Biomedical subjects

H Shibuta

Publications and source records attributed to H Shibuta.

At least 19 recordsLinked to original sources

PubMed1998

Magnetic resonance imaging of the shoulder in abduction.

To simulate the painful arm position in subacromial impingement syndrome, magnetic resonance imaging of the shoulder was done with the arms at the sides and at 80 degrees abduction in two groups of patients. Group A had 20 shoulders in 20 patients with subacromial impingement syndrome and Group B had 19 shoulders in 19 patients without impingement syndrome. When arm position was changed from adduction to abduction, an increase in signal intensity of the rotator cuff tendon was seen more often in Group A (45%) than in Group B (26%). Group A shoulders showed encroachment of the acromion or the acromioclavicular joint to the rotator cuff in 25% of the shoulders with the arms at the sides, and in 75% with the arms in abduction. Similar findings were observed in only 14% and 21% of Group B shoulders in the respective arm positions. Abduction of the arms seemed to cause encroachment of the overlying structure, especially of the acromioclavicular joint, to the rotator cuff in patients with subacromial impingement syndrome, even if there were no such findings in the images with the arms at the sides.

Acromioclavicular Joint↗

PubMed1995

Protein factors required for in vitro transcription of Sendai virus genome.

To elucidate the mechanism of transcription and replication of Sendai virus, we developed an efficient and faithful in vitro transcription system using purified virus particles. The in vitro RNA synthesis was almost entirely dependent on the addition of eukaryotic cell extracts, including those from various cultured mammalian cells, mammalian tissues, and even from plant cells. The RNA products were almost identical to authentic mRNA species synthesized in the infected cells, in their size distribution, the presence of 3'-poly(A) tail and the presence of methylated 5'-cap structure (m7GpppAm). Ribonuclease protection experiments after annealing the in vitro RNA with viral genomic RNA (vRNA) indicated that the virion-associated RNA-dependent RNA polymerase transcribes correct regions of the RNA genome in vitro. The active component(s) that is required for Sendai virus mRNA synthesis was partially purified from bovine brain and was separated into at least two complementary fractions, one of which could be replaced by highly purified cellular tubulin. When viral ribonucleoprotein complexes were used instead of virus particles in the in vitro transcription, only Sendai virus-infected cell extracts supported mRNA synthesis, and extracts from uninfected cells or cells infected with other viruses were found to be inert. These results suggest that, in addition to the general factors which are present ubiquitously in eukaryotic cells, a factor(s) specific to Sendai virus-infection is required for Sendai virus transcription.

Animals↗

PubMed1992

RNA packaging signal of human immunodeficiency virus type 1.

Cells infected with a recombinant vaccinia virus carrying the gag and pol regions of the human immunodeficiency virus type 1 genome (Vac-gag/pol) released human immunodeficiency virus (HIV)-like particles containing HIV-specific RNA. However, cells infected with another recombinant vaccinia, Vac-gag/pol-dP, derived through the deletion of an 85-base region (nucleotide positions 679-763) of the HIV genome between the primer binding site and the gag initiation codon of Vac-gag/pol, produced HIV-like particles devoid of the HIV-specific RNA. This 85-base deletion was suggested to cause the collapse of a stable stem-loop structure of 46 bases (751-796) around the gag initiation codon. To examine the role of the stem-loop structure in the packaging of RNAs, we constructed a vaccinia vector plasmid that carried this 46-base sequence followed by the Sendai virus nucleocapsid (NP) gene. When both Vac-gag/pol-dP and this plasmid were introduced into cells, HIV-like particles released from the cells contained the NP gene RNA. However, another vaccinia vector plasmid, which carried the 46-base sequence in the midst of the NP gene, could not supply RNA for incorporation into HIV-like particles. Computer analysis of this plasmid sequence suggested that the 46-base sequence cannot form the stem-loop structure. These findings suggest that the stem-loop structure formed by the 46-base sequence is crucial as a packaging signal.

Base Sequence↗

PubMed1992

The induction of cataracts by HIV-1 in transgenic mice.

