction of electricity requirements as well as the partial reuse of methanol., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although the physiological impact of the actinide elements as nuclear toxicants has been widely investigated for half a century, a description of their interactions with biological molecules remains limited. It is however of primary importance to better assess the determinants of actinide speciation in cells and more generally in living organisms to unravel the molecular processes underlying actinide transport and deposition in tissues. The biological pathways of this family of elements in case of accidental contamination or chronic natural exposure (in the case of uranium rich soils for instance) are therefore a crucial issue of public health and of societal impact. Because of the high chemical affinity of those actinide elements for phosphate groups and the ubiquity of such chemical functions in biochemistry, phosphate derivatives are considered as probable targets of these cations. Among them, nucleotides and in particular adenosine mono- (AMP) and triphosphate (ATP) nucleotides occur in more chemical reactions than any other compounds on the earth's surface, except water, and are therefore critical target molecules. In the present study, we are interested in trans-plutonium actinide elements, in particular americium and curium that are more rarely considered in environmental and bioaccumulation studies than early actinides like uranium, neptunium and plutonium. A first step in this strategy is to work with chemical analogues like lanthanides that are not radioactive and therefore allow extended physical chemical characterization to be conducted that are difficult to perform with radioactive materials. We describe herein the interaction of lutetium(III) with adenosine AMP and ATP. With AMP and ATP, insoluble amorphous compounds have been obtained with molar ratios of 1:2 and 1:1, respectively. With an excess of ATP, with 1:2 molar ratio, a soluble complex has been obtained. A combination of spectroscopic techniques (IR, NMR, ESI-MS, EXAFS) together with quantum chemical calculations has been implemented in order to assess the lutetium coordination arrangement for the two nucleotides. In all the complexes described in the article, the lutetium cation is coordinated by the phosphate groups of the nucleotide plus additional putative water molecules with various tridimensional arrangements. With AMP 1:2 and ATP 1:1 solid-state compounds, polynuclear complexes are assumed to be obtained. In contrast, with ATP 1:2 soluble compound, the Lu coordination sphere is saturated by two ATP ligands, and this favors the formation of a mononuclear complex. In order to further interpret the EXAFS data obtained at the Lu LIII edge, model structures have been calculated for the 1:1 and 1:2 ATP complexes. They are discussed and compared to the EXAFS best fit metrical parameters.

Aqueous and organic fractions from the crude extracts of 17 sponge species collected at Boca de Calderas, Havana, Cuba were analysed. The organic fractions of Mycale laxissima, Clathria echinata and Agelas cerebrum exhibited values of concentrations causing 50% inhibition of in vitro growth of Plasmodium berghei (IC50) of 42.3 ± 5.1, 52 ± 9.7 and 60.3 ± 10.6 μg/mL, respectively, while their selectivity indexes for fibroblast cell lines were 9.45, 4.24 and 8.7, correspondingly. These fractions reduced parasitemia of infected Balb/c mice as well. Selective cytotoxicity indexes against tumour HeLa cells focused an interest on the aqueous fraction of M. laxissima (>7.12) and organic fractions of Polymastia nigra (5.95), A. cerebrum (5.48) and Niphates erecta (>4.2). Triterpenoids/steroids and alkaloids detected in the organic fractions of M. laxissima, C. echinata and A. cerebrum should be isolated for future investigation.

In this paper, we show the effect of crambescidin-816, -800, and -830 on Saccharomyces cerevisiae viability. We determined that, of the three molecules tested, crambescidin-816 was the most potent. Based on this result, we continued by determining the effect of crambescidin-816 on the cell cycle of this yeast. The compound induced cell cycle arrest in G2/M followed by an increase in cell DNA content and size. When the type of cell death was analyzed, we observed that crambescidin-816 induced apoptosis. The antifungal effect indicates that crambescidins, and mostly crambescidin-816, could serve as a lead compound to fight fungal infections.

The extraction and purification of the bioactive extract of Cystodytes violatinctus (Solomon Islands) led to the isolation and identification of six pyridoacridine alkaloids. The structures of four new members of this family, shermilamine F (1), dehydrokuanoniamine F (2), and arnoamines C (3) and D (4), were elucidated on the basis of NMR and MS data and by comparison with data of known compounds isolated from this genus. A general hypothetical biogenetic pathway is then proposed for pyridoacridine alkaloids that contain a fused pyrrole ring. Comparison of the biological properties of the isolated alkaloids is also discussed.

