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: 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: 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: 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: One third of a collection of cloned Stylonychia pustulata micronuclear DNA PstI fragments were found to be of a similar size, consistent with their being members of a repetitious sequence family with a repeat size of about 160 base pairs. Cross-hybridization experiments confirmed that these small cloned fragments are related by sequence homology. Hybridization of the cloned repetitious sequences to PstI digested micronuclear DNA revealed a “ladder” of bands (step size = 160 base pairs), indicating that the repeats are found in tandem arrays. This is the first demonstration of highly repetitious, tandemly repeated sequences in a ciliated protozoan.

Notes: The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of 3H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.

Notes: Bloodstream trypomastigote and culture procyclic (insect midgut) forms of a cloned T. rhodesiense variant (WRATat 1) were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), soybean agglutinin (SBA), fucose binding protein (FBP), wheat germ agglutinin (WGA), and castor bean lectin (RCA). Fluorescence-microscopic localization of lectin binding to both formalin-fixed trypomastigotes and red cells was determined with fluorescein isothiocyanate (FITC)-conjugated Con A, SBA, FBP, WGA, RCA, PNA (peanut agglutinin), DBA (Dolichos bifloris), and UEA (Ulex europaeus) lectins. Electron microscopic localization of lectin binding sites on bloodstream trypomastigotes was accomplished by the Con A-horseradish peroxidase-diaminobenzidine (HRP-DAB) technique, and by a Con A-biotin/avidin-ferritin method. Trypomastigotes, isolated by centrifugation or filtration through DEAE-cellulose or thawed after cryopreservation, were agglutinated by the lectins Con A and PP with agglutination strength scored as Con A 〈 PP. No agglutination was observed in control preparations or with the lectins WGA, FBA or SBA. Red cells were agglutinated by all the lectins tested. Formalin-fixed bloodstream trypomastigotes bound FITC-Con A and FITC-RCA but not FITC-WGA, -SBA, -PNA, -UEA or -DBA lectins. All FITC-labeled lectins bound to red cells. Con A receptors, visualized by Con A-HRP-DAB and Con A-biotin/avidin-ferritin techniques, were distributed uniformly on T. rhodesiense bloodstream forms. No lectin receptors were visualized on control preparations. Culture procyclics lacked a cell surface coat and were agglutinated by Con A and WGA but not RCA, SBA, PP and FBP. Procyclics were not agglutinated by lectins in the presence of competing sugar at 0.25 M. The expression of lectin binding cell surface saccharides of T. rhodesiense WRATat 1 is related to the parasite stage. Sugars resembling α-D-mannose are on the surface of bloodstream trypomastigotes and culture procyclics; n-acetyl-D-galactosamine and D-galactose residues are on bloodstream forms; and n-acetyl-D-glucosamine-like sugars are on procyclic stages.

Notes: The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.

Notes: Distinctive organic-walled resting cysts of at least three different types with a highly conservative morphology appear to characterize specific orders or groups of genera within the Class Polyhymenophorea (Protozoa, Ciliophora), contrasting markedly with the great diversity of form seen in trophic stages. Polyhymenophorean ciliates have been considered in the past to form a cohesive class within the Phylum Ciliophora and, possibly, to represent the pinnacle of ciliate evolution. Evidence from cysts challenges the cohesive nature of the class, suggesting that the hypotrichs should be subdivided and that they have a different phylogenetic origin from the heterotrichs, tintinnids, and oligotrichs.

Notes: Yellow-brown, algal symbionts varying in diameter from approximately 5 μ m to 20 μ m, associated with solitary Radiolaria with spongiose skeletons (i.e. Spongodrymus sp.), exhibit fine structural features resembling the Prymnesiida (botanical class, Prymnesiophyceae). A large central vacuole is surrounded by a thin layer of cytoplasm containing plastids with lamellae composed of three thylakoids and granular pyrenoids with internal tubules immersed between the thylakoids. The pyrenoids lack internal thylakoid membranes. The nucleus is surrounded by a dilated cisterna of the nuclear envelope that also encloses the plastids and gives rise to saccules of the endoplasmic reticulum. The algal symbionts appear coccoid; hence no flagella nor surface scales were observed. The symbiont fine structure is compared to similar yellow-brown symbionts associated with Acantharia. Thus far, three kinds of algal symbionts have been observed to be associated with solitary Radiolaria: dinoflagellate, prasinomonad, and this apparent prymnesiomonad.

