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 beta-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.(ABSTRACT TRUNCATED AT 250 WORDS)

The degradation of three polycyclic aromatic hydrocarbons (PAH), pyrene (PYR), benz[a]anthracene (BAA), and benzo[a]pyrene (BaP), by Mycobacterium sp. strain RJGII-135 was studied. The bacterium was isolated from an abandoned coal gasification site soil by analog enrichment techniques and found to mineralize [ 14 C]PYR. Further degradation studies with PYR showed three metabolites formed by Mycobacterium sp. strain RJGII-135, including 4,5-phenanthrene-dicarboxylic acid not previously isolated, 4-phenanthrene-carboxylic acid, and 4,5-pyrene-dihydrodiol. At least two dihydrodiols, 5,6-BAA-dihydrodiol and 10,11-BAA-dihydrodiol, were confirmed by high-resolution mass spectral and fluorescence analyses as products of the biodegradation of BAA by Mycobacterium sp. strain RJGII-135. Additionally, a cleavage product of BAA was also isolated. Mass spectra and fluorescence data support two different routes for the degradation of BaP by Mycobacterium sp. strain RJGII-135. The 7,8-BaP-dihydrodiol and three cleavage products of BaP, including 4,5-chrysenedicarboxylic acid and a dihydro-pyrene-carboxylic acid metabolite, have been isolated and identified as degradation products formed by Mycobacterium sp. strain RJGII-135. These latter results represent the first example of the isolation of BaP ring fission products formed by a bacterial isolate. We propose that while this bacterium appears to attack only one site of the PYR molecule, it is capable of degrading different sites of the BAA and BaP molecules, and although the sites of attack may be different, the ability of this bacterium to degrade these PAH is well supported. The proposed pathways for biodegradation of these compounds by this Mycobacterium sp. strain RJGII-135 support the dioxygenase enzymatic processes reported previously for other bacteria. Microorganisms like Mycobacterium sp. strain RJGII-135 will be invaluable in attaining the goal of remediation of sites containing mixtures of these PAH.

Various Brassica species accumulate Se into the thousands of ppm. This suggests some of them as candidates for Se phytoremediation. Brassica juncea (Indian mustard) was used to accumulate selenium by growing with sodium selenite as the selenium source under hydroponic conditions resulting in Se accumulation of up to hundreds of ppm in various parts of the plant. To date, few selenium speciation studies have been done in plants, with most studies reporting total selenium concentration in various parts of the plant. Se species extraction was evaluated by several digestion/extraction procedures, including the use of HCl, Tris-HCl buffer, and enzymatic hydrolysis (using proteinase K and protease XIV). The best extraction was obtained with proteinase K (extracting approximately 75% of the total Se present in the plant). Some of the species produced by the plant, such as selenomethionine, can be identified at ppb levels by RP-HPLC-ICPMS, since standards are readily available. Others needed to be further characterized by ES-MS. Enzymatic hydrolysis releases mostly Se-methionine from juncea leaves, although other Se-containing species can also be observed by HPLC-ICPMS. In this initial study, the possible identification (by ES-MS) of a small chromatographic peak containing a Se-S bridged seleno amino acid with a structure similar to cystine is suggested.

Pneumocystis causes a type of pneumonia in immunodeficient mammals, such as AIDS patients. Mammals cannot alkylate the C-24 position of the sterol side chain, nor can they desaturate C-22. Thus, the reactions leading to these sterol modifications are particularly attractive targets for the development of drugs against fungal and protozoan pathogens that make them. In the present study, the definitive structures of 43 sterol molecular species in rat-derived Pneumocystis carinii were elucidated by nuclear magnetic resonance spectroscopy. Ergosterol, ⌬ 5,7 sterols, trienes, and tetraenes were not among them. Most (32 of the 43) were 24-alkylsterols, products of S -adenosyl-L -methionine:C-24 sterol methyl transferase (SAM:SMT) enzyme activity. Their abundance is consistent with the suggestion that SAM:SMT is highly active in this organism and that the enzyme is an excellent anti-Pneumocystis drug target. In contrast, the comprehensive analysis strongly suggest that P. carinii does not form ⌬ 22 sterols, thus C-22 desaturation does not appear to be a drug target in this pathogen. The lanosterol derivatives, 24-methylenelanost-8-en-3 ␤ -ol and ( Z )-24-ethylidenelanost-8-en-3 ␤ -ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci , were also detected among the sterols of the rat-derived P. carinii organisms. Pneumocystis pneumonia (PcP) remains among the most prevalent opportunistic infections in immunocompromised individuals such as AIDS patients. It has becoming evident that the high incidence of PcP in AIDS patients is global and that the organism can infect other immunodeficient people such as patients undergoing chemotherapy for cancer or solid organ transplant (1, 2), children prior to becoming fully immunocompetent, and the elderly with diminished immune systems. Pneumocystis can also transiently colonize normal, healthy people and animals without causing overt symptoms of respiratory disorder. The combination of trimethoprim and sulfamethoxazole and other agents (e.g., pentamidine, atovaquone) used for prophylaxis and for clearing PcP has successfully reduced the number of deaths directly attributed to PcP. However, AIDS and other patients with prolonged immunodeficiency experience recurrent Pneumocystis jiroveci ( Pneumocystis carinii f. sp. hominis ) (3, 4) infections. Also, some individuals cannot tolerate these drugs and suffer undesirable side effects. The development of drug-resistant pathogen populations is of serious concern, making it imperative to develop a larger armamentarium of diverse drugs to circumvent these problems (5 Ϫ 7). Although the organism can be maintained in long-term, small-volume axenic cultures (8), growth is very slow and insufficient numbers of organisms are obtained for most biochemical studies. Thus, Pneumocystis remains an organism considered difficult to manipulate for experimental work. Despite this difficulty, much is now known about the organism's lipids from analyses performed on preparations purified from infected rat lungs (9 Ϫ 11...

Pneumocysterol [(24Z)-ethylidenelanost-8-en-3␤-ol], a rare sterol detected in the opportunistic pathogenCommunicated by William Trager, The Rockefeller University, New York, NY, November 2, 1998 (received for review February 4, 1998 ABSTRACTPneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct ⌬ 7 , 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C 32 sterol were found in humanderived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C 31 and C 32 sterols were identified as euphorbol (24-methylenelanost-8-en-3␤-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3␤-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.Sterols and their biosyntheses are excellent targets for chemotherapeutic attack against infectious microbes, especially the fungi. Polyene antibiotics such as amphotericin B bind avidly to ergosterol in fungal cell membranes. After the sterol-drug complexes aggregate, large pores in the membranes are formed, dissipating ion gradients. Fluconazole and some other compounds routinely used clinically for systemic mycosis target ergosterol biosynthesis at nuclear demethylation steps. Ergosterol was not detected in Pneumocystis carinii carinii that was isolated and purified from the lungs of corticosteroid-immunosuppressed rats. In this respect, the pathogen appears to be unlike higher fungi. However, the organism synthesizes its own distinct sterols, e.g., fungisterol (24-methylcholest-7-en-3␤-ol and 24-ethylcholest-7-en-3␤-ol; refs. 1-4). Parasites generally scavenge sterols (e.g., cholesterol) from the host and utilize them for membrane formation and other cell functions. If host sterols do not fulfill the precise stereochemi...