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Biomedical subjects

H Akashi

Publications and source records attributed to H Akashi.

At least 19 recordsLinked to original sources
PubMed2007

Sequence determination and functional analysis of the Akabane virus (family Bunyaviridae) L RNA segment.

Akabane virus (AKAV) causes epizootic congenital deformities in cattle, sheep, and goats. Due to the lack of a complete genome sequence, the molecular biological properties of this virus are not known. We have cloned and sequenced the functional large (L) RNA segment of AKAV, and shown that it has polymerase activity using a minireplicon system with RNA polymerase I. The complete L RNA segment is 6868 nucleotides long and encodes an L protein of 2251 amino acids, which functions as an RNA-dependent RNA polymerase. A minireplicon reporter plasmid was constructed by flanking either the firefly luciferase or the green fluorescent protein gene in the antisense orientation with the 5'- and 3'-terminal noncoding regions of the small RNA segment. HmLu-1 cells were transfected with the reporter plasmid, and the L protein and nucleoprotein (N protein) expression plasmids. The reporter activity was upregulated in a dose-dependent manner with increasing concentration of either the L or N protein expression plasmid. Furthermore, the reporter activity could be downregulated by the AKAV NSs protein as well as by other orthobunyaviruses. These results show that the AKAV minireplicon system is a powerful tool for studying transcription and for rescuing infectious viruses from cloned cDNAs.

Animals↗
PubMed2006

Genetic analysis of calicivirus genomes detected in intestinal contents of piglets in Japan.

Enteric caliciviruses, noroviruses, and sapoviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this study, swine enteric caliciviruses were detected in ten samples of intestinal contents from 24 piglets in Japan by reverse transcription-polymerase chain reaction using a broadly reactive primer pair (P290/289) that targeted the highly conserved RNA polymerase regions of the enteric caliciviruses. From the positive samples, the entire viral genome of strain K7/JP and 3'-end parts of the genomes of strains K5/JP and K10/JP were cloned and sequenced. K7/JP had an RNA genome of 7144 bases, excluding its 3' poly (A) tail. The K7/JP genome possessed two open reading frames and characteristics common to sapoviruses. In phylogenetic analysis using amino acid sequences of VP1, K5/JP was demonstrated to be close to the noroviruses previously detected in pigs, and K7/JP and K10/JP were considered to be classified as a new genogroup of sapoviruses.

Animals↗
PubMed2006

Screening and determination of gene function using randomized ribozyme and siRNA libraries.

Rapid progress in the sequencing of the genomes of model organisms, such as the mouse, rat, nematode, fly, and Arabidopsis, as well as the human genome, has provided abundant sequence information, but functions of long stretches of these genomes remain to be determined. RNA-based technologies hold promise as tools that allow us to identify the specific functions of portions of these genomes. In particular, catalytic RNAs, known also as ribozymes, can be engineered for optimization of their activities in the intracellular environment. The introduction of a library of active ribozymes into cells, with subsequent screening for phenotypic changes, can be used for the rapid identification ofa gene function. Ribozyme technology complements another RNA-based tool for the determination of gene function, which is based on libraries of small interfering RNAs (siRNAs).

Animals↗
PubMed2006

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype.

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Base Sequence↗
PubMed2005

Genetic recombination at different points in the Npro-coding region of bovine viral diarrhea viruses and the potentials to change their antigenicities and pathogenicities.

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain KS86-1 cp was isolated from a cow persistently infected with non-cytopathogenic (ncp) BVDV strain KS86-ncp after development of mucosal disease by superinfection with cp BVDV strain Nose. cp BVDV strains 799cp and 839cp were also isolated from independent cattle that developed mucosal disease by superinfection with cp BVDV KS86-1cp. In the present study, genetic analysis revealed that the genes of cp BVDV strains 799cp and 839cp were chimeras between the genes of the persisting ncp BVDVs and that of superinfecting KS86-1cp. The genetic recombination that generates 799cp occurred between the identical points in the N(pro) gene region, whereas genetic recombination that generates 839cp occurred between different points in the N(pro) gene region. Both 799cp and 839cp were inherited Jiv gene of KS86-1cp strain and envelope protein genes of the persisting viruses. In addition, neutralization test disclosed that antigenicities of 799cp, 839cp, and KS86-1cp were also similar to each persisting virus. These findings indicate that exogenous cp BVDV containing insertion of Jiv gene in the 5 terminal region can induce genetic recombination with the original ncp BVDV at different points in the N(pro) gene region, and those viruses have high potential to change those antigenicities and pathogenicities by RNA recombination.

Animals↗
PubMed2005

Enhancement of apoptosis via an extrinsic factor, TNF-alpha, in cells infected with cytopathic bovine viral diarrhea virus.

