Biomedical subjects

Yuichi Oba

Publications and source records attributed to Yuichi Oba.

At least 19 recordsLinked to original sources

PubMed2006

Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178.

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.

Adenine↗

PubMed2006

Phylogenetic relationships of click beetles (Coleoptera: Elateridae) inferred from 28S ribosomal DNA: insights into the evolution of bioluminescence in Elateridae.

Although the taxonomy of click beetles (family Elateridae) has been studied extensively, inconsistencies remain. We examine here the relationships between species of Elateridae based on partial sequences of nuclear 28S ribosomal DNA. Specimens were collected primarily from Japan, while luminous click beetles were also sampled from Central and South America to investigate the origins of bioluminescence in Elateridae. Neighbor-joining, maximum-parsimony, and maximum-likelihood analyses produced a consistent basal topology with high statistical support that is partially congruent with the results of previous investigations based on the morphological characteristics of larvae and adults. The most parsimonious reconstruction of the "luminous" and "nonluminous" states, based on the present molecular phylogeny, indicates that the ancestral state of Elateridae was nonluminous. This suggests that the bioluminescence in click beetle evolved independent of that of other luminous beetles, such as Lampyridae, despite their common mechanisms of bioluminescence.

Animals↗

PubMed2006

Stereoselective incorporation of isoleucine into Cypridina luciferin in Cypridina hilgendorfii (Vargula hilgendorfii).

The emission of light in the marine ostracod Cypridina hilgendorfii (presently Vargula hilgendorfii) is produced by the Cypridina luciferin-luciferase reaction in the presence of molecular oxygen. Cypridina luciferin has an asymmetric carbon derived from isoleucine, and the absolute configuration is identical to the C-3 position in L-isoleucine or D-alloisoleucine. To determine the stereoselective incorporation of the isoleucine isomers (L-isoleucine, D-isoleucine, L-alloisoleucine, and D-alloisoleucine), we synthesized four (2)H-labeled isoleucine isomers and examined their incorporation into Cypridina luciferin by feeding experiments. Judging by these results, L-isoleucine is predominantly incorporated into Cypridina luciferin. This suggests that the isoleucine unit of Cypridina luciferin is derived from L-isoleucine, but not from D-alloisoleucine.

Animals↗

PubMed2006

Biosynthesis of 2'-O-methylmyxalamide D in the myxobacterium Cystobacter fuscus: a polyketide synthase-nonribosomal peptide synthetase system for the myxalamide D skeleton and a methyltransferase for the final O-methylation.

The biosynthetic gene cluster for the polyene antifungal antibiotic, 2'-O-methylmyxalamide D, was cloned from myxobacterium Cystobacter fuscus AJ-13278. A sequence analysis of the 12.8-kb region in the gene cluster revealed the presence of two type I polyketide synthase genes, mmxB and mmxC. The involvement of these two genes in the biosynthesis of 2'-O-methylmyxalamide D was confirmed by a gene disruption experiments. In addition, an S-adenosylmethionine-dependent methyltransferase gene (mmxM) was found downstream of the gene cluster and demonstrated, by a gene disruption analysis, to be responsible for converting the known unmethylated precursor, myxalamide D, into 2'-O-methylmyxalamide D.

Chromatography, High Pressure Liquid↗

PubMed2005

Identification of paralogous genes of firefly luciferase in the Japanese firefly, Luciola cruciata.

Two homologous genes of firefly luciferase, LcLL1 and LcLL2, were cloned from the Japanese firefly Luciola cruciata, and were expressed and characterized. The gene product of LcLL1 had long-chain fatty acyl-CoA synthetic activity, but not luciferase activity. The other gene product of LcLL2 did not show enzymatic activities of acyl-CoA synthetase and luciferase. RT-PCR analysis showed that the transcript of LcLL1 was abundant in larva but very low in adult, while LcLL2 was expressed in both larva and adult. Phylogenetic analysis indicated that LcLL1 and LcLL2 are paralogous genes of firefly luciferase. Recently, we found that CG6178 in Drosophila melanogaster is an orthologue of firefly luciferase and shows fatty acyl-CoA synthetic activity, but not luciferase activity. These results suggest that firefly luciferase might be evolved from a fatty acyl-CoA synthetase by gene duplication in insects.

Amino Acid Sequence↗

PubMed2005

Zooxanthellamide Cs: vasoconstrictive polyhydroxylated macrolides with the largest lactone ring size from a marine dinoflagellate of Symbiodinium sp.

Zooxanthellamide Cs (ZAD-Cs), C(128)H(220)N(2)O(53)S(2) (ca. 2.7 kDa), was obtained from a cultured marine dinoflagellate of the genus Symbiodinium as an inseparable isomeric mixture of polyhydroxylated 61- to 66-membered macrolides. The chemical structures of the components were clarified by detailed 2D NMR analysis to be the macrolactonized analogues of zooxanthellamide A (ZAD-A), which had been previously isolated from the same microalgae. Chemical lability of ZAD-Cs suggests that ZAD-A is an artifact derived from ZAD-Cs during the isolation steps. Three of the components possess the largest (63-, 64-, and 66-membered) ring sizes found to date among the natural macrolides. ZAD-Cs exhibited higher vasoconstrictive activity than that of the zooxanthellatoxins, the first vasoconstrictive macrolides from Symbiodinium sp. The structure-activity relationship suggests that the huge macrolactone structure is important for biological activity. The relationship between the structures of the polyol metabolites and the phylogenetic systematics of Symbiodinium sp. is also discussed.

Animals↗

PubMed2005

Construction of a bacterial artificial chromosome library for a myxobacterium of the genus Cystobacter and characterization of an antibiotic biosynthetic gene cluster.

A bacterial artificial chromosome (BAC) library was constructed to isolate the biosynthetic gene cluster for the polyketide/peptide hybrid-type antibiotic cystothiazole A from the myxobacterium Cystobacter fuscus strain AJ-13278. Sequence analysis of a 63.9 kb contiguous region that encompasses the biosynthetic gene cluster (cta) led to the identification of a polyketide synthase (PKS)/nonribosomal peptide synthetase (NRPS) hybrid gene cluster 32.1 kb in size, which consists of six open reading frames (ORFs), ctaB to ctaG, as well as downstream genes ctaJ and ctaK (1.0 and 0.9 kb, respectively) responsible for the final biosynthetic steps. The genes ctaB, ctaE, and ctaF encode PKSs, the genes ctaC and ctaG encode NRPSs, and ctaD encodes an NRPS-PKS hybrid enzyme. Disruption of ctaD impaired cystothiazole A production. Additionally, two downstream genes, ctaJ and ctaK, which encode a nitrilase and an O-methyltransferase, respectively, must be responsible for the final methyl ester formation in the cystothiazole A biosynthesis.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2005

Novel relationship between the antifungal activity and cytotoxicity of marine-derived metabolite xestoquinone and its family.

Xestoquinone and related metabolites (the xestoquinone family) occur in marine sponges and are known to show a variety of biological activities. In this study, the first comprehensive evaluation of antifungal activity was performed for xestoquinone and nine natural and unnatural analogues in comparison with their cytotoxicity. The cytotoxicity against two human squamous cell carcinoma cell lines, A431 and Nakata, indicated that the terminal quinone structure of the polycyclic molecules was important (xestoquinone, etc.) and that the presence of a ketone group at C-3 of the opposite terminus dramatically diminished the activity (halenaquinone, etc.). In contrast, a ketone group at C-3 enhanced the antifungal activity against the plant pathogen, Phytophthora capsici, regardless of the presence of a quinone moiety. The cytotoxicity and antifungal activity of the xestoquinone family were negatively correlated with each other.

Animals↗

PubMed2005

Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-CoA synthetase.

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.

Acyl Coenzyme A↗

PubMed2005

Purification and characterisation of blarinasin, a new tissue kallikrein-like protease from the short-tailed shrew Blarina brevicauda: comparative studies with blarina toxin.

