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: SYNOPSIS. The ultrastructure of interphase and mitotic nuclei of the epimastigote form of Trypanosoma cyclops Weinman is described. In the interphase nucleus the nucleolus is located centrally while at the periphery of the nucleus condensed chromatin is in contact with the nuclear envelope. The nucleolus fragments at the onset of mitosis, but granular material of presumptive nucleolar origin is often recognizable in the mitotic nucleus. Peripheral chromatin is in contact with the nuclear envelope throughout mitosis, and it seems reasonable to assume that the nuclear envelope is involved in its segregation to the daughter nuclei. Spindle microtubules extend between the poles of the dividing nucleus and terminate close to the nuclear envelope. The basal body and kinetoplast divide before the onset of mitosis and do not appear to have any morphologic involvement in that process. Spindle pole bodies, kinetochores, and chromosomal microtubules have not been observed.

Notes: SYNOPSIS. Developing 2nd- and 3rd-generation schizonts of Eimeria tenella were found in the ceca of chicks infected orally with sporulated oocysts. Several free 2nd-generation schizonts, which varied in diameter from 11 to 21.6 μm, were found on the epithelial surface of the cecum. Some schizonts appeared to have lost merozoites. Other schizonts were intact, one of which was surrounded by an unbroken membrane that followed the contours of the merozoites. Third-generation schizonts, much smaller than 2nd-generation schizonts and with fewer merozoites, were found only on cut or fractured surfaces of the cecal tissue. Third-generation merozoites appeared shorter and thicker than those of the 2nd-generation and were attached to the schizont residuum. A form with conical protuberances and another with 4 triangular segments were found; they were believed to be developing stages 3rd-generation schizonts.

Notes: SYNOPSIS. Endosymbiotic algae from Paramecium bursaria when added to the culture medium are ingested by Chlorella-bearing P. bursaria at a rate of 2,000 algae/organism/day. That the ingested algae are digested and assimilated by the ciliates is suggested by the more rapid growth of Paramecium when algae are added to the medium (G= 40 hr with algae compared to 190 hr without). The digestion by the ciliates of exogenous algae contrasts with the survival of these algae under normal growth conditions. It is suggested that the protection of the endogenous algae is related to their location in peripheral perialgal vacuoles.

Notes: SYNOPSIS. Toxoplasma ranae sp. n. is described from the brain of a leopard frog, Rana pipiens, probably from Mexico. Its pseudocysts were 72(55-106) × 48(29-70) μm in fixed sections. They contained an average of ∼ 4,000 slightly curved elongate zoites measuring 4–5 × 0.5 μm, with a central, spherical, vesicular nucleus.

Notes: Book reviewed in this article:Pennycuick, C. J. 1974. Handy Matrices of Unit Conversion Factors for Biology and Mechanics.

Notes: SYNOPSIS. Egestion of carmine particle-containing food vacuoles from the cytoproct of Tetrahymena pyriformis has been analyzed by high-speed cinemicrography. The vacuole may enter into position in the cytoproct ∼ 7 sec before ejection, and forms a distinct bulge beyond the outline of the cell surface for over 2 sec prior to ejection. The ejection process itself requires 20–80 msec.

Notes: SYNOPSIS. Blood films were examined from 1477 birds of Taiwan (193 species, 49 families). Haemoproteus Kruse was by far the commonest parasite, with Leucocytozoon Danilewski a not very close second. It is probable that some of the Haemoproteus infections represented new species, and 1 occurring in the Bamboo Partridge (Bambusicola thoracica sonorivox Gould) seemed characteristic enough to justify recognition as such; the name Haemoproteus bambusicolae sp. n. is proposed for this organism. Malaria was found in 77 birds, the greatest number of infections occurring in the Bamboo Partridge. Most of them were caused by Plasmodium juxtanucleare Versiani & Gomes, a pathogen of chickens, but a number were due to an undetermined species of Plasmodium. The Bamboo Partridge may be a reservoir host of the former. A few other identified species (P. rouxi Sergent & Sergent, P. hexamerium Huff, P. tenue Laveran & Mesnil) were seen, as well as some unidentified ones. Plasmodium tenue was seen in Garrulax canorus taewanus Swinhoe, a babbler: until now it was known only from the Pekin Robin (Leiothrix luteus Scopoli), also a babbler, in which we have found it extremely common. Sixty-four microfilarial infections were identified; they were especially frequent in the Button Quail (Turnix suscitator rostrata Swinhoe).

