Biomedical subjects

Takashi Aoki

Publications and source records attributed to Takashi Aoki.

At least 73 records · Page 4Linked to original sources

PubMed2004

A new method for detecting single nucleotide polymorphism using GFP-display.

The single nucleotide polymorphism (SNP) of aldehyde dehydrogenase-2 (ALDH2) codon 487, GAA (Glu) or AAA (Lys), was examined using green fluorescent protein (GFP)-display, an electrophoretic detection method for single amino acid changes. Although no shift in migration between the GFP-ALDH (Glu487) and GFP-ALDH (Lys487) fusion proteins was observed on SDS/urea gel, the two migrated to different positions when tagged with Asp. The SNP analysis was performed with GFP-ALDH-Asp3, and GFP-ALDH-Asp3 constructed from donors having the codon GAA/GAA, GAA/AAA or AAA/AAA was detected as different patterns as expected. GFP-display is potentially a unique method in SNP analysis, which does not require any special equipment or chemicals.

Aldehyde Dehydrogenase↗

PubMed2004

A pore-forming protein, perforin, from a non-mammalian organism, Japanese flounder, Paralichthys olivaceus.

A perforin cDNA of Japanese flounder, Paralichthys olivaceus, was cloned from a cDNA library of kidney stimulated with ConA/PMA. The full-length cDNA is 2,157 bp, which encodes 587 amino acids. The Japanese flounder perforin gene consists of five exons and four introns, with a length of approximately 3 kb. The amino acid sequence of the Japanese flounder perforin is 36% identical to that of rat perforin and 37% identical to amino acid sequences of mouse and human perforin. The Japanese flounder perforin also showed low homology to human and mouse complement components (C6, C7, C8 and C9), ranging from 19% to 24%. However, the membrane attack complex/perforin domain is conserved. A phylogenetic analysis placed the Japanese flounder perforin in the same cluster with other known mammalian perforins. RT-PCR analysis revealed that the perforin gene was expressed in the peripheral blood leukocytes, head kidney, trunk kidney, spleen, heart, gill and intestine of healthy fish. Recombinant perforin produced in insect cells using the baculovirus expression system showed calcium-dependent hemolytic activity.

Amino Acid Sequence↗

PubMed2004

Genes of the constant regions of functional immunoglobulin heavy chain of Japanese flounder, Paralichthys olivaceus.

IgM and IgD genes of the Japanese flounder were cloned and characterized from a genomic bacterial artificial chromosome (BAC) library. The micro gene contained four constant region exons (Cmi1-Cmi4), and two transmembrane exons (miTM1 and miTM2), which conforms to the organizational pattern of all other vertebrate micro-chain genes examined so far. In the same BAC clone, the delta gene, which is homologous to the IgD gene in mammals and teleost fish, was found immediately (0.9 kb) downstream of the IgM gene. This gene encoded seven exons (Cdelta1-Cdelta7) and two deltaTM exons (deltaTM1 and deltaTM2) and had no duplication of Cdelta1-Cdelta2, as found in Atlantic cod, or Cdelta2-Cdelta3-Cdelta4, as found in Atlantic salmon and channel catfish. Phylogenetic and sequence analyses strongly suggest that teleost delta is more closely related to non-IgM isotypes than IgM isotypes. The heavy chain (IgH) locus of Japanese flounder, which encodes microm, micros and deltam, was found to be fully functional.

Amino Acid Sequence↗

PubMed2004

Species identification of the tropical abalone (Haliotis asinina, Haliotis ovina, and Haliotis varia) in Thailand using RAPD and SCAR markers.

A randomly amplified polymorphic DNA (RAPD) analysis was used to identify the species- and population-specific markers of abalone; Haliotis asinina, H. ovina, and H. varia in Thai waters. Fifteen species-specific and six population-specific RAPD markers were identified. In addition, an 1650 bp band (UBC195) that was restricted to H. ovina from the Gulf of Thailand (east) was also found. All of the specific RAPD markers were cloned and sequenced. Twenty pairs of primers were designed and specificity-tested (N = 12 and 4 for target and non-target species, respectively). Seven primer pairs (CUHA1, 2, 4, 11, 12, 13, and 14) were specifically amplified by H. asinina DNA, whereas a single pair of primers showed specificity with H. ovina (CUHO3) and H. varia (CUHV1), respectively. Four primer pairs, including CUHA2, CUHA12, CUHO3, and CUHV1, were further examined against 216 individuals of abalone (N = 111, 73, and 32, respectively). Results indicated the species-specific nature of all of them, except CUHO3, with the sensitivity of detection of 100 pg and 20 pg of the target DNA template for CUHA2 and CUHA12 and CUHV1, respectively. The species-origin of the frozen, ethanol-preserved, dried, and boiled H. asinina specimens could also be successfully identified by CUHA2.

