Biomedical subjects

Takashi Aoki

Publications and source records attributed to Takashi Aoki.

At least 37 records · Page 2Linked to original sources

PubMed2006

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.

DNA-Binding Proteins↗

PubMed2005

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA and gene in Japanese flounder, Paralichthys olivaceus.

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily signaling involves myeloid differentiation factor 88 (MyD88) that acts as an important adapter protein. A Japanese flounder (Paralichthys olivaceus) MyD88 (jfMyD88) cDNA and gene were cloned, and found to have lengths of 1.5 and 3.01 kb, respectively. The ORF encodes 285 amino acids that contain a death domain and a Toll/IL-1 receptor domain. The gene is composed of 5 exons and 4 introns. The jfMyD88 gene is highly expressed in organs involved in immune functions, including the gills, intestines, kidney, skin and spleen. Three days after a fish was infected with Edwardsiella tarda, staining with anti-jfMyD88 polyclonal antibody revealed an increased population of MyD88-positive cells in the kidney and spleen. These results imply that MyD88 has an important role in the innate immune system in Japanese flounder.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Genetic vaccines protect red seabream, Pagrus major, upon challenge with red seabream iridovirus (RSIV).

We have investigated the protective effect of immunization of juvenile red seabream, Pagrus major, with DNA plasmids encoding the viral major capsid protein (MCP) and an open reading frame (ORF) containing a transmembrane domain against red seabream iridovirus (RSIV). The expression of the MHC class I transcript in the DNA-vaccinated fish was significantly upregulated at the 15th day post-vaccination and the relative level of expression was maintained until the 30th day post-vaccination. This pattern of expression was similar in fish vaccinated with a commercially prepared formalin-inactivated RSIV vaccine. In vaccine efficiency tests, the relative percentage survival (RPS) of fish receiving the DNA vaccines and their combination ranged from 42.8 to 71.4% in two experimental runs, and these were significantly different from the control groups. Our results clearly demonstrate that DNA vaccines are able to induce robust protection in fish against RSIV infection, and a cellular immune response as shown by the upregulation of the MHC class I transcript after vaccination, which may be associated with such protection.

Animals↗

PubMed2005

Development of gene transfer technology for black tiger shrimp, Penaeus monodon.

An effective foreign gene transfer method for shrimp would have several potential uses in the shrimp culture industry, such as in preventing infectious diseases. We evaluated two gene transfer methods and used black tiger shrimp, Penaeus monodon, as a model target species. For a promoter, we used the 1,592-bp promoter region of the EF-1alpha gene, a house-keeping gene, of kuruma shrimp Marsupenaeus japonicus. The promoter region was linked to either the gene for green fluorescence protein (GFP) or the gene for chloramphenicol acetyl transferase (CAT). The fusion genes were designated pJEF-GFP and pJEF-CAT, respectively. The pJEF-GFP gene was introduced into fertilized eggs of black tiger shrimp by microinjection and particle gun bombardment. The survival rate of the microinjected eggs was 17.6%, and 1.0% of the treated embryos were found to be GFP-positive. However, the GFP-positive embryos were damaged and embryogenesis did not progress. The survival rate of the particle-bombarded eggs was 60.6%, and 0.42% of the treated embryos were found to be GFP-positive. Ubiquitous GFP expression was observed from 8 hr post-fertilization and these embryos developed and hatched normally. The pJEF-CAT gene was introduced into fertilized eggs of black tiger shrimp using the optimized conditions of the particle gun bombardment. CAT activity was observed from 1 to 7 days post-fertilization, with the highest activities being observed at 5 and 7 days post-hatching.

Animals↗

PubMed2005

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum.

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.

Animals↗

PubMed2005

Characterization and expression of a CD40 homolog gene in Japanese flounder Paralichthys olivaceus.

A CD40 homolog cDNA encoding 300 amino acid residues was isolated from a Japanese flounder leukocyte cDNA library. The amino acid sequence identities of Japanese flounder CD40 and previously reported CD40s of cow, human, mouse, and chicken range from 32 to 35%. The positions of cysteine residues, CD40 ligand binding amino acid residues, and four cysteine-rich domains are well conserved in Japanese flounder CD40. The Japanese flounder CD40 gene is composed of nine exons and eight intervening introns spread over 6 kb. The nucleotide sequence of the 5'-flanking region of this gene revealed the presence of several regulatory regions, including a TATA-like box, AP-1-, CEBPB-, IRF-1-, LYF-1-, NF-kappaBp-, SP-1-, and Stat-1-like motifs. In healthy fish, reverse transcriptase-polymerase chain reaction (RT-PCR) detected constitutive expression of CD40 in all tested tissues (leukocytes, kidney, spleen, liver, intestine, brain, gill, and skin). The mRNA of CD40 was predominantly expressed in several tissues that contained lymphocytes (leukocytes, kidney, spleen, intestine, and gill). Expression of the Japanese flounder CD40 molecule was induced in peripheral blood leukocytes from 1 to 6 h following Con A (50 microg/ml)/phorbol myristate acetate (PMA) (0.35 microg/ml) stimulation, with a peak at 1 h after stimulation, and increased at 1, 3, and 6 h after induction by lipopolysaccharide (LPS) (500 microg/ml) compared with the control.

