ALBERT

Notes: SYNOPSIS. Ten new species of colorless Eugleninae are described. One of them is the type of a new genus (Calycimonas physaloides nov. gen. nov. sp.). Especially remarkable forms are: Astasia acus, a species related to the green Euglena acus but without chromatophores and stigma and considerably smaller; Astasia edax, a species with animal-like nutrition and peculiar ecological adaptations; Petalomonas striata, a form which is not spirally but transversely striated; and finally Hyalophacus caecus, which was observed already by Klebs and Pochmann but was not sufficiently described. The new genus Calycimonas (ex fam. Peranemacearum) is related to Tropidoscyphus on the one hand (nearly the same shape as e.g. Tr. octocostatus, but possessing only a single flagellum) and to Petalomonas on the other hand (mode of nutrition and of swimming, formation of the reservoir, rigidity: however in transverse section not flattened).The Astasiae with zootrophic nutrition are collected in a new subgenus Astasiae devorantes or Phytophaga which comprises besides A. edax three species already described by Skuja.

Notes: SYNOPSIS. H. canis (James) is reported from three of 36 stray dogs at Singapore and one of 34 palm civets (Paradoxurus hermaphroditus Pallas) from Jeram, Selangor. H. muris (Balfour) was found at Singapore in 10 of 61 examples of Rattus norvegicus (Erxleben) and five of 23 of R. rattus diardi (Jentinck), and is also recorded from six of 139 examples of R. r. jarak (Bonhote) from Pulau Jarak, Straits of Malacca. Neutrophils of P. hermaphroditus containing gametocytes of H. canis exhibit pronounced karyorrhexis, while most of the parasites themselves show signs of pyknosis.

Notes: SYNOPSIS. A total of 155 clones of Stylonychia putrina collected in nature were tested for mating type. Two sexually isolated varieties were found among them: Variety I with 12 mating types and Var. II with 11. Five of the clones isolated in the laboratory belonged to 3 additional mating types of Var. I making a total of 15 mating types in this variety.Crosses were made among 6 of the mating types of Var. I, and 114 clones of progeny were raised and tested for mating type. No simple genetic explanation of the results was evident.An abnormal type of conjugation with fusion of the conjugants and subsequent fission was studied. Both parental mating types sometimes segregated during the first two fissions of a fused animal. Mating type determination in the progeny of the fused animals seemed to parallel that of the progeny of the normal conjugants in that in both cases certain mating types tended to be dominant over others.

Notes: SYNOPSIS. The cytochemical procedure of Sen was used to demonstrate urease activity in Tetrahymena pyriformis S.

Notes: SYNOPSIS. The chimpanzee is shown to behave like man to infection with Babesia divergens or bovis: the intact animal is totally resistant, whereas the splenectomized animal develops a fulminating infection accompanied by blackwater. The splenectomized rhesus monkey reacts in the same way also, but splenectomized rabbits are insusceptible. In the chimpanzee the typical accolé position of the “divergens” organisms (as seen in cattle) is absent, but occurs in the rhesus. It is suggested that latent piroplasmosis in man may exist on a large scale in rural populations in infected localities.

Notes: SYNOPSIS. Studies cf 3 conjugating strains of Tetrahymena pyriformis grown in a bacterized medium indicated the presence of a system of alternative immobilizing surface antigens. Each strain was found to have the potential for the expression of 3 serologically unrelated antigens. These consisted of a “high temperature” antigen (expressed in the range of 20–35d̀C), a “low temperature” antigen (exhibited in cultures at 10d̀), and an antigen induced by growth in the presence of the above-mentioned “high temperature” antiserum. Normally, by the immobilization reaction, only one of these antigens could be detected as present on any one organism at any given time. The “high temperature” antigens of two of the strains were serclogically related while that of the 3rd strain did not cross-react with antisera to the other two. The “low temperature” antigens of the 3 strains were serologically related as were the antigens induced by growth in antiserum.Studies of inbred hybrids of two of the strains indicated a potential for still more alternative antigens as well as for the spontaneous expression of the antigen which in the parental strains could only be induced to appear by the presence of the specific antiserum.Study of the 3 strains in axenic medium indicated the existence of a more complex system and the possibility that two or more immobilizing antigens may be present simultaneously.

Notes: SYNOPSIS. The establishment of Eimeria tenella sporozoites in the cecal mucosa of the chicken is described. The invasion process was similar to that reported for E. necatrix by Van Doorninck and Becker. Sporozoites were found to pass through the surface epithelium of the cecal mucosa into the lamina propria. Within the lamina propria the sporozoites were engulfed by macrophages and transported to the cells of the glands of Lieberkühn. Development of the sporozoites ensues within the gland epithelium.

Notes: SYNOPSIS. The morphology of the vegetative phase and the sexual process in Pandorina morum have been studied in detail under controlled laboratory conditions. A number of strains from various areas of the United States, all essentially indistinguishable on morphological grounds, were analyzed for sexual compatibility. Forty-seven heterothallic clones were found to represent 15 separate pairs of mating types, or a minimum of 15 syngens (sensu Sonneborn, 1957). Two clones proved to be homothallic. Nine additional clones, which were not observed to mate with any strain, can be classified only after further collecting. The separate heterothallic pairs of mating types are sexually isolated by factors acting at the stage of gamete production; incompatible mating types produce no gametes when they are mixed together. The geographical distribution of the sets of mating types is very incompletely defined as yet, but they are not strictly endemic forms. Thirty heterothallic strains, representing each of the 15 sexually isolated sets of mating types of Pandorina morum, have been deposited with the Culture Collection of Algae maintained at Indiana University, Bloomington, Indiana, U.S.A.

Notes: SYNOPSIS. When grown in CPLM medium or in a similar medium containing glucose instead of maltose as its sugar, Tritrichomonas foetus, Strain O, was much more sensitive to injury when frozen to -21d̀ C. in the presence. of 1 m glycerol during the initial and logarithmic phases of its population growth curve than at its peak and for some time thereafter. In 7 experiments in which the population peak occurred an average of 28.1 hours after inoculation, the average culture age at which the protozoa first survived freezing was 20.3 hours, at which time the population had reached 52.6% of its peak. The optimum culture age for survival after 1 day of freezing averaged 37.7 hours at which time the population averaged 75.6% of its peak. The optimum culture age for survival after 7 to 15 days of freezing averaged 32.3 hours, at which time the population averaged 82.7% of its peak. Better survival upon freezing was obtained in those experiments in which the population peak was reached relatively rapidly.

Notes: SYNOPSIS. Specimens of Blepharisma undulans were transferred from young clone cultures to a rotocompressor with 3 mm3 of culture medium. The organisms were slightly compressed and photographed at various intervals with dark field illumination. The sequence of macronuclear and cytoplasmic changes was compared with similarly followed Feulgen preparations.The cycle falls into several phases: (1) an interphase (12–24 hr.) in which the organism increases in size while cytoplasmic and macronuclear appearances remain unchanged. During this phase, the macronucleus consists of 3 to 5 nodes of various sizes connected by strands. (2) a pre-condensation phase (1 hr.) in which a new posterior peristome and cytopyge appear without visible macronuclear change. (3) a condensation phase (10–20 min.) in which the macronuclear nodes coalesce into a round mass without dissolution of the central nodes or strands. (4) a postcondensation phase (1 hr.) characterized by: (a) elongation of the condensed macronucleus into a rod-like shape followed by typical nodal formation; or (b) elongation of the condensed macronucleus into a form resembling the letter “J”, followed by nodal formation, and resulting in a double row of nodes in one daughter and a single row in the other. Separation occurs at this time, initiating the new interphase. The development of the j-form macronucleus. the lack of obliteration of the central nodes, and the characteristic interphase condition distinguish this strain of B. undulans from others described elsewhere.

Notes: SYNOPSIS. Six species of astomatous Infusoria, 4 new, are described from the alimentary tract of Oligochaeta from Ochrida Lake. Two belong to Ochridanus, characterized by a cytoskeleton whose “V”-shaped basal piece bears on its branches two articulated hooks brought together by a skeletal blade. This genus found in Ochridanous Tubificidae represents the counterpart of Anthonyella in the Lumbriculidae of Lake Baïkal. The presence of Ochridanus in these worms concurs with the almost complete absence of representatives of Radiophrya of which we have found but one species. A species of Metaradiophrya, a genus unknown to date from the Lumbricidae, was noticed in Glossoscolecidae. The different species of Ochridanus, Anthonyella, Metaradiophrya, and Radiophrya compose a very homogeneous group of Radiophryinae. The study of the ciliary rows and the cytoskeleton of 2 species of Juxtaradiophrya, parasitic in the Lumbriculidae of Ochrida, shows that, in the morphology of these ciliates, as well as in the same forms from Lake Baïkal, many transition characteristics exist between the Radiophryinae and Hoplitophryinae or Mesnilellinae. These 3 sub-families, with their undeniable genetic kinship give coherence to the family Hoplitophryidae.

Notes: SYNOPSIS. Histochemical techniques were used to demonstrate the intracellular distribution of some hydrases, hydrolases, oxidases, and dehydrogenases in Stylonychia pustulata. The hydrase, aconitase, was confined to the mitochondria. Zymohexase activity occurred in the cytoplasm and probably in the mitochondria. The hydrolases, acid and alkaline phosphatases, lipase, and urease, were localized in the mitochondria. Lactic and glutamic dehydrogenases were confined to the mitochondria. Peroxidase and glycerophosphate dehydrogenase were absent.

Notes: SYNOPSIS. When two strains cf T. pyriformis that do not require exogenous pyridoxine are crossed, all progeny grow without the vitamin. Offspring from crosses of two pyridoxine requiring clones require pyridoxine with the exception of a few which will grow without pyridoxine. The ratio is approximately 3:1 favoring the pyridoxine requiring category. In matings involving the homozygous dominant pyridoxine requiring clones with the double recessive mutant, that is +/+ X p/p, all of the resulting progeny need pyridoxine. Test crossing these heterozygotes (+/p) with the parental pyridoxine non-requiring clones (p/p) gives offspring approximating a 1:1 ratio. Matings between two heterozygotes derived from breeding experiments also yield progeny in approximately 3 pyridoxine requiring: 1 pyridoxine non-requiring. All data indicate selection for the heterozygote in the population and a possible selection against either homozygote. The great abundance of heterozygotes and rarity of recessive homozygotes in natural habitats corroborates these findings. The genetic evidence supports a single gene hypothesis although the possibility of multiple closely linked genes cannot be ignored. There is also the possibility that a dominant suppressor gene may function in blocking the activity of the pyridoxine mutant genes. Moreover, if this gene exists it may be incompletely dominant since the heterozygote grows slightly on deficient media.

Notes: SYNOPSIS. Trichodina urinicola was found in newts, Triturus cristatus and T. taeniatus, in three localities in Czechoslovakia. The ciliate populations showed important differences on the basis of which they were separated as three new forms of this species: T. urinicola f. typica, T. urinicola f. bohemica, both from Triturus cristatus, and T. urinicola f. taeniatus from Triturus taeniatus. The great variability of trichodinids is evident from the literature as well as from our own observations, so that these new forms are to be regarded as provisional ones until it is possible to decide on the basis of a large number of observations the extent of specificity and variation of individual endozoic species of Trichodina. A detailed description of these forms is given as well as a comparison with the known species of trichodinids inhabiting the urinary tract of amphibians. A brief comment on the present taxonomy of the Urceolaridae in general is outlined.The need for a uniform description of these ciliates is emphasized; in connection with this, the taxonomic value of individual body characters is discussed. Special attention is paid to the adhesive disk of Trichodina, the structure of which is of greatest importance in the taxonomy of this group. On the basis of Dogel's and Fauré-Fremiet's descriptive methods employed in study of trichodinids, a proposal of a uniform description of Trichodina is made which involves all the important features of these protozoa.

