Biomedical subjects
Publications and source records attributed to M Shinagawa.
Influences of 38 monoclonal antibodies (mAbs) on the growth of bovine T lymphoma cells (BTL-27) were examined. One of them, S37, inhibited the cell growth strikingly when it was added to the cultures. S37-treated cells showed DNA fragmentation containing approximately 200-base pair multiple patterns and chromatin condensation. This evidence suggested that mAb S37 induced apoptosis in BTL-27 cells.
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We have cloned genome fragments of canine parvovirus strain Y1 from replicative-form DNA and double-stranded DNA synthesized from virion DNA in vitro, and constructed a recombinant plasmid containing a full-length Y1 genome (pCPVY 1). When this recombinant plasmid was transfected into cell cultures, an infectious virus could be recovered. To characterize this pCPVY 1-derived virus, its biological properties were compared with those of the parental strain. No difference was observed between them in antigen expression, viral DNA replication, hemagglutination ability, and virus multiplication, indicating that the virus derived from the infectious plasmid inherited the biological properties of the authentic Y1 strain. Therefore, this recombinant plasmid appears to be useful for reverse genetics of canine parvovirus.
A five-month-pregnant Suffolk sheep histologically diagnosed as spontaneous scrapie was studied. Western blot analysis was performed with rabbit serum against the sheep scrapie-associated fibrils (SAF). In the proteinase K (pk)-treated parental brain and spleen samples, three major bands (15 K, 18 K, and 23 K) were detected. These major bands were not detected from the placenta. Infectious agents were isolated in mice from the brain samples but not from the placental homogenates. In another case of a three-month-pregnant Corriedale sheep without any clinical sign of, but histologically diagnosed as scrapie, was also studied in a similar approach. In the parental brain samples, three major bands (15 K, 18 K and 23 K) were detected. SAF protein was not detected in the parental spleen and placenta. No bands reactive with the antiserum were detected in any other samples from the fetal brain and spleen in both cases. However, infectious agents were isolated in mice from both brain and placental homogenates. Since the placenta is an important site of natural infection, it is worthwhile to study these tissues for the epidemiological study of scrapie infection.
Two familial scrapie cases successively occurred in 29-month-old and 30-month-old Corriedale dams and in three of their offspring as early as 10-19 months of age in a small flock. They showed ataxy but no pruritus. Histopathological and immunochemical examinations of those sheep revealed vacuolation of nerve cells in brain tissue and presence of PrPsc, a component of scrapie-associated fibrils, in brain, spleen and lymph node tissues. We discuss about shortened incubation periods and lacking pruritus in the present scrapie cases.
Expression of cellular oncogenes in 3 lymphoid cell lines, BTL-PC3 (BoCD2-, BoCD4-, BoCD8-, BoWC1+), BLS1 (BoCD2+, BoCD4-, BoCD8-, BoWC1+) and BLT2 (BoCD2-, BoCD4-, BoCD8-, BoWC1-), which have been established from calf, skin, and thymic types of lymphosarcomas, respectively, were analyzed by DNA-RNA (northern blot) hybridization. To determine specific expression of oncogenes involved in malignant transformation of the lymphoid cells, cellular RNA was isolated from bovine tumor cell lines, BTL-PC3, BLS1, and BLT2, and from Madin Darby bovine kidney cells used as a control for bovine cell lines. The RNA was hybridized against 5 viral oncogene probes (v-jun, v-myc, v-erbB, v-erbA and v-fes), 6 human cellular oncogene probes (N-ras, c-Blym-1 c-erbB-2, c-fos, c-myb and c-abl), human p53 tumor suppressor gene, and bovine LDH-A gene probes. Line BTL-PC3 expressed 2.4-kilobase (kb) c-myc and 4.0- and 3.6-kb c-myb transcripts, and line BLT2 expressed a 3.8-kb c-myb transcript, but line BLS1 expressed no message for the oncogenes tested. Specific transcripts of p53 were found in BTL-PC3 and BLT2 lines, but not in BLS1. Madin Darby bovine kidney cell line expressed multiple cellular oncogenes, c-jun, c-myc, and c-fos, and p53 genes. Southern blot hybridization did not reveal abnormal DNA rearrangements associated with the expressed oncogenes (c-myc and c-myb) in the 3 bovine tumor lines. (ABSTRACT TRUNCATED AT 250 WORDS)
Infectious amyloid filaments designated as prion rods or scrapie associated fibrils (SAF) present in brain tissues affected by transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS) and kuru of humans, and scrapie of sheep. A hydrophobic glycoprotein, PrPSc is a major component of SAF, and is known to be associated with the infectivity of these diseases. Both PrPSc and the normal isoform of this glycoprotein, PrPC are encoded by a single host gene, PrP gene, and the conversion of PrPC to PrPSc is a posttranslational event. Several mutations on the PrP gene are associated with variations of the phenotype and the occurrence in familial CJD and GSS.