Notes: SYNOPSIS. Short pulse-labeling of log phase Crithidia fasciculata cells with [3H]thymidine allowed the autoradiographic visualization of 2 sites of replication of kinetoplast DNA situated at the periphery of the networks and separated by 180°. Longer pulse-labeling led to the previously reported total peripheral labeling pattern. Pulse-labeled networks possess an intermediate density in ethidium bromide-CsCl equilibrium gradients between the densities characteristic of closed networks and open or linear DNA. Removal of ethidium bromide by several methods and treatment of intermediate band networks with RNase and pronase had no effect on the equilibrium rebanding pattern. Closed minicircles of Leishmania tarentolae are not labeled by a short pulse of intact cells with [3H]thymidine. A chase of ∼ 3–4 hr is required for the appearance of radioactivity in closed minicircles, a time delay which implies the existence of intermediate events between replication and eventual covalent closure of the minicircles.

Notes: SYNOPSIS. Epizootic outbreaks of red-sore disease in several reservoirs in the southeastern United States have been reported to cause heavy mortality among several species of fish having sport and commercial value. The etiologic agent is said to be the peritrich ciliate Epistylis sp.; secondary infection by the gram-negative bacterium Aeromonas hydrophila produces hemorrhagic septicemia which results in death. However, in recent studies on the largemouth bass Micropterus salmoides, Epistylis sp. could be isolated from only 35% of 114 lesions from 114 fish, while A. hydrophila was found in 96% of the same lesions. Transmission and scanning electron microscopy of lesions associated with red-sore disease indicate that neither the stalk nor the attachment structure of Epistylis sp. have organelles capable of producing lytic enzymes. Since other investigators have shown that A. hydrophila produces strong lytic toxins, and in absence of evidence to the contrary, it is concluded that Epistylis sp. is a benign ectocommensal and that A. hydrophila is the primary etiologic agent of red-sore disease.

Notes: Weiser, Jaroslav. 1977. An Atlas of Insect Diseases, 2nd ed. Dr. W. Junk, B. V., Publishers, P. O. Box 3713, The Hague, Holland and Academia Publishing House, Czechoslovak Academy of Sciences, Prague, Czechoslovakia. 81 + 240 pp. 75 Dutch Guilders.

Notes: SYNOPSIS. The structure and cytochemistry of spores of Myxobolus sp. from plasmodia which occur in the gill filaments of the common shiner Notropis cornutus were studied by light microscopy and by scanning and transmission electron microscopy. The thin-walled valves of the pyriform spores are thickened in the lateral sutural and apical regions. Mucous material is associated predominantly with the posterior end of many spores. The plasmodium is surrounded by a syncytial wall bounded by 2 membranes. Pinocytotic channels are formed by the inner membrane and numerous dense vesicles are pinched off at the distal ends of the channels. Sporogenesis is initiated by the envelopment of one vegetative cell by another. The larger, enveloped cell divides to form a disporous pansporoblast, which contains 2 pairs of capsulogenic and valvogenic cells and 2 binucleate sporoplasm cells. Each capsular primordium and connecting external tubule gives rise to a polar capsule which houses a helically coiled polar tubule. The apical end of each polar capsule is plugged by a stopper. The valvogenic cells surround the capsulogenic and posteriorly situated sporoplasm cells to form the spore valves. Iodinophilic (glycogen) inclusions were not seen in spores stained with iodine or Best's carmine. A darkly stained band was observed around the posterior region of most spores stained with Best's carmine. In the electron microscope large aggregates of β glycogen particles were seen in the cytoplasm of sporoplasm cells in mature spores.

Notes: SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [3H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [3H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G1, S, G2, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.

Notes: SYNOPSIS. The activity and sedimentation of acid phosphatase (APase), acid deoxyribonuclease (DNase), and acid ribonuclease (RNase) were investigated throughout growth and encystment in Acanthamoeba castellanii. The activities/mg protein of all 3 hydrolases are high in young cultures and decrease to constant levels in postlog cells. The RNase activity/ ameba decreases 50% during growth, whereas the activity/cell of both APase and DNase remains constant. The percent sedimentation at 20,000 g of all 3 enzymes gradually increases from about 40% in midlog to a plateau of 80% in postlog cells. During encystment, the sedimentation behavior of RNase differs from that of APase and DNase. Encystment is characterized by a differential decrease in the activity/cell of the 3 hydrolases, with RNase decreasing most rapidly and APase least rapidly. APase is unique in that a transient increase of its specific activity is noted during encystment, even though its activity/cell is decreasing.

Notes: SYNOPSIS. Eighteen strains of algae, including 17 exsymbiotic from Paramecium bursaria, were tested for infectivity for P. bursaria, syngen 2 aposymbiotes, and Concanavalin A (Con A) agglutinability. All 6 infective algal strains were relatively resistant to agglutination by Con A, suggesting that algal surface characteristics are correlated with infectivity. Among the noninfective strains, high and low agglutinability were about equally represented, indicating that the Con A titer alone is not a sufficient indicator of infectivity. It is suggested that noninfective algal strains are the progeny of mutations occurring within the endozoic population and fortuitously selected by the external culture medium.

Notes: SYNOPSIS. Studies on sexual compatibility among populations of different origins of axenically cultivated, heterotrophic, homothallic dinoflagellates resembling Crypthecodinium cohnii have been undertaken using motility mutants and complementation testing. We showed previously that one interbreeding group or biologic species can be found on the East and West coasts of the United States, thus far associated only with Fucus. In addition, we found several strains of these organisms each of which, though fertile in intrastrain crosses, appeared infertile with all other isolates. A continuation of these studies has revealed another biologic species of wide geographic distribution (Taiwan, California, Florida, New York) and apparently associated with sea weeds other than Fucus.

Notes: SYNOPSIS. The effect of pretreatment with Isospora felis and bacillus Calmette-Guérin (BCG) on the reexcretion of Toxoplasma gondii occysts was studied in 16 coccidia-free cats. The following conclusions were drawn: (A) Chronically T. gondii-infected cats reexcreted T. gondii oocysts after superinfection with I. felis, and this reexcretion was prevented in cats infected with I. felis before T. gondii infection. (B) Administration of BCG before Toxoplasma infection had no apparent effect on the outcome of the infection.

Notes: SYNOPSIS. In cultures of Platymonas subcordiformis Hazen, grown in appropriate light-dark cycles, as many as 75% of the cells adhered to the surface of the glass culture vessel toward the end of the light period of each day. Cell division occurred primarily while the cells were attached. Subsequently, motile daughter cells were released into the growth medium by the rupture of the mother cell theca. The settling behavior appears to be an integral part of the life cycle being synchronized to the same extent as cell division.

Notes: SYNOPSIS. The coccidian parasite Eimeria gadi was found in the haddock, Melanogrammus aeglefinus, taken from the Nova Scotian fishing banks. The haddock infection rates ranged from a high of 58% on Emerald Bank to a low of 4% on Georges Bank, the average being 32%. There was no relationship between sex and degree or prevalence of infection. Although the probability of an occurrence of infection increased with size, small fish with heavy infections were observed. The degree of infection had no apparent effect on the condition factor (length/weight) of the fish. The infection rate reached a maximum in the fall of the year while the heaviest infections were observed in the spring. It is evident from the data that the infection is fatal.The parasite mass, appearing as a creamy viscous to a yellow semisolid material in the swimbladder, consisted of various parasite stages, fibrous and cellular debris, and lipid material. Some aspects of the sporocyst stage are described.No other gadoids from the Nova Scotian banks were found to be infected; however, a single specimen of the fourbeard rockling, Enchelyopus cimbrius, from St. John's, Newfoundland, was found to be heavily infected with E. gadi.

Notes: SYNOPSIS. Synthesis of RNA in the macronucleus and appearance of RNA in the cytoplasm were studied in heat synchronized Tetrahymena pyriformis GL and compared to those found under conditions of logarithmic growth (28 C) and during heat shocks (34 C).In macronuclei of logarithmically growing cells precursors were processed to 2 rRNA species (25S and 17S). In addition, another RNA (15S), more homogeneous than the RNA (8-15S) in the cytoplasm, was observed in the macronucleus. Both 17S and 25S rRNA species were found in the cytoplasm, 17S rRNA appearing more rapidly than 25S rRNA. Synthesis of rRNA was suppressed at 34 C in cells subjected to heat synchronization; 8-15S RNA synthesis appeared to be inhibited to a lesser extent.During the time preceding the first synchronized division, the synthesis of rRNAs in the macronucleus slowly recovered. Early in the cycle, almost no newly synthesized rRNAs were extracted. By 30 min after the last heat shock (EH), most of the RNA synthesized was not identified as rRNA. By 60 min after EH, the pattern of RNA synthesis had not returned to that observed in logarithmically growing cells.

