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M Shinagawa

Publications and source records attributed to M Shinagawa.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization of the bovine prion protein gene: the expression requires interaction between the promoter and intron.

We cloned the part of the bovine PrP gene which contains the 5'-flanking region, exon 1, exon 2 and intron 1 to analyze its promoter region. The 5' non-coding region of the bovine PrP gene consisted of three exons and two introns, and its organization was similar to that of the mouse, rat and sheep PrP genes. The 5'-flanking region of the bovine PrP gene from the transcription start site to nucleotide position -88 was (G + C)-rich (78%) and contained three potential binding sites for the transcription factor Sp1, but no CCAAT-box or TATA-box. This region showed high homology (89%) with that of the sheep PrP gene, but relatively low homology (approximately 46-62%) with the same region of the mouse, rat, hamster and human PrP genes. The position from -88 to -30 within the 5'-flanking region of the bovine PrP gene showed major promoter activity. However, this region was able to function properly only in collaboration with the region at +123 to +891 of intron 1 of the bovine PrP gene.

Animals↗
PubMed1996

Predominant p53 mutations in enzootic bovine leukemic cell lines.

The role of the p53 tumor suppressor gene in bovine lymphosarcomas, a fragment of about 100 bp corresponding to approximately 97% of the open reading frame of the p53 gene was first amplified from single-strand cDNA originated from calf thymus by polymerase chain reaction PCR) and sequenced to obtain the bovine wild-type p53 gene. At the amino acid level, the omologies of the bovine p53 gene with the human, mouse, chicken and cat p53 genes were 0.9%, 72.8%, 52.7% and 82.3%, respectively. Moreover, eight bovine leukemic cells lines were studied for alterations in the p53 gene. These lines showed no significant somatic alterations in southern blot analysis, and expressed 2.5 kb p53-specific transcripts in Northern blot analysis. In mutation analysis using the reverse transcriptase-PCR technique, we detected three missense point mutations in four of these bovine leukemic cell lines. These mutations occurred in the 'hotspots' of the p53 gene. Thus p53 mutations predominantly occur in BLV-transformed cell lines and seem to be necessary for development of enzootic bovine leukosis (EBL).

Amino Acid Sequence↗
PubMed1996

Improvement of PrPSc-detection in mouse spleen early at the preclinical stage of scrapie with collagenase-completed tissue homogenization and Sarkosyl-NaCl extraction of PrPSc.

Scrapie in sheep has recently become again a target of control measures and eradication programs. Crucial for the effectiveness of these measures is the detection of infected sheep during the long and potentially hazardous incubation period. However, routine-diagnosis is mostly limited to clinical examinations when disease becomes apparent, and to postmortem investigations. Through the detection of the scrapie-specific isoform of the prion protein (PrPSc) by Western blot in the spleen and lymph nodes from scrapie-infected mice and sheep, we have shown previously that diagnosis during the preclinical stage is possible. We introduce here an improved method for the diagnosis of mouse scrapie shortly after infection. Through a homogenization procedure that includes a collagenase digestion step, and through extraction and salting-out of PrPSc by Sarkosyl and NaCl, respectively, we were able to detect PrPSc in spleen tissue of intraperitoneally infected mice seven days postinfection. Moreover, the new protocol makes sample-handling easier and reduces the hands-on time. We also successfully enriched PrPSc from spleen tissue through immobilized metal affinity chromatography (IMAC); however, for the diagnosis at the earliest stage of infection, extraction of PrPSc by Sarkosyl and NaCl was more effective.

Animals↗
PubMed1996

Differentiation of vaccine virus from field isolates of feline panleukopenia virus by polymerase chain reaction and restriction fragment length polymorphism analysis.

