Evidence for the Presence of "Metabolic Sterols" in Pneumocystis: Identification and Initial Characterization of Pneumocystis carinii Sterols

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"     

Ultrastructural Differences Between Species of Trypanosomatids With and Without Endosymbionts1

Species of trypanosomatids without endosymbionts (Leptomonas seymouri, L. collosoma, L. samueli, Crithidia fasciculata, C. luciliae, C. acanthocephali, Herpetomonas megaseliae, H. mariadeanei, H. samuelpessoai, H. muscarum muscarum, Trypanosoma cruzi) and species of trypanosomatids with endosymbionts (Crithidia deanei, C. oncopelti, Blastocrithidia culicis) were comparatively studied by means of electron microscopy. Artificially aposymbiotic strains derived from species with symbiont were also included in the survey. Species with symbiont were found to differ in some ultrastructural aspects from the group of species without symbiont. Paraxial rods of flagella or intraflagellar structure were found exclusively in species without symbiont. Peripheral branching of mitochondria, accompanied by absence of subpellicular microtubules in sites where the mitochondrial branches are appressed to the cell membrane, were found exclusively in species with symbiont. Networks of kinetoplast DNA fibrils were found to be larger and looser in species with symbiont. Symbiont-free strains of species with symbiont retained the same morphological characteristics of their parental species.     

The effect of salinity in the growth medium on carbohydrate metabolism in pea root tips

The effect of chloride and sulphate types of salinity on respiratorymechanism of pea roots was studied. Both types of salinity depressedthe absorption of external glucose and the rate of respirationand changed the relative part of the metabolic pathways. Thepercentage of the pentosephosphate pathway was increased byincreasing levels of NaCl salinity while NaaSOi salinity practicallydid not affect it. Chloride salinity depressed CO₂ evolutionfrom C-6 but did not affect the CO₂ evolution from C-1. Sulphatesalinity depressed both. Most of the glycolytic enzymes studiedwere depressed in roots grown in saline medium except glucosephosphateisomerase which was not affected by sulphate salinity but wasincreased more than ten fold by chloride salinity. Phosphogluconatedehydrogenase was not affected by salinity. Glucose-6-phosphatedehydrogenase was found in the mitochondria as well as in thesupernatant and both the mitochondrial and the supernatant enzymeswere active with NADP and NAD. Salinity of both types increasedthe NADP linked activity in the soluble fraction. The informationgained however is still not sufficient to explain adequatelythe effect of salinity on plant metabolism. (Received July 31, 1967; )     

The Occurrence of Biogenic Calcian Struvite, (Mg, Ca)NH4PO4.6H2O, as Intracellular Crystals in Paramecium

ABSTRACT. Intracellular crystals are conspicuous refractile "inclusion bodies" commonly found in many protozoans, but very few have been identified mineralogically. We have isolated crystals from axenically grown mass cultures of Paramecium tetraurelia , and purified them using differential centrifugation. The crystals' structure and chemistry were analyzed using x-ray powder diffraction and energy-dispersive electron microprobe techniques. The morphology was studied by means of scanning electron microscopy. The crystals were identified as the orthorhombic mineral, calcian struvite, (Mg, Ca)NH₄PO₄.6H₂O. Struvite from P. tetraurelia exhibited a variety of crystal habits, including hemimorphic forms, epitactic overgrowths and several types of twins. A linear correlation between computed hydration number and Mg content suggests that the crystal composition may reflect the range of conditions under which struvite nucleation and growth occur. The mineral struvite also occurs in association with guano and other rich organic products, and can be biologically induced to precipitate extracellularly. Extracellular struvite has been well characterized in pathogenic calculi (kidney stones) of humans and cats, where precipitation is enhanced by bacterial urease activity that produces ammonia in the urinary tract. This is the first study demonstrating that struvite is also biologically controlled to form as an intracellular mineral. These crystals may have formed within lipid-rich, membrane-bound vesicles in Paramecium .     

Schizogonic Development of Leucocytozoon smithi

ABSTRACT The schizogonic development of Leucocytozoon smithi in the liver of experimentally infected turkey poults was examined by electron microscopy. Following intraperitoneal injection, sporozoites migrated to the liver and entered hepatic cells to become intracellular trophozoites. Three to four days post inoculation (PI), trophozoites underwent asexual multiple fission known as merogony or schizogony. Two generations of schizonts were observed. The primary or first generation schizonts, abundant on day 4 PI, appeared as interconnected cytoplasmic masses (pseudocytomeres). Each pseudocytomere was enclosed by a membranous vacuole and contained varying numbers of nuclei. As nuclear division and growth of the schizonts continued, larger discrete cytoplasmic masses or cytomeres were formed with rhoptries and multiple nuclei in various stages of division. Synchronous multiple cytoplasmic cleavage of the schizont resulted in the formation of numerous uninucleate merozoites. Second generation schizonts, which developed from hepatic merozoites released from primary schizonts, were abundant in hepatocytes on day 6 PI. Although tissue samples from liver, lung, spleen, kidney, intestine, brain, blood vessels and lymph nodes were examined, schizogonous forms were observed in liver only. No megaloschizonts were detected in any host tissue examined. Schizogonic development was completed by day 7 PI as merozoites developed into gametocytes within mononuclear phagocytes.     

Sporogonic Development of Leucocytozoon smithi

The sporogonic development of Leucocytozoon smithi in its black fly vector was studied by light and electron microscopy and was compared with that of other haemosporidians. Within 18 to 24 h after ingestion of gametocytes by black flies, ookinetes passing through the midgut epithelium were observed. Intracellular migration of ookinetes resulted in the apparent disruption and degeneration of host cells. Intercellular migration also occurred as was evidenced by the presence of ookinetes between midgut cells. Transformation of ookinete to spherical oocyst occurred extracellularly in three different sites. Although most oocysts were found between the host cell basal membrane and the basal lamina, large numbers also were found attached to the external surface of the basal lamina, projecting into the hemocoel. Ectopic development of oocysts in the midgut epithelium between cells was observed much less frequently than development on the basal side of the midgut. The oocyst wall of dense granules, believed to be of parasite origin, was distinguishable from the basal lamina of the host's midgut epithelium. As in other Leucocytozoidae, the cytoplasm of the oocyst differentiated into a single sporoblastoid from which 30–50 sporozoites were formed. Beginning on the third day post infection, elongation of segregated dense sporoblastoid material associated with pellicle thickening led to the formation of the finger-like sporozoite buds which projected into the oocyst cavity. Sporozoites within mature oocysts and salivary glands were structurally similar to sporozoites as described for other haemosporidians.     

The control of postharvest decay in table grapes using acetaldehyde vapours

Grapes ( Vitis vinifera L. cv. 'Sultanina') harvested at the end of the 1985–1988 seasons, received postharvest application of acetaldehyde (AA) vapours for 24–40 h. Treatment with AA vapour at 20 °C or 0 °C reduced significantly the decay caused by several fungi: Botrytis cinerea, Rhizopus stolonifer, Aspergillus niger and Alternaria alternata . In grapes treated with 0.5% AA for 24 h, no R. stolonifer was found after 8 days of storage at 20 °C. Treatment with 0.25% AA vapour for 40 h of grapes cv. 'Perlette' inoculated with R. stolonifer reduced the decay by 89%.     

Composition of Pneumocystis carinii Neutral Lipids and Identification of Coenzyme Q10 as the Major Ubiquinone Homolog

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.     

Observations on the Ultrastructure of Uronema spp., Marine Scuticociliates*

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.