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Biomedical subjects

M Kohara

Publications and source records attributed to M Kohara.

At least 73 records · Page 4Linked to original sources
PubMed1994

The position of the mandibular condyle at maximal mouth opening in normal subjects.

PURPOSE: To investigate the position of the mandibular condyle at maximal mouth opening in individuals 19 to 30 years of age with no functional disorders of the masticatory system. PATIENTS AND METHODS: Measurements were made in 153 subjects (99 men, 54 women), and 41 men and 44 women had transcranial radiographs taken of their right and left temporomandibular joints in the closed and maximal opening positions. RESULTS: In all subjects the condyle moved forward beyond the summit of the articular eminence, and in more than half the subjects the condyle was located inferior to the eminence at maximal opening. CONCLUSION: The correlation between maximal opening and anterior shift of the condyle from the summit of the eminence was significant, but the correlation with vertical shift of the condyle was not significant. The correlation between the form of the glenoid fossa (length and depth) and anterior or vertical shift of the condyle from the summit of the eminence also was not significant.

Adult↗
PubMed1994

Optimization for the detection of hepatitis C virus antigens in the liver.

To optimize the detection of hepatitis C viral antigens in liver tissue, cryostat and formalin-fixed, paraffin-embedded liver sections from 21 patients with chronic hepatic C viral infection were studied. For cryostat sections, six different fixatives were compared. Sixteen primary antibodies were tested: nine different mouse monoclonal anti-hepatitis C virus-core antibodies, a human monoclonal anti-hepatitis C virus-non-structural 4, and six rabbit polyclonals directed against synthetic peptides of the hepatitis C virus core, envelope, and non-structural 3, non-structural 4, non-structural 5. Three detection systems, 3- and 5-step peroxidase-antiperoxidase and avidin-biotin complex, were examined. In cryostat sections, acetone/chloroform formation consistently produced the best signal-to-background ratio. Five anti-hepatitis C virus-core monoclonals which recognize amino acid sequence 26-45 of the hepatitis C virus-core region consistently detected the viral antigen, but not the monoclonals directed against 39-74 of the hepatitis C virus-core region. The human anti-hepatitis C virus-non-structural 4, which reacts to amino acid sequence 1700-1705, also regularly detected viral antigen. The rabbit polyclonals produced either negative or nonspecific staining. The 5-step peroxidase-antiperoxidase provided the strongest signal and the avidin-biotin system produced high background consistently. Overall, hepatitis C virus core and non-structural 4 antigens were detected in 71% and 57% of the patients studied. Of the 16 patients seropositive for hepatitis C virus RNA, 75% and 69% had detectable hepatitis C virus core and non-structural 4, in contrast to 60% and 20% of the five hepatitis C virus RNA seronegative patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Viral↗
PubMed1994

Hepatitis C virus particle detected by immunoelectron microscopic study.

To clarify the morphology of hepatitis C virus (HCV), an indirect immunogold electron microscopic study was carried out on two plasma samples with high HCV RNA titres using polyclonal and monoclonal antibodies specific to the putative HCV envelope protein. Spherical virus-like particles, 55 to 65 nm in diameter with spike-like projections, were found in 1.14 to 1.16 g/ml fractions after sucrose density gradient centrifugation. These particles were found only in HCV-infected blood donors and had morphological features similar to those of flaviviruses. Moreover, these particles specifically reacted with the polyclonal and monoclonal antibodies to the putative HCV envelope protein. This is the first known report in which the morphology of the HCV particle is clearly shown.

Antibodies↗
PubMed1994

Significance of specific antibody assay for genotyping of hepatitis C virus.

Group I and II hepatitis C virus genotypes were determined by a newly developed serological genotyping assay. This assay detected antibodies against group-specific recombinant proteins in the putative NS4 protein region (amino acid no. 1676-1760) by an enzyme-linked immunosorbent assay. This region of the hepatitis C virus peptide has many group-specific amino acids; fewer than 50% of these amino acids are identical between groups I and II. Genotypes determined by the serological genotyping assay were compared with those determined by a method in which the polymerase chain reaction was used in 91 chronic hepatitis patients. The group-specific polymerase chain reaction was performed within the genome region corresponding to the putative NS5 protein, where the group II hepatitis C virus genome is 57 nucleotides longer than that of group I. Among 91 chronic hepatitis C patients who had positive results in the second-generation hepatitis C virus antibody (core and NS3 region) assay, hepatitis C virus RNA was detected in 80 patients by polymerase chain reaction in the 5' untranslated region and in 78 patients by this group-specific polymerase chain reaction. As a result, in 76 of 91 patients (84%) genotypes determined by the serological genotyping assay showed complete agreement with those determined by the group-specific polymerase chain reaction, and none of the patients revealed a group opposite to that of hepatitis C virus genotype. The detection rate of the serological genotyping assay (89 of 91; 98%) was even higher than that of the polymerase chain reaction assay (78 of 91; 86%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[Specific antibody assay for genotyping of hepatitis C virus].

