Yuichi Oba

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.

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

Abstract 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.

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, Mg2+ and coenzyme A (CoA). A long chain fatty acyl‐CoA (C16–C20), 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 (C18:3) and arachidonic acid (C20:4) 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.

Ovarian Carbonyl Reductase-Like 20β-Hydroxysteroid Dehydrogenase Shows Distinct Surge in Messenger RNA Expression During Natural and Gonadotropin-Induced Meiotic Maturation in Nile Tilapia

Abstract Meiotic maturation in fish is accomplished by maturation-inducing hormones. 17α,20β-Dihydroxy-4-pregnen-3-one (17α,20β-DP) was identified as the maturation-inducing hormone of several teleosts, including Nile tilapia. A cDNA encoding 20β-hydroxysteroid dehydrogenase (20β-HSD), the enzyme that converts 17α-hydroxyprogesterone to 17α,20β-DP, was cloned from the ovarian follicle of Nile tilapia. Genomic Southern analysis indicated that 20β-HSD probably exists as a single copy in the genome. The Escherichia coli-expressed cDNA product oxidized both carbonyl and steroid compounds, including progestogens, in the presence of NADPH. Carbonyl reductase-like 20β-HSD is broadly expressed in various tissues of tilapia, including ovary, testis, and gill. Northern blot and reverse transcription polymerase chain reaction analyses during the 14-day spawning cycle revealed that the expression of 20β-HSD in ovarian follicles is low from Day 0 to Day 8 after spawning and is not detectable on Day 11. Distinct expression was evident at Day 14, the day of spawning. In males, 20β-HSD expression was observed continually in mature testes but not in immature testes of 30-day-old fish. In vitro incubation of postvitellogenic immature follicles (corresponding to Day 11 after spawning) with hCG induced the expression of 20β-HSD mRNA transcripts within 1–2 h, followed by the final meiotic maturation of oocytes. In tissues such as gill, muscle, brain, and pituitary, however, hCG treatment did not induce any changes in the levels of mRNA transcripts. Actinomycin D blockade of hCG-induced 20β-HSD expression and final oocyte maturation demonstrated the involvement of transcriptional factors. The carbonyl reductase-like 20β-HSD plays an important role in the meiotic maturation of tilapia gametes.

The Chemical Basis of Fungal Bioluminescence

Many species of fungi naturally produce light, a phenomenon known as bioluminescence, however, the fungal substrates used in the chemical reactions that produce light have not been reported. We identified the fungal compound luciferin 3-hydroxyhispidin, which is biosynthesized by oxidation of the precursor hispidin, a known fungal and plant secondary metabolite. The fungal luciferin does not share structural similarity with the other eight known luciferins. Furthermore, it was shown that 3-hydroxyhispidin leads to bioluminescence in extracts from four diverse genera of luminous fungi, thus suggesting a common biochemical mechanism for fungal bioluminescence.

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.

The role of doublesex in the evolution of exaggerated horns in the Japanese rhinoceros beetle

Male‐specific exaggerated horns are an evolutionary novelty and have diverged rapidly via intrasexual selection. Here, we investigated the function of the conserved sex‐determination gene doublesex (dsx) in the Japanese rhinoceros beetle (Trypoxylus dichotomus) using RNA interference (RNAi). Our results show that the sex‐specific T. dichotomus dsx isoforms have an antagonistic function for head horn formation and only the male isoform has a role for thoracic horn formation. These results indicate that the novel sex‐specific regulation of dsx during horn morphogenesis might have been the key evolutionary developmental event at the transition from sexually monomorphic to sexually dimorphic horns.

Biosynthesis of coelenterazine in the deep-sea copepod, Metridia pacifica

Coelenterazine is an imidazopyrazinone compound (3,7-dihydroimidazopyrazin-3-one structure) that is widely distributed in marine organisms and used as a luciferin for various bioluminescence reactions. We have used electrospray ionization-ion trap-mass spectrometry to investigate whether the deep-sea luminous copepod Metridia pacifica is able to synthesize coelenterazine. By feeding experiments using deuterium labeled amino acids of l-tyrosine and l-phenylalanine, we have shown that coelenterazine can be synthesized from two molecules of l-tyrosine and one molecule of l-phenylalanine in M. pacifica. This is the first demonstration that coelenterazine is biosynthesized from free l-amino acids in a marine organism.

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.

Identification and characterization of a luciferase isotype in the Japanese firefly, Luciola cruciata, involving in the dim glow of firefly eggs.

We isolated the cDNA of a luciferase isotype (LcLuc2) from the Japanese firefly, Luciola cruciata (Lampyridae, Coleoptera). The gene product of LcLuc2 (LcLuc2) showed 59% amino acid identity with firefly luciferase LcLuc1, which was previously identified in L. cruciata. The recombinant LcLuc2 showed both luminescence activity and fatty acyl-CoA synthetic activity comparable to those of LcLuc1. The spectral maxima of the luminescence by LcLuc1 and LcLuc2 were 554 and 543 nm, respectively. Reverse transcription-PCR analysis showed that the transcripts of LcLuc1 were abundant in the lanterns of larva, adult male, and adult female, whereas both LcLuc1 and LcLuc2 were expressed in eggs. The luminescence spectra of the lantern extracts from larva, adult male, and adult female were in good agreement with that of recombinant LcLuc1. On the other hand, the emission maximum of the extract from eggs was between those of LcLuc1 and LcLuc2. These results suggest that L. cruciata possesses two luciferases: LcLuc1 is responsible for the major luminescence in larva and adult, whereas LcLuc1 and LcLuc2 are responsible for the dim glow in firefly eggs.