Animals↗

PubMed2004

Characterization of gene structure and expression of two toll-like receptors from Japanese flounder, Paralichthys olivaceus.

We cloned the cDNAs and genes of two different types of toll-like receptors from Japanese flounder. The results of homology searches suggested that these genes (designated JF-TLR2 and JF-TLR22) are homologues of human TLR2 and fugu TLR22, respectively. The cDNAs of JF-TLR2 and JF-TLR22 encoded 818 and 961 amino acid residues, respectively. JF-TLR2 and JF-TLR22 contained two distinct structural/functional motifs of the TLR family, such as a leucine-rich repeat (LRR) domain in the extracellular portion and a toll/interleukin-1 receptor (TIR) domain in the intracellular portion. The genes of JF-TLR2 and JF-TLR22 consisted of 12 exons (4.9 kb in total length) and four exons (4.3 kb in total length), respectively. The first exon of each gene is a non-coding exon. Southern blot hybridization indicated that both JF-TLR2 and JF-TLR22 exist as single copies in the genome. These genes were mainly expressed in peripheral blood leukocytes (PBLs) and weakly expressed in PBL-rich organs such as kidney, spleen and gill. Expression of these genes was induced by both peptidoglycan and polyI:C, although the number of JF-TLR-expressing cells were not changed after induction. All of these results suggest that they are involved in the innate immune system.

Amino Acid Sequence↗

PubMed2004

Identification of a novel Japanese flounder (Paralichthys olivaceus) CC chemokine gene and an analysis of its function.

A cDNA of Japanese flounder (Paralichthys olivaceus) CC chemokine designated as Paol-SCYA104 was cloned and sequenced. The cDNA contains an opening reading frame of 315 nucleotides encoding 104 amino acid residues. The full gene was cloned and sequenced from a BAC library. It has a length of approximately 750 bp from the start codon to the stop codon and is composed of four exons and three introns. Four cysteine residues are conserved in the same positions as those of mammalian and fish CC chemokines. Paol-SCYA104 gene was expressed in several organs, including peripheral blood leukocytes (PBLs), head kidney, trunk kidney, and spleen. The recombinant Paol-SCYA104 was expressed in Escherichia coli and the expressed protein was partially purified. The recombinant Paol-SCYA104 was able to attract Japanese flounder PBLs in a microchemotaxis chamber. On the other hand, a negative control, the fraction of the control cells carrying an expression vector lacking the Paol-SCYA104 cDNA, did not show chemotactic activity. These results indicate that Paol-SCYA104 probably acts as a CC chemokine.

Amino Acid Sequence↗

PubMed2004

Molecular cloning and expression analysis of alpha 2-macroglobulin in the kuruma shrimp, Marsupenaeus japonicus.

The cDNA encoding the kuruma shrimp, Marsupenaeus japonicus alpha(2)-macroglobulin (alpha(2)M) was obtained by screening a haemocyte cDNA library and 5' RACE PCR amplification. The full length cDNA of 4748 bp contains an open reading frame of 4518 nucleotides that translates into a 1505-amino acid putative peptide, with a 5'untranslated region (UTR) of 59 bp and a 3'UTR of 171 bp. The open reading frame encodes an N-terminal signal sequence of 17 residues and a mature protein of 1488 residues. The entire amino acid sequence is similar to the alpha(2)M sequences of arthropods (30-31% identity), mammals (26-27% identity) and fish (25-28% identity). The M. japonicus alpha(2)M sequence contains putative functional domains including a bait region, an internal thiol ester site, and a receptor-binding domain, which are present in mammalian alpha(2)Ms. In a healthy shrimp, the mRNA of alpha(2)M was mainly expressed in haemocytes. In addition, the expression level of alpha(2)M mRNA was dramatically increased by through time upon oral administration of peptidoglycan (PG), which is an immune stimulant. The highest expression of alpha(2)M mRNA was observed 7 days after feeding with PG. These results suggest that the shrimp alpha(2)M is an important molecule in immune system.

Amino Acid Sequence↗

PubMed2004

Development of a DNA vaccine against hirame rhabdovirus and analysis of the expression of immune-related genes after vaccination.

