Evidence of sexual reproduction in the protozoan parasite Leishmania (Kinetoplastida: Trypanosomatidae).

Parasitic protozoa of the genus Leishmania (Kinetoplastida: Trypanosomatidae) are generally thought to multiply by binary fission; however, data from quantitative microspectrophotometry indicate that nuclear fusion or sexual reproduction takes place in the intracellular amastigote form. Among several different Leishmania species, the mean +/- SD nuclear DNA content of all promastigotes (extracellular form) and of some amastigotes (intracellular form) in macrophages was 0.098 +/- 0.032 relative units; in contrast, other amastigotes within the same macrophage had a mean +/- SD nuclear DNA content of 0.219 +/- 0.050. The latter population of amastigotes are apparently produced when the nuclei of a pair of 0.098 amastigotes fuse. These 0.219 amastigotes later go through what is probably the typical meiotic cycle to reform the 0.098 condition we observed among promastigotes. The demonstration of sexual reproduction in Leishmania has important implications for the future direction of research on this medically important parasite.

Chemical and physiological effects of Candida albicans toxin on tissues.

The prevalence of immunologically suppressed patients, including those infected with the AIDS virus, with cancer, and those having had transplant surgery to name a few, has provided an avenue for the rapid proliferation among these patients of the virulent yeast Candida albicans. Previous studies have determined that a potent toxin is produced by C. albicans which may cause extensive tissue damage. The extent of the tissue damage has never been determined, neither has the mechanism been explained. The present work shows that intraperitoneal inoculation of C. albicans produces numerous tumour-like lesions and abscesses on the major organs of experimental laboratory rats. The results demonstrate that damage is caused by the initial release of lysosomal enzymes by the affected tissues.

Quantitative cytophotometric and autoradiographic analysis of the cell cycle kinetics during growth and development of the plasmodium in the myxomycete Didymium iridis.

Nuclear changes in basic protein, RNA, and DNA in plasmodia of Didymium iridia at 4, 8, and 12 days during their progression through the growth cycle prior to sporulation were analysed. Relative determinations of basic protein and RNA were made using naphthol yellow-S/azure B in conjunction with absorption microspectrophotometry. DNA was quantified by quantitative cytophotometric measurements of Feulgen-stained nuclei. Autoradiographic techniques provided information on periods of DNA synthesis. A net decrease in protein and RNA to a point of stabilization was shown which correlated with increased age of the organism and decreased nutrient supply in the plasmodia. The 4C DNA content of the 4 day plasmodia indicated rapid growth and high metabolic activity while in the 8 day plasmodia a 2C DNA was observed consistent with the presence of a G1 period of some duration.

Evidence for an induced plasma membrane mating receptor in the myxomycete Didymium iridis.

Haploid myxamoeboid mating types, CR5-11A11 and HON 1-2A1, of the acellular myxomycete Didymium iridis, were isolated and grown in liquid culture. Previously it has been postulated that a molecule, possibly a hormone, is responsible for the induction of mating competency through receptor site synthesis. The intention of this study was to isolate the molecule and to determine its mode of action. Experiments were designed in order to learn whether it acts only upon the cells which produce it (self-induction) or does it have an effect only on opposite mating types (cross-induction). It was found that plasmodia can only form on solid media, although zygotes are readily formed in liquid media. Moreover, competency of cells to fuse, or to form receptors required that they be haploid and in a specific phase of the cell cycle, namely G1 of interphase. The use of high pressure liquid chromatography techniques was also employed in order to investigate the inducer molecule. It was demonstrated that the inducer molecule was similarly produced by both mating types, and its production was essentially paralleled by cell numbers. It was also discovered that mating competency is self-induced and these results are discussed.

Evidence for a plasma membrane mating receptor in the myxomycete Didymium iridis.

Haploid isolates Panamanian 2-7A8 and Honduran 1-2A1 of the heterothallic myxomycete Didymium iridis were investigated in order to determine whether membrane mating factors or receptors were present and necessary in order that cell fusion could occur and zygote formation take place. Haploid and diploid controls grown on half strength cornmeal media, and experimental crosses grown on the same media plus concanavalin A, were analysed by quantitative cytophotometry employing the Feulgen nuclear reaction. Nuclear DNA analysis demonstrated that Con A treated cells when crossed did not produce zygotes or plasmodia, while those left untreated did so almost all of the time. On rare occasion plasmodia did develop in treated cultures (1.8% or two out of 110). These plasmodia demonstrated low viability and upon analysis were shown to be haploid. It was concluded that they had developed by apogamy, and therefore did not require plasma membrane bound mating receptors, cell fusion, or zygote formation.