The marine environment offers both economic and scientific potential which are relatively untapped from a biotechnological point of view. These environments whilst harsh are ironically fragile and dependent on a harmonious life form balance. Exploitation of natural resources by exhaustive wild harvesting has obvious negative environmental consequences. From a European industry perspective marine organisms are a largely underutilised resource. This is not due to lack of interest but due to a lack of choice the industry faces for cost competitive, sustainable and environmentally conscientious product alternatives. Knowledge of the biotechnological potential of marine organisms together with the development of sustainable systems for their cultivation, processing and utilisation are essential. In 2010, the European Commission recognised this need and funded a collaborative RTD/SME project under the Framework 7-Knowledge Based Bio-Economy (KBBE) Theme 2 Programme 'Sustainable culture of marine microorganisms, algae and/or invertebrates for high value added products'. The scope of that project entitled 'Sustainable Production of Biologically Active Molecules of Marine Based Origin' (BAMMBO) is outlined. Although the Union is a global leader in many technologies, it faces increasing competition from traditional rivals and emerging economies alike and must therefore improve its innovation performance. For this reason innovation is placed at the heart of a European Horizon 2020 Strategy wherein the challenge is to connect economic performance to eco performance. This article provides a synopsis of the research activities of the BAMMBO project as they fit within the wider scope of sustainable environmentally conscientious marine resource exploitation for high-value biomolecules., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The chemical investigation of the recently described Mediterranean Homoscleromorpha sponge Oscarella balibaloi revealed an original family of five closely related glucosylated sesterterpenes 1-4, named balibalosides. Their structure elucidation was mainly inferred from NMR and HRMS data analyses. Balibalosides differ by the pattern of acetyl substitutions on the three sugar residues linked to the same aglycone sesterterpenoid core. From a biosynthetic perspective, these compounds may represent intermediates in the pathways leading to more complex sesterterpenes frequently found in Dictyoceratida, a sponge Order belonging to Demospongiae, a clade which is phylogenetically distinct from the Homoscleromorpha. While steroid and triterpenoid saponins were already well known from marine sponges, balibalosides are the first examples of glycosilated sesterterpenes.

Coatline A (1) and α-epi-coatline A (4) co-occur in the trunk extract of Andira coriacea. Inspection of their chiroptical properties led to intriguing results. After a careful examination of the experimental data used for the previously reported absolute configuration of these compounds, some uncertainties were identified. A combined theoretical approach including conformational analyses and calculation of electronic circular dichroism (ECD) spectra, in addition with experimental data obtained for schoepfin A (5) and the new schoepfin D (6) isolated from Senna quinquangulata, allowed the revision of the absolute configuration of coatlines A (1) and B (2)., (Copyright © 2012 Wiley Periodicals, Inc.)

Three new triterpene glycosides, named ulososide F (1), urabosides A (2) and B (3), together with the previously reported ulososide A (4), were isolated from the Caribbean marine sponge Ectyoplasia ferox. Their structures were elucidated using extensive interpretation of 1D and 2D-NMR data, as well as HRESIMS. The aglycon of all compounds is a rare 30-norlonastane and the sugar residues were identified after acid hydrolysis and GC analyses. Cytotoxicities of the isolated compounds were evaluated against Jurkat and CHO cell lines by a MTT in vitro assay as well as a hemolysis assay. Unexpectedly, all these saponin derivatives showed very low activity in our bioassays.

A new chlorinated sesquiterpenoid analogue of fumagillin, ligerin (1), was isolated from a marine-derived strain of Penicillium, belonging to the subgenus Penicillium, along with the known compounds penicillic acid (2), orcinol, and orsellinic acid. Chemical structures were established by an interpretation of spectroscopic data including IR, UV, and HRESIMS, together with analyses of 1D and 2D NMR spectra and X-ray analysis for the determination of the absolute configuration. Ligerin (1) displayed strong inhibitory activity against an osteosarcoma cell line. This is the first report of the isolation of a fumagillin analogue from a marine-derived Penicillium strain.

Crambescins and crambescidins are two families of guanidine alkaloids from the marine sponge Crambe crambe. Although very little information about their biological effect has been reported, it is known that crambescidin 816 (Cramb816) blocks calcium channels in a neuroblastoma X glioma cell line. Taking this into account, and the fact that ion channels are frequent targets for natural toxins, we examined the effect of Cramb816 and three compounds from the crambescin family, norcrambescin A2 (NcrambA2), crambescin A2 (CrambA2), and crambescin C1 (CrambC1), in the main voltage-dependent ion channels in neurons: sodium, potassium, and calcium channels. Electrophysiological recordings of voltage gated sodium, potassium, and calcium currents, in the presence of these guanidine alkaloids, were performed in cortical neurons from embryonic mice. Different effects were discovered: crambescins inhibited K(+) currents with the following potency: NcrambA2 > CrambC1 > CrambA2, while Cramb816 lacked an effect. Only CrambC1 and Cramb816 partially blocked Na(+) total current. However, Cramb816 partially blocked Ca(2+) , while NcrambA2 did not. Since the blocking effect of Cramb816 on calcium currents has not been previously reported in detail, we further pharmacologically isolated the two main fractions of HVA Ca(2+) channels in neurons and investigated the Cramb816 effect on them. Here, we revealed that Cav1 or L-type calcium channels are the main target for Cramb816. These two families of guanidine alkaloids clearly showed a structure-activity relationship with the crambescins acting on voltage-gated potassium channels, while Cramb816 blocks the voltage-gated calcium channel Cav1 with higher potency than nifedipine. The novel evidence that Cramb816 partially blocked CaV and NaV channels in neurons suggests that this compound might be involved in decreasing the neurotransmitter release and synaptic transmission in the central nervous system. The findings presented here provide the first detailed approach on the different effects of crambescin and crambescidin compounds in voltage-gated sodium, potassium, and calcium channels in neurons and thus provide a basis for future studies.

The marine sponge Haliclona fulva (previously described as Reniera fulva) is widespread in the Mediterranean Sea. The chemical study of the sponge led to the isolation and identification of a new compound: 3-epi-cladocroic acid (1) alongside the previously reported cladocroic acid (2) and some other known compounds previously isolated. The structure was fully determined on the basis of extensive analysis by 1D and 2D NMR, as well as GC-MS/MS. The absolute configuration was determined by comparison of the experimental electronic circular dichroism (ECD) spectra with theoretically calculated spectra; these results may be extended to other asymetric cyclopropane carboxylic acids.