Intramuscular injection of Japanese flounder, Paralichthys olivaceus (average weight approximately 2 g) with 1 and 10 microg of a plasmid DNA vaccine encoding the hirame rhabdovirus (HIRRV) glycoprotein gene (pCMV-HRVg) was found to provide strong protection against HIRRV. We also conducted a real-time PCR analysis to quantify immune-related genes, e.g. MHC class Ialpha, IIalpha, IIbeta, TCR-alpha, beta1, beta2 and delta, to characterize the immune response at 1 and 7 days after DNA vaccination. In general, the copy numbers were at least 2-fold higher than those of the non-vaccinated fish. Interestingly, the gene expression of TCR beta1 and beta2 increased 1 day post-DNA vaccination, after which their copy numbers returned to levels similar to those before vaccination. These results suggest that the immune system of Japanese flounder was activated immediately after DNA immunization.

Animals↗

PubMed2004

Rapid detection of a fish iridovirus using loop-mediated isothermal amplification (LAMP).

A loop-mediated isothermal amplification (LAMP) procedure is described to detect the genomic DNA molecule of red seabream iridovirus (RSIV), a fish iridovirus belonging to the Iridoviridae family. The RSIV DNA was amplified using DNA extracts obtained from spleen of infected red seabream, Pagrus major and from various RSIV isolates. The method was at least 10 times more sensitive than conventional PCR in detecting for the presence of RSIV. A striking feature of the LAMP reaction is its ability to synthesize a large amount of DNA leading to the production of a white precipitate, magnesium pyrophosphate, as a by-product. The presence or absence of this white precipitate facilitates easy detection of the RSIV genomic DNA without the use of gel electrophoresis. A strong correlation exists between the amount of input viral DNA copy and the corresponding turbidity reading at the end of the reaction; hence, the LAMP reaction may be used potentially to quantify RSIV particles in the infected fish.

Animals↗

PubMed2003

In vitro inhibition of fish rhabdoviruses by Japanese flounder, Paralichthys olivaceus Mx.

A homologous fish cell line stably expressing the recombinant Japanese flounder Mx (JFMx) was infected with hirame rhabdovirus (HIRRV) and viral hemorrhagic septicemia virus (VHSV), both of which are negative single-stranded RNA viruses belonging to the Rhabdoviridae family. Analysis of primary transcription of the two rhabdoviruses showed that there was lower expression level and copy number of the viral nucleoprotein transcript in the JFMx-transfected cell line than the infected, control cells, although no significant difference was observed. This suggests that JFMx may not be a potent inhibitor of rhabdoviral primary transcription. Kinetics of rhabdovirus expression by RT-PCR and quantitative real-time RT-PCR showed reduced levels of the rhabdoviral glycoprotein and nucleoprotein transcripts over time, indicating the possible role of JFMx in blocking rhabdoviral replication by interfering with the transcription of the viral subgenomic mRNAs. Significant inhibition in rhabdovirus replication consequently resulted in the synthesis of fewer viral particles. This may explain why JFMx-expressing cells are less susceptible to virus-induced cell lysis, and thus, why they would have a significantly higher survival than the infected, control cells. These results provide direct evidence that JFMx has an antiviral effect in vitro.

Animals↗

PubMed2003

Cutting edge: a potent adjuvant effect of ligand to receptor activator of NF-kappa B gene for inducing antigen-specific CD8+ T cell response by DNA and viral vector vaccination.

The ligand to receptor activator of NF-kappaB (RANK-L)/RANK interaction has been implicated in CD40 ligand/CD40-independent T cell priming by dendritic cells. In this report, we show that the coadministration of the RANK-L gene with a Trypanosoma cruzi gene markedly enhances the induction of Trypanosoma Ag-specific CD8(+) T cells and improves the DNA vaccine efficacy. A similarly potent adjuvant effect of the RANK-L gene on the induction of Ag-specific CD8(+) T cells was also observed when recombinant influenza virus expressing murine malaria Ag was used as an immunogen. In contrast, the coadministration of the CD40L gene was not effective in these systems. Our results demonstrated, for the first time, the potent immunostimulatory effect of the RANK-L gene to improve the CD8(+) T cell-mediated immunity against infectious agents.

Adjuvants, Immunologic↗

PubMed2003

Involvement of ICOS-B7RP-1 costimulatory pathway in the regulation of immune responses to Leishmania major and Nippostrongylus brasiliensis infections.

