ALBERT

Notes: The in vitro cultivation of Pneumocystis carinii in chick lung cell culture made it possible to observe the organism proceeding through its life cycle. It provided the foundation for extensive serocpidcmiologic studies, for in vitro drug screening, and for essential biological, metabolic, and morphologic research. In vitro culture made possible the discovery of P. carinii antigenemia, its biochemical nature, and its relevance to subclinical and clinical infection. Its utility in the presumptive diagnosis of P. carinii pneumonia and in monitoring responses to drug therapy illustrate the value and clinical application of basic research.

Notes: This study describes an approach to cultivation of Pneumocystis carinii (Pc) on 2 cell lines derived from lung (A549, human and L2, rat) with emphasis on the organisms which adhered to the cells. Immunofluorescent staining was used for growth assays

Notes: Pneumocystis infection in athymic nude mice lungs showed a particularly high trophozoite to cyst ratio. A similar observation was obtained from a study of a patient with lymph node infection with Pneumocystis. Eosinophilic foamy masses in these sites were observed by light microscopy . With the electron microscope, the masses were seen to be composed of large aggregates of trophozoites. Cystic forms (precyst, cyst and empty cyst) were extremely scarce in comparison with the huge numbers of trophozoites. These cystic forms were mostly undergoing degeneration. These observations indicate that the mode of proliferation in both situations was predominantly asexual, that is, proliferation by trophozoites, suggesting that certain conditions may enhance asexual reproduction or depress the formation of cysts.

Notes: Homogenates of trophozoites of Entamoeba histolytica released glucose 1-phosphate from amylopectin, glycogen, and amylose in a ratio of 100:78:74 at glucopolysaccharide concentrations of 0.1%. By use of self-generating Percoll gradients this activity was shown to be particulate and associated with glycogen. The phosphorylase was extracted from the 40,000 g pellet in aqueous medium and purified to homogeneity by gel filtration on Fractogel TSK HW-55(F) followed by chromatography on Blue Sepharose CL-6B. The purified enzyme was active not only against the glucopolysaccharides but also on dextrins with more than 3 glucose moieties, which were primarily formed by the action of amoebic amylases. At substrate concentrations of 1 mM nonreducing ends of each glucan, the phosphorolysis rate of the branched polysaccharides was about 1.75 × 104 times higher than those of the maltodextrins. By means of HPLC the sequential degradation of 4-nitrophenyl-maltoheptaoside (G7-pNP) was studied. Native phosphorylase exhibited a relative molecular mass of Mr= 200,000 by gel filtration and gel electrophoresis. The SDS electrophoresis, under reducing conditions, indicated that the native enzyme was a dimer. Optimal degradation of the polysaccharides and dextrins was achieved at pH values of 7.5 and 7.0, respectively.

Notes: An in vitro assay was developed to study the recognition mechanism for attachment of Leishmania flagella to sand fly midgut epithelium. Frozen sections of sand fly guts were incubated with Hagella preparations, and probed with a flagella-specific monoclonal antibody. Tissue-specific adhesion of flagella to midgut epithelium was demonstrated by indirect immunofluorescence. None of the 13 sugars, screened to test for possible lectin-mediation, appeared to significantly inhibit the adhesion of flagella to gut sections. Similarly no inhibition was achieved by incubating flagella with pep 63 which inhibits the promastigote-macrophage recognition mechanism. Significant inhibition was attained by incubating flagella preparations with a monoclonal antibody which binds to a flagellar membrane-component. The possible relevance of the described mechanism for the biology of Leishmania in their sand fly hosts, is discussed.

Notes: Flagella-specific proteins of Leishmania have been identified employing the monoclonal antibody technique. Six monoclonal antibodies recognized 3 different proteins. A doublet of protein of Mr 69,000 and 74,000 Da identified by monoclonal antibodies F-3, F-4 and F-6 is continuously distributed along the flagellum by immunofluorescence. Immunocytochemical electron microscopic studies localize these molecules to the paraxial rod of the flagellum. A single protein of Mr 13,200 Da is recognized by monoclonal antibodies F-1, F-2 and F-5. The distribution of the Mr 13,200 protein appears irregular, occurring in localized patches along the length of the flagellum, especially at the flagellar tip. Immunocytochemical electron microscopic experiments show that the Mr 13,200 molecule is associated with the membrane of the flagellum. Indirect immunofluorescence experiments demonstrated these monoclonal antibodies cross-reacted with members of the Kinetoplastida family (Endotrypanum, Trypanosoma, Leishmania) suggesting that these molecules may be evolutionarily conserved.

Notes: . Phorbol 12-myristate 13-acetate increased the number of gametocytes by 50 to 100% in well or petri dish cultures of the HB-3 clone of Plasmodium falciparum. Phorbol dibutyrate had a similar effect. The optimal concentration for each of these agents was 20 ng/ml or approximately 30 nM. No effect of forskolin was found, other than a general inhibition of growth at concentrations over 10 μM. An inhibitor of phosphodiesterase, 8-bromo cyclic adenosine monophosphate (at concentrations of 0.1 and 1.0 μM) also significantly increased the number of gametocytes formed by this clone.

Notes: Characterization of a cytochalasin D-resistant mutant of the human parasite Entamoeba histolytica capable of growing at 10 μM cytochalasin is described. The mutant cells also show resistance to 5 mM colchicine and 100 μM cytochalasin B, drugs proved deleterious for wild type trophozoites. The mutants show increased osmotic fragility and electric mobility but reduced phagocytic activity, and agglutination by Concanavalin A. On the other hand pinocytic activity remains unaltered when compared with the wild type cells. Polymerized actin, seen by staining with phalloidin, often appears polarized to one end of the trophozoites and forms few of the endocytic invaginations found in wild type amebas. An altered distribution of part of the actin could explain the differences in surface properties and motility observed in the mutant amebas.

Notes: While the mating structure of unmated mating type minus (mt-) gametes of Chlamydomonas reinhardtii has few intramembrane particles (IMPs), activation results in movement of IMPs to its center. Analysis of freeze-fractured replicas of wild type (wt) mt- and 3 mt- fusion-defective mutants, gam-1, gam-10 and gam-11, before and after activation with wt+ flagella, provides a basis for suggesting that some of the IMPs in mt- mating structures, particularly a subset of particles that partitions to the E face, may be fusion-controlling molecules. Unmated gametes of gam-10 show a full range of images, from particle-free to fully activated, with both the P and E face of the mating structure revealing approximately twice as many IMPs as those observed on wt. Unactivated gametes of gam-1 and gam-11 appear identical to wt-. After activation, the mating structures of all of these gametes appear to have approximately the same number of IMPs. If the sizes of particles for these mutants are compared to wild type at the restrictive temperature, all 3 mutants have significantly smaller IMPs on the E face; before mating, in the plasma membrane and after mating, in the mating structure. At 34° C, the gam-1-II mating structure appears to be missing most of the particles from 15.5 to 16.5 nm in diameter, while all gametes with the ability to fuse have an equivalent percentage of their mating structure particles in this size range. The possibility that an IMP in this size range represents a protein that may be responsible for gamete fusion is discussed.