In an attempt to distinguish feline panleukopenia virus (FPLV) live vaccine strains from FPLV field isolates in Japan, we compared restriction fragment length polymorphisms (RFLP) of polymerase chain reaction (PCR)-amplified fragments of live FPLV vaccine strains with those of FPLV Japanese field isolates. On the basis of nucleotide sequence differences between PLI-IV, a live vaccine strain, and FPV-483, a recent field isolate, two restriction enzymes, Dra I and Afa I, were selected for PCR-RFLP analysis of nucleotide (nt) differences at nt 3695 and 4508, respectively. Three live vaccine strains including the PLI-IV strain could be distinguished from the Japanese field isolates by their PCR-RFLP patterns by Afa I, but one live vaccine strain was indistinguishable from the Japanese isolates when Dra I and Afa I were used. The Japanese field isolates were divided into two groups by the profile of PCR-RFLP patterns generated by Dra I and Afa I, suggesting that PCR-RFLP analysis using several enzymes provides a good genetic estimate of strain differentiation. No isolate that shows a Dra I-negative/Afa I-negative pattern has emerged in Japan, indicating the possibility that the live vaccine viruses with a Dra I-negative/Afa I-negative pattern, such as the PLI-Iv strain, are candidates for use as live FPLV vaccine strain in Japan where they can be genetically distinguished from field strains.

Animals↗
PubMed1996

Potential transforming-ability by internal deletion of the bovine c-myb gene.

Two kinds of bovine c-Myb, the 75 kDa full-length (FL) c-Myb from the thymic type of sporadic bovine lymphosarcoma (SBL) cells, and 65 kDa deleted (DEL) c-Myb, which is an internally deleted form (85 amino acids corresponding to the negative regulatory domain) from calf-type SBL were found. To investigate the relationship between internal deletion and transforming capacity, we constructed retroviral vectors carrying cDNA that encoded either FL- or DEL-Myb protein, and transfected them to mouse fetal liver cells. In spite of the high trans-activating capacity of DELMyb, DELMyb and FLMyb showed no significant difference in oncogenic capability by measurement of colony formation in soft agar or in methylcellulose medium. Thus, the internal deletion of the c-myb gene is competent for transactivation but not directly relevant to transformation of mouse hemopoietic cells.

3T3 Cells↗
PubMed1995

Sonoelastic determination of human skeletal muscle elasticity.

It is not currently practical to directly measure viscoelastic parameters in human muscles in situ. Methods used in vitro cannot readily be applied, and motion analysis provides only a gross estimate. We report on the application of a hybrid approach, sonoelastography, which uses ultrasound to measure the propagation of shear waves induced by externally applied vibrations. Because shear waves predominate in incompressible viscoelastic media at low frequencies, sonoelastic data should be comparable to those obtained using conventional means. We recorded vibration propagation speeds as a function of applied load in the quadriceps muscles of ten volunteers as they underwent a series of static contractions. Data collection during dynamic contractions, not possible with the current equipment, will be the subject of future experimentation. Although statistically significant correlations were not uniformly obtained above 60 Hz nor for propagation perpendicular to the muscle fibers, this is felt to have resulted from deviations from the applied plane wave model. Calculated values of Young's modulus for 30 Hz propagation parallel to the muscle fibers were 7 +/- 3, 29 +/- 12 and 57 +/- 37 x 10(3) Nm-2 for applied loads of 0, 7.5 and 15 kg, respectively. The corresponding values at 60 Hz were 25 +/- 6, 75 +/- 61 and 127 +/- 65. These values were statistically significant and linearly correlated with the applied load, as expected. Our data represent the first in situ human measurements of their kind. It is anticipated that sonoelastography will provide a useful adjunct to the study of human biomechanics.

Adult↗
PubMed1995

Amino acid polymorphisms of PrP with reference to onset of scrapie in Suffolk and Corriedale sheep in Japan.