The ICOS-B7RP-1-mediated T cell costimulatory pathway has been implicated crucial for T cell activation and differentiation. In this study, we investigated the role of this costimulation in the regulation of immune responses to parasitic infections by using blocking antibody against B7RP-1 as well as ICOS-deficient mice. The administration of anti-B7RP-1 monoclonal antibody (mAb) significantly suppressed the footpad swelling in susceptible BALB/c mice upon Leishmania major infection. The observation was consistent not only with the significant suppression of IL-4, IL-5 and IL-10 secretion from lymph node cells, which were derived from L. major-infected mice, but also with the significant reduction of total serum IgE and IgG(1) in anti-B7RP-1 mAb-treated BALB/c mice. Infection of ICOS-deficient mice with L. major also suggested the impaired Th2 immune responses in the absence of this costimulation. The immunological function of ICOS-B7RP-1 costimulatory pathway in infection was further confirmed by infecting anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice with Nippostrongylus brasiliensis. The characteristic elevation of total serum IgE and eosinophilia upon N. brasiliensis infection was suppressed by blocking this costimulation. Moreover, the protection to N. brasiliensis adult worms was suppressed in anti-B7RP-1 mAb-treated wild type or ICOS-deficient mice. These results suggest the crucial role of this costimulatory pathway in the regulation of Th2-biased T cell differentiation and in host immune responses against L. major and N. brasiliensis infections.

Animals↗

PubMed2003

Characterization and function of kuruma shrimp lysozyme possessing lytic activity against Vibrio species.

Lysozyme cDNA was isolated from a kuruma shrimp, Marsupenaeus japonicus, hemocyte cDNA library. The cDNA consists of 1055 base pairs (bp) and encodes a chicken-type (c-type) lysozyme with a deduced amino acid sequence of 156 residues. The kuruma shrimp lysozyme has a high identity (79.7%) with pacific white shrimp lysozyme, and low to moderate identities (33.3-43.0%) with lysozymes of insects and vertebrates. Comparisons with other c-type lysozymes from invertebrates and vertebrates showed that the two catalytic residues (Glu58 and Asp75) and the eight cysteine residue motif were completely conserved. Two novel insertion sequences were also observed in the kuruma and pacific white shrimp lysozyme amino acid sequences. Interestingly, phylogenetic analysis revealed that the kuruma shrimp lysozyme was more closely related to vertebrate c-type lysozymes. Expression of the cDNA in insect cells, using a baculovirus expression system, yielded a recombinant lysozyme with optimum activity at pH 7.5 and 50 degrees C, as evaluated by a lysoplate assay. The kuruma shrimp lysozyme displayed lytic activities against several Vibrio species and fish pathogens, including Vibrio penaeicida (a pathogenic bacteria to the kuruma shrimp) and suggested that shrimp lysozyme affects a greater variety of pathogens.

Amino Acid Sequence↗

PubMed2003

Occurrence of two distinct types of tissue inhibitor of metalloproteinases-2 in teleost fish.

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.

Amino Acid Sequence↗

PubMed2003

Diversity of aspartate carbamoyltransferase genes of Trypanosoma cruzi.

The pyrimidine-biosynthetic (pyr) gene cluster, a tandem array of pyr1-pyr3-pyr6/5-pyr2(ACT)-pyr4 from the 5' terminus, encodes all the six enzymes of de novo pyrimidine biosynthesis and occurs as a polycistronic transcription unit in Trypanosoma cruzi. The gene encoding aspartate carbamoyltransferase (ACT), the second enzyme of the pathway, was characterised using a laboratory-reared Tulahuen strain and Tulahuen-derived clones of T. cruzi. Three loci with different restriction maps that contain ACT1, ACT2, and ACT3 were identified. ACT1 and ACT2 are involved in the pyr gene cluster on two different chromosomal DNA molecules of 1,000 and 800 kb, respectively, whereas ACT3 is linked with pyr4 alone. There are 29 nucleotide substitutions out of 981 positions in these three ACTs, yielding 13 amino acid replacements, and a deletion of triplet nucleotides in ACT1 entails a lack of single amino acid residue. Transcription of the three ACTs takes place in the three developmental stages of the parasite, epimastigotes, trypomastigotes, and amastigotes. Pulsed field gel electrophoresis and Southern blot analyses demonstrated that the cloned T. cruzi stocks, Y-02, CAN III/1, Sylvio-X10/4, and possibly Esmeraldo/3, also possess two complete sets of the pyr gene cluster including ACT, accompanied by additional incomplete clusters. These results suggest that the marked intra-species diversity in the copy number and chromosomal localisations of ACT and other pyr genes may have resulted from partial duplications and subsequent translocations of the polycistronic pyr gene cluster.

Amino Acid Sequence↗

PubMed2003

General properties of GFP-display, an electrophoretic analysis for single amino acid changes in target polypeptides.