Notes: Ultrastructural observations of the cortically-located mitochondria of Tetrahymena thermophila revealed associations not only between the mitochondria and certain of the cortical microtubule bands, but also between the mitochondria and the epiplasm of the cortex. Most of the distal mitochondrial surface is close and parallel to the epiplasm; favorable views show bridge-like structures spanning the 20–10 nm gap between the mitochondrion and the epiplasm.Previous studies have shown that the placement of mitochondria in the cortex appears to be determined by certain of the cortical microtubule bands. This study, however, shows that mitochondrion-microtubule interactions account for only a small proportion of the total mitochondrial area associated with the cortex; the rest is accounted for by the epiplasm. A possible analogue of the spectrin layer of erythrocyte membranes, the epiplasm may be important in helping to arrange the intricately organized components of the ciliate cortex. Its involvement in apparently helping to “moor” mitochondria to their cortical sites is the first suggestion of any role in cell patterning played by the epiplasm.

Notes: Ce Tetradimorpha, rencontre en eau douce se présente soit sous forme sphérique pourvue de quatre flagelles et d'axopodes rayonnants, soit sous forme allongée avec a l'avant quatre flagelles associes a quatre axopodes et a l'arriére six a huit axopodes divergents. L'etude ultrastructurale révèle un cytosquelette axopodial de type centroplastidie comprenant un centroplaste lenticulaire homogéne, centre organisateur des quatre axopodes anterieurs et des six a huit axopodes posterieurs, auquel s'ajoutent les quatre cinetosomes des flagelles anterieurs. En outre, un deuxiéme éleément cytosquelettique incluant un microtubule associe chacun des quatre cinetosomes a l'axopode antérieur correspondant. Des cordons microfibrillaires réunissent axopodes et cinetosomes au niveau du centroplaste, puis a quelque distance du centroplaste les axopodes posterieurs. Les axonémes des axopodes comprenant de 5 a 30 microtubules sont constitues de triades, lorsqu'on peut détecter une organisation. Le noyau, a nucléole central est coince dans le cone axopodial posterieur, lui-méme entouré des dictyosomes. Par l'organisation du cytosquelette, par la structure des kinétocystes, par la structure des flagelles dépourvus de mastigonémes tubulaires, Tetradimorpha différe nettement de Ciliophrys marina. Comme le prévoyait Davidson (1975), il represönte bien un des chainons dans la série évolutive des Héliozoaires centrohélidiens. Mais il ne présente guère d'affinites avec les Chrysomonadines considerees comme la souche des Héliozoaires. L'intéret de ce Protiste dans l'étude de la differentiation et de l'evolution du cytosquelette est également présente.〈section xml:id="abs1-2"〉〈title type="main"〉ABSTRACTThis freshwater species of Tetradimorpha has a spherical body with four flagella and radiating axopods; it transforms into a pear-shaped cell that anteriorly has four flagella intercalated between four axopods and posteriorly has six to eight divergent axopods. Ultrastructural study reveals an axopodial cytoskeleton of the centrohelidan type comprising an homogeneous lenticular centroplast which acts as MTOC for axopodial microtubules. A second skeletal element is a microtubular linkage between the kinetosomes and the axonemes of anterior axopods. A microtubule embedded in dense material diverges from near the base of each kinetosomes and parallels the distal portion of the axoneme of each anterior axopod. A microfibrillar envelope around the centroplast links the axopodial bases to the kinetosomes situated just above. Close to the centroplast, microfibrillar strands link the axopodial axonemes to the kinetosomes. Axopodial axonemes are composed of 5 to 30 microtubules irregularly arranged except for some that form equilateral triangles. The nucleus containing a central nucleolus is constrained within a cone formed by the axonemes of the posterior axopods and surrounded by dictyosomes. By the cytoskeletal organization, the structure of kinetocysts, and flagella wthout tubular mastigonemes, Tetradimorpha differs obviously from Ciliophrys marina. As Davidson (1975) predicted, Tetradimorpha is an intermediate link in the centrohelidan lineage: however, it lacks the characteristics of chrysomonads, the supposed ancestors of Heliozoa. The contribution of this genus to the study of the differentiation and the evolution of the cytoskeleton is also presented and discussed.

