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N Kitamoto

Publications and source records attributed to N Kitamoto.

At least 37 records · Page 2Linked to original sources
PubMed1996

Antigenic mapping of the recombinant Norwalk virus capsid protein using monoclonal antibodies.

Norwalk virus (NV) is the prototype strain of a group of noncultivatable caliciviruses that infect humans and cause outbreaks of epidemic acute nonbacterial gastroenteritis. The NV virion is composed of 180 copies of a single structural protein that, when expressed in insect cells infected with a recombinant baculovirus, assembles into empty recombinant Norwalk virus-like particles (rNV VLPs) which are morphologically and antigenically similar to native NV. We have begun to dissect the antigenic structure of the rNV particles using monoclonal antibodies made to the rNV VLPs. Ten MAbs made to rNV particles were characterized for their reactivity as detector antibodies by ELISA, as capture antibodies in an ELISA to detect NV in stools, by Western blot, and by immunoprecipitation. Seven of the MAbs recognize discontinuous epitopes, requiring the rNV capsid protein to remain at least partially folded, while the other three recognize continuous epitopes. Eight of the MAbs map to the C-terminal half of the capsid protein as they react by Western blot and by immunoprecipitation with a 32K trypsin cleavage product of the full-length 58K capsid protein, suggesting that the C-terminal half of the capsid protein may contain the immunodominant epitopes. The three MAbs that recognize continuous epitopes map to the extreme C terminus of the capsid protein, between amino acids 457 and 530, in a region that is relatively conserved among different human calicivirus capsid proteins. These MAbs which were assigned into three antigenic groups will be useful as tools to further dissect the structural and antigenic topography of the NV virion, and as unlimited reagents to detect NV in diagnostic assays.

Animals↗
PubMed1996

Molecular cloning, purification and characterization of two endo-1,4-beta-glucanases from Aspergillus oryzae KBN616.

Two endo-1,4-beta-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 degrees C.

Amino Acid Sequence↗
PubMed1996

Streptococcus pneumoniae in the nasal cavity of mice causes lower respiratory tract infection after airway obstruction.

A model of persistent colonisation in the nasal cavity of mice by Streptococcus pneumoniae has been established. S. pneumoniae NNP-4 was introduced to the lungs of CBA/J mice at a density of c. 10(4) cfu/lung by an aerosol method and a high dose of ampicillin was administered 1 h after infection. This antibiotic eliminated bacteria from the lungs and trachea, but did not affect the bacterial counts in the nasal cavity. In mice given ampicillin, the bacteria were recovered from the nasal cavity only more than 2 weeks after infection, but IgG antibody against the colonising organisms was produced in sera around day 8 after infection. Airway obstruction was induced by intratracheal injection of formalin 2% into mice. Organisms appeared in the lungs in greater numbers when formalin was injected before the antibody production than when the immunity was established. In the early stages of infection, 10(3)-10(4) cfu appeared in the lungs 6 h after the formalin injection and the bacterial counts increased to c. 10(6) cfu within 24 h. When ampicillin was administered again 1 h after formalin was given, no bacteria were recovered from lungs 6 h later. However, in some of the mice given ampicillin after formalin, bacteria appeared in the lungs on the next day and the bacterial counts increased thereafter. These results suggest that S. pneumoniae in the nasal cavity invade the lower respiratory tract and that these organisms can localise and proliferate in lungs in the event of damage to the airway.

Airway Obstruction↗
PubMed1996

Attachment and entry of recombinant Norwalk virus capsids to cultured human and animal cell lines.

