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Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes characteristic of virtually all eukaryotic cells and a 35-kilobase circular extrachromosomal DNA. In situ hybridization techniques were used to localize the 35-kilobase DNA of Toxoplasma gondii to a discrete organelle surrounded by four membranes. Phylogenetic analysis of the tufA gene encoded by the 35-kilobase genomes of coccidians T. gondii and Eimeria tenella and the malaria parasite Plasmodium falciparum grouped this organellar genome with cyanobacteria and plastids, showing consistent clustering with green algal plastids. Taken together, these observations indicate that the Apicomplexa acquired a plastid by secondary endosymbiosis, probably from a green alga.

Malaria and related apicomplexan parasites have two highly conserved organellar genomes: one is of plastid (pl) origin, and the other is mitochondrial (mt). The organization of both organellar DNA molecules from the human malaria parasite Plasmodium falciparum has been determined, and they have been shown to be tightly packed with genes. The 35-kb circular DNA is the smallest known vestigial plastid genome and is presumed to be functional. All but two of its recognized genes are involved with genetic expression: one of the two encodes a member of the clp family of molecular chaperones, and the other encodes a conserved protein of unknown function found both in algal plastids and in eubacterial genomes. The possible evolutionary source and intracellular location of the plDNA are discussed. The 6-kb tandemly repeated mt genome is the smallest known and codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two bizarrely fragmented rRNAs. The organization of the mt genome differs somewhat among genera. The mtDNA sequence provides information not otherwise available about the structure of apicomplexan cytochrome b as well as the unusually fragmented rRNAs. The malarial mtDNA has a phage-like replication mechanism and undergoes extensive recombination like the mtDNA of some other lower eukaryotes.

Determined efforts are being made to explore the non-photosynthetic plastid organelle of Plasmodium falciparum as a target for drug development. Certain antibiotics that block organellar protein synthesis are already in clinical use as antimalarials. However, all the indications are that these should be used only in combination with conventional antimalarials. The use of antibiotics such as doxycycline and clindamycin may reduce the development of drug resistant parasites and such means to avoid drug resistance should be explored hand-in-hand with drug development. Genomic information predicts that fatty acid type II (FAS II) and isoprenoid biosynthetic pathways are localized to the plastid. However, clinical trials with fosmidomycin (a specific inhibitor of DOXP reductase in the non-mevalonate pathway for isoprenoids) suggest it too should only be used in drug combinations. Prospects for more potent antimalarial compounds have emerged from studies of several of the enzymes involved in the FAS II pathway. Lead antibiotics such as thiolactomycin (an inhibitor of beta-ketoacyl-ACP synthase) and triclosan (a specific inhibitor of enoyl-ACP reductase) have led to structurally similar, active compounds that rapidly kill ring- and trophozoite-stage parasites. The FAS II pathway is of particular interest to the pharma-industry.

All eight enzymes required for de novo heme biosynthesis have been predicted from the nuclear genome of the human malaria parasite Plasmodium falciparum. We have studied the subcellular localization of three of these using a GFP reporter in live transfected parasites. The first enzyme in the pathway delta-aminolevulinic acid synthase (ALAS) is targeted to the mitochondrion, but the next two enzymes porphobilinogen synthase (PBGS) and hydroxymethylbilane synthase (HMBS) are targeted to the plastid. An enzymatically active recombinant version of PBGS from P. falciparum was over-expressed and its activity found to be stimulated by Mg2+ (and enhanced by Mn2+) but not by Zn2+. A hypothetical scheme for the exchange of intermediates in heme biosynthesis between the mitochondrion and plastid organelle, as well as organelle attachment is discussed.