Notes: SYNOPSIS. Several substrains of Euglena gracilis var. bacillaris made chlorotic by treatment with pyribenzamine or streptomycin, or by growth at high temperature (35–36°C.), have been examined for their carotenoid content. They differ from the normal green strain both qualitatively and quantitatively. Some strains produce no detectable carotenoids while the carotenoid concentration in the strains producing most is at best only one-fifth that of the normal strain. In all substrains producing carotenoids, the carotene fraction consists of β-carotene accompanied by some members of the phytofluene series. In only two of these substrains, HB-G and PBZ-G3, are xanthophylls produced in significant amounts. In HB-G, the main pigment is echinenone, and in PBZ-G3 it is zeaxanthin. The significance of these findings is briefly discussed.

Notes: SYNOPSIS. Association of gamonts of Pyxinia crystalligera takes place in the midgut of its beetle host, Dermestes vulpinus. At 25°C. the development of gametocysts to the point of liberation of sporocysts is completed between about 15 hours and 27 hours after the gametocysts are deposited with fecal material. Dehiscence is favored by relative humidities of 0% to 90%, but is not favored by a relative humidity of 100%. During the early development of the gametocysts outside the host, the crystals and paraglycogen granules in the cytoplasm of the associated gamonts become concentrated in large masses. The gametes are formed at the periphery of the gamonts. After fusion of the gametes takes place and the sporoblasts begin to develop, the residual cytoplasm containing the inclusions moves outward to form a continuous layer next to the gametocyst envelope, so that the sporoblasts become crowded into a central core. A few hours before dehiscence is initiated a clear area appears on the upper side of the gametocyst. The contents of the gametocyst begin to shrink away from the envelope except in the region of the clear area. Eventually the sporocysts emerge through the clear area and press against the envelope of the gametocyst, causing formation of a conical papilla in the envelope. With continued pressure from the sporocysts, the papilla ruptures at its tip, and the sporocysts emerge in a continuous thread until dehiscence is completed. The thread of sporocysts may attain a length of about 11 mm.

Notes: SYNOPSIS. The steps leading to purine ring closure were examined in Strigomonas oncopelti. The flagellate has an obligate adenine requirement (hypoxanthine and guanine are inert) when grown without p-aminobenzoic acid. The imidazole counterpart of adenine but not the imidazole counterpart of hypoxanthine was active. A pathway for purine biosynthesis compatible with these results is sketched.

Notes: SYNOPSIS. The Nuttallia of the gerbil is transmitted by Rhipicephalus secundus only if the infected meal is taken by the larva, and the subsequent nymphal stage is the only one which is able to infect. There is no transovarial transmission.Infective trophozoites remain in the larval caeca for about 12 hours after gorging. The infective nymph is able to give rise to new infections at various times after it has been allowed to feed, but never later than the third day after disengagement from the host.

Notes: SYNOPSIS. The process of autogamy in unassociated individuals of Paramecium polycaryum was reported by the author in 1954. In May, 1955, conjugation was first seen in this species in cultures collected by me at Annamalainagar, South India, thus removing it from the list of non-conjugating species. This appears to be the first instance in which the process of autogamy was detected prior to observation of conjugation in the same species. Autogamy occurs in singles of the Indian race and appears to be similar, cytologically, to that of American races. The details of the micronuclear behavior in conjugation parallel those of autogamy in singles. In fact, the conjugation process seems to be one of double autogamy (cytogamy), rather than of reciprocal gametic interchange. Paroral cones, often of fair size, are formed but breakdown of the cones to permit micronuclear passage has not been observed. In conjugation there are the usual three pregamic divisions; the first shows four characteristic crescents. The resulting nuclei may all participate in the second division. Fertilization occurs in the paroral cone area. Frequently, separation of the conjugants takes place immediately after the first division of the synkaryon. The old macronucleus undergoes very little change prior to the last postzygotic micronuclear division in the ex-conjugant, when it goes into a skein condition. Four macronuclear and four micronuclear anlagen are formed in the ex-conjugants at the completion of reorganization. On occasion giant individuals of P. polycaryum were observed to have ingested numbers of Tetrahymena pyriformis. The presence of an unidentified rod-like organism in the cytoplasm of the paramecia (non-conjugating) was detected in one collection from Bangalore, India.