Isolates of bovine viral diarrhea virus (BVDV) are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. Calves persistently infected with ncp BVDV are known to develop lethal mucosal disease (MD) after superinfection by cp BVDV. Although the UV-irradiated supernatant of cp BVDV-infected cells has been reported to have no capacity to induce cell death, we found that it could enhance cell death through apoptosis. Up-regulation of tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) mRNAs was detected specifically in cp BVDV-infected primary cell cultures. Suppression of TNF-alpha via antisense oligonucleotide transfection or incubation with a polyclonal antibody against TNF-alpha resulted in attenuation of apoptosis induced by cp BVDV, suggesting that TNF-alpha participates in apoptosis execution. Although TNF-alpha is one of the iNOS-inducible factors, the iNOS up-regulation was not regulated by TNF-alpha. And iNOS was revealed to serve as anti-apoptotic factor, contrary to our expectation. In addition, the expression level of both TNF-alpha and iNOS mRNAs in the ncp BVDV-infected cells was kept lower than that in the mock-infected cells, suggesting that ncp BVDV reduced or interfered with the factor triggering the expression of both mRNAs. These characteristic mRNA transcriptions would help to explain why BVDV acts differently in cells as well as in vivo, depending on its biotype. To elucidate viral factors inducing TNF-alpha and iNOS may be critical to understand the mechanism of MD development, which closely correlates with cp BVDV-induced apoptosis.

Animals↗
PubMed2004

A novel antigenic variant of Canine parvovirus from a Vietnamese dog.

Nine isolates of Canine parvovirus (CPV) were obtained from Vietnamese dogs and cats. One canine isolate showed a unique antigenic property which indicates a novel antigenic variant of CPV-2b when examined with hemagglutination inhibition tests using our monoclonal antibodies, 21C3 and 19D7, which were recently developed. This isolate had an amino acid substitution of residue 426, Asp to Glu, and the same substitution has recently been found in CPV from Italian dogs. This study first showed that such substitution caused an antigenic difference demonstrable by monoclonal antibodies and that a similar evolution may have occurred in CPV in Vietnam.

Animals↗
PubMed2002

Obstruction of St Jude medical valves in the aortic position: a consideration for pathogenic mechanism of prosthetic valve obstruction.

Between 1995 and 2000, 8 patients with St. Jude Medical (SJM) valves in the aortic position required 9 redo valve replacement for prosthetic valve obstruction. Obstruction of the prosthetic valve was diagnosed by simultaneous echocardiography and cineradiography, and process of restricted leaflet movement that progressed to hemodynamic impairment was observed by serial studies in three recent patients. An oral anticoagulation was considered to be adequate in all patients except one patient who had withdrawal of warfrain. Pannus was the sole cause of valve obstruction in seven events in 6 patients, and both thrombus and pannus in 2 patients. Pannus overgrowth was found on the inflow aspect of the SJM valve, and involved the ends of the straight edge of the leaflets over pivot guards. These results suggest that pannus might play the primary role in development of obstruction of aortic SJM valves in patients on adequate oral anticoagulation.

Aged↗
PubMed2002

[Long-term results of aortic root replacement for Marfan syndrome].

Between January 1974 and December 2000, aortic root replacement was performed for 46 patients with Marfan syndrome. The hospital mortality rate amounts to 8.6% (4 patients out of 46). Fifteen (35.7% of survivor) of 42 survivor died during the period of follow-up. Eighteen of 42 survivor were developed cardiovascular events of 21 times and 11 patients had to be reoperated on at least once for a total of 12 reoperations. Nine of 15 late death patient died in relation to aortic dissection and pseudoaneurysm of distal anastomosis and coronary anastomosis. Actuarial survival rate was satisfactory for 10 years after aortic root replacement, but sharply decreased after postoperative 10 years. The use of non-sealed graft, classical Bentall method, reoperation and inclusion technique were risk factors for the late death.

Adolescent↗
PubMed2001

Cardio-aortic operation in octogenarians.

OBJECTIVE: Aging of the population is a current phenomenon in Japan, and life expectancy at 80 years old is getting longer. So we reviewed cardio-aortic operations on octogenarians at our institution. SUBJECTS AND METHODS: Thirty-three consecutive octogenarian patients who had undergone cardio-aortic operations from 1992 to 1998 were studied. There were 14 men and 19 women. The mean age was 81.9 years. Of the 33, 19 patients (58%) were in New York Heart Association class IV, and 21 patients (64%) were operated on urgently or in emergency. The procedures undergone were operation for coronary artery disease in 17 patients, operation for valvular disease in 7 patients, operation for thoracic-aorta in 7 patients, and others in 2 patients. RESULTS: The hospital mortality rate was 27% (9 patients). However, 89% of patients experiencing hospital death were in New York Heart Association class IV preoperatively and had required an emergency/urgent operation. On the other hand, there was only one hospital death (1/12, 8.3%) among the elective patients. The statistically significant risk factors for hospital death were renal insufficiency, shock, New York Heart Association class IV, intra-aortic balloon pumping, and longer cardiopulmonary bypass time. The one-, three-, and five-year-survival rate was 73%, 68%, and 55%, respectively. Of the survivors, 77% were in class I or II. CONCLUSION: Although octogenarians' hospital mortality was still very high, the mid-term results were acceptable and the survivors' quality of life was satisfactory. These data suggested that we should operate on cardio-aortic patients before they reach a very serious state, especially in octogenarians.

Age Factors↗
PubMed2001

Measurement of circulating biliary glycoprotein (CD66a) in liver diseases.

PURPOSE: Biliary glycoprotein (BGP), a member of the carcinoembryonic antigen (CEA) gene family, is produced by hepatocytes, and is suggested to function as a cell adhesion molecule, mouse hepatitis virus receptor, and tumor suppressor. Our aim was to establish an enzyme immunoassay for circulating BGP and to study its significance in liver diseases. METHODS: For enzyme immunoassay, a monoclonal antibody (mAb), TS135, which recognizes BGP was used as a catcher, and biotin-labeled polyclonal anti-CEA antibodies were used as a tracer. Seventy-six serum specimens obtained from patients with various liver diseases were submitted to the assay. RESULTS: The incidence of positivity for antigen TS135 in the serum samples of the 76 patients was 57.9%. The most significant correlation among conventional liver function tests was found between antigen TS135 and gamma-glutamyl transpeptidase (gamma-GTP). However, among the 56 patients whose serum antigen TS135 and gamma-GTP levels could be measured simultaneously, 5 were antigen TS135-positive and gamma-GTP-negative (8.9%) and 6 were antigen TS135-negative and gamma-GTP-positive (10.7%). The increased serum level of antigen TS135 in 6 cholangiocellular carcinoma (CCC) patients led us to the immunohistochemical study of CCC, in which 8 of the 8 tissue specimens tested were positive for mAb TS135, indicating the production of the antigen from CCCs. CONCLUSIONS: This preliminary study suggests that the circulating antigen TS135 level correlates with gamma-GTP in liver diseases, but that TS135 may also have a unique significance, different from that of gamma-GTP, as a liver function test.

Adult↗
PubMed2001

Surgical management of a graftenteric fistula: a modified method of in situ reconstruction.

Between December 1989 and May 1998, we performed a modified method of in situ reconstruction on three of seven patients with graftenteric fistulas (GEFs) at the Kurume University Hospital. The modification involved performing an anastomosis of the infrarenal abdominal aorta and running a new prosthesis through the left side of the descending colon in the retroperitoneal cavity, and wrapping the proximal anastomosis and the proximal site of the prosthesis in the greater omentum. Good results were achieved in all three patients. We describe herein this modified method of in situ reconstruction for a GEF and summarize the case reports of these three patients.

Adult↗
PubMed2001

Genomic and serological diversity of bovine viral diarrhea virus in Japan.

Genomic properties of 62 field isolates of bovine viral diarrhea virus (BVDV) collected from 1974 to 1999 in Japan were investigated. The 5' untranslated region (UTR) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and the 244 to 247 base nucleotide sequences were determined. Serological properties were also characterized by the cross-neutralization test using antisera against the representative strain of the classified genotype. Using phylogenetic tree analysis, BVDV 1 was subdivided into two major clusters, BVDV-1a (29 isolates) and BVDV-1b (27 isolates). In group 1a, 3 differed from the other viruses, and were classified in a branch assigned as 1a'. However, 4 isolates (So CP/75, 190 CP, 190 NCP and KS86-1-NCP) could not be assigned to group 1a or 1b. In comparison with the published sequence data, KS86-1-NCP, 190 CP and 190 NCP were similar to the Southern Africa isolates that have recently been proposed as BVDV 1c. The 5' UTR sequence of So CP/75 was unique among those of BVDV 1, suggesting that the isolate should be classified into a new genotype. Only 2 out of 62 isolates collected in 1989 and 1990 were identified as BVDV 2. The results of the cross-neutralization test strongly supported these data, suggesting a close correlation between the 5' UTR sequence and the antigenicity of BVDV.

5' Untranslated Regions↗
PubMed2001

Gene expression and molecular evolution.

The combination of complete genome sequence information and estimates of mRNA abundances have begun to reveal causes of both silent and protein sequence evolution. Translational selection appears to explain patterns of synonymous codon usage in many prokaryotes as well as a number of eukaryotic model organisms (with the notable exception of vertebrates). Relationships between gene length and codon usage bias, however, remain unexplained. Intriguing correlations between expression patterns and protein divergence suggest some general mechanisms underlying protein evolution.

Animals↗
PubMed2001

Suppression of gene expression by RNA interference in cultured plant cells.

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Animals↗
PubMed2001

A simple and rapid system for the quantitation of RNA interference in plant cultured cells.

The phenomenon known as RNA interference (RNAi) by double-stranded RNA (dsRNA) that was reported recently in the nematode Caenorhabditis elegans has been shown to operate by a mechanism that is widely conserved among species including plant cells. No quantitative analysis of the effects of RNAi on the expression of specific genes in plant cultured cells has been reported. An RNAi effect was observed 24 h after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Cell Line↗
PubMed2001

Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field.

Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.

Animals↗