The genome of the malaria parasite, Plasmodium falciparum, contains a myosin gene sequence, which bears a close homology to one of the myosin genes found in another apicomplexan parasite, Toxoplasma gondii. A polyclonal antibody was generated against an expressed polypeptide of molecular mass 27,000, based on part of the deduced sequence of this myosin. The antibody reacted with the cognate antigen and with a component of the total parasite protein on immunoblots, but not with vertebrate striated or smooth muscle myosins. It did, however, recognise two components in the cellular protein of Toxoplasma gondii. The antibody was used to investigate stage-specificity of expression of the myosin (here designated Pf-myo1) in P. falciparum. The results showed that the protein is synthesised in mature schizonts and is present in merozoites, but vanishes after the parasite enters the red cell. Pf-myo1 was found to be largely, though not entirely, associated with the particulate parasite cell fraction and is thus presumably mainly membrane bound. It was not solubilised by media that would be expected to dissociate actomyosin or myosin filaments, or by non-ionic detergent. Immunofluorescence revealed that in the merozoite and mature schizont Pf-myo1 is predominantly located around the periphery of the cell. Immuno-gold electron microscopy also showed the presence of the myosin around almost the entire parasite periphery, and especially in the region surrounding the apical prominence. Labelling was concentrated under the plasma membrane but was not seen in the apical prominence itself. This suggests that Pf-myo1 is associated with the plasma membrane or with the outer membrane of the subplasmalemmal cisterna, which forms a lining to the plasma membrane, with a gap at the apical prominence. The results lead to a conjectural model of the invasion mechanism.

We have attempted to determine whether the parasitophorous vacuole membrane, in which the malaria parasite (merozoite) encapsulates itself when it enters a red blood cell, is derived from the host cell plasma membrane, as the appearance of the invasion process in the electron microscope has been taken to suggest, or from lipid material stored in the merozoite. We have incorporated into the red cell membrane a haptenic phospholipid, phosphatidylethanolamine, containing an NBD (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) group, substituted in the acyl chain, and allowed it to translocate into the inner bilayer leaflet. After invasion of these labelled cells by the parasite, Plasmodium falciparum, immuno-gold electron microscopy was used to follow the distribution of the labelled lipid; this was found to be overwhelmingly in favour of the host cell membrane relative to the parasitophorous vacuole. Merozoites of P. knowlesi were allowed to attach irreversibly to red cells without invasion, using the method of pretreatment with cytochalasin. The region of contact between the merozoite and the host cell membrane was in all cases devoid of the labelled phosphatidylethanolamine. These results lead us to infer that the parasitophorous vacuole membrane is derived wholly or partly from lipid preexisting in the merozoite.

An extract of blood from Aotus monkeys infected with Plasmodium falciparum contained all the clones of malaria precipitins so far identified in human blood.We are grateful to the Medical Research Council and Overseas Development Administration of Great Britain and to the World Health Organization for their financial assistance.

Methotrexate inhibited the growth of D. viviparus by acting either on the moult from the third- to the fourth-stage larva or on the growth of fourth-stage larva itself. Despite these effects of the drug, the early developmental stages of the worms primed the immune mechanism of the host; the inductive phase of antibody production was completed in less than 5 days. An effective immune response, however, was blocked by the continued presence of methotrexate which resulted in delayed elimination of a superinfection and the absence of anaphylactic antibody in the serum. Immune elimination appeared to require a quantitative threshold of worm antigen and host antibody.This work was made possible by the generous supply of infective helminth material from Messrs Allen and Hanbury Ltd, Ware, Herts. I should also like to thank Dr J. H. Humphrey and Dr S. R. Smithers of N.I.M.R. for their helpful discussions.

The problem considered here is that of the displacement produced by an impulsive torsional body force situated within an elastic half-space which is bonded to a half-space of different material properties. (Author)

Summary Homocytotropic antibodies produced by the rat in response to infection with N. brasiliensis were found to be heat-labile and susceptible to inactivation by 2-mercaptoethanol. On electrophoresis in agar at pH 8.6, H.A. migrated faster than the bulk of γG-, γA- and γM-globulins. The molecular size of H.A. appeared to be somewhat greater than that of 7 S, but smaller than 19 S globulins. On diethylaminoethyl cellulose chromatography, H.A. was eluted with the peak concentration of γA-globulins. H.A. activity was removed from rat antiserum by absorption with rabbit antiserum to rat immunoglobulins. Although available evidence suggests that H.A. constitutes a unique rat immunoglobulin class analogous to human γE, final assignment should be delayed until the characteristics of the heavy polypeptide chains of rat H.A. have been determined.

Owl monkeys infected with the Malayan Camp strain of Plasmodium falciparum were found to have malarial antigens in their serum at the height of infection. The antigens were identical with certain malarial S-antigens present in the sera of West African (Gambian) children acutely infected with P. falciparum. The infected owl monkey thus appears to provide an experimental model which is relevant to the study of the serology of human malaria.

Relative to discussion by D. W. Bishopp of a paper by I. G. Gass, presents evidence that the diabase of Cyprus is intrusive rather than a series of highly folded lava flows.

