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Notes: The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ10 was the predominant homolog with lesser amounts of CoQ9 present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ9 and lesser amounts of CoQ8, but no detectable CoQ10. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ10 (if not both CoQ110 and CoQ9) in P. carinii as not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.

Notes: Mixed life cycle stages of rat-derived Pneumocystis carinii were isolated from host lungs and their sterols were compared with those present in lungs from normal and immunosuppressed uninfected rats. Gas-liquid chromatography consistently detected, resolved, and quantified 9, 10, and 20 sterol components in the total nonsaponifiable neutral lipid fraction of lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii preparations, respectively. In all samples, cholesterol was the most abundant sterol present, comprising 97%, 93%, and 78% of total sterols in lungs from normal rats, lungs from immunosuppressed uninfected rats, and P. carinii, respectively. Tentative identifications of several rat lung and P. carinii minor sterols were made based on gas-liquid chromatogram retention times and fragmentation patterns from mass spectral analyses. Campesterol (ergost-5-en-3-ol), cholest-5-en-3-one, and β-sitosterol (stigmast-5-en-3-ol) were among the minor components present in both types of lung controls, and were also components of P. carinii sterols. In contrast to lung controls, the sterols of P. carinii were enriched in C28 and C29 sterols with one or two double bonds, and a hydroxyl group at C-3 (ergost-5-en-3-ol, ergost-7-en-3-ol, ergosta-dien-3-ol, stigmast-5-en-3-ol, stigmast-7-en-3-ol and stigmasta-dien-3-ol). Steryl esters of P. carinii, probably stored in cytoplasmic lipid droplets, were dominated by those present in the host lung. In separate studies. 3-hydroxy-3-methylglutaryl coenzyme A activity, a key enzyme in the regulation of sterol biosynthesis, was detected in purified P. carinii preparations and incorporation of radiolabeled squalene and mevalonate was observed. Together, these results suggest that the parasite readily takes up and incorporates host sterols, and that the organism synthesizes some of its own “metabolic sterols”

Notes: The major ester-linked fatty acids of the total lipids extracted from Pneumocystis carinii isolated from the lungs of corticosteroid-trcaicd rats were 16:0. 18:0, 18:1, 18:2 and 20:4. Others detected included 14:0, 16:1 and 22:4. The major sphingolipid fatty acids were 16:0, 18:0,22:0,24:0 and 24:1; others included 14:0, 18:1, 20:0, 23:0, 24:2 and 26:0. The total lipid fatty acid compositions of preparations from appropriate lung controls were similar to those of the organism.

Notes: . Serine is an important amino acid that is utilized in the biosyntheses of proteins and lipids. It is directly incorporated into the head group of phosphatidylserine, which in turn can be converted to other phospholipids. Also, it is required for the formation of long chain bases, precursors of sphingolipids. Uptake and incorporation of radiolabeled serine into both lipids and acid-precipitable material were demonstrated in Pneumocystis carinii carinii organism preparations freshly isolated from infected rat lungs. Radioactivity in proteins was about double that observed in lipids. Liquid scintillation spectrometry of metabolically radiolabeled lipids separated by thin-layer chromatography showed 53% of the total radioactivity were in phosphatidylserine, 12% in phosphatidylethanolamine, 24% in ceramides, and 11% in long chain bases and other compounds. Four long chain bases were detected by thin-layer chromatography in hydrolyzed P. carinii ceramides metabolically labeled with radioactive serine. Phytosphingosine and dihydrosphingosine were tentatively identified by their migrations on thin-layer plates. Radiolabeled ethanolamine was incorporated into P. carinii phosphatidylethanolamine, but relatively low incorporation of radiolabeled choline into phosphatidylcholine occurred. The observations made in this study indicated that P. carinii has the biosynthetic capacity to metabolize phospholipid head groups and to de novo synthesize sphingolipids. L-Cycloserine and β-CI-D-alanine, inhibitors of long chain base synthesis, reduced the incorporation of serine into P. carinii long chain bases and ceramides, which supported the conclusion that the pathogen synthesizes sphingolipids.