Notes: SUMMARY. The mating behavior of 16 stocks of Paramecium multimicronucleatum from 12 states has been studied. Mating always follows a decline in nutritive conditions. The evidence indicates that there exists one set of four interbreeding mating types. The period of subculture strongly influences the mating reactions, many stocks mating only long after isolation from nature and culture in the laboratory under the restricted conditions employed. Selfing was observed to occur in many races, usually after long periods of subculture. It first occurred in only a small proportion of the population, later in a larger proportion. The similarities and differences between P. multimicronucleatum and other species of protozoa showing multiple mating types are discussed.

Notes: SUMMARY. A study of the temperature-pressure relationship in oxygen poisoning of Paramecium caudatum was undertaken, as the initial step in defining some of the major factors in in vivo oxygen poisoning. Paramecium was selected because it was relatively simple to culture in a pureline clone, large numbers were readily obtained, and it was large enough to be clearly visible under low magnification. The protozoa were exposed to oxygen pressures of 0 (100%), 15, 30, 45, 60, 75, 90, 105, 120 pounds/inch2 gauge pressure at each of the following temperatures: 1, 5, 10, 15, 20, and 27°C. Exposure was accomplished in a transparent, high-pressure lucite tank which permitted visual observation with aid of a dissecting microscope. It was found at temperatures above 5°C. that oxygen toxicity varied directly with pressure, but below this temperature, with oxygen tensions of 1–2 atmospheres (absolute), oxygen toxicity varied inversely as the temperature. The possibility is advanced that oxygen may be affecting two cellular processes (perhaps enzymatic), one of which is temperature-limited below 5°C. and would, therefore, decrease the death time as the temperature is decreased. Several experiments performed at 3°C. produced a death time intermediate between results obtained at 1° and 5°C.

Notes: SUMMARY. A stock of Paramecium bursaria is described in which the peniculus contains only 8 columns of cilia and associated “granules”. The gullet system is composed of organelle complexes almost exclusively, and incorporates no underlying or transecting extensions of “pellicular fibrillar systems”. A brief discussion is presented on the value of the peniculus as a taxonomic criterion in separating “aurelia” and “bursaria” groups in Paramecium.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Notes: SUMMARY. Strains E, S and W of Tetrahymena pyriformis were examined for their ability to carry out the reactions of the Krebs-Henseleit urea cycle, using growth and enzyme studies. None of the strains was able to grow on either citruliine or ornithine in place of arginine, and proline was as active as citrulline or ornithine in sparing arginine. So little citrulline or arginine was synthesized by cell-free preparations as to be of no significance in the growth or nitrogen metabolism of the ciliates. Slight arginase activity could be detected in homogenates, but no urea was found in cultures. No urease activity could be detected using urea-C14.

Notes: SUMMARY. An eimerian coccidian is described from the flying squirrel, Glaucomys volans, in Florida. It is identified as the same eimerian described by Roudabush from the flying squirrel in Iowa as Eimeria sciurorum. Evidence is presented that Roudabush incorrectly identified the organism. It is renamed as E. parasciurorum nov. sp. Mature oocysts have mean measurements of 29 × 16°, an index of 1.82, are cylindrical with rounded ends, have a dual membrane, and no extra residual body. Oocysts are without micropyle. Four egg-shaped, mature sporocysts in the oocyst have mean measurements of 11.2 × 6.2°, an index of 1.81, contain an oval, granular, intraresidual body and two pyriform sporozoites 10 × 3.2°, index 3.11.

