Masatsugu Kimura

Identification of the four species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene

Abstract A polymorphic region of the small subunit rRNA gene of the four human malaria parasite species was sequenced to see intraspecies variations. Two new variant sequences were found in P. ovale and P. malariae. Based on these sequences together with those published, we have designed species-specific primers that identify the four species of human malaria parasites by nested PCR following amplification of the polymorphic region with interspecies conserved primers. Our method detected the P. ovale variant which had a sequence undetected by a microtiter plate hybridization (MPH) method and had higher detection level in P. malariae species than MPH.

Molecular Analysis of Plasmodium ovale Variants

Sequence analyses of six isolates from Southeast Asia supports dividing species into types

Stable Patterns of Allelic Diversity at the Merozoite Surface Protein‐1 Locus of Plasmodium falciparum in Clinical Isolates from Southern Vietnam

ABSTRACT The extent of allelic diversity at the Merozoite Surface Protein‐1 locus of Plasmodium falciparum (PfMSP‐1) was examined in isolates collected from symptomatic patients living in a mesoendemic area in southern Vietnam. The variable blocks 2, 4 and 10 were typed by polymerase chain reaction and 24 PfMSP‐1 gene types were defined as unique combinations of allelic types detected in each variable block. Nineteen PfMSP‐1 gene types were identified and 182 parasite populations were fully typed among 102 isolates. Forty‐eight (47%) patients harbored more than one typed parasite population, and one patient had at least eight genetically distinct subpopulations. As previously shown in the same endemic area, recombination between blocks 4 and 10 was significantly less frequent than expected from random assortment of allelic types. The distribution of PfMSP‐1 gene types, however, did not differ significantly from that observed in isolates collected in the same area 17‐24 mo before the present study. Furthermore, the prevalence of the most common gene types and the average number of different gene types harbored by the same host did not decrease with age. This argues against the prominence of frequency‐dependent immune selection of PfMSP‐1 polymorphisms in this parasite population.

UNUSUAL PLASMODIUM MALARIAE-LIKE PARASITES IN SOUTHEAST ASIA

During malaria surveys in Myanmar, 2 peculiar forms of Plasmodium malariae-like parasites were found. The morphologies of their early trophozoite stages were distinct from that of the typical P. malariae, resembling instead that of Plasmodium vivax, var. minuta, reported by Emin, and Plasmodium tenue, reported by Stephens, both in 1914. Two polymerase chain reaction (PCR)-based diagnoses, which target the same regions in the small subunit ribosomal RNA (SSUrRNA) genes, indicated that these parasites were new variant forms of P. malariae and that they could be separated into 2 genetic types that correlated with the 2 morphological types. Sequence analysis of the SSUrRNA and the circumsporozoite protein genes revealed that they were distinct both from each other and from other known P. malariae isolates and that the P. tenue-like type was closer to a monkey quartan malaria parasite, Plasmodium brasilianum. These results illustrate that the microscopic appearance of human P. malariae parasites may be more varied than previously assumed and suggest the value of molecular tools in the evaluation of malaria morphological variants.

RBM10 regulates alternative splicing.

RBM10, originally called S1‐1, is a nuclear RNA‐binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis‐related genes, Fas and cFLIP. In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl‐x genes. RBM10 promotes exon skipping of Fas pre‐mRNA as well as selection of an internal 5′‐splice site in Bcl‐x pre‐mRNA. We propose a consensus RBM10‐binding sequence at 5′‐splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.

Characteristic Age Distribution of Plasmodium vivax Infections after Malaria Elimination on Aneityum Island, Vanuatu

ABSTRACT Resurgence is a major concern after malaria elimination. After the initiation of the elimination program on Aneityum Island in 1991, microscopy showed that Plasmodium falciparum disappeared immediately, whereas P. vivax disappeared from 1996 onward, until P. vivax cases were reported in January 2002. By conducting malariometric surveys of the entire population of Aneityum, we investigated the age distribution of individuals with parasites during this epidemic in the context of antimalarial antibody levels and parasite antigen diversity. In July 2002, P. vivax infections were detected by microscopy in 22/759 individuals: 20/298 born after the beginning of the elimination program in 1991, 2/126 born between 1982 and 1991, and none of 335 born before 1982. PCR increased the number of infections detected to 77, distributed among all age groups. Prevalences were 12.1%, 16.7%, and 6.0%, respectively (P < 0.001). In November, a similar age pattern was found, but with fewer infections: 6/746 and 39/741 individuals were found to be infected by microscopy and PCR, respectively. The frequencies of antibody responses to P. vivax were significantly higher in individuals born before 1991 than in younger age groups and were similar to those on Malakula Island, an area of endemicity. Remarkably low antigen diversity (h, 0.15) of P. vivax infections was observed on Aneityum compared with the other islands (h, 0.89 to 1.0). A P. vivax resurgence was observed among children and teenagers on Aneityum, an age distribution similar to those before elimination and on islands where P. vivax is endemic, suggesting that in the absence of significant exposure, immunity may persist, limiting infection levels in adults. The limited parasite gene pool on islands may contribute to this protection.

Cloning and structural analysis of the gene for cAMP-dependent protein kinase catalytic subunit from Plasmodium yoelii.

We isolated the gene encoding the cAMP-dependent protein kinase catalytic subunit (cAPK[C]) from Plasmodium yoelii by screening a genomic library for the DNA fragment as produced by the polymerase chain reaction. The deduced protein of 341 amino acids conserves residues that are important for the function of serine/threonine protein kinases and shows the highest homology to cAPK[C]s of other organisms. However, P. yoelii cAPK[C] has 8 residues, which are perfectly conserved in cAPK[C]s of other organisms, radically replaced with residues having different side-chain properties. It is stressed that two radical replacements occur in regions for the binding with a regulatory subunit and/or a heat-stable inhibitor protein.

Recombinant Plasmodium falciparum dihydrofolate reductase-based in vitro screen for antifolate antimalarials

We describe the system for screening the effective antifolate antimalarials that uses the recombinant Plasmodium falciparum DHFR domain of the bifunctional DHFR-TS expressed in Escherichia coli, and were designed with amino acid alterations found in the DHFR genes of the antifolate resistant strains. The validity of the screen was verified by the subsequent examination of several substituted pyrrolo[2,3-d]pyrimidines for their antimalarial activity. Among the 120 chemical derivatives, 5 compounds were identified by their preferential inhibition of the drug sensitive pfDHFR to that of the mammalian isoenzyme. As compared to the sensitive enzyme, the decrease in response of the cycolguanil-resistant and pyrimethamine-resistant enzymes to the selected compounds were relatively moderate. This gave folds decrease in sensitivity of 0.8-7.5 and 3.6-29, respectively, while those for cycloguanil and pyrimethamine were 400 and 308. The compounds inhibited the growth of drug-sensitive cultured P. falciparum with 50% effective concentrations of the ranged 0.17-30 nM. As contrasted with the sensitive strain, the fold decrease in sensitivity of the resistant parasites were 0.9-2 and 15-50 in the case of the test compounds, while those for cycloguanil and pyrimethamine were 690 and 20,500. Moreover, the most selective pyrrolo-pyrimidine (P-1) showed in vivo activity against P. berghei in mice.

Alterations and diversity in the cytoplasmic tail of the fusion protein of subacute sclerosing panencephalitis virus strains isolated in Osaka, Japan.

We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan, from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain, five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed.

Allelic Diversity at the Merozoite Surface Protein-1 (MSP-1) Locus in Natural Plasmodium falciparum Populations: a Brief Overview

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed.