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Biomedical subjects

A Totsuka

Publications and source records attributed to A Totsuka.

At least 19 recordsLinked to original sources
PubMed1999

Hepatitis A virus proteins.

The RNA genome of hepatitis A virus (HAV) shares common characteristics of the picornavirus family. However, the nucleotide or amino acid sequences are distantly related with other members of the family. Like other picornaviruses, HAV proteins are cleaved from a large polyprotein (PO), but the processing and some products are quite different. The 3C protein is the sole processing enzyme, and the primary cleavage takes place at the 2A/2B site. Several VP1-2A sites are proposed. In some strains, the intermediate VP1-2A polypeptides are assembled in the virion. The VP4 is very small and not detected in the mature virion. Some mutations in 2B, 2C and 3A proteins are identified to enhance viral replication or to induce cytopathogenic effects in the viruses adapted to cell cultures.

Amino Acid Sequence↗
PubMed1997

Quantitation of group-specific a antigen in hepatitis B vaccines by anti-HBs/a monoclonal antibody.

Balb/c mice were immunized with aluminium hydroxide [alum, Al (OH)3]-adjuvanted hepatitis B (HB) vaccines of subtypes adr, ayw or adw. Spleen cells from the immune animals were fused with SP2/O cells. Eight hybridoma clones producing antibodies specific for HB surface antigen (HBsAg) were selected. Monoclonal antibodies (mAbs) of four clones were specific for group-specific antigen/a, and the other of four clones were specific for subtype antigen/d, y, r, or w. The anti-HBs/a mAbs were classified into three non-competitive groups. Quantitation of group-specific determinant a of HBsAg (HBsAg/a) was performed by sandwich enzyme-linked immunosorbent assay (ELISA), in which a solid phase of anti-HBs guinea-pig polyclonal antibodies (pAb), the HBsAg for testing, anti-HBs/a mouse mAb and horseradish peroxidase (HRP)-conjugated anti-mouse IgG were used. The unadsorbed HBsAg was used to establish the standard curve of HBsAg/a. The lower detection limits were 0.5 to 1 ng/ml of HBsAg. Methods of solubilization of alum were investigated to quantify HBsAg/a in adsorbed HB vaccines. The recovery rate was more than 60% if vaccines were prediluted. The recovery of HBsAg/a in HB vaccines produced by the same manufacturer showed the similar recovery rate, and the contents of HBsAg/a in adsorbed HB vaccines could be estimated by the recovery rate determined for adsorbed HB vaccines.

Animals↗
PubMed1997

The latest seroepidemiological pattern of hepatitis A in Japan.

Age-specific prevalence of anti-hepatitis A virus antibody (anti-HAV) was surveyed with 2,708 sera collected in 1994 in various areas of Japan. By age-group analyses, we found strong association of anti-HAV antibody with higher age group. The prevalence ratios of antibody in the groups of 30-34, 35-39, 40-44, 45-49, 50-54, 55-59, 60-64 and 65 years or older were 0, 4.2, 22.0, 44.8, 57.6, 76.4, 84.5 and 91.4%, respectively. Geometric mean titers of anti-HAV antibody in the positive age groups were approximately 6,000 mIU/ml. The seropositives among older population were ascribed to the infections more than 40 years ago and the high anti-HAV titers have been maintained since that time. In Japan, people younger than 40 years of age are extremely risky to HAV infection, since 99% have no antibody. Those in forties are also risky since two-thirds of them are seronegative. In Japan, an inactivated vaccine was licensed in 1994. Vaccination may be recommended for such high-risk groups as travelers going to endemic areas, patients who have received blood product medication and child-care staffs.

Adolescent↗
PubMed1996

Functional analysis of Glu380 and Leu383 of soybean beta-amylase. A proposed action mechanism.

Soybean beta-amylase, comprising a (beta/alpha)8-barrel core with a mobile loop, similar to that of triose phosphate isomerase, was mutated by site-directed mutagenesis at residues Glu380 and Leu383. X-ray crystallographic findings suggest that Glu380 is the counterpart of the catalytic site (Glu186) and that Leu383, located near the active-site cavity, forms an inclusion complex with cyclomaltohexaose. Separate substitutions of Glu380 by Gln and Asp completely eliminated the activity without inducing any significant changes in the circular dichroic spectra nor in the binding affinity for cyclomaltohexaose. Glu380, in cooperation with Glu186, therefore, is clearly indispensable for the liberation of beta-maltose from starch. Substitutions of Leu383 by Ile and Gln, in contrast, led to remarkable increases in the Km values of both mutants when compared to that of the non-mutant enzyme. The mutants also showed marked reductions in their binding affinities to cyclomaltohexaose. Overall, it would appear that the kcat/Km of soybean beta-amylase increases in proportion to the length of the substrate molecule, and depends also on the characteristics of the side chain of the residue at position 383. Leu383, therefore, may be important for both substrate penetration and subsequent retention at the active site. Based on the foregoing, we propose an action mechanism of soybean beta-amylase involving the interactions of three essential amino acid residues (Asp101, Glu186 and Glu380) in concert with Leu383, and assumed an indispensable role for Asp101.

Amino Acid Sequence↗
PubMed1996

Identification of a surface glycoprotein on African green monkey kidney cells as a receptor for hepatitis A virus.

Very little is known about the mechanism of cell entry of hepatitis A virus (HAV), and the identification of cellular receptors for this picornavirus has been elusive. Here we describe the molecular cloning of a cellular receptor for HAV using protective monoclonal antibodies raised against susceptible African green monkey kidney (AGMK) cells as probes. Monoclonal antibodies 190/4, 235/4 and 263/6, which reacted against similar epitopes, specifically protected AGMK cells against HAV infection by blocking the binding of HAV. Expression cloning and nucleotide sequence analysis of the cDNA coding for epitope 190/4 revealed a novel mucin-like class I integral membrane glycoprotein of 451 amino acids, the HAV cellular receptor 1 (HAVcr-1). Immunofluorescence analysis indicated that mouse Ltk- cells transfected with HAVcr-1 cDNA gained limited susceptibility to HAV infection, which was blocked by treatment with monoclonal antibody 190/4. Our results demonstrate that the HAVcr-1 polypeptide is an attachment receptor for HAV and strongly suggest that it is also a functional receptor which mediates HAV infection. This report constitutes the first identification of a cellular receptor for HAV.

Amino Acid Sequence↗
PubMed1994

Residues essential for catalytic activity of soybean beta-amylase.

To determine which amino acid residues are essential for the catalytic activity of soybean beta-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in beta-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean beta-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in beta-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50 degrees C to 30 degrees C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.

Amino Acid Sequence↗
PubMed1993

Expression and mutation of soybean beta-amylase in Escherichia coli.

The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence. Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively. All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E. coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.

Amino Acid Sequence↗
PubMed1993

Affinity purification of beta-amylases originating from plant using cyclomaltohexaose-immobilized Sepharose 6B in the presence of ammonium sulfate.

This paper reports a novel method of affinity purification of soybean and barley beta-amylase on cyclomaltohexaose-immobilized Sepharose. Until now, it has been shown that sweet potato beta-amylase can be purified using the above absorbent but beta-amylases from soybean and barley seeds cannot. We found that soybean and barley beta-amylase becomes adsorbed specifically on the above absorbent if it is in solution with 1 to 2 M ammonium sulfate, and the adsorbed enzyme can be easily eluted with a buffer containing no ammonium sulfate. Employing this procedure, soybean beta-amylase was demonstrated to be purified about 10-fold to homogeneity as judged from analysis of both a sodium dodecyl sulfate-gel electrophoresis and the specific activity, using a crude enzyme preparation (sp act 95 U/mg) as a starting material. The specific activity of this highly purified enzyme (950 U/mg) was almost the same as that of crystallized soybean beta-amylase at 37 degrees C.

Ammonium Sulfate↗
PubMed1993

[Molecular epidemiology of hepatitis A virus].

The genome of hepatitis A virus (HAV) is a linear plus-strand RNA molecule of 7,500 nucleotides and it shares common strategies of construction and function of the picornavirus family. Since it has a unique nucleotide sequence homology, HAV has been classified in the genus of hepatovirus, newly added to the family. Nucleotide sequence of the putative VP1/2A junction area was found variable and a 168 nucleotide portion of the region has been compared with many HAV sequences obtained from all over the world. It was found that HAV strains could be identified and classified into 7 genotypes or 9 subgenotypes. Analyses of the nucleotide sequence homology of this particular region is useful, not only in the study of epidemiology of hepatitis A, but also in the study of the molecular epidemiology of HAV.

