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Biomedical subjects

K Okochi

Publications and source records attributed to K Okochi.

At least 19 recordsLinked to original sources
PubMed2001

Detection of serum thermolabile beta-2 macroglycoprotein (Hakata antigen) by enzyme-linked immunosorbent assay using polysaccharide produced by Aerococcus viridans.

Although a serum thermolabile beta-2 macroglycoprotein (TMG) may play a role in host defense as a lectin, little is known of its related physiological functions, mainly due to a lack of appropriate methods for tracing the functions of TMG. We identified a polysaccharide from Aerococcus viridans, PSA, which reacts with TMG, and based on this finding, we developed an enzyme-linked immunosorbent assay to trace the functions of TMG. Using ethanol precipitation and DEAE-Sepharose and Sephacryl S-400 column chromatographies, we isolated PSA from cultured medium of A. viridans, and it exhibited specific binding against TMG in blood samples. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the isolated PSA showed ladder bands that implied the existence of repeating units composed of D-glucose, N-acetyl-D-glucosamine, D-mannose, and D-xylose, as confirmed by gas chromatography-mass spectrometry. SDS-PAGE and immunochemical analysis, using rabbit anti-TMG antibody, showed that PSA specifically binds solely to intact serum TMG but not to TMG heated at 56 degrees C for 30 min, a condition under which antigenicity is lost. TMG in serum samples bound to PSA in a dose-dependent manner, and this binding was clearly suppressed by addition of PSA. These observations indicate that PSA is a useful adsorbent to TMG and can be used to develop appropriate methods for tracing the functions of TMG.

Antibodies, Bacterial↗
PubMed2000

Four putative subtypes of human parvovirus B19 based on amino acid polymorphism in the C-terminal region of non-structural protein.

The nucleotide sequence of 10 isolates of human parvovirus B19 (B19) were determined and compared throughout 96.3% of the open reading frames (4145 nucleotides from nt. 509-4653). In the 4145 nucleotides, 122 mutation sites were found, of which 24 were accompanied by amino acid displacement. Furthermore, the polymorphism of the amino acids was seen in about 110 bases near the carboxy terminal of the non-structural protein, ranging from nt. 2011 to 2123, where four amino acid mutation points were found to exist. Based on the amino acid polymorphism of these four mutation sites in this area, 10 isolates of the B19 parvovirus could be divided into 4 subtypes (subtypes A, B, C, and D). The frequency of isolation of the subtypes depended on the time and location of collection of the B19 viremic blood specimens.

Amino Acid Sequence↗
PubMed2000

Biological response to ionizing radiation in mouse embryo fibroblasts with a targeted disruption of the DNA polymerase beta gene.

Base excision repair (BER) is carried out by two distinct pathways in mammalian cells, one dependent on DNA polymerase beta (Polb) and the other on proliferating cell nuclear antigen (Pcna). We studied whether the Polb-dependent pathway plays an important role in BER in vivo after exposure to ionizing radiation. For this purpose, we used mouse embryo fibroblasts derived from wild-type and Polb gene knockout littermates. Both cell lines had essentially the same clonogenic cell survival and low levels of apoptosis as determined by a colony formation assay and by a change in mitochondrial membrane potential, respectively. No significant cleavage of protein kinase C delta (Pkcd) in vivo, which is a substrate for caspase 3, was detected, and intact Pkcd was retained in both cell lines for at least 72 h after irradiation. Similar significant increases in caspase 3-like activities as measured by Asp-Glu-Val-Asp (DEVD) cleaving activity in vitro were observed in both cell lines after irradiation. Radiation induced cell cycle arrest in the form of a G(2)-phase block, and G(2)/M-phase fractions reached a peak approximately 10 h after irradiation and decreased thereafter with a similar time course in both cell lines. Similar levels of chromatin-bound Pcna were observed immediately after irradiation in non-S-phase cells of both cell lines and disappeared by 4 h after irradiation. We conclude that the deficiency in Polb does not have a significant influence on the radiation responses of these cells. Together with evidence accumulated in vitro, these results strongly support the idea that the Pcna-dependent pathway predominantly acts in BER of radiation-induced DNA damage in vivo.

Animals↗
PubMed1999

Infection of the erythroid cell line, KU812Ep6 with human parvovirus B19 and its application to titration of B19 infectivity.