OBJECTIVE: To elucidate the tissue specificity of the expression of HIV-1 genes in an animal and its pathological effects on these tissues. DESIGN AND METHODS: Transgenic mice carrying a defective HIV-1 genome were bred in order to overcome the host-range barrier of this virus. RESULTS: mRNA specific to the transgene was detected in the eyes and the spleen, and, in smaller quantities, in the thymus and the brain. Interestingly, many of the transgenic mice developed cataracts at 3-6 months of age. Swelling and vacuolation of the lens fiber cells were marked, but the epithelial cells of the lens were less affected. HIV antigens were detected in the lens fiber cells and the retina by immunological staining. Accumulation of large amounts of p24 Gag antigen was demonstrated in the affected lens by immunoblot analysis, while negligible Env or other viral proteins was detected. Although accumulation of the Gag protein was also detected in the skin and the brain, no apparent abnormality was observed in these tissues. CONCLUSIONS: Preferential expression of the HIV genes in the eyes, skin, brain and lymphoid tissues was demonstrated. The accumulation of the Gag protein is suggested to have detrimental effects on lens fiber cells, causing cataracts.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Syncytium formation of human and non-human cells by recombinant vaccinia viruses carrying the HIV env gene and human CD4 gene.

We examined the ability of the various human and non-human cell lines to form syncytia upon coinfection with recombinant vaccinia viruses each carrying the HIV env and the human CD4 gene. We found that three human cell lines and one of three monkey cell lines exhibited syncytium formation, but that one human cell line, two monkey cell lines and all the rabbit and mouse cell lines examined did not. This indicated that factors other than HIV env and CD4 were participating in syncytium formation and that distribution of these factors was restricted by species and by the cell type in a species.

Animals↗

PubMed1991

Immune response of mice infected with recombinant vaccinia viruses carrying the HIV gag gene.

We examined mouse immune response to 4 kinds of recombinant vaccinia viruses carrying the HIV gag gene, including vac-gag/pol, which produces HIV-like particles with processed gag proteins; vac-gag, which also produces HIV-like particles but with unprocessed gag protein; and vac-gag-pol-fuse and vac-es-gag/pol, neither of which produces such particles but releases reverse transcriptase and gag protein, respectively, from infected cells. Although infection of mice with recombinant vaccinia viruses induced production of the anti-p24 antibody in all mice, vac-gag/pol and vac-es-pol induced higher production than the other two recombinants. Increase in [3H]thymidine uptake by splenic lymphocytes following p24 antigen stimulation was most evident in mice infected with vac-gag/pol. Thus, the highest immune reaction, both humoral and cellular, was elicited by vac-gag/pol, indicating that among those tested, this recombinant vaccinia virus is the best candidate for a vaccine that induces anti-HIV gag immunity.

Animals↗

PubMed1990

Molecular analysis of structural protein genes of the Yamagata-1 strain of defective subacute sclerosing panencephalitis virus. II. Nucleotide sequence of a cDNA corresponding to the P plus M dicistronic mRNA.

The nucleotide sequence of a cloned cDNA corresponding to the P + M dicistronic mRNA of a subacute sclerosing panencephalitis (SSPE) virus was determined and compared with data of measles virus (MV). The dicistronic mRNA of the SSPE virus consisted of the 3' proximal 626 nucleotides of P mRNA, intercistronic trinucleotides, a full length of M mRNA, and 75 poly A nucleotides. The part encoding the P protein had a high homology to MV, except at the noncoding region. The terminating consensus sequence of the P gene and the intercistronic trinucleotides of the SSPE virus were CTAC(A)6 and CCT; in MV they are TTAT(A)6 and CTT, respectively. In the M gene, the starting consensus sequence was exactly the same as MV, but at the 5' proximal end, one third of this gene was different: The first ATG codon of the MV M gene signaling opening of the reading frame was changed to ACG in the SSPE virus and one long open reading frame started from the third ATG codon. The stop codon (TAG) of the MV M gene was also changed to CAG in the SSPE virus. Thus, the deduced SSPE-virus M protein lacked 50 amino acids at the amino terminal and had 15 extra amino acids at the carboxyl end when compared with the MV M protein.

