Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbagen.2023.130342

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Abstract

Orbital cavernous venous malformation (OCVM) is a sporadic vascular anomaly of uncertain etiology characterized by abnormally dilated vascular channels. Here, we identify a somatic missense mutation, c.121G > T (p.Gly41Cys) in GJA4, which encodes a transmembrane protein that is a component of gap junctions and hemichannels in the vascular system, in OCVM tissues from 25/26 (96.2%) individuals with OCVM. GJA4 expression was detected in OCVM tissue including endothelial cells and the stroma, through immunohistochemistry. Within OCVM tissue, the mutation allele frequency was higher in endothelial cell-enriched fractions obtained using magnetic-activated cell sorting. Whole-cell voltage clamp analysis in Xenopus oocytes revealed that GJA4 c.121G > T (p.Gly41Cys) is a gain-of-function mutation that leads to the formation of a hyperactive hemichannel. Overexpression of the mutant protein in human umbilical vein endothelial cells led to a loss of cellular integrity, which was rescued by carbenoxolone, a non-specific gap junction/hemichannel inhibitor. Our data suggest that GJA4 c.121G > T (p.Gly41Cys) is a potential driver gene mutation for OCVM. We propose that hyperactive hemichannel plays a role in the development of this vascular phenotype.

DOI： 10.1007/s10456-022-09846-5

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Other Link： https://link.springer.com/article/10.1007/s10456-022-09846-5/fulltext.html

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Pannexin (PANX) family proteins form large-pore channels that mediate purinergic signaling. We analyzed the cryo-EM structures of human PANX1 in lipid nanodiscs to elucidate the gating mechanism and its regulation by the amino terminus in phospholipids. The wild-type channel has an amino-terminal funnel in the pore, but in the presence of the inhibitor probenecid, a cytoplasmically oriented amino terminus and phospholipids obstruct the pore. Functional analysis using whole-cell patch-clamp and oocyte voltage clamp showed that PANX1 lacking the amino terminus did not open and had a dominant negative effect on channel activity, thus confirming that the amino-terminal domain played an essential role in channel opening. These observations suggest that dynamic conformational changes in the amino terminus of human PANX1 are associated with lipid movement in and out of the pore. Moreover, the data provide insight into the gating mechanism of PANX1 and, more broadly, other large-pore channels.

DOI： 10.1126/scisignal.abg6941

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Royal Society  

Skin patterns are the first example of the existence of Turing patterns in living organisms. Extensive research on zebrafish, a model organism with stripes on its skin, has revealed the principles of pattern formation at the molecular and cellular levels. Surprisingly, although the networks of cell–cell interactions have been observed to satisfy the ‘short-range activation and long-range inhibition’ prerequisites for Turing pattern formation, numerous individual reactions were not envisioned based on the classical reaction–diffusion model. For example, in real skin, it is not an alteration in concentrations of chemicals, but autonomous migration and proliferation of pigment cells that establish patterns, and cell–cell interactions are mediated via direct contact through cell protrusions. Therefore, the classical reaction–diffusion mechanism cannot be used as it is for modelling skin pattern formation. Various studies are underway to adapt mathematical models to the experimental findings on research into skin patterns, and the purpose of this review is to organize and present them. These novel theoretical methods could be applied to autonomous pattern formation phenomena other than skin patterns.


This article is part of the theme issue ‘Recent progress and open frontiers in Turing's theory of morphogenesis’.