A human parvovirus B19 (B19) infectivity assay was developed using the erythroid cell line, KU812Ep6. KU812Ep6 was cloned for high efficiency infection with B19 in vitro, in the presence of erythropoietin by a limiting dilution method from the parent cell line, KU812. B19 was effectively propagated in KU812Ep6 and was detected for B19 antigens, VP1 and VP2. The titers of B19 positive sera measured with KU812Ep6 cells were in the range of 10(6) to 10(8) TCID50 ml. This KU812Ep6 infectivity assay had a 10(3)-10(4.5) higher sensitivity than the colony forming unit-erythroid (CFU-e) injury assay. It was calculated that one TCID50 needed 10(3) B19 genome copies, judging from the infectivity assay and semi-quantitative PCR. The KU812Ep6 infectivity assay was also used to determine infectivity of B19 in vitro, and to evaluate inactivation, as well as clearance of the virus. The inactivation of B19 by heating was carried out and infectivity declined from 10(4) TCID50 ml to < 10 TCID50 ml (lower limit of detection) at 60 degrees C for 3 h or at 70 degrees C for 30 min, but only decreased to 10(2.5) TCID50 ml at 50 degrees C for 8 h.

Antigens, Surface↗
PubMed1999

Efficacy of donor screening for HTLV-I and the natural history of transfusion-transmitted infection.

BACKGROUND: It has been 10 years since the implementation in Japan of donor blood screening for human T-cell lymphotropic virus type I (HTLV-I). This report reviews the effectiveness of screening in preventing transmission of HTLV-I through blood transfusion and the current status of patients with confirmed seroconversion due to transfusions given before the implementation of screening. STUDY DESIGN AND METHODS: Patients who received blood at Kyushu University Hospital from 1990 to 1997 were followed. Serum samples were collected before transfusion and 60 days or more after transfusion. Seroconversion was determined by a second-generation particle agglutination test. Confirmation tests were an immunofluorescence assay, enzyme-linked immunosorbent assay, and immunoblotting. Confirmed seroconverted patients were followed by a search of hospital records. RESULTS: Seroconversion was found in one of 4672 transfused patients, but the donor was identified and confirmed to be negative for anti-HTLV-I and virus genome by nested polymerase chain reaction. A total of 23,323 red cell concentrates and 17,237 platelet concentrates were transfused to these 4672 patients. Therefore, the anti-HTLV-I prevalence in blood for transfusion after screening was estimated at 1 in 45,560 (0.0022%; the upper 95% CI was 0.0080%). One hundred two seroconverted patients who were transfused before donor screening for HTLV-I were followed. One patient developed HTLV-I-associated myelopathy, diagnosed 18 weeks after seroconversion, and another patient developed uveitis 1 month after seroconversion. No patients developed adult T-cell lymphoma, and the survival rate of seroconverted patients was 92.5 percent 15 years after transfusion. CONCLUSION: This study confirmed that the present donor screening program for HTLV-I by the new particle agglutination test can almost completely prevent virus transmission by transfusion. Complications of HTLV-I transmission were at lower rates than expected.

Adolescent↗
PubMed1999

Screening of blood donors for human parvovirus B19 and characterization of the results.

BACKGROUND AND OBJECTIVES: Human parvovirus B19 (B19 virus) can be transmitted through blood transfusion and plasma-derived products. In a previous report, we utilized the simple hemagglutination method based on the interaction between the B19 virus and P antigen on human erythrocytes in order to screen the blood donors. We called this method receptor-mediated hemagglutination (RHA) [Lancet 1995;346:1237-1238]. In this paper, we report on a large-scale screening of the B19 virus by RHA and discuss the results. MATERIALS AND METHODS: Donor sera from September 1995 to March 1997 and seroconversion panels were enrolled. Donor sera were examined by RHA for large-scale screening. The positive sera in the first screening were then further investigated by the RHA inhibition test, countercurrent immunoelectrophoresis (CIE), an enzyme-linked immunosorbent assay, and polymerase chain reaction (PCR). We also evaluated the infectivity and neutralizing activity of various kinds of sera by the erythroid colony forming unit (CFU-e) assay. To examine the detection limits of the B19 virus by RHA, B19-viremic sera were purified by sucrose gradient ultracentrifugation. RESULTS: Among 257,710 sera specimens, 293 sera (0.11%) gave a positive reaction in the first screening using RHA. Out of these 293 sera specimens, 31 were positive for PCR, of which 28 were also RHA inhibition-positive, and 25 of the 28 CIE-positive. In the CFU-e injury assay, all the RHA inhibition (+) sera showed a decrease in the number of erythroid colonies. The RHA inhibition (-) PCR (+) B19 antibody (+) sera did not affect the erythroid colony formation and protected CFU-e from injury by the B19 virus. By measuring the amount of purified B19 protein and its RHA titer, the detection limit of the B19 virus by RHA was calculated to the 0.37+/-0.03 ng/ml. CONCLUSION: These results suggest that the RHA(+) RHA inhibition (+) sera were infectious in vitro. The combination of RHA and the RHA inhibition test is considered to be useful for the large-scale screening of infectious B19 virus in blood donors with high specificity.

Blood Donors↗
PubMed1998

Cloning and characterization of the Hakata antigen, a member of the ficolin/opsonin p35 lectin family.