Amino Acid Sequence↗

PubMed1990

Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV.

We constructed a recombinant vaccinia virus carrying the entire gag and pol genes of human immunodeficiency virus type 1 (HIV-1). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of 100-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pol proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pol gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production.

Animals↗

PubMed1990

Antigenic variation of human and bovine parainfluenza virus type 3 strains.

Three human and six bovine parainfluenza virus type 3 (PIV3) strains were examined by the use of 60 monoclonal antibodies (MAbs). Fifty-three MAbs to the human C243 strain were directed against six, four, nine and seven epitopes of the haemagglutinin-neuraminidase (HN), fusion (F), nucleocapsid (N) and matrix proteins, respectively. Seven MAbs to the bovine strain were directed against three epitopes of the HN protein and three epitopes of the F protein. Each strain was characterized in ELISA and immunofluorescence tests with all MAbs and in a haemagglutination inhibition assay with the anti-HN MAbs. There were marked differences between human and bovine viruses, primarily in the HN protein where five epitopes differed. One epitope of the F and one of the N protein also differed. Bovine PIV3 was found to be a homogeneous subtype and distinct from human PIV3.

Animals↗

PubMed1989

Rescue of Sendai virus from viral ribonucleoprotein-transfected cells by infection with recombinant vaccinia viruses carrying Sendai virus L and P/C genes.

The Sendai virus ribonucleoprotein (RNP) showed only very low plaque-forming titers upon transfection and the virus yields after one-step growth were quite limited. We tried to enhance the Sendai virus yield by supplying the viral L and P/C gene products through vaccinia vectors. A combination of the recombinant vaccinia viruses carrying the L gene (Vac-HL) and the P/C gene (Vac-HPC), both of which were driven by the promoter of the vaccinia virus 7.5K protein gene, enhanced the yield only a little whereas another combination of Vac-HLd7.5, the L gene insert of which was driven by the promoter of the vaccinia virus thymidine kinase gene in place of the 7.5K promoter, and Vac-HPC greatly enhanced the Sendai virus yield. This seemed to correlate with the fact that the Vac-HL interfered with Sendai virus growth markedly while the Vac-HLd7.5 did not. These results strongly suggest that the L and P/C gene products act in cooperation as the RNA polymerase, and overproduction of the L protein is inhibitory for Sendai virus growth. This system seems to be of value as a tool for analyzing the functions of L and P/C genes of Sendai virus.

Animals↗

PubMed1989

Syncytium formation by recombinant vaccinia viruses carrying bovine parainfluenza 3 virus envelope protein genes.

The highly syncytium-inducing M strain and the weakly syncytium-inducing SC strain of bovine parainfluenza 3 virus differ by a single amino acid substitution in each of the hemagglutinin-neuraminidase (HN) and membrane (M) proteins, while their fusion (F) proteins are identical (T. Shioda, S. Wakao, S. Suzu, and H. Shibuta, Virology 162:388-396, 1988). We constructed recombinant vaccinia viruses which express separately the M virus HN (Vac-MHN), SC virus HN (Vac-SCHN), M virus M (Vac-MM), SC virus M (Vac-SCM), and common F (Vac-F) proteins. CV-1 cells were infected with the recombinants, singly or in combination, and implanted onto indicator MDBK cells for syncytium formation. Combinations of Vac-MHN plus Vac-F and Vac-SCHN plus Vac-F induced extensive and weak syncytium formation, respectively. Vac-F alone did not induce syncytium formation, and both Vac-MM and Vac-SCM had no effect on syncytium formation. These findings indicated that the syncytium formation by bovine parainfluenza 3 virus requires both the F and HN proteins and that the extensive syncytium formation by the M virus is due to the M virus HN protein. MSC, another weakly syncytium-inducing virus variant, newly isolated from the M virus, was identical to the M virus in the primary structure of the HN and M proteins but differed from the M virus by a single amino acid residue in the F protein. The combination of the recombinant vaccinia virus expressing the MSC virus F protein and Vac-MHN resulted in weak syncytium formation.