The first phytochemical inspection of the Marquesan endemic plant Rauvolfia nukuhivensis led to the isolation and structure characterization of two new indolo[2,3-a]quinolizinium derivatives named nukuhivensium and N₁₂-methyl-nukuhivensium. They feature an aromatic indolo[2,3-a]quinolizinium core, substituted at C-2 by a n-propyl group, which is unusual in this family of alkaloid derivatives. The structure elucidation was performed on the basis of NMR spectroscopy and especially by interpretation of 2D HMBC correlations. A biosynthetic pathway is proposed on the basis of known enzymatic transformations for this family of natural products. These compounds exhibited low antimicrobial activities.

While marine organisms such as bivalves, seagrasses and macroalgae are commonly used as biomonitors for the environment pollution assessment, widely distributed sponges received little attention as potential helpful species for monitoring programmes. In this study, the trace element and radionuclide bioaccumulation and retention capacities of some marine sponges were estimated in a species-comparative study using radiotracers technique. Six Mediterranean species were exposed to background dissolved concentrations of (110m)Ag, (241)Am, (109)Cd, (60)Co, (134)Cs, (54)Mn, (75)Se and (65)Zn allowing the assessment of the uptake and depuration kinetics for selected elements. Globally, massive demosponges Agelas oroides, Chondrosia reniformis and Ircinia variabilis displayed higher concentration factor (CF) than the erectile ones (Acanthella acuta, Cymbaxinella damicornis, Cymbaxinella verrucosa) at the end of exposure, suggesting that the morphology is a key factor in the metal bioaccumulation efficiency. Considering this observation, two exceptions were noted: (1) A. acuta reached the highest CF for (110m)Ag and strongly retained the accumulated metal without significant Ag loss when placed in depuration conditions and (2) C. reniformis did not accumulate Se as much as A. oroides and I. variabilis. These results suggest that peculiar metal uptake properties in sponges could be driven by specific metabolites or contrasting biosilification processes between species, respectively. This study demonstrated that sponges could be considered as valuable candidate for biomonitoring metal contamination but also that there is a need to experimentally highlight metal-dependant characteristic among species., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The aqueous ethanol extract of Thalassia testudinum leaves (BM-21) is now being developed in Cuba as an herbal medicine due to its promising pharmacological properties. Although some interesting biological activities of BM-21 have already been reported, its chemical composition remains mostly unknown. Thus, we now describe the qualitative and quantitative analyzes of BM-21 using standard phytochemical screening techniques, including colorimetric quantification, TLC and HPLC analyses. Phytochemical investigation of BM-21 resulted in the isolation and identification of a new phenolic sulfate ester (1), along with ten previously described phenolic derivatives (2-11), seven of which have never been previously reported from the genus Thalassia. The structures of these compounds were established by analysis of their spectroscopic (1D and 2D NMR) and spectrometric (HRMS) data, as well as by comparison of these with those reported in the literature. Furthermore, BM-21 was found to exhibit strong antioxidant activity in four different free radical scavenging assays (HO*, RO2*, O2-* and DPPH*). Consequently, this is the first study which highlights the phytochemical composition of BM-21 and demonstrates that this product is a rich source of natural antioxidants with potential applications in pharmaceutical, cosmetic and food industries.

Since more than 50 years, sponges have raised the interest of natural product chemists due to the presence of structurally original secondary metabolites. While the main objective were first to discover new drugs from the Sea, a large number of interrogations arose along with the isolation and structure elucidations of a wide array of original architectures and new families of natural products not found in the terrestrial environment. In this chapter, we focus on the results obtained during this period on the following questions. A preliminary but still unresolved issue to be addressed will be linked to the role of the microbiota into the biosynthesis of these low-weight compounds. Our knowledge on the biosynthetic pathways leading to plant secondary metabolites is now well established, and this background will influence our comprehension of the biosynthetic events occurring in a sponge. But is the level of similarity between both metabolisms so important? We clearly need more experimental data to better assess this issue. This question is of fundamental interest because sponges have a long evolutionary history, and this will allow a better understanding on the transfer of the genetic information corresponding to the biosynthesis of secondary metabolites. After the how, the why! The question of the ecological role of these metabolites is also of high importance first not only because they can serve as synapomorphic characters but also because they may represent chemical cues in the water environment. Even if most of these compounds are considered as defensive weapons for these sessile invertebrates, they may also be linked to physiological characters as the reproduction. Finally, a metabolomic approach can appear as a complementary tool to give additional information on the sponge fitness. All the new developments in molecular biology and bioanalytical tools will open the way for a better comprehension on the complex field of sponge secondary metabolites., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The chemical composition of the aerial parts of the Cape Verdean endemic shrub Artemisia gorgonum Webb (Asteraceae) was careful investigated, which led to the isolation and identification of six known furfuran lignans: eudesmin (1), magnolin (2), epimagnolin A (3), aschantin (4), kobusin (5), sesamin (6) and a flavone: artemetin (7). Compounds 1-7 were evaluated in vitro for their cytotoxicity in a screening panel consisting of various mammalian tumor cell lines, for their antimalarial activity against chloroquine-resistant Plasmodium falciparum (FcB1 strain) and for their cytotoxicity against murine normal cells (CFU-GM). While no promising cytotoxicity against human tumor cells were noticed, marginal potency and selectivity was found for compounds 1-5 against murine colon 38. Besides, compounds 2-7 showed mild antiplasmodial activities, 6 and 7 being the most active compounds (IC(50) 3.37 and 3.50 μg/ml respectively) without noticeable toxicity on mammalian normal cells. This is the first report of antiplasmodial activity for furfuran lignans and the first isolation of 1-7 from Artemisia gorgonum., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Six new steroidal saponins, pandarosides K-M (1-3) and their methyl esters (4-6), were isolated as minor components, after a careful chemical reinvestigation of the Caribbean sponge Pandaros acanthifolium. Their structures were established on the basis of spectroscopic analyses and comparison with the data obtained from previous metabolites of this family. All new compounds showed moderate to weak activity against four parasitic protozoa. Additionally, these compounds and previously reported pandarosides and acanthifoliosides were tested on three human tumour cell lines, and their haemolytic and liposome permeabilizing activity were assessed. Two pandarosides exhibited moderate to strong cytotoxic effect, while three acanthifoliosides showed strong haemolytic activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chemical investigation of the Mediterranean gorgonian Paramuricea clavata resulted in the isolation of two new alkaloids, 2-bromo-N-methyltryptamine (1) and 3-bromo-N-methyltyramine (2), together with nine known compounds (3-10 and linderazulene). The bromoindole derivative 3 is reported herein for the first time from a natural source. The chemical structures of these compounds were assigned by spectroscopic analyses and comparison with literature values. The antifouling activity and toxicity of compounds 1-10 were assessed using three marine biofilm bacteria and the Microtox assay. In contrast to commercial antifoulants, bufotenine (5) and 1,3,7-trimethylisoguanine (10) showed significant antiadhesion activity against one bacterial strain while being nontoxic.