Notes: SUMMARY Two sterols in autopsied whole lung specimens obtained from Pneumocystis carinii pneumonia patients were detected by gas-liquid chromatography and their structures were elucidated by mass spectrometry and nuclear magnetic resonance spectrometry. Both were in the lanosterol series; the C31 sterol, with a methyl group at C-24, was identified as euphorbol, and the more abundant C32 sterol, with an ethyl group at C-24, is given the trivial name pnemocysterol.

Notes: Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was 〉 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (〈 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated 〉 99.5% purity. The total non-P. carinii protein in the final preparation (〈 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 108-109 organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.

Notes: . The biochemical characterizations of lipophosphoglycans from various Leishmania species reported by other workers may or may not contain several types of lipophosphoglycan molecules. This is the first report in which a specific lipophosphoglycan has been defined by both its antigenie and electrophoretic properties. Furthermore, a purification procedure for this specific lipophosphoglycan is described and some biochemical characterizations are presented. Phospholipase C and the so-called phosphatidylinositol-specific phospholipase C of Bacillus cereus convert the amphipathic form of the lipophosphoglycan antigen to the hydrophilic form. Under equivalent incubation conditions, other phospholipases tested were not effective in conversion of the amphipathic to the hydrophilic form. Since the amphipathic form is present in conditioned media, antigen shedding cannot be explained by phospholipase C digestion of the amphipathic form, which would result in the release of only the hydrophilic form into the medium. Both the pellet and the supernatant fractions of conditioned media contained both forms of the antigen and did not differ in the relative amounts of the two. This observation rules out membrane blebbing as the major mechanism for the release of the amphipathic form.

Notes: Several divalent cation-dependent ATP phosphohydrolases associated with cilia, ciliary axonemes, ciliary membranes, pellicles, trichocysts, nuclei, mitochondria, microsomes, and soluble peripheral cell surface fractions of Paramecium tetraurelia were resolved in this study. Fifteen different activity bands were detected in whole cell sonicates or subcellular fractions by Triton polyacrylamide gel electrophoresis and ATPase activity staining. The ciliary surface membrane contained two major ATPase activities that were distinct from the enzymes associated with all other cell fractions.

Notes: SYNOPSIS. Observations of the ultrastructure of marine scuticociliatids, tentatively assigned to the genus Uronema, were made by light, transmission electron, and scanning electron microscopy. Giant, cortically oriented mitochondria filled the subpellicular, intermeridional areas, and were in close association with the epiplasm immediately under the inner alveolar sac membranes. Reconstructions of serial sections of the posterior poles of ciliates indicated that the intermeridional mitochondria could fuse at that point and the entire chondriome might at times be a single organelle. A system of tubules was observed to be intimately associated with the mitochondria in the posterior region. The tubules anastomosed and were directed posteriorly into the region of the nephridial-contractile vacuole system. The outer surfaces were coated with projections arranged in helical patterns. The system may be regarded as a fluid segregation organelle. The tripartite nature of the polar basal body complex observed by silver impregnation was confirmed by transmission electron microscopy. The 3 structures were the basal body of the caudal cilium and 2 parasomal sacs. A prominent ring around the caudal cilium was observed by scanning electron micrcscopy; it is probably responsible for the silver deposition surrounding the polar basal body complex that can be seen by light microscopy of silver-impregnated specimens. The ultrastructure of the nonmotile caudal cilium and its kinetosome was unremarkable, being like that of the motile, somatic cilia. The micronuclear and macronuclear outer membranes were continuous at several sites. Such interconnections explain the intimate physical relationship between the nuclei during interphase in many ciliates, and could be a structural basis for chemical communication between the 2 nuclear types. Within the cytoplasm surrounding the opening of the cytoproct, numerous clear vesicles were observed. Their position and appearance suggested that the cytoproct may be involved in the elimination of solutions as well as solids. Food vacuoles, cortical microtubules, lamellar vesicles, disc-shaped vesicles, mucocysts, and a contractile vacuole and its pore were also observed.