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Biomedical subjects

A Nomoto

Publications and source records attributed to A Nomoto.

At least 37 records · Page 2Linked to original sources
PubMed1999

Interaction of poliovirus with its receptor affords a high level of infectivity to the virion in poliovirus infections mediated by the Fc receptor.

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcgamma receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.

Animals↗
PubMed1999

Dual stem loops within the poliovirus internal ribosomal entry site control neurovirulence.

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5' nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.

5' Untranslated Regions↗
PubMed1999

Intracellular redistribution of truncated La protein produced by poliovirus 3Cpro-mediated cleavage.

The La autoantigen (also known as SS-B), a cellular RNA binding protein, may shuttle between the nucleus and cytoplasm, but it is mainly located in the nucleus. La protein is redistributed to the cytoplasm after poliovirus infection. An in vitro translation study demonstrated that La protein stimulated the internal initiation of poliovirus translation. In the present study, a part of the La protein was shown to be cleaved in poliovirus-infected HeLa cells, and this cleavage appeared to be mediated by poliovirus-specific protease 3C (3Cpro). Truncated La protein (dl-La) was produced in vitro from recombinant La protein by cleavage with purified 3Cpro at only one Gln358-Gly359 peptide bond in the 408-amino-acid (aa) sequence of La protein. The dl-La expressed in L cells was detected in the cytoplasm. However, green fluorescence protein linked to the C-terminal 50-aa sequence of La protein was localized in the nucleus, suggesting that this C-terminal region contributes to the steady-state nuclear localization of the intact La protein in uninfected cells. The dl-La retained the enhancing activity of translation initiation driven by poliovirus RNA in rabbit reticulocyte lysates. These results suggest that La protein is cleaved by 3Cpro in the course of poliovirus infection and that the dl-La is redistributed to the cytoplasm. dl-La, as well as La protein, may play a role in stimulating the internal initiation of poliovirus translation in the cytoplasm.

3C Viral Proteases↗
PubMed1999

Isolation and molecular characterization of a poliovirus type 1 mutant that replicates in the spinal cords of mice.

The Mahoney strain of poliovirus type 1 (OM) is generally unable to cause paralysis in mice. We isolated a mouse-adapted mutant, PV1/OM-SA (SA), from the spinal cord of a mouse that had been intracerebrally inoculated with OM. SA showed mouse neurovirulence only with intraspinal inoculation, and the infected mice developed a flaccid paralysis, which was indistinguishable from that observed in poliovirus-sensitive transgenic mice inoculated with OM. SA antigens were detected in neurons of the spinal cords of the infected mice. Nucleotide (nt) sequence analysis revealed 9 nt changes on the SA genome, resulting in three amino acid (a.a.) substitutions, i.e., one each in the capsid proteins VP4 and VP1 and in the noncapsid protein 2C. To identify the key mutation site(s) for the mouse neurovirulence, virus recombinants between OM and SA were constructed by using infectious cDNA clones of these two viruses and tested for their mouse neurovirulence after inoculation via an intraspinal route. The results indicated that a mutation at nt 928 (replacement of A with G), resulting in a substitution of Met for Ile at a.a. 62 within VP4, was responsible for conferring the mouse neurovirulence phenotype of the mutant SA. The mutation in VP4 may render the virus accessible to a molecule that acts as a virus receptor and is located on the surfaces of neurons of the mouse spinal cord. This molecule appears not to be expressed in the mouse brain.

Adaptation, Physiological↗
PubMed1998

Retrograde transport of intact poliovirus through the axon via the fast transport system.