Notes: Pneumocystis carinii is a pathogen which, causes fatal pneumonia in patients with the acquired immune deficiency syndrome (AIDS). To facilitate the basic study of P. carinii, we have analyzed its major surface proteins by both immunochemical and biochemical methods. The major protein components of both cysts and trophozoites are a group of proteins called “P115” with apparent masses of 105–120 kd. It includes 6 isoelcclric variants. A monoclonal antibody raised against cysts recognizes all 6 variants and reacts with epitopes located in the cell wall indicating that P115 is an immunorcactive surface component. The isoelectric variants contain identical or closely related protein components and they are mannose-rich glycoproteins. The isoelectric variation may be due primarily to differences in glycosylation. The majority of sera from humans with diagnosed pneumocystosis that were tested reacted strongly with the P115 proteins. To develop probes for DNA diagnosis and to facilitate molecular studies, a genomic DNA library of P. carinii has been constructed. Some of these clones were used for DNA hybridization analysis of rat and human lungs.

Notes: . The behavior of nuclear envelopes during mitosis in Amoeba proteus was studied by means of indirect immunofluo-rescence staining using a monoclonal antibody against a 220-kD membrane-associated protein of amoebae in conjunction with DAPI staining of chromatin. The antibody selectively recognized antigens on nuclear envelopes during interphase but did not react with the nuclear membranes during mitosis until after cytokinesis had been completed. Thus, it appeared that the membrane-associated protein reacting with the monoclonal antibody and normally present on the nuclear membranes was absent from fragmented nuclear membranes or nuclear membranes that were continuous but did not have the honey-comb lamina. The findings suggested that the 220-kD nuclear-membrane protein may be involved in the dissolution and reformation of the honey-comb lamina during mitosis in amoebae.

Notes: Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the-capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary.The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.

Notes: The two unit membranes which envelope the endosymbiont of the trypanosomatid protozoon, Blastocrithidia culicis, were studied using the freeze-fracture technique. The distribution of the intramembranous particles on both fracture faces of the inner and outer membrane of the endosymbiont was analyzed in the replicas. The protoplasmic face of the inner membrane (PFi) had a higher density of membrane particles than that observed on the extracellular face (EFi), a pattern typical of plasma membranes. The extracellular face of the outer membrane (EFo) presented a density of membrane particles much higher than that observed on the P face of the outer membrane (PFo) a distribution significantly different from that found in the inner membrane of the endosymbiont and in the plasma membrane of the protozoon, but similar to that observed in Gram-negative bacteria. The data obtained support the idea that the endosymbiont of trypanosomatids represents a Gram-negative bacterium-like microorganism enveloped by two unit membranes and lacking a peptidoglycan layer and which lives in direct contact with the cytoplasm of the protozoon.

Notes: Paramecium caudatum syngen 3 could not reproduce in the defined medium (DM) which had been developed for P. octaurelia stock 299, but we succeeded in culturing it in DM supplemented with glycogen. The number of food vacuoles which formed in 5 and 10 min at 25°C in the DM alone was greater in comparison with the DM supplemented with glycogen. These results showed that the high molecular weight substance which needed to be added to a defined medium for the cultivation of Paramecium did not always support cell reproduction by stimulating food vacuole formation.

Notes: Ribosomes from Trypanosoma brucei rhodesiense and from Leishmania infantum were isolated and optimal conditions for in vitro translation were established. The effect of ribosome-inactivating proteins extracted from several plants was then assessed in order to identify those suitable for the preparation of immunotoxins against these organisms. Ribosomes from both species were inactivated by some ribosome-inactivating proteins (dianthins, saporins, pokeweed antiviral proteins, and the ribosome-inactivating chain of abrin). The similarity of the effects on the ribosomes from the two species examined indicates that ribosome-inactivating proteins should also be effective in a similar way on ribosomes from other species of Trypanosoma and Leishmania.

Notes: The Euplotes cytoskeleton obtained by treatment with Triton X-100 in a microtubule-stabilizing buffer has been studied by electron microscopy and SDS-polyacrylamide electrophoresis. An antiserum has been prepared using the tubulin band from preparative gels of Euplotes cytoskeletons as an antigen. This antiserum reacts with the tubulin from different ciliated protozoa but fails to recognize the vertebrate tubulin by immunoblotting. Immunoblotting studies have demonstrated a slower electrophoretic mobility for the α-tubulin of Euplotes and Oxytricha than that of Paramecium and Tetrahymena. A cytoplasmic microtubular network in Euplotes has been revealed by indirect immunoftuorescence using both an anti-α-tubulin monoclonal antibody directed against chick brain tubulin and an antiserum raised against the tubulin of Euplotes.

Notes: Some physical and chemical properties of DNA isolated from the dinoflagellate Woloszynskia bostoniensis were determined. Analytical cesium chloride gradient centrifugation gave a major component and a minor component banding at 1.719 and 1.693 g/cm, respectively. Thermal denaturation in 0.1 SSC showed a broad transition with a Tm of 70.5° C. Derivation of this curve indicated that two components were present having Tm values of 66° C and 70° C. Base composition analysis showed a GC content of 48.1% and a high degree of thymine replacement by 5-hydroxymethyluracil. Two minor bases, identified as 5-methylcytosine and N6-methyladenine, were also detected. Reassociation kinetics showed a typical eukaryotic reassociation pattern with 45% repetitive and 55% single copy sequences.

Notes: BIOMINERALIZATION is the process by which living organisms assemble structures from naturally occurring inorganic compounds. Mineral deposition is common and widespread amongst Protozoa and in most instances the mineralized structures provide skeletal support and protection for softer organic parts [10]. The 2 most common minerals to be deposited by Protozoa are silica and calcium carbonate. Groups of Protozoa that deposit silica, which we are concerned with here, include the diatoms, chrysophytes, choanoflagellates, Radiolar-ia, Heliozoa and testate amoebae [10].In the majority of silica-depositing protista, silica is taken up from the medium in the form of monomelic orthosilicic acid Si(OH)4 (soluble reactive silicate) and deposited as amorphous, polymerised biogenic silica or opal within membrane-bounded vesicles known as silica deposition vesicles (SDV). Often biogenic silica is characteristically patterned and ornamented and for most protozoan groups the morphology of silicified parts is of prime taxonomic importance.By far the most extensively studied group of silica-depositing organisms are the diatoms [1, 12, 13]. To date most of our knowledge of silica metabolism in protists has been based on investigations into this group. Diatoms require silica for the production of their frustules. Uptake and deposition of silica occurs within a closely denned portion of the cell cycle, between nuclear division and cell separation. It occupies about ± of the cell cycle and without an adequate supply of silica diatoms are unable to produce new frustule valves with the result that cell division cannot be completed. Diatoms, therefore, have an obligate requirement for silica and without this nutrient they cease to grow [11].In contrast to diatoms a number of other silica-depositing protistan groups, such as loricate choanoflagellates and certain chrysophytes, have a facultative requirement for silica. In the past decade the ultras true ture, physiology and ecology of loricate choanoflagellates have been extensively studied by a number of different workers [7] and the significance of these studies to our understanding of the mechanisms, controls and dynamics of silica secretion is summarised and discussed here.

Notes: . Trypanosoma eudyptulae n. sp. was present in 9 blood smears from 57 Little Penguins (Eudyptula minor Forster) from Tasmania. Trypanosoma eudyptulae is long and slender (with the kinetoplast situated close to the nucleus) with a long and attenuated posterior end. This is the first report of a trypanosome from a penguin.

Notes: . Cartwheel complexes reassembled in a fraction derived by treating isolated oral apparatuses from Tetrahymena with 1.0 M KC1 for 12 h. Approximately 40% of the KCl-soluble protein reassembled into cartwheel complexes. The reassembly reaction was protein-concentration dependent, and reassembled cartwheels were stable at 3° C. Sucrose gradient centrifugation resolved 3 high molecular mass protein complexes from the KCl-soluble fraction. Each of the 3 complexes has a different mass, but each contains the same 5 polypeptides, 2 of which arc probably tubulins. When these complexes were removed from the KCl-soluble fraction by high speed centrifugation, cartwheel reassembly did not occur. The 5 polypeptides in the high molecular mass complexes were among several other polypeptides resolved from reassembled cartwheels by 2-dimensional gel electrophoresis. The high molecular mass complexes are probably essential for cartwheel formation. The electrophorctic data also show that several polypeptides in the KCL-soluble fraction do not appear to be incorporated into cartwheels. These polypeptides are probably non-essential for cartwheel formation.

Notes: .We have analyzed the macronuclear DNA of Paramecium tetraurelia using orthogonal-field-altemation gel electrophoresis. The mean size of the linear macronuclear DNA molecules is approximately 450 kb. Less than 6% of the macronuclear DNA is larger than 800 kb. Using pulse times of 20, 40, 60 and 90 s we show that the macronuclear fragment containing the A type variable surface antigen gene migrates reproducibly as a 320-kb linear DNA. Over the same pulse times we describe the unusual migration of the ribosomal RNA gene (rDNA) of P. tetraurelia. At pulse times of 20 and 40 s the rDNA migrates at limit mobility (300 and 500 kb, respectively) whereas with 60- and 90-s pulse times, 2 components of rDNA are observed; 1 fraction independent of pulse time migrating at limit mobility, and a 2nd component migrating between 100-kb and 400-kb linear markers. Based upon previous electron micrographic studies of Paramecium rDNA as well as data presented here we conclude that the majority of Paramecium rDNA molecules are a circular DNA form.

Notes: The cytoplasmic 5S ribosomal RNA sequence from Pneumocystis carinii was determined and compared with those of 382 eukaryotes and an evolutionary tree was constructed to establish the phylogenetic position of Pneumocystis. The data suggest that Pneumocystis is associated with the Rhizopoda/Myxomycota/ Zygomycota group but not with common fungi, such as Ascomycota or Basidiomycota, nor with other protozoa.

Notes: Uluastraclurally, the cyst wall of Pneumocystis carinii consists of an electron-dense outer layer, an electron-lucent middle layer, and an innermost plasmalemma. This is similar in appearance to the cell wall of some yeasts, e.g. Saccharomyces cerevisiae, which consists of an outer dense layer of mannan, a middle lucent layer of β-1,3-glucan (yeast glucan) and an innermost plasmalemma. The cyst wall of P. carinii, as well as the cell wall of S. cerevisiae, can be labeled by a variety of methods which stain polysaccharides, such as Gomori's methenamine silver (GMS) and by Aniline blue, a dye which selectively stains β-l,3-glucan. The treatment of P. carinii cysts with Zymolyase, which the key enzyme is β1-l,3-glucan laminari-pentaohydrolase, results in lysis of the outer 2 layers of the cyst wall and the loss of positive staining by both GMS and Aniline blue. The lysis of elements of the cyst wall of P. carinii is achieved under the same conditions and concentration at which Zymolyase lyses the outer 2 layers of the cell wall of viable cells of S. cerevisiae. These observations indicate that a major component of the cyst wall of P. carinii is β-l,3-glucan.

Notes: .Trophozoites grown in vitro were shown to undergo binary fission by transmission electron microscopy (TEM). Standard fixation with subsequent embedding in Spun- was employed using 3% glularaldehyde and 1% osmium tetroxide with 5% sucrose added to both fixatives and 0.1 M cacodylate buffer washes. Trophozoites were grown on WI-38 cells in vitro. Trophozoites were found in various stages of fission. The dividing trophozoite has daughter cells that arc rounder than the pleomorphic, non-dividing trophozoites. Tubular forms external to the dividing trophozoites were decreased in number, tubular forms when present were concentrated around the forming sepia. Nuclear material was sometimes, but not always, well defined in both daughter cells. There was no concentration of nuclear material at the poles. Vacuoles without membrane were present in the dividing forms. Separate nuclear regions were sometimes found in the dividing trophozoites. These observations suggest that binary fission does occur in culture; however, the significance of binary fission to the life cycle of Pneumocystis carinii (Pc) is not yet clear.

Notes: . Cysts of Giardia microti, isolated from feces and intestinal contents of Microtus ochrogaster, were examined by light and electron microscopy. These cysts differed morphologically from cysts of other G. duodenalis morphological types in that these cysts often contained two apparently differentiated trophozoites with mature ventral discs. Cysts more closely resembling those reported for G. lamblia and G. muris were in greater abundance in preparations made from intestinal contents and were interpreted as immature cysts. “Multiple fission” cysts, reported in G. muris and G. microti by earlier workers, were not observed; however, endosymbiotic bacteria were found in the cysts of G. microti and may have been responsible for reports of multiple fission in the cysts of Giardia.