We investigated the relationships between amino acid polymorphisms of the prion protein (PrP), restriction fragment length polymorphisms (RFLP) of the PrP gene and the incidence of natural scrapie in Japan. Six variant alleles of the PrP gene were found in healthy sheep. Based on the substitutions at codons 112, 136, 154 and 171, these allelic variants were designated PrPMARQ, PrPTARQ, PrPMVRQ, PrPMAHQ, PrPMARR and PrPMARH. Each RFLP haplotype (e1h2, e1h2 or e3h1) consisted bo multiple alleles including PrPMARQ. Three of these variant alleles were found in scrapie-affected Suffolk sheep. PrPMARQ was associated with high disease incidence, PrPTARQ and PrPMARR were associated with low disease incidence. We found that one scrapie-affected Suffolk was homozygous for PrPMARR and four PrPSc-positive Suffolks carried PrPMVRQ. Both of two scrapie-affected Corriedales and two out of three scrapie-affected cross-breeds between Suffolk and Corriedale carried PrPMARH, suggesting that this allele associates with high incidence of scrapie in Corriedale and its cross-breeds.

Alleles↗
PubMed1995

A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep.

A cellular form of the prion protein (PrPC) is thought to be a substrate for an abnormal isoform of th eprion protein (PrPSc) in scrapie. PrPC is abundant in tissues of the central nervous system, but little is known about the distribution of PrPC in non-neuronal tissues of sheep, the natural host of scrapie. This study investigated the tissue distribution of PrPC in sheep. Although PrPC was abundant in neuronal tissues, it was detected in non-neuronal tissues such as spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, proventriculus, abomasum and mammary gland. Neither PrPC nor PrP mRNA was detected in the liver. The tissue distribution of PrPC appears to be inconsistent with the tissues which possess scrapie infectivity, suggesting that factor(s) specific to certain cell types may be required to support multiplication of the scrapie agent.

Amino Acid Sequence↗
PubMed1995

Restriction endonuclease analysis of bovine herpesvirus type 1 isolates from calves with fatal encephalitis: comparison with vaccine virus.

Meningo-encephalitis in feedlot cattle sporadically occurred in the Tokachi area in northern Japan. The calves had been vaccinated intranasally with a mixed live-vaccine (infectious bovine rhinotracheitis virus, bovine viral diarrhea-mucosal disease virus, and parainfluenza 3 virus) for which intramuscular inoculation was indicated. Two additional live vaccines, bovine adenovirus type 7 and bovine respiratory syncytical virus, had been inoculated simultaneously. Eleven isolates of bovine herpesvirus type 1 were plaque-purified from two brains with fatal encephalitis; their viral DNAs were examined by restriction endonuclease analysis (REA) using PstI and HindIII. The REA patterns of the virus clones were almost identical to those of the vaccine strains 758-43, suggesting that the isolates from this outbreak of fatal encephalitis originated in the abnormally administered vaccine.

Administration, Intranasal↗
PubMed1995

Hantavirus infection among Rattus norvegicus in Japan.

Seroepizootiological study of hantavirus infection among 393 urban rats (Rattus norvegicus) captured in six regions in Japan during the period from 1990 to 1994 was carried out by the indirect fluorescent antibody (IFA) test and Western blot (WB). Fifteen out of 393 (3.8%) rat sera were antibody-positive by IFA, i.e., Tokyo Port (12.8%, 6/47), Shimizu Port (5.7%, 2/35), Otaru Port (1.5%, 1/65) and Nagoya City (3.6%, 6/167). In two other regions, i.e., Kasai Seaside Park and Haneda Airport, rat sera were antibody-negative. One serum with a lower IFA titer, 1:64, from Otaru Port was confirmed to be antibody-positive by WB, while two sera from Shimizu Port (IFA titer, 1:32 and 1:64) were not. In Nagoya City, one out of four sera (IFA titer, 1:32) and one of two sera (IFA titer, 1:64) were also confirmed to be antibody-positive by WB. Continuous hantavirus infection among rats in Tokyo Port, Shimizu Port and Nagoya City and the existence of hantavirus among rats in Otaru Port were demonstrated.

Animals↗
PubMed1994

A spontaneous internal deletion of the c-myb protooncogene enhances transcriptional activation in bovine T lymphoma cells.

