Makoto Uchikawa

Basigin is a receptor essential for erythrocyte invasion by Plasmodium falciparum

Erythrocyte invasion by Plasmodium falciparum is central to the pathogenesis of malaria. Invasion requires a series of extracellular recognition events between erythrocyte receptors and ligands on the merozoite, the invasive form of the parasite. None of the few known receptor–ligand interactions involved are required in all parasite strains, indicating that the parasite is able to access multiple redundant invasion pathways. Here, we show that we have identified a receptor–ligand pair that is essential for erythrocyte invasion in all tested P. falciparum strains. By systematically screening a library of erythrocyte proteins, we have found that the Ok blood group antigen, basigin, is a receptor for PfRh5, a parasite ligand that is essential for blood stage growth. Erythrocyte invasion was potently inhibited by soluble basigin or by basigin knockdown, and invasion could be completely blocked using low concentrations of anti-basigin antibodies; importantly, these effects were observed across all laboratory-adapted and field strains tested. Furthermore, Oka− erythrocytes, which express a basigin variant that has a weaker binding affinity for PfRh5, had reduced invasion efficiencies. Our discovery of a cross-strain dependency on a single extracellular receptor–ligand pair for erythrocyte invasion by P. falciparum provides a focus for new anti-malarial therapies.

Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent : application to a human single-chain Fv fragment

An improved and efficient refolding system for a single-chain antibody fragment (scFv) from inclusion bodies expressed in Escherichia coli was developed. Stepwise removal of denaturing reagent and controlled addition of oxidizing reagent were found to be the most effective conditions to achieve for almost complete recovery of functional monomeric scFv from inclusion bodies. Adding L-arginine to the refolding solution also increased the yield of refolded functional scFv. The single-chain Fv fragments of both a mouse anti-lysozyme monoclonal antibody, HyHEL10, and a human monoclonal antibody against the D antigen of the Rh blood group, D10, in solubilized inclusion bodies could be refolded under these conditions with yields of up to 95%. The refolding procedures developed in this study will contribute to providing a stable supply of large amounts of human single-chain Fv fragments.

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A1 (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.

A comprehensive analysis of DEL types: partial DEL individuals are prone to anti-D alloimmunization

BACKGROUND: The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion‐ and pregnancy‐induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption‐elution techniques, with so far only poorly defined antigenic properties.

Prevalence of Borna disease virus RNA in peripheral blood mononuclear cells from blood donors

The presence of Borna disease virus (BDV) in peripheral blood mononuclear cells (PBMC) of 100 blood donors from Sapporo and 72 blood donors from Tokyo was examined using nested reverse transcriptase/polymerase chain reaction amplification with specific-primers for BDV p24. Anti-BDV p24 antibodies in the plasma of the 100 blood donors from Sapporo also were studied by enzyme-linked immunosorbent assay and by Western blot. BDV RNA was detected in 3 (4.2%) of the 72 PBMC samples from Tokyo, and in 5 (5%) of the 100 PBMC samples from Sapporo. In contrast, anti-p24 antibodies were found in only 1 (1%) of the donors from Sapporo. These results suggest that BDV infection in humans may be more widespread than previously thought.

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes

Abstract. We identified five different alleles, tentatively named ABO*O301, *O302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *O301 and *O302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-O2 and B-O2, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.

Different alleles cause an imbalance in A2 and A2B phenotypes of the ABO blood group

Background and Objectives: In several populations, including the Japanese, the frequency of the A2B phenotype is significantly higher than expected based on the A2 phenotype frequency. To understand the genetic basis of this ‘excess’ of A2B, we examined ABO alleles in individuals with A2‐related phenotypes. Materials and Methods: ABO alleles were identified by means of polymerase chain reaction single‐strand conformation polymorphism (SSCP) and nucleotide sequence analyses. Results: The frequencies of A2‐related alleles (*A105, *A106, *A107, *A111 and *R101) were clearly different between the A2 and A2B phenotypes. In particular, a putative recombinant allele, *R101, was uncommon in the A2 but common in the A2B phenotype individuals. This allele was also detected in 4 of 401 (1%) unrelated A1 phenotype (AO genotype) individuals. Conclusion: *R101 is presumably expressed as phenotype A1 in *R101/*O heterozygous individuals, but as phenotype A2 in *R101/*B heterozygotes, thus giving rise to a high A2B phenotype frequency.

Evaluation of Macaca mulatta as a model for genotoxicity studies.

We have investigated the use of peripheral blood from the nonhuman primate (NHP) rhesus monkey (Macaca mulatta) as a model system for mutation detection. The rhesus monkey is metabolically closer to humans than most common laboratory animals, and therefore may be a relevant model for hazard identification and human risk assessment. To validate the model, conditions were determined for in vitro selection and expansion of 6-thioguanine-resistant (6-TGr) HPRT mutant and proaerolysin-resistant (ProAERr) PIG-A mutant lymphocytes from peripheral blood obtained by routine venipuncture. Also, flow cytometric methods were developed for the rapid detection of PIG-A mutant erythrocytes. The flow cytometric analysis of PIG-A mutant erythrocytes was based on enumerating cells deficient in surface markers attached to the cellular membrane via glycosylphosphatidyl inositol (GPI) anchors. Mutant cells were enumerated over an extended period of time in peripheral blood of male monkeys receiving daily doses of the electrolyte replenisher Prangtrade mark (a common carrier for oral delivery of drugs in NHPs), and in the blood of one male monkey treated with a single i.p. dose of 50mg/kg of N-ethyl-N-nitrosourea at approximately 2 years of age and another similar injection at approximately 3.5 years of age. The spontaneous PIG-A and HPRT T-cell mutant frequency (MF) was low in animals receiving Prang (0-8x10(-6)), and treatment with ENU resulted in a clearly detectable increase in the frequency of ProAERr and 6-TGr lymphocytes (up to approximately 28x10(-6) and approximately 30x10(-6), respectively). Also, the ENU-treated animal had higher frequency of GPI-deficient erythrocytes (46.5x10(-6) in the treated animal vs. 7.8+/-4.2x10(-6) in control animals). Our results indicate that the rhesus monkey can be a valuable model for the identification of agents that may impact upon human health as mutagens and that the PIG-A gene can be a useful target for detection of mutation in both white and red blood cells.

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.

A new blood group system, RHAG: three antigens resulting from amino acid substitutions in the Rh-associated glycoprotein

Background and Objectives  Rh‐associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ola) have serological characteristics suggestive of expression on RhAG.