Sponge natural product biosynthesis: A highly sensitive in vivo protocol based on (14)C radiolabeled precursors and beta-imager autoradiography allowed the unraveling of the origin of the pyrrole 2-aminoimidazole-containing key biosynthetic intermediate oroidin. Proline and lysine are now proposed as the early precursors of the pyrrole and the 2-aminoimidazole moieties of oroidin respectively., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The apolar fraction F1 of Thalassia testudinum was chemically characterized by gas chromatography-mass spectrometry, which led to the identification of 43 metabolites, all of them reported for the first time in the genus Thalassia. More than 80% of the F1 composition was constituted by aromatic metabolites including the major components 1,1-bis(p-tolyl)ethane (6.0%), 4,4'-diisopropylbiphenyl (4.8%) and a 1,1-bis(p-tolyl)ethane isomer (4.7%). This lipophilic fraction was assayed for its antioxidant effects and skin protective action. In vitro assays showed that F1 strongly scavenged DPPH* (IC(50) 312.0 ± 8.0 μg mL(-1)), hydroxyl (IC(50) 23.8 ± 0.5 μg mL(-1)) and peroxyl radical (IC(50) 6.6 ± 0.3 μg mL(-1) ), as well as superoxide anion (IC(50) 50.0 ± 0.7 μg mL(-1)). Also, F1 markedly inhibited the spontaneous lipid peroxidation (LPO) in brain homogenates (IC(50) 93.0 ± 6.0 μg mL(-1)) and the LPS-stimulated nitrite generation on RAW624.7 macrophages (58.6 ± 3.2%, 400 μg mL(-1)). In agreement with these findings, its topical application at 250 and 500 μg cm(-2) strikingly reduced skin damage on mice exposed to acute UVB radiation by 45% and 70%, respectively and significantly attenuated the LPO developed following the first 48 h after acute exposure to UVB irradiation, as manifested by the decreased malondialdehide level and by the increased of reduced gluthatione content. Our results suggest that F1 may contribute to skin repair by attenuating oxidative stress due to its antioxidant activity., (© 2011 The Authors. Photochemistry and Photobiology © 2011 The American Society of Photobiology.)

The chemical composition of Tornabenea bischoffii J. A. Schmidt essential oil from Cape Verde was studied by GC and GC/MS. Sixty volatile compounds were identified of which myristicin was the major constituent (33.6%). Using the 2,2-diphenyl-2-picrylhydrazyl free-radical scavenging method and the in vitro assay for prevention of lipid peroxidation by thiobarbituric reactive species, significant antioxidant activities were evidenced.

Citharoxazole (1), a new batzelline derivative featuring a benzoxazole moiety, was isolated from the Mediterranean deep-sea sponge Latrunculia (Biannulata) citharistae Vacelet, 1969, together with the known batzelline C (2). This is the first chemical study of a Mediterranean Latrunculia species and the benzoxazole moiety is unprecedented for this family of marine natural products. The structure was mainly elucidated by the interpretation of NMR spectra and especially HMBC correlations., (Copyright © 2011 John Wiley & Sons, Ltd.)

Lysophospholipids (LPLs) are recognized as important signaling molecules in metazoan cells. LPLs seem to be widely distributed among marine invertebrates, but their physiological role remains poorly known. Marine sponges produce original phospholipids and LPLs whose isolation and structural elucidation rarely have been reported. Two LPLs were isolated for the first time from the Mediterranean Homoscleromorph sponge Oscarella tuberculata: a bioactive lyso-PAF already identified in some other sponge species; and the new lysophosphatidylethanolamine C20:2 (LPE 1). The expression of LPL metabolites was investigated over time to determine their baseline variations and to relate them to the sponge reproduction pattern in order to better understand their putative role in the sponge life cycle. Expression levels of both compounds appeared to be highly correlated displaying significant seasonal fluctuations with maximal values in summer and minimal in winter. A significant higher LPL content was detected in reproductive sponges and especially in females, with a peak occurring during embryogenesis and larval development. The results suggest that LPLs could play a role of mediators in sponge embryogenesis and morphogenesis.