Amino Acids↗

PubMed1988

Differences in bovine parainfluenza 3 virus variants studied by sequencing of the genes of viral envelope proteins.

By determining gene nucleotide sequences we compared the primary structures of the membrane (M), fusion (F), and hemagglutinin-neuraminidase (HN) proteins of bovine parainfluenza 3 virus strains, M, SC, and MR which are substrains derived from a wild strain YN. The M and SC viruses are indistinguishable in having very weak hemagglutination (HA) and neuraminidase (NA) activities, but M virus' syncytium-inducing (SI) activity is considerably higher than that of the SC virus. However, the results showed that the amino acid sequence of the F protein was identical in M and SC viruses, demonstrating that M virus' high SI activity was not due to alteration of its F protein. Two differences in M and SC viruses' other proteins then seemed to be important, although their significance in the SI activity is not clear at present; the first being the 70th amino acid residue of the M protein, which was Asp in the M virus and Gly in the SC virus, and the other being the 539th residue of the HN protein, which was Tyr in the M virus and His in the SC virus. The nucleocapsid proteins of both M and SC viruses were identical. The MR virus, which is a variant derived from the M virus and has high HA and NA activities but very weak SI activity, was different from the M virus at only one site throughout the M, F, and HN proteins; the 193rd amino acid residue of the HN protein was Leu in the MR virus and Phe in the M virus. This result strongly suggested that the substitution of Leu with Phe at this particular site was closely linked to the drastic reduction in both HA and NA activities.

Amino Acid Sequence↗

PubMed1987

Nucleotide sequence of the bovine parainfluenza 3 virus genome: its 3' end and the genes of NP, P, C and M proteins.

We present the nucleotide sequence of bovine parainfluenza 3 virus (BPIV3) genome from its 3' end to the opening region of the F gene, through the NP, P plus C, and M genes. Comparison of the sequence with those reported for other paramyxoviruses indicated that BPIV3 was most similar to human parainfluenza 3 virus (HPIV3), and also very similar to Sendai virus in the structural make-up of its genome and the amino acid sequences of its gene products, suggesting that these three viruses constitute a paramyxovirus subgroup from which Newcastle disease and measles viruses are separable. In BPIV3 and Sendai virus, the NP and M proteins, the main structural elements, were more highly conserved than the functionally important P and C proteins. This tendency was also observed even in BPIV3 and HPIV3. Virus-specific amino acid sequences of the NP and M proteins were found at the carboxyl and amino terminal regions, respectively. BPIV3 M mRNA was found to have aberrations in its poly A attachment site.

Amino Acid Sequence↗

PubMed1987

Nucleotide sequence of the bovine parainfluenza 3 virus genome: the genes of the F and HN glycoproteins.

By analysing complementary DNA clones constructed from genomic RNA of bovine parainfluenza 3 virus (BPIV3), we determined the nucleotide sequence of the region containing the entire F and HN genes. Their deduced amino acid sequences showed about 80% homologies with those of human parainfluenza 3 virus (HPIV3), about 45% with those of Sendai virus, and about 20% with those of SV5 and Newcastle disease virus (NDV), indicating, together with the results described in the preceding paper on the NP, P, C and M proteins of BPIV3, that BPIV3, HPIV3 and Sendai virus constitute a paramyxovirus subgroup, and that BPIV3 and HPIV3 are very closely related. The F and HN proteins of all these viruses, including SV5 and NDV, however, were shown to have protein-specific structures as well as short but well-conserved amino acid sequences, suggesting that these structures and sequences are related to the activities of these glycoproteins.

Amino Acid Sequence↗

PubMed1987

Single amino acid substitution of Sendai virus at the cleavage site of the fusion protein confers trypsin resistance.

