Isolation and {\it in vitro}-immunosuppressive characterization of a novel cyclosporine metabolite

Cyclosporine (CsA) has shown great benefit to organ transplant recipients, as an immunosuppressive drug. To optimize CsA immunosuppressive therapy, pharmacodynamic evaluation of serial patient serum samples after CsA administration, using mixed lymphocyte culture (MLC) assays, revealed in vitro serum immunosuppressive