DOI： 10.1098/rsta.2020.0274

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Other Link： https://royalsocietypublishing.org/doi/full-xml/10.1098/rsta.2020.0274

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

<title>Abstract</title>Mitochondrial translation appears to involve two stalled-ribosome rescue factors (srRFs). One srRF is an ICT1 protein from humans that rescues a “non-stop” type of mitochondrial ribosomes (mitoribosomes) stalled on mRNA lacking a stop codon, while the other, C12orf65, reportedly has functions that overlap with those of ICT1; however, its primary role remains unclear. We herein demonstrated that the <italic>Saccharomyces cerevisiae</italic> homolog of C12orf65, Pth3 (Rso55), preferentially rescued antibiotic-dependent stalled mitoribosomes, which appear to represent a “no-go” type of ribosomes stalled on intact mRNA. On media containing a non-fermentable carbon source, which requires mitochondrial gene expression, respiratory growth was impaired significantly more by the deletion of <italic>PTH3</italic> than that of the <italic>ICT1</italic> homolog <italic>PTH4</italic> in the presence of antibiotics that inhibit mitochondrial translation, such as tetracyclines and macrolides. Additionally, the in organello labeling of mitochondrial translation products and quantification of mRNA levels by quantitative RT-PCR suggested that in the presence of tetracycline, the deletion of <italic>PTH3</italic>, but not <italic>PTH4</italic>, reduced the protein expression of all eight mtDNA-encoded genes at the post-transcriptional or translational level. These results indicate that Pth3 can function as a mitochondrial srRF specific for ribosomes stalled by antibiotics and plays a role in antibiotic resistance in fungi.

DOI： 10.1038/s42003-021-01835-6

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Other Link： http://www.nature.com/articles/s42003-021-01835-6

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.bbadva.2021.100006

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Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in the ancestor of goldfish and common carp. We conducted whole-genome sequencing of 27 domesticated goldfish strains and wild goldfish. We identified more than 60 million genetic variations and established a population genetic structure of major goldfish strains. Genome-wide association studies and analysis of strain-specific variants revealed genetic loci associated with several goldfish phenotypes, including dorsal fin loss, long-tail, telescope-eye, albinism, and heart-shaped tail. Our results suggest that accumulated mutations in the asymmetrically evolved subgenomes led to generation of diverse phenotypes in the goldfish domestication history. This study is a key resource for understanding the genetic basis of phenotypic diversity among goldfish strains.

DOI： 10.1016/j.cub.2020.04.034

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Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.

DOI： 10.1126/sciadv.aax3157

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

Connexin39.4 (Cx39.4) and Connexin41.8 (Cx41.8), two gap-junction proteins expressed in both melanophores and xanthophores, are critical for the intercellular communication among pigment cells that is necessary for generating the stripe pigment pattern of zebrafish. We previously characterized the gap-junction properties of Cx39.4 and Cx41.8, but how these proteins contribute to stripe formation remains unclear; this is because distinct types of connexins potentially form heteromeric gap junctions, which precludes accurate elucidation of individual connexin functions in vivo. Here, by arranging Cx39.4 and Cx41.8 expression in pigment cells, we identified the simplest gap-junction network required for stripe generation: Cx39.4 expression in melanophores is required but expression in xanthophores is not necessary for stripe patterning, whereas Cx41.8 expression in xanthophores is sufficient for the patterning, and Cx41.8 expression in melanophores might stabilize the stripes. Moreover, patch-clamp recordings revealed that Cx39.4 gap junctions exhibit spermidine-dependent rectification property. Our results suggest that Cx39.4 facilitates the critical cell-cell interactions between melanophores and xanthophores that mediate a unidirectional activation-signal transfer from xanthophores to melanophores, which is essential for melanophore survival.

DOI： 10.1242/dev.181065

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Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fphys.2019.00080

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1111/dgd.12564

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Epithelial tissues line the lumen of tracts and ducts connecting to the external environment. They are critical in forming an interface between the internal and external environment and, following assault from environmental factors and pathogens, they must rapidly repair to maintain cellular homeostasis. These tissue networks, that range from a single cell layer, such as in airway epithelium, to highly stratified and differentiated epithelial surfaces, such as the epidermis, are held together by a junctional nexus of proteins including adherens, tight and gap junctions, often forming unique and localised communication compartments activated for localised tissue repair. This review focuses on the dynamic changes that occur in connexins, the constituent proteins of the intercellular gap junction channel, during wound-healing processes and in localised inflammation, with an emphasis on the lung and skin. Current developments in targeting connexins as corrective therapies to improve wound closure and resolve localised inflammation are also discussed. Finally, we consider the emergence of the zebrafish as a concerted whole-animal model to study, visualise and track the events of wound repair and regeneration in real-time living model systems.

