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Biomedical subjects

A Nomoto

Publications and source records attributed to A Nomoto.

At least 73 records · Page 4Linked to original sources
PubMed1993

Development of candidates for new type 2 and type 3 oral poliovirus vaccines.

Monkey neurovirulence tests on in vitro recombinant viruses between the virulent Mahoney and the attenuated Sabin 1 strains of type 1 poliovirus revealed that a strong neurovirulence determinant(s) resided in the 5' non-coding sequence of the genome and that the surface structure of the virion particle had a little correlation with the neurovirulence or attenuation phenotype. The results suggested that new and safer vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively, because the Sabin 1 strain is the safest vaccine among the Sabin strains. We have constructed recombinant viruses, PV1/2 (SS)BB and PV1/3(SS)BN, as vaccine candidates for type 2 and type 3 oral poliovirus vaccines respectively. These recombinant viruses were fully viable and showed antigenicities and immunogenicities identical to those of type 2 and type 3 polioviruses respectively. The monkey neurovirulence tests performed on these recombinant viruses suggest that the recombinant viruses are possible candidates for new type 2 and type 3 poliovirus vaccine strains.

Animals↗
PubMed1993

Pharmacological profile of FR139317, a novel, potent endothelin ETA receptor antagonist.

The effects of FR139317 on the cardiovascular system were investigated in cultured cells, isolated organs and whole animals. FR139317 inhibited the specific binding of [125]endothelin(ET)-1 to porcine aortic microsomes in a concentration-dependent, monophasic fashion with an IC50 of 0.53 nM. In contrast, FR139317 showed low affinity for [125I]ET-1 specific binding sites in porcine kidney (IC50, 4.7 microM). In isolated rabbit aorta, FR139317 shifted the ET-1-induced concentration-contractile response curve to the right with a pA2 value of 7.2 and lacked agonist activity. A single (i.v.) bolus dose of FR139317 completely inhibited the pressor response to ET-1 in vivo, but had no effect on the initial depressor response in conscious normotensive rats. These data indicate that FR139317 is a potent, highly specific ETA receptor antagonist. In addition, FR139317 also inhibited ET-1 induced [3H]thymidine incorporation in cultured vascular smooth muscle cells from rat aorta (IC50, 4.1 nM), suggesting that ET-1-induced mitogenesis is mediated only by the ETA receptor. FR139317 could become a useful tool for investigating the physiological and pharmacological actions of ET.

Animals↗
PubMed1992

Crystallization and preliminary X-ray diffraction studies of coxsackievirus B1.

Preparations of coxsackievirus B1 (CVB1) derived from an infectious cDNA clone have been crystallized in multiple crystal forms. Using high intensity synchrotron radiation, an orthorhombic form of the crystals was shown to diffract X-rays to at least 2.9 A resolution. The unit cell has a primitive lattice with dimensions a = 323 A, b = 450 A, and c = 522 A. A crystallographic asymmetric unit of these CVB1 crystals probably contains an entire virus particle, implying the presence of 60-fold non-crystallographic redundancy. This CVB1 crystal form appears to be suitable for high-resolution structure determination by X-ray crystallography.

Crystallography↗
PubMed1992

Adenovirus E1A proteins stimulate inositol phospholipid metabolism in PC12 cells.

To study the influence of nuclear oncogenes on inositol phospholipid metabolism, we examined the various parameters of inositol phospholipid metabolism in PC12 cells expressing adenovirus type 12 or adenovirus type 5 E1A. Although the inositol 1,4,5-trisphosphate content was increased only slightly, the diacylglycerol content was 2.4-fold higher in E1A-expressing PC12 cells. Furthermore, we found that the activity of phospholipase C, one of the key enzymes in inositol phospholipid metabolism, was increased at least five- to eightfold. Diacylglycerol kinase activity in the membrane fraction was 10 to 15% of that in parental PC12 cells. Overall protein kinase C activities in E1A-expressing PC12 cells were decreased, but the activity of membrane-bound protein kinase C was significantly increased. These observations clearly indicate that inositol phospholipid metabolism is stimulated in cells producing E1A and suggest that nuclear oncogene E1A has the ability to stimulate inositol phospholipid metabolism.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

A second gene for the African green monkey poliovirus receptor that has no putative N-glycosylation site in the functional N-terminal immunoglobulin-like domain.

Using cDNA of the human poliovirus receptor (PVR) as a probe, two types of cDNA clones of the monkey homologs were isolated from a cDNA library prepared from an African green monkey kidney cell line. Either type of cDNA clone rendered mouse L cells permissive for poliovirus infection. Homologies of the amino acid sequences deduced from these cDNA sequences with that of human PVR were 90.2 and 86.4%, respectively. These two monkey PVRs were found to be encoded in two different loci of the genome. Evolutionary analysis suggested that duplication of the PVR gene in the monkey genome had occurred after the species differentiation between humans and monkeys. The NH2-terminal immunoglobulin-like domain, domain 1, of the second monkey PVR, which lacks a putative N-glycosylation site, mediated poliovirus infection. In addition, a human PVR mutant without N-glycosylation sites in domain 1 also promoted viral infection. These results suggest that domain 1 of the monkey receptor also harbors the binding site for poliovirus and that sugar moieties possibly attached to this domain of human PVR are dispensable for the virus-receptor interaction.

Amino Acid Sequence↗
PubMed1992

Internal ribosome entry site within hepatitis C virus RNA.

The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.

Base Sequence↗
PubMed1991

Functional domains of the poliovirus receptor.

A number of mutant cDNAs of the human poliovirus receptor were constructed to identify essential regions of the molecule as the receptor. All mutant cDNAs carrying the sequence coding for the entire N-terminal immunoglobulin-like domain (domain I) confer permissiveness for poliovirus to mouse L cells, but a mutant cDNA lacking the sequence for domain I does not. The transformants permissive for poliovirus were able to bind the virus and were also recognized by monoclonal antibody D171, which competes with poliovirus for the cellular receptor. These results strongly suggest that the poliovirus binding site resides in domain I of the receptor. Mutant cDNAs for the sequence encoding the intracellular peptide were also constructed and expressed in mouse L cells. Susceptibility of these cells to poliovirus revealed that the entire putative cytoplasmic domain is not essential for virus infection. Thus, the cytoplasmic domain of the molecule appears not to play a role in the penetration of poliovirus.

Animals↗
PubMed1991

Transgenic mice susceptible to poliovirus.

Poliovirus-sensitive transgenic mice were produced by introducing the human gene encoding cellular receptors for poliovirus into the mouse genome. Expression of the receptor mRNAs in tissues of the transgenic mice was analyzed by using RNA blot hybridization and the polymerase chain reaction. The human gene is expressed in many tissues of the transgenic mice just as in tissues of humans. The transgenic mice are susceptible to all three poliovirus serotypes, and the mice inoculated with poliovirus show clinical symptoms similar to those observed in humans and monkeys. Rabbit antipoliovirus serum detects the antigens mainly in motor neurons in the anterior horn of the spinal cord and in nerve cells in the medulla oblongata and pons of the paralyzed transgenic mice. Therefore, cell types sensitive to poliovirus in the central nervous system of the transgenic mice appear to be identical to those of humans and monkeys. Furthermore, many more doses of oral poliovirus vaccine strains than of the virulent strains are required to cause paralysis in the transgenic mice. This may reflect the observation that the virulent strain multiplies more efficiently in the central nervous system than the attenuated strain. Thus, the transgenic mice may become an excellent new animal model to study molecular mechanisms of pathogenesis of poliovirus and to assess oral poliovirus vaccines.

Animals↗
PubMed1991

A second group of hepatitis C viruses.

cDNA clone 11-7 was isolated by immunoscreening a cDNA library that was prepared from a pooled plasma of non-A non-B hepatitis (NANBH) patients using expression vector lambda gt11. This cDNA corresponds to known nucleotide positions 3983-4745 of the genome of hepatitis C virus (HCV). This clone was used as a probe for screening the HCV-related cDNAs in a cDNA library similarly prepared by using lambda gt10. As a result, six more cDNA clones were isolated and analyzed for their nucleotide sequences. The results strongly suggested that there are at least two groups of HCV, group I and group II. According to our classification, the prototype HCV and clone 11-7 belong to group I HCV, and their nucleotide and deduced amino acid sequences were diverged from those of group II HCV. Genetic variation observed in the nucleotide and the amino acid sequences between the two groups resembles that in the NS3 region of the genome between Japanese encephalitis virus and West Nile fever virus. Polypeptides produced in Escherichia coli carrying a clone 11-7 or a group II cDNA clone E reacted with antibodies in the blood of 12 or 4 out of 14 individual chronic NANBH patients, respectively. Our data clearly indicate the existence of a second group of HCV.

Amino Acid Sequence↗
PubMed1991

Identification of mutations that occurred on the genome of Japanese encephalitis virus during the attenuation process.