Three double negative (BoCD4-, BoCD8-) bovine T cell lines, BTL-PC3, BLT2, and Pr2181, which have been established from bovine lymphosarcomas, were examined for expression and molecular function of bovine c-myb genes. BTL-PC3 expressed 4.0- and 3.6-kilobase c-myb transcripts, and BLT2 and Pr2181 expressed a 3.8-kilobase c-myb message. The c-Myb protein (75 kDa) was detected in Pr2181 but not as a clear band in BLT2, while BTL-PC3 exhibited a 65-kDa c-Myb band in immunoprecipitation tests with anti-Myb antiserum. Nucleotide sequences for c-myb cDNA clones from BTL-PC3 and BLT2 indicated that the predicted bovine wild-type c-Myb from BLT2 consists of 640 amino acids whereas that from BTL-PC3 consists of 555 amino acids lacking 85 internal amino acids. This deleted DNA region (255 base pairs consisting of 85 amino acids) corresponds to the human genomic exon 9 encoding a negative regulatory domain in the c-myb gene. Upon cotransfections with reporter plasmids containing myb binding sites, the internally deleted c-Myb exhibited a 3-fold higher transcriptional activity than the wild-type c-Myb in chloramphenicol acetyltransferase assays. These results indicate that internal DNA deletion in the c-myb gene is directly involved in the enhancement of transcriptional activation in bovine T lymphoma cells.

Amino Acid Sequence↗
PubMed1994

Detection of PrPSc in sheep at the preclinical stage of scrapie and its significance for diagnosis of insidious infection.

A field sheep insidiously infected with natural scrapie was diagnosed at the preclinical stage through detection of the core fragment of PrpSc (PrPcore) in peripheral lymph nodes by the biopsy method. Three out of 32 euthanized healthy sheep were found positive for PrPcore in the spleen and lymph nodes. Mice that were inoculated with spleen homogenate of one of these sheep showed clinical signs of scrapie and were positive for PrPcore in their brain samples. These results suggest that the detection of PrPcore is significant for diagnosis in the preclinical phase of scrapie.

Animals↗
PubMed1994

Putative bovine B cell lineage tumor in sporadic bovine leukosis.

Two calves (C924 and C928) clinically identified as suffering from the calf type of sporadic bovine leukosis (SBL) were examined for immunophenotypical distribution of their peripheral blood lymphocytes (PBL) with T cell or B cell specific monoclonal antibodies (mAbs). Flow cytometric profile analysis indicated that both calves possessed a high population of MHC class II positive cells and a low population carrying T cell associated markers. More than 93% of the PBL of C928 were positive for MHC class II and BoCD5, but were not IgM positive, indicating that clonal tumor cells (MHC-II+, BoCD5+ and IgM-) accounted for most of the PBL of C928. A tumor cell line, BLC3, established from calf C924 in nude mice was examined for immunophenotype, DNA rearrangement of immunoglobulin (Ig) genes and expression of cellular oncogenes c-abl, c-myc and c-myb. BLC3 showed a characteristic profile, being MHC class I and MHC class II positive, but without T cell associated markers. Although the rearranged configuration in the Ig loci was not clearly found in Southern blot analysis using sheep Ig probes, steady-state levels of c-myb transcripts were detected in tumor cell line BLC3 by Northern blot hybridization. Taken together, our data show that the calf type of SBL found in these two calves is induced by immature pre-B cells, including tumor cell line BLC3 established in nude mice.

Animals↗
PubMed1994

Differentiation analysis of bovine T-lymphosarcoma.

The tumor cells from three different types (calf, skin and thymic) of bovine lymphosarcoma identified as sporadic bovine leukosis (SBL) were transplanted to nude mice and established as lymphoid tumor lines. To clarify the T-cell lineage, clonality and differentiation stage, these cell lines were examined for the immunophenotypic profile, the expression and the gene rearrangement of T-cell receptor (TCR) genes. The tumor cells from all three types showed alpha-naphthyl acetate esterase-positive staining and the absence of surface immunoglobulin (sIg). Moreover, the tumor cells appeared to be all BoCD5+ BoCD6+ BoCD4- BoCD8- cells in the FACS analysis with mAbs. Rearrangement of TCR-beta and -delta, but not of TCR-alpha and -gamma, genes was found to be unique for each tumor line showing T-cell clonality. Mature or truncated TCR-beta, -gamma and -delta transcripts were detected in the tumor cells, but all of them were found to be non-functional in cDNA cloning analysis. These results suggested that tumor cells from SBL are immature double-negative T-cells expressing non-functional TCR gamma delta transcripts to somewhat differentiated degrees.