Based on their nucleotide sequences and the deduced amino acid sequences, most hepatitis C virus (HCV) isolates have been classified into two genotypes, type 1 (subtype 1a and 1b) and type 2 (subtype 2a and 2b). Using part of the putative NS4 protein region (amino acids No. 1676-1760) as antigens, an enzyme-linked immunosorbent assay (ELISA) was developed. The results of genotyping of HCVs in chronic hepatitis patients by this ELISA system were almost completely consistent with those by PCR. The genotype 1 HCV infected patients showed a poor response to interferon (IFN) therapy. In contrast, genotype 2 HCV infected patients showed good response. The results suggest that genotype 1 and 2 HCVs possess different biological properties.

Amino Acid Sequence↗
PubMed1994

Fetal heart rate recorder for long-duration use in active full-term pregnant women.

We sought to assess the signal consistency and accuracy of a long-duration fetal heart rate (FHR) recorder (a 1-MHz ultrasonic Doppler transducer composed of six miniprobes and an autocorrelation technique) in active, full-term pregnant women. The FHR data of 15 normal full-term fetuses were obtained every 250 milliseconds in various maternal positions using the new Doppler system or a direct scalp electrocardiographic (ECG) transducer. Differences between simultaneous Doppler and ECG measures were small and within acceptable limits except for short-term variability. Signal loss, assessed in 15 subjects in various positions, was less than 10% except during sitting or walking. The mean (+/- standard deviation) percentages of FHR signals lost over 2 hours in bed, 1 hour out of bed, and 24 hours in and out of bed were 1.2 +/- 2.1, 12.9 +/- 16.2, and 3.8 +/- 4.4, respectively. This system for recording FHR is suitable for clinical research and routine FHR monitoring, and provides data comparable to Holter ECG monitoring in patients with heart disease.

Electrocardiography↗
PubMed1993

[Antigenicities of groups I and II hepatitis C virus].

HCV genomes are considerable heterogeneities in nucleotide and amino acid sequences among individual isolates. The primary structure of the putative core and NS3 protein regions are relatively conserved among HCV isolates, while those of envelope proteins (E1 and E2) and NS4 are variable. On the basis of nucleotide sequence homology of parts of HCV genomes, several research groups have reported the possible existence of multiple subtypes of HCV isolates. From comparative sequence studies on HCV cDNA clones corresponding to NS3 and NS4 regions followed by diagnostic studies using these clones, have shown that there are at least two groups of HCV, group I and group II. The peptide produced in E. coli, carrying group I and II cDNA clones (A.A. positions 1676-1736 of NS4 region) are recognized by circulating antibodies specific to group I and II HCV. These group specific antigens of NS4 region are highly reliable in detecting (over 90%) circulating antibodies against either groups of HCV. In this study, most of the HCV isolates used were shown to be classified into group I or II HCV based on their amino acid differences and phylogenic analysis within the putative 5'UTR, core and NS3-4 regions. Biological significance of these two groups of HCV is suggested by the observation that the group I HCV was resistance to the IFN therapy (10-20% showing a good response), on the other hand the group II HCV showed a good response (70-90%).

Amino Acid Sequence↗
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↗
PubMed1992

Expression and characterization of glycoprotein gp35 of hepatitis C virus using recombinant vaccinia virus.

Complementary DNA clones corresponding to one of the putative structural regions of the hepatitis C virus (HCV) genome were obtained from sera of non-A non-B hepatitis patients. The putative envelope gene was expressed by using a recombinant vaccinia virus carrying this region of the HCV genome. In cells infected with the recombinant vaccinia virus, a glycosylated protein with an M(r) of about 35K (gp35) was specifically detected by convalescent sera from hepatitis C patients. The sera from rabbits immunized with this recombinant vaccinia virus reacted to the gp35 produced in insect cells and also to gp35 which was translated in vitro in the glycosylated and processed form. The gp35 was used to detect antibodies in sera of only 7 to 23% of HCV patients at various stages of HCV disease. These results suggest that the gp35 of HCV may not have high antigenicity in humans.

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↗
PubMed1992

Performance of an enzyme-linked immunosorbent assay system for antibodies to hepatitis C virus with two new antigens (c11/c7).

