Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

Identification of inheritance modes of mitochondrial diseases by introduction of pure nuclei from mtDNA-less HeLa cells to patient-derived fibroblasts.

A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.

Variations in the C-terminal repeats of the knob-associated histidine-rich protein of Plasmodium falciparum.

The knob-associated histidine rich protein (KAHRP) of Plasmodium falciparum plays an important role in the pathophysiology of cerebral malaria. In the present study, the immunogenic C-terminal repeat domain of the KAHRP gene was amplified, cloned and sequenced from the Indian (RJ181) and Honduran (HB3) isolates of P. falciparum. Based on the number and types of repeats in the domain, we report here the presence of three unique variant forms of KAHRP among these isolates. The Indian isolate (RJ181) contained four units of the decapeptide repeats whereas the Honduran isolate (HB3) contained two forms i.e. one form containing four decapeptide repeats plus a tetrapeptide subunit and the other form containing three decapeptide repeats plus a tetrapeptide subunit. Thus, all together, the number of KAHRP variants is increased to five which includes previously described two variants, each containing either 3 or 5 decapeptide repeats. This high rate of variability in the antigenic domain of the KAHRP gene via deletion or addition of whole or part of the decapeptide units could be involved in the evasion of host immune system possibly by providing the speculative complementarity to the vargene product. The results of the present study will be useful in designing the suitable molecular therapeutic reagents for cerebral malaria.

Sequence comparison between the flavoprotein subunit of the fumarate reductase (complex II) of the anaerobic parasitic nematode, Ascaris suum and the succinate dehydrogenase of the aerobic, free-living nematode, Caenorhabditis elegans.

Complex II in adult mitochondria of the parasitic nematode, Ascaris suum, exhibits high fumarate reductase activity and plays a key role in the anaerobic electron-transport observed in these organelles. In the present study, cDNAs for the flavoprotein (Fp) subunits of complex II have been isolated, cloned and sequenced from both A. suum and the aerobic, free-living nematode, Caenorhabditis elegans. Additional sequence at the 3' end of the mRNAs was determined by the Rapid Amplification of cDNA Ends (RACE). Nucleotide sequence analysis of the A. suum cDNAs revealed a 22-nucleotide trans-spliced leader sequence characteristic of many nematode mRNAs, an open reading frame of 1935 nucleotides and a 3' untranslated region of 616 nucleotides including a poly (A) tail from a polyadenylation signal (AATAAA). The open reading frame encoded a 645 amino acid sequence, including a 30 amino acid mitochondrial presequence. The amino acid sequences for the Fp subunits from both organisms were very similar, even though the ascarid enzyme functions physiologically as a fumarate reductase and the C. elegans enzyme a succinate dehydrogenase. The ascarid sequence was much less similar to the Escherichia coli fumarate reductase. The sensitivity of other Fp subunits to sulfhydryl reagents appears to reside in a cysteine immediately preceding a conserved arginine in the putative active site. In both nematode sequences, this cysteine is replaced by serine even though the succinate dehydrogenase activity of both enzymes is still sensitive to sulfhydryl inhibition. A cysteine six residues upstream of the serine may be involved in the sulfhydryl sensitivity of the nematode enzymes. Surprisingly, in contrast to succinate dehydrogenase activity, the fumarate reductase activity of the ascarid enzyme was not sensitive to sulfhydryl inhibition, suggesting that the mechanism of the two reactions involves separate catalytic processes.