Sterol composition of Pneumocystis jirovecii with blocked 14alpha-demethylase activity

Several drugs that interact with membrane sterols or inhibit their syntheses are effective in clearing a number of fungal infections. The AIDS-associated lung infection caused by Pneumocystis jirovecii is not cleared by many of these therapies. Pneumocystis normally synthesizes distinct C28 and C29 24-alkylsterols, but ergosterol, the major fungal sterol, is not among them. Two distinct sterol compositional phenotypes were previously observed in P. jirovecii. One was characterized by delta7 C28 and C29 24-alkylsterols with only low proportions of higher molecular mass components. In contrast, the other type was dominated by high C31 and C32 24-alkylsterols, especially pneumocysterol. In the present study, 28 molecular species were elucidated by nuclear magnetic resonance analysis of a human lung specimen containing P. jirovecii representing the latter sterol profile phenotype. Fifteen of the 28 had the methyl group at C-14 of the sterol nucleus and these represented 96% of the total sterol mass in the specimen (excluding cholesterol). These results strongly suggest that sterol 14alpha-demethylase was blocked in these organisms. Twenty-four of the 28 were 24-alkylsterols, indicating that methylation of the C-24 position of the sterol side chain by S-adenosyl-L-methionine:sterol C-24 methyl transferase was fully functional.     

Comprehensive and definitive structural identities of Pneumocystis carinii sterols

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, Delta(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 Delta(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 beta-ol and (Z)-24-ethylidenelanost-8-en-3 beta-ol (pneumocysterol), previously identified in human-derived Pneumocystis jiroveci, were also detected among the sterols of the rat-derived P. carinii organisms.     

The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.     

Is Pneumocystis a Plant?

Pneumocystis, an AIDS-associated opportunistic pathogen of the lung has some unusual features. This article focuses on work done by my group to understand the organism's distinct sterols. Although Pneumocystis is closely related to fungi, it lacks the major fungal sterol, ergosterol. Several delta(7) 24-alkysterols synthesized by P. carinii are the same as those reported in some basidiomycete rust fungi. The 24-alkylsterols are synthesized by the action of S-adenosyl-L-methionine:C-24 sterol methyl transferase (SAM:SMT). Fungal SAM:SMT enzymes normally transfer only one methyl group to the C-24 position of the sterol side chain and the cells accumulate C28 24-alkylsterols. In contrast, the P. carinii SAM:SMT and those of some plants catalyze one or two methyl transfer reactions producing both C28 and C29 24-alkylsterols. However, unlike most fungi, plants, and the kinetoplastid flagellates Leishmania and Trypanosoma cruzi, P. carinii does not appear to form double bonds at C-5 of the sterol nucleus and C-22 of the sterol side chain. Furthermore, the P. carinii SAM:SMT substrate preference for C30 lanosterol differs from that of homologous enzymes in any other organisms studied. C31 24-Methylenelanosterol and C32 pneumocysterol, products of SAM:SMT activity on lanosterol, can accumulate in high amounts in some, but not all, human-derived Pneumocystis jiroveci populations.     

Incorporation In Vivo and In Vitro of Radiolabeled Sphingolipid Precursors into Paramecium tetraurelia Lipids

ABSTRACT Paramecium tetraurelia contains high concentrations of ethanolamine sphingolipids, especially in its ciliary membrane. Three ethanolamine sphingophospholipids with different long chain bases (dihydrosphingosine, sphingosine and phytosphingosine), and their phosphonyl analogs, were previously identified and characterized. In the present study, radiolabeling experiments on lag- and log-phase cells were performed to investigate the extent of sphingolipid biosynthetic capacities of the ciliate. Long chain bases of sphingolipids are formed by an initial condensation reaction of serine with a fatty-coenzyme A. Thus, radiolabeled palmitic acid, stearic acid and serine were used as precursor compounds in these experiments. The results indicated that (1) sphingolipid precursors were incorporated into every major lipid fraction. (2) ethanolamine sphingophosphonolipids accumulated faster than the ethanolamine sphingophospholipids, (3) in contrast to these sphingolipids, the glycerolipid, phosphatidyethanolamine. accumulated faster than its phosphono analog, and (4) palmitic acid, but not stearic acid, was incorporated into the long chain bases of ethanolamine sphingophospho- and sphingophosphonolipids. consistent with an earlier report demonstrating that these lipids contain only C,g long chain bases. Since P. tetraurelia takes up serine and other water-soluble substrates very slowly, and catabolizes fatty acids rapidly, label is randomized in intact cells. Thus, cell-free protocols provide useful experimental systems for studies of sphingolipid biosynthesis than do intact organisms, when the uptake of precursor substrates are slow.     

Ligand-dependent differences in estrogen receptor beta-interacting proteins identified in lung adenocarcinoma cells corresponds to estrogenic responses

Background

A recent epidemiological study demonstrated a reduced risk of lung cancer mortality in breast cancer patients using antiestrogens. These and other data implicate a role for estrogens in lung cancer, particularly nonsmall cell lung cancer (NSCLC). Approximately 61% of human NSCLC tumors express nuclear estrogen receptor β (ERβ); however, the role of ERβ and estrogens in NSCLC is likely to be multifactorial. Here we tested the hypothesis that proteins interacting with ERβ in human lung adenocarcinoma cells that respond proliferatively to estradiol (E₂) are distinct from those in non-E₂-responsive cells.

Methods

FLAG affinity purification of FLAG-ERβ-interacting proteins was used to isolate ERβ-interacting proteins in whole cell extracts from E₂ proliferative H1793 and non-E₂-proliferative A549 lung adenocarcinoma cell lines. Following trypsin digestion, proteins were identified using liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). Proteomic data were analyzed using Ingenuity Pathway Analysis. Select results were confirmed by coimmunoprecipitation.

Results

LC-MS/MS identified 27 non-redundant ERβ-interacting proteins. ERβ-interacting proteins included hsp70, hsp60, vimentin, histones and calmodulin. Ingenuity Pathway Analysis of the ERβ-interacting proteins revealed differences in molecular and functional networks between H1793 and A549 lung adenocarcinoma cells. Coimmunoprecipitation experiments in these and other lung adenocarcinoma cells confirmed that ERβ and EGFR interact in a gender-dependent manner and in response to E₂ or EGF. BRCA1 interacted with ERβ in A549 cell lines and in human lung adenocarcinoma tumors, but not normal lung tissue.

Conclusion

Our results identify specific differences in ERβ-interacting proteins in lung adenocarcinoma cells corresponding to ligand-dependent differences in estrogenic responses.

     

Level of daily physical activity in individuals with COPD compared with healthy controls

Background

Persons with Chronic Obstructive Pulmonary Disease (COPD), performing some level of regular physical activity, have a lower risk of both COPD-related hospital admissions and mortality. COPD patients of all stages seem to benefit from exercise training programs, thereby improving with respect to both exercise tolerance and symptoms of dyspnea and fatigue. Physical inactivity, which becomes more severe with increasing age, is a point of concern in healthy older adults. COPD might worsen this scenario, but it is unclear to what degree. This literature review aims to present the extent of the impact of COPD on objectively-measured daily physical activity (DPA). The focus is on the extent of the impact that COPD has on duration, intensity, and counts of DPA, as well as whether the severity of the disease has an additional influence on DPA.

Results

A literature review was performed in the databases PubMed [MEDLINE], Picarta, PEDRO, ISI Web of Knowledge and Google scholar. After screening, 11 studies were identified as being relevant for comparison between COPD patients and healthy controls with respect to duration, intensity, and counts of DPA. Four more studies were found to be relevant to address the subject of the influence the severity of the disease may have on DPA. The average percentage of DPA of COPD patients vs. healthy control subjects for duration was 57%, for intensity 75%, and for activity counts 56%. Correlations of DPA and severity of the disease were low and/or not significant.

Conclusions

From the results of this review, it appears that patients with COPD have a significantly reduced duration, intensity, and counts of DPA when compared to healthy control subjects. The intensity of DPA seems to be less affected by COPD than duration and counts. Judging from the results, it seems that severity of COPD is not strongly correlated with level of DPA. Future research should focus in more detail on the relation between COPD and duration, intensity, and counts of DPA, as well as the effect of disease severity on DPA, so that these relations become more understandable.     

GObar: A Gene Ontology based analysis and visualization tool for gene sets

Background  

Microarray experiments, as well as other genomic analyses, often result in large gene sets containing up to several hundred genes. The biological significance of such sets of genes is, usually, not readily apparent.     

Neutral lipids, their fatty acids, and the sterols of the marine ciliated protozoon, Parauronema acutum

The neutral lipids and their fatty acids and the sterol fractions of the marine ciliated protozoon, Parauronema acutum, were characterized. The neutral lipids consisted of triglycerides (30%), sterols (29%), free fatty acids (24%), steryl esters (9%), and diglycerides (8%) and small amounts of fatty alcohols. The fatty acid profiles of these lipids were very similar although quantitative differences were detected. Saturated fatty acids, primarily 14:0, 16:0, and 18:0 constituted 20-30% of the total. Unsaturated fatty acids containing one to three double bonds, primarily 18:1(9), 18:2 (9,12), 18:3 (9, 12, 15) and 20:3 (11, 14, 17), constituted 35-50% of the total. Highly unsaturated fatty acids, 18:4 (6, 9, 12, 15), 20:5 (5, 8, 11, 14, 17) and 22:6 (4, 7, 10, 16, 19), constituted 16-25% of the total. The fatty alcohols consisted of 14:0 (2%), 16:0 (66%), 18:0 (3%), 20:0 (8%), and 22:0 (21%). The sterols of Parauronema acutum consisted of cholesterol (53%), campesterol (32%), desmosterol (7%), and beta-sitosterol (8%).