Jean-Michel Robert

Highly branched isoprenoids (HBIs): identification of the most common and abundant sedimentary isomers

Abstract Tri- and tetraunsaturated highly branched isoprenoid (HBI) alkenes are widespread sedimentary geochemicals but few have been isolated from sediments in sufficient quantities for rigorous identification. However, two C25 trienes, four C25 tetraenes and two C25 pentaenes have now been isolated from the diatom Pleurosigma intermedium following bulk scale culture, and these have been purified by column chromatography and fully characterised by NMR spectroscopy and mass spectrometry. The compounds have been used to identify the previously unknown, but common and abundant HBIs found previously in many studies of sediments, particles and biota from around the world. These HBIs are structurally different to those reported from other diatoms. For example, unlike HBIs from the diatoms Haslea ostrearia and Rhizosolenia setigera, the alkenes in P. intermedium are unsaturated at the major branch point of the carbon skeleton and E/Z isomerism is observed for one of the trisubstituted double bonds. There is no evidence for the presence of configurational diastereoisomerism. The distributions of HBIs in P. intermedium (including the E/Z ratios) also show a dependence on the growth conditions within the five cultures studied. The positions of the double bonds in the HBIs of P. intermedium, and by inference, of the sediments, are consistent with the positions of sulphur incorporation in some of the HBI thiolanes and thiophenes which have been reported previously in some sediments and oils.

Diatom cultivation and biotechnologically relevant products. Part I: Cultivation at various scales

Abstract. Biotechnological applications of diatoms are still in development. Further development at the industrial scale will depend on optimisation of the culture process with the aim of reducing costs. Because of the photoautotrophic status of the majority of diatoms, microalgal cultures suffer from the limitation of light diffusion, which requires the development of suitable photobioreactors. Thus, genetically engineered microalgae that may be cultivated in heterotrophic conditions present a new opportunity. Other limiting factors, such as nutrients (phosphate, nitrogen, silicon), pH, temperature, bioturbation and many more must be taken into account. Most of the time, metabolic stress conditions lead to an overproduction of the products of interest, with a decrease in biomass production as a consequence. Outdoor cultures in open ponds are usually devoted to aquaculture for the feeding of shrimps and bivalve molluscs (commercial production), while closed axenic indoor/outdoor photobioreactors are used for biotechnological compounds of homogeneous composition (still at the laboratory scale). In addition to the optimum culture conditions that have to be taken into account for photobioreactor design, the localisation of produced metabolites (intra- or extracellular) may also be taken into account when choosing the design. Microalgal cell immobilisation may be a suitable technique for application to benthic diatoms, which are usually sensitive to bioturbation and/or metabolites which may be overexpressed.

Structural characterisation of widespread polyunsaturated isoprenoid biomarkers: A C25 triene, tetraene and pentaene from the diatom Haslea ostrearia simonsen

Abstract Three highly branched isoprenoid (HBI) polyenes have been isolated from the diatom Haslea ostrearia and characterised by nmr spectroscopy

Effects of tumour necrosis factor-α synthesis inhibitors on rat trinitrobenzene sulphonic acid-induced chronic colitis

The fact that tumour necrosis factor-alpha (TNF-alpha) is clearly involved in the pathogenesis of intestinal bowel disease, especially Crohns disease, suggests that TNF-alpha synthesis inhibitors could be beneficial for treatment. The present study assessed the effect of chronic oral gavage of two in vitro TNF-alpha synthesis inhibitors, JM 34 maleate or [N-(4,6-dimethylpyridin-2-yl)-furane-2-carboxamide)] maleate and XC 21 or (N-betapicolyl-tetrafluorophtalimide), on colonic inflammation in trinitrobenzene sulphonic acid-induced colitis in rats. Rats received JM 34 maleate (100 mg/kg) and XC 21 (50 mg/kg) 1 h before colitis induction and then daily for 8 days by oral gavage. The colon was removed on day 8 and processed for clinical score, myeloperoxidase activity, and soluble TNF-alpha release. Treatment with XC 21, as well as dexamethasone and sulphasalazine, reduced colonic damage and decreased (except with dexamethasone) the incidence of diarrhoea. JM 34 maleate failed to improve the clinical signs of chronic colitis. After trinitrobenzene sulphonic acid-induced colitis, myeloperoxidase activity and TNF-alpha colonic mucosal production were substantially increased compared to the control (saline instillation). Both of these inflammatory indicators were then significantly decreased (P< or =0.05) after the four chronic treatments (JM 34 maleate, XC 21, sulphasalazine, and dexamethasone). XC 21 appeared to be as efficient as sulphasalazine in improving colonic inflammation.