Background: Acid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats., Results: Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21., Conclusions: To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

The intra-specific diversity in secondary metabolites can provide crucial information for understanding species ecology and evolution but has received limited attention in marine chemical ecology. The complex nature of diversity is partially responsible for the lack of studies, which often target a narrow number of major compounds. Here, we investigated the intra-specific chemical diversity of the Mediterranean sponge Spongia lamella. The chemical profiles of seven populations spreading over 1200 km in the Western Mediterranean were obtained by a straightforward SPE-HPLC-DAD-ELSD process whereas the identity of compounds was assessed by comparison between HPLC-MS spectra and literature data. Chemical diversity calculated by richness and Shannon indexes differed significantly between sponge populations but not at a larger regional scale. We used factor analysis, analysis of variance, and regression analysis to examine the chemical variability of this sponge at local and regional scales, to establish general patterns of variation in chemical diversity. The abundance of some metabolites varied significantly between sponge populations. Despite these significant differences between populations, we found a clear pattern of increasing chemical dissimilarity with increasing geographic distance. Additional large spatial scale studies on the chemical diversity of marine organisms will validate the universality or exclusivity of this pattern.

Nine bromotyrosine-derived compounds were isolated from the Caribbean marine sponge Verongula rigida. Two of them, aeroplysinin-1 (1) and dihydroxyaerothionin (2), are known compounds for this species, and the other seven are unknown compounds for this species, namely: 3,5-dibromo-N,N,N-trimethyltyraminium (3), 3,5-dibromo-N,N,N, O-tetramethyltyraminium (4), purealidin R (5), 19-deoxyfistularin 3 (6), purealidin B (7), 11-hydroxyaerothionin (8) and fistularin-3 (9). Structural determination of the isolated compounds was performed using one- and two-dimensional NMR, MS and other spectroscopy data. All isolated compounds were screened for their in vitro activity against three parasitic protozoa: Leishmania panamensis, Plasmodium falciparum and Trypanosoma cruzi. Compounds 7 and 8 showed selective antiparasitic activity at 10 and 5 μM against Leishmania and Plasmodium parasites, respectively. Cytotoxicity of these compounds on a human promonocytic cell line was also assessed.

Secondary metabolite production is assumed to be costly and therefore the resource allocation to their production should be optimized with respect to primary biological functions such as growth or reproduction. Sponges are known to produce a great diversity of secondary metabolites with powerful biological activities that may explain their domination in some hard substrate communities both in terms of diversity and biomass. Oscarella balibaloi (Homoscleromorpha) is a recently described, highly dynamic species, which often overgrows other sessile marine invertebrates. Bioactivity measurements (standardized Microtox assay) and metabolic fingerprints were used as indicators of the baseline variations of the O. balibaloi secondary metabolism, and related to the sponge reproductive effort over two years. The bioactivity showed a significant seasonal variation with the lowest values at the end of spring and in early summer followed by the highest bioactivity in the late summer and autumn. An effect of the seawater temperature was detected, with a significantly higher bioactivity in warm conditions. There was also a tendency of a higher bioactivity when O. balibaloi was found overgrowing other sponge species. Metabolic fingerprints revealed the existence of three principal metabolic phenotypes: phenotype 1 exhibited by a majority of low bioactive, female individuals, whereas phenotypes 2 and 3 correspond to a majority of highly bioactive, non-reproductive individuals. The bioactivity was negatively correlated to the reproductive effort, minimal bioactivities coinciding with the period of embryogenesis and larval development. Our results fit the Optimal Defense Theory with an investment in the reproduction mainly shaping the secondary metabolism variability, and a less pronounced influence of other biotic (species interaction) and abiotic (temperature) factors.

The chemical composition of the Caribbean sponge Pandaros acanthifolium was reinvestigated and led to the isolation of 12 new steroidal glycosides, namely, pandarosides E-J (1-6) and their methyl esters (7-12). Their structures were determined on the basis of extensive spectroscopic analyses, including two-dimensional NMR and HRESIMS data. Like the previously isolated pandarosides A-D (13-16), the new compounds 1-12 share an unusual oxidized D-ring and a cis C/D ring junction. The absolute configurations of the aglycones were assigned by interpretation of CD spectra, whereas the absolute configurations of the monosaccharide units were determined by chiral GC analyses of the acid methanolysates. The majority of the metabolites showed in vitro activity against three or four parasitic protozoa. Particularly active were the compounds 3 (pandaroside G) and its methyl ester (9), which potently inhibited the growth of Trypanosoma brucei rhodesiense (IC(50) values 0.78 and 0.038 microM, respectively) and Leishmania donovani (IC(50)'s 1.3 and 0.051 microM, respectively).

A method involving flash chromatography, semi-preparative phenylhexyl RP HPLC-DAD-ELSD combined with analytic polar-RP HPLC-DAD, was applied to separate and purify six highly nitrogenated bases and a bicyclic amidine alkaloid, the major components of the marine sponge Niphates digitalis. Their structures were identified as 1,8-diazabicyclo[5.4.0]undec-7-ene (1), deoxycytidine (2), phenylalanine (3), adenosine (4), deoxyguanosine (5), adenine (6) and thymidine (7) on the basis of spectroscopic data analyses. This is the first report of these compounds in a marine sponge belonging to the Niphates genus and the first evidence of the presence of 1 from a natural source.

A new footprinting method for mapping protein interactions has been developed, using tritium as a radioactive label. As residues involved in an interaction are less labeled when the complex is formed, they can be identified via comparison of the tritium incorporation of each residue of the bound protein with that of the unbound one. Application of this footprinting method to the complex formed by the histone H3 fragment H3(122-135) and the protein hAsf1A(1-156) afforded data in good agreement with NMR results.

The chemical composition of Artemisia gorgonum Webb essential oil from Cape Verde was analyzed by GC and GC/MS. A total of 111 volatile compounds, accounting for 94.9% of the essential oil, were identified by GC and GC/MS. The major compounds were camphor (28.7%), chrysanthenone (10.8%), lavandulyl 2-methylbutanoate (9.5%), alpha-phellandrene (5.5%), lavandulyl propanoate (4.2%), camphene (4.0%), and p-cymene (3.4%). The volatile oil of this endemic plant, which is used in Cape Verdean folk medicine against several ailments, was tested for its antioxidant and antimalarial properties, and was found to exhibit free-radical scavenging on 1,1-diphenyl-2-picrylhydrazyl (DPPH), prevention of lipid peroxidation-in vitro by TBARS (thiobarbituric acid reactive species) assay, and antiplasmodial activity.

The chemical composition of the Caribbean sponge Pandaros acanthifolium was investigated and led to the isolation of seven new steroidal glycosides namely pandarosides A-D (1, 3, 4 and 6) along with the three methyl esters of pandarosides A, C, and D (2, 5 and 7). Their structures were characterized as 3beta-[beta-glucopyranosyl-(1-->2)-beta-glucopyranosyloxyuronic acid]-16-hydroxy-5alpha,14beta-poriferast-16-ene-15,23-dione (1) and its methyl ester (2), 3beta-[beta-glucopyranosyloxyuronic acid]-16-hydroxy-5alpha,14beta-poriferast-16-ene-15,23-dione (3), 3beta-[beta-glucopyranosyl-(1-->2)-beta-glucopyranosyloxyuronic acid]-16-hydroxy-5alpha,14beta-cholest-16-ene-15,23-dione (4) and its methyl ester (5), 3beta-(beta-glucopyranosyloxyuronic acid)-16-hydroxy-5alpha,14beta-cholest-16-ene-15,23-dione (6) and its methyl ester (7) on the basis of detailed spectroscopic analyses, including 2D NMR and HRESIMS studies. Pandarosides A-D and their methyl esters (1-7) are all characterized by a rare 2-hydroxycyclopentenone D-ring with a 14beta configuration. The absolute configuration of the aglycon part of pandaroside A (1) was assigned by comparison between experimental and TDDFT calculated circular dichroism spectra on the more stable conformer.

Seven new guanidine alkaloids (1-7) together with the known batzelladines A, F, H, and L, ptilomycalin A, and fromiamycalin were isolated from the Caribbean marine sponges Monanchora arbuscula and Clathria calla. Molecular structures were assigned on the basis of detailed analysis of 1D and 2D NMR spectra and mass spectrometry data, and bioactivities of the alkaloids were evaluated against human cancer cell lines and malaria protozoa.

The chemical composition of essential oil from the air-dried aerial parts of Satureja forbesii (Benth.) Briq. from Cape Verde was studied by GC and GC/MS. Thirty-nine volatile compounds were identified of which geranial (42.0%) and neral (31.2%) were the major constituents. Using the 2,2-diphenyl-2-picrylhydrazyl free-radical scavenging method and the in vitro assay for prevention of lipid peroxidation by thiobarbituric reactive species, significant activities were evidenced.

Five new hydantoin alkaloids, named parazoanthines A-E (1-5), were isolated as the major constituents of the Mediterranean sea anemone Parazoanthus axinellae. Their structural elucidation was achieved through NMR spectroscopic and mass spectrometric analyses. The absolute configuration of the chiral compounds 1 and 4 was determined by comparison between experimental and TDDFT-calculated CD spectra. The configuration of the trisubstituted double bond of 2, 3, and 5 was deduced from the (3)J(H6-C4) coupling constant value. This family of alkaloids represents the first example of natural 3,5-disubstituted hydantoins that do not exhibit a methyl at N-3. All compounds were tested for their natural toxicity (Microtox assay), and parazoanthine C (3) exhibited the highest natural toxicity.

Leaves and flowers of Artemisia gorgonum (Asteraceae) collected in Fogo, Cape Verde islands, were phytochemically investigated and resulted in isolation and characterization of three guaianolides 1, 2, 5, and a secoguainolide 4, in addition to eight known guaianolides 6-11 and two known germacranolides 12, 13. Structures were elucidated by 1D and 2D NMR experiments. Careful examination of the (13)C NMR spectrum led to revision of the structure of a previously described guaianolide from 2 to 3. Most compounds exhibited mild antiplasmodial activities, ridentin (13) being the most interesting with an IC(50) of 3.8+/-0.7microgml(-1) against Plasmodium falciparum FcB1 and weak cytotoxicity in a vero cell line (IC(50) 71.0+/-3.9microgml(-1)).

