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Biomedical subjects

A Machida

Publications and source records attributed to A Machida.

At least 37 records · Page 2Linked to original sources
PubMed1992

Two distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core protein.

Four distinct genotypes of hepatitis C virus types I, II, III and IV have been identified by comparison of nucleotide sequences of isolates from different areas of the world. We examined the possibility that hepatitis C virus may have serologically definable subtypes. Enzyme-linked immunosorbent assay systems were prepared by use of two synthetic peptides deduced from the putative core protein of hepatitis C virus. The following are the two peptides that were used: (a) IPKARRPEGRTWAQPGY (subtype-1) conserved in hepatitis C virus isolates with type I and type II genotypes; and (b) IPKDRRSTGKSWGKPGY (subtype-2) conserved in type III and type IV genotypes. With the enzyme-linked immunosorbent assays, the subtype-1 antibodies were detected in 26 (68%) of 38 subjects whose hepatitis C virus RNA had been genotyped as type I or type II, whereas subtype-2 antibodies were not detected. Inversely, the subtype-2 antibodies were detected in 10 (56%) of 18 subjects with hepatitis C virus RNA genotypes III or IV, whereas subtype-1 antibodies were detected in none of them. These results suggest that hepatitis C virus has two serologically distinguishable core antigen subtypes, corresponding to either genotype I/II or genotype III/IV. Subtyping of HCV by serological methods would contribute to tracking transmission routes of the virus, especially in cases where serum samples were not stored under conditions to preserve RNA or in infected hosts who have cleared the virus and therefore have only antibodies remaining to identify the infection.

Amino Acid Sequence↗
PubMed1992

Hepatitis B surface antigen particles of subtypes adw and adr, and compound subtype (adwr) in symptom-free carriers in Japan.

Of sera from 1,878 Japanese blood donors who carried hepatitis B surface antigen (HBsAg), 420 were subtyped as adw (22.4%) and 1,443 as adr (76.8%); only 15 (0.8%) contained HBsAg of subtype ayw or ayr. Sera with HBsAg/adr had higher HBsAg titres than those with HBsAg/adw (geometric mean of haemagglutination titre: 10.1 +/- 2.4 vs. 9.7 +/- 2.4, p less than 0.01), and a higher prevalence of hepatitis B e antigen (24% vs. 13%, p less than 0.001). Carriers of HBsAg/adr progressively predominated over those of HBsAg/adw with increasing age. Of sera from 1,863 carriers of HBsAg/adw or HBsAg/adr, 182 (9.8%) contained HBsAg particles with both subtypic determinants in the w/r allele. The presence of w and r determinants on the same particles was ascertained by sandwiching them between monoclonal antibody with the specificity for w and that with the specificity for r. HBsAg particles of compound subtype (adwr) were found more often in sera with hepatitis B e antigen than those without it (145/403 [36.0%] vs. 37/1,460 [2.5%], p less than 0.001). Sera with HBsAg/adwr particles had HBsAg titres higher than those without them (12.4 +/- 1.9 vs. 9.7 +/- 2.3, p less than 0.001). HBsAg/adwr particles arise from phenotypic mixing of the S-gene product of wild-type virus and that of mutants with point mutations for subtypic changes. The results obtained indicated that HBV strains of subtype adr have a higher replicative activity than those of adw, and suggested that mutations in the S gene for subtypic changes would be associated with an active replication of hepatitis B virus.

Adult↗
PubMed1992

Intra- and extracellular localization of hyaluronic acid and proteoglycan constituents (chondroitin sulfate, keratan sulfate, and protein core) in articular cartilage of rabbit tibia.

