Partial purification of mitochondrial DNA topoisomerase II from Plasmodium falciparum and its sensitivity to inhibitors.

Mitochondria of Plasmodium falciparum (K1 strain) were isolated by differential centrifugation. Mitochondrial DNA topoisomerase II from P. falciparum was partially purified using fast protein liquid chromatography(FPLC). Parasite mitochondria contained approximately 8% of DNA topoisomerase II activity compared with its nuclear fraction. The effects of fluoroquinolones, inhibitors of bacterial DNA topoisomerase II or DNA gyrase, against partially purified P. falciparum mitochondrial DNA topoisomerase II were investigated using a decatenation assay. Minimum inhibitory concentrations (MIC) of ofloxacin, ciprofloxacin and norfloxacin were > 1, 10 and 100 mM, compared with that of >0.5 and 10 mM for eukaryotic DNA topoisomerase II inhibitor etoposide (VP-16) and amsacrine, respectively. The results indicate that partially purified mitochondrial DNA topoisomerase II was insensitive to fluoroquinolones and it is suggested that their inhibitory effects on P. falciparum growth may be directed against plastid DNA topoisomerase II.

Molecular defect of PKD1 gene resulting in abnormal RNA processing in a Thai family.

Autosomal dominant polycystic kidney disease (ADPKD) is a common human autosomal disorder caused mainly by mutations of the PKD1 gene. In analysis of PKD1 transcripts by long RT-PCR and nested PCR procedures, we observed PKD1-cDNA fragments from three ADPKD siblings from the same family with a size approximately 250 base pairs (bp) shorter than normal. Further investigations showed that the PKD1 transcripts from these patients had been abnormally processed, the nucleotide sequence of exon 43 containing 291 nt was missing from the transcripts, which would result in an abnormal polycystin-1 with an in-frame deletion of 97 amino acids. This splicing defect did not result from a mutation that disrupted the splice donor or acceptor sites adjacent to exon 43 or the branch sites in flanking introns but was most likely due to 20-bp deletion observed in intron 43. The intronic deletion was present in 8 affected members but absent in 11 unaffected members, corresponding with the results of genetic linkage analysis using 5 polymorphic markers in the PKD1 region. Molecular diagnosis of PKD1 in this family could, therefore, be carried out by genomic DNA amplification to directly detect the PKD1 intronic deletion.

Large scale culture technique for pure Plasmodium falciparum gametocytes.

A large scale technique for pure gametocyte cultures of Plasmodium falciparum was established in culture flasks by using treated erythrocytes and RPMI medium supplemented with 15% human plasma instead of serum. Pure gametocyte cultures were successfully obtained following treatment with 5% sorbitol on day 8 and 9 of cultivation. This method resulted in approximately 97% reduction of asexual parasites and provided pure gametocytes in culture. The highest numbers of gametocytes were obtained from cultures starting with 2% parasitemia and 2% erythrocyte suspension. On day 12 of cultivation, approximately 35 x 10(6) gametocytes per 100 ml of cell suspension could be harvested.

Genotypic and phenotypic implications in paroxysmal nocturnal hemoglobinuria (PNH): a preliminary investigation.

The genetic and biochemical defects underlying paroxysmal nocturnal hemoglobinuria (PNH) have recently been elucidated. The deficiency of the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins caused by a somatic mutation of the PIG-A gene, an X-chromosomal gene that participates in the first step of the GPI anchor synthesis, has been shown to be responsible for PNH in all patients. The mutations of PIG-A studied to date are highly heterogeneous. They are however mainly of the frameshift type (61.5%). The characteristic abnormalities of PNH phenotypes has also been shown especially by DAF- and/or CD59-based fluorescent immunocytometry. A great degree of heterogeneity in the patterns and levels of expression of GPI-anchored proteins in various cell types was demonstrated indicating a discrepancy of lineage involvement. In this investigation, major blood cell populations, i.e erythrocytes and granulocytes were analyzed immunophenotypically, the mutations of PIG-A were identified by heteroduplex analysis and nucleotide sequencing and the consequences of PIG-A mutations were observed. All the mutations identified in 9 patients with PNH resulted in complete loss of function as clones of affected granulocytes completely negative for CD59 expression were shown in all patients. Interestingly, granulocytes in these patients contained variable proportions of affected cells varied from 50% to 100% and four of the patients had erythrocytes with diminished expression of GPI-anchored DAF and CD59 coexisting with normal and completely negative cells. Immunophenotypic analysis of reticulocytes in peripheral blood of patients with PNH demonstrated the conserved patterns of DAF and CD59 expression in circulating erythroid cells and the discrepancies between granulocytic and erythroid lineages. These findings suggested that the characteristics of abnormal phenotypes which appear to be highly variable between different hematopoietic lineages are not solely caused by mutation of PIG-A but are influenced by other factor(s).