A general strategy to localize and quantify carbon-centered radicals within proteins is described. The methodology was first exemplified on amino acids and then on a peptide. This method is applicable to any protein system regardless of size, and the site of hydrogen abstraction by *OH on all residues within proteins is easily and accurately detected.

The bioassay-guided fractionation of the crude extract of the marine sponge Axinella weltneri led to the isolation and the identification of a new triterpene named sodwanone S (1), with an uncommon oxepane-cyclohexane system, along with the known sodwanones A and G. The structure was elucidated using spectroscopic data, and the biological activity was evaluated against 13 human tumor cell lines. A biogenetic pathway of this new compound is also proposed.

The new iso-, nor-, and dinor-spiculoic acids A (1, 2, and 3, respectively) with a rare spiculane skeleton were isolated from the marine sponge Plakortis zyggompha, collected in the waters south of Martinique. The structural determination of the compounds was based on 1D and 2D NMR studies and mass spectral determinations. Compounds 1 and 2 showed weak cytotoxicity against the two tumor cell lines A549 and HT29.

In previous studies with feed-restricted broiler breeders, it was found that supplementation with dietary Trp decreases aggressive activity more in socially dominant males than in socially subordinate males. Although it is probable that this effect is mediated centrally by serotonin, an alternative possibility is that it is associated with other metabolic products of Trp such as niacin. The objectives of this study were to determine whether 1) supplemental dietary niacinamide decreases aggression in broiler breeder males, 2) elevated blood niacin levels are associated with a decrease in aggression, and 3) social status influences any of these effects on behavior. Using a randomized complete block design, a control (0.19% Trp, 22 mg niacin/kg), Trp (1.5% Trp, 22 mg niacin/kg), or niacinamide (0.19% Trp, supplemented with 140 mg niacinamide/kg) diet was assigned to each pen. There were 16 birds per pen and three pens per treatment. Birds were maintained on skip-a-day feed restriction throughout, and behavioral observations were conducted until the social hierarchy in each pen was determined. Treatment diets were then fed from 15 through 18 wk of age, and the number of aggressive pecks and threats per pen recorded in three 20-min observation periods/wk. Blood samples were collected from dominant and subordinate birds and assayed for niacin using Tetrahymena pyriformis. Supplementing the diet with Trp significantly (P < 0.05) decreased aggression. Niacinamide-treated birds did not differ from either Trp-treated or control-treated birds, and no decreases in aggression were seen in birds with elevated blood niacin levels. Blood levels of niacin were, however, higher in dominant niacin-treated birds than in dominant control birds (P < 0.05), although there were no differences among subordinate or Trp-treated birds. Thus, the modulatory effect of Trp on aggression does not appear to be mediated by increased niacin synthesis.

Two experiments were conducted to study the effect of feeding raw or treated common vetch seeds (V) on BW, feed intake (FI), egg production (EP), feed conversion (FC), egg weight (EW), shell thickness (ST), yolk color score (YC), and Haugh unit score (HU) of Single Comb White Leghorn hens for 56 d. In Experiment 1, diets contained 0, 7.5, 15, and 22.5% raw V. Compared with the control, the 22.5% V diet decreased (P < .05) BW, FI, and EP. In Experiment 2, intact or ground (G) V were either soaked (S) in water (1:5) for 24 h or autoclaved (A) at 103.5 x 10(3) Pa for 8 h, and then dried (D) at 55 C for 24 h. Eight diets were used, a corn soybean (control) and seven others, each containing 25% V, previously subjected to the forementioned treatments as follows: untreated V (UV), SDV, GSDV, ADV, GADV, SADV, and GSADV. Compared with the control, the UV diet decreased (P < .05) FI (75 vs 98 g) and EP (47.3 vs 88.7%), increased FC (1.99 vs 1.4 kg feed per dozen eggs), and induced BW loss (-63 vs 49 g). Soaking intact V improved (P < .05) FI (85 g) and EP (69%). Remaining treatments resulted in further improvement. Hens fed all V diets produced eggs with HU score 13 points better than that of the control (P < .05). In both experiments, EW, YC, and ST were not different among treatments. Results indicated that autoclaved V at 25% level was not detrimental to layers' performance.

Using force-feeding and pair-feeding techniques, two experiments were conducted to investigate the problem of leg abnormality associated with feeding a Val-deficient diet as compared with a diet deficient in all the branched chain amino acids (BCAA) or a Val-supplemented diet. The BCAA-deficient diet contained .96, .52, and .63% of Leu, Ile, and Val, respectively. The Val-deficient diet contained 1.37 Leu, .82 Ile, and .63% Val. The Val-supplemented diet contained 1.37 Leu, .82 Ile, and .83% Val. In both studies birds fed the Val-supplemented gained more weight than birds in the other two groups. Although birds on Val-deficient and BCAA-deficient diets had similar weight gain, the former were lethargic and showed feather and leg abnormalities. Bone ash and bone calcium for Val-deficient birds were the lowest among the three groups studied (P < .05). The levels of the BCAA in plasma reflected those of the treatment diets. Plasma hydroxyproline was lowest in birds fed the Val-deficient diet (P < .05), indicating a reduction in bone collagen breakdown. Kidney function measurements were the same for birds on the Val-supplemented and Val-deficient diets. Fractional excretion of calcium in Val-deficient birds (.13%), however, was three times higher than that of Val-supplemented birds (P < .05). The results indicated that Val deficiency per se increased calcium excretion in urine and induced leg abnormality in young chickens.