Notes: Two-dimensional gel electrophoresis was used to identify the patterns of protein synthesis during initiation, and the patterns of membrane protein expression following initiation, in all of the mating types of the Tetrahymena thermophila B family. In addition, one-dimensional analysis was used to survey 125I-Concanavalin A-binding proteins. Although a large number of proteins was identified by each technique, no variation among the mating types was observed.

Notes: Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.

Notes: The rapid, synchronous differentiation of N. gruberi from amoebae to flagellates is a useful paradigm to study aspects of cell differentiation, including regulation of the synthesis of proteins that are related to the changes in cell shape and motility, which occur during differentiation. The differentiation requires synthesis of new RNA and protein molecules to accomplish defined morphogenetic events. Specific new proteins, including the tubulins that form the flagellar microtubules, are synthesized at various times during differentiation, and particular mRNA species appear and disappear. The time course of the synthesis of the α and β subunits of flagellar tubulin is paralleled by the programmed appearance and disappearance of flagellar tubulin mRNAs. The evidence supports the hypothesis that the synthesis of flagellar tubulin is regulated by the transcription, and subsequent disappearance, of flagellar tubulin mRNA. Translatable mRNAs for two calmodulin-like calcium-binding proteins appear and disappear contemporaneously with those for flagellar tubulin. During differentiation the synthesis of actin, the major protein of amoebae, is selectively shut down, and translatable actin mRNA rapidly disappears. This description of the orderly appearance, utilization, and disappearance of the mRNAs for actin, calcium-binding proteins, and flagellar tubulin during differentiation provides means and motivation to investigate the mechanisms that regulate these events.

Notes: Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.

Notes: During an electron microscopic study of Glugea stephani, three morphologically distinct tubular appendages that are continuous with the sporoblast plasmalemma were observed. The tubules were designated as: type I, 45–50 nm in diameter and 600–900 nm in length; type II, 25–35 nm in diameter, averaging 1300 nm in length; type III, 50–70 nm in diameter and with an indeterminate length, which often exceeds 3000 nm. Type III tubules contain regularly spaced, electron-dense particles that are approximately 30 nm in diameter. Since many genera of microsporida have some type of appendage, which may eventually be utilized for taxonomic purposes, we propose the formation of a system of serially numbered detailed descriptions of these structures to promote uniformity and clarity in future publications.

Notes: Cells of Paramecium tetraurelia, stock hrd, cultured in a micro-capillary containing 1 μl fresh culture medium, expressed mating activity through the whole cell cycle. Mating-reactive G2 phase cells can conjugate with cells of other phases. The G2 phase cells, which have double (4C) the normal micronuclear DNA content, undergo pre-meiotic DNA synthesis when conjugated with G1 phase cells. The micronucleus of the progeny from the cross between a G1 and a G2 cell becomes triploid.

Notes: Buffered solutions of KCl and NaCl were tested for their stimulatory effect on the germination of variously-aged spores of Vavraia culicis. Germination was optimal in 0.2 M KCl, pH 6.5 for one isolate, and, for another isolate, peaks of germination occurred at pH 7.0 and 9.5. Spores incubated for several hours in suboptimal solutions became unable to germinate under optimal conditions. After being returned to water, they regained their ability to germinate. Calcium chloride, magnesium chloride, and ammonium chloride inhibited germination. After ingestion by mosquito larvae, spores germinated near the posterior end of the midgut.

Notes: . Leishmania tropica promastigotes transport α-aminoisobutyric acid (AIB), the nonmetabolizable analog of neutral amino acids, against a substantial concentration gradient. AIB is not incorporated into cellular material but accumulates within the cells in an unaltered form. Intracellular AIB exchanges with external AIB. Various energy inhibitors (amytal, HOQNO, KCN, DNP, CCCP, and arsenate) and sulfhydryl reagents (NEM, pCMB, and iodoacetate) severely inhibit uptake. The uptake system is saturable with reference to AIB-and the Lineweaver-Burk plots show biphasic kinetics suggesting the involvement of two transport systems. AIB shares a common transport system with alanine, cysteine, glycine, methionine, serine, and proline. Uptake is regulated by feedback inhibition and transinhibition.