Notes: The 115 kd band in polyacrylamide gels is a major antigen of Pneumocystis carinii. Data obtained from treatment with enyzmes, binding to lectins, and labelling the surface with biotin suggest that this moiety is a glycoprotein containing mannosyl/glucosyl and N-acetylglucosamine residues, and that it is located on the cell wall of the organism. Other rat and human P. carinii antigens also are glycoproteins but differ in specific protein or carbohydrate residues or location on the organism.

Notes: The binding characteristics of a panel of commercially available hi lC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infected lung homogenates were incubated with FTTC-conjugated lectins in a series of concentrations, counterstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acetylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simplicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Madura pomifera and Dolichos biflorus agglutinins and Giffonia simplicifolia I lectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffonia simplicifolia 1-β4 lectin had not effect. The organisms reacted weakly with Ulex europeus 1 agglutinin which is specific for fucose and did not react with Limax flavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.

Notes: Cochlosoma anatis Kotlán (Zoomastigophorea, Retortamonadida, Cochlosomidae), isolated from the large intestines of domestic Rouen ducks, and Cochlosoma soricis n. sp., isolated from the small intestines of shrews, were observed by light and scanning electron microscopy. In both organisms, a single flagellum inserted on the dorsal surface at the same level as the insertion of 4 other flagella on the ventral surface. The 4 ventro-lateral flagella emerged from the left side of the anterior attachment disk below the margin and just above the lateral groove which extended the length of the organism. A 6th flagellum emerged from the margin of the attachment disk. The proximal ends of the flagella formed a bundle with the distal ends becoming unraveled like a rope. During motility, the bundle portion extended straight out from the cell and the free ends of the flagella produced a whipping motion. In C. anatis, the dorsal surface was covered with knob-like lumps and small pits and the cells had an axostyle that emerged slightly to the right of the midline in the posterior 1/3 of the body. The axostylar tip was shorter and thicker than the flagella and in most cells it also had an irregular, knobby appearance. The irregular cell surface and axostyle were absent from C. soricis. The margin of the attachment disk curved toward the center and terminated in C. anatis as a straight edge while in C. soricis it continued as a spiral. Indentations in the mucosal brush border similar to those produced by Giardia, but distinctly belonging to Cochlosoma, were interpreted as points of attachment to the host.

Notes: Culture forms of Trypanosoma rangeli could be agglutinated with Canavalia ensiformis (Con A) lectin and, less effectively with Pisum sativum agglutinin (PEA), at a concentration of 200 μg/ml. Ricinus communis agglutinin I (RCA I) agglutinated trypanosomes only if they were not previously washed with physiological Ringer's solution. Three other lectins did not react with the same parasite forms. Direct or indirect lectin-gold labeling techniques were applied to LR-White embedded thin sections of T. rangeli culture forms and to forms in the gut, hemolymph, and salivary glands of Rhodnius prolixus. Under these conditions, Con A was the only lectin out of 9 that bound to the surface of trypanosomes from culture and from the bug hemolymph. Con A did not react with any midgut or salivary gland forms. The preservation of the biological activity of the lectin-gold complexes that did not bind to the parasite surface was confirmed by reactions with structures of the invertebrate host.

Notes: This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating 〉 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of 〉48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.

Notes: 13C-nuciear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeIed substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-l/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [l,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%. This is consistent with rapid equilibration of alanine with pyruvate derived from glucose and yet little decrease in the specific activity of the large alanine pool.

Notes: A recent analysis of sequence variations in ribosomal RNA's from 31 species of tetrahymenine ciliates groups them into 9 sets referred to as “ribosets.” These species associations are not well correlated with the distributions of distinctive morphological characteristics. The phylogenetic structure suggests that modem “pyriform” tetrahymenines may be paraphyletic survivors of primitive design and that the morphologically distinctive forms may include examples of convergent evolution of derived forms. Alternatively, the common ancestor may have been a polymorphic species that has lost its plasticity in some derived lineages. In an attempt to test the ribosomal phylogeny, we here compare it with a phytogeny based on isozymic variation. The main features of the ribosomal and isozymic phylogenies are similar. The carnivorous (macrostome-forming) species are widely scattered in both, as are the bacteriophagous pyriform species. Isozymic and ribosomal analyses are optimally useful, however, in different contexts. Isozymic variations can distinguish species that are ribosomally identical. Ribosomal variations provide more secure evaluations of distant relationships.

Notes: Large numbers of Pneumocystis carinii (2 × 1010 nuclei) were isolated and separated from the lungs of immunosupprcsscd rats by an enzymaric (collagcnasc, hyaluronidasc and DN'asc) digestion procedure. The nucleic acid isolated from this P. cartnii-cnnchcd preparation was characterized by melting point analysis and RNA-sizing gels. The GC content of P. carinii DNA was approximately 33% while the rat DNA was 41.4%. In addition, RNA isolated from the P. curmii-enrichcd preparation showed unique ribosomal RNA bands of 3.4 kb and 1.8 kb as compared with uninfected rat lung ribosoma! RNA. which banded at 4.8 and 1.9 kb. Following isolation and fragmentation by sonicaüon, the P. carinii D.VA fragments were inserted into the vector, λ gt-11. The resultant library contained 1.1 × 105 phage, of which 40–45% hybridized to P. carinii DNA but not to rat DNA.

Notes: In this study, the presence of actin in cultured trypanosomatids was investigated using polyclonal antibodies to heterologous actin. Polyclonal antisera to rabbit muscle actin and a monospecific anti-actin antibody react with a 43-kDa polypeptide in extracts of Trypanosoma cruzi, Herpetomonas samuelpessoai and Leishmania mexicana amazonensis on protein immunoblots. The 43-kDa polypeptide co-migrates with skeletal muscle actin and is retained within trypanosomatid cytoskeletons. Attempts to isolate H. samuelpessoai actin through DNase I affinity chromatography showed that the 43-kDa polypeptide did not bind to the column. Instead, low yields of a 47-kDa polypeptide were obtained indicating that the trypanosomatid actin displays unusual DNase I binding behavior when compared to actins from higher eukaryotes. Immunofluorescence studies confirmed that cytoskeletons retain the actin-like protein. In H. samuelpessoai, actin is localized in the region close to the flagellum, whereas in T. cruzi it is more homogeneously distributed. The data presented here show that trypanosomatid actin displays biochemical characteristics similar to actins of other protozoa.

Notes: Toial RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity. An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method. Host cells were eliminated by hypotonic lysis and a series of microfiltrations. Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents. The major ribosomal species of P. carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA. The 28s-like species migrated well ahead of rat and A549 cell rRNA and weli behind the prokaryotic large rRNA species.

