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We developed a computing method to identify linear cationic α-helical antimicrobial peptides (LCAMPs) in the genome of Ciona intestinalis based on its structural and physicochemical features. Using this method, 22 candidates of Ciona LCAMPs, including well-known antimicrobial peptides, were identified from 21,975 non-redundant amino acid sequences in Ciona genome database, Ghost database. We also experimentally confirmed the antimicrobial activities of five LCAMP candidates, and three of them were found to be active in the presence of 500 mM NaCl, nearly equivalent to the salt concentration of seawater. Membrane topology prediction suggested that salt resistance of Ciona LCAMPs might be influenced by hydrophobic interactions between the peptide and membrane. Further, we applied our method to Xenopus tropicalis genome and found 11 LCAMP candidates. Thus, our method may serve as an effective and powerful tool for searching LCAMPs that are difficult to find using conventional homology-based methods.
Yukio Ohtsuka; Hidetoshi Inagaki. In silico identification and functional validation of linear cationic α-helical antimicrobial peptides in the ascidian Ciona intestinalis. Scientific Reports 2020, 10, 1 -9.
AMA StyleYukio Ohtsuka, Hidetoshi Inagaki. In silico identification and functional validation of linear cationic α-helical antimicrobial peptides in the ascidian Ciona intestinalis. Scientific Reports. 2020; 10 (1):1-9.
Chicago/Turabian StyleYukio Ohtsuka; Hidetoshi Inagaki. 2020. "In silico identification and functional validation of linear cationic α-helical antimicrobial peptides in the ascidian Ciona intestinalis." Scientific Reports 10, no. 1: 1-9.
We determined the complete mitochondrial genome sequence of the Japanese forest green tree frog (Rhacophorus arboreus). The mitochondrial genome is 22,236 bp in length, which encodes 13 protein-coding genes, 2 rRNA, and 22 tRNA genes, and two control regions (D-loops). The whole gene arrangement of R. arboreus was the same as that of Rhacophorus omeimontis and Rhacophorus schlegelii.
Hidetoshi Inagaki; Yoshikazu Haramoto; Hiroshi Y. Kubota; Yasushi Shigeri. Complete mitochondrial genome sequence of Japanese forest green tree frog (Rhacophorus arboreus). Mitochondrial DNA Part B 2020, 5, 3347 -3348.
AMA StyleHidetoshi Inagaki, Yoshikazu Haramoto, Hiroshi Y. Kubota, Yasushi Shigeri. Complete mitochondrial genome sequence of Japanese forest green tree frog (Rhacophorus arboreus). Mitochondrial DNA Part B. 2020; 5 (3):3347-3348.
Chicago/Turabian StyleHidetoshi Inagaki; Yoshikazu Haramoto; Hiroshi Y. Kubota; Yasushi Shigeri. 2020. "Complete mitochondrial genome sequence of Japanese forest green tree frog (Rhacophorus arboreus)." Mitochondrial DNA Part B 5, no. 3: 3347-3348.
We previously identified 92 toxin-like peptides and proteins, including pilosulin-like peptides 1–6 from the predatory ant Odontomachus monticola, by transcriptome analysis. Here, to further characterize venom components, we analyzed the venom and venom sac extract by ESI-MS/MS with or without trypsin digestion and reducing agent. As the low-molecular-mass components, we found amino acids (leucine/isoleucine, phenylalanine, and tryptophan) and biogenic amines (histamine and tyramine) in the venom and venom sac extract. As the higher molecular mass components, we found peptides and proteins such as pilosulin-like peptides, phospholipase A2s, hyaluronidase, venom dipeptidyl peptidases, conotoxin-like peptide, and icarapin-like peptide. In addition to pilosulin-like peptides 1–6, we found three novel pilosulin-like peptides that were overlooked by transcriptome analysis. Moreover, pilosulin-like peptides 1–6 were chemically synthesized, and some of them displayed antimicrobial, hemolytic, and histamine-releasing activities.