Ammonium thresholds for simultaneous uptake of ammonium and nitrate by oyster-pond algae

Abstract Natural microalgal populations and axenic algal isolates from oyster ponds have been grown either in situ or in controlled conditions, in the presence of ammonium and nitrate as nitrogen sources. Both ions were added at various concentrations, up to 50 μg-at. N·l −1 ; other nutrients were in excess. In one experiment, urea was also added. Uptake of nitrate was followed by measuring disappearance of nitrate from the medium, and by incorporation of 15 NO 3 ; nitrate reductase (NR) activity and the intracellular nitrate pool were also measured. The uptake of nitrate was prevented by the presence of ammonium above a concentration which varied according to species. The ammonium threshold (in units of μg-at. N·l −1 ) was ≈ 30 for natural populations and the diatom Navicula ostrearia Bory, ≈ 21 for Nitzschia ovalis (sensu Hustedt), and ≈ 44 for Amphora coffeaeformis (sensu Hendey). Nitrate uptake started at a rate which was ≈ 39% of the eventual maximum rate observed for the natural populations, and from 11 to 17% for cultured strains. The initial low rate was maintained until the ambient ammonium concentration had decreased to ≈ 7.5 μ g-at. N·l −1 , except for A. coffeaeformis which shifted from slow to fast nitrate uptake at 23.5μg-at. N·l −1 . The nitrate uptake system then operated at a slightly higher rate than the one for ammonium (ammonium uptake/nitrate uptake = 0.86). Cultures with an initial ammonium concentration lower than the threshold values did not show an initial low rate or a lag phase for uptake of nitrate. NR activity was detectable even in the presence of ≈ 30 μ g-at. NH 4 -N·l −1 in the external medium. When urea and nitrate were presented simultaneously, urea was not taken up preferentially, as reported elsewhere; uptake was initially at a reduced rate until the external nitrate concentration decreased to 3.7 μg-at. N·l −1 . Then the rate of urea uptake increased to a maximum. It is suggested that because oyster-pond algae have evolved in an environment where concentrations of ammonium, nitrate, and organic nitrogen are continuously high, the threshold of inhibition by ammonium of uptake of these compounds is much higher than for similar pelagic and neritic species, so that they are able to assimilate other sources of nitrogen, such as nitrate and urea, simultaneously with ammonium.

New anti-inflammatory N-pyridinyl(alkyl)phthalimides acting as tumour necrosis factor-α production inhibitors

This paper describes the synthesis of N-pyridinyl(alkyl)phthalimides related to N-phenyl-4,5,6,7-tetrafluorophthalimides known to be inhibitors of tumour necrosis factor-alpha (TNFalpha) production. Pharmacomodulation at the phthalimidic nitrogen led to the selection of two pharmacophoric fragments (2,4-lutidinyl and beta-picolyl), allowing significant inhibition of TNFalpha production (compounds 12 and 17). Variation of the substituents linked to the homocycle of their phthalimide scaffold indicated that high (TNFalpha production) inhibitory potency could be achieved, notably by 5-fluoro, 4- or 5-nitro, 5-amino and especially tetrafluoro substitution. The most active compound, N-(pyridin-3-ylmethyl)-4,5,6,7-tetrafluorophthalimide (32) (84% inhibition at 10 microM), also produced an anti-oedematous effect in the PMA-induced mouse-ear swelling test. Although less active than dexamethasone, it exerted a marked reduction in ear thickness after oral administration (63% vs. 85% for dexamethasone at 0.2 mMkg(-1)) and remained efficient after topical application (46% vs. 96% for the dexamethasone). It also induced potent inhibition in the rat carrageenan foot oedema test with an ID(50) (0.14 microMkg(-1)) comparable with that of N-(2,6-diisopropylphenyl)phthalimide (4) (0.15 microMkg(-1)).