Notes: The cytoplasmic 58 ribosomal RNA sequence from Pneumocystis carinii was determined and compared with those of 382 eukaryotes and an evolutionary tree was constructed to establish the phylogenetic position of Pneumocystis. The data suggest that Pneumocystis is associated with the RhizopodaAlyxomycota/ Zygomycota group but not with common fungi, such as Ascomycota or Basidiomycoia, nor with other protozoa.

Notes: The antimicrobial agent novobiocin, an inhibitor of the bacterial enzyme topoisomerase II (DNA gyrase), is known to antagonize Trypanosoma cruzi amastigotes growing in cell-free medium. To determine sites of antagonism of novobiocin, the effects of drug on parasite ultrastructure and incorporation of radiolabeled precursors of DNA, RNA and protein into macromolecules were determined. The predominant ultrastructural abnormality seen after exposure to 0.40 mM novobiocin for 24 h was the presence of electron-dense clumps in the mitochondrion-kinetoplast organelle in 95 of 257 (37%) of cells, in comparison to no clumps seen in 110 drug-free cells. In addition, in the nucleus, the karyosome was less distinct than in control cells and appeared to merge with the chromatin. In the radiolabcling studies, incorporation of thymidine was inhibited in a dose-dependent fashion by novobiocin (0.16–0.80 mM) in a range of drug concentrations that also inhibited parasite growth. For 0.16 and 0.24 mM novobiocin, incorporation of thymidine was inhibited up to 65% relative to drug-free control cells while uptake of uridine and leucine was unaltered. We interpret these ultrastructure and precursor-incorporation studies as suggesting that (i) the mitochondrion-kinetoplast and possibly the nucleus are sites of novobiocin antagonism of T. cruzi amastigotes and (ii) that novobiocin appears to antagonize DNA synthesis within these organisms. Whether the drug target is topoisomerase II, however, is as yet unknown.

Notes: We have previously shown that nonimmune human milk kills Giardia lamblia trophozoites in vitro. Killing requires a bile salt and the activity of the milk bile salt-stimulated lipase. We now show that human small-intestinal mucus protects trophozoites from killing by milk. Parasite survival increased with mucus concentration, but protection was overcome during longer incubation times or with greater milk concentrations. Trophozoites preincubated with mucus and then washed were not protected. Protective activity was associated with non-mucin CsCl density gradient fractions. Moreover, it was heat-stable, non-dialyzable, and non-lipid. Whereas whole mucus inhibited milk lipolytic activity, protective mucus fractions did not inhibit the enzyme. Furthermore, mucus partially protected G. lamblia trophozoites against the toxicity of oleic acid, a fatty acid which is released from milk triglycerides by lipase. These studies show that mucus protects G. lamblia both by inhibiting lipase activity and by decreasing the toxicity of products of lipolysis. The ability of mucus to protect G. lamblia from toxic lipolytic products may help to promote intestinal colonization by this parasite.

Notes: The new microsporidium (Microsporida: Pereziidae), Perezia dichroplusae n. sp., infects the epithelial cells of the Malpighian tubules of the Argentine grasshopper Dichroplus elongatus. Characteristics of the pathogen include the following: development in direct contact with the host cell cytoplasm; bi-, tetra-, and sometimes multinucleate diplokaryotic meronts, rounded or elongate in shape; unikaryotic sporonts and sporogonial plasmodia, elongate in shape; sporoblasts and spores uninucleated; spores highly variable in size (1.6–6.7 by 1.0–2.7 μm, x̄= 3.5 ± 0.09 by 1.5 ± 0.02, n = 100) with eight or fewer polar tube coils and showing a posterior electron-dense inclusion body.

Notes: Trophozoites grown in vitro were shown to undergo binary fission by transmission electron microscopy (TEM). Standard fixation with subsequent embedding in Spurr was employed using 3% gluuraldehydc and 1% osmium tetroxide with 5% sucrose added to bom fixatives and 0.1 M cacodylate buffer washes. Trophozoites were grown on WI-38 cells in vitro. Trophozoites were found in various stages of fission. The dividing trophozoite has daughter cells that arc rounder than the pleomorphic, non-dividing trophozoites. Tubular forms external to the dividing trophozoites were decreased in number; tubular forms when present were concentrated around the forming septa. Nuclear material was sometimes, but not always, well defined in both daughter cells. There was no concentration of nuclear material at the poles. Vacuoles without membrane were present in the dividing forms. Separate nuclear regions were sometimes found in the dividing trophozoites. These observations suggest that binary fission does occur in culture; however, the significance of binary fission to the life cycle of Pneumocystis carinii (Pc) is not yet clear.

Notes: Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26, 142 nucleotides (8, 714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs (Oxytricha and Stylonychia) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the “preferred” codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.

Notes: Two new phenomena were observed during macronuclear development in E. patella. During the formation of giant chromosomes, the number of chromosomes decreased while individual chromosomes gradually became longer and thicker. Immediately before macronuclear elongation, ring-like anlagen appeared, which did not contain chromatin at their centers. The course of macronuclear development in Euplotes is reconsidered in light of these findings.

Notes: Pneumocystis carinii-specitic immune complexes were detected by immunoblot and enzyme-linked immunosorbent assay (ELISA) in 53% of sera from Acquired Immunodeficiency Syndrome (AIDS) patients with P. carinii pneumonia (PCP). Resolution of glycoprotein antigenemia (50–55 kd = dominant species) appears to correlate with successful PCP drug therapy and recovery. An epitope map has been constructed from im-munoblots of P. carinii hydrolysates and from human and murine scrum containing P. carinii antigens.

Notes: An ultrastructural study of life cycle stages of Pneumocystis carinii in infected rat lungs in situ was undertaken utilizing 8 different modes of fixation. Three of the fixatives employed gave good fixation of cysts and intracystic bodies, but for the trophic forms fixation was only fair. Both the trophic forms and intracystic bodies have nuclear pores. The mitochondria of the organism have cristac that appear lamellar. One of the fixation modes revealed a thin, electron-dense layer on the outer surface of the cell wall, a “fuzzy coat” that had not been described previously. This material appears to mediate tight adhesion of trophic forms with other trophic forms, cysts, and with pneumocytcs of the lung alveolus.

Notes: Pneumocystis carinii-parasitizcd lung explains were obtained from corticoid-lreated rabbits and maintained in vilro. Twenty-one days after the beginning of explant cultures, the ullrastructura! morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ui-trastrucuirc of P. carinii from infected rabbit. However, after the 36th day, only altered forms of P. carinii were observed. Lung tissue showed only minor alterations. fntracytoplasmic lamellar inclusions were observed in type 2 alveolar cells from which they were released. While the total number of parasites increased approximately 4-fold from day 0 to day 41, trophozoite counts increased approximately 6 times. Pneumocystis cells from inocula and supcmaics of cultures with and without Vera cells showed important ullrastructural alterations.

Notes: The 115 kd band in polyacrylamide gels is a major antigen of Pneumocystis carinii. Data obtained from treatment with enyzmes, binding to lectins, and labelling the surface with biotin suggest that this moiety is a glycoprotein containing mannosyl/glucosyl and N-acetylglucosamine residues, and that it is located on the cell wall of the organism. Other rat and human P. carinii antigens also are glycoproteins but differ in specific protein or carbohydrate residues or location on the organism.

Notes: The binding characteristics of a panel of commercially available FITC-conjugated lectins to Pneumocystis carinii (Pc) were assessed by fluorescence microscopy and flow cytometry. Rat Pc obtained from infecteding homogenates were incubated with FTTC-conjugated lectins in a series of concentrations, counlerstained with propidium iodide, and analyzed for percent fluorescence and fluorescence intensity. All organisms bound concanavalin A and Wisteria floribunda agglutinin, 2 representatives of the glucose/mannose-binding group. From the lectin group specific for N-acctylglucosamine, Pc reacted more strongly with wheat germ agglutinin than with Solanum tuberosum agglutinin or Griffonia simpiicifolia II lectin. Pneumocystis treated with lectins specific for N-acetyl-D-galactosamine and galactose exhibited much variation; the cells reacted moderately well to soybean agglutinin and less to Bauhinia purpurea, Madura pomifera and Dolichos biflorus agglutinins and Giffonia simpiicifolia Hectin. Arachis hypogaea agglutinin, Viscum album agglutinin and Griffon'ui simpiicifolia I—β Section had not effect. The organisms reacted weakly with Ulex europeus I agglutinin which is specific for fucose and did not react with Limax ftavus lectin, which is specific for sialic acid. Competitive inhibition studies using relevant carbohydrates were performed to indicate that the positive reactions were specific. These studies should help to elucidate the mechanisms of attachment and pathogenesis of this organism.

Notes: The major ester-linked fatty acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid-trcaicd rats were 16:0. 18:0, 18:1, 18:2 and 20:4. Others detected included 14:0, 16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0,22:0,24:0 and 24:1; others included 14:0, 18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Notes: Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2′- deoxycytidine and 5-fluoro-2′-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2′-fluoro-2′-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.

Notes: . Lagenophrys anticthos n. sp. resembles L. aegleae Mouchet-Bennati; however, the two species are distinguished from one another by differences in the structure of the lorica aperture. Similarities in the shape of the lorica and macronucleus indicate a close phylogenetic relationship between L. anticthos and L. aegleae. The size of L. anticthos varies greatly within a population, and it is unclear whether this can be attributed to genetic differences or to environmental factors. In L. anticthos, variation in the form of the lips of the lorica aperture is correlated with variation in size. The brown, iron-rich incrustations observed around the loricae of L. aegleae by an earlier worker were not seen, indicating that the incrustations do not play a role in the symbiosis between L. aegleae and its host as was previously thought.

Notes: Morphology and locomotive behavior in the marine amoeba, Paramoeba pemaquidensis Page, was examined under different environmental conditions. Paramoeba requires a minimum surface negative charge density for adhesion of amoebae to substrata. Once adhesion to the substratum has been attained, however, surface negative charge density has no effect on morphology or locomotive rate. Divalent cations are not required for adhesion, but external calcium is required for normal locomotion. In the presence of calcium, Paramoeba often assumes a locomotive form with a broad, well-developed anterior hyaline region and truncate posterior region. Locomotive forms vary from those with only a well-developed hyaline region (Flabellula-like) to forms with long digitiform sub-pseudopodia (Vexillifera-like), with intermediate morphotypes. Locomotive rates decrease and anteroposterior polarity disappears in the presence of living or heat-killed bacteria, indicating that phagocytosis temporarily interferes with locomotion and alters form.

Notes: The vertical distributions of two pond-dwelling zoochlorellae-bearing ciliates (Euplotes daidaleos Diller & Kounaris, 1966 and Frontonia vernalis Ehrenberg, 1838) were monitored over a 24-h period. Both species maintained peak abundance at a low O2 level (usually 〈 1 mg/liter). They did not migrate in response to the changing light level. Experiments with laboratory cultures indicated that the characteristic distribution in an O2 gradient in the dark was largely controlled by the oxygen tension. The increased motility in anoxia and high pO2 was independent of large changes in pCO2 and pH. Ciliates living in anoxia or a very low pO2 would migrate out of the dark and into the dimly lit (10 μE m-2 sec-1) part of a glass cell because there they could photosynthesize, produce O2, and create a suitable oxygenated microenvironment; a further increase in the light level caused a slow migration out of the light. Similar migrations were observed when the light level remained low but the pO2 was artificially raised. Ciliates suspended in 1 μM DCMU (an inhibitor of photosynthetic O2 evolution) took longer to migrate into the light and they did not avoid high light levels (〉 100, μE m-2 sec-1)- Frontonia suspended in water with a pO2 of 1% aggregated at a low light level (1 μE m-2 sec-1); peak daytime abundance in the pond occurred at about this light level. Frontonia vernalis tends to swim vertically upwards (anterior end up) when suspended in anoxic water. This apparent negative geotaxis compensates for the high sedimentation velocity (0.36 mm sec-1) of this large ciliate and facilitates its aggregation at the metalimnion. The O2 tension appears to be the principal factor controlling the vertical distributions of both species. Occasional, enhanced convection within the metalimnion has a secondary influence. Light influences the vertical profile only if it promotes photosynthesis and increases the intracellular pO2.

Notes: Population dynamics of round and elongate gametocytes of Leucocytozoon in wild and captive blue grouse (Dendragapus obscurus (Say)) from Hardwicke Island, British Columbia, were studied from 1980 to 1982. Blue grouse chicks were sampled weekly throughout each transmission season. Three patterns in the type of gametocyte produced during primary infection were observed in naturally-infected captive and wild blue grouse chicks. Such variation in the expression of the gametocyte stage within a single host population suggests a different interpretation than has been previously reported for species of Leucocytozoon. The data from the primary patterns and profiles coupled with reexposure data and the asynchronous appearance of round and elongate gametocytes can be best interpreted as infection with two concurrent species of Leucocytozoon in blue grouse. More detailed research on the life cycle is necessary to confirm if two species of Leucocytozoon exist in blue grouse.

Notes: We report a method that allows us to grow and maintain the freshwater ciliate Euplotes octocarinatus in large quantities. Frequent exchange of culture fluid proved more effective than aeration in obtaining high cell densities (4200 cells/ml) and reasonable doubling times in large-scale cultures. For harvesting gamone 1, the cell density was raised to 10,000 cells/ml. Under these conditions, the cells continued to produce and secrete gamone; they were slightly starved, but they no longer divided. Cell-free fluid with a steady and relatively high yield of gamone was obtained from two such cultures over a period of five months. We isolated gamone 1 also from cell homogenates and compared it with secreted gamone 1, but found no differences in the gamones from these two sources.

Notes: Because heavy branchial infestations are thought to interfere with respiration, we examined the attachment of three stalked ciliates commonly found in the branchial chambers of Louisiana crawfish. Attachments by Cothurnia sp., Epistylis sp. and Acineta sp. differ in their fine structure. Stalks of the peritrichous ciliates. Cothurnia and Epistylis, contain striated tubules that differ in their arrangement, diameter, and in the periodicity of their striations. In both species the striated tubules branch within the basal disk and attach to a pad of adhesive material secreted by the organism during initial attachment to the gill surface. The stalk of the suctorian Acineta is composed of a striated honeycomb-like matrix. Within the basal disc the matrix is disorganized; however, striated elements anchor the stalk to a pad of adhesive material. Attachment sites also differ in the amount of secretory material deposited. Cothurnia forms a multi-layered, granular pad; Epistylis forms an indistinct, microfibrillar layer, and Acineta deposits a thick mucoid pad. None of the ciliates appear to damage the gill epicuticle nor is there an obvious host response. Harmful effects are probably limited to a decrease in respiratory surface area and disruption of normal water flow patterns. This may impair respiration sufficiently to increase the susceptibility of crawfish to low dissolved oxygen concentrations encountered periodically in commercial crawfish ponds.

Notes: Leucocytozoon caulleryi sporozoites that had been stored at - 196° C or -80° C for 6 or 12 months in Eagle's minimum essential medium or Medium 199 supplemented with 5% glycerol and 10% chicken serum showed infectivity to chickens. Glycerol at a concentration of 10% and dimethyl sulfoxide at 10% and 5% were found to be ineffective cryoprotective agents for the low temperature preservation of sporozoites. Sporozoites isolated from the intact females of Culicoides arakawae, which had been stored at -80° C for 6 or 12 months without cryoprotective agents, retained their infectivity. No differences were observed in the prepatent period, duration of parasitemia, and presence of serum-soluble antigens between chickens infected with frozen sporozoites and those infected with fresh sporozoites.

Notes: Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.

Notes: A total of 169 cross-transmission attempts has been made with 44 (11.8%) of the 372 named species of Eimeria of rodents. Of these, 161 were rodent-to-rodent, 6 rodent-to-lagomorph, and 1 each rodent-to-carnivore and rodent-to-bird. None of the last three categories was successful. In the rodent-to-rodent combinations, 39 (80%) of the 49 attempts to transmit a coccidian species from one rodent species to another of the same genus were successful, and only 14 (12.5%) of the 112 attempts to transmit a coccidium to a rodent of a different genus were successful. Eight of the successful attempts were with E. chinchillae, which was the only truly euryxenous species of Eimeria in the group. Two successful attempts were between the closely related rodent genera Spermophilus and Cynomys, and two were both of E. separata from Rattus norvegicus to some genetic strains but not to others of Mus musculus. One attempt with E. vermiformis from Mus musculus to Rattus norvegicus required treatment of the rat with the immunosuppressant dexamethasone to succeed. More cross-transmission studies are needed to determine the host-spectra of the species of Eimeria and other coccidian genera, and to determine the roles of genetics and immunosuppression in their transmission.

Notes: Cortical morphogenesis during excystment in Histriculus similis has been studied by light microscopy of preparations impregnated with silver proteinate (protargol). This morphogenesis shows clear differences from cortical morphogenesis during division. The oral, paroral, and fronto-ventro-transverse primordia originate from an extensive field of kinetosomes. The marginal cirral primordia and the dorsal bristle primordia appear at an early stage of morphogenesis. The left marginal cirral primordium originates from the oral primordium.

Notes: Using restriction enzyme analysis, mitochondrial DNA fragment patterns from seven strains of pathogenic and nonpathogenic Naegleria and one strain of Vahlkampfia were compared to estimate nucleotide sequence divergence. Significantly high levels of estimated genetic variation between strains of N. gruberi, N. fowleri, and N. jadini support the current taxonomic level of the individual Naegleria species and suggest a distinct phylogeny for each group. Naegleria lovaniensis, strain TS, was shown to have significant nucleotide sequence homology with N. gruberi, strain EGs, suggesting that the two groups share a close taxonomic relationship. The pathogenic strain MB-41 of N. fowleri exhibited distinct genetic divergence from the highly homologous, pathogenic strain Nf66 and the drug-cured strain 6088. Morphologically distinct strains EGs and 1518/la of N. gruberi exhibited significantly large sequence divergence consistent with a more distant taxonomic relationship. Amoebae from the genus Vahlkampfia expressed genetic similarity with strains of N. gruberi.

Notes: Bodo saltans was isolated from a chalk stream and fed with pure cultures of seven bacteria obtained from the same river. The flagellates were allowed to migrate into suspensions of either of two bacterial species in a T-maze at 20–22°C. There was a significant difference (P 〈 0.01) between the numbers of flagellates which migrated into suspensions of different bacteria, which were subsequently arranged in an order of “attractiveness” to the flagellate. Bodo saltans grew successfully in monoxenic suspensions of all seven bacterial strains, but more rapid growth occurred with non-flagellated than with flagellated bacteria; this may be because while feeding, B. saltans tends to associate with surfaces where non-flagellated bacteria may also congregate. The efficiency with which B. saltans is able to utilize different bacteria may be influenced by the motility or secretory activities of the bacteria. There was no incontrovertible evidence that B. saltans responds to specific bacterial attractants.

Notes: Thirteen eastern moles, Scalopus aquaticus, collected in West Texas were examined for coccidian oocysts; 11 (85%) were infected and eight (73%) of these had multiple infections representing two or more species. One cyclosporan, three eimerians, and two isosporans were studied and all are described as new species. Sporulated oocysts of Cyclospora megacephali n. sp. were subspheroidal, 18.5 × 15.7 (14–21 × 12–18) μm; they had sporocysts pointed at one end with Stieda bodies nearly as wide as the sporocysts themselves, and were 15.0 × 7.2 (11–17 × 6–9) μm; C. megacephali was found in four (31%) hosts. Sporulated oocysts of Eimeria scalopi n. sp. were spheroidal to subspheroidal, 13.6 × 12.6 (11–17 × 11–15) μm with sporocysts lemon-shaped, 8.7 × 5.5 (7–10 × 4–7) μm; it was found in six (46%) hosts. Sporulated oocysts of Eimeria aquatici n. sp. were asymmetrically ellipsoidal, 17.0 × 10.6 (14–20 × 9–14) μm, with sporocysts elongately ovoidal, 9.0 × 5.2 (8–11 × 4–6) μm; it was found in two (15%) hosts. Sporulated oocysts of Eimeria motleiensis n. sp. were subspheroidal, 17.0 × 15.3 (15–20 × 13–18) μm with sporocysts ovoidal, 10.7 × 6.8 (10–13 × 6–8) μm; it was found in seven (54%) hosts. Sporulated oocysts of Isospora motleiensis n. sp. were spheroidal to subspheroidal, 13.6 × 12.0 (10–17 × 8–15) μm with sporocysts broadly ovoidal, 9.5 × 6.7 (7–11 × 4–8) μm; it was found in nine (69%) hosts. Sporulated oocysts of Isospora aquatici n. sp. were subspheroidal, 20.9 × 18.4 (15–24 × 13–21) μm with sporocysts ellipsoidal, 11.8 × 9.0 (9–14 × 7–11) μm; it was found in two (15%) hosts.

Notes: Irradiation with X rays, UV irradiation after incorporation of bromodeoxyuridine (BU) into the DNA, and cis-platinum (cis-Pt) treatment each cause the loss of micronuclei of Stylonychia lemnae while the macronuclei are not severely affected. The abilities of both nuclei to repair DNA were investigated. Unscheduled DNA synthesis could not be demonstrated after X-ray irradiation, but it was found after treatment with BU/UV and cis-Pt in macro- and micronuclei. The extent of the repair process in the micro- and macronuclei was alike, as indicated by grain counts of [6-3H]thymidine-treated cells. One reason for the different sensitivity of both nuclei to DNA-damaging treatment may be the different number of gene copies in the macro- and micronuclei.

Notes: Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 57% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ˜20 protein bands. The major bands are at 31 and 30 kD (G1), 27 and 26.5 kD (G2), 25 kD, 23 kD, and six bands at 15–20 kD (G3). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca2+ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G1 and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G3 proteins. On two-dimensional gels of trichocysts, ˜40 acidic protein spots are resolved with pI's of 4.6–6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G3, in two spots at 23 kD, in all five spots of G1, and in seven spots over 35 kD.

Notes: Goussia girellae n. sp. is described from the opaleye fish, Girella nigricans. Merogonic stages were observed in the apices of intestinal epithelial cells, in the lamina propria, and in extra-intestinal sites including liver, gills, and spleen. Gamonts were observed in the intestinal epithelial cells. Only unsporulated oocysts were detected in the intestine, and sporulation occurred when feces containing oocysts were incubated for 48 h in seawater at 21°C. Oocysts are elongated (24.8 × 14.7 μm) with a wall about 200 nm thick and have no residuum, micropyle, or polar granule. Sporocysts are ellipsoid (8.5 × 4.5 μm), have a thin two-layered wall approximately 30 nm thick, and consist of two valves joined by a suture. Although moribund opaleye were also infected with Gyrodactylus sp., Cryptobia sp., Cardicola sp., and epitheliocystis organisms (chlamydia), all fish were heavily infected with G. girellae and morbidity was thus attributed to the coccidium.

Notes: Trimyema compressum is a species included in the family Trimyemidae with the single genus Trimyema. This species has 50–60 somatic kinetics and three rows of kinetosomes surrounding the oral cavity. Two isolated groups of kinetosomes can also be observed on the right side of the oral region. The morphogenesis of bipartition is telokinetal; all the new infraciliary structures of the opisthe come from the longitudinal and postero-anterior proliferation of the last kinetosome of all the somatic kinetics. In the proter there is a reorganization of the oral infraciliature.As a result of our observations, we suggest that the systematic position of the genus Trimyema be changed from the subclass Vestibulifera to the subclass Gymnostomata. We also consider that this genus must be included in the suborder Trimyemina Jankowski, 1980.

Notes: Specimens of Pelomyxa palustris from five collecting sites had numerous nonmotile flagella. The structures are called flagella because of morphological similarities to flagella and because P. palustris has affinities with amoeboid flagellates. Flagella were photographed on living cells and studied by transmission and scanning electron microscopy. From 64 to 742 flagella per cell were estimated from scanning electron microscopy of ten cells 204 to 1269 μm in length. The nonmotile flagella arise from basal granules which were, in one strain, surrounded by radiating electron-dense microtubules. This strain also had excess axonemal microtubules. Abundant cytoplasmic microtubules were arranged in several different patterns. In about half of the P. palustris cells in which nuclei were studied, microtubules were either apposed to the nuclear membrane in a parallel alignment (with some also radiating) or radiating from the nuclear membrane (with none parallel). Bacteria associated with nuclei were of three characteristic types: Gram-negative rods, Gram-positive rods, and large rods. All nuclei within a given trophozoite had similar perinuclear features. Recent proposals for separation of Pelomyxa to its own phylum (based on its proposed primitive, unique nature) can not be justified. Pelomyxa is a complex, highly specialized organism adapted to live in a specific fresh-water environment. Mastigamoebid amoeboid flagellates of the genera Mastigamoeba, Mastigella, Mastigina, and possibly Dinamoeba are placed with Pelomyxa within the order Pelobiontida Page, 1976, emend., containing two families. Pelomyxidae Schulze, 1877, and Mastigamoebidae Goldschmidt, 1907.

Notes: Utilizing the previously reported inter-clonal differences in total DNA/organism, flow cytometry was used to analyze the population dynamics of Trypanosoma cruzi clone mixtures growing in liquid medium or vertebrate cells. The growth of clone mixtures in liquid medium can be described by unique parameters reflecting exponential growth rate (r), stationary phase population density (1/k), and the interaction between the clones (h). The relative numbers of each clone in the population change rapidly with time and the results arc in quantitative agreement with mathematical models of competitive population growth. The relationship between the parameters for T. cruzi is such that, in general, there is no dynamic equilibrium with coexistence of clones with different growth rates; under all culture protocols, the faster growing clone will prevail. A computer simulation of the vertebrate cell cycle of T. cruzi suggests that clone mixtures grow relatively independently; the basic attributes of the model were substantiated experimentally. Although wide fluctuations in the proportion of each clone released occurred, the faster growing clone again predominated. Finally, these results underline the importance of working with well-defined clones in the laboratory to avoid inconsistencies and paradoxical results and stress the importance of the rapid isolation of single cell clones from clinical specimens when studying the relationship of the parasite to human disease.

Notes: Stimulation of phagocytosis by serotonin and catecholamincs in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being ∼0.1 to 1.0 μM. The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the β- and α-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis. A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena. Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine. Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.