Naoki Tani; Kohei Kazuma; Yukio Ohtsuka; Yasushi Shigeri; Keiichi Masuko; Katsuhiro Konno; Hidetoshi Inagaki. Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components. Toxins 2019, 11, 50 .
AMA StyleNaoki Tani, Kohei Kazuma, Yukio Ohtsuka, Yasushi Shigeri, Keiichi Masuko, Katsuhiro Konno, Hidetoshi Inagaki. Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components. Toxins. 2019; 11 (1):50.
Chicago/Turabian StyleNaoki Tani; Kohei Kazuma; Yukio Ohtsuka; Yasushi Shigeri; Keiichi Masuko; Katsuhiro Konno; Hidetoshi Inagaki. 2019. "Mass Spectrometry Analysis and Biological Characterization of the Predatory Ant Odontomachus monticola Venom and Venom Sac Components." Toxins 11, no. 1: 50.
Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae, Dolichoderinae, and members of other subfamilies, most ant species have a sting with venom. The venoms are composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Unlike the venoms of other animals such as snakes and spiders, ant venoms have seldom been analyzed comprehensively, and their compositions are not yet completely known. In this study, we used both transcriptomic and peptidomic analyses to study the composition of the venom produced by the predatory ant species Odontomachus monticola. The transcriptome analysis yielded 49,639 contigs, of which 92 encoded toxin-like peptides and proteins with 18,106,338 mapped reads. We identified six pilosulin-like peptides by transcriptomic analysis in the venom gland. Further, we found intact pilosulin-like peptide 1 and truncated pilosulin-like peptides 2 and 3 by peptidomic analysis in the venom. Our findings related to ant venom peptides and proteins may lead the way towards development and application of novel pharmaceutical and biopesticidal resources.
Kohei Kazuma; Keiichi Masuko; Katsuhiro Konno; Hidetoshi Inagaki. Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola. Toxins 2017, 9, 323 .
AMA StyleKohei Kazuma, Keiichi Masuko, Katsuhiro Konno, Hidetoshi Inagaki. Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola. Toxins. 2017; 9 (10):323.
Chicago/Turabian StyleKohei Kazuma; Keiichi Masuko; Katsuhiro Konno; Hidetoshi Inagaki. 2017. "Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola." Toxins 9, no. 10: 323.
Mass spectrometry-guided venom peptide profiling is a powerful tool to explore novel substances from venomous animals in a highly sensitive manner. In this study, this peptide profiling approach is successfully applied to explore the venom peptides of a Japanese solitary carpenter bee, Xylocopa appendiculata (Hymenoptera: Apoidea: Apidae: Anthophila: Xylocopinae: Xylocopini). Although interesting biological effects of the crude venom of carpenter bees have been reported, the structure and biological function of the venom peptides have not been elucidated yet. The venom peptide profiling of the crude venom of X. appendiculata was performed by matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. The venom was purified by a reverse-phase HPLC. The purified peptides were subjected to the Edman degradation, MS/MS analysis, and/or molecular cloning methods for peptide sequencing. Biological and functional characterization was performed by circular dichroism analysis, liposome leakage assay, and antimicrobial, histamine releasing and hemolytic activity tests. Three novel peptides with m/z 16508, 1939.3, and 1900.3 were isolated from the venom of X. appendiculata. The peptide with m/z 16508 was characterized as a secretory phospholipase A2 (PLA2) homolog in which the characteristic cysteine residues as well as the active site residues found in bee PLA2s are highly conserved. Two novel peptides with m/z 1939.3 and m/z 1900.3 were named as Xac-1 and Xac-2, respectively. These peptides are found to be amphiphilic and displayed antimicrobial and hemolytic activities. The potency was almost the same as that of mastoparan isolated from the wasp venom. We found three novel biologically active peptides in the venom of X. appendiculata and analyzed their molecular functions, and compared their sequential homology to discuss their molecular diversity. Highly sensitive mass analysis plays an important role in this study.