Amino acid sequences of fusion (F) proteins of two trypsin-resistant mutants of Sendai virus, TR-2 and TR-5, were deduced from nucleotide analysis of cDNA encoding the F gene and were compared with that of the trypsin-sensitive wild-type Sendai virus. In both mutants, amino acid substitutions were found at residues 116 (Arg----Ile), the cleavage site of the F protein, and 109 (Asn----Asp). Two trypsin-sensitive revertants, TSrev-52 and TSrev-58, derived from TR-5 were both activated by trypsin similarly to the wild-type virus and had a single amino acid reversion from Ile to Arg at residue 116, leaving Asp as before at residue 109. These results indicate that the trypsin sensitivity of Sendai virus can be changed by a single amino acid substitution at the cleavage site of the F protein and a mutation from Arg to Ile is responsible for the acquisition of resistance to trypsin.

Amino Acid Sequence↗

PubMed1987

Interferon induction by transfection of Sendai virus C gene cDNA.

To elucidate the mechanism of interferon (IFN) induction on virus infection, we constructed two types of plasmids by inserting a part of the cDNA of the Sendai virus into a simian virus 40-derived expression vector (pSV2-0). One, pSV2-PC, contained the P + C gene, which codes for the P and C proteins in overlapping reading frames, and the other, pSV2-C, contained only the C gene. After transfecting the plasmids into mammalian cells, we determined the IFN activity in the culture medium. We found that the level obtained with pSV2-PC was significantly positive but very low, whereas that obtained with pSV2-C was as high as or even higher than that observed in the culture medium after Sendai virus infection. By cleaving pSV2-C between the simian virus 40 promotor and the C gene or by inserting a stop codon within the C gene [pSV2-C(stop)], induction of IFN was greatly diminished. In Northern blot analyses of the transcripts obtained from the cells transfected with the plasmids with cDNA to the P + C gene as a probe, the transcript having the expected size was detected with both pSV2-C and pSV2-C(stop), whereas none was detected with cleaved pSV2-C or pSV2-0. The results indicate that both transcription and translation of the C gene seem to be required for IFN induction after Sendai virus infection.

Animals↗

PubMed1986

Determination of the complete nucleotide sequence of the Sendai virus genome RNA and the predicted amino acid sequences of the F, HN and L proteins.

We previously determined the 3' proximal 5,824 nucleotides of the Sendai virus genome RNA (Nucleic Acids Res. 11, 7317-7330, 1983; Nucleic Acids Res. 12, 7965-7973, 1984), and present here the sequence of the remaining 5' proximal 9,559 nucleotides. Thus, this is the first paramyxovirus to have its genome organization elucidated. The set of complementary DNA clones used was prepared by the method of Okayama and Berg from polyadenylylated viral genome RNA. We sequenced the region containing the 5' proximal half of the F gene, and the subsequent HN and L genes, and predicted the complete amino acid sequence of the products of these genes. Sequence analyses confirmed that all the genes are flanked by consensus sequences and suggest that the viral mRNAs are capable of forming stem-and-loop structures. Comparison of the F and HN glycoproteins of Sendai virus with those of simian virus 5 strongly suggests that the cysteine residues are highly important for maintenance of the molecular structures of these glycoproteins.

Amino Acid Sequence↗

PubMed1986

Differences in bovine parainfluenza 3 virus variants studied by monoclonal antibodies against viral glycoproteins.

We previously showed that of three bovine parainfluenza 3 virus strains the M strain, which is neurovirulent for young mice, has an extensive syncytium-inducing activity, whereas avirulent SC and 910N strains are weak in this activity. It was also demonstrated that both M and SC strains have very low hemagglutination and neuraminidase activities, while the 910N strain shows these activities to high levels. In the present study, monoclonal antibodies (Mabs) were raised against the glycoproteins of the 910N strain, and utilized to further characterize these three viral strains. Five Mabs against the hemagglutinin-neuraminidase protein, which were classified into four different epitope-recognizing groups, neutralized the M strain much more effectively than the 910N and SC strains, while the Mabs showed lower hemagglutination inhibition (HI) titers against the M and SC strains than the 910N strain. Three Mabs against the fusion protein neutralized the M strain but not the 910N and SC strains, while they showed no HI activity against any of these strains. These findings suggested that the M strain is considerably different from other strains in the structure of the viral envelope proteins.

Animals↗