Intramuscularly inoculated poliovirus is thought to spread to the central nervous system through neural pathways in humans, monkeys, and the transgenic (Tg) mice carrying the human poliovirus receptor (PVR) gene. To gain insight into molecular mechanisms for the retrograde axonal transport of poliovirus, resulting in the expression of neurovirulence, a poliovirus-sensitive ICR-PVRTg21 mouse line (Tg21) was used as an animal model for poliomyelitis. We detected poliovirus antigens in axons of the sciatic nerve. All of the Tg21 mice, which had been inoculated into the calves with 1 x 10(6) pfu of the Mahoney strain of type 1 poliovirus, showed symptoms of paralysis in the inoculated limbs (initial paralysis) within 48 h after the inoculation. The appearance of this initial paralysis was observed in mice whose sciatic nerves were transected at various times after virus inoculation. The results were indicators of the velocity of poliovirus transportation through the sciatic nerves under analysis. Poliovirus-related materials recovered from the sciatic nerve were mainly composed of intact 160S virion particles. The amount of 160S particle recovered was greatly reduced by coinjection with anti-PVR monoclonal antibody. These results suggest that one of the fast retrograde axonal transport systems is involved in poliovirus dissemination through the sciatic nerve and that IM-inoculated poliovirus is incorporated into the sciatic nerve as intact particles in a PVR-dependent manner, as it is in humans.

Animals↗
PubMed1998

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage.

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1alpha (SDF-1alpha) and 1beta (SDF-1beta), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34(+) progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1alpha confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1alpha and SDF-1beta likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4(+) cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1alpha and SDF-1beta are involved.

Animals↗
PubMed1998

Determination of functional domains in polypyrimidine-tract-binding protein.

Polypyrimidine-tract-binding protein (PTB) is involved in pre-mRNA splicing and internal-ribosomal-entry-site-dependent translation. The biochemical properties of various segments of PTB were analysed in order to understand the molecular basis of the PTB functions. The protein exists in oligomeric as well as monomeric form. The central part of PTB (amino acids 169-293) plays a major role in the oligomerization. PTB contains several RNA-binding motifs. Among them, the C-terminal part of PTB (amino acids 329-530) exhibited the strongest RNA-binding activity. The N-terminal part of PTB is responsible for the enhancement of RNA binding by HeLa cell cytoplasmic factor(s).

Amino Acid Sequence↗
PubMed1998

Crm1 (XpoI) dependent nuclear export of the budding yeast transcription factor yAP-1 is sensitive to oxidative stress.

BACKGROUND: The yAP-1 transcription factor is crucial for the oxidative stress response of the budding yeast Saccharomyces cerevisiae; its activity is induced in response to oxidative stress, and as a consequence the expression of a number of target genes is enhanced. We have shown previously that yAP-1 is mainly found in the cytoplasm, but that upon the imposition of oxidative stress it localizes to the nucleus. In this study, we addressed the mechanism through which yAP-1 nuclear localization is regulated. RESULTS: Here we show that yAP-1 localization is mediated by active export from the nucleus, resulting from the activity of Crm1 (XpoI), a conserved protein that functions as an export receptor which recognizes the nuclear export signal (NES). When Crm1 expression was repressed, yAP-1 was localized in the nucleus and induced the expression of a yAP-1 dependent target gene. Our results also suggest that the cysteine rich domain (CRD), at the C-terminus of yAP-1, functions as an export recognition sequence. yAP-1 and Crm1 interact in vivo and this interaction is reduced in response to oxidative stress. CONCLUSIONS: These results suggest a novel regulatory mechanism of nucleocytoplasmic transport which is dependent upon a redox sensitive nuclear export pathway.

Bacterial Proteins↗
PubMed1998

A new internal ribosomal entry site 5' boundary is required for poliovirus translation initiation in a mouse system.

Four mutants of the virulent Mahoney strain of poliovirus were generated by introducing mutations in nucleotides (nt) 128 to 134 of the genome, a region that contains a part of the stem-loop II (SLII) structure located within the internal ribosomal entry site (IRES; nt 120 to 590) (K. Shiroki, T. Ishii, T. Aoki, Y. Ota, W.-X. Yang, T. Komatsu, Y. Ami, M. Arita, S. Abe, S. Hashizume, and A. Nomoto, J. Virol. 71:1-8, 1997). These mutants (SLII mutants) replicated well in human HeLa cells but not in mouse TgSVA cells that had been established from the kidney of a poliovirus-sensitive transgenic mouse. Their neurovirulence in mice was also greatly attenuated compared to that of the parental virus. The poor replication activity of the SLII mutants in TgSVA cells appeared to be attributable to reduced activity of the IRES. Two and three naturally occurring revertants that replicated well in TgSVA cells were isolated from mutants SLII-1 and SLII-5, respectively. The revertants recovered IRES activity in a cell-free translation system from TgSVA cells and returned to a neurovirulent phenotype like that of the Mahoney strain in mice. Two of the revertant sites that affected the phenotype were identified as being at nt 107 and within a region from nt 120 to 161. A mutation at nt 107, specifically a change from uridine to adenine, was observed in all the revertant genomes and exerted a significant effect on the revertant phenotype. Exhibition of the full revertant phenotype required mutations in both regions. These results suggested that nt 107 of poliovirus RNA is involved in structures required for the IRES activity in mouse cells.