To demonstrate the intra- and extracellular localization of hyaluronic acid (HA) in articular cartilage of the rabbit tibia, biotinylated HA binding region, which specifically binds to the HA molecule, was applied to the tissue. In comparison with the localization of HA, that of chondroitin sulfate (CS), keratan sulfate (KS), and the protein core (PC) of the proteoglycan was examined by immunohistochemistry. Strong positive staining for HA was detected in chondrocytes located in the transition between the superficial and middle zones of the tissue. Pre-treatment with chondroitinase ABC, keratanase II, or trypsin enhanced the stainability for HA in peri- and intercellular matrices. Immunohistochemistry with or without enzymatic pre-treatment demonstrated that immunoreactivity for CS, KS, and PC was distinctly discerned in chondrocytes and in the extracellular matrix located in the middle and deep zones. In particular, the immunoreactivity for KS and PC was augmented by pre-treatment with chondroitinase ABC not only in chondrocytes but in the extracellular matrix located in the middle and deep zones. Microbiochemical analysis corresponded well with histochemical and immunohistochemical results. These results suggest that HA is abundantly synthesized and secreted in chondrocytes located in the transition between the superficial and middle zones.

Aggrecans↗
PubMed1992

Mutations within the S gene of hepatitis B virus transmitted from mothers to babies immunized with hepatitis B immune globulin and vaccine.

A variant of hepatitis B virus (HBV) having a specific mutation within the S gene has been found to infect vaccinees. To know whether similar variants were involved in Japan, we analyzed two cases of maternal transmission of HBV in infants immunized with hepatitis B immune globulin and hepatitis B vaccine. DNA clones of HBV S genes were propagated from patients and family members and sequenced. In one family, the DNA clones from the baby patient had a Gly-to-Arg mutation at the 145th codon of the S gene, whereas those from her mother had no such mutations. In the other family, all the DNA clones obtained from the two infected children had the 145th codon intact, but they had a missense mutation at the 126th codon of the S gene, causing an amino acid substitution of Asn for Thr or Ile. This same mutation was observed in 12 of 17 clones of DNA obtained from their mother. In comparison with the wild type HBV-derived hepatitis B surface antigen, the two types of S gene mutations, either at the 145th or the 126th codon, were associated with a significant decrease in the antigenicity of some determinants on the hepatitis B surface antigen, measured by MAb. Amino acid substitution at these sites, therefore, would have induced the escape from conventional vaccines that were S gene products of wild type HBV and also from hepatitis B immune globulin, whose main components were probably also antibodies against the S gene products expressed by wild type HBV.

Adult↗
PubMed1991

Molecular heterogeneity of e antigen polypeptides in sera from carriers of hepatitis B virus.

Hepatitis B e Ag (HBeAg) was isolated from pooled sera of carriers, without abnormalities in liver function, by affinity column chromatography with mAb against HBeAg. HBeAg polypeptide with an estimated molecular size of 20,000 Da (p20e) was detected, in addition to regular HBeAg polypeptides (p17e/p18e). p20e, as well as p17e/p18e, did not bind with mAb against the carboxyl-terminal domain of the C-gene product. p20e disclosed an N-terminal sequence of MQLFHLXLII- (X unknown), whereas p17e had that of SKLXLGXLXGMDIDPXKEFG- (X's unknown). By comparing them with the amino acid sequence encoded by the precore region and C gene of hepatitis B virus DNA, p20e was deduced to possess amino acids 1 to 19 of the precore-region product at the N-terminus, which contains signal sequence and usually removed before the secretion of HBeAg. p17e had amino acids 20 to 29 of the precore-region product that continued to the C-gene product. Inasmuch as p20e was invariably detected in HBeAg preparations from carriers without evidence for liver disease, it would not have been released into the circulation from destructed hepatocytes. HBeAg polypeptide bearing an uncleaved signal sequence would help in further understanding the mechanism of HBeAg secretion.

Amino Acid Sequence↗
PubMed1991

The effect of sodium hyaluronate on the migration of rabbit corneal epithelium. II. The effect of topical administration.