Morphological alterations and apoptosis of endothelial cells induced by thalassemic serum in vitro.

Vascular complications such as lung thromboembolism and leg ulcer have been observed in thalassemic patients. Recently, our group has reported impaired proliferation of endothelial cells (ECs) after exposure to alpha- and beta-thalassemic sera in a culture system. This study was undertaken to detect apoptotic phenomena of ECs in the presence of alpha- and beta-thalassemic serum. ECs from normal human umbilical cord vein were exposed to 30% thalassemic serum in vitro and morphological changes were observed by using phase contrast, fluorescence and scanning electron microscopy. Such treated ECs presented morphological characteristics of apoptosis as shown by the appearance of compact cytosol, membrane blebbing, margination of nuclear matrix, condensed nuclei, and fragmented bodies. The majority of apoptotic cells was in the floating population. Similar morphological changes were also observed by treating ECs with hydrogen peroxide in the concentration range of 0.1-10 mM.

Development of cultivation technique for pure Plasmodium falciparum gametocytes.

Pure gametocyte culture of Plasmodium falciparum, isolate KT3, from Kanchanaburi Province, Thailand, was successfully established by 5% sorbitol treatment on days 9, 10 and 11 following initiation of culture. Using medium supplemented with 15% human plasma and activated erythrocytes, daily medium change was not required during the cultivation. There was 99% reduction in the numbers of asexual parasites in the culture but the numbers of gametocytes were not affected. Furthermore, the gametocytes could undergo their usual morphological development with retention of function as demonstrated by the appearance of exflagellating microgametocytes, macrogametocytes and of oocyst formation in midgut of infected mosquito.

Molecular mechanisms of thalassemia in southeast Asia.

Hemoglobinopathies are the most common genetic disorders in Southeast Asia. alpha-Thalassemia is most often due to a alpha-globin gene deletion. Hb Constant Spring (CS) occurs from the mutation at the termination codon of the alpha-globin gene resulting in an elongated polypeptide; alpha(CS)-globin mRNA is also unstable and only small amounts of Hb CS are produced. Thus Hb CS has an alpha-thalassemia 2-like effect. beta-Thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions or deletions or insertions of one to four nucleotides. Hemoglobin E occurs from a Glu --> Lys substitution at position 26 of the beta-globin chain. The abnormal gene also results in reduced amounts of beta E-mRNA and hence of beta E-globin chains. Therefore, Hb E has a mild beta + thalassemia phenotype. Homozygous beta-thalassemia and beta-thalassemia/Hb E are the major beta-thalassemic syndromes in Southeast Asia. In spite of seemingly identical genotypes, severity of beta-thalassemia/Hb E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. A number of genetic factors have been shown to determine the differences in severity of anemia in beta-thalassemia/Hb E, including co-inheritance of alpha-thalassemia determinants and co-inheritance of other determinants which elevate Hb F expression. A correlation between the extent of beta E-globin mRNA cryptic splicing and the severity of anemia in beta(zero)-thalassemia/Hb E patients has been observed. Complete characterization of mutations causing hemoglobinopathies will help to bolster the establishment of prenatal diagnosis of these genetic disorders in the region.

Role of alternatively spliced beta E-globin mRNA on clinical severity of beta-thalassemia/hemoglobin E disease.

