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.     

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%).     

Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture

Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O₂ (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm² flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O₂ in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O₂, respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O₂ and 500 μl (1,480-μm depth) at 5% O₂ in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.     

Detection of two distinct transporter systems for 2-deoxyglucose uptake by the opportunistic pathogen Pneumocystis carinii.

Since the opportunistic pathogen Pneumocystis carinii grows only slowly in vitro, the mechanism of glucose uptake was investigated to better understand how the organism transports nutrients. Using the non-metabolizable analogue 2-deoxyglucose, two uptake systems were detected with Q(10) values of 2.12 and 2.09, respectively. One had a high affinity (K(m)=67.5 microM) and the other a low affinity (K(m)=5.99 mM) for 2-deoxyglucose uptake. Glucose or deoxyglucose phosphate products from transported radiolabeled substrates were not detected during the incubation times used in this study. Both systems were inhibited by mannose, galactose, fructose, galactosamine, glucosamine, and glucose but not by allose, 5-thioglucose, xylose, glucose 6-phosphate and glucuronic acid. Salicylhydroxamate, KCN, iodoacetate, and 2,4-dinitrophenol inhibited the high-affinity transporter, suggesting it required ATP. Ouabain, monensin, carbonyl cyanide m-chlorophenylhydrazone, and N,N'-dicyclohexylcarbodiimide also inhibited deoxyglucose uptake, as did the replacement of Na(+) in the incubation medium with choline, indicating requirements for Na(+) and H(+). The high-affinity system was also inhibited by the protein synthesis inhibitors cycloheximide and chloramphenicol. In contrast, the low-affinity system transported deoxyglucose by facilitated diffusion mechanisms. Unlike the human erythrocyte glucose transporter GLUT1, the P. carinii transporters recognized fructose and galactose and were relatively insensitive to cytochalasin B, suggesting that the P. carinii glucose transporters may be good drug targets.     

A seroepidemiologic survey of human T-cell lymphotropic virus type I in two Hawaiian hematologic-oncologic practices.

A seroepidemiologic survey for antibodies to the human T-cell lymphotropic virus type I (HTLV-I) was carried out in two Hawaiian hematologic-oncologic practices. Specimens of serum or plasma from 215 donors were assayed using the ELISA technique, followed by the Western blot technique to confirm antibody specificity to HTLV-I. Of 214 seropositive donors, 16 (7.5%) were positive. Of 172 donors of Japanese ancestry, 16 (9.3%) were seropositive; none were white, Chinese, Filipino, or Pacific Islander. One donor contracted the virus through blood transfusions. The results suggest that HTLV-I was introduced to Hawaii with the Japanese immigration.     

Fast Technology Analysis Enables Identification of Species and Genotypes of Latent Microsporidia Infections in Healthy Native Cameroonians

Several enteric microsporidia species have been detected in humans and other vertebrates and their identifications at the genotype level are currently being elucidated. As advanced methods, reagents, and disposal kits for detecting and identifying pathogens become commercially available, it is important to test them in settings other than in laboratories with “state‐of‐the‐art” equipment and well‐trained staff members. In the present study, we sought to detect microsporidia DNA preserved and extracted from FTA (fast technology analysis) cards spotted with human fecal suspensions obtained from Cameroonian volunteers living in the capital city of Yaoundé to preclude the need for employing spore‐concentrating protocols. Further, we tested whether amplicon nucleotide sequencing approaches could be used on small aliquots taken from the cards to elucidate the diversity of microsporidia species and strains infecting native residents. Of 196 samples analyzed, 12 (6.1%) were positive for microsporidia DNA; Enterocytozoon bieneusi (Type IV and KIN‐1), Encephalitozoon cuniculi, and Encephalitozoon intestinalis were identified. These data demonstrate the utility of the FTA cards in identifying genotypes of microsporidia DNA in human fecal samples that may be applied to field testing for prevalence studies.