The effect of topically administered sodium hyaluronate (Na-HA) on the healing of corneal epithelial defect was investigated using rabbit eyes. The corneal epithelium was removed surgically or with iodine vapor or n-heptanol, and saline was administered to one eye as the control, and 0.1% or 0.25% Na-HA with a molecular weight of 87.3 x 10(4) to the other eye once daily. The area of epithelial defect was measured once daily before a topical administration, and the healing rate of epithelial defect was calculated. When the corneal epithelium was removed with iodine vapor, a topical administration of 0.1% or 0.25% Na-HA did not significantly accelerate the epithelial healing. But when removed surgically or with n-heptanol, the healing rates of the corneas treated with 0.25% Na-HA significantly exceeded those of the control eyes. When the epithelium was removed surgically, treatment with 0.1% Na-HA also significantly accelerated the healing. To determine why the effect of Na-HA differed in these three models with the epithelial defect, the amount of fibronectin (FN) produced by the cornea were investigated. The amount of FN produced was determined from the concentration of FN in the medium obtained after incubation of the corneo-scleral section with corneal epithelial defect, and the amount of Na-HA retained on that cornea was estimated from radioactivity detected in tears and cornea after a topical administration of 14C-labeled Na-HA (14C-Na-HA). The corneo-scleral section whose corneal epithelium had been removed surgically, or with n-heptanol, produced a significantly larger amount of FN than that whose corneal epithelium had been removed with iodine vapor. In addition, the amount of 14C-Na-HA retained on the cornea of the first or second model also significantly exceeded that on the cornea of the third model. The topical administration of Na-HA would thus appear to accelerate the healing of the epithelial defect producing a larger amount of FN or retaining a larger amount of Na-HA.

Administration, Topical↗
PubMed1991

Nucleotide sequence of the genomic RNA of hepatitis C virus isolated from a human carrier: comparison with reported isolates for conserved and divergent regions.

The complete nucleotide sequence of a hepatitis C virus derived from plasma of a human carrier in Japan was determined. The cDNA of the isolate (HC-J6) contained 9481 nucleotides and an additional T stretch of 30 to 108 nucleotides at the 3' end, and had one large open reading frame coding for a polyprotein of 3033 amino acids. It differed by 31.8 to 32.1% in the nucleotide sequence and by 27.4 to 27.7% in the amino acid sequence from an American isolate and two Japanese isolates previously reported. Among these four isolates, the 5' non-coding region of 329 to 341 nucleotides was well conserved (greater than 93% identity), whereas the 3' non-coding region of 39 to 45 nucleotides (T stretches not included) was more variable (greater than 30% identity). An excellent degree of conservation of the 5' non-coding region would reflect its pivotal role in replication, and primers deduced from this region could be applied for the sensitive and specific detection of viral RNA by polymerase chain reaction. Due to a high degree of similarity in the amino acid sequence of the putative core protein (greater than 90%), antigen probes deduced from it would be suitable for the serological diagnosis of HCV infection. Low sequence similarity in the putative envelope protein (greater than 53% identity), however, would have to be taken into account in considering the immunoprophylaxis of HCV infection.

Amino Acid Sequence↗
PubMed1991

Phosphorylation in the carboxyl-terminal domain of the capsid protein of hepatitis B virus: evaluation with a monoclonal antibody.

The capsid protein of hepatitis B virus (p21c) is made of 183 amino acids coded for by the C gene. By using p21c isolated from Dane particles (hepatitis B virus) as an immunogen, a monoclonal antibody (no. 2212) which recognized an epitope dependent on the phosphorylation of p21c was raised. The binding of no. 2212 antibody to authentic p21c was completely inhibited by a synthetic undecapeptide with a sequence of RRRSQSPRRRR, representing amino acids 165 to 175 of p21c, only when the peptide was phosphorylated. Either or both of Ser-168 and Ser-170 were phosphorylated in p21c in vivo, therefore, and contributed to the manifestation of the epitope. No. 2212 antibody bound to p21c from core particles derived from Dane particles or hepatocellular carcinoma tissues (PLC/342) propagated in nude mice but did not bind to p21c from core particles expressed in Escherichia coli or yeast cells, indicating different states of phosphorylation in them. Nonphosphorylated p21c showed a higher affinity for the viral DNA than did phosphorylated p21c. Since the serum from an asymptomatic carrier, with a high titer for antibody to hepatitis B core antigen, specifically bound to phosphorylated undecapeptide (amino acids 165 to 175), the epitope would stimulate humoral antibody responses in the human host.