Hiroko Kawakami; Shin G. Goto; Kazuya Murata; Hideaki Matsuda; Yasushi Shigeri; Tomohiro Imura; Hidetoshi Inagaki; Tetsuro Shinada. Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata. Journal of Venomous Animals and Toxins including Tropical Diseases 2017, 23, 29 .
AMA StyleHiroko Kawakami, Shin G. Goto, Kazuya Murata, Hideaki Matsuda, Yasushi Shigeri, Tomohiro Imura, Hidetoshi Inagaki, Tetsuro Shinada. Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata. Journal of Venomous Animals and Toxins including Tropical Diseases. 2017; 23 (1):29.
Chicago/Turabian StyleHiroko Kawakami; Shin G. Goto; Kazuya Murata; Hideaki Matsuda; Yasushi Shigeri; Tomohiro Imura; Hidetoshi Inagaki; Tetsuro Shinada. 2017. "Isolation of biologically active peptides from the venom of Japanese carpenter bee, Xylocopa appendiculata." Journal of Venomous Animals and Toxins including Tropical Diseases 23, no. 1: 29.
Homeostasis of the body’s biochemical reactions is maintained by the balance of specific proteases and their counterparts, protease inhibitors. Snake venom protease inhibitors disrupt this balance and are thereby utilized by snakes as weapons to prey on other animals. Snake venom protease inhibitors primarily disrupt blood coagulation and blood pressure regulation. The resulting blood loss, blood clotting, or hypotension leads to immobilization or death of the prey. This review describes the protease inhibitors that are thought to be distributed among many snake species, Kunitz-type protease inhibitors, bradykinin-potentiating peptides, and cystatins. In addition, as many components of snake venoms have diverse functions, the protease inhibitors that share motifs other than the protease inhibitory domains, i.e., phospholipase A2-like proteins and three-finger toxins, are described. The majority of these toxins function as enzymes and neurotoxins, respectively. Finally, recent studies including genome, transcriptome, and proteome analyses have identified protease inhibitor-like proteins as new components of snake venoms; however, none of these have yet been proven to exhibit protease inhibitory activities. In this regard, waprin and Kazal-type protease inhibitor-like proteins will be introduced.
Hidetoshi Inagaki. Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future. Organotypic Models in Drug Development 2017, 161 -186.
AMA StyleHidetoshi Inagaki. Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future. Organotypic Models in Drug Development. 2017; ():161-186.
Chicago/Turabian StyleHidetoshi Inagaki. 2017. "Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future." Organotypic Models in Drug Development , no. : 161-186.
Homeostasis of the body’s biochemical reactions is maintained by the balance of specific proteases and their counterparts, protease inhibitors. Snake venom protease inhibitors disrupt this balance and are thereby utilized by snakes as weapons to prey on other animals. Snake venom protease inhibitors primarily disrupt blood coagulation and blood pressure regulation. The resulting blood loss, blood clotting, or hypotension leads to immobilization or death of the prey. This review describes the protease inhibitors that are thought to be distributed among many snake species, Kunitz-type protease inhibitors, bradykinin-potentiating peptides, and cystatins. In addition, as many components of snake venoms have diverse functions, the protease inhibitors that share motifs other than the protease inhibitory domains, i.e., phospholipase A2-like proteins and three-finger toxins, are described. The majority of these toxins function as enzymes and neurotoxins, respectively. Finally, recent studies including genome, transcriptome, and proteome analyses have identified protease inhibitor-like proteins as new components of snake venoms; however, none of these have yet been proven to exhibit protease inhibitory activities. In this regard, waprin and Kazal-type protease inhibitor-like proteins will be introduced.
Hidetoshi Inagaki. Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future. Organotypic Models in Drug Development 2015, 1 -26.
