Determination of FVIIa-sTF Inhibitors in Toxic Microcystis Cyanobacteria by LC-MS Technique

The blood coagulation cascade involves the human coagulation factors thrombin and an activated factor VII (fVIIa). Thrombin and fVIIa are vitamin-K-dependent clotting factors associated with bleeding, bleeding complications and disorders. Thrombin and fVIIa cause excessive bleeding when treated with vitamin-K antagonists. In this research, we explored different strains of toxic Microcystis aeruginosa and cyanobacteria blooms for the probable fVIIa-soluble Tissue Factor (fVIIa-sTF) inhibitors. The algal cells were subjected to acidification, and reverse phase (ODS) chromatography-solid phase extraction eluted by water to 100% MeOH with 20%-MeOH increments except for M. aeruginosa NIES-89, from the National Institute for Environmental Studies (NIES), which was eluted with 5%-MeOH increments as an isolation procedure to separate aeruginosins 89A and B from co-eluting microcystins. The 40%–80% MeOH fractions of the cyanobacterial extract are active against fVIIa-sTF. The fVIIa-sTF active fractions from cultured cyanobacteria and cyanobacteria blooms were subjected to liquid chromatography-mass spectrometry (LC-MS). The 60% MeOH fraction of M. aeruginosa K139 exhibited an m/z 603 [M + H]+ attributed to aeruginosin K139, and the 40% MeOH fraction of M. aeruginosa NIES-89 displayed ions with m/z 617 [M − SO3 + H]+ and m/z [M + H]+ 717, which attributed to aeruginosin 89. Aeruginosins 102A/B and 298A/B were also observed from other toxic strains of M. aeruginosa with positive fVIIa-sTF inhibitory activity. The active fractions contained cyanobacterial peptides of the aeruginosin class as fVIIa-sTF inhibitors detected by LC-MS

Nostosin G and Spiroidesin B from the Cyanobacterium Dolichospermum sp. NIES-1697

Chemical investigation of the cyanobacterium Dolichospermum sp. NIES-1697 afforded nostosin G (1), a linear tripeptide, spiroidesin B (2), and two known compounds, anabaenopeptins I (3) and J (4). Planar structures and absolute configurations for 1 and 2 were determined by 2D NMR, HRMS, Marfey's methodology, chiral-phase HPLC, and enzymatic degradation. Nostosin G (1) is a unique example of a linear peptide containing three subunits, 4-hydroxyphenyllactic acid (Hpla), homotyrosine (Hty), and argininal, with potent trypsin inhibitory properties. The biosynthetic gene clusters for nostosin G (1) and spiroidesin B (2) were investigated based on the genome sequence of Dolichospermum sp. NIES-1697

Microbial Degradation of Amino Acid-Containing Compounds Using the Microcystin-Degrading Bacterial Strain B-9

Strain B-9, which has a 99% similarity to Sphingosinicella microcystinivorans strain Y2, is a Gram-negative bacterium with potential for use in the degradation of microcystin-related compounds and nodularin. We attempted to extend the application area of strain B-9 and applied it to mycotoxins produced by fungi. Among the tested mycotoxins, only ochratoxin A was completely hydrolyzed to provide the constituents ochratoxin α and l-phenylalanine, and levels of fumonisin B1 gradually decreased after 96 h. However, although drugs including antibiotics released into the aquatic environment were applied for microbial degradation using strain B-9, no degradation occurred. These results suggest that strain B-9 can only degrade amino acid-containing compounds. As expected, the tested compounds with amide and ester bonds, such as 3,4-dimethyl hippuric acid and 4-benzyl aspartate, were readily hydrolyzed by strain B-9, although the sulfonamides remained unchanged. The ester compounds were characteristically and rapidly hydrolyzed as soon as they came into contact with strain B-9. Furthermore, the degradation of amide and ester compounds with amino acids was not inhibited by the addition of ethylenediaminetetraacetic acid (EDTA), indicating that the responsible enzyme was not MlrC. These results suggest that strain B-9 possesses an additional hydrolytic enzyme that should be designated as MlrE, as well as an esterase

Microbial isolate P129-1 from a marine sponge inhibitory to Straphylococcus aureus ATCC 12600 is an Ochrobactrum sp.

Marine microorganisms are complex and diverse. They thrive in conditions of extreme pressure, salinity, and temperature as commensals, epibionts, or symbionts. Their potential to produce bioactive metabolites is a reflection of their versatile metabolism and physiology char enabled them to survive the complex marine environment. They may occur as free-Jiving entities or in associa­tion with eukaryotes such as the marine invertebrates. They inhabit sur­faces, tissues, and internal spaces of host organisms. In this study, the cultural and morphological features of the microbial isolate P129-1, which manifested an inhibitory activity against S aureus ATCC 12600, was determined with the end view of ascertaining the identity and phylogenetic relationship of the isolate with other bacteria

Thrombin Inhibitors from the Freshwater Cyanobacterium <i>Anabaena compacta</i>

Bioassay-guided investigation of the cyanobacterium <i>Anabaena compacta</i> extracts afforded spumigin J (<b>1</b>) and the known thrombin inhibitor spumigin A (<b>2</b>). The absolute configuration of <b>1</b> was analyzed by advanced Marfey’s methodology. Compounds <b>1</b> and <b>2</b> inhibited thrombin with EC₅₀ values of 4.9 and 2.1 μM, and 0.7 and 0.2 μM in the cathepsin B inhibitory assay, respectively. The MM-GBSA methodology predicted spumigin A with 2<i>S</i>-4-methylproline as the better thrombin inhibitor

FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139

The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF) have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE) from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC50 10.62 µM, which was more efficient than thrombin inhibition of EC50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF) and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors