Bulk isolation of immune response-related genes by expressed sequenced tags of Japanese flounder Paralichthys olivaceus leucocytes stimulated with Con A/PMA.
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Amino Acid Sequence↗
Biomedical subjects
Takashi Aoki
Publications and source records attributed to Takashi Aoki.
Upper extremity interaction with a deploying side airbag: a characterization of elbow joint loading.
Computer simulations, dummy experiments with a new enhanced upper extremity and small female cadaver experiments were used to analyze the small female upper extremity response under side airbag loading. After establishing a worst case initial position, three tests were performed with the fifth percentile female hybrid III anthropometric test dummy and six experiments with small female cadaver subjects. A new fifth percentile female enhanced upper extremity was developed for the dummy experiments that included a two-axis wrist load cell in addition to the existing six-axis load cells in both the forearm and humerus. Forearm pronation was also included in the new dummy upper extremity to increase the biofidelity of the interaction with the handgrip. Instrumentation for both the cadaver and dummy tests included accelerometers and MHD angular rate sensors on the forearm, humerus, upper and lower spine. In order to quantify the applied loads to the cadaver hand and wrist from the door mounted handgrip, the handgrip was mounted to the door through a five-axis load cell and instrumented with accelerometers for inertial compensation. All six of the cadaver tests resulted in upper extremity injuries including comminuted mid-shaft humerus fractures, osteochondral fractures of the elbow joint surfaces, a transverse fracture of the distal radius and an osteochondral fracture of the lunate carpal bone. The results from the 6 cadaver tests presented in this study were combined with the results from 12 previous cadaver tests. A multivariate logistic regression analysis was performed to investigate the correlation between observed injuries and measured occupant response. Using inertially compensated force measurements from the dummy mid-shaft forearm load cell, the linear combination of elbow axial force and shear force was significantly (P=0.05) correlated to the observed elbow injuries.
Air Bags↗
Cloning and expression analysis of rainbow trout Oncorhynchus mykiss interferon regulatory factor 1 and 2 (IRF-1 and IRF-2).
Two rainbow trout (Oncorhynchus mykiss) genes for interferon regulatory factors (IRF) 1 and 2 have been cloned and sequenced. The IRF-1 cDNA contains an open reading frame (ORF) of 996 nucleotides that translates into a 331 amino-acid putative peptide, with a 5' untranslated region (UTR) of 145bp and a 3' UTR of 481bp. The IRF-2 cDNA contains a 1035bp ORF that translates into a 344 amino-acid putative peptide, with a 5' UTR of 146bp and a 3' UTR of 925bp. In vivo, IRF-1 and IRF-2 are constitutively expressed in head kidney, gill and spleen but not liver. Both genes were induced in all the tissues examined. IRF-1 but not IRF-2 expression was significantly increased at the site of injection 1 week after DNA vaccination against viral haemorrhagic septicaemia virus. In vitro, IRF-1 and IRF-2 transcripts are present in unstimulated rainbow trout gonad cells and are up-regulated by poly I/C.
Amino Acid Sequence↗
Cloning and characterization of cDNAs for two distinct tumor necrosis factor receptor superfamily genes from Japanese flounder Paralichthys olivaceus.
Tumor necrosis factor receptor (TNFR) superfamily regulates diverse biologic functions, including cell proliferation, differentiation, and survival, in addition to providing costimulatory signals for programmed cell death or apoptosis. In this study, cDNA fragments for two distinct TNFR homologues were obtained from a Japanese flounder, Paralichthys olivaceus, cDNA library. Full-length cDNAs of TNFR-1 and TNFR-2 homologues were obtained by using these cDNA fragments as probes. The cDNA for the Japanese flounder TNFR-1 homologue predicts a peptide of 395 amino acids that is 35% identical to the extracellular region of mouse TNFR-1, whereas the cDNA of the Japanese flounder TNFR-2 homologue predicts a peptide of 483 amino acids that is 40% identical to the extracellular region of human TNFR-2. The cytoplasmic domain contains a sequence that has the consensus motif of the death domain of the Japanese flounder TNFR-1 homologue. In a healthy fish, the mRNAs of both TNFR homologues were predominantly expressed in leukocytes, kidney, gill, and spleen. Expression of the Japanese flounder TNFR-1 homologue was induced in peripheral blood lymphocytes (PBLs) after stimulation with LPS (500 microg/ml) for 1 h, and TNFR-2 homologue was strongly induced in PBLs after stimulation with Con A (50 microg/ml) and PMA (0.35 microg/ml) for 3 h. The different expression patterns of the two distinct TNFR homologues may be critical in determining whether binding with TNF-alpha or TNF-beta have activating, proliferative, or apoptotic effects on target cells.
Amino Acid Sequence↗
Isolation and characterization of the stage-specific cytochrome b small subunit (CybS) of Ascaris suum complex II from the aerobic respiratory chain of larval mitochondria.
We recently reported that Ascaris suum mitochondria express stage-specific isoforms of complex II: the flavoprotein subunit and the small subunit of cytochrome b (CybS) of the larval complex II differ from those of adult enzyme, while two complex IIs share a common iron-sulfur cluster subunit (Ip). In the present study, A. suum larval complex II was highly purified to characterize the larval cytochrome b subunits in more detail. Peptide mass fingerprinting and N-terminal amino acid sequencing showed that the larval and adult cytochrome b (CybL) proteins are identical. In contrast, cDNA sequences revealed that the small subunit of larval cytochrome b (CybS(L)) is distinct from the adult CybS (CybS(A)). Furthermore, Northern analysis and immunoblotting showed stage-specific expression of CybS(L) and CybS(A) in larval and adult mitochondria, respectively. Enzymatic assays revealed that the ratio of rhodoquinol-fumarate reductase (RQFR) to succinate-ubiquinone reductase (SQR) activities and the K(m) values for quinones are almost identical for the adult and larval complex IIs, but that the fumarate reductase (FRD) activity is higher for the adult form than for the larval form. These results indicate that the adult and larval A. suum complex IIs have different properties than the complex II of the mammalian host and that the larval complex II is able to function as a RQFR. Such RQFR activity of the larval complex II would be essential for rapid adaptation to the dramatic change of oxygen availability during infection of the host.
Aerobiosis↗
Cloning and characterisation of a cDNA encoding Japanese flounder Paralichthys olivaceus IgD.
A cDNA containing the gene for Japanese flounder IgD consisted of 3240 bp encoding 998 amino acid residues. The amino acid sequence of the constant region of Japanese flounder IgD shares 38-80% identity with the sequences of previously reported teleost IgDs. The structure of the constant region of Japanese flounder IgD, which contains the micro1, delta1, delta2, delta3, delta4, delta5, delta6, delta7, and TM regions, is similar to the structures of the constant regions of the IgDs of channel catfish and Atlantic salmon. Southern blot hybridisation showed that the Japanese flounder IgD gene exists as a single locus. The Japanese flounder IgD gene was mainly detected in peripheral blood leucocytes (PBLs) and small amounts were detected in the spleen, head and trunk kidney, although IgM mRNA was detected in similar amounts in PBLs, the head kidney, and spleen. The copy number of IgM mRNA in Japanese flounder PBL was 56-fold higher than that of IgD.
Amino Acid Sequence↗
Characterization of polymer-adhesive interfaces on a nanometre scale by elemental mapping and image EELS using EFTEM.
Energy-filtering transmission electron microscopy (EFTEM) was applied for investigating interfaces between a polymer and an adhesive. The sample employed in this work is polybutylene terephtharate (PBT) sheets laminated with an epoxy adhesive. It was found that heat aging of the PBT at 180 degrees C in air for > 9 h prior to adhesion decreases the adhesion strength drastically. To investigate this unfavourable aging effect on the adhesion strength, we performed elemental mapping and image EELS using EFTEM. A weak boundary layer with a thickness of < 50 nm was visualized at the PBT-adhesive interface by elemental mapping in the sample subjected to the heat aging and image EELS revealed the origin of this layer. Thus, we clearly correlated the nanoscale interfacial structure with the adhesion strength by EFTEM.
Journal Article↗
Activation of natural killer T cells by alpha-galactosylceramide impairs DNA vaccine-induced protective immunity against Trypanosoma cruzi.
Innate immunity as a first defense is indispensable for host survival against infectious agents. We examined the roles of natural killer (NK) T cells in defense against Trypanosoma cruzi infection. The T. cruzi parasitemia and survival of CD1d-deficient mice exhibited no differences compared to wild-type littermates. NK T-cell activation induced by administering alpha-galactosylceramide (alpha-GalCer) to T. cruzi-infected mice significantly changed the parasitemia only in the late phase of infection and slightly improved survival when mice were infected intraperitoneally. The combined usage of alpha-GalCer and benznidazole, a commercially available drug for Chagas' disease, did not enhance the therapeutic efficacy of benznidazole. These results suggest that NK T cells do not play a pivotal role in resistance to T. cruzi infection. In addition, we found that the coadministration of alpha-GalCer with DNA vaccine impaired the induction of epitope-specific CD8(+) T cells and undermined the DNA vaccine-induced protective immunity against T. cruzi. Our results, in contrast to previous reports demonstrating the protective roles of NK T cells against other infectious agents, suggest that these cells might even exhibit adverse effects on vaccine-mediated protective immunity.
Animals↗
Critical contribution of CD28-CD80/CD86 costimulatory pathway to protection from Trypanosoma cruzi infection.
The CD28-CD80/CD86-mediated T-cell costimulatory pathway has been variably implicated in infectious immunity. In this study, we investigated the role of this costimulatory pathway in resistance to Trypanosoma cruzi infection by using CD28-deficient mice and blocking antibodies against CD80 and CD86. CD28-deficient mice exhibited markedly exacerbated T. cruzi infection, as evidenced by unrelenting parasitemia and 100% mortality after infection with doses that are nonlethal in wild-type mice. The blockade of both CD80 and CD86 by administering specific monoclonal antibodies also exacerbated T. cruzi infection in wild-type mice. Splenocytes from T. cruzi-infected, CD28-deficient mice exhibited greatly impaired gamma interferon production in response to T. cruzi antigen stimulation in vitro compared to those from infected wild-type mice. The induction of T. cruzi antigen-specific CD8(+) T cells was also impaired in T. cruzi-infected, CD28-deficient mice. In addition to these defects in natural protection against T. cruzi infection, CD28-deficient mice were also defective in the induction of CD8(+)-T-cell-mediated protective immunity against T. cruzi infection by DNA vaccination. These results demonstrate, for the first time, a critical contribution of the CD28-CD80/CD86 costimulatory pathway not only to natural protection against primary T. cruzi infection but also to DNA vaccine-induced protective immunity to Chagas' disease.
Animals↗
Construction and initial analysis of bacterial artificial chromosome (BAC) contigs from the sex-determining region of the platyfish Xiphophorus maculatus.
Despite the major importance of sex determination in aquaculture, no master sex-determining gene has been identified so far in teleost fish. In the platyfish Xiphophorus maculatus, this master gene is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene responsible for melanoma formation in some Xiphophorus interspecific hybrids, and its proto-oncogenic counterpart. Both Xmrk genes, which have already been characterised at the molecular level, delimit a region of about 1 Mb that contains other gene loci involved in sexual maturity, pigmentation and melanoma formation. We have constructed a genomic bacterial artificial chromosome (BAC) library of X. maculatus with a tenfold coverage of the haploid genome and walked on both X and Y sex chromosomes starting from both Xmrk genes. This led to the assembly of BAC contigs from the sex-determining region covering approximately 950 kb of the X and 750 kb of the Y chromosome. To our knowledge, these are the largest contigs reported so far for sex chromosomes in fish. Molecular analysis suggests that the sex-determining region of X. maculatus frequently undergoes retrotranspositions and other kinds of rearrangements. This genomic plasticity might be related to the high genetic variability observed in Xiphophorus for sex determination, sexual maturity, pigmentation and melanoma formation, which are encoded by gene loci located in the sex-determining region.
Animals↗
"GFP-display," an easy detection method for single amino acid changes in a target polypeptide: application to random mutagenesis.
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Amino Acid Substitution↗
Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach.
Gene expression in haemocytes of the kuruma prawn (Penaeus japonicus) was investigated using an expressed sequence tag (EST) approach. Partial nucleotide sequences of cDNA library clones constructed from normal and white spot syndrome virus (WSSV)--infected P. japonicus haemocytes were determined. Of 635 clones obtained from the normal library, 284 (44.7%) significantly matched sequences in GenBank, and of 370 clones obtained from WSSV-infected library, 174 (47.0%) significantly matched sequences in the database. One hundred fifty-two deduced proteins were newly identified. Of these, 28 types were involved in biodefence. For the prophenoloxidase system, there are prophenoloxidase, coagulation factor G-beta chain precursor, factor D, Masquarade-like protease, transglutaminase (TGase), clottable protein and eight types of protease inhibitors (two types of antileukoproteinase, alpha-2-macroglobulin, chelonianin, elastase inhibitor, two types of Kazal inhibitor and Kunitz-type inhibitor). For antibacterial peptides, there are bactinecin 11, penaeidin-2 precursor and lysozyme c type. The others defence-related proteins are basophil leukocyte interleukin-3-regulated protein, natural killer enhancing factor (NK-EF), integral membrane protein (CD34+), ESM-1, Notch homologue and Drac homologue. For the adhesion proteins, there are beta-integrin, cell adhesion molecule (CAM) and three types of collagens. All ESTs representing protease inhibitors and tumour-related proteins were found only in the WSSV-infected library. Those encoding for apoptotic peptides were expressed at high levels in infected library. The putative defence proteins accounted for 2.7% of total ESTs in a normal shrimp library and 15.7% of the total ESTs in an infected library.
Amino Acid Sequence↗
Differential expression and cellular localization of activin and inhibin mRNA in the rainbow trout ovary and testis.
An inhibin cDNA from rainbow trout consisted of 1305 bp, which coded for 352 amino acid residues. The deduced amino acid sequence of mature inhibin was 50 to 60% identical to mammalian sequences. Distribution of inhibin alpha and activin beta A and beta B in different ovarian and testis compartments was studied in rainbow trout by in situ hybridization with complementary RNA probes. In testis tissue, inhibin alpha and activin beta A and beta B were expressed only in the testicular interstitia between the seminal lobules, where Sertoli cells and Leydig cells are distributed. The localizations and intensities of the reactions were constant throughout the maturation of the testis. Within ovarian tissue, the theca cell layers of follicles showed strong reactions of Dig-labeled antisense mRNA probes hybridizing against inhibin alpha and activin beta A and beta B in all samples over the same sampling period. In regressing oocytes, a positive reaction was observed in the granular cell layer of the follicles.
Activins↗
Molecular cloning and characterization of Plasmodium falciparum transportin.
A cDNA encoding transportin, a protein involved in the nuclear import of M9 nuclear localization signal-bearing proteins, has been cloned from the malaria parasite Plasmodium falciparum. The complete cDNA consists of 3,667 bp encoding 1,136 amino acid residues. Amino acid sequence analysis revealed that Ran-GTP and M9 binding domains are highly conserved in P. falciparum, suggesting that the transportin-mediated nuclear transport pathway exists in this protozoan parasite. Southern blot analysis revealed that the transportin gene exists as a single copy in the malarial genome.
Active Transport, Cell Nucleus↗
Identification of immune-related genes in hemocytes of black tiger shrimp (Penaeus monodon).
An expressed sequence tag (EST) library was constructed from hemocytes of the black tiger shrimp (Penaeus monodon) to identify genes associated with immunity in this economically important species. The number of complementary DNA clones in the constructed library was approximately 4 x 10(5). Of these, 615 clones having inserts larger than 500 bp were unidirectionally sequenced and analyzed by homology searches against data in GenBank. Significant homology to known genes was found in 314 (51%) of the 615 clones, but the remaining 301 sequences (49%) did not match any sequence in GenBank. Approximately 35% of the matched ESTs were significantly identified by the BLASTN and BLASTX programs, while 65% were recognized only by the BLASTX program. Of the 615 clones, 55 (8.9%) were identified as putative immune-related genes. The isolated genes were composed of those coding for enzymes and proteins in the clotting system and the prophenoloxidase-activating system, antioxidative enzymes, antimicrobial peptides, and serine proteinase inhibitors. Three full-length ESTs encoding antimicrobial peptides (antilipopolysaccharide and penaeidin homologues) and a heat shock protein (cpn10 homologue) are reported.