Notes: . Oxytricha strains used in biochemical studies have traditionally been grown in unaerated, unagitated culture tubes or Fernbach flasks. These cultures are limited in volume to about one liter and have a very nonuniform distribution of cells, with the majority of the cells at the very top or bottom of the medium. We have found conditions in which Oxytricha can be grown in 50-liter fermentation vats. The cultures grow to a uniform density of about 6000 cells/ml.

Notes: . The fine structure of the tomite of Foettingeria actiniarum (Claparède) was examined and compared with that of other apostome tomites. This stage in the life cycle has a unique configuration of kineties that form a spiral through the cytoplasm in the interior of the body. The structure and behavior of this internal spiral were evaluated as a mechanism for the storage of kinetosomes, an adaptation to the ciliate's two-host life cycle. The spiral is composed of nine ribbons of laterally compressed kinetosomes that are in contact with a thin electron-dense fibril. Paralleling the kineties of the spiral are conspicuous, swollen lamellae of the rough endoplasmic reticulum; these lamellae contain moderately electron-dense material. The spiral is associated with the large contractile vacuole and winds about the macronucleus. The tomite of Foettingeria possesses a single, robust, caudal cilium located in a pit, along with the nozzle-like pore of the contractile vacuole. The walls of the pit contain several trichocysts arranged radially about the caudal cilium and aimed into the pit.

Notes: . Fine structural studies of a specialized vesicle system associated with the endoplasmic reticulum (ER) of exo-erythrocytic Plasmodium berghei suggest that this system may be the equivalent of a Golgi apparatus. Patches of ER, randomly distributed in the cytoplasm of developing parasites, are formed of smooth and ribosome-studded cisternae intermingled with each other. The vesicle systems are located between as well as at the edges of ER aggregates and appear to be in different stages of budding from the cisternae. Prolonged osmication reveals distinct staining of the nuclear envelope and ER of the parasites as well as part of the Golgi apparatus of the hepatocytes. However, the small vesicles associated with the parasite's ER are unstained, as are the coated vesicles in the Golgi region of the liver cell. These sites in the parasite cytoplasm seem comparable to the concave surface of the Golgi apparatus in liver cells. The pinched-off vesicles fuse with others to form the prominent peripheral vacuolization characteristic of the nearly mature exo-erythrocytic form. The formation of these peripheral vacuoles and their subsequent fusion with the parasite membrane may be an exocytosis mechanism supplying the rapidly expanding parasite with new plasma membrane material.

Notes: . Ultrastructural cytochemical techniques were used to analyze the nucleus and the kinetoplast of epimastigotes of Trypanosoma cruzi. With the use of ethanolic phosphotungstic acid, which detects basic proteins, reaction product was seen in the chromatin and at the periphery of the kinetoplast. Thallium alcoholate, which interacts with DNA, stained strongly the whole kinetoplast and the chromatin. With the use of a silver impregnation method that detects acidic nucleolar proteins, silver granules were seen preferentially located in the central region of the nucleolus. With the EDTA method, which reveals the presence of ribonucleoproteins, staining was observed in the nuclear pores. Also 6–8 nm fibrils, 25 nm and 40 nm granules, which correspond to the perichromatin fibers, interchromatin granules and the perichromatin granules, respectively, were identified in the nucleus. The EDTA method also revealed the presence of 40 nm granules in the kinetoplast. These granules were seen mainly at the two extremities of the kinetoplast. Freeze-fracture images indicate that the nuclear membrane contains ca. 9 pores/μm2 of nuclear surface area. The mean diameter of the pores was 80 nm. All these results suggest that epimastigotes of T. cruzi have a very active nucleus and a high rate of nucleocytoplasmic interchange.