Amino Acid Sequence↗
PubMed1990

Synthetic oligopeptides bearing a common or subtypic determinant of hepatitis B surface antigen.

Two determinants of hepatitis B surface Ag (HBsAg), identified by mAb raised against polypeptide components, were characterized immunochemically. One was expressed on HBsAg irrespective of the four major subtypes, i.e., adw, adr, ayw, and ayr, whereas the other was subtypic but not identical to any of d, y, w, and r determinants. The common determinant was generated by a synthetic pentadecapeptide with a sequence of Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-Ile-Pro-Ala-Gln representing amino acids 115-129 of the S gene product, and detected invariably in 366 HBsAg samples in sera from asymptomatic carriers in Japan. The activity of the S gene product, as well as the peptide (115-129), to bind with the mAb was not affected by alkylation alone, but was completely lost after reductive alkylation. The antigenic activity was lost when the S gene product was severed between Lys122 and Thr123 by trypsin. A microconformation maintained by the -Cys121-Cys124 bond, therefore, would be required for the common determinant. The other mAb identified an epitope of HBsAg that was mimicked by a synthetic tetradecapeptide with a sequence of Thr-Cys-Thr-Ile-Pro-Ala-Gln-Gly-Thr-Ser-Met-Phe-Pro-Ser, representing amino acids 123-136 of the S gene product. Among 16 HBsAg samples with known S gene sequences, 5 with Ile126 possessed this subtypic determinant, but the remaining 11 with Thr126 did not. The 5 hepatitis B virus genomes encoding the subtypic determinant differed less than 5.6% from each other in the entire nucleotide sequence, but by 8.0% or more from any of the other 11 genomes without the capacity to encode it.

Alkylation↗
PubMed1990

The effect of sodium hyaluronate on the migration of rabbit corneal epithelium. I. An in vitro study.

The effect of sodium hyaluronate (Na-HA) on the migration of corneal epithelium was investigated in vitro using corneo-scleral sections of rabbits. The effect on migration was evaluated by the length of migrating epithelium on the cut surface of corneal stroma after incubation in media containing Na-HA of various molecular weights or concentrations. The medium concentration of Na-HA was 400 micrograms/ml, and the molecular weights were 0.4 x 10(4), 2 x 10(4), 10 x 10(4), 68 x 10(4), 102 x 10(4) and 216 x 10(4). Regarding Na-HA with a molecular weight of 102 x 10(4); media containing Na-HA at concentrations of 50, 100, 200, 400, 800, 2000 and 4000 micrograms/ml were also investigated. When the concentration of Na-HA was 400 micrograms/ml, Na-HA with molecular weights of over 68 x 10(4) accelerated significantly the epithelial migration. Regarding Na-HA with a molecular weight of 102 x 10(4), acceleration was observed at concentrations of 200, 400 and 800 micrograms/ml. Furthermore, the concentration of fibronectin (FN) in medium containing the effective Na-HA was significantly lower than that in the control medium after the incubation. These results suggest a possibility that the accelerative effect of Na-HA depends on the reduction of FN release from the cornea or the facilitation of FN deposition on the cornea.

Animals↗
PubMed1990

Gamma-interferon production in response to hepatitis B core protein and its synthetic peptides in patients with chronic hepatitis B virus infection.