The Hakata antigen is a novel, thermolabile beta2-macroglycoprotein that reacts with sera from patients suffering from systemic lupus erythematosus. In this study we present the structure and the function of the Hakata antigen. We have identified cDNA clones encoding the Hakata antigen and analyzed its function. The cDNA included a possible open reading frame of 897 nucleotides, encoding 299 amino acids. The Hakata antigen consisted of a collagen-like domain in the middle section and a fibrinogen-like domain in the COOH terminus, both of which are homologous to human ficolin-1 and opsonin P35, indicating that these three molecules form a distinct family. The molecular mass of the Hakata antigen expressed in transfected cells was 35 kDa under reduced conditions, and it formed ladder bands under nonreducing conditions compatible with the previous result that the Hakata antigen exists in serum as homopolymers. Purified Hakata antigen sustained lectin activity, showing affinity with GalNAc, GlcNAc, D-fucose as mono/oligosaccharide, and lipopolysaccharides from Salmonella typhimurium and Salmonella minnesota. These results suggest that the Hakata antigen, a new member of the ficolin/opsonin P35 family, plays a role in the serum exerting lectin activity under physiological conditions.

Amino Acid Sequence↗
PubMed1995

Human parvovirus B19 infection in blood donors.

Using an immunodiffusion assay, we tested all of the blood units donated at the Fukuoka Red Cross Blood Center from June 1991 to July 1994 for B19 antigen. Over this 3-year trial period, we detected 16 viremic cases out of approximately 560,000 blood donors. Interestingly, most of the viremic donors (15 out of 16) were detected between February 1992 and January 1993, which coincided with a local erythema infectiosum epidemic in the Fukuoka area (December 1991 to August 1992). In particular, we detected 4 cases of viremia in March 1992, which was the peak of the erythema infectiosum epidemic. The incidence of B19 viremia in this peak period was approximately 1/4,000. The viremic donors ranged in age from 17 to 45 years, and most (11/16) were between 31 and 39 years old. By ELISA, using virus particles purified from viremic donor plasma as antigen, we analyzed the prevalence of B19-specific antibody among blood donors. The antibody-positive rate was approximately 40% in donors 16-30 years old, gradually increased in middle age, and reached a peak of 92% in donors more than 61 years old.

Adolescent↗
PubMed1993

Quantitation of HTLV-I provirus among seropositive blood donors: relation with antibody profile using synthetic peptides.

To study the pathophysiological state of HTLV-I carriers and the quantitative aspect of the risk of HTLV-I infection, we measured the absolute amounts of HTLV-I provirus in 39 seropositive blood donors. The amount of provirus varied from less than one to more than 10(4) molecules per 10(5) peripheral-blood mononuclear cells (PBMC). The average amount of HTLV-I provirus among seropositive blood donors was estimated as 3.7 x 10(3) molecules per 10(5) PBMC. The titer of the antibody against only the synthetic peptide (aa 100 to 130) of the gag p19 region of the HTLV-I provirus showed a weak but significant correlation with the amount of the provirus.

Adult↗
PubMed1992

Gly145 to Arg substitution in HBs antigen of immune escape mutant of hepatitis B virus.

A Japanese child born to an HBeAg-positive carrier mother received anti-HBs immunoglobulins and a plasma-derived HBs vaccine with a poor anti-HBs-antibody response. The child, who is now 3 years old, is presently suffering from chronic hepatitis with unusual serological findings that are positive for HBsAg, anti-HBs and HBeAg, since being infected with a measles virus at 12 months of age. The nucleotide sequences of the S region of HBV DNA obtained from the patient, the mother and an HBeAg-positive brother were completely identical except for one nucleotide at position 587 (mother and brother: guanosine, patient: adenosine), giving an amino acid change: Gly - greater than Arg at position 145 of the major HBs protein.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a thermolabile beta-2 macroglycoprotein ('thermolabile substance' or 'Hakata antigen') detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus.

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.

Adult↗
PubMed1991

Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies.

We identified and mapped the regions responsible for neutralization in the human parvovirus B19 structural protein by using region-specific human antibodies derived from seropositive blood donors. The region-specific antibodies were purified by using affinity columns coupled with synthetic peptides of the hydrophilic regions including the beta-turn structure deduced by the predicted secondary structure of VP2. Fifteen highly specific antibodies against the synthetic peptides were obtained. Ten of them were able to precipitate the radiolabeled virus. Six of them proved to be able to protect the colony-forming unit erythroid cells in human bone marrow cell cultures from injury by the virus. The sequences recognized by the six neutralizing antibodies were sites corresponding to amino acids 253 to 272, 309 to 330, 325 to 346, 359 to 382, 449 to 468, and 491 to 515 from the amino-terminal portion of VP2. These observations suggest that the neutralizing epitopes were distributed in the region from amino acid 253 in the amino-terminal portion of VP2 to the carboxyl terminus of VP2.

Amino Acid Sequence↗