Notes: . This essay considers the responses of Paramecium and other ciliates to the inorganic ion environment from an elec-trophysiological point of view. In reviewing data from published and unpublished sources it is shown that ions affect the cellular behaviour in multiple ways because the transmembrane potential can change due to the alteration of equilibrium potentials, ion conductances and surface charges of the membrane. Sensory input including effects from the ionic environment converge upon the membrane potential which has a temporal and spatial summing function. Hyperpolarizing and depolarizing potential shifts from the set point are near-simultaneously and omnidirectionally transmitted along the membrane including the ciliary boundaries. The membrane potential regulates ciliary motility via an intraciliary messenger, Ca2+, which can enter, and presumably leave, the cytosol directly adjacent to the ciliary motor. Integration of the responses of thousands of cilia occurs in accordance with the electrical and structural provisions of the cell. Potential-regulated motor and behavioural responses attenuate with time. This phenomenon, which has been loosely termed adaptation, has an electrophysiological basis in analogy to membrane accommodation following sustained stimulus input. The mechanisms of adaptation serve to restore, in principle, the membrane resting state and, thereby, the sensitivity to depolarizing and hyperpolarizing shifts of the membrane potential and the cell's responsiveness to environmental stimuli, respectively. For the inorganic ions involved in chemosensation the terms attractant and repellent are not applicable. They should be reserved to signalling substances which per se can define the behaviour of the cell.

Notes: The ultrastructure of sporozoites and zoites of Hammondia heydorni was studied in cultured bovine cells. In addition to ultrastructural features typical of coccidian parasites, H. heydorni sporozoites and zoites contain rhoptries that are located posteriorly as well as anteriorly. Also, sporozoites contain a posteriorly located crystalloid body (1.2 μm in diameter); a small crystalloid body (0.5 μm in diameter) was occasionally seen in the anterior end. Zoites resulting from the 1st division of endodyogeny contain a posteriorly located crystalloid body, which is absent in zoites formed by subsequent divisions. Zoites contain posteriorly located amylopectin granules and a relatively large anterior vacuole which is not present in sporozoites. During penetration, the host cell plasmalemma ballooned laterally around the sporozoite creating a large cavity, which later disappeared. Sporozoites and zoites undergoing cell penetration usually exhibit partially empty anterior rhoptries; no changes occur in posterior rhoptries. Lysosomes fuse with the par-asitophorous vacuole surrounding killed sporozoites but not live sporozoites.

Notes: The effects of the 2 ornithine decarboxylase inhibitors α-difluoromethylomithine (DFMO) and α-monofluoromethyl-dehydroormithine methyl ester (ΔMFMO-ME) on growth ofBabesia bovis blood stages in vitro were tested. The DFMO had no apparent effects on in vitro growth of B. bovis nor on the morphology of the parasite at concentrations up to 20 mM. In contrast, ΔMFMO-ME had cytotoxic effects on B. bovis at 0.5 mM which were more pronounced at 5 mM. ΔMFMO-ME caused both a decrease in percentage parasitized erythrocytes and a degeneration of parasites after 12 h exposure, and the magnitude of both effects was dose-dependent. The effects of ΔMFMO-ME were not reversible for B. bovis precultured for 12 h (5 mM) or 24 h (0.5 mM) in drug before culturing the parasite in drug-free medium. Unexpectedly, 300 μM putrescine did not reverse the effects of ΔMFMO-ME on B. bovis blood stage, raising the possibility that inhibition of omithine decarboxylase is not responsible for these effects.

Notes: Pneumocystis carinii is an opportunistic pathogen of man, carried as a commensal in healthy subjects. It frequently causes a fatal pneumonia in the immunosupprcssed host. It is a major complication of HIV-1 infection in man (AIDS). Using surface radioiodination of rat-denved P. carinii trophozoites obtained from in vitro culture, a major surface glycoprotein (gp120) has been identified. The glycoprotein exhibits adherent behavior similar to that of the intact organism. Purification of gp120 by conventional methods was unsuccessful as the glycoprotein irreversibly bound lo numerous column matrices. A combination of gel chromatography and hydroxyapatile chromatography in sodium dodecylsulfaie v. as utilized to purify the glycoprotein. Some preliminary characterization of the glycoprotein is presented.

Notes: . Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC I. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania.