The nucleocapsid of hepatitis B virus (HBV) is an efficient immunogen in activating T cells to produce interferon-gamma (IFN-gamma) in patients with chronic HBV infection. We investigated hepatitis B core antigen (HBcAg)-specific T cell recognition, which seems to be implicated in the pathogenesis of chronic liver disease. IFN-gamma production by peripheral-blood mononuclear cells of patients with chronic HBV infection [25 patients with chronic active hepatitis (CAH) and 14 asymptomatic carriers of HBV (ASCs)] was measured by an enzyme-linked immunosorbent assay. P19 polypeptide, which is derived from recombinant HBcAg particle (rHBcAg), increased IFN-gamma production in patients with CAH, but its effect was weaker than that of rHBcAg. P19 had no stimulating effect on T cells from ASCs. The fine specificity of T cell recognition of HBcAg was examined using 8 kinds of synthetic peptides. T cells from the patients who responded against P19 polypeptide recognized the sites within the common sequences of HBcAg and HBeAg (p72-90, P90-99, P108-122 and P126-146). These results suggest that HBcAg and P19 are cross-reactive at the T cell level, and that these T cells recognize the sites within the common sequences of HBcAg and HBeAg in HBV-infected patients.

Adult↗
PubMed1990

Common and subtypic determinants of hepatitis B surface antigen particles: susceptibility to reduction and/or alkylation evaluated with monoclonal antibodies.

The specificity of five monoclonal antibodies, three raised against hepatitis B surface antigen (HBsAg) particles and two against envelope polypeptides, was tested for on a panel of 366 sera containing HBsAg of various subtypes (131 adw, 146 adr, 39 ayw and 50 ayr). Three monoclonals bound to HBsAg irrespective of subtypes, and therefore, were directed to the common antigenic determinants of HBsAg. Of these, two raised against particles (No. 824 and No. 7922) did not bind with reduced HBsAg particles. The other raised against peptides (No. 5124) bound to reduced HBsAg particles. It did not, however, bind to reduced and alkylated HBsAg particles, thereby indicating that it was directed to an epitope involving cysteine residues not contributing to the conformation. The remaining two monoclonals were directed to subtypic determinants not identical to any of d, y, w and r determinants. The subtypic determinant detectable by one of them (No. 4403), raised against HBsAg polypeptides, markedly increased after reduction of HBsAg particles with or without alkylation. In contrast, the subtypic determinant, detectable by the other monoclonal (No. 2155) raised against particles, substantially decreased after reduction. Non-identity of common or subtypic determinants detectable by the five monoclonals were established by blocking tests in which labeled antibody was competed by non-labeled antibody, of a homologous or heterologous specificity, for the binding with HBsAg. These monoclonals would be useful in studies for immunochemical configuration of HBsAg particles and epidemiology of novel subtypic determinants.

Alkylation↗
PubMed1990

Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region.

The 5'-noncoding region of hepatitis C virus (HCV) genomes is highly conserved. A two-stage polymerase chain reaction (PCR), involving two pairs of primers deduced from the 5'-noncoding region of the HCV genome, was developed for a sensitive and specific detection of HCV RNA. The first stage of PCR was performed for 35 cycles with primers capable of multiplying fragments of 221 base pairs. PCR products in samples negative for HCV RNA were subjected to the second stage of PCR for 30 cycles with primers located internal to those employed in the first stage of PCR. The two-stage PCR detected up to 10 chimpanzee infectious doses/ml of HCV, and HCV RNA in 11 (92%) of 12 sera from patients with chronic non-A, non-B hepatitis without detectable antibodies to HCV by a commercial assay kit. Primers from the 5'-noncoding region of the HCV genome would be suitable for detecting HCV RNA by PCR, since the other regions of the HCV genome diverge extensively in sequence because of its nature as an RNA virus.

Animals↗
PubMed1989

Antigenic sites on the arginine-rich carboxyl-terminal domain of the capsid protein of hepatitis B virus distinct from hepatitis B core or e antigen.