New selective nonsteroidal aromatase inhibitors: Synthesis and inhibitory activity of 2,3 or 5-(α-azolylbenzyl)-1H-indoles

Six azolyl substituted indoles were synthesized and tested for their activity to inhibit two P450 enzymes: P450 arom and P450 17a. It was observed that the introduction of alpha-imidazolylbenzyl chain at carbon 3 or 5 on indole nucleus led to very active molecules. Compounds 22, 23 and especially 33 demonstrate very high potential against P450 arom. Under our assay conditions of high substrate concentration the IC50 are 0.057, 0.0785 and 0.041 microM, respectively. These compounds are moderate inhibitors against P450 17alpha.

Highly branched C25 isoprenoids in axenic cultures ofHaslea ostrearia

Abstract The hydrocarbon compositions of axenic cultures of the diatom Haslea ostrearia grown in the presence of penicillin, streptomycin and kanamycin were examined at lag,exponential and stationary growth phases. The production of highly branched isoprenoid (HBI)C 25 trienes to pentaenes with the 2,6,10,14-tetramethyl-7- (3-methylpentyl)pentadecane carbonskeleton was demonstrated at all three phases (2300–7000 fg cell −1 ). Of the ten HBI trienes tohexaenes reported previously from non-axenic cultures of H. ostrearia , four were present in theaxenic samples. In addition, two novel trienes and a pentaene were found. The most abundant ofthe new trienes was isolated from a larger, non-axenic batch culture and identified from 13 C- and 1 H-NMR data as 2,6,10,14-tetramethyl-7- (3-methylpent-4-enyl)pentadec-5,9-diene. Interestingdifferences in HBI isomer distributions were observed among the three growth phases. Forexample, the newly identified, non-methylenic triene above, only occurred in the exponentialgrowth phase. As a primary producer of these alkenes, several of which have demonstratedcytostatic activity, Haslea ostrearia , and perhaps related Haslea species, is clearly worthy offurther study.

Synthesis of 3,5-bis(2-indolyl)pyridine and 3-((2-indolyl)-5-phenyl)- pyridine derivatives as CDK inhibitors and cytotoxic agents

We here report the synthesis and biological evaluation of new 3,5-bis(2-indolyl)pyridine and 3-[(2-indolyl)-5-phenyl]pyridine designed as potential CDK inhibitors. Indole, 5-hydroxyindole, and phenol derivatives were used to generate three substitutions of the pyridine. The resulting skeletons were successively exploited to introduce various dimethylaminoalkyl side chains by Williamson type reactions. The synthesis includes Stille or Suzuki type reactions, which were realized on the 3,5-dibromopyridine. The preparation and the use of stannylindoles in mono or bis cross-coupling reactions were also described and each step was optimized and detailed. Kinase assays were realized and shown that nude compounds 7, 18, and 25 inhibited CDK1 in the 0.3-0.7 micromolar range with a good selectivity over GSK-3. Cytotoxicity against CEM human leukemia cells was evaluated with IC(50) values in the 5-15 micromolar range. Precise structure-activity relationships were delineated. Molecular modeling and docking solutions were proposed to complete the studies and to explain the observed SAR in the CDK assays.

Identification of a C25 highly branched isoprenoid triene in the freshwater diatom Navicula sclesvicensis

Abstract The first example of a freshwater diatom species that biosynthesises the widespread organic geochemicals known as highly branched isoprenoids (HBIs) is identified. As a result, diatoms known to produce HBIs now include benthic, planktonic, marine and freshwater species. Laboratory cultures of a number of freshwater diatoms isolated from Swanpool, UK and Paimpont, France were examined for highly branched isoprenoid (HBI) alkenes. A C 25 HBI triene was identified in Navicula sclesvicensis , and characterised using GC–MS, by comparison with an authentic standard.