Norwalk virus (NV) is the prototype strain of a group of noncultivable human caliciviruses responsible for epidemic outbreaks of acute gastroenteritis. While these viruses do not grow in tissue culture cells or animal models, expression of the capsid protein in insect cells results in the self-assembly of recombinant Norwalk virus-like particles (rNV VLPs) that are morphologically and antigenically similar to native NV. We have used these rNV VLPs to examine virus-cell interactions. Binding and internalization of VLPs to cultured human and animal cell lines were studied in an attempt to identify potentially susceptible cell lines for virus propagation in vitro and to determine if early events in the replication cycle were responsible for the narrow host range and restriction of virus growth in cell culture. Radiolabeled VLPs specifically bound to a saturable number of binding molecules on the cell surface of 13 cell lines from different origins, including human intestine (differentiated and undifferentiated Caco-2) and insect (Spodoptera frugiperda 9) ovary. Differentiated Caco-2 cells bound significantly more rNV VLPs than the other cell lines. Variations in the amount of bound VLPs among the different cell lines did not correlate with the tissue or species of origin. VLP binding was specific, as determined by competition experiments with unlabeled rNV VLPs; however, only 1.4 to 6.8% of the specifically prebound radiolabeled VLPs became internalized into cells. Blocking experiments using polygonal and monoclonal anti-rNV sera and specific antipeptide sera were performed to map the domains on rNV VLPs involved in binding to cells. One monoclonal antibody (NV8812) blocked binding of rNV VLPs to human and animal cell lines. The binding site of monoclonal antibody NV8812 was localized to the C-terminal 300 to 384 residues of the capsid protein by immunoprecipitation with truncated and cleaved forms of the capsid protein. These data suggest that the C-terminal region of the capsid protein is involved in specific binding of rNV VLPs to cells.

Animals↗
PubMed1995

Detection of immunoreactive antigen, with a monoclonal antibody to measles virus, in tissue from a patient with Crohn's disease.

Using immunofluorescence (IF), we investigated reactive antigens present in Crohn's disease patients with monoclonal antibodies derived from cells infected with measles virus, but not with the subacute sclerosing panencephalitis virus. During immunoblotting, one monoclonal antibody (mAb 86) reacted with a polypeptide with a molecular weight of 36,000 dalton (M; matrix protein) in measles virus-infected cells. This monoclonal antibody displayed a positive reaction only with tissues from patients with Crohn's disease by the IF test. It did not react with samples from patients with other chronic inflammatory bowel diseases, such as ulcerative colitis. Other monoclonal antibodies to the measles virus protein, and monoclonal antibodies to Herpes simplex virus type 1 did not react with the same tissue samples. The role of measles virus infection and/or a viral antigen (possibly the M protein) as a causative agent in Crohn's disease poses a challenging avenue for further research.

Antibodies, Monoclonal↗
PubMed1995

Serum antibody against Helicobacter pylori assayed by a new capture ELISA.

We developed a highly specific detection technique for serum antibody, using a monoclonal antibody to a specific antigen of Helicobacter pylori. A monoclonal antibody preparation that reacted with the 54-kDa molecule of H. pylori antigens was obtained. Using this preparation, an antigen-capture enzyme-linked immunosorbent assay (ELISA) was established by fixation of the monoclonal antibody, followed by reaction with sonicated whole cell antigens. The serum antibody titers of patients with gastritis and peptic ulcers were significantly higher than control titers, and the antibody titer correlated with the histological severity of gastritis. Patients positive for H. pylori by bacterial culture had higher titers than H. pylori-negative patients. Our new ELISA may be useful for the diagnosis of H. pylori infections and for evaluation of the severity of gastric inflammation.

Antibodies, Bacterial↗
PubMed1995

The nitrate reductase gene from a shoyu koji mold, Aspergillus oryzae KBN616.

A niaD gene encoding nitrate reductase was isolated from Aspergillus oryzae KBN616 and sequenced. The structural gene comprises 2973 bp and 868 amino acids, which showed a high degree of similarity to nitrate reductases from other filamentous fungi. The coding sequence is interrupted by six introns varying in size from 48 to 98 bp. The intron positions are all conserved among the niaD genes from A. oryzae, Aspergillus nidulans, and Aspergillus niger. A homologous transformation system was developed for an industrial shoyu koji mold, A. oryzae KBN616, based on the nitrate reductase (niaD) of the nitrate assimilation pathway.

Amino Acid Sequence↗
PubMed1993

Isolation and nucleotide sequences of the genes encoding killer toxins from Hansenula mrakii and H. saturnus.

