Differentiation-Dependent Decline of DNA Synthetic Activities in Naegleria gruberi

SYNOPSIS. DNA of Naegleria gruberi strain NEG, grown in axenic culture, forms a band at a density of 1.6912 in CsCl gradient and has a GC content of 31.8%. Incorporation of [³H]thymidine into DNA is much reduced in differentiating Naegleria immediately after the stimulation to transforms, primarily because of the reduction in thymidine uptake by differentiating cells. In addition, there is a marked decrease in the rate of incorporation of [³H]thymidine and [³H]uracil into DNA at from 45 to 60 min after the stimulation for differentiation. This decrease in the rate of precursor incorporation into DNA appears to be due to the differentiation-dependent cessation of nuclear DNA synthesis. The differentiated phenotype (the flagellate) emerges at ∼ 70 min after the stimulation, and over 90% of the population differentiates within the next 30 min. Synthesis of mitochondrial DNA is detectable until 190 min after the stimulation. Since the S phase of Naegleria lasts ∼ 180 min, some cells in the population must cease synthesizing nuclear DNA in the middle of the S phase.     

Nutritional Study of Three Strains of Naegleria gruberi*

Naegleria gruberi strains cloned from amebas isolated from a Vero cell culture (“TS”), a sewer drainage ditch (“PD”), and an established laboratory line (“S”) were morphologically identical except for differences in size and flagellate transforming ability. Cultivation on a Trypticase-yeast extract-glucose medium (“TYG”) fortified with autoclaved E. coli resulted in increased cell size of 2 strains. Differences also were noted in growth rates and optimal growth temperatures. The autoclaved E. coli in TYG medium was replaceable with serum only for strains TS and PD. A basal salts medium + autoclaved E. coli supported growth of all 3 strains, but the basal salts medium + serum would not support growth of any of the strains.     

Cell Size,Macromolecular Composition,and O2 Consumption During Agitated Cultivation of Naegleria gruberi*

SYNOPSIS. Cell size, macromolecular composition, carbohydrate utilization patterns, and O₂ concentrations were measured throughout the growth stages of Naegleria gruberi in agitated cultures in a complex medium. Biphasic logarithmic growth occurred during the initial 83 hr of growth and the mean generation time was 7.0 hr and 19 hr during initial and secondary log growth stages, respectively. The maximum yield was 5 × 10* amebaeJml. The pH rose rapidly (1 pH unit) during the secondary log growth phase (52-83 hr) and continued into the stationary growth phase (83-120 hr). Dry weight, total protein, carbohydrate, and RNA per ameba increased just before the secondary log growth phase. RNA increased 31% to 35% per ameba at the end of each phase of log growth. DNA increased ～ 2-fold throughout the different growth phases. Average cell size increased 90% during biphasic log growth then decreased during stationary phase. O₂ tension decreased from 100% to 18% of saturation during the biphasic growth phase, then increased during stationary growth to near 100% saturation. Glucose and total carbohydrate assays showed little utilization of those substrates throughout the growth stages. Naegleria gruberi presumably has a predominantly aerobic metabolism, also its metabolism may change during the different growth phases.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

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.     

Cellular and Environmental Variables Determining Numbers of Flagella in Temperature-shocked Naegleria*

SYNOPSIS Naegleria gruberi amebae normally transform into biflagellated cells. When subjected to high temperatures during flagellate differentiation, populations develop an average of 4–5 flagella/flagellate. Attempts to maximize this phenomenon by altering cellular and environmental variables revealed that: (a) few Naegleria isolates become multiflagellated: strain NB-1 gives the greatest response to heat shocks: (b) temperature is the most critical variable: highest numbers of flagella are obtained only if cells are temperature-shocked at precisely 38.2 ± 0.1 C, then returned to 19–22 C to complete differentiation; (c) although pH alone does not affect numbers of flagella. a pH optimum of 5.5–7.0 exists for temperature-shocked cells; and (d) single cells in microdrops become multiflagellated, but the population response is density-dependent. Optimal conditions are described for growing, washing, and transforming amebae to generate reproducibly highest numbers of flagella.     

Respiratory Chain Components of Leishmania tropica Promastigotes*

Synopsis. Crude preparations of kinetoplast vesicles were used to investigate the respiratory chain components in Leishmania tropica promastigotes. In difference spectra from enzymically and chemically reduced preparations, cytochrome b was the predominant component. By utilizing special assays designed to minimize the influence of cytochrome b on difference spectra, cytochromes a, a₃, and c₃₃₃ were demonstrated. Difference spectra from chemically reduced preparations indicated that pyridine nucleotides (NADH) and flavoproteins were also part of the respiratory chain. The presence of these components as well as their response to respiratory inhibitors and ascorbate provide evidence for the presence of a typical trypanosomatid respiratory chain in L. tropica promastigotes.     

Comparative Study of Six Strains of Naegleria with Special Reference to Nonpathogenic Variants of Naegleria fowleri*

SYNOPSIS. Naegleria fowleri strains HB-1 and KUL, pathogenic for humans, Naegleria gruberi strain 1518/1e, and 3 strains (Vm1, LvH₁, and LvH₂) of Naegleria isolated from a body of water polluted with thermal effluents were compared in an attempt at specific identifications of the latter strains. The 3 environmental isolates were morphologically almost identical with N. fowleri and had almost the same temperature tolerance, although at 37 and 42 C the growth rates of LvH₁ and LvH₂ were higher than those of the human pathogen, N. fowleri, and of isolate Vm1, which was pathogenic for mice. Serologic examinations by indirect fluorescent antibody method revealed a very close relationship of the new isolates with the human pathogens. While Vm1 was indistinguishable from N. fowleri, LvH₁ and LvH₂ were not, when cross-absorbed antisera were used. Of all the strains examined, only the 2 LvH isolates were not inhibited by amphotericin B, while only N. gruberi was not inhibited by fumagillin. The cytopathic effect in Vero cell cultures suggested that the LvH strains could have a certain degree of virulence, although this was not confirmed by intranasal and intracerebral inoculations of mice. The cytopathic effects of the human pathogens and of the isolate pathogenic for mice were related to their virulence for mice. It is concluded that there exists an intermediate form between N. gruberi and N. fowleri, with a strong relationship to the latter species. We refer to such strains as nonpathogenic variants of N. fowleri. Further research is needed to reveal their place in the taxonomy.     

Starch Gel Electrophoresis: An Effective Method for Separation of Pathogenic and Nonpathogenic Naegleria Strains*

SYNOPSIS. Isoenzyme electrophoresis of 7 different enzyme systems was used to compare 24 strains of Naegleria fowleri and 6 strains of N. gruberi. The 30 strains could be grouped into 4 distinct categories based upon zymogram patterns. No interstrain band variation in all enzyme systems was demonstrated in pathogenic strains of N. fowleri. Three nonpathogenic high temperature-tolerant strains of Naegleria had similar zymograms. Four of the 5 remaining nonpathogenic Naegleria strains had no interstrain band variation. Based upon zymograms, the 22 pathogenic strains constitute a homogenous species. Similarly the high temperature-tolerant nonpathogenic strains formed a cohesive group. The remaining nonpathogenic strains could be separated into 2 groups.     

Observations on Gametogenesis in Plasmodium falciparum from Continuous Culture*

Synopsis. Gametocytes of Plasmodium falciparum were produced in continuous cultures but eventually declined in numbers after 3–4 months in vitro. Their development progressed in a consistent pattern, from small rounded, through triangular, to ellipsoidal, and finally after 8 days to crescentic forms. Morphologic maturity occurred at 8–9 days, but the gametocytes would not exflagellate in vitro, even after 14–18 days of development. Thus, current culture methods cannot produce a continuous supply of functional gametes for further studies.     

Isolation,Enumeration and Identification of Amebae from a Nebraska Lake*

A plaque technic was evaluated and used for the isolation and enumeration of small, free-living amebae in lake-bottom samples which were collected each month for one year from a Nebraska lake. Several culture media were evaluated, and a simple glucose-salts medium was chosen. The most frequent ameba in the lake-bottom samples was Acanthamoeba polyhaga which underwent marked increases and decreases in population densities during the collection period. This pattern was not correlated with water temperature, bacterial counts, and nitrate or phosphate levels. Other species of amebae of the genera Hartmannella, Vahlkampfia, Naegleria, Paratetramitus and Echinamoeba were isolated. Most of these were either found infrequently or remained at relatively low, constant densities throughout the year. In addition, 4 species of Acrasieae of the genera Dictyostelium and Polysphondylium were isolated from the lake-bottom samples.     

Partial Purification and Characterization of a Bacteriolytic Enzyme Secreted by Tetrahymena*

Synopsis. Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3–4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded “C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacterio-lytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.     

