Yasuto Okubo

Glycophorin B as an EBA-175 independent Plasmodium falciparum receptor of human erythrocytes

Invasion of erythrocytes by malaria parasites involves multiple receptor-ligand interactions. To elucidate these pathways, we made use of four parasite clones with differing specificities for invasion, erythrocytes that are mutant for either glycophorin A or B, and enzyme modification of the erythrocyte surface with neuraminidase and trypsin. Neuraminidase alone abolishes invasion of two parasite clones (Dd2, FCR3/A2); these invade after trypsin treatment alone. A third clone (7G8) is unable to invade trypsin-treated erythrocytes. The fourth clone (HB3) can invade after either neuraminidase or trypsin treatment. The receptor for invasion of trypsin-treated erythrocytes was explored in two ways: treatment of trypsin-treated normal cells with neuraminidase, and trypsin treatment of glycophorin B-deficient cells. Both treatments eliminated invasion by all clones, indicating that the trypsin-independent pathway uses sialic acid and glycophorin B. To identify parasite proteins involved in the different pathways, erythrocyte binding assays were performed with soluble parasite proteins from each clone. Based on binding assays using erythrocytes that lack glycophorin A, the parasite protein known as EBA-175 appears to bind predominantly to glycophorin A. In contrast, the glycophorin B pathway does not appear to involve EBA-175, as binding of EBA-175 was similarly reduced to trypsin-treated normal and trypsin-treated glycophorin B-deficient erythrocytes. Thus, the glycophorin B-dependent, sialic acid-dependent invasion of trypsin-treated normal erythrocytes uses a different parasite ligand, indicating two or more sialic-dependent pathways for invasion. Clone 7G8, which cannot invade trypsin-treated erythrocytes, may be missing the ligand for invasion via glycophorin B.(ABSTRACT TRUNCATED AT 250 WORDS)

The RHD gene is highly detectable in RhD-negative Japanese donors.

Recent molecular studies on the Rh blood group system have shown that the Rh locus of each haploid RhD-positive chromosome is composed of two structural genes: RHD and RHCE, whereas the locus is made of a single gene (RHCE) on each haploid RhD-negative chromosome. We analyzed the presence or absence of the RHD gene in 130 Japanese RhD-negative donors using the PCR method. The RhD-negative phenotypes consisted of 34 ccEe, 27 ccee, 17 ccEE, 26 Ccee, 19 CcEe, 1 CcEE, and 6 CCee. Among them, 36 (27.7%) donors demonstrated the presence of the RHD gene. Others showed gross or partial deletions of the RHD gene. These results were confirmed by Southern blot analysis. Additionally, the RHD gene detected in the RhD-negative donors seemed to be intact through sequencing of the RhD polypeptide cDNA and the promoter region of RHD gene. The phenotypes of these donors with the RHD gene were CC or Cc, but not cc. It suggested that there is some relationship between the RHD gene and the RhC phenotypes in RhD-negative individuals. In Caucasian RhD-negative individuals, the RHD gene has not been found outside of the report of Hyland et al. (Hyland, C.A., L.C. Wolter, and A. Saul. 1994. Blood. 84:321-324). The discrepant data on the RHD gene in RhD-negative donors between Japanese and Caucasians appear to be derived from the difference of the frequency of RhD-negative and RhC-positive phenotypes. Careful attention is necessary for clinicians in applying RhD genotyping to clinical medicine.

Gene frequencies of human platelet antigens on glycoprotein IIIa in Japanese

Background: Polymorphism of glycoprotein IIIa on human platelets is one of the factors in alloimmunization that causes neonatal alloimmune thrombocytopenia and refractoriness to platelet transfusion.

Prevalence of second generation antibody to hepatitis C virus among voluntary blood donors in Osaka, Japan

To clarify the demographic characteristics of the prevalence of hepatitis C virus (HCV) infection in Osaka, Japan, where hepatocellular carcinoma is common, we investigated the screening data of antibody to HCV (anti-HCV, DAINABOTHCVPHA, second generation assay) in 197,600 voluntary blood donors residing in Osaka. The study found that age-standardized prevalence of anti-HCV was significantly higher than that of HBsAg (2.25cf 0.86 percent among males,P<0.001; 2.17cf 0.55 percent among females,P<0.001. It was much higher in the blood donors aged 55–64 years than in those aged 16–54 years (8.49cf 1.32 percent among males,P<10−5; 7.26cf 1.42 percent among females,P<10−5). The prevalence of anti-HCV among males was significantly higher than that of females in the younger (25–34 years) generations (1.02 to 1.49 percentcf 0.71 to 1.13 percent,P<0.05). A similar tendency was observed in the prevalence of high-titer (≧212) anti-HCV. The number of coinfection (both HBsAg and anti-HCV seropositive) was very small, and it was not statistically different from the expected number.