The HMK gene, encoding a killer toxin (HMK) of Hansenula mrakii strain IFO 0895, and the HSK gene, encoding a killer toxin (HSK) of H. saturnus strain IFO 0117, were cloned and sequenced. The HMK and HSK genes encode precursors to killer toxins of 125 amino acids (aa) and 124 aa, respectively. Both precursors have an N-terminal signal sequence of 37 aa which may be removed by a signal peptidase, and a propeptide which may be cleaved off by a KEX2-like protease. There is extensive homology between the aa sequences of HMK and HSK with the exception of the addition of one aa residue in HMK. The HMK and HSK genes were placed, separately, downstream from the yeast GAL10 promoter and introduced into a mutant of Saccharomyces cerevisiae that was resistant to the HMK. The transformants were capable of killing sensitive yeasts in medium that contained galactose with killing spectra similar to those of the donor strains of the toxins. These observations suggest that both killer toxins were synthesized and secreted from S. cerevisiae cells and killed sensitive yeasts, perhaps by the same mechanism as that associated with the donor strains and, moreover, that the difference in primary structure between the two toxins is responsible for the difference in their killing spectra.

Amino Acid Sequence↗
PubMed1993

Structural features of a polygalacturonase gene cloned from Aspergillus oryzae KBN616.

A genomic gene encoding a polygalacturonase from Aspergillus oryzae, used in soy sauce production, was cloned and sequenced. The structural gene comprises 1227 bp coding for 363 amino acids with a putative prepropeptide of 28 amino acids and the open reading frame is disrupted by two short introns of 57 bp and 81 bp. The deduced amino acid sequence of the mature protein showed 63, 63, 63 and 64% homology with those of Aspergillus niger polygalacturonase I, Aspergillus niger polygalacturonase II, Aspergillus tubingensis polygalacturonase II and Cochliobolus carbonum polygalacturonase, respectively. There is, however, little homology among fungal, plant and bacterial polygalacturonases.

Amino Acid Sequence↗
PubMed1993

Alterations in the sequence of the gene 4 from a human rotavirus after multiple passages in HepG2 liver cells.

The human Wa strain of rotaviruses, initially unable to grow in liver cells, was adapted by multiple passages to grow in HepG2 cells. The genome segment 4 of both the parental and passaged strains was cloned and sequenced. Five amino acid differences (residues 38, 120, 421, 525, and 618) were found in the HepG2-passaged variant compared to the parental Wa strain. Our results support the hypothesis that viral variants that have improved capabilities for infecting liver cells can be generated during infection.

Amino Acid Sequence↗
PubMed1991

Comparative growth of different rotavirus strains in differentiated cells (MA104, HepG2, and CaCo-2).

The production of viral antigen after infection of MA104, HepG2 (derived from human liver), and CaCo-2 (derived from human colon) cells with various cultivatable human and animal rotavirus strains was compared using immunofluorescence tests. All rotavirus strains examined expressed antigen in CaCo-2 cells and MA104 cells, but only some virus strains, namely, SA11-Cl3 (simian), RRV (simian), CU-1 (canine), and Ty1 (turkey), produced antigen in numbers of infected HepG2 cells comparable to infections in MA104 and CaCo-2 cells. Fl-14 (equine), OSU (porcine), NCDV (bovine), and Ch2 (chicken) strains were found to infect moderate numbers of HepG2 cells. Most human rotaviruses (representing viruses in serotypes 1, 2, 3, 4, 8, and 9), a simian rotavirus variant (SA11-4F), lapine (Ala, C-11 and R-2) viruses and porcine (Gottfried) virus infections resulted either in no detectable antigen or antigen synthesis in a low percentage of HepG2 cells. Human rotavirus isolates obtained from the stool specimens of an immunocompromised child with rotavirus antigen in his liver showed two different patterns of replication in HepG2 cells. Examination of the replication of a subset of viruses in the liver and intestinal tissues of orally infected suckling mice showed the CU-1 and Ty1 strains replicated well, while the OSU and human rotavirus strains did not. These results indicate that growth restriction in HepG2 cells is not serotype-specific, and growth of a virus in HepG2 cells does not necessarily correlate with the hepatotropic potential of a virus strain. Factors that may influence these differences of virus infectivity in HepG2 cells are discussed.

Animals↗
PubMed1990

Virus-specific early antigen expressed in the nucleus of cowpox virus-infected cells.