Animals↗
PubMed1998

Interaction of poliovirus with its purified receptor and conformational alteration in the virion.

Polypeptides of amino acids 1 to 241 (PVR241) and 1 to 330 (PVR330) of the human poliovirus receptor (hPVR) were produced in a baculovirus expression system. PVR241 contained extracellular domains 1 and 2 of hPVR, and PVR330 contained extracellular domains 1, 2, and 3. These peptides were purified by immunoaffinity column chromatography with an anti-hPVR monoclonal antibody (MAb). After the purification, PVR241 and PVR330 appeared to retain their native conformation as judged by reactivity with an anti-PVR MAb that recognized domain 1 of hPVR in a conformation-dependent manner. The virulent Mahoney strain of poliovirus type 1 was mixed with the purified PVRs in various concentrations. An average of at least 43 PVR330 molecules were able to bind to one virion particle under the conditions used. The equilibrium dissociation constant between the PVR330 molecule and the PVR binding site (canyon) on the virion was determined to be 4.50 +/- (0.86) x 10(-8) M at 4 degrees C. Higher rates of conformational change of the virus (160S) to 135S and 80S particles were observed as the concentration of PVR330 was increased. In this in vitro system, the ratio of the amount of the 135S particle to that of the 80S particle seemed to be always constant. After the disappearance of the 160S particle, the amount of the 80S particle was not increased by further incubation at 37 degrees C. These results suggested that the 80S particle was not derived from the 135S particle under the conditions used in this study.

Animals↗
PubMed1998

Expression and microvillar localization of scavenger receptor, class B, type I (a high density lipoprotein receptor) in luteinized and hormone-desensitized rat ovarian models.

Steroidogenic cells in rats and mice obtain most of their cholesterol for steroid production and cholesteryl ester (CE) storage via the selective uptake pathway in which high density lipoprotein CE (HDL-CE) is taken into the cell without the uptake and degradation of the HDL particle. A number of recent studies show that the scavenger receptor, class B, type I (SR-BI) can mediate HDL-CE selective uptake in cultured cells and suggest that this receptor may be responsible for HDL-CE selective uptake in steroidogenic cells in vivo. In the current study we examine the relationship between SR-BI expression and HDL-CE selective uptake in the gonadotropin-primed, luteinized rat ovary and in the ovary that is desensitized by multiple gonadotropin treatments. Results from this study demonstrate a tight association between expression of SR-BI and measurements of HDL-CE selective uptake regardless of the steroidogenic state of the ovary. Thus, in the luteinized ovary (which is actively producing progestins), HDL-CE selective uptake is high, as is the expression of SR-BI. In the desensitized ovary (where CE content is reduced by 90% and progestin production is virtually absent), HDL-CE selective uptake and SR-BI are induced 2- to 3-fold compared with those in the luteinized ovary. These data argue that SR-BI can be regulated by the cholesterol status of the luteal cell independently of gonadotropic stimulation. Immunostaining at the light microscopic level showed strong expression of SR-BI specifically on the surface of luteal cells in the luteinized and desensitized ovary. Immunolocalization at the electron microscopic level showed that SR-BI was associated with microvilli and microvillar channels of the luteal cell surface. This result supports the hypothesis that microvilli and microvillar channels represent a cell surface compartment that is specialized for the selective uptake of lipoprotein cholesterol into steroidogenic cells.

Animals↗
PubMed1998

Simultaneous induction of an HDL receptor protein (SR-BI) and the selective uptake of HDL-cholesteryl esters in a physiologically relevant steroidogenic cell model.