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

Antigens were released in vitro from human red cells infected with Plasmodium falciparum only when parasites reached a late stage of schizogony (probably at rupture of infected red cells during reinvasion). Immune complexes formed if antibody was present. Serum from immune Aotus monkeys opsonized parasite debris from artificially disrupted infected Aotus red cells.

Summary Ring-stage asexual parasites of P. falciparum were collected from six Gambian children and the S-antigens radiolabeled by 3H-glycine uptake during in vitro culture up to rupture of infected cells and merozoite release. Ouchterlony double diffusion of boiled culture supernatants against a panel of adult Gambian sera identified one S-antigen precipitin arc for five isolates and two precipitin arcs for one isolate. Five of the six isolates were serologically distinct. Analysis of S-antigens by comparison of SDS-polyacrylamide gel electrophoresis patterns of heat-treated soluble proteins revealed a more complex pattern of 3H-labelled S-antigens that was different for each isolate. There were between two and six different 3H-labelled bands for each isolate in the size range of molecular weight 137 000 to 285 000. This result confirms the large size range of S-antigens identified with culture adapted P. falciparum. Several bands were relatively weakly labelled with 3H-glycine, suggesting that natural isolates contain one or two predominant S-antigen phenotypes and several other S-antigen phenotypes expressed by minor parasite subpopulations. Immunoprecipitation was performed using a panel of sera from Gambian adults, or, acute and 3 week convalescent sera from the same patients used for S-antigen radiolabelling. Adult sera generally immunoprecipitated some of the S-antigens in each isolate, including antigens that must represent extremely minor parasite subpopulations since they could not be seen in the patterns of non-immunoprecipitated heat-stable proteins. Sera from convalescent children were generally negative on immunoprecipitation, even with the homologous isolate. In one case we observed the acquisition of specific immuoprecipitating antibody to one of the homologous S-antigens during the convalescent period. The antigenic and structural complexity of S-antigens in natural isolates that have not been submitted to the selection pressure of adaptation for in vitro culture is clearly greater than for culture adapted P. falciparum.

SummaryExtracts of rhesus monkey erythrocytes infected withPlasmodium knowlesiwere fractionated by polyacrylamide gel electrophoresis (PAGE) and several zones of endopeptidase activity were demonstrated by an imprint-digest method. The enzymes were active only under acid conditions; activity was detected at pH 3·2 but not between pH 6·4 and 8·9 using haemoglobin, albumin or erythrocyte lysate as the substrate. Optimized PAGE conditions separated highly active parasite enzymes withRfvalues of 73, 63 and 53 (± 7%), as well as a red cell endopeptidase,Rf44. Of two other minor bands of activity, one was associated with platelets.

IT has been reported that neonatal thymectomy causes a defect in the production of “IgA” antibody in the rat. Arnason et al.1 have shown that thymectomized rats immunized with bovine serum albumin (BSA), diphtheria toxoid, or heat-killed Salmonella typhosa produce comparatively small amounts of anti-BSA precipitating antibody and anti-diphtheria toxin (both largely IgA antibodies) but produce average amounts of anti-typhoid “O” agglutinin (IgM antibody) and of anti-BSA haemagglutinin (IgG antibody). In the present investigation, the production of anaphylactic antibody was compared in neonatally thymectomized and sham-operated rats infected with the intestinal nematode, Nippostrongylus brasiliensis. This infection normally results in high titres of reagin-like antibody to worm antigen2. The object of the present experiments was to determine whether the immunological deficit produced by neonatal thymectomy affects the production of anaphylactic antibody and the acquisition of immunity to i0nfection with N. brasiliensis.

The antimalarial effects of rifampin in 60 adults with Plasmodium vivax malaria were assessed. There were three treatment groups: rifampin (20 and 15 mg/kg of body weight per day for 1 and 4 days, respectively; n = 5); rifampin followed by primaquine (15 mg of base per day for 14 days; n = 25), and chloroquine (25 mg of base per kg over 3 days) followed by primaquine (n = 30). All patients were hospitalized till clearance of fever and parasites, and 45 patients stayed in the hospital for 1 month. Despite initial clearance of fever in all patients and a > or = 6-fold reduction in parasitemia per 48-h life cycle, rifampin alone was not effective: all five patients had subsequent R2-like parasitological responses. All patients treated with rifampin-primaquine cleared both fever and parasitemia, but the therapeutic responses were slower than those following treatment with chloroquine-primaquine. Final fever clearance times were significantly longer (mean [standard deviation] = 43 [35] versus 27 [19] h; P = 0.046), and the parasite clearance times (to 50 and 90% of admission parasite counts and to a level undetectable in a peripheral blood smear) were also significantly greater (P = 0.053 to < 0.001). However, reappearance of infection occurred in only one patient treated with rifampin-primaquine. The results of this study suggest that rifampin at the usual therapeutic doses has partial activity against blood stages of P. vivax in humans but that used alone it is insufficient for cure. Rifampin might therefore be of value in combination antimalarial therapy.