Plasmodium falciparum in Culture: Improved Continuous Flow Method*

SYNOPSIS. A new design of flow vessel provides a method for continuous culture of P. falciparum in a settled layer of human erythrocytes with a slow flow of culture medium over them. The parasitemia is kept fluctuating from ? 1%, just after addition of fresh erythrocytes. to ? 10%, 2 or 3 days later. Each vessel provides each week 3 harvests, each containing ? 0.6–1 × 10⁹ parasites.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

Regulation of Macronuclear DNA Content in Tetrahymena thermophila*†

Synopsis.
Unequal macronuclear division in Tetrahymena thermophila introduces variance into G1 macronuclei; unless eliminated such variance would result in continuous variation in DNA content. Analysis of G1 and G2 macronuclear variances reveals that the added variance is eliminated by action on the extremes of macronuclear DNA content. In this model (Model II), macronuclei with small amounts of DNA have an additional complete S phase, while those with large amounts of DNA skip S. From available data, chromatin extrusion is shown not to contribute significantly, if at all, to the elimination of variance. Computer simulations utilizing haploid subunits indicate that model II predictions apply reasonably well to experimental data in terms of coefficients of variation, mean DNA content, and frequency of additional and skipped S phases. The simulations reveal also that within certain constraints, particularly the thresholds for additional and skipped S phases, macronuclear assortment is unaffected by Model II regulation. The relationships between Model II and other aspects of the cell cycle are briefly discussed.     

In Vitro Binding of Trypanosoma congolense to Erythrocytes*

Synopsis. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin. Trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.     

Transport of L-Proline and its Regulation in Leishmania tropica Promastigotes*

Leishmania tropica promastigotes transport L-proline through an active uptake system that has saturation kinetics, temperature dependence, a requirement for metabolic energy and transport against a concentration gradient. In experiments lasting 10 min, less than 10% of the proline transported is incorporated into macromolecules. The remainder is largely unaltered proline with an intracellular concentration nearly 60 times that in the reaction mixture. The uptake system has a relatively broad specificity; it is competitively inhibited by D-proline as well as by alanine, methionine, valine, azetidine-2–carboxylate, thioproline, 3,4–dehydroproline, hydroxyproline and α-aminoisobutyric acid. Pre-established intracellular proline pools exchange with external proline as well as compounds that compete with it for uptake. Evidence is presented that feedback inhibition and transinhibition may regulate proline uptake in this organism.     

Genetic Evidence Concerning the Structure of the Tetrahymena thermophila Macronucleus*†

Synopsis.
A satisfactory model of the Tetrahymena thermophila macronucleus must explain its genetic behavior in terms of its constituent molecules. Particular genetic phenomena requiring explanation are (a) phenotypic assortment , here interpreted as resulting from allelic disjunction rather than from differential gene expression; (b) unequal allelic input for some loci , interpreted as a consequence of unequal and selective replication of some alleles during early macronuclear development; (c) delayed assortment at some loci , interpreted as an effect of inequality of allelic input combined with a generalized elevation of DNA content during early clonal history; (d) linkage disruption , probably reflecting continuous somatic recombination rather than dissolution of chromosomes into small repliconic units; (e) assortment depression , brought about by the occasional association of homologous replicons (chromosomes) or else by a differential increase in some classes of replicons; (f) ploidy-related developmental differences in macronuclear primordia are interpreted on the basis of quantitative differences in DNA rather than in terms of an early perception of genic imbalance, (g) Ploidy independent macronuclear DNA content is consistent with several models of size regulation.     

Acid and Neutral Hydrolases in Trypanosoma cruzi. Characterization and Assay*

Twelve acid hydrolases, 4 near-neutral hydrolases and alkaline phosphatase were demonstrated in 0.34 M sucrose homogenates of Trypanosoma cruzi strain Y: p-nitrophenylphosphatase and α-naphthylphosphatase, with optimum pH at ? 6.0; α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-glucosaminidase, cathepsin A and peptidase I and III, with optimum pH between 5.0 and 6.0: and arylsulfatase cathepsin D, α-arabinase and α-mannosidase with optimum pH at ? 4.0 α-Glucosidase, gluccse-6-phosphatase and peptidase II had optimum pH at ? 7.0. β-Glycerophcsphatase had a broad pH-activity curve from 4.0 to 7.4, with maximum activity at pH 7.0. The main kinetic characteristics of these enzymes and their quantitative assay methods were studied. No activity was detected for α-fucosidase, β-xylosidase, β-glucuronidase, elaidate esterase. acid lipase, and alkaline phospho-diesterase.     

An Infectious Agent Associated with Amebas of the Genus Naegleria*

A study of amebas of the genera Naegleria, Acanthamoeba, Polysphondylium, and Didymium shows that a cytopathogenic agent that is filterable and passageable is present only in the strains of the Naegleria whether they are obtained free-living from soil samples (N. gruberi) or as pathogens from humans (N. fowleri). The agents obtained from the different Naegleria strains are similar in amount and in their cytopathogenic interaction with chick cultures. The agent has characteristics that distinguish it from the known viruses.