Journal Article↗
Characterisation and expression of the Fasciola gigantica cathepsin L gene.
The gene structure of a cathepsin L from Fasciola gigantica was characterised. The gene spans approximately 2.0 kb and comprises four exons and three introns and is a compact gene as in the cases of crustaceous and platyhelminth cathepsins L. Southern blot analysis suggested that a few copies of the genes are sparsely organised in the genome. Of the three intron insertion positions, two of which are in the same position as in the mammalian cathepsin L gene. Phylogenetic analysis revealed that F. gigantica cathepsin L forms a clade with those from Fasciola hepatica, but not with those from Spirometra erinacei and schistosomes. Putative TATA-boxes were found upstream of a transcription initiation site. The sequence analysis of the 5'-upstream of the transcript revealed that the cathepsin L gene is transcribed by cis-splicing fashion. Furthermore, the experiments using recombinant F. gigantica procathepsin L showed that it was processed to an enzymatically active cathepsin L by pH-dependent autocatalysis. However, the pro-peptide deleted cathepsin L showed no enzyme activity, indicating that the pro-region of F. gigantica procathepsin L is essential for the folding and/or refolding of functional cathepsin L. These results are consistent with the observations in mammalian cathepsin L and papain.
Amino Acid Sequence↗
Molecular cloning and expression of CCAAT/enhancer binding proteins in Japanese flounder Paralichthys olivaceus.
The findings of this study represent the first report, to the authors' knowledge, of CCAAT/enhancer binding protein (C/EBP) cDNA sequence in a fish species. C/EBP epsilon of Japanese flounder was 1861 bp in length (ORF of 822 bp) encoding for 274 amino acids, with a calculated molecular weight of 30 kDa. Japanese flounder C/EBP beta was found to be 1561 bp in length (ORF of 1041 bp), encoding for 347 amino acids and a calculated molecular weight of 39 kDa. These genes were expressed in various fish organs, tissues and secretions. C/EBP epsilon was detected by Northern blot from total RNA of head and posterior kidney, heart and spleen. However, RT-PCR also detected C/EBP epsilon in brain, spleen and peritoneal cavity fluid and peripheral blood leucocyte cDNA. C/EBP beta was detected by Northern blot analysis in the head and posterior kidney, spleen, intestine, liver, brain, heart, gill and testis and further found by RT-PCR to be detected in mucus, peritoneal cavity fluid, peripheral blood leucocytes and eye cDNA. Phylogenetic analysis placed the Japanese flounder C/EBP beta within the same cluster as previously reported C/EBP beta sequences. However, Japanese flounder C/EBP epsilon sequence data were not found to cluster with the three reported mammalian C/EBP epsilon sequences currently available. Understanding C/EBP transcriptional gene control in commercially important fish species may lead to a better control of disease.
Amino Acid Sequence↗
Identification and analysis of an interleukin 8-like molecule in rainbow trout Oncorhynchus mykiss.
An interleukin 8 (IL-8) homologue has been identified in the rainbow trout Oncorhynchus mykiss. The transcript contains an open reading frame of 294 nucleotides that translates into a 97 amino acid putative peptide, with 5' and 3' untranslated regions (UTR) of 171 and 453 nucleotides, respectively. As with previously sequenced lamprey and flounder genes, the trout amino acid sequence lacks the typical ELR motif upstream of the first pair of cysteines, where DLR is present. The trout IL-8 gene contains four exons divided by three short introns of 341, 247 and 292bp, and occupies 1824bp of genomic DNA. RT-PCR reveals a low level constitutive expression of the IL-8 homologue in many tissues, including spleen, heart, liver, head kidney and gill. Expression was not detectable in the brain. Whilst no apparent affect of lipopolysaccharide (LPS) on IL-8 expression was observed in vivo, stimulation of a trout macrophage cell line (RTS-11) with either LPS or poly I:C did result in clear up-regulation of IL-8 expression, detectable by RT-PCR and Northern blot analysis.
Amino Acid Sequence↗