Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several Δ⁷ 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-l-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C₂₈ 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C₂₈ and C₂₉ 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C₃₀ lanosterol differs from that of homologous enzymes in any other organisms studied. C₃₁ 24-Methylenelanosterol and C₃₂ pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.

Paramecium tetraurelia, like some other ciliate species, uses an alternative nuclear genetic code where UAA and UAG are translated as glutamine and UGA is the only stop codon. It has been postulated that the use of stop codons as sense codons is dependent on the presence of specific tRNAs and on modification of eukaryotic release factor one (eRF1), a factor involved in stop codon recognition during translation termination. We describe here the isolation and characterisation of two genes, eRF1-a and eRF1-b, coding for eRF1 in P. tetraurelia. The two genes are very similar, both in genomic organization and in sequence, and might result from a recent duplication event. The two coding sequences are 1,314 nucleotides long, and encode two putative proteins of 437 amino acids with 98.5% identity. Interestingly, when compared with the eRF1 sequences either of ciliates having the same variant genetic code, or of other eukaryotes, the eRF1 of P. tetraurelia exhibits significant differences in the N-terminal region, which is thought to interact with stop codons. We discuss here the consequences of these changes in the light of recent models proposed to explain the mechanism of stop codon recognition in eukaryotes. Besides, analysis of the expression of the two genes by Northern blotting and primer extension reveals that these genes exhibit a differential expression during vegetative growth and autogamy.

Host growth factors induce proliferation of Trypanosoma cruzi amastigotes by mechanisms that remain poorly defined. Here we examined human epidermal growth factor (EGF) for its ability to bind to the mammalian multiplicative forms of T. cruzi and to induce growth of the parasites. EGF stimulated incorporation of [³H] thymidine into DNA and growth of amastigotes both in a concentration-dependent manner. Radiolabeled EGF was found to bind to amastigotes in a concentration-dependent and saturable manner but it did not bind to trypomastigotes. Scatchard analysis showed a single class of receptors with a K_d of 0.8 nM and numbering 3.1 × 10³ per amastigote. Results from internalization experiments provided evidence of receptor-mediated endocytosis of EGF. Northern analysis showed a 3.0-kb transcript for the putative EGF receptor (EGFR) homologue in amastigotes, but not trypomastigotes. Binding of EGF to amastigotes induced signal transduction events. EGF induced “in vitro” kinase activity as determined by γ;en[³²P] ATP incorporation into amastigote proteins. EGF also increased protein kinase C activity in a concentration-dependent manner and Mitogen Activated Protein (MAP) kinase activity in a time- and concentration-dependent manner. A specific inhibitor (AG14782) of the EGFR and a MAP kinase inhibitor (PD98059) decreased EGF-dependent T. cruzi MAP kinase activity. These results describe a novel mechanism used by amastigotes to regulate their proliferation mediated by an EGF-dependent signal transduction pathway.

Gregarines in the genus Ascogregarina are not known to develop in sabethine mosquitoes, but we successfully infected larvae of Wyeomyia smithii with Ascogregarina taiwanensis in the laboratory. Ascogregarina taiwanensis is a natural parasite of the exotic Asian tiger mosquito, Aedes albopictus. Only 18% to 70% of the W. smithii larvae had visible trophozoites, with a range of 1–92 per larva. Trophozoites persisted in the midgut for more than 37 d, and one adult female W. smithii had gametocysts in its Malphigian tubules, which indicated that A. taiwanensis might fully develop in W. smithii. After 50 d, gregarines were not found in W. smithii larvae.

The microsporidian Vittaforma corneae has been reported as a pathogen of the human stratum corneum, where it can cause keratitis, and is associated with systemic infections. In addition to this direct role as an infectious, etiologic agent of human disease, V. corneae has been used as a model organism for another microsporidian, Enterocytozoon bieneusi, a frequent and problematic pathogen of HIV-infected patients that, unlike V. corneae, is difficult to maintain and to study in vitro. Unfortunately, few molecular sequences are available for V. corneae. In this study, seventy-four genome survey sequences (GSS) were obtained from genomic DNA of spores of laboratory-cultured V. corneae. Approximately, 41 discontinuous kilobases of V. corneae were cloned and sequenced to generate these GSS. Putative identities were assigned to 44 of the V. corneae GSS based on BLASTX searches, representing 21 discrete proteins. Of these 21 deduced V. corneae proteins, only two had been reported previously from other microsporidia (until the recent report of the Encephalitozoon cuniculi genome). Two of the V. corneae proteins were of particular interest, reverse transcriptase and topoisomerase IV (parC). Since the existence of transposable elements in microsporidia is controversial, the presence of reverse transcriptase in V. corneae will contribute to resolution of this debate. The presence of topoisomerase IV was remarkable because this enzyme previously had been identified only from prokaryotes. The 74 GSS included 26.7 kilobases of unique sequences from which two statistics were generated: GC content and codon usage. The GC content of the unique GSS was 42%, lower than that of another microsporidian, E. cuniculi (48% for protein-encoding regions), and substantially higher than that predicted for a third microsporidian, Spraguea lophii (28%). A comparison using the Pearson correlation coefficient showed that codon usage in V. corneae was similar to that in the yeasts, Saccharomyces cerevisiae (r = 0.79) and Shizosaccharomyces pombe (r = 0.70), but was markedly dissimilar to E. cuniculi (r = 0.19).