Amino Acid Sequence↗

PubMed2005

Immunogenicity, retention and protective effects of the protein derivatives of formalin-inactivated red seabream iridovirus (RSIV) vaccine in red seabream, Pagrus major.

A formalin-inactivated virus was previously found to be efficient in protecting fish against challenge with red seabream iridovirus (RSIV), a DNA virus belonging to the Iridoviridae family. In the present study, we determined the amount of the virus in the vaccine in terms of the number of copies of the gene for the major capsid protein (MCP) gene by quantitative real-time PCR and examined the longevity and types of immune response generated after intramuscular vaccination. We also tested whether the protein components of the vaccine are able to mount a protective immune response in fish. The vaccine contained 10(7) MCP copies per microliter of vaccine, and was detected in blood, kidney and spleen of vaccinated fish up to 15 days post-vaccination. Fish vaccinated with either the intact formalin-inactivated vaccine or its protein derivatives had increased serum neutralization antibodies and enhanced expression of MHC class I, although the kinetics of expression varied among groups. However, only those vaccinated with the intact vaccine survived the virus challenge, and this indicates that serum neutralization antibodies have scarce role in protecting the fish against RSIV. We hypothesize that the cell-mediated immunity, particularly the MHC class I pathway is responsible for such protection.

Analysis of Variance↗

PubMed2005

Anticonvulsant and neuroprotective effects of the novel nootropic agent nefiracetam on kainic acid-induced seizures in rats.

Nefiracetam is a novel pyrrolidone-type nootropic agent, and it has been reported to possess a potential for antiepileptic therapy as well as cognition-enhancing effects. We investigated the anticonvulsant and neuroprotective effects of nefiracetam in kainic acid-induced seizures of rats, compared with levetiracetam and standard antiepileptic drugs. Subcutaneous injection of kainic acid (10 mg/kg) induced typical behavioral seizures such as wet dog shakes and limbic seizures and histopathological changes in the hippocampus (degeneration and loss of pyramidal cells in CA1 to CA4 areas). Nefiracetam (25, 50 and 100 mg/kg po) had no effect on the behavioral seizures and dose-dependently inhibited the hippocampal damage. In contrast, levetiracetam, a pyrrolidone-type antiepileptic drug, inhibited neither. Valproic acid and ethosuximide prevented the hippocampal damage without attenuating the behavioral seizures as nefiracetam. Zonisamide and phenytoin did not inhibit the behavioral seizures, while zonisamide enhanced the hippocampal damage and phenytoin increased the lethality rate. Carbamazepine inhibited the behavioral seizures at 50 mg/kg and enhanced that at 100 mg/kg, and it completely inhibited the hippocampal damage at both doses. We have previously reported that anticonvulsant spectrum of nefiracetam paralleled that of zonisamide, phenytoin or carbamazepine in standard screening models. However, the pharmacological profile of nefiracetam was closer to valproic acid or ethosuximide than that of zonisamide, phenytoin or carbamazepine in this study. These results suggest that anticonvulsant spectrum and mechanism of nefiracetam are distinct from those of standard antiepileptic drugs, and nefiracetam possesses a neuroprotective effect that is unrelated to seizure inhibition.

Animals↗

PubMed2005

Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms.

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.

Animals↗

PubMed2005

Expression, purification and crystallization of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with orotate.

Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate, the fourth step and the only redox reaction in the de novo biosynthesis of pyrimidine. DHOD from Trypanosoma cruzi (TcDHOD) has been expressed as a recombinant protein in Escherichia coli and purified to homogeneity. Crystals of the TcDHOD-orotate complex were grown at 277 K by the sitting-drop vapour-diffusion technique using polyethylene glycol 3350 as a precipitant. The crystals diffract to better than 1.8 A resolution using synchrotron radiation (lambda = 0.900 A). X-ray diffraction data were collected at 100 K and processed to 1.9 A resolution with 98.2% completeness and an overall Rmerge of 7.8%. The TcDHOD crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 67.87, b = 71.89, c = 123.27 A. The presence of two molecules in the asymmetric unit (2 x 34 kDa) gives a crystal volume per protein weight (VM) of 2.2 A3 Da(-1) and a solvent content of 44%.