The nucleotide sequence of the citC coding for the citrate carrier in several Salmonella serovars has been determined, and the amino acid sequence of the carrier protein was deduced. The predicted citrate carrier from Salmonella pullorum and Salmonella enteritidis consists of 446 amino acids with a molecular weight of 47,621, whereas that from Salmonella dublin is the same 446 amino acids with the slightly different molecular weight of 47,591, because 1 amino acid residue was substituted. The predicted proteins are highly hydrophobic (69% nonpolar amino acids). The hydropathy profile suggests that the proteins are composed of 11-12 hydrophobic membrane-spanning segments with two hydrophilic cores in the middle of the protein sequence. No homology in the nucleotide and amino acid sequences was found in the molecular structures of citA, citP, and tctI genes. The citC-coding citrate transport activity is Na(+)-dependent and specific for citrate only. The transcript from the citC gene was not detected in the total RNA from several Salmonella serovars except S. dublin in Northern blot analysis, although the promoter of the citC genes appeared to be functional in Escherichia coli and Salmonella typhimurium strains using the lacZ fusion assay. These results suggested that the citC gene-coding citrate carrier is probably a TctIII system such as that identified previously in S. typhimurium.
Nine bovine T-cell receptor (Tcr) gamma chain (Tcrg) and three Tcr delta chain (Tcrd) cDNA clones were isolated from the cDNA libraries constructed from peripheral blood lymphocytes and thymocytes. Of nine Tcrg cDNA clones, only four were rearranged and contained specific V, J, and C gene segments, but the remaining five contained specific J and C or only C gene segments without the V gene segment. Three kinds of Tcrg-C, which were highly related at the nucleotide and amino acid levels, were found and designated as Tcrg-C1, Tcrg-C2, and Tcrg-C3. Compared with human and mouse Tcrg-C, bovine Tcrg-C sequences are much longer, with about 27-55 amino acids corresponding to the hinge and connector regions, where the characteristic repetitive 5-amino acid motif (TTEPP or TTKPP) exists in sheep Tcrg-C as previously reported. From three Tcrd cDNA clones, two Tcrd-V and three Tcrd-J segments were isolated. The nucleotide and deduced amino acid sequences of bovine Tcrd-C, especially the transmembrane and cytoplasmic domains, are well conserved among species. As in bovine Tcrg-C, diversity of amino acid residues in the Tcrd-C region is concentrated in the hinge regions. Southern blot analysis showed that there are at least three Tcrg-C genes and one Tcrd-C gene in the bovine genome. The analysis also revealed the presence of Tcrg-C- and Tcrd-C-associated restriction fragment length polymorphisms among bovine breeds.
We have investigated restriction fragment length polymorphism (RFLP) on the PrP gene and the frequencies of RFLP patterns in 35 healthy Suffolk sheep randomly collected. According to the combinations of PrP encoding DNA fragments generated by restriction enzymes Eco RI and Hind III, the RFLP patterns were classified into six types and designated as types I to VI. The frequencies of these types were as follows: I, 8.6%; II, 11.4%; III, 17.6%; IV, 11.4%; V, 28.6%; and VI, 22.9%. In 10 sheep diagnosed as having natural scrapie, RFLP types, I, III, IV, and V were determined. To examine the correlation between the RFLP type and the occurrence of scrapie, the frequencies of RFLP types in sheep infected with natural scrapie were compared with those in healthy sheep. It was found that the frequency of type I in the sheep with natural scrapie was 70%, about eight times higher than that in randomly collected healthy sheep. In the 13 experimentally infected sheep that had been used for other purposes, however, no relationship between the RFLP type and onset of scrapie was found.