The morphology and infraciliature of a new hypotrichous ciliate, Euplotidium smalli n. sp., isolated from eutrophic coastal water in Korea, were observed in living cells and investigated using the protargol impregnation technique. This new ciliate bears 13–14 frontoventral cirri, 7 transverse cirri, and 5–6 dorsal kineties. Neither left marginal cirrus nor caudal cirrus is present. The new species differs from the related species, Euplotidium agitatum Noland, 1937 in the different number of frontoventral and transverse cirri and different body shape. With the exception of Euplotidium agitatum, the known species of the genus Euplotidium Noland, 1937 with the presence of left marginal cirrus are assigned to a new genus, Paraeuplotidium n. g. Diagnosis of Paraeuplotidium is: Gastrocirrhidae with funnel-shaped buccal cavity; with frontoventral and transverse cirri; left marginal cirrus present. Paraeuplotidium itoi (Ito, 1958) n. comb. is designated here the type species. Four additional species are included: Paraeuplotidium psammophilus (Vacelet, 1961) n. comb., Paraeuplotidium arenarium (Magagnini & Nobili, 1964) n. comb., Paraeuplotidium helgae (Hartwig, 1980) n. comb., and Paraeuplotidium prosaltans (Tuffrau, 1985) n. comb. An improved generic diagnosis of Euplotidium is suggested based on morphology and infraciliature characters: marine hypotrichs with a funnel-shaped buccal cavity; with frontoventral and transverse cirri; neither left marginal cirrus nor caudal cirri present.

The plasma membrane potential (ΔΨ) of procyclic and bloodstream trypomastigotes of Trypanosoma brucei was studied using the potentiometric fluorescent dye bisoxonol. Our results suggest that a proton pump plays a significant role in the regulation of ΔΨ in procyclic and bloodstream forms, as evidenced by depolarization of the plasma membrane by H -ATPase inhibitors (e.g. dicyclohexylcarbo-diimide, N-ethylmaleimide, diethylstilbestrol, and bafilomycin A₁). In bloodstream stages the plasma membrane was significantly depolarized by ouabain only when the cells were incubated in sodium-rich buffers indicating that a sodium pump was being inhibited. In contrast, ouabain had no effect on the ΔΨ of the procyclic stages in a sodium-rich buffer. However, it induced an additional significant depolarization in these stages when their plasma membrane was already partially depolarized by the H -ATPase inhibitor dicyclohexylcarbo-diimide, indicating the presence of an ouabain-sensitive sodium pump whose activity is masked by the H -ATPase. Unlike procyclic forms, the ΔΨ of bloodstream-stage trypomastigotes was markedly sensitive to extracellular Na and K concentrations. Thus, there are significant differences between procyclic and blooodstream forms in the maintenance of the ΔΨ and in their permeability to cations.

Perkinsus atlanticus cultures were established either with trophozoites isolated from fresh gills, with hypnospores isolated from tissues incubated in fluid thioglycollate medium, or directly from infected hemocytes of carpet shell clams Tapes decussatus from Algarve (Southern Portugal), using a culture medium and conditions optimized for Perkinsus marinus. Perkinsus atlanticus isolates were cloned by limiting dilution, and their identity unequivocally established by PCR-based species-specific diagnostic assays, and by sequencing the complete rRNA gene cluster. The rRNA gene cluster is 7.5-kb in length including 5S, IGS, SSU, ITS1, 5.8S, ITS2, LSU, and an inter–cluster spacer. rDNA sequences of the P. atlanticus clone were between 98.3–100% identical to P. atlanticus sequences previously obtained from clam tissue (non-clonal) isolates. Based on the IGS sequences available from Perkinsus species, a set of primers was designed to amplify P. atlanticus and the two clonally cultured Perkinsus species (P. marinus and P. andrewsi) currently available from a recognized repository. This Perkinsus “genus-specific” PCR-based assay complements the species-specific assays developed earlier and strengthen the detection of Perkinsus species for which specific detection assays are not yet available.

A marine planktonic ciliate, belonging to the genus Askenasia Blochmann, 1895, is described with notes on its distribution and ecology. Specimens of this new species were collected from four coastal sites across the northern hemisphere. Samples were collected between March and December, from depths of 1–20 m. Relatively low (< 1 ml⁻¹) densities were recorded from Atlantic and Pacific Oceans and North Sea coastal sites, whereas samples from the English Channel suggested that this species has the ability to form blooms. Specimens were protargol-stained, revealing the key features of the genus, including the diagnostic sub-equatorial kinety belt consisting of three unciliated monokinetids. However, the species is larger than other Askenasia with a single distinctive, rope-like, scalloped macronucleus. This species also appears to have an extra somatic kinety belt, which may be present but has not been observed in other Askenasia species. Based on size, kinety structure, kinety number, and macronuclear shape a new species has been established.

Phytate, the storage form of phosphate in seeds and grains, is a major form of environmental phosphate loading from fertilizer inputs and agricultural runoff. We have investigated the ability of Tetrahymena populations to grow on phytate as their sole phosphate source. Populations grew equally well in chemically defined medium with phosphate and medium in which the phosphate was replaced with phytate in comparable concentrations between 0.5 mM and 6 mM. Intracellular phytate concentrations of cells grown in phytate showed a 4–6-fold increase over those grown in phosphate when measured during the late stage of exponential growth. These results demonstrate that phytate can provide a source of adequate phosphate for sustained growth in phytate-rich environments.