Aminomethyltransferase↗

PubMed2005

Comparative immune responses in Japanese flounder, Paralichthys olivaceus after vaccination with viral hemorrhagic septicemia virus (VHSV) recombinant glycoprotein and DNA vaccine using a microarray analysis.

A viral hemorrhagic septicemia virus (VHSV), recombinant glycoprotein vaccine (VHSVg) and a DNA vaccine (pCMV-VHSg) were injected in Japanese flounder (Paralichthys olivaceus). Each fish was injected with 10 microg vaccine dissolved in 50 microl phosphate buffer saline (PBS). One month after the vaccination, the fish were challenged intraperitoneally with either 1 x 10(2) or 1 x 10(3) TCID(50) of virus. Fish that received the VHSg DNA vaccine were highly protected against virus infection over a 21-day observation period, with cumulative mortalities ranging from 4 to 10%. However, the recombinant protein vaccine group appeared to have low survival rates. Using microarray analysis, humoral defense-related genes such as complement component C3, complement regulatory plasma proteins, IgM, IgD, MHC class II-associated invariant chain and CD20 receptor were observed to be up-regulated by the VHSg recombinant protein vaccine at 1 or 21 days post vaccination. On the other hand, cellular defense-related genes such as CD8 alpha chain, T-cell immune regulator, MIP1-alpha and apoptosis-associated protein were not detected. Specific antibodies against VHSVg protein were detected in both vaccinated groups at a titer of 1:40 at 28 days post vaccination.

Animals↗

PubMed2005

Gene expression profiles in response to Fas stimulation in Trypanosoma cruzi-infected host cells.

To determine the molecular mechanism by which apoptosis is inhibited in Trypanosoma cruzi-infected host cells, we used human cDNA apoptosis chips to compare the gene expression profiles in response with 'death ligands target' (Fas) stimulation in infected and uninfected cells. Of the 164 apoptosis-related genes examined, 20, including those encoding both pro- and anti-apoptotic proteins, were highly up-regulated in the infected group. Genes encoding caspases and apoptosis inhibitors were optimally expressed 10-30 min after induction of apoptosis, whereas genes involved in transcriptional regulation and cell proliferation were up-regulated after 2-24 h. These results suggest that host anti-apoptotic gene(s) may play a crucial role in the inhibition of Fas-mediated apoptosis in T. cruzi-infected cells.

Animals↗

PubMed2005

A homogeneous assay for relative affinity of binding proteins using a green fluorescent protein tag and membrane disk.

When the association between a ligand immobilized on a membrane disk and a fluorescence-labeled analyte was monitored with a fluorescent microplate reader, the time-dependent increase in fluorescence intensity of the reaction mixture was observed. A novel assay system for the specific interaction based on this phenomenon was designated the homogeneous assay for fluorescence concentrated on membrane (HAFCOM). In this study, streptococcal protein G (SpG) and glycogen-binding subunit R5 of protein phosphatase 1 (PPP1R5) tagged by green fluorescent protein (GFP) were used as the fluorescence-labeled analytes, and the affinity change caused by various amino acid substitutions was measured with HAFCOM. From the site-directed mutagenesis of SpG and PPP1R5, it was clarified that (i) the association rate constant of the Lys454Pro/Glu456Gln mutant of SpG to goat immunoglobulin G was almost equivalent to that of the wild-type but its dissociation rate constant was about 2.7 times that of the wild-type and (ii) the amino acid substitutions of Phe180 in PPP1R5 reduced glycogen-binding by 30-50%. Since HAFCOM using the GFP-tagged analyte requires no special chemicals and instruments, this system can easily and economically assay the specific interaction between target protein and ligand.

Animals↗

PubMed2005

Four novel hemolysin genes of Vibrio anguillarum and their virulence to rainbow trout.

Four nucleotide sequences showing homology to known hemolysin genes were cloned and sequenced from V. anguillarum strain H775-3. The four genes, vah2, vah3, vah4 and vah5, have open reading frames encoding polypeptides of 291, 690, 200 and 585 amino acid residues, respectively, with predicted molecular masses of 33, 75, 22 and 66KDa, respectively. VAH2 is most closely related to a putative hemolysin of Vibrio vulnificus YJ016 (89% identity). VAH3 is most closely related to a hemolysin-related protein in Vibrio cholerae O1 (68% identity). VAH4 is most closely related to a thermostable hemolysin in V. cholerae O1 (72% identity). VAH5 is most closely related to a putative hemolysin in V. cholerae O1 (73% identity). The purified hemolysin proteins showed hemolytic activities against erythrocyte of fish, sheep and rabbit. Four strains of V. anguillarum mutants were constructed, each deficient in one of the hemolysin genes. Each mutant was less virulent than V. anguillarum H775-3 to juvenile rainbow trout (Oncorhynchus mykiss), indicating that each hemolysin gene contributes to the virulence of V. anguillarum H775-3.

Amino Acid Sequence↗

PubMed2005

Characterization of promoter activities of four different Japanese flounder promoters in transgenic zebrafish.

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5' flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.

Animals↗

PubMed2005

Trypanosoma cruzi posttranscriptionally up-regulates and exploits cellular FLIP for inhibition of death-inducing signal.

Intracellular persistence of the protozoan parasite, Trypanosoma cruzi, is an aggravating cause of Chagas' disease, involving that the protozoan infection specifically inhibits death receptor-mediated apoptosis of host cells. Here we demonstrate that the parasite dramatically up-regulates cellular FLICE inhibitory protein (c-FLIP), the only known mammalian inhibitor specific for death receptor signaling, in infected cells by an unusual, posttranscriptional stabilization of the short-lived protein. We also show that c-FLIP is accumulated in T. cruzi-infected mouse heart muscle cells in vivo. Stimulation of death receptor Fas in infected cells induces recruitment of c-FLIP to block the procaspase-8 activation at the most upstream caspase cascade. c-FLIP knock-down with a small interfering RNA significantly restores Fas-mediated apoptosis in infected cells. Taken together, our findings indicate that T. cruzi posttranscriptionally up-regulates and exploits host c-FLIP for the inhibition of death-inducing signal, a mechanism that may allow parasites to persist in host cells.

Animals↗

PubMed2005

Induction of Japanese flounder tnf promoter activity by lipopolysaccharide in zebrafish embryo.

A recombinant plasmid containing the 2381-bp promoter region of Japanese flounder tumor necrosis factor (TNF) and green fluorescence protein (GFP) was introduced into zebrafish fertilized eggs by microinjection. GFP was expressed in 2 transgenic zebrafish lines in the heart and around the pharynx under unstressed condition. When embryos were exposed to lipopolysaccharide (LPS), GFP was expressed in the whole zebrafish embryonic surface, and Western blot analysis also showed that the level of the expressed GFP protein in zebrafish embryo was higher at an LPS concentration of 140 microg/ml than at LPS concentrations of 0 and 70 microg/ml. Stimulation with either concanavalin A or phorbol myristate acetate induced weak GFP expression, but stimulation with both of them induced strong expression similar to that induced by LPS. GFP expression peaked 1 hour after stimulation, then gradually decreased. These results indicate that transcription regulated by the Japanese flounder TNF promoter could be under the control of the LPS-recognition system in zebrafish embryos.

Animals↗

PubMed2005

Interchromosomal duplication of major histocompatibility complex class I regions in rainbow trout (Oncorhynchus mykiss), a species with a presumably recent tetraploid ancestry.

Salmonid fishes are among the few animal taxa with a probable recent tetraploid ancestor. The present study is the first to compare large (>100 kb) duplicated genomic sequence fragments in such species. Two contiguous stretches with major histocompatibility complex (MHC) class I genes were detected in a rainbow trout BAC library, mapped and sequenced. The MHC class I duplicated regions, mapped by fluorescence in situ hybridization (FISH), were shown to be located on different metaphase chromosomes, Chr 14 and 18. Gene organization in both duplications is similar to that in other fishes, in that the class I loci are tightly linked with the PSMB8, PSMB9, PSMB10 and ABCB3 genes. Whereas one region, Onmy-IA, has a classical MHC class I locus (UBA), Onmy-IB encodes only non-classical class Ib proteins. The nucleotide diversity between the Onmy-IA and Onmy-IB noncoding regions is about 14%. This suggests that the MHC class I duplication event has occurred about 60 mya close to the time of an hypothesized ancestral tetraploid event. The present article is the first convincing report on the co-existence of two closely related MHC class I core regions on two different chromosomes. The interchromosomal duplication and the homology levels are supportive of the tetraploid model.

Amino Acid Sequence↗