The total nucleotide sequences of the genomes of two Japanese encephalitis virus (JEV) strains, the attenuated vaccine strain SA14-14-2 and its parental virulent strain SA14, were determined by using the molecular cloning technique. The sequence analysis revealed that both virion RNA molecules were 10,976 nucleotides long with 95 and 585 flanking bases at the 5' and 3' untranslated sequences, respectively. A single, long open reading frame spanning 10,296 nucleotides was observed to encode a polyprotein of 3432 amino acid residues. When these sequences were compared with each other, 57 nucleotide substitutions were found to be scattered all over the genome. Of these, 24 resulted in amino acid changes within viral proteins. Structural proteins C and E contain one and eight amino acid changes, respectively. Of the nonstructural proteins, NS1 contains three, NS2a two, NS2b two, NS3 four, NS4a one, NS4b one, and NS5 two amino acid substitutions. The 5'- and 3'-terminal untranslated regions contain one- and two- point mutations, respectively. These data and comparative studies with other JEV strain genomes provide a molecular basis for investigating attenuation mechanisms of JEV.

Amino Acid Sequence↗
PubMed1991

Preparation of Japanese encephalitis virus nonstructural protein NS1 obtained from culture fluid of JEV-infected Vero cells.

The Japanese encephalitis virus (JEV) nonstructural protein NS1 was released efficiently into culture fluid of JEV-infected Vero cells. The JEV NS1 protein in the infected culture fluid was found almost as a high-molecular-weight form, probably a dimer form of NS1, and was converted to a monomer by boiling. Large amounts of NS1 protein were accumulated in the infected culture fluid. The NS1 protein, separated from JE virions by centrifugation through sucrose layer, could be obtained in large quantities.

Animals↗
PubMed1991

Role of inflammatory responses in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery.

Immediately after a cuff-sheathing of rabbit carotid artery, a large number of leukocytes adhered to injured endothelium then infiltrated into the media. These inflammatory responses were followed by an atherosclerotic change, intimal thickening, of the artery. A simultaneous injection of dexamethasone (10 mg/kg i.m.) inhibited the leukocyte accumulation by 74% when evaluated 18 h thereafter. Similarly, 39% inhibition was obtained with the same dose of FR110302, a potent 5-lipoxygenase inhibitor. On the other hand, the same dose of indomethacin, a cyclooxygenase inhibitor, had little effect on the leukocyte accumulation. The intimal thickening which was evaluated 3 weeks after the cuff-treatment was attenuated by a daily dose (10 mg/kg i.m.) of dexamethasone or FR110302 but not by one of indomethacin. The inhibition by the two former drugs were 91 and 58%, respectively. In vitro, the three drugs in concentrations up to 10 microM hardly affected endothelial adhesion of PMN which was induced by LPS or IL-1. Though 10 microM of FR110302 and indomethacin significantly decreased PMN chemotaxis induced by LTB4, the decreases were less than that at 10 microM dexamethasone. These results confirm a possible linkage between inflammation and atherosclerosis, and suggest that 5-lipoxygenase products contribute to the initiation and development of atherosclerosis.

Animals↗
PubMed1991

Nucleotide sequences important for translation initiation of enterovirus RNA.

An infectious cDNA clone was constructed from the genome of coxsackievirus B1 strain. A number of RNA transcripts that have mutations in the 5' noncoding region were synthesized in vitro from the modified cDNA clones and examined for their abilities to act as mRNAs in a cell-free translation system prepared from HeLa S3 cells. RNAs that lack nucleotide sequences at positions 568 to 726 and 565 to 726 were found to be less efficient and inactive mRNAs, respectively. To understand the biological significance of this region of RNA, small deletions and point mutations were introduced in the nucleotide sequence between positions 538 and 601. Except for a nucleotide substitution at 592 (U----C) within the 7-base conserved sequence, mutations introduced in the sequence downstream of position 568 did not affect much, if any, of the ability of RNA to act as mRNA. Except for a point mutation at 558 (C----U), mutations upstream of position 567 appeared to inactivate the mRNA. In the upstream region, a sequence consisting of 21 nucleotides at positions 546 to 566 is perfectly conserved in the 5' noncoding regions of enterovirus and rhinovirus genomes. These results suggest that the 7-base conserved sequence functions to maintain the efficiency of translation initiation and that the nucleotide sequence upstream of position 567, including the 21-base conserved sequence, plays essential roles in translation initiation. A deletion mutant whose genome lacks the nucleotide sequence at positions 568 to 726 showed a small-plaque phenotype and less virulence against suckling mice than the wild-type virus. Thus, reduction of the efficiency of translation initiation may result in the construction of enteroviruses with the lower-virulence phenotype.

Animals↗
PubMed1990

The poliovirus receptor protein is produced both as membrane-bound and secreted forms.