It has been shown that the entry of the malaria parasite into the red blood cell requires the presence of ATP in the host cell cytoplasm1,2. In red blood cell ghosts that contain no ATP the receptor on the extracellular surface remains in place and parasites will bind to the membrane, but will not enter3. ATP is thus necessary for one of the steps4 in the invasion sequence that follows recognition and attachment. The process of entry appears to involve the active participation of the host cell membrane cytoskeleton5. We have suggested2 that the function of the intracellular ATP may be to regulate phosphorylation of the cytoskeleton. We now present evidence that the activity of the membrane-associated cyclic AMP-independent kinase of the red blood cell is inseparable from invasion; the active substrate may be spectrin.

In raw sugar analysis, the apparent sucrose content is determined as its polarisation value, a measure of the rotation of plane-polarised light passing through a solution of the sugar, which is directly proportional to sucrose concentration when the impurity level is low.Before taking a reading with a polarimeter, a “normal” raw sugar solution (26·000 g per 100 ml) is clarified by addition of basic lead acetate solution to precipitate impurities, which are removed by simple filtration; about 50 ml of filtrate are needed for analysis. When the solution is filtered, the sucrose concentration increases because of preferential absorption of water by the filter-paper. Initially in the current study, the magnitude of the absorption has been examined, and it has been determined that the first 10 ml of filtrate should be discarded to minimise this effect.Differences of 1 part in 40,000 in the polarisation value of sugar solutions, however, can now be detected by using modern photo-electric polarimeters and the refined analytical techniques described in this paper. This compares with 1 part in 2000 when using older visual instruments and standard techniques. Therefore, an insight into evaporation and humidity effects has also been gained, and a comparison of filtration and centrifugation, an alternative method of clarification, made.Centrifugation has practical advantages over filtration in laboratories in which large numbers of analyses are performed. In addition, it is not subject to preferential absorption and evaporation errors, and currently appears to give the best estimate of polarisation value, as defined and specified by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA).

A worm antigen which reacted with rat homocytotropic antibody to N. brasiliensis was demonstrated in culture fluids of adult worms. Production of antigen by adult worms in vitro declined during the first 4 days of maintenance in serum-free medium but was more prolonged in serum-supplemented medium. Six antigens were demonstrated in worm extract using rabbit antisera of restricted immunological specificity. Four of these antigens were present also in culture fluids of adult worms. On preparative agar electrophoresis of worm extract, the antigen which combined with rat homocytotropic antibody to N. brasiliensis migrated towards the cathode with a mobility resembling that of rat -yA-globulin. Limited evidence from physicochemical studies suggested that the antigen is a conjugated protein with a molecular weight between 12,000 and 15,000. Sera of rats infected with the intestinal nematode, Nippostrongylus brasiliensis, frequently contain high titers of homocytotropic antibody (Ogilvie, 1964). The term "homocytotropic antibody" has been proposed (Becker and Austen, 1966) as a replacement for rat "mast cell sensitizing" antibody (Mota, 1963) or "anaphylactic antibody" (Binaghi and Benacerraf, 1964) and will be used to describe a unique homologous immunoglobulin which sensitizes rat mast cells so that they release histamine upon contact with antigen. In the present study, homocytotropic antibody was assayed by passive cutaneous anaphylaxis. Attempts have been made to assess the role of homocytotropic antibody in immunity to infection with N. brasiliensis (Wilson, Jones, and Leskowitz, 1967; Ogilvie and Jones, 1967) and to charactize it physicochemically (Wilson, Bienenstock, and Bloch, 1966). The present studies, however, were undertaken in order to investigate some of the physicochemical characteristics of the worm antigen which reacts with homocytotropic antibody produced in infected rats. The production in culture fluids of an active but less complex antigenic mixture than whole worm extract is also described. MATERIALS AND METHODS