DOI： 10.3390/ijms19051354

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Frontiers Media SA  

DOI： 10.3389/fcell.2017.00013

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (other academic)  

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1074/jbc.m116.720110

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DOI： 10.1074/jbc.m115.673129

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Authorship：Lead author   Publishing type：Research paper (scientific journal)   Publisher：American Association for the Advancement of Science (AAAS)  

Mahalwar et al . (Reports, 12 September 2014, p. 1362) observed the onset of pigment pattern formation in zebrafish. They concluded that their data do not support our Turing mechanism–based model and presented an essentially different mechanism. Here, we clarify their misunderstanding that may have caused their conclusion and explain past experimental data that do not support their proposed mechanism.

DOI： 10.1126/science.1261947

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DOI： 10.1016/j.tig.2014.11.005

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DOI： 10.1111/pcmr.12328

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Authorship：Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：The Company of Biologists  

The skin pigment pattern of zebrafish is a good model system in which to study the mechanism of biological pattern formation. Although it is known that interactions between melanophores and xanthophores play a key role in the formation of adult pigment stripes, molecular mechanisms for these interactions remain largely unknown. Here, we show that Delta/Notch signaling contributes to these interactions. Ablation of xanthophores in yellow stripes induced the death of melanophores in black stripes, suggesting that melanophores require a survival signal from distant xanthophores. We found that deltaC and notch1a were expressed by xanthophores and melanophores, respectively. Moreover, inhibition of Delta/Notch signaling killed melanophores, whereas activation of Delta/Notch signaling ectopically in melanophores rescued the survival of these cells, both in the context of pharmacological inhibition of Delta/Notch signaling and after ablation of xanthophores. Finally, we showed by in vivo imaging of cell membranes that melanophores extend long projections towards xanthophores in the yellow stripes. These data suggest that Delta/Notch signaling is responsible for a survival signal provided by xanthophores to melanophores. As cellular projections can enable long-range interaction between membrane-bound ligands and their receptors, we propose that such projections, combined with direct cell-cell contacts, can substitute for the effect of a diffusible factor that would be expected by the conventional reaction-diffusion (Turing) model.

DOI： 10.1242/dev.099804

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Authorship：Last author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Summary

Skin pigment pattern formation in zebrafish requires pigment‐cell autonomous interactions between melanophores and xanthophores, yet the molecular bases for these interactions remain largely unknown. Here, we examined the dali mutant that exhibits stripes in which melanophores are intermingled abnormally with xanthophores. By in vitro cell culture, we found that melanophores of dali mutants have a defect in motility and that interactions between melanophores and xanthophores are defective as well. Positional cloning and rescue identified dali as tetraspanin 3c (tspan3c), encoding a transmembrane scaffolding protein expressed by melanophores and xanthophores. We further showed that dali mutant Tspan3c expressed in HeLa cell exhibits a defect in N‐glycosylation and is retained inappropriately in the endoplasmic reticulum. Our results are the first to identify roles for a tetraspanin superfamily protein in skin pigment pattern formation and suggest new mechanisms for the establishment and maintenance of zebrafish stripe boundaries.