In spite of seemingly identical genotypes, severity of beta-thalassemia/hemoglobin (Hb) E patients can vary greatly. Some may have a severe clinical disorder approaching that seen in homozygous beta-thalassemia. Since mutation in codon 26 of the beta E-globin gene can lead to an alternative splicing, Hb E acts like a mild beta(+)-thalassemia. Variation in the amount of beta E-globin mRNA may also govern the difference in severity of anemia in beta-thalassemia/Hb E patients who otherwise have the same genetic determinants. We have determined the percentage of the alternatively spliced beta E-globin mRNA by the RT-PCR technique in 14 patients and found that the amount of abnormal spliced beta E-globin mRNA in those patients with severe symptoms ranged between 2.9 to 6.1%, whereas those with milder symptoms had the values which ranged between 1.6 to 2.6%. The extent of beta E-globin mRNA cryptic splicing was better associated with clinical severity of the patients than did the patterns of the Xmn I polymorphism at position -158 of the G gamma-globin gene or levels of Hb F.

Field evaluation of simplified radioactive and nonradioactive DNA probe methods for the diagnosis of falciparum malaria.

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.

Encapsidation defectiveness of herpes simplex virus type 2 during replication at acid pH condition.

The maximal yield of herpes simplex virus type 2 (HSV-2) grown at pH 6.5 decreased 10(2)-10(3) fold compared to that recovered at pH 7.5. Electron microscopic observation of the infected cells maintained at these 2 pH conditions indicated that approximately equal amounts of immature virions were synthesized 6 hours after infection. However, at 18 hours post infection the majority of viruses present in the nucleus of infected cells maintained at pH 6.5 were empty or partially cored capsids with some particles enveloped and present in the cytoplasm, whereas at pH 7.5 mature virions already appeared at the cytoplasmic membrane. Analysis of viral polypeptides by radioimmunoprecipitation indicated that the synthesis of p40, a family of polypeptides closely involved in viral DNA encapsidation, was significantly impaired in infected cells maintained at pH 6.5.

Partial purification and characterization of DNA topoisomerase II from Plasmodium falciparum.

DNA topoisomerase II from Plasmodium falciparum was partially purified by FPLC using three columns: Econo-Pac Q, heparin-agarose and Mono Q. The enzyme showed ATP- and Mg2 +/- dependent activities in a decatenation assay, with optimum concentrations of 0.5 and 10 mM, respectively. Furthermore, highest activity was detected in the presence of 100 mM KCI. Enzyme decatenation activity was not inhibited by the DNA topoisomerase I inhibitor, camptothecin, but was sensitive to both prokaryotic and eukaryotic DNA topoisomerase II inhibitors.

The thalassemic red cell membrane.

The underlying cause of pathology in thalassemia is the premature destruction of red cells, both in the bone marrow and by the reticuloendothelial system. It is generally accepted that the presence of unpaired excess globin chains is the primary circumstance leading to such membrane alterations as oxidation of phospholipids, modification of cytoskeletal proteins and their interactions, reduced membrane-associated ATPase activities, and enhanced permeability of cations. Such perturbations in turn result in the exposure of outer surface neoantigens, enhanced binding of autoantibodies and complement fixation to the outer red cell surface. These factors contribute to the observed distinctive morphologies, increased rigidity and decreased deformability of the thalassemic red cells. In alpha-thalassemic red cells, excess beta-globin chains form homotetramers, Hb H, which are relatively stable and will only damage red cell membrane when precipitated as inclusion bodies, whereas excess alpha-globin chains cannot form such homotetramers and upon synthesis rapidly bind to the cytoplasmic side of the beta-thalassemic red cell membrane, even in young erythroblasts. This difference in properties of the excess globin chains may offer an explanation for the variation in clinical severity observed between these two forms of thalassemia.

A simple technique for large scale in vitro culture of Plasmodium falciparum.

The large scale in vitro cultivation method of Fairlamb et al (1985) was modified to contain human plasma-supplemented medium in HEPES buffer. After 4 days with no change of medium nor agitation of the culture flask, 27- to 50-fold increase in the starting parasitemia of trophozoites were obtained.

Detection of Plasmodium falciparum using a cloned DNA probe: a simple procedure suitable for field application.

A simple procedure was developed for spotting blood samples directly onto nylon membrane filter, without the necessity to treat samples with pronase or proteinase K, followed by hybridizing with 32P-labelled DNA probe, pUNK1-45. This probe detected specifically P. falciparum DNA and did not cross react with DNA from man, P. knowlesi, P. chabaudi or P. cynomolgi. The probe was sensitive to detect a parasitemia of 0.001% in 20 microliters of blood.