Notes: SYNOPSIS. Cell-free preparations of Acanthamoeba castellanii trophozoites transfer glucose from UDP-[U-14C]glucose to a chloroform-soluble form. This radioactive material has been isolated by thin-layer chromatography; it contains an alkali-labile and an alkali-stable (unsaponifiable) component. Treatment of the enzymic product with 0.1 N KOH for 15 min at 0 C or 20 C releases radioactivity into the aqueous phase as glucose. During this treatment, 30–60% of the original glycolipid remains chloroform-soluble. It is considered to be an alkali-stable glycolipid because no further loss of radioactivity occurs during an additional 45-min of treatment with 0.1 N KOH. During incubation with 0.1 N HCI at 100 C glucose is released quantitatively from both the untreated glycolipid and the alkali-stable glycolipid with a half-time of 6 min. Glycolipid formation is inhibited by UDP and is reversible; extracts catalyze the formation of UDP-glucose from the alkali-stable glucolipid and UDP.The chemical and physical properties of the alkali-stable glycolipid are consistent with a glucosyl phosphoryl polyprenol structure. Extracts prepared from cysts catalyze the formation of glycolipids aiso, but the glucosyltransferase activity/cell decreases during the course of encystment. Radioactivity is incorporated into the fraction insoluble in chloroform-methanol-water (1:1:1:) during these incubations when UDP-[U-14C]glucose or [14C]glycolipid is the substrate.

Notes: SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.

Notes: SYNOPSIS. Although large hemoglobin inclusions are observed in intraerythrocytic Babesia microti parasites, they are absent from parasites freed of hamster red cells by immune lysis with antihamster erythsocyte serum. Babesia microti has no cytostome. This parasite, therefore, does not appear to feed by phagocytosis of large boluses of hemoglobin, as does Plasmodium. To determine whether Babesia can pinocytose protein, free parasites were fed ferritin in an in vitro system. Ferritin was taken up from the entire cell surface into narrow channels within 15 min at 37 C. Only merozoites, with their pellicular complex, failed to take up the protein. By 60 min, the ferritin was highly concentrated in many channels and vesicles, which formed interconnecting stacks. The ferritin-containing channels became associated with membrane whorls of the multimembranous structure. Membrane whorls were also observed in the process of extrusion in samples incubated for longer times. These events may represent steps in the digestion and excretion of the pinocytosed protein. Empty channels formed when Babesia was fed albumin. The diaminobenzidine reaction for hemoprotein was positive for the channels in both free and intraerythrocytic babesias. The staining reaction was completely inhibited by cyanide, but not at all by aminotriazole. These results further suggest that Babesia pinocytoses hemoglobin in vivo. Plasmodium lophurae parasites freed of red cells by immune lysis are surrounded by 2 membranes and apparently can ingest ferritin only through the cytostome. Extracellular cytostomal feeding involves both membranes, as it does in vivo. Ferritin was found in food vacuoles, some of which contained hemoglobin ingested before parasite isolation, connected to or near the cytostome. In both Plasmodium and Babesia low temperature inhibited ferritin uptake.

Notes: SYNOPSIS. During the course of infection in the rat, Trypanosoma lewisi produces 2 antigenic variants: the 1st represents the initial, reproducing population of cells; and the 2nd the nonreproducing, ablastin-inhibited adult population. The specificities of the agglutinins elicited by the variants were studied by adsorption and agglutination methods and the newer immunoelectroadsorption technic. It was found that the reproducing variant has a surface antigen that reacts with the agglutinin specific for the adult variant, but this antigen does not become immunogenic until transformation to the adult variant occurs. It was also found, with fractions of immune sera obtained by gel filtration, that the agglutinin specific for the reproducing variant is IgG and that specific for the adult variant, IgM. The antigenic variants of pathogenic and nonpathogenic trypanosomes are compared, and the roles of trypanocidal and ablastic antibodies in the induction of antigenic variation are discussed.