DOI： 10.1111/pcmr.12192

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：S. Karger AG  

Cichlid fishes in the African Great Lakes are known as a spectacular example of adaptive radiation in vertebrates. Four linkage maps have been constructed to identify the genes responsible for adaptation and speciation, and the genetic linkages of those genes are assumed to play an important role during adaptive evolution. However, it is difficult to analyze such linkages because the linkage groups of one species do not match well with those of the other species. Chromosome markers are a powerful tool for the direct identification of linkage homology between different species. We used information about the linkage map of the Lake Malawi cichlid (Labeotropheus fuelleborni/Metriaclima zebra) to isolate bacterial artificial chromosome (BAC) clones from the BAC library of Paralabidochromis chilotes, Lake Victoria. We identified 18 of 22 P. chilotes chromosomes by single- and multi-color BAC fluorescence in situ hybridization using 19 BAC clones. Comparative mapping with the chromosome markers of P. chilotes in Astatotilapia burtoni (2n = 40) from Lake Tanganyika revealed the chromosome rearrangements that have occurred in this lineage. These chromosome markers will be useful for delineating the process of genome and chromosome evolution in African species. (C) 2013 S. Karger AG, Basel

DOI： 10.1159/000356128

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DOI： 10.1371/journal.pgen.1002899

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Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1038/srep00473

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Other Link： https://www.nature.com/articles/srep00473.pdf

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

Summary

The skin patterns of animals are very important for their survival, yet the mechanisms involved in skin pattern formation remain unresolved. Turing’s reaction–diffusion model presents a well‐known mathematical explanation of how animal skin patterns are formed, and this model can predict various animal patterns that are observed in nature. In this study, we used transgenic zebrafish to generate various artificial skin patterns including a narrow stripe with a wide interstripe, a narrow stripe with a narrow interstripe, a labyrinth, and a ‘leopard’ pattern (or donut‐like ring pattern). In this process, connexin41.8 (or its mutant form) was ectopically expressed using the mitfa promoter. Specifically, the leopard pattern was generated as predicted by Turing’s model. Our results demonstrate that the pigment cells in animal skin have the potential and plasticity to establish various patterns and that the reaction–diffusion principle can predict skin patterns of animals.

DOI： 10.1111/j.1755-148x.2012.00984.x

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The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

DOI： 10.1371/journal.pgen.1002203

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DOI： 10.1016/j.ydbio.2010.05.063

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Lake Victoria harbors hundreds of cichlid species, which have aggressively developed their morphological features through their evolution. In particular, the shapes of jaws and teeth have diverged markedly, and correlate with feeding ecology. These species are believed to have explosively arisen within the last 15,000 years and are therefore of particular interest to evolutionary biologists. Previously, we reported expressed sequence tags (ESTs) from two cichlid species, Haplochromis chilotes and H. sp. "redtailsheller", and provided a comparative EST analysis. In this study, we further examined the molecular basis for diversification of Lake Victoria cichlids by generating EST from another cichlid species, H. sp. "Matumbi hunter"; 9,219 clones were applied for sequence determination and 16,795 sequences were newly obtained from this EST set. Comparative analysis of the 68 genes common to the three cichlid species revealed that the degrees of genetic distance and the degrees of genetic polymorphism were highly similar for each pair-wise comparison, suggesting that the protein coding regions of the three cichlid genomes are essentially the same. Because the three species used for the comparative analysis were particularly diverged from each other in morphological and also ecological features, we concluded that the uniformity of transcript sequences among cichlid species in Lake Victoria is a global feature.

DOI： 10.1016/j.gene.2008.11.023

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DOI： 10.1016/j.gene.2007.04.024

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DOI： 10.1371/journal.pbio.0040433

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Lake Victoria harbors more than 300 species of cichlid fish, which are adapted to a variety of ecological niches with various morphological species-specific features. However, it is believed that these species arose explosively within the last 14,000 years and transcripts among Lake Victoria cichlid species are almost identical in sequence. These data prompted us to develop a DNA chip assay to compare patterns of gene expression among cichlid species. We prepared a DNA chip spotted with 6240 elements derived from cichlid expressed sequence tag (EST) clones and successfully characterized gene expression differences between the cichlid species Haplochromis chilotes and Haplochromis sp. "rockkribensis". We identified 14 transcripts that were differentially expressed between these species at an early developmental stage, 15 days post-fertilization (dpf), and several were further analyzed using quantitative real-time PCR (qPCR). One of these differentially expressed transcripts was a homolog of microfibril-associated glycoprotein 4 (magp4), a putative causative gene for the human inherited disease, Smith-Magenis syndrome (SMS), for which facial defects are among the phenotypic features. Further analysis of magp4 expression showed that magp4 was expressed in the jaw portion of cichlid fry and that expression profiles between Haplochromis chilotes and Haplochromis sp. "rockkribensis" differed during development. These data suggest that the differential expression of a gene associated with human cranial morphogenesis may be involved in the diversification of cichlid jaw morphs.