Notes: SUMMARY. Two centrioles, an old one and a new one, are always present in the resting cell. From prophase onward two new ones and two old ones are present. Beginning with the resting stage, five types of centriole life cycles are described and compared with one another: In type 1, both centrioles are elongate; in type 2, the old one is long and the new one, which is short, elongates in prophase; in type 3, both are short, both elongate in prophase, and both, except for their anterior tips, degenerate in late telophase; in type 4, both are long but in prophase their distal ends become free of the rest of the centrioles, these ends migrate to center or posterior end of cell, where, after they produce the achromatic figure and it completes its function in nuclear division, they degenerate; in type 5, both are short and neither elongates at any stage of its life cycle.New centrioles are produced by the anterior ends of old ones. In their first generation, centrioles produce only extranuclear organelles (flagella, parabasals, axostyles, etc.); in their second and later generations, they produce only the achromatic figure (gametogenesis in Trichonympha and reorganization in Barbulanympha and Rhynchonympha are exceptions to this rule).The distal ends of centrioles in some types of cycles are surrounded by centrosomes; in others they are not. In one type of centriole life cycle a small central spindle is present in the resting cell in two genera; in the other types this is not the case.

Notes: SUMMARY. The endogenous development of the life cycle of Eimeria alabamensis Christensen, 1941, occurs in the nucleus of the intestinal cells of cattle. Calves were killed at various intervals after inoculation with infective oöcysts to study the endogenous cycle. Excysted sporozoites were found in the contents or scrapings from the walls of rumen, omasum, small intestine, cecum, and colon. They were found in the cytoplasm of intestinal epithelium at 2 days. Schizonts were found in the nuclei beginning at 2 days, but the number was low by the 8th day. Merozoite numbers usually ranged between 16 and 32. Some host nuclei contained as many as 48 or more, but these appeared to be the result of more than one schizont merging in the same host nucleus. Merozoites were slender, spindle-shaped bodies while still in the schizont walls, but were short with bluntly rounded tips when found in intracellular spaces and crypts. Gametocytes were found as early as the 4th day. Most of the stages of gametogenesis were limited to the lowest third of the small intestine, but in heavy infections some were also found in the cecum and upper colon. Microgametocytes were multinucleate and were more densely stained than the uninucleate macrogametocytes. The ratio of macrogametocytes to microgametocytes in 100 gametes was 78: 22. Oöcysis with “shells” were found in sections of the lower 20 feet of the ileum on the 6th day, which coincided with the shortest prepatent period reported previously. As many as three schizonts or microgametocytes or four or five macrogametocytes or oöcysts could be found in the same host nucleus. The variations in shape of the oöcysts appeared to be dependent on the number of oöcysts crowded into each nucleus.

Notes: SUMMARY. The role of centrioles in achromatic figure production is considered when the number present varies from 1–8. Each centriole after it becomes elongate produces astral rays from its distal end. Some of these rays remain free; some, by joining centromeres, become chromosomal fibers; and some, by joining and growing along those produced by one or more other centrioles, produce the central spindle portion of the achromatic figures. Thus, one centriole may function cooperatively with one to several others in the production of central spindles. But at least two centrioles must be present, and in the proper spacial relation to each other, to form a central spindle; one by itself can form only free astral rays, no central spindle or chromosomal fibers.The flagellated areas (to which the centrioles are anchored anteriorly) play an important role in determining the position of the distal ends of the centrioles with respect to one another, and the position of these ends, in turn, in a large measure, determines the types of achromatic figures produced, particularly the number of central spindles.

Notes: SUMMARY. Observations on binary fission of Lacrymaria olor show that it is a transverse fission. It involves probable intranuclear division of the micronucleus. Both micro- and macronucleus elongate in preliminary stages. Each is ultimately divided as cytoplasmic constriction cuts the spindle fibers of the former, and the connecting, nucleoplasmic thread of the latter.Surging movements of cytoplasm after fission elongate the daughter organisms and move new nuclei to normal, central sites. The anterior proboscis of the posterior daughter regenerates suddenly, complete with coronal cilia. Metachronal waves along ciliary meridians, strongly reversed on the posterior daughter, cause an oscillating movement which pulls the two apart, except for a slender, pellicular thread, ultimately severed. Until broken, this thread connects the rear tip of the anterior animal to the forward end of the proboscis of the posterior one. The organism is semi-quiescent, with proboscis retracted (except spasmodically) throughout fission. After fission the anterior animal quickly begins feeding movements and soon swims away. The posterior animal requires about half an hour before being able to begin feeding and swimming movements.The anterior contractile vacuole of the original animal becomes the primarily active vacuole of the anterior daughter; the posterior one that of the posterior daughter. Missing vacuoles are regenerated by the daughters in about one hour after fission. The division process requires about one hour for completion at 22.4°C.