Notes: SYNOPSIS. Living Trypanosoma musculi bloodstream trypomastigotes were agglutinated specifically with concanavalin A (ConA), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose-binding protein (FBP). The agglutination with these lectins of living cells from which the coat was removed by trypsinization was the same as with intact trypanosomes. Glutaraldehyde or formalin fixation did not affect the results with regard to agglutination with WGA, SBA, and FBP, but lower agglutination with ConA was observed upon fixation. By using a dense iron-dextran marker many fewer ConA marker particles were localized at the fine structural level in the intact than in trypsin-treated trypanosomes. On the basis of the results obtained by agglutination and electron microscopy, it is likely that fixation cross-links intact surface-coat components associated with the ConA binding sites. It is evident from the studies in which lectins were employed that ligands containing α-D-mannose, N-acetylglucosamine, N-acetylgalactosamine, and α-L-fucose are randomly distributed in the outer surface of the pellicular and flagellar membranes of T. musculi trypomastigotes. Results obtained with α-amylase- and dextranase-treated trypanosomes suggested that lectin-binding sugar ligands in the cell surface were not directly associated with α-1,4 or repetitive α-1,6 glucan-bonded polysaccharide moieties. Similar conclusions can be drawn on the basis of neuraminidase treatment with regard to N-acetylated neuraminic acids.After thorough washing, intact, but not trypsin-treated trypomastigotes were agglutinated specifically with antisera against whole mouse serum and against mouse IgG. Evidently, adsorbed constituents of mouse serum are regular components of the T. musculi surface coat. After incubation in dilute whole mouse serum or in mouse IgG solutions, also the trypsinized cells were agglutinated by the 2 antisera. No such results were obtained with trypsinized cells incubated in serum-free buffers. It was concluded that mouse serum proteins were readily readsorbed on, and firmly bound to the trypsinized cells' surfaces. Specific agglutinations were obtained with trypsinized cells after incubation in dilute rat, rabbit, bovine, and human sera and in solutions of rat and rabbit IgG in reactions with the corresponding antisera. It seems, therefore, that the host serum proteins are adsorbed nonspecifically to the cell surface of trypsinized T. musculi bloodstream forms.When examined by electron microscopy, the intact trypomastigotes were covered by an ununiform, slightly granular, fibrillar extracellular coat, applied to the entire outer lamina of the pellicular and flagellar membranes. No indication of such a coat was noted in the trypsinized organisms. Flocculent surface coat-like matrix could, however, be discerned in cells which, after trypsinization, were incubated in various sera.

Notes: SYNOPSIS. Leptomonas pessoai from the reduviid hemipteron Zelus leucogrammus, was cloned. The original strain (ATCC 30252) and clones can differentiate from promastigote to opisthomastigote. Differentiation is faster at high temperature (37 C) and in a defined medium as compared with a complex medium. It is suggested that Leptomonas pessoai be designated Herpetomonas samuelpessoai.

Notes: Book reviewed in this article: Journal of Toxicology and Environmental Health. Vol. 1, no. 1, Sept. 1975. Hemisphere Publishing Corp.Maloine, S. A., ed. 1974. Diagnostique Immunologique des Parasitoses a Protozoaires et Helminthes.

Notes: SYNOPSIS. Haemogregarina bigemina was found in all Blennius pholis which exceeded 5.0 cm in length, but in none measuring less than 3.5 cm. No exoerythrocytic development was recorded. The first B. pholis eggs hatched in May while the first patent infections of H. bigemina occurred from September onward in metamorphosed fish. Consequently, if the life cycle of H. bigemina includes a vector, that organism is active between May and September at least. Circumstantial evidence indicates that the hematophagous isopod, Gnathia maxillaris and not leeches, could be a vector of H. bigemina. Developmental stages of sporozoa were found in a small number of the isopods which had fed on infected B. pholis but the parasites could not be identified as H. bigemina with certainty. Subcellular organization, typical of sporozoa, was recorded by electron microscopy of H. bigemina.

Notes: SYNOPSIS. Leptomonas pessoai promastigotes cultivated in a synthetic medium were agglutinated with concanavalin A (Con A). Agglutination was predominantly of the flagellar-flagellar type, and was inhibited with sucrose or α-methyl-D-mannoside.Cell surface polysaccharides and/or glycoproteins were demonstrated by several cytochemical methods at the fine-structural level. A Con A-horseradish peroxidase-diaminobenzidine (DAB) technic was used to detect Con A receptors in the pellicular membrane of the flagellar pocket region and in the flagellar membrane proper. Somewhat less of the Con A-DAB reaction product was observed in the pellicular membrane enclosing the rest of the cell.

Notes: SYNOPSIS. Phylogenetic relations are ultimately determined by homologous relationships, including both structural and functional data. Following the establishment of those relationships, the direction of evolutionary change must be determined using paleontologic, developmental, and especially morphocline, data. From that perspective the direction of subsequent development becomes clearer and the problems of origins become more explicit.Using the foregoing methodology it has been possible to identify plausibly monophyletic groups of animal protists or protozoa. Allowing for attendant difficulties, there nevertheless emerges certain fairly convincing generalities: (a) the predominantly pseudopodial forms, with a few minor exceptions, have direct origins from apochlorotic algae; (b) the predominantly kinetidal forms (zooflagellates and ciliates), though also derived originally from apochlorotic algae, give evidence of extended evolutionary development with the especially noteworthy emergence of a permanent ingestatory structure; (c) both groups have increased size, a tendency towards multinuclearity and polyploidy, cytoplasmic differentiations of various sorts, and complex life cycles.In terms of further evolution, namely the emergence of multicellular animals, the pseudopodial forms are almost certainly a dead end while the kinetidal forms are arguably the ancestors of at least 2 metazoan phyla.