Both genomic and complementary DNA clones encoding poliovirus receptors were isolated from genomic and complementary DNA libraries prepared from HeLa S3 cells, respectively. Nucleotide sequence analysis of these cloned DNAs revealed that the poliovirus receptor gene is approximately 20 kb long and contains seven introns in the coding region, and that at least four mRNA isoforms referring to the coding sequence are generated by alternative splicing and appear to encode four different molecules, that is, PVR alpha, PVR beta, PVR gamma and PVR delta. The predicted amino acid sequences indicate that PVR alpha and PVR delta, corresponding to the previously described cDNA clones H20A and H20B, respectively, are integral membrane proteins while the other two molecules described here for the first time lack a putative transmembrane domain. Mouse cell transformants carrying PVR alpha were permissive for poliovirus infection, but those carrying PVR beta were hardly permissive. In contrast to PVR alpha, PVR beta was not detected on the surface of the mouse cell transformants but was detected in the culture fluid by an immunological method using a monoclonal antibody against poliovirus receptor. Three types of splicing products for PVR alpha, PVR beta and PVR gamma were detected by polymerase chain reactions using appropriate primers in poly(A)+ RNAs of the brain, leukocyte, liver, lung and placenta of humans; the choice of primers used did not permit detection of PVR delta. In situ hybridization using a cDNA fragment as a probe demonstrated that the PVR gene is located at the band q13.1----13.2 of human chromosome 19.

Amino Acid Sequence↗
PubMed1989

Purification of enzymatically active poliovirus proteinase 3C produced in Escherichia coli.

A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.

3C Viral Proteases↗
PubMed1989

Strong inclination toward transition mutation in nucleotide substitutions by poliovirus replicase.

A viable insertion mutant of the Sabin strain of type 1 poliovirus was constructed. The mutant carried an insertion sequence of 72 nucleotides at nucleotide position 702 in the 5' non-coding region (742 nucleotides long) of the genome of the Sabin strain. This mutant showed a small-plaque phenotype, as compared with the parental virus. Indeed, the final yield of the mutant in a single cycle of infection was tenfold fewer than that of the parental virus. Many large-plaque variants that are easily generated from the insertion mutant appeared to regain efficient viral replication and have single nucleotide changes. All nucleotide changes observed were limited to within three nucleotides of an AUG sequence in the insertion sequence. The result indicates strongly that the AUG sequence itself in this genome region functions in reducing the plaque size of the parental Sabin type 1 virus. The insertion mutant with a small-plaque phenotype may be the first in vitro mutant of poliovirus whose viability is lowered only by a primary sequence inserted into the 5' non-coding region of the genome. Base substitutions to alter the AUG sequence should largely be the result of errors of the virus-specific replicase, since variants with base substitutions must be subject to only minimum selection pressure. Accordingly, nucleotide sequence analysis of the genome region containing the AUG sequence was performed on a number of genomes of large-plaque variants to investigate types of nucleotide substitutions caused by characteristic errors in RNA replication. Only one transversion mutation was detected in the genomes of 44 independently isolated large-plaque variants with single base changes in the AUG sequence. This result suggests strongly that transition mutations occur predominantly as nucleotide substitutions caused by characteristic errors of poliovirus replicase.

Animals↗
PubMed1989

Construction of less neurovirulent polioviruses by introducing deletions into the 5' noncoding sequence of the genome.

Viral attenuation may be due to lowered efficiency of certain steps essential for viral multiplication. For the construction of less neurovirulent strains of poliovirus in vitro, we introduced deletions into the 5' noncoding sequence (742 nucleotides long) of the genomes of the Mahoney and Sabin 1 strains of poliovirus type 1 by using infectious cDNA clones of the virus strains. Plaque sizes shown by deletion mutants were used as a marker for rate of viral proliferation. Deletion mutants of both the strains thus constructed lacked a genome region of nucleotide positions 564 to 726. The sizes of plaques displayed by these deletion mutants were smaller than those by the respective parental viruses, although a phenotype referring to reproductive capacity at different temperatures (rct) of viruses was not affected by introduction of the deletion. Monkey neurovirulence tests were performed on the deletion mutants. The results clearly indicated that the deletion mutants had much less neurovirulence than with the corresponding parent viruses. Production of infectious particles and virus-specific protein synthesis in cells infected with the deletion mutants started later than in those infected with the parental viruses. The rate at which cytopathic effect progressed was also slower in cells infected with the mutants. Phenotypic stability of the deletion mutant for small-plaque phenotype and temperature sensitivity was investigated after passaging the mutant at an elevated temperature of 37.5 degrees C. Our data strongly suggested that the less neurovirulent phenotype introduced by the deletion is very stable during passaging of the virus.

Animals↗