Experiments were designed to investigate the effect of feeding diets deficient in one or more of the three branched-chain amino acids (BCAA) on the performance of 3-wk-old male broilers. In the first experiment, levels of .96 and 1.46% Leu, .52 and .82% Ile, and .65 and .95% Val were used. Feeding the lowest combination of the three BCAA resulted in weight gain (WG) and feed conversion ratio (FC) of 344 g and 1.59 g:g, respectively. These parameters were not improved by adding dietary increments of the three BCAA individually. The greatest response, however, for both WG (435 g) and FC (1.41 g:g) was obtained by the addition of the three BCAA simultaneously. Chicks fed the low-Val diets in combinations with added levels of Ile and Leu exhibited feather and leg abnormalities. A second experiment was designed to investigate the effect of Val deficiency on feather protein, feather amino acids, and calcium content of the bone. Three treatments were used: a BCAA-deficient diet with .96% Leu, .52% Ile, and .63% Val; a Val-deficient diet, which contained 1.37, .82, and .63% of Leu, Ile, and Val, respectively; and a Val-supplemented diet, which was the same as the previous diets except that the Val content was .83%. Valine deficiency significantly decreased WG (243 g), FC (1.69 g:g), bone calcium (134 mg/g dry bone), and feather protein (82.7% of wet weight). Valine deficiency also decreased the level of Cys in feathers, but increased those of Asp, Glu, Met, Tyr, His, and Lys.(ABSTRACT TRUNCATED AT 250 WORDS)

The edible alga Spirulina platensis was uniformly labeled with 13C by growth in an atmosphere of pure 13CO2. The labeled biomass was then incorporated into the diet of a laying hen for 27 days. The isotopic enrichment of individual amino acids in egg white and yolk proteins, as well as in various tissues of the hen at the end of the feeding period, was analyzed by negative chemical ionization gas chromatography/mass spectrometry. The amino acids of successive eggs showed one of two exclusive enrichment patterns: complete preservation of the intact carbon skeleton or extensive degradation and resynthesis. The same observation was made in tissue proteins. These patterns were cleanly divided according to known nutritional amino acid essentiality/nonessentiality but revealed differences in labeling among the nonessential amino acids: most notable was that proline accretion was derived entirely from the diet. Feeding uniformly 13C-labeled algal protein and recovering and analyzing de novo-synthesized protein provides a useful method to examine amino acid metabolism and determine conditional amino acid essentially in vivo.

Broiler breeders display high levels of aggressive activity as a result of feed restriction. The objective of this study was to determine if aggressive activity in developing and mature broiler breeder males is influenced by increased dietary L-tryptophan (Trp). Broiler breeder males were raised using a skip-a-day feed restriction regimen. In Experiment 1, three levels of dietary Trp, .19 (control), .75, and 1.5%, were fed from the start of feed restriction at 4 wk of age and throughout development. All occurrences of aggressive behavior were recorded during three 20-min observation sessions per week on mornings on which feed was not delivered through Week 20. The Trp significantly decreased (P less than .05) aggressive pecking in birds fed supplemental Trp as compared to controls. No differences in threatening behavior were found. In the second part of Experiment 1, the effect of continued Trp supplementation on mature flocks of male broiler breeders and time-of-day effects on aggression were investigated. A depressing effect of supplemental Trp on pecking and threatening was observed. Maximum aggressive activity in all treatment groups occurred on mornings when feed was delivered. In Experiment 2, four levels of Trp, .19 (control), .38, .75, and 1.5%, were fed to developing birds as in Experiment 1. Birds fed supplemental Trp pecked less (P less than .05) than controls and .38% Trp was as effective in decreasing pecking as was 1.5%. It was concluded that increased levels of dietary Trp decrease aggression in both developing and mature, socially stable flocks of broiler breeder males.

Growth studies were conducted with 3-wk-old, male broiler chicks to determine their requirements for the branched-chain amino acids (BCAA) leucine, isoleucine, and valine during the starter period. The central-composite, rotatable design was applied. The data were analyzed using regression methodology. The results showed that the linear term of Leu and the cross-product terms of Leu with Ile or Val were nonsignificant (P greater than .05). Second-order equations described the relationship between the BCAA, weight gain (WG), and feed conversion (FC). The coefficient of determination values indicated that 80% or more of the variation in WG and FC was explained by the polynomial equations. The results suggested that the Leu requirement does not exceed 1.16% of a diet calculated to contain 3,200 kcal of metabolizable energy per kilogram. The optimum response, maximum in the case of WG (444 g) and minimum for FC (1.40), would be obtained by feeding a diet containing 1.16, .80, and .90% of Leu, Ile, and Val, respectively. The values of Ile and Val are the actual values obtained when the diets were hydrolyzed for 72 hours. For Leu, however, the hydrolysis time was 24 hours.

BAY Vi7533 (2-chloro-N-[[[4-(trifluoromethoxy)phenyl] amino] carbonyl] benzamide) was fed to White Leghorn and Vedette Mini-broiler breeder hens at concentrations ranging from 0 to 25 ppm in the ration. Concentrations of 15 and 25 ppm resulted in greater than 95% mortality of house flies, Musca domestica L., seeded as larvae into the droppings. Concentration of 25 ppm reduced egg production in the White Leghorn but not in the Vedette Mini-broiler hens. At each concentration (except 5 ppm for Vedette Mini-broilers) residues of the compound were detected in the eggs. These residues, however, had no adverse effects on fertility or hatchability of the eggs.