A cDNA clone encoding bovine scrapie-associated fibril protein, PrP, from a bovine brain cDNA library and six amplified genomic DNA clones of bovine PrP were characterized. These clones possessed specific characteristics observed in other animal PrP genes. However, the bovine PrP was divided into two types by the number of repeats. One possessed four octapeptide repetitive sequences, like other animal PrP genes, and consisted of 256 amino acids; the other had five such repetitive sequences and 264 amino acids. The amino acid sequence of the former bovine PrP agreed with that of sheep PrP up to the 165th amino acid from the N-terminus. Bovine PrP cDNA introduced into mouse L-929 cells were stably expressed. The expression level of recombinant bovine PrP in the cells judged by immunofluorescence was higher than that of authentic mouse PrP. The recombinant PrP comigrated with authentic bovine PrP in SDS-polyacrylamide gel electrophoresis, suggesting that the recombinant product was fully glycosylated in L-929 cells. Distinct bundles of the intermediate filaments were frequently seen at the perinuclear region of the cells.
Feline panleukopenia virus (FPLV), mink enteritis virus (MEV), and canine parvovirus (CPV) are classified as a host-range variants. They show different host-range specificity in vivo and host-cell specificity in vitro. For instance, FPLV and MEV cannot grow or can grow only inefficiently in canine cell lines such as MDCK and the canine fibroma cell line A72. Here we have studied the mechanism(s) by which the different cell tropism is mediated in vitro. When FPLV or MEV was inoculated to A72 cells, viral DNA replicated slightly, few viral-antigen-positive cells were detected, and the culture fluid contained the threshold level of infectivity. On the other hand, when an infectious molecular clone of MEV (pMEV) was introduced into A72 cells, viral DNA replicated efficiently, and the culture fluid of pMEV-transfected cells contained much higher infectivities than that of MEV-infected cells. In spite of the restrictive growth in A72 cells, MEV could bind to A72 cells as efficiently as CPV. No detectable viral RNA was produced in MEV-infected A72 cells. In contrast, efficient viral transcription occurred in pMEV-transfected A72 cells. These results suggest that the restrictive infections of MEV and FPLV in A72 cells are not mediated by the attachment of the virus to the cells or by the events occurring after the viral transcription. It appears to be caused by the stage(s) in the virus replication cycle, which exists between a postadsorptional step required for virus penetration and the initiation of viral transcription.
Subfractions, a nucleic acid fraction and a PrP fraction consisting of PrP17-25, a core fragment of PrPsc, were prepared from the scrapie-associated fibril-enriched fraction from scapie-affected mouse brains. The nucleic acid fraction consisted mainly of variously fragmented DNA and no scrapie-specific nucleic acid was detected in the fraction by SDS polyacrylamide gel electrophoresis. To examine the biological activity, the nucleic acid fraction was either first introduced into mouse L-929 cells before or after nuclease treatments, then transfected cell lysates prepared 2 weeks later were inoculated into mice, or directly inoculated into mice with or without the PrP fraction. The PrP fraction alone was also inoculated into mice. Mice inoculated with the transfected cell lysates or with the nucleic acid fraction alone showed no scrapie signs during their lifespan or the observation period. While 60% of the mice inoculated with the PrP fraction alone and 67% of those inoculated with the fraction together with the nucleic acid fraction showed clinical signs of scrapie. A nucleic acid molecule bound covalently to PrP17-25 was not detected. The results obtained by the present procedures so far suggest scrapie infectivity to be associated with PrPsc, which does not contain any detectable scrapie-genome molecule as either free or covalently bound nucleic acid.
The usefulness of detecting the scrapie-associated fibrillar protein (PrP) in the lymphoreticular organs of sheep as a diagnostic tool was investigated. The PrP was detected by means of a rabbit-anti-sheep PrP polyclonal antibody by Western blot analysis. PrP was detected in samples from the central nervous system (CNS) of five of six sheep showing clinical signs of natural scrapie infection, in spleen samples from four of the six sheep and in lymph node samples taken from three of the sheep. PrP was detected in the spleen and lymph node samples, but not in the CNS samples from one of the six sheep that was clinically and histopathologically abnormal. This animal appeared to be in the early clinical stage of the disease. A total of 47 clinically normal sheep were examined for the presence of PrP. It was detected in spleen samples from three of the 47 sheep and in lymph node samples from three of the 39 sheep tested. Similarly, PrP was detected in a sample of lymph node obtained surgically from one of three experimentally infected sheep 14 months after inoculation. The PrP-positive sheep and one of the remaining PrP-negative sheep showed clinical signs of scrapie six and five months later respectively. One sheep euthanased 18 months after experimental infection was positive for PrP in the CNS, spleen and lymph node, but five other sheep which were killed or died two, eight, 16, 18 and 21 months after infection were negative or doubtful for the detection of PrP.
