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BackgroundMicroalgae are gaining importance as sustainable production hosts in the fields of biotechnology and bioenergy. A robust biomass accumulating strain of the genus Monoraphidium (SAG 48.87) was investigated in this work as a potential feedstock for biofuel production. The genome was sequenced, annotated, and key enzymes for triacylglycerol formation were elucidated.ResultsMonoraphidium neglectum was identified as an oleaginous species with favourable growth characteristics as well as a high potential for crude oil production, based on neutral lipid contents of approximately 21% (dry weight) under nitrogen starvation, composed of predominantly C18:1 and C16:0 fatty acids. Further characterization revealed growth in a relatively wide pH range and salt concentrations of up to 1.0% NaCl, in which the cells exhibited larger structures. This first full genome sequencing of a member of the Selenastraceae revealed a diploid, approximately 68 Mbp genome with a G + C content of 64.7%. The circular chloroplast genome was assembled to a 135,362 bp single contig, containing 67 protein-coding genes. The assembly of the mitochondrial genome resulted in two contigs with an approximate total size of 94 kb, the largest known mitochondrial genome within algae. 16,761 protein-coding genes were assigned to the nuclear genome. Comparison of gene sets with respect to functional categories revealed a higher gene number assigned to the category “carbohydrate metabolic process” and in “fatty acid biosynthetic process” in M. neglectum when compared to Chlamydomonas reinhardtii and Nannochloropsis gaditana, indicating a higher metabolic diversity for applications in carbohydrate conversions of biotechnological relevance.ConclusionsThe genome of M. neglectum, as well as the metabolic reconstruction of crucial lipid pathways, provides new insights into the diversity of the lipid metabolism in microalgae. The results of this work provide a platform to encourage the development of this strain for biotechnological applications and production concepts.

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Nitric oxide (NO) production from exogenous NG-hydroxy-L-arginine (OH-L-Arg) was investigated in rat aortic smooth muscle cells in culture by measuring nitrite accumulation in the culture medium. As well, the interaction between OH-L-Arg and L-arginine uptake via the y+ cationic amino acid transporter was studied. In cells without NO-synthase activity, OH-L-Arg (l-1000 ,uM) induced a dose-dependent nitrite production with a half-maximal effective concentration (EC,,) of 18.0 f 1.5 PM (n = 4-7). This nitrite accumulation was not inhibited by the NO-synthase inhibitor No-nitro+arginine methyl ester, L-NAME (300 PM). In contrast, it was abolished by miconaxole (100 pM), an inhibitor of cytochrome PJ5@ Incubation of vascular smooth muscle cells with LPS (10 &ml) induced an L-NAME inhibited nitrite accumulation, but did not enhance the OH-L-Arg induced nitrite production. OH-L-Arg and other cationic amino acids, L-lysine and L-ornithine, competitively inhibited [3H]-L-arginine uptake in rat aortic smooth muscle cells, with inhibition constants of 195 f 23 ,uM (a = 12), 260 & 4OpM (n = 5) and 330 f 10pM (n = 5), respectively. These results show that OH-L-Arg is recognized by the cationic ~-amino acid carrier present in vascular smooth muscle cells and can be oxidized to NO and nitrite in these cells in the absence of NO-synthase, probably by cytochrome P4M or by a reaction involving a cytochrome P4r,, byproduct.

Upon agonist activation, the nicotinic acetylcholine receptor undergoes allosteric transitions leading to channel opening and sodium ion influx. The molecular structure of the agonist binding site has been mapped previously by photoaffinity labeling, but most photosensitive probes used for this purpose interact only with closed receptor states (resting or desensitized). We have synthesized two novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as cholinergic agonist candidates, with the objective of identifying structural changes at the acetylcholine binding site associated with receptor activation. One of these ligands, 9b, is a functional agonist at muscle acetylcholine receptors in human TE 671 cells. In photolabeling experiments with 9b, up to 35% inactivation of agonist binding sites was observed at Torpedo acetylcholine receptors. Tritiated 9b was synthesized, and photolabeling was found to occur mainly on the alpha-subunit in a partially protectable manner. This novel radiolabeled photoprobe appears to be suitable for future investigation of the molecular dynamics of allosteric transitions occurring at the active acetylcholine receptor binding site.