Effects of chemistry, manufacturing and concentration of the dye basic fuchsin regarding its use in quantitative cytophotometry.

Four separately manufactured preparations of basic fuchsin were compared to determine the effects of concentration, chemistry, and the manufacturing process for their quantitative value in the nuclear Feulgen reaction. In order to make the necessary comparisons, the two wavelength method of quantitative cytophotometry was employed to analyse each stain application regarding DNA measurements. Chicken erythrocytes, and myxamoebae and plasmodia of Didymium iridis were employed as experimental tissues. Results indicated that all four preparations of the stains yielded acceptable and valid quantitative data in relative DNA values. Differing manufacturers and dye concentrations had no appreciable effect on relative quantitative measurements. However, the maximum staining intensity was affected by differences both in the chemical structure of the individual stains and by the products from various manufacturers. The maximum dye intensity and accurate quantitative absolute values for DNA measurement were best obtained by the use of basic fuchsin having the same colour index (Cl) 42510 as that manufactured by Fisher Scientific Incorporated.

Quantitative cytophotometric determination of DNA, RNA and lysine bound protein in relationship to zygote formation and protein synthesis in myxamoebae and swarm cells of Didymium iridis.

Quantitative cytochemical determinations were made of the DNA of zygotes formed from myxamoebae and swarm cells of Didymium iridis. The nuclear and cytoplasmic RNA and lysine bound protein of these cells were also measured. Significant zygote formation in myxamoebae crosses began at 20-30 min, while swarmers required 35-40 min. Myxamoebae, however, demonstrated a greater ability to form zygotes. The total cytoplasmic RNA and protein bound lysine for myxamoebae was higher than that of the swarmer cells. This observed decrease in swarmers may be due to reduced protein synthesis. Values for nuclear RNA were higher in the myxamoebae, but nuclear lysine bound protein was higher in the swarmers. The data presented suggest that prefusion swarmers, after replicating their DNA, go into a period of G2 arrest and remain in this condition postfusion. In contrast, prefusion myxamoebae readily divide after DNA replication, and continue to synthesize nuclear DNA, and to divide after fusion.

Isolation of haploid clones bearing a mating allele from a diploid apomictic isolate of Didymium iridis.

Clones of myxamoebae which fail to undergo a direct morphogenetic change into plasmodia have been isolated from cultures of the Philippine-1 isolates of Didymium iridis. These clones have been maintained continuously for a period of more than 4 years without producing plasmodia. It has been demonstrated by Feulgen-DNA cytophotometry that the mean nuclear DNA content of such clones is equivalent to the haploid replicated (2C) value. Conversely, myxamoebae which produce plasmodia directly have a mean nuclear DNA content which is equivalent to the diploid replicated (4C) value. The haploid Philippine-1 clones have been demonstrated to be cross compatible with isolates bearing the mating alleles A1, and A3 through A11, and incompatible with isolates bearing the mating allele A2. Furthermore, plasmodia which result from the crosses Ph-1n (A2) Hon 1-2 (A1), (A1), Ph-1n (A2) x Pan 2-4 (A7) and Ph-1n (A2) x Pan 2-7 (A8) have mean nuclear DNA values which are equivalent to the replicated diploid value, and produce spores which bear the mating alleles in the predicted ratios. Such data demonstrate that the Ph-1n clones are sexually compatible with haploid myxamoebae of heterothallic isolates. The significance of the isolation of these haploid clones with regard to a recent hypothesis related to the evolution of developmental systems in the Myxomycetes is discussed.

A rapid method for isolation of the plasma membrane of the myxamoebae and swarm cells of the myxomycete Didymium iridis.

A method for the isolation of the plasma membranes of the acellular slime mould, Didymium iridis in the myxamoebae and swarm cell stages was developed using a modified dextranpolyethylene glycol aqueous two-phase polymer system. It was found to be far superior to the widely accepted technique of density gradient centrifugation concerning this cellular system. Chemical and enzymatic assays performed on the plasma membranes and other cell fractions as well as microscopic examination were used as a basis for positive identification and assessment of purity. Results of the chemical and enzymatic assays indicate that plasma membranes are recovered with high yield and purity using the modified two-phase polymer technique. The method is both rapid and effective and can be performed using low-speed centrifugation.