The migrating position of green fluorescent protein (GFP)-fused polypeptide varied on an SDS/urea gel by a single amino acid change in the fused polypeptide segment. An easy detection method for a single amino acid change based on this observation was called "GFP-display." Using various target polypeptides, staphylococcal protein A (SpA), Ras, p53, and human beta3 adrenergic receptor (AR), and their mobility-shift patterns resulting from the single amino acid changes, several important properties of GFP-display were revealed as follows: (i). since the binding of dodecyl sulfate ions to acidic or hydrophilic amino acids is weaker than that to basic or hydrophobic amino acids, the ions bound weakly to the fused polypeptide segment are forced to come off by high concentrations of urea prior to the ions bound strongly, resulting in the mobility shift, (ii). the mobility shift is estimated to a certain extent using a new parameter called the "GD value" calculated from the isoelectric point, hydrophilicity, and number of fused amino acids, and (iii). the fluorescence intensity of GFP-fused polypeptide tends to increase with the average hydrophilicity of the fused polypeptide segment. GFP-display will be a helpful technique for many kinds of gene or protein studies related to amino acid substitutions such as the random mutagenesis in a gene of interest.

Amino Acid Substitution↗

PubMed2003

Heterologous expression of Ascaris suum cytochrome b5 precursor protein: a histidine-tagged full-length presequence is correctly processed to transport the mature protein to the periplasm of Escherichia coli.

The cytochrome b(5) of the body wall of adult Ascaris suum, a porcine parasitic nematode, is a novel type of cytochrome b(5). It is a soluble protein that lacks the COOH-terminal membrane-anchoring domain found in erythrocyte cytochrome b(5), but possesses an NH(2)-terminal extension (presequence) of 30 amino acids that are missing from the 82-residue protein purified from the nematode tissues [Yu, Y., Yamasaki, H., Kita, K., and Takamiya, S., 1996, Arch. Biochem. Biophys. 328, 165-172]. The nematode cytochrome b(5) is, therefore, probably synthesized as a precursor protein whose presequence is cleaved to form a mature protein, but the localization of the mature protein is still unknown. To investigate the processing of the putative precursor protein, the wild-type precursor of nematode cytochrome b(5) with a complete presequence (b5wt) and its NH(2) terminus-truncated derivatives, b5Delta18 and b5Delta28, with 18 and 28 residues deleted, respectively, were expressed using pET-28a(+) vector in Escherichia coli. As expected, all transformants, tb5wt, tb5Delta18, and tb5Delta28, produced recombinant proteins with a histidine-tagged NH(2)-terminal extension. However, only the recombinant protein with the full-length presequence, produced in tb5wt, was correctly processed and transported to the periplasm, from which the majority of the induced product was purified as a mature protein chemically and functionally identical to the native protein purified from the nematode body wall. These results clearly show that the nematode histidine-tagged presequence functions as a signal peptide in E. coli.

Amino Acid Sequence↗

PubMed2003

The four TCR genes of teleost fish: the cDNA and genomic DNA analysis of Japanese flounder (Paralichthys olivaceus) TCR alpha-, beta-, gamma-, and delta-chains.

We have isolated and identified all four TCR alpha, beta, gamma, and delta cDNAs and genomic clones from a Japanese flounder leukocyte cDNA library and bacterial artificial chromosomal genomic library. Numerous TCR transcripts were sequenced to examine the variability against antigenic peptide, and were shown hypervariability on their complementarity-determining region 3 (CDR3) loops. Among CDR3s, CDR3 delta showed a long and broad length distribution, indicating greater similarity to that of Ig. From cDNA sequences and genomic gene analysis of each chain, we found that flounder TCR beta, gamma, and delta have two different C gene segments, while the TCR alpha C region exists as a single segment. The flounder C gammas and C deltas showed different lengths in the connecting peptide (CP) region between the different types of polypeptides. The C delta 1 gene consists of two exons, one that encodes an extracellular Ig-like domain (exon 1) and the other that encodes either a very short or possibly a lacking CP region, a transmembrane region, and a cytoplasmic tail (exon 2); these are located within TCR alpha gene locus. Southern blot analysis, using the bacterial artificial chromosomal genomic DNA clones, revealed that the C delta 2 gene segment, which has a long CP region and different genomic organization to the C delta 1 gene, exists on same gene locus as the TCR gamma-chain. This suggests that the flounder possesses very unique genomic DNA organization and gene loci for TCR, C alpha/C delta 1, and C gamma/C delta 2.

Amino Acid Sequence↗