Further analysis of Del (D-elute) using polymerase chain reaction (PCR) with RHD gene-specific primers

Del (D‐elute) in the Rh blood group system is a variant with very weak D antigen and no agglutination is found by the indirect antiglobulin test. This variant is characterized by the presence of anti‐D eluate obtained after an adsorption‐elution test using anti‐D antibodies. We studied here the molecular genetic status of Del by using polymerase chain reaction with sequence‐specific primers (PCR‐SSP).

Simultaneous DNA Typing of Human Platelet Antigens 2, 3 and 4 by an Allele‐Specific PCR Method

We developed an allele‐specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy‐three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA‐2, ‐3 and ‐4 in parallel without the use of platelets, platelet‐specific alloantibodies or restriction enzymes.

Distribution of abo genotypes and allele frequencies in a korean population

SummaryThe genotypes of the ABO blood group system were investigated in Korean living in Kangwon-Do area by PCR-RFLP analysis of the seven polymorphic nucleotide positions 261, 467, 526, 646, 703, 796 and 803 of the cDNA from A1 transferase. In 253 unrelated Korean individuals, 15 genotypes were found and the allele frequencies of A(Pro), A(Leu), B, O(T) and O(A) were 0.022, 0.209, 0.209, 0.360 and 0.200, respectively, with no deviation from Hardy-Weinberg expectations (Χ 2=2.145, d.f.=6, 0.90<p<0.95). As for the distribution of allele frequencies, a significant difference was noticed between the Korean and a Japanese (Χ 2=30.87, d.f.=4, p<0.001) and a German (Χ 2=127.76, d.f.=4, p<0.001) populations.

Phenotype i Associated with Congenital Cataract in Japanese

A Japanese family with two siblings of phenotype i is presented. Both had a past history of surgical treatment for congenita] cataract. In Japan, 18 individuals of phenotype i, including our case, have been found in ten unrelated families. Seventeen of them had congenital cataract. Cataract was not found in any of the 45 phenotype I members in these families. It is briefly discussed why these two linked and quite rare genes were found in combination only in Japanese persons.

Heterogeneity of the phenotype Jk(a-b-) found in Japanese.

Fourteen Jk(a‐b‐) persons were detected by testing 638,460 Osaka blood donors with an automated 2 M urea technique. Two of these 14 Jk(a‐b‐) samples were quite different from the hitherto reported Jk(a‐b‐) phenotype, and a family study showed that the mode of the inheritance was dominant. The red cell membranes of these Jk(a‐b‐) samples were studied by polyacrylamide gel electrophoresis and unusual protein bands (apparent mw, 67,000 d) were detected.

Molecular basis for Rhnull syndrome: Identification of three new missense mutations in the Rh50 glycoprotein gene

Rhnull is a rare autosomal recessive disorder characterized by an absence of Rh antigens and a varying degree of hemolytic anemia and spherostomatocytosis. We report studies of two Japanese Rhnull cases and describe three new missense mutations of RHAG, the locus that encodes Rh50 glycoprotein and modulates Rh antigen expression. In Rhnull(HT), RHAG harbored in exon 6 two G→A transitions, GTT→ATT and GGA→AGA, which cause Val270→Ile and Gly280→Arg substitutions, respectively. These missense mutations were cotransmitted from the propositus to the children and were predicted to reside in endoloop 5 and transmembrane (TM) segment 9, respectively. In Rhnull(WO), RHAG contained in exon 9 a single G→T transversion, GGT→GTT, which caused a Gly380→Val missense change in TM12 segment. The G→T transversion, which is located at the +1 position of exon 9, had also affected pre‐mRNA splicing and caused partial exon skipping. Although both Rhnull cases had a structurally normal RH antigen locus, hemagglutination and immunoblotting showed no expression of Rh antigens or proteins. These results correlate each mutation with a structural defect in the respective TM domain of Rh50 glycoprotein. Am. J. Hematol. 62:25–32, 1999.