A new tissue kallikrein-like protease, blarinasin, has been purified from the salivary glands of the short-tailed shrew Blarina brevicauda. Blarinasin is a 32-kDa N-glycosylated protease with isoelectric values ranging between 5.3 and 5.7, and an optimum pH of 8.5 for enzyme activity. The cloned blarinasin cDNA coded for a pre-pro-sequence and a mature peptide of 252 amino acids with a catalytic triad typical for serine proteases and 43.7-54.0% identity to other mammalian tissue kallikreins. Blarinasin preferentially hydrolysed Pro-Phe-Arg-4-methylcoumaryl-7-amide (MCA) and N-tert-butyloxycarbonyl-Val-Leu-Lys-MCA, and preferentially converted human high-molecular-weight kininogen (HK) to bradykinin. The activity of blarinasin was prominently inhibited by aprotinin (K(i) =3.4 nM). A similar kallikrein-like protease, the lethal venom blarina toxin, has previously been purified from the salivary glands of the shrew Blarina and shows 67.9% identity to blarinasin. However, blarinasin was not toxic in mice. Blarinasin is a very abundant kallikrein-like protease and represents 70-75% of kallikrein-like enzymes in the salivary gland of B. brevicauda.

Amino Acid Sequence↗

PubMed2004

Identification of the luciferin-luciferase system and quantification of coelenterazine by mass spectrometry in the deep-sea luminous ostracod Conchoecia pseudodiscophora.

The bioluminescence system of the ostracod Conchoecia pseudodiscophora, which is abundant in the Sea of Japan, has been characterized. The luminescence (lambda(max)=463 nm) is produced by a luciferin-luciferase reaction, and the luciferin has been identified as coelenterazine. Coelenterazine, coelenteramide, and coelenteramine from C. pseudodiscophora were quantified by LC-ESI-MS/MS analysis. The coelenterazine content was estimated to be approximately 230 pg per animal by using a calibration curve of synthetic coelenterazine. The reaction between homogenates of C. pseudodiscophora and synthetic coelenterazine showed luminescence activity; this suggests that a coelenterazine-type luciferase is present.

Animals↗

PubMed2004

Blarina toxin, a mammalian lethal venom from the short-tailed shrew Blarina brevicauda: Isolation and characterization.

Venomous mammals are rare, and their venoms have not been characterized. We have purified and characterized the blarina toxin (BLTX), a lethal mammalian venom with a tissue kallikrein-like activity from the submaxillary and sublingual glands of the short-tailed shrew Blarina brevicauda. Mice administered BLTX i.p. developed irregular respiration, paralysis, and convulsions before dying. Based on the amino acid sequence of purified protein, we cloned the BLTX cDNA. It consists of a prosequence and an active form of 253 aa with a typical catalytic triad of serine proteases, with a high identity with tissue kallikreins. BLTX is an N-linked microheterogeneous glycoprotein with a unique insertion of 10 residues, L(106)TFFYKTFLG(115). BLTX converted kininogens to kinins, which may be one of the toxic pathogens, and had dilatory effects on the blood vessel walls. The acute toxicity and proteolytic activity of BLTX were strongly inhibited by aprotinin, a kallikrein inhibitor, suggesting that its toxicity is due to a kallikrein-like activity of the venom.

Amino Acid Sequence↗

PubMed2004

Characterization of CG6178 gene product with high sequence similarity to firefly luciferase in Drosophila melanogaster.

This is the first identification of a long-chain fatty acyl-CoA synthetase in Drosophila by enzymatic characterization. The gene product of CG6178 (CG6178) in Drosophila melanogaster genome, which has a high sequence similarity to firefly luciferase, has been expressed and characterized. CG6178 showed long-chain fatty acyl-CoA synthetic activity in the presence of ATP, CoA and Mg(2+), suggesting a fatty acyl adenylate is an intermediate. Recently, it was revealed that firefly luciferase has two catalytic functions, monooxygenase (luciferase) and AMP-mediated CoA ligase (fatty acyl-CoA synthetase). However, unlike firefly luciferase, CG6178 did not show luminescence activity in the presence of firefly luciferin, ATP, CoA and Mg(2+). The enzymatic properties of CG6178 including substrate specificity, pH dependency and optimal temperature were close to those of firefly luciferase and rat fatty acyl-CoA synthetase. Further, phylogenic analyses strongly suggest that the firefly luciferase gene may have evolved from a fatty acyl-CoA synthetase gene as a common ancestral gene.

Adenosine Monophosphate↗

PubMed2004

Zooxanthellamide B, a novel large polyhydroxy metabolite from a marine dinoflagellate of Symbiodinium sp.

Zooxanthellamide B, C(128)H(220)N(2)O(53)S(2), a polyhydroxy secondary metabolite, was isolated from a cultured marine dinoflagellate of the genus Symbiodinium. A detailed 2D NMR analysis revealed the chemical structure as a delta-lactone analogue of zooxanthellamide A, which had previously been isolated from the same dinoflagellate by us. The relative configuration of the delta-lactone moiety was determined by NOE experiments and a coupling constant analysis, and that of other ring systems was found to be the same as zooxanthellamide A by the chemical correlation between zooxanthellamides A and B.

Deuterium Oxide↗

PubMed2003

Synthesis of biotinylated xestoquinone that retains inhibitory activity against Ca2+ ATPase of skeletal muscle myosin.

Xestoquinone isolated from a marine sponge binds to skeletal muscle myosin and inhibits its Ca(2+) ATPase activity. In this study, we first examined xestoquinone and its analogues to assess the relationships between structure and myosin Ca(2+) ATPase inhibitory activity. On the basis of the resultant data, we then designed a biotinylated xestoquinone analogue. Xestoquinone and its analogues were derived from extracts of the marine sponge Xestospongia sapra. Four xestoquinone analogues with a quinone structure significantly inhibited Ca(2+) ATPase activity. In contrast, four xestoquinone analogues in which the quinone structure was converted to a quinol dimethyl ether did not inhibit Ca(2+) ATPase activity. This suggests that the quinone moiety is essential for inhibitory activity. Then, we synthesized a biotinylated xestoquinone in which a biotin tag was introduced to a site far from the quinone moiety, and this molecule exhibited stronger inhibitory activity than that of xestoquinone. This biotinylated xestoquinone could be useful as a probe in studies of the xestoquinone-myosin binding mode.

Animals↗

PubMed2003

Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase.

Firefly luciferase can catalyze the formation of fatty acyl-CoA via fatty acyl-adenylate from fatty acid in the presence of ATP, Mg(2+) and coenzyme A (CoA). A long chain fatty acyl-CoA (C(16)-C(20)), produced by luciferase from a North American firefly (Photinus pyralis) and a Japanese firefly (Luciola cruciata), was isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Of a number of substrates tested, linolenic acid (C(18:3)) and arachidonic acid (C(20:4)) appear to be suitable for acyl-CoA synthesis. This evidence suggests that firefly luciferase within peroxisomes of the cells in the photogenic organ may be a bifunctional enzyme, catalyzing not only the bioluminescence reaction but also the fatty acyl-CoA synthetic reaction.

Acyltransferases↗

PubMed2003

Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization.

The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Actin Cytoskeleton↗

PubMed2002

Synergistic expression of Ad4BP/SF-1 and cytochrome P-450 aromatase (ovarian type) in the ovary of Nile tilapia, Oreochromis niloticus, during vitellogenesis suggests transcriptional interaction.

Involvement of Ad4BP/SF-1 in the ovarian cytochrome P-450 aromatase (oP450arom) gene expression was investigated using ovarian follicles of the Nile tilapia, possessing an average 14-day spawning cycle. The promoter region (5' flanking region) of oP450arom gene cloned from tilapia contains two Ad4 binding sites. Subsequently, a cDNA encoding Ad4BP/SF-1 was cloned from the ovarian follicles. It is expressed in gonadal tissues, brain, and kidney. Oligonucleotide probes containing putative orphan nuclear receptor binding motifs (derived from promoter region of the aromatase gene) formed complexes with in vitro-translated Ad4BP/SF-1 and nuclear extracts of tilapia ovarian (midvitellogenic) follicles, indicating that Ad4BP/SF-1 is one of the transcriptional regulators for aromatase gene expression. Northern blot analysis revealed that the expression of both oP450arom and Ad4BP/SF-1 increased in parallel with ovarian growth from Day 0 to Day 5 after spawning and declined sharply from Day 8 to Day 11. On the day of spawning (Day 14), the expression of both correlates became undetectable. In vitro incubation of post vitellogenic full-grown immature follicles (corresponding to Day 11 after spawning) with hCG purged both oP450arom and Ad4BP/SF-1 messenger RNA transcripts at 18 h. Conversely, in vitro incubation of late vitellogenic follicles (corresponding to Day 8 after spawning) with hCG retained Ad4BP/SF-1 messenger RNA transcripts more or less steadily and up-regulated oP450arom. Ad4BP/SF-1 probably acts as a transcriptional modulator to implement the paradoxical actions of gonadotropins on oP450arom gene.

Animals↗