The capsid protein of hepatitis B virus (P19) is made of 183 amino acids and carries the antigenic sites of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) on the amino-terminal domain. The carboxyl-terminal domain of P19 (amino acids 150-183) is arginine-rich (47%) and faces the interior of the nucleocapsid for the binding with DNA. Monoclonal antibody was raised against an antigenic site on this protamine-like region of P19, which was distinct from HBcAg or HBeAg sites, and the novel antigenic site(s) was provisionally designated as hepatitis B inner core antigen (HBicAg). When P19 in a low concn (150 ng/ml) was immobilized on the solid surface, HBicAg sites were preserved, while HBcAg or HBcAg sites were no longer available on it. This allowed the detection of antibodies against HBicAg (anti-HBic), by sandwiching them between immobilized P19 and anti-IgG labeled with horseradish peroxidase. Anti-HBic was detected in sera from HBsAg carriers, typically those seropositive for antibody to HBeAg. A synthetic arginine-rich decapeptide, with a sequence of Arg-Arg-Arg-Gly-Arg-Ser-Pro-Arg-Arg-Arg, representing amino acids 150-159 of P19 and conserved in the majority of reported hepatitis B virus, absorbed the activity to bind with P19 in seven (44%) out of 16 sera containing anti-HBic. These results indicate that the decapeptide carries an HBicAg epitope and the remaining amino acid sequence of the arginine-rich carboxyl terminal domain (160-183) may be responsible for the other HBicAg epitopes.

Amino Acid Sequence↗
PubMed1988

Antibody against the translation product of the pre-S2 region of hepatitis B virus and the resolution of infection after accidental needlestick exposure.

During a 5-yr period before the introduction of post-exposure immunoprophylaxis of hepatitis B virus (HBV), 363 medical personnel contracted infection after exposure to a contaminated needlestick. Among them, five (1.4%) developed a persistent carrier state, and they were followed for antibodies against translation product of the pre-S2 region (anti-pre-S2) by the enzyme immunoassay with native viral polypeptides. The consecutive seven cases in whom infection was terminated served as controls. In the acute phase, the levels of IgM and IgG anti-pre-S2 were significantly higher in the seven in whom infection resolved than in the five in whom infection persisted (mean sample:normal ratio, 11.1 +/- 4.4 vs 2.0 +/- 1.3 and 8.7 +/- 7.0 vs 3.0 +/- 1.1, respectively, p less than 0.05). In convalescence, 6 months after the normalization of serum transaminase, IgG anti-pre-S2 was detected in a high titer in the staff members with resolved infection (27.4 +/- 38.5), but was not detectable in those with persistent infection. On the basis of these observations, the intensity of humoral antibody response against the pre-S2 region product is closely correlated with the resolution of infection after exposure to HBV, and anti-pre-S2 persists in the circulation much longer than previously reported.

Accidents, Occupational↗
PubMed1987

A synthetic peptide coded for by the pre-S2 region of hepatitis B virus for adding immunogenicity to small spherical particles made of the product of the S gene.

Small spherical particles produced in the non-permissive phase of hepatitis B virus infection, when the viral genome is integrated into the chromosome of hosts, are rich in the product of the S gene, but poor in the product of the pre-S2 region. For the purpose of adding immunogenicity to spherical particles deficient in the pre-S2 region product, they were conjugated with a synthetic peptide of 19 amino acid residues. The peptide reproduced a hydrophilic area of the pre-S2 region product encoded by viral genomes of subtypes adr, ayw and ayr. The spherical particles supplemented with the pre-S2 peptide raised antibody to the pre-S2 region product in mice, in addition to antibody to the product of the S gene. Antibody to pre-S2 region product, prepared from sera of immunized mice by absorption with the S gene product, bound to spherical particles bearing pre-S2 region product, irrespective of adr, adw, ayw or ayr subtype, and agglutinated hepatitis B virions in immune electron microscopy. Based on the results obtained, the synthetic peptide may prove useful in adding protective efficacy to small spherical particles poor in pre-S2 region product.

Absorption↗