The membrane fraction from scrapie infected mouse brains was dissolved in saturated urea, centrifuged on a 10 to 50% glycerol gradient at 35,000 rpm for 24 h, and fractionated from the bottom of the tube into 11 fractions. PrP was detected throughout the gradient. However, the relative PrP concentrations of fractions 4 and 8 were the highest. The relative PrP concentration versus protein concentration of fractions 1 to 4 was higher than that of the other fractions. Scrapie infectivity also was detected in all fractions. Fractions 2, 3, 4, 7, and 8 produced the shortest incubation periods. Positively stained filamentous aggregates with sizes varying from about 40 x 60 nm to more than 4 microns were observed in fractions 2 and 4 by negative staining. These resembled amyloid filaments. Congo red-stained aggregates showed birefringence under polarized light. Aggregation of the filamentous aggregates was observed by incubation with anti-mouse SAF serum. Fine fibrils 10-18 nm in width were partially dissociated from the aggregates by brief exposure to the detergent Sarkosyl. These facts suggest that SAF are not products of self-assembly from subunit structures liberated from membranes by exposure to detergent, but exist as aggregates of amyloid-like filaments from which SAF are dissociated by detergent extraction.
A cryptic citrate transport gene (citA) from Salmonella typhimurium chromosome was cloned and its nucleotide sequence was determined. The cloned plasmid conferred citrate-utilizing ability on wild-type Escherichia coli, which cannot grow on citrate as the sole source of carbon. The resultant E. coli transformant was able to transport citrate. A 1,302-base-pair open reading frame with a preceding ribosomal binding site was found in the cloned DNA fragment. The 434-amino-acid protein that could be translated from this open reading frame is highly hydrophobic (69% nonpolar amino acid residues), consistent with the fact that the transport protein is an intrinsic membrane protein. The molecular weight of this protein was calculated to be 47,188. The gene sequence determined is highly homologous to those of Cit+ plasmid-mediated citrate transport gene, citA, from E. coli, the chromosomal citA gene from Citrobacter amalonaticus and the chromosomal cit+ gene from Klebsiella pneumoniae. The hydropathy profile of the deduced amino acid sequence suggests that this carrier has 12 hydrophobic segments, which may span the membrane lipid bilayer.
We have cloned the replicative form (RF-) DNA of mink enteritis virus (MEV), constructed an infectious recombinant plasmid containing MEV DNA and determined the nucleotide sequence of the cloned MEV DNA. RF-DNAs were detected and infectious virus was generated when the recombinant plasmid containing the entire MEV genome was introduced into feline kidney cell cultures. The MEV genome was 5094 nucleotides (nt) in length; the 3' end of the virion strand contained a 205 nt palindromic sequence and the 5' end a 62 nt palindromic sequence that could assume Y- and U-shaped configurations, respectively. The 5' end of the virion strand had a direct repeat of 61 nt at the carboxyl terminus of the capsid protein gene. The organization of the MEV genome is similar to those of canine parvovirus (CPV) and feline panleukopenia virus (FPLV); there are two large open reading frames (ORFs), one in the 3' half and the other in the 5' half of the genome, with coding capacities of 668 and 722 amino acid residues, respectively. Both are in the same reading frame and no significant ORFs are apparent in the virion strand (negative-sense strand). Possible functional promoter motifs are located at map unit (m.u.) 4.5 and m.u. 40, and a possible functional poly(A) signal is located at m.u. 96. The nucleotide and amino acid sequence homology with CPV and FPLV is greater than 98%, consistent with the hypothesis that MEV and CPV are host-range variants of FPLV.
A total of 122 adenovirus type 4 (Ad4) strains isolated from patients with epidemic keratoconjunctivitis at an eye clinic in Sapporo, Japan, from 1985 to 1989 were identified as Ad4a. By the use of HinfI and TaqI restriction endonucleases, these Ad4a strains were classified into 12 subgenome types. During this observation period, two epidemics of Ad4 infection relating to epidemic keratoconjunctivitis occurred. During the Ad4a epidemic in 1985, three subgenome types became predominant successively. However, except for one strain, the prevalent strains isolated during the epidemic of Ad4a in 1988 all belonged to a single subgenome type. In the years when an Ad4a epidemic was not observed, such Ad4a strains were new or previous subgenome types. The prevalent subgenome types tended to change within several years.