Notes: Garcia, L. & Bruckner, D. 1988. Diagnostic Medical Parasitology

Notes: . Two strains of the Leishmania braziliensis complex have been adapted to grow extracellularly at elevated temperature as amastigote-like forms in a cell-free medium. These parasites can be serially cultivated and maintained at 32°C for L. panamensis (WR442; L. braziliensis panamensis) and at 28°C for L. braziliensis (M5052; L. braziliensis braziliensis). Several observations are presented that the forms adapted at elevated temperature are amastigote-like. Morphologically, the amastigote-like organisms appear rounded to ovoid and are immotile and smaller than promastigotes; the flagellum of the amastigote-like forms does not extend beyond the flagellar pocket. In comparison, the promastigotes are very elongated, with a nucleus at mid-cell length and a very long flagellum. By electron microscopy, the short flagellum of the amastigote-like form is within a distended flagellar pocket; the 9 + 2 axonemal configuration is present but the paraxial rod is not observed. By contrast, the flagellum of the promastigote has a paraxial rod which extends from the axosome level. In addition, these amastigote-like forms of Leishmania are able to infect, to survive and to divide within the macrophage cell line J774.

Notes: Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to 〉200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.

Notes: Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas rein-hardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter±SD of 95±17 nm, n=300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.

Notes: The micronuclei of the ciliated protozoan Pseudourostyla cristata were eliminated by amputation shortly before binary fusion. The amicronucleate cell lines derived from regenerants were maintained for more than a year. They exhibited a lower viability and reduced vigor in asexual propagation. There was some improvement in the growth of the cell lines 1 mo after operation, but the growth rate remained subnormal even up to 1 yr of culture. The exact cause of the poor growth and survival in the first 3 wk after operation, whether the loss of the micronucleus or operational damage, remains to be determined. It is nevertheless clear that the micronucleus is important for subsequent asexual propagation. The amicronucleate cell lines were permanently crippled in morphogenesis, unlike the situation in Paramecium amicronucleates in which stomatogenesis returned to near-normal during asexual propagation. They always included some cells with a characteristically defective adoral zone of membranelles, reduced number of frontal-ventral-transverse cirri, and reduced body length. They were also reluctant to encyst. It is evident that the micronucleus is important for maintaining normality of the oral apparatus. It is postulated that the permanent stomatogenic crippling of amicronucleates might be related to genomic reduction in the developing macronucleus in sexual reproduction, as exhibited by other hypotrichs. The morphological defects associated with the adoral zone of membranelles may be rationalized as arising from the spreading of a zone of degeneration in the cortex affecting the left edge of the membranelles.

Notes: .Using fixed sporozoites in a 3-layer immunofluorescence assay (TLIFA), class-specific, parasite-specific antibody responses in chicks to single-pulse infection with Eimeria tenella have been studied in gut contents and bile as well as plasma and feces. After infection with 103 oocysts, IgA antibody was first detected in the duodenal lumen, then in bile, plasma, cecum, and the distal small intestine. The kinetics of the bile IgA response correlated with that in plasma and peaked 9 days post-infection (d.p.i.); IgM was detected in gut contents and bile as well as plasma, and IgG was occasionally detected in gut contents, especially in the duodenum. In some experiments, IgA was detected in gut contents and bile to at least 21 d.p.i. Infection with 103 oocysts resulted in an earlier and increased response and relatively high IgG titers in cecal contents. Coproantibody was detected inconsistently and at low titer. When sporozoites that excysted in vitro were incubated in specific, antibody-positive (9 d.p.i.) cecal contents, some complement-mediated IgG-associated anti-sporozoite effects were observed; however, the major effect of cecal contents and the only effect of bile was a non-lethal agglutination of living sporozoites. By fractionation of cecal contents and imtnunoblotting this was confirmed to be IgA mediated; IgA antibodies in cecal contents and bile after infection were shown to bind to sporozoite membrane antigens by surface fluorescence as well as agglutination. Agglutination detected anti-sporozoite antibody in gut contents and bile up to 21 d.p.i., peaking between 7 and 13 d.p.i., corresponding with TLIFA results. The immunofluorescence studies reveal the extremely labile nature of the sporozoite surface antigens and support the hypothesis that naturally elaborated IgA antibodies are involved in the modulation of complexed sporozoite surface antigens.