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Biomedical subjects

T Shioda

Publications and source records attributed to T Shioda.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1992

Thiamin triphosphate does not affect contraction of skinned fibers.

Effects of thiamin triphosphate (TTP) on contraction of chemically skinned fibers prepared from the extensor digitorum longus muscle of guinea pigs were investigated. The addition of TTP at 40 microM concentration affected neither Ca2+ uptake nor Ca(2+)-induced Ca2+ release by sarcoplasmic reticulum. Contraction of myofibril was not affected by TTP either.

Animals↗
PubMed1991

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides.

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.

Adenine Nucleotides↗
PubMed1991

Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase.

To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined. E. coli harboring the vector plasmid was used as a control. Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG). TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG. TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG. Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains. These results indicate that chicken AK1 expressed in E. coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.

Adenosine Diphosphate↗
PubMed1991

Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene.

Strains of human immunodeficiency virus type 1 (HIV-1) display a high degree of biological heterogeneity which may be linked to certain clinical manifestation of AIDS. They vary in their ability to infect different cell types, to replicate rapidly and to high titre in culture, to down-modulate the CD4 receptor, and to cause cytopathic changes in infected cells. Some of these in vitro properties correlate with pathogenicity of the virus in vivo. To map the viral determinants of the cellular host range of HIV-1, recombinant viruses were generated between biologically active molecular clones of HIV-1 isolates showing differences in infection of primary peripheral blood macrophages and established T-cell lines. We report here that a specific region of the envelope gp120 gene representing 159 amino-acid residues of glycoprotein gp120 seems to determine macrophage tropism, whereas an overlapping region representing 321 amino-acid residues determines T cell-line tropism. These studies provide a basis for relating functional domains of the HIV-1 env gene to pathogenic potential.

Amino Acid Sequence↗
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↗
PubMed1991

Host range, replicative, and cytopathic properties of human immunodeficiency virus type 1 are determined by very few amino acid changes in tat and gp120.

Human immunodeficiency virus type 1 (HIV-1) isolates display differences in a variety of in vitro biological properties, including the ability to infect different cell types, the kinetics of replication, and cytopathicity in the infected cells. Studies with isolates obtained from the same individual over time have shown that these in vitro properties of the viral isolates correlate with pathogenicity in the host. The later isolates, recovered when disease has developed, display a wider cellular host range, replicate rapidly and to high titers in the infected cells, and induce syncytia in these cells. In the present studies, the genomic determinants of these biological properties were defined with recombinant viruses generated between two HIV-1 isolates recovered sequentially from the same individual. The results show that the rate of HIV-1 replication in the HUT 78 T-cell line is controlled by the first coding exon of tat. Infection of T-cell and monocytic cell lines is determined by two specific regions in the envelope gp120, one of which also confers the ability of an isolate to induce syncytia. Amino acid sequence comparison of the regions identified revealed minor differences between the two viral isolates: 2 amino acids in the tat gene product and 10 and 12 amino acids in the two regions of envelope gp120. These data suggest that small changes in the tat and env proteins can have dramatic effects on the pathogenic potential of HIV-1.

Acquired Immunodeficiency Syndrome↗
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

Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle.

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
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↗
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

Membrane modification during secretory granule formation in rat somatotrophs.

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.

Animals↗