Notes: SUMMARY. The multiplication rate of Tetrahymena pyriformis HS in proteose peptone medium was measured at 12 temperatures between 18.4°C. and 36.6°C. At the temperature optimum, 32.5°C., the generation time is 2.25 hours. The upper lethal temperature lies between 36.6°C. and 38.0°C. Similarly, a study of Tetrahymena pyriformis GL revealed a temperature optimum for multiplication of 29°C. with a generation time of 3.70 hours. The upper lethal temperature falls between 34.6°C. and 35.4°C. At all temperatures employed the HS strain of organisms multiplies more rapidly than strain GL. Under identical conditions, the two strains have distinctly different growth optima, upper lethal temperatures and growth rates.As measured by multiplication rate the readjustment to a sudden change in temperature (from 18.4°C. to 27.7°C.) is completed very rapidly, with an effective lag time of about 1 hour. Such a shift in temperature gives rise to a small degree of division synchrony during the first and second population doublings which follow. Subsequently, all traces of division synchrony are lost.

Notes: SUMMARY. Test tube cultures of Tetrahymena pyriformis GL in the early maximum stationary phase of growth were used as inocula. After the elapse of a short lag phase a rapid transition to the logarithmic phase of growth was observed in cultures grown on peptone media(4,6). During the early phase of exponential multiplication more cells (as expressed on a percentual basis of the population number) were in the visible stage of cytoplasmic fission than after the elapse of 5 to 6 generations. Analysis of comprehensive data suggests that the higher division index in the early logarithmic phase of growth is not the expression of a synchronization of cells in metabolic respects but rather indicates a prolongation of the stage of cell fission during this phase.

Notes: SUMMARY. Ochromonas malhamensis (Pringsheim strain) can be grown above 35.5°C.; below 35°, the previous chemically defined medium supports dense growth. The B12 and thiamine requirements rise steeply with temperature, and growth promotion by folic acid emerges; folic acid spares the enhanced B12 requirement. B12 is spared also, perhaps wholly bypassed, by purines + pyrimidines + amino acids (below 35°, exogenous purines, pyrimidines, and folic acid have little effect). Requirements also emerge for glycine (spared by serine), valine and isoleucine (their ratio is critical; leucine and threonine assist in maintaining a good balance), and, at very slightly higher temperatures, phenylalanine, tryptophan, cystine, and lysine. Requirements for Mg, Fe, Zn, and Mn appear to rise steeply with temperature; metal toxicities have to be circumvented carefully. The proportion of histidine + arginine to carbohydrate has to be increased, and a Krebs-cycle component such as succinic acid becomes stimulatory. At 36.3–36.7°, a further supplement of crude natural materials such as an autoclaved suspension of Ochromonas cells is needed. Relevance of these findings to fever stress in vertebrates, general mitochondrial function, and repair of radiation damage, is discussed.

Notes: SUMMARY. According to Kahl the trichostome genus Trichopelma may be considered to include five species: T. sphagnetanim, T. eurystoma, T. euglenivora, T. opaca, and T. torpens. A sixth species is added in the present account. The following constant characteristics distinguish it from the five earlier described ones: total lack of trichocysts; differences in the morphology of the body surfaces; dissimilarity of the upper and lower surfaces in shape (one plane, the other convex), in ciliation, and in number of cuticular furrows; unique location of the contractile vacuole.

Notes: SUMMARY. Isospora citelli n. sp. is described from the rock squirrel, Citellus variegatus Utah, from Grand Canyon National Park, Arizona. Its oocysts are subspherical, 22.4 by 21.5 μ, with a smooth, two-layered wall, an oocyst refractile globule and a sporocyst residuum, but without a micropyle or oocyst residuum.

