THE ULTRASTRUCTURE OF COELOMOCYTES OF THE SEA STAR DERMASTERIAS IMBRICATA

Volume: 159Start Page: 295End Page: 31

Ubiquinone Synthesis and its Regulation in Pneumocystis carinii

The opportunistic pathogen Pneumocystis causes a type of pneumonia in individuals with defective immune systems such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), is effective in clearing mild to moderate cases of the infection. Rat-derived Pneumocystis carinii was the first organism in which CoQ synthesis was clearly demonstrated to occur in both mitochondrial and microsomal subcellular fractions. Atovaquone inhibits microsomal CoQ synthesis with no effect on mitochondrial CoQ synthesis. We here report on additional studies evaluating CoQ synthesis and its regulation in the organism. Buparvaquone also inhibited CoQ synthesis and it reduced the synthesis of all four CoQ homologs in the microsomal but not the mitochondrial fraction. Glyphosate, which inhibits a reaction in the de novo synthesis of the benzoquinone moiety of CoQ reduced cellular ATP levels. Bacterial and plant quinones, and several chemically synthesized phenolics, flavanoids, and naphthoquinones that inhibit electron transport in other organisms were shown to reduce CoQ synthesis in P. carinii. The inhibitory action of naphthoquinone compounds appeared to depend on their molecular size and structural flexibility rather than redox potential. Results of experiments examining the synthesis of the polyprenyl chain of CoQ were consistent with negative feedback control of CoQ synthesis. These studies on P. carinii suggest that cellular sites and the control of CoQ synthesis in different organisms and cell types might be more diverse than previously thought

Quantitation of Absolute Pneumocystis Carinii Nuclear DNA Content. Trophic and Cystic Forms Isolated From Infected Rat Lungs Are Haploid Organisms

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4\u27,6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37°C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism\u27s chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms

Ubiquinone Synthesis in Mitochondrial and Microsomal Subcellular Fractions of Pneumocystis spp.: Differential Sensitivities to Atovaquone

The lung pathogen Pneumocystis spp. is the causative agent of a type of pneumonia that can be fatal in people with defective immune systems, such as AIDS patients. Atovaquone, an analog of ubiquinone (coenzyme Q [CoQ]), inhibits mitochondrial electron transport and is effective in clearing mild to moderate cases of the infection. Purified rat-derived intact Pneumocystis carinii cells synthesize de novo four CoQ homologs, CoQ(7), CoQ(8), CoQ(9), and CoQ(10), as demonstrated by the incorporation of radiolabeled precursors of both the benzoquinone ring and the polyprenyl chain. A central step in CoQ biosynthesis is the condensation of p-hydroxybenzoic acid (PHBA) with a long-chain polyprenyl diphosphate molecule. In the present study, CoQ biosynthesis was evaluated by the incorporation of PHBA into completed CoQ molecules using P. carinii cell-free preparations. CoQ synthesis in whole-cell homogenates was not affected by the respiratory inhibitors antimycin A and dicyclohexylcarbodiimide but was diminished by atovaquone. Thus, atovaquone has inhibitory activity on both electron transport and CoQ synthesis in this pathogen. Furthermore, both the mitochondrial and microsomal fractions were shown to synthesize de novo all four P. carinii CoQ homologs. Interestingly, atovaquone inhibited microsomal CoQ synthesis, whereas it had no effect on mitochondrial CoQ synthesis. This is the first pathogenic eukaryotic microorganism in which biosynthesis of CoQ molecules from the initial PHBA:polyprenyl transferase reaction has been unambiguously shown to occur in two distinct compartments of the same cell