Amino Acid Sequence↗
PubMed1992

Genetic relatedness of hepatitis A virus strains recovered from different geographical regions.

A pairwise comparison of the nucleic acid sequence of 168 bases from 152 wild-type or unique cell culture-adapted strains of hepatitis A virus (HAV) revealed that HAV strains can be differentiated genetically into seven unique genotypes (I to VII). In general, the nucleotide sequence of viruses in different genotypes differs at 15 to 25% of positions within this segment of the genome. Viruses from four of the genotypes (I, II, III and VII) were recovered from cases of hepatitis A in humans, whereas viruses from the other three genotypes (IV, V and VI) were isolated only from simian species developing a hepatitis A-like illness during captivity. Among non-epidemiologically related human HAV strains, 81 were characterized as genotype I, and 19 as genotype III. Within each of these major genotypes, there were two distinct groups (subgenotypes), which differed in sequence at approximately 7.5% of base positions. Each genotype and subgenotype has a characteristic amino acid sequence in this region of the polyprotein, with the most divergent genotypes differing at 10 of 56 residues. Strains recovered from some geographical regions belonged to a common (endemic) genotype, whereas strains from other regions belonged to several, probably imported, genotypes. Thus, HAV strains recovered in North America were for the most part closely related at the nucleotide sequence level, whereas in other regions, such as Japan and Western Europe, HAV strains were derived from multiple genotypes or sub-genotypes. These data indicate that patterns of endemic transmission can be differentiated from situations in which infections are imported due to travel.

Amino Acid Sequence↗
PubMed1992

[Family-acquired hepatitis A--prevalence of hepatitis A among the family in Aichi Prefecture, 1990].

We studied the transmission of hepatitis A virus (HAV) in 45 families, which members were diagnosed as hepatitis A in 8 hospitals in 1990. Feces and sera from 50 patients and their 126 family members were tested for HAV-specific antigen and IgM antibody by ELISA or polymerase chain reaction (PCR) technology. From the interval of the onset of hepatitis or detection of HAV antigen in feces, HAV transmission was recognized in 11 (24.4%) of 45 families. The transmission was found to be concerned with contacts of the children and that from children to parents was found in 4 families and the reverse in 2. HAV antigen was detected from feces of 4 family members before onset of icterus by ELISA and furthermore, 3 by PCR. It was indicated that these methods would be used to prevent the transmission in a family, day-care centers, or institutions for the mentally retarded.

Adolescent↗
PubMed1987

Expression of soybean glycinin subunit precursor cDNAs in Escherichia coli.

As the cDNAs encoding A1aB1b and A2B1a subunit precursors of the glycinin A2 subfamily contain a unique NcoI site sequence, (A)CCATGG, occurring at their translation initiation sites, plasmids were constructed to direct the synthesis of those precursor proteins by inserting NcoI/PstI fragments derived from those cDNA clones into the NcoI/PstI-pKK233-2 expression vector in Escherichia coli MV1190, respectively. The resultant plasmids directed the expression of 57-kDa protein components that have molecular masses in agreement with those of the in vitro translation products directed by glycinin A2 subfamily mRNAs, by the addition of isopropyl beta-D-thiogalactoside. These proteins, which comprised as much as 1% of the total bacterial protein, are immunoprecipitable with rabbit antibodies specific for glycinin subunits. This procedure makes glycinin subunits available as a model for studying structure-function relationships in seed proteins using site-directed mutagenesis. This is the first expression of glycinin-like storage protein in E. coli.

DNA↗
PubMed1981

Strain-specific aggregation of enterovirus by dextran sulfate.

Dextran sulfate aggregates several enteroviruses depending not only on the pH, the ionic strength of the medium, but also on the protein content of the fluids and on strain specificities of the viruses. The aggregation effect was measured by filtration experiments, by sedimentation in the ultracentrifuge and by electron microscopy. The well known inhibiting effect of dextran sulfate on plaque formation may be due to its aggregating effect: A very strong inhibition of the release of matured virions from the infected cells is observed in medium containing dextran sulfate, whereas the adsorption process is inhibited much less compared with PBS controls. The maximal effect on virus aggregation, plaque size and virus release is observed at the same concentration of dextran sulfate.

Cytopathogenic Effect, Viral↗