Notes: SUMMARY. Leptomonads of Leishmania tarentolae were grown continuously in a defined medium containing: inorganic salts', glucose, hemin, 17 amino acids, purines and pyrimidines, and a mixture of vitamins of the B group. In this medium the population of organisms reached about 20 to 50 million per ml. alter 1 week at 27°C. Only slightly better growth occurred in a partially defined medium containing bovine plasma fraction V. In earlier experiments, however, omission of the plasma fraction resulted in decreased growth, and under these circumstances cholesterol or lecithin had growth-stimulating effects. In later experiments in the fully-defined medium no effect of these lipids could be found. The leptomonads were shown to require at least the following substances: inorganic salts; a source of purines and pyrimidines; tryptophan and the nine other amino acids essential for the growth of rats, glutamic acid, tyrosine, proline, serine, one or more of the group alanine, glycine and aspartic acid; folic acid, biotin, pantothenic acid, nicotinamide, riboflavm, thiamine, and either pyridoxine plus choline or pyridoxal or pyridoxamine. Choline at 2 × 10−5 m gave optimal growth in the presence of pyridoxine at 1 × 10−5 m. In a medium with a suboptimal concentration of choline (0.4 × 10−5 m) the leptomonads grew through nine transfers but they were mostly somewhat rounded and aflagellate.

Notes: SUMMARY. Trichopus lachmanni n. sp. possesses, in common with other members of the family Dysteriidae, a fixation organelle composed of vesicles and a secretory ampoule. But no “foot-like” appendix exists in this species, and the ampoule opens at the bottom of an antapical pit which bears a short fringe of membranelles derived from the somatic ciliature. The vibratile fringe participates in the spinning of the glutinous secretion which temporarily fastens the organism to the substrate. This specialized ciliature is characteristic of the genus Trichopus which was created by Claparède & Lachmann for a species, T. dysteria, which, though insufficiently described, is certainly different from T. lachmanni.

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: SUMMARY. Various species of ciliates are characterized by the formation and accumulation in the cytoplasm of mineral concretions which are refringent, isotropic or anisotropic. These cytoplasmic inclusions most often are composed of calcium carbonate; in several species, however, their nature remains partially or even totally undetermined. The isotropic calcium-containing concretions often exhibit a definite shape; the calcium carbonate in this case appears to be bound to an organic substrate. The physiological role of the calcic concretions is not known; their characteristic presence in a given species is not necessarily related to ecological conditions. In a few species the calcification is localized in definite structures: spicules, skeletal plates, or otoliths of organelles supposedly sensory in nature.

Notes: SUMMARY. The combination Tween 80 and cholesterol replaced blood serum as a requirement for the cultivation of Tritrichomonas foetus. Choline + potassium glycerophosphate enhanced growth in the presence of Tween 80 + cholesterol. A method for ensuring adequate cholesterol suspension-an essential factor for consistent growth-is described.

Notes: SUMMARY. High variability within cultures of Tritrichomonas batrachorum (from Bufo boreas halophilus, Rana catesbeiana, and Rana pipiens) casts doubt on the validity of length measurements for species designation.The morphology differs from that described previously. The axostylar capitulum is a complex structure with a preblepharoplastic portion and a perinuclear cup spreading laterally in curved membranes ending in filaments. The parabasal apparatus is Y-shaped with a very short base.Two types of amoeboid activity occur. Filopodia are used for anchorage. Their bases migrate and the filopodia may fuse and reseparate as in foraminifers. Ingestion is an amoeboid process utilizing lobopodia. Food adherent to a lobopod is invested in a food cup at the surface. Ingrowth of the lip of the food cup forms the food vacuole, or in cases of cannibalism, constricts the prey and forms a food tube pushing a vacuole before it. Repeated, this produces an alternating series of vacuoles and tubes. There is a pseudo-amoeboid degenerative process in which the flagella, withdrawn into the cytosome, continue to move and produce non-functional lobopod-like protrusions.An actual cytostome has not been observed, but there is a region of reduced staining intensity ventral to the axostylar capitulum.

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