This study addresses the question of whether the level of expression of SR-BI (an HDL receptor) is linked to the expression of selective lipoprotein-cholesteryl ester delivery in a steroidogenic cell model. Rat ovarian granulosa cells are physiologically normal cells which show no selective uptake of HDL-cholesteryl esters and no progestin production until luteinized by trophic hormones or adenylate cyclase stimulators, after which expression of the selective cholesterol pathway and production of steroid hormone is dramatically up-regulated. The current study demonstrates that at every cell stage studied, the protein content and level of expression of SR-BI mRNA are linked to changes that occur in HDL-cholesteryl ester uptake; i.e., SR-BI is not present in basal (non-luteinized) cells, develops slowly (from 6-9 h) after hormone treatment, increases robustly from 9-48 h after stimulation, and remains high after incubation with HDL. In contrast, another structural protein, caveolin, did not follow this pattern; caveolin expression showed an inverse relationship to selective cholesteryl ester uptake, and was most prominent in basal cells and least prominent in luteinized, HDL-incubated cells. Morphologically, SR-BI appears to be associated with cell surface sites showing high levels of cholesteryl ester uptake (after luteinization and/or incubation with HDL labeled with fluorescent cholesteryl esters), and at the electron microscope level, SR-BI is most clearly associated with microvillar regions on the cell surface which also bind HDL-labeled with colloidal gold. Thus, induction of the SR-BI receptor system and induction of the HDL-selective cholesterol uptake pathway in rat granulosa cells appear to be linked morphologically, biochemically, and functionally.

Amino Acid Sequence↗
PubMed1997

A novel dicistronic AAV vector using a short IRES segment derived from hepatitis C virus genome.

Adeno-associated virus (AAV) vectors have a limited capacity for packaging DNA. To insert both a therapeutic gene and a selectable marker gene in the same AAV vector efficiently, we developed a novel dicistronic AAV vector containing a 230 base pairs (bp) internal ribosome entry site (IRES) element derived from hepatitis C virus (HCV) genome and a 420 bp blasticidin S-resistance gene (bsr) as a small selectable marker in the second cistron. The 650 bp HCV IRES-bsr construct was placed downstream of the 3' end of the luciferase gene (Luc) under the control of the human cytomegalovirus (CMV) promoter. This dicistronic gene conferred blasticidin S-resistance to 293 cells besides luciferase activity, when examined not only by transfection but also by transduction using AAV vectors. The dicistronic AAV vector harbouring HCV IRES-bsr is capable of expressing a therapeutic gene of up to 3.6 kilobases (kb) (including promoter/enhancer elements) as well as a selectable marker gene. If a selectable marker gene is not necessary, this vector is able to incorporate two different kinds of therapeutic genes more easily than that containing EMCV IRES. The dicistronic AAV vector described here is useful for expressing many kinds of cDNA besides a selectable marker.

Animals↗
PubMed1997

Mouse homolog of poliovirus receptor-related gene 2 product, mPRR2, mediates homophilic cell aggregation.

Poliovirus receptor (PVR) is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. Although MPH was initially reported as the mouse homolog of human PVR, recent data strongly suggest that MPH is the mouse homolog of human PRR2, a PVR-related gene 2 product, and not that of human PVR. Thus MPH is renamed mPRR2 in this study. Physiological functions of the PVR-related gene products have not been elucidated, although PVR has been well characterized as the poliovirus receptor. In this study, a possible function of mPRR2 (MPH), which is not a functional receptor for poliovirus, was investigated. Mouse L cells expressing mPRR2 were prepared. Those mouse cells showed a higher activity of cell aggregation than the parental mouse L cells. Enhancement of cell aggregation was also observed for insect Sf9 cells infected with recombinant baculovirus carrying mPRR2 cDNA. On the other hand, L cells expressing human PVR or monkey PVR (AGM alpha1 or AGM alpha2) did not show increased cell aggregation. The cell aggregation activity of L cells expressing mPRR2 was inhibited by the addition of anti-mPRR2 monoclonal antibodies or a soluble mPRR2 molecule produced by the baculovirus expression system. An immunofluorescence study revealed that mPRR2 protein was localized to the cell-cell contact sites between cells expressing mPRR2. A similar localization of mPRR2 was observed for intrinsic mPRR2 molecules of the mouse neuroblastoma cell line NS20Y. The contact site-specific localization of mPRR2 was not observed on the border between mPRR2-expressing and nonexpressing HeLa cells. Furthermore, mPRR2 proteins directly bound to each other in vitro. mPRR2 was detected on various types of cultured cells of mouse origin and in various mouse tissues. These results suggest that mPRR2 is an intercellular adhesion molecule with a homophilic binding manner.