Amino Acid Sequence↗
PubMed1994

Mapping of determinants of the host range for canine cells in the genome of canine parvovirus using canine parvovirus/mink enteritis virus chimeric viruses.

Feline panleukopenia virus (FPLV), mink enteritis virus (MEV) and canine parvovirus (CPV) are more than 98% similar in DNA and predicted amino acid sequences, but they show different host-cell specificities; CPV is able to replicate in canine cells in culture, whereas FPLV and MEV cannot or replicate only to a low titre. To map the genomic region responsible for the host range of CPV in vitro, CPV/MEV chimeric viruses were generated by transfecting infectious CPV/MEV chimeric plasmids into a cultured feline kidney cell line, and their host cell ranges were analysed. The 60 to 91 map units (m.u.) region of the CPV genome, which contains a part of the capsid protein (VP) gene encoding from amino acid 91 (in the VP2 sequence) to the carboxy terminus of VP protein, was required to impart the ability to replicate in canine cells to MEV, although the chimeric virus containing the 60 to 91 m.u. region of the CPV genome in the MEV background did not replicate in canine cells as efficiently as did CPV derived from the infectious plasmid of CPV. Not only the VP gene, but also a part of the NS gene of CPV were considered to participate in the full expression of the ability to replicate in canine cells. Within the 60 to 91 m.u. region, five of nine amino acid changes between MEV-Abashiri and CPV-Y1 were thought to be phylogenetically CPV-common; however, a recombinant virus containing all five amino acid changes of CPV in the MEV background replicated minimally in canine cells.

Animals↗
PubMed1994

Prolonged occurrence of Moraxella bovis infection on a restricted heifer farm.

An epizootiologic study was conducted for infectious bovine keratoconjunctivitis (IBK) on a large grassland farm rearing a total of 1,300-1,400 heifers during May to October in both 1991 and 1992. Heifers were examined for infection with Moraxella bovis, and isolation of M. bovis from the affected eyes and from Asian face flies (Musca bezzii Patton et Cragg) swarming on the heifer face was carried out. During the observation period, 10.7% of the heifers in 1991 and 5.3% of the heifers in 1992 were affected with IBK. Eight M. bovis isolates were isolated from the ocular swabs of affected heifers with IBK for 2 years and identified by biochemical examinations and Southern DNA-DNA hybridization, but no M. bovis was obtained from 331 Asian face flies tested. Plasmid profile analysis showed that two plasmids (35-kb and 4.0-kb) were commonly found in all 8 M. bovis isolates. This indicates that M. bovis isolates with the same plasmid profile were widely disseminated on this farm for 2 years.

Animals↗
PubMed1994

Characterization of monoclonal antibodies against sporadic bovine leukosis cell lines.

We report here distributions of antigens expressed on sporadic bovine leukosis (SBL) cells by a total of 38 monoclonal antibodies (mAbs) directed against three different types of SBL-cell lines, BLT2 (thymic type), BTL-PC3 (calf type) and BLS1 (skin type). Most mAbs had high reactivities with some bovine lymphoma cell lines and less reactivity with normal lymphocytes except for some peripheral blood mononuclear cells (PBM) in cattle and certain other species. They were more reactive with cultured T-cell line BTL-PC3 and B-cell lines, BL312 and KU-1, than naturally occurring lymphoid tumor cells. Among the 38 mAbs, 27 was determined the molecular weights of their recognized antigens in Western blot analyses. A mAb C419 had high reactivity with lymphoma cell lines but no reactivity with normal lymphocytes, indicating that it recognizes tumor-associated antigens of SBL.

Animals↗