Two monoclonal antibodies (MAbs) were obtained from BALB/c mice immunized with cowpox virus (CPV)-infected cells cultured in the presence of cytosine-1-beta-D-arabinofuranosyl-HCl (Ara C). In the immunofluorescence test, the specific antigen reacting with these MAbs was restricted almost entirely to the nucleus in CPV-infected cells in the presence of Ara C at any time. On the other hand, in CPV-infected cells in the absence of Ara C, the antigen was first detectable within the nucleus 2 h after infection and migrated to the cytoplasm as the infection proceeded. On immunoblotting, only one component with an Mr of 27K was detected 2 to 24 h after infection of cells with CPV in either the presence or absence of Ara C. The antigen was also detected in vaccinia virus-infected cells, but not in any mock-infected cells nor in any virions purified from infected cells. These results suggest that the antigen reacting with the MAbs is virus-specific and a non-structural early antigen.

Animals↗
PubMed1988

Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes.

A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined. The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues. The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167. The amino acid sequence of the C. thermosulfurogenes beta-amylase showed 54, 32, and 32% homology with those of the Bacillus polymyxa, soybean, and barley beta-amylases, respectively. Twelve well-conserved regions were found among the amino acid sequences of the four beta-amylases. To elucidate the mechanism rendering the C. thermosulfurogenes beta-amylase thermophilic, its amino acid sequence was compared with that of the B. polymyxa beta-amylase. The C. thermosulfurogenes beta-amyulase contained more Cys residues and fewer hydrophilic amino acid residues than the B. polymyxa beta-amylase did. Several regions were found in the amino acid sequence of the C. thermosulfurogenes beta-amylase, where the hydrophobicity was remarkably high as compared with that of the corresponding regions of the B. polymyxa beta-amylase.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies to cowpox virus: polypeptide analysis of several major antigens.

Monoclonal antibodies directed against major antigens induced by cowpox virus (CPV) were produced. The specificities of these antibodies were established by immunoprecipitation, immunoblotting and several serological analyses, and from the cross-reactivities of these antibodies with cells infected with various other poxviruses, ectromelia virus (EV), vaccinia virus and Shope fibroma virus. The antibodies defined included ones reacting with each of the known major antigens of poxviruses, i.e. the common antigen of all poxviruses (probably NP antigen), the Orthopoxvirus-specific antigen (probably LS antigen), the haemagglutinin, the cell surface antigen, the common A-type inclusions in CPV and EV, and the antigen involved in neutralization.

Animals↗
PubMed1986

Polypeptide analysis with monoclonal antibodies of A type inclusion bodies induced by cowpox virus.

The antigen in A type inclusion bodies (ATIB) induced by cowpox virus (CPV) was examined by immunofluorescent staining with monoclonal antibodies and polypeptide analysis. In the immunofluorescence (IF) test, these monoclonal antibodies reacted only with cytoplasmic inclusion bodies in cells infected with CPV. The fluorescence became detectable in the cells 6-9 hours after infection with CPV. No fluorescence was detectable in cells infected with CPV in the presence of cytosine-I-beta-D-arabinofuranosyl-HCl (Ara C) or in cells infected with other poxviruses, such as vaccinia virus (VV) or Shope fibroma virus (SFV). On Western blotting and immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), only one component with a molecular weight of about 160,000 (160 K) was detected in CPV-infected cells. This 160 K polypeptide was first detectable 12 hours after infection of cells with CPV, and was not detectable in infected cells in the presence of Ara C. The 160 K polypeptide was not detected in cells infected with VV or SFV, or in virions purified from CPV-, VV- or SFV-infected cells.

Animals↗
PubMed1986

Cross-reactivity among cowpox, ectromelia and vaccinia viruses with monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies.

Several monoclonal antibodies recognizing distinct antigenic determinants in A-type inclusion bodies (ATIB) induced by cowpox virus (CPV) were obtained to examine the cross-reactivity among various strains of poxviridae, comprising CPV, ectromelia virus (EV), vaccinia virus (VV) and Shope fibroma virus (SFV). The monoclonal antibodies were classified into at least 3 groups on the basis of the results of an immunofluorescence test and immunoblotting; i. e., strain-specific, CPV and EV-specific and Orthopoxvirus (CPV, EV and VV)-specific antibodies. Differences were found between the antigenic determinants of ATIB of LB strains (LB red and LB white) and other strains (Amsterdam, 53, 58 and 60) of CPV and also between those of ATIB of CPV and EV. An interesting finding was that VV also produces the antigen analogous to that associated with ATIB in CPV- and EV-infected cells despite the absence of morphologically defined ATIB.

Antibodies, Monoclonal↗