AMA StyleHidetoshi Inagaki. Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future. Organotypic Models in Drug Development. 2015; ():1-26.
Chicago/Turabian StyleHidetoshi Inagaki. 2015. "Snake Venom Protease Inhibitors: Enhanced Identification, Expanding Biological Function, and Promising Future." Organotypic Models in Drug Development , no. : 1-26.
Kunitz-type protease inhibitors, which consist of around 60 amino acid residues and three distinctive disulfide bridges, exhibit a broad range of physiological functions such as protease inhibitor and ion channel blocker. In this study, we identified cDNAs encoding Kunitz-type protease inhibitors, Pr-mulgins 1, 2 and 3, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequences of the Pr-mulgins are 92.4-99.3% identical with their orthologs in Australian P. australis. Pr-mulgin proteins were recombinantly prepared and subjected to inhibitory assays against proteases. Pr-mulgin 1 significantly affected matrix metalloprotease (MMP) 2; Pr-mulgins 2 and 3 showed potent inhibition to trypsin and plasma plasmin; and Pr-mulgin 2 inhibited α-chymotrypsin. Pr-mulgins 1, 2, and 3, however, had essentially no effect on Drosophila K(+) channels (Shaker) and rat K(+) channels (K(v) 1.1).
Hidetoshi Inagaki; Hikari Kimoto; Yoko Yamauchi; Michihisa Toriba; Tai Kubo. Functional characterization of Kunitz-type protease inhibitor Pr-mulgins identified from New Guinean Pseudechis australis. Toxicon 2012, 59, 74 -80.
AMA StyleHidetoshi Inagaki, Hikari Kimoto, Yoko Yamauchi, Michihisa Toriba, Tai Kubo. Functional characterization of Kunitz-type protease inhibitor Pr-mulgins identified from New Guinean Pseudechis australis. Toxicon. 2012; 59 (1):74-80.
Chicago/Turabian StyleHidetoshi Inagaki; Hikari Kimoto; Yoko Yamauchi; Michihisa Toriba; Tai Kubo. 2012. "Functional characterization of Kunitz-type protease inhibitor Pr-mulgins identified from New Guinean Pseudechis australis." Toxicon 59, no. 1: 74-80.
Snake Phospholipase A(2) (PLA(2)) exhibits diverse pharmacological effects, such as hemolysis, myotoxicity, and neurotoxicity. In this study, we identified 10 novel PLA(2)-like protein cDNAs, which we named Pr 1-10, from the venom gland cDNA library of Papuan pigmy mulga snake (New Guinean Pseudechis australis). The deduced amino acid sequence of Pr 1, which ortholog has not been reported in mulaga snake (Australian P. australis) yet, shows 78.8% identity with the ortholog in Australian tiger snake (Notechis scutatus scutatus). The amino acid sequences of Pr 2-10 are 92.4-99.3% identical with their orthologs and paralogs in Australian P. australis.
Hidetoshi Inagaki; Yoko Yamauchi; Michihisa Toriba; Tai Kubo. Regional divergence of phospholipase A2-like protein cDNAs between New Guinean and Australian Pseudechis australis. Toxicon 2010, 56, 637 -639.
AMA StyleHidetoshi Inagaki, Yoko Yamauchi, Michihisa Toriba, Tai Kubo. Regional divergence of phospholipase A2-like protein cDNAs between New Guinean and Australian Pseudechis australis. Toxicon. 2010; 56 (4):637-639.
Chicago/Turabian StyleHidetoshi Inagaki; Yoko Yamauchi; Michihisa Toriba; Tai Kubo. 2010. "Regional divergence of phospholipase A2-like protein cDNAs between New Guinean and Australian Pseudechis australis." Toxicon 56, no. 4: 637-639.
TRPA1 is a calcium-permeable, nonselective cation channel expressed in the dorsal root ganglion and trigeminal ganglia nociceptive neurons. It is activated by the pungent compounds in mustard oil (AITC, allyl isothiocyanate), cinnamon (cinnamaldehyde), garlic (allicin), and is believed to mediate the inflammatory actions of environmental irritants and proalgesic agents. Thiosulfinate (allicin) and isothiocyanate (AITC) compounds contain reactive electrophilic chemical groups that react with cysteine residues within the TRPA1 channel N terminus, leading to channel activation. Ajoene also contains reactive electrophilic chemical groups likely to target TRPA1 channel. Here, we have used voltage-clamp recordings to show that TRPA1-responses are enhanced by ajoene application in a Xenopus oocyte expression system. Though ajoene alone did not activate TRPA1, subsequent application of ajoene enhanced the AITC-, allicin- and depolarization-induced responses of TRPA1. Moreover, when increasing concentrations of ajoene were applied along with constant concentrations of allicin or AITC, stronger responses were elicited. These findings suggest that ajoene is a novel TRPA1 channel enhancer, operating in a channel-opening-dependent manner.
Ricardo Tsuneo Yassaka; Hidetoshi Inagaki; Tsuchiyoshi Fujino; Kei Nakatani; Tai Kubo. Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative. Neuroscience Research 2010, 66, 99 -105.
AMA StyleRicardo Tsuneo Yassaka, Hidetoshi Inagaki, Tsuchiyoshi Fujino, Kei Nakatani, Tai Kubo. Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative. Neuroscience Research. 2010; 66 (1):99-105.
Chicago/Turabian StyleRicardo Tsuneo Yassaka; Hidetoshi Inagaki; Tsuchiyoshi Fujino; Kei Nakatani; Tai Kubo. 2010. "Enhanced activation of the transient receptor potential channel TRPA1 by ajoene, an allicin derivative." Neuroscience Research 66, no. 1: 99-105.
Venom of the Australian ant species Myrmecia pilosula contains a number of allergenic peptides including pilosulins. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of M. pilosula species complex was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulins. We isolated a cDNA clone encoding the novel bioactive peptide pilosulin 5. Tandem mass analysis was entirely consistent with the cDNA derived sequence, and indicated that pilosulin 5 is connected by a single disulfide bridge to create a dimmer peptide of 8546Da. Synthetic pilosulin 5 peptide caused a significant histamine release in a dose-dependent manner, and the mastoparan homologous region of pilosulin 5 was responsible for the activity.
Hidetoshi Inagaki; Masaaki Akagi; Hirotami T. Imai; Robert W. Taylor; Michael D. Wiese; Noel Davies; Tai Kubo. Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant). Archives of Biochemistry and Biophysics 2008, 477, 411 -416.
AMA StyleHidetoshi Inagaki, Masaaki Akagi, Hirotami T. Imai, Robert W. Taylor, Michael D. Wiese, Noel Davies, Tai Kubo. Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant). Archives of Biochemistry and Biophysics. 2008; 477 (2):411-416.
Chicago/Turabian StyleHidetoshi Inagaki; Masaaki Akagi; Hirotami T. Imai; Robert W. Taylor; Michael D. Wiese; Noel Davies; Tai Kubo. 2008. "Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant)." Archives of Biochemistry and Biophysics 477, no. 2: 411-416.
Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.
Hidetoshi Inagaki; Masaaki Akagi; Hirotami T. Imai; Robert W. Taylor; Tai Kubo. Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia. Archives of Biochemistry and Biophysics 2004, 428, 170 -178.
AMA StyleHidetoshi Inagaki, Masaaki Akagi, Hirotami T. Imai, Robert W. Taylor, Tai Kubo. Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia. Archives of Biochemistry and Biophysics. 2004; 428 (2):170-178.
Chicago/Turabian StyleHidetoshi Inagaki; Masaaki Akagi; Hirotami T. Imai; Robert W. Taylor; Tai Kubo. 2004. "Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia." Archives of Biochemistry and Biophysics 428, no. 2: 170-178.