Animals↗
PubMed1997

Genetic analysis of internal ribosomal entry site on hepatitis C virus RNA: implication for involvement of the highly ordered structure and cell type-specific transacting factors.

Hepatitis C virus (HCV) carries an internal ribosomal entry site (IRES) within the 5' portion of the RNA. To identify structures that influence efficiency of the translation initiation, relative activities of modified IRESs were examined by using engineered bicistronic mRNAs, between the two cistrons of which various mutant IRESs were inserted. An IRES derived from genotype 2b is at least two times more efficient than one from genotype 1b in cultured cells. Activity ratios of genotype 2b IRES to 1b IRES differ in magnification among cultured cells, suggesting the difference in assortment of IRES-related host factors among individual cell types. Recombinant IRESs between the genotypes show similar or higher activities compared with 2b IRES in cell-free systems and show intermediate activities in cultured cells. Patterns of relative activities of those IRESs indicate that the IRES activity is not regulated by defined structure(s), although a cluster of different nucleotides is observed in the genome region of nucleotides 176-224 between the two alleles. The results suggest that a highly ordered structure formed by the entire 5' portion of the RNA is important for the IRES activity. The 5' border of HCV IRES was examined by using a series of deletion RNAs in various systems. The results strongly suggest that the border resides between nucleotide positions 28 and 45. Patterns of relative activities of the deletion IRESs differ in translation systems or cell types. These results imply that interactions of HCV RNA with the related transacting factor(s) may differ in the translation systems or cell types.

Base Sequence↗
PubMed1997

Cross-family interaction between the bHLHZip USF and bZip Fra1 proteins results in down-regulation of AP1 activity.

Heterodimerization among the basic-leucine zipper (bZIP) proteins or among the basic-helix-loop-helix-leucine zipper (bHLHZip) proteins confers a multitude of combinational activities to these transcription factors. To further examine the function of the bHLHZip protein, USF, we screened for cellular proteins which could directly interact with USF using the yeast two-hybrid system. A bZip protein, Fra1, was found to efficiently interact with USF. USF specifically interacts with Fra1 but not with other closely related family members, c-Fos, Fra2, FosB, or with c-Jun. Both the bHLHZip and the N-terminal regions of Fra1 are required for efficient interaction with USF. In vivo association between USF and Fra1 has been demonstrated by co-immunoprecipitation. Expression of exogenous USF led to a decrease in AP1-dependent transcription in F9 cells. Co-expression of exogenous Fra1 restored the AP1 activity in a dose-dependent manner. These data show that USF and Fra1 physically and functionally interact demonstrating that cross-talk occurs between factors of distantly related transcription families.

Animals↗
PubMed1997

Regulation of yAP-1 nuclear localization in response to oxidative stress.

The YAP1 gene of Saccharomyces cerevisiae encodes a bZIP-containing transcription factor that is essential for the normal response of cells to oxidative stress. Under stress conditions, the activity of yAP-1 is increased, leading to the induced expression of a number of target genes encoding protective enzymes or molecules. We have examined the mechanism of this activation. Upon imposition of oxidative stress, a small increase in the DNA-binding capacity of yAP-1 occurs. However, the major change is at the level of nuclear localization; upon induction the yAP-1 protein relocalizes from the cytoplasm to the nucleus. This regulated localization is mediated by a cysteine-rich domain (CRD) at the C-terminus, its removal resulting in constitutive nuclear localization and high level activity. Furthermore, the CRD of yAP-1 is sufficient to impose regulated nuclear localization of the GAL4 DNA-binding domain. Amino acid substitutions indicated that three conserved cysteine residues in the CRD are essential for the regulation. We suggest therefore, that these cysteine residues are important in sensing the redox state of the cell and hence regulating yAP-1 activity.

Amino Acid Sequence↗