Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.     

Species differences for stereoselective hydrolysis of propranolol prodrugs in plasma and liver

Species differences and substrate specificities for the stereoselective hydrolysis of fifteen O-acyl propranolol (PL) prodrugs were investigated in pH 7.4 Tris-HCl buffer and rat and dog plasma and liver subfractions. The (R)-isomers were preferentially converted to propranolol (PL) in both rat and dog plasma with the exception of isovaleryl-PL in rat plasma, although the hydrolytic activities of prodrugs in rat plasma were 5–119-fold greater than those in dog plasma. The prodrugs with promoieties (C(=O)CH(R)CH₃) based on propionic acid showed marked preference for hydrolysis of the (R)-enantiomers in plasma from both species (R/S ratio 2.5–18.2). On the other hand, the hepatic hydrolytic activities of prodrugs were greater in dog than rat, especially in cytosolic fractions. The hydrolytic activity was predominantly located in microsomes of the liver in rat, while the cytosol also contributed to hepatic hydrolysis in dog. Hepatic microsomal hydrolysis in dog showed a preference for the (R)-isomers except acetyl- and propionyl-PL. Interestingly, in rat liver all types of prodrugs with substituents of small carbon number showed (S)-preference for hydrolysis. The hydrolyses of (R)- and (S)-isomers of straight chain acyl esters in rat liver microsomes were linearly and parabolically related with the carbon number of substituents, respectively, while these relationships were linear for both isomers in dogs. Chirality 9:661–666, 1997. © 1997 Wiley-Liss, Inc.     

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals () generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2-ethoxycarbonyl-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (EMPO). The findings show that the presence of normal-physiological serum concentrations of IS (0.1-10 μM) resulted in decreased formation of EMPO-superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell-derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal-physiological conditions.     

6β-Hydroxyursolic acid and other triterpenoids of Enkianthus cernuus

From the leaves of Enkianthus cernuus forma rubens, a new triterpene, 6β-hydroxyursolic acid, was isolated together with several known compounds     

Molecular diversity of endosymbiotic Nephroselmis (Nephroselmidophyceae) in Hatena arenicola (Katablepharidophycota)

Hatena arenicola (Katablepharidophycota) is a single-celled eukaryote that temporarily possesses a chlorophyte alga of the genus Nephroselmis as an intracellular symbiont. In the present study, we investigated the molecular diversity of the endosymbiont Nephroselmis in a natural population of the host H. arenicola. We sequenced the host’s 18S rRNA gene and the endosymbiont’s plastid-encoded 16S rRNA gene. The results indicated that almost identical strains of the host harbored at least three distinct strains of the algal endosymbiont affiliated to the clade Nephroselmis rotunda. This finding supports our previous hypothesis that H. arenicola and its symbiotic alga are in an early stage of secondary endosymbiosis.     

Molecular characteristics of a single and novel form of carp (Cyprinus carpio) monoamine oxidase

Two mammalian monoamine oxidases (MAO), MAO-A and MAO-B, are similar in primary structures but have unique substrate/inhibitor selectivities. Carp (Cyprinus carpio) contains a MAO enzyme (C-MAO) with properties different from MAO-A and MAO-B. To determine the molecular characteristics of C-MAO and its phylogenetic relationship with other fish and mammalian MAOs, the primary structure of C-MAO was estimated. The putative C-MAO cDNA encodes 526 amino acids with 59.001 Da, and the deduced amino acid sequence showed as much as 68.9% homology with some mammalian MAO-A proteins, 69.8% homology with some mammalian MAO-B proteins, and as much as 92.4% homology with some fish MAOs. Comparison of two regions in the polypeptide sequence of C-MAO determining possible substrate/inhibitor preferences of MAO-A and MAO-B showed both 79.5% homologies.     

Chain length-dependent binding of fatty acid anions to human serum albumin studied by site-directed mutagenesis

Human serum albumin is the most abundant protein in the circulatory system, and one of its principal functions is to transport fatty acids. Binding of octanoate, decanoate, laurate and myristate was studied by a rate-of-dialysis technique. The primary association constants increased, but not linearly, with chain length. The number of high-affinity sites also increased with chain length; octanoate and decanoate bind to one such site, whereas laurate and myristate most probably bind to two sites. Albumin is composed of three homologous helical domains (I-III), which can be subdivided into two subdomains (A and B). For getting information about the positions of the high-affinity sites we produced 13 recombinant isoforms mutated in four different subdomains. Results obtained with these albumins are in accordance with the following model: octanoate and decanoate bind to a single site in subdomain IIIA, laurate binds to sites in subdomains IIIA and IIIB, whereas myristate binds in subdomains IB and IIIB. The results also showed that primary fatty acid binding is sensitive to amino acid substitutions in other parts of the protein. This is in contrast to the effect of amino acid substitutions of genetic albumin variants (alloalbumins). Usually these substitutions, which are situated at the surface of the protein, have no effect on fatty acid binding. Binding of fatty acid anions to different high-affinity sites and the sensitivity of these sites to amino acid substitutions elsewhere in the protein (and perhaps also to other types of modifications) are important factors that could effect simultaneous binding of other ligands, e.g. in patients treated with albumin-binding drugs.     

Involvement of organic anion transporters in the efflux of uremic toxins across the blood-brain barrier

Renal failure causes multiple physiological changes involving CNS dysfunction. In cases of uremia, there is close correlation between plasma levels of uremic toxins [e.g. 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippurate (HA) and indoleacetate (IA)] and the degree of uremic encephalopathy, suggesting that uremic toxins are involved in uremic encephalopathy. In order to evaluate the relevance of uremic toxins to CNS dysfunction, we investigated directional transport of uremic toxins across the blood-brain barrier (BBB) using in vivo integration plot analysis and the brain efflux index method. We observed saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA, which was inhibited by probenecid. For all uremic toxins evaluated, apparent efflux clearance across the BBB was greater than apparent influx clearance, suggesting that these toxins are predominantly transported from the brain to blood across the BBB. Saturable efflux transport of [(3)H]CMPF, [(14)C]HA and [(3)H]IA was completely inhibited by benzylpenicillin, which is a substrate of rat organic anion transporter 3 (rOat3). Taurocholate and digoxin, which are common substrates of rat organic anion transporting polypeptide (rOatp), partially inhibited the efflux of [(3)H]CMPF. Transport experiments using a Xenopus laevis oocyte expression system revealed that CMPF, HA and IA are substrates of rOat3, and that CMPF (but not HA or IA) is a substrate of rOap2. These results suggest that rOat3 mediates brain-to-blood transport of uremic toxins, and that rOatp2 is involved in efflux of CMPF. Thus, conditions typical of uremia can cause inhibition of brain-to-blood transport involving rOat3 and/or rOatp2, leading to accumulation of endogenous metabolites and drugs in the brain.