We developed an enzyme-linked immunosorbent assay (ELISA) system for antibodies to the hepatitis C virus (HCV), using two new recombinant antigens (c11 and c7) derived from the HCV genome. The performance of this ELISA system (Imucheck HCV Ab) was examined. The CV values for both intra-assay precision and reproducibility of identifying HCV antibody in the panel sera ranged from 3.5% to 6.4%. The blood elements in serum and anticoagulants did not interfere in this ELISA system. The specificity of Imucheck HCV Ab to samples from patients with non-A, non-B (NANB)-type chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma was 93.7%, 93.5%, and 81.4%, respectively. These results are more sensitive than those obtained by the first-generation anti-HCV ELISA system. In the samples from patients with NANB-type acute hepatitis, Imucheck HCV Ab enabled detection of HCV antibodies at an early stage. This system increased the sensitivity for blood donor screening and for monitoring patients with acute hepatitis.

Antibody Specificity↗
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

Immunological and virological characterization of improved construction of recombinant vaccinia virus expressing rinderpest virus hemagglutinin.

We constructed a recombinant vaccinia virus (RVV) expressing rinderpest virus (RPV) hemagglutinin (H) by modifying the promoter region of the original RVV. The promotor region was modified at three points, i.e., an outframe ATG was eliminated, the sequence between the promoter and initiation codon was shortened and the base sequence just upstream of the initiation codon was changed. As compared with the original RVV, the modified RVV was found to produce a remarkably large amount of H protein in infected rabbit kidney cells cultured in vitro and to induce high titers of anti-RPV-H antibodies in rabbits. The median protective doses in rabbits of the modified and of the original RVVs were 10(2) pfu and 10(3.5) pfu, respectively, indicating that the modified RVV was at least 10-times more effective in protection than the original. The neurovirulence of the modified RVV and the parental LC16mO strain was roughly at the same level, and was much lower than that of WR strain. The modified RVV was as heat-stable as the original one. These results indicate that the modified RVV could be a candidate rinderpest vaccine for further examinations including cattle.

Animals↗
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

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↗
PubMed1989

Determinants in the 5' noncoding region of poliovirus Sabin 1 RNA that influence the attenuation phenotype.

A number of recombinants between the virulent Mahoney and attenuated Sabin strains of type 1 poliovirus were constructed by using infectious cDNA clones of the two strains. To identify a strong neurovirulence determinant(s) residing in the genome region upstream of nucleotide position 1122, these recombinant viruses were subjected to biological tests, including monkey neurovirulence tests. The results of the monkey neurovirulence tests suggested the important contribution of an adenine residue (Mahoney type) at position 480 to the expression of the neurovirulence phenotype of type 1 poliovirus. This nucleotide, however, had only a minor effect, if any, on viral temperature sensitivity. Monkey neurovirulence tests on the recombinant virus whose genome had a guanine residue (Sabin type) at position 480 and variants generated from this recombinant virus in the central nervous system of monkeys strongly suggested that only one nucleotide change, from adenine to guanine, was not sufficient for full expression of the attenuation phenotype encoded by this genome region. These results suggest that the expression of the attenuation phenotype depends on the highly ordered structure formed in the 5' noncoding sequence and that the formation of such a structure is possibly influenced by the nucleotide at position 480. Furthermore, in vitro biological tests performed on viruses recovered from the central nervous system of monkeys injected with a temperature-sensitive recombinant virus showing the small-plaque and d phenotypes revealed that most of the recovered viruses had even higher temperature sensitivities and that all of the recovered viruses that had acquired the large-plaque phenotype had lost the d phenotype to some extent. These results indicate that there may be an unknown selection pressure(s) in the central nervous system and that common determinants might be involved in the expression of the small-plaque and d phenotypes.

Animals↗
PubMed1988

Strategy for construction of live picornavirus vaccines.

A number of recombinant viruses between the virulent Mahoney and the attenuated Sabin 1 strains of type 1 poliovirus were constructed in vitro using their infectious cDNA clones. Monkey neurovirulence tests on these recombinant viruses revealed that the surface structure of the virion particle had a little correlation with the neurovirulent phenotype, and that the strong neurovirulence determinant(s) resided in the 5' noncoding sequence. These results in turn led to two possible strategies for constructing live attenuated picornavirus strains. One is to use the Sabin 1 strain as a vector carrying foreign antigenicities. The other is the construction of the attenuated picornaviruses by introducing mutations into the 5' noncoding sequences. The antigenicity of the Sabin 1 strain was successfully changed to those of other poliovirus serotypes without loss of vaccine quality. Furthermore, it was proved that introduction of deletion mutation into the 5' noncoding sequence of genomes of the Sabin 1 and Mahoney strains resulted in construction of viruses with a less neurovirulent phenotype.

Chromosome Deletion↗