DOI： 10.1016/j.gene.2006.01.016

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DOI： 10.1371/journal.pgen.0020197

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DOI： 10.1016/j.gene.2004.09.013

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Genetics Society of Japan  

Cichlid fishes in Lake Victoria are model organisms for studying rapid radiation and speciation. On the way to examine the molecular basis of how these cichlid fishes achieved such a remarkable morphological diversification, we constructed a bacterial artificial chromosome (BAC) library derived from a cichlid species, Haplochromis chilotes, from Lake Victoria. The library includes 157,056 clones with the average insert size of 128 kb, corresponding to a 10-fold coverage of the H. chilotes genome. Given that the cichlid fishes endemic to Lake Victoria are closely related to one another phylogenetically and their genomes are nearly identical, this BAC library can be utilized to isolate genes from the more than 200 Haplochromine cichlid species in Lake Victoria.

DOI： 10.1266/ggs.78.103

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Archaeosine tRNA-guanine transglycosylase (ArcTGT) catalyzes the exchange of guanine at position 15 in the D-loop of archaeal tRNAs with a free 7-cyano-7-deazaguanine (preQ(0)) base, as the first step in the biosynthesis of an archaea-specific modified base, archaeosine (7-formamidino-7-deazaguanosine). We determined the crystal structures of ArcTGT from Pyrococcus horikoshii at 2.2 A resolution and its complexes with guanine and preQ(0), at 2.3 and 2.5 A resolutions, respectively. The N-terminal catalytic domain folds into an (alpha/beta)(8) barrel with a characteristic zinc-binding site, showing structural similarity with that of the bacterial queuosine TGT (QueTGT), which is involved in queuosine (7-[[(4,5-cis-dihydroxy-2-cyclopenten-1-yl)-amino]methyl]-7-deazaguanosine) biosynthesis and targets the tRNA anticodon. ArcTGT forms a dimer, involving the zinc-binding site and the ArcTGT-specific C-terminal domain. The C-terminal domains have novel folds, including an OB fold-like "PUA domain", whose sequence is widely conserved in eukaryotic and archaeal RNA modification enzymes. Therefore, the C-terminal domains may be involved in tRNA recognition. In the free-form structure of ArcTGT, an alpha-helix located at the rim of the (alpha/beta)(8) barrel structure is completely disordered, while it is ordered in the guanine-bound and preQ(0)-bound forms. Structural comparison of the ArcTGT.preQ(0), ArcTGT.guanine, and QueTGT.preQ(1) complexes provides novel insights into the substrate recognition mechanisms of ArcTGT.

DOI： 10.1016/s0022-2836(02)00090-6

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Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

DOI： 10.1128/jb.183.20.6009-6016.2001

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DOI： 10.1074/jbc.m005043200

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DOI： 10.1074/jbc.272.32.20146

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

It is proposed that arylmalonate decarboxylase (AMDase)-catalyzed decarboxylation proceeds via a thiol ester intermediate. Kinetics and CD spectra indicated that α-bromophenylacetate is a competitive inhibitor. TOF mass data indicated that the inhibitor bound with the enzyme through a thiol ester bond which was formed between a cysteine residue of the enzyme and the carboxyl group of the inhibitor. This result was also supported by reactivation of the enzyme by the addition of 2-mercaptoethanol, which is expected to cleave the enzyme–inhibitor bond via nucleophilic attack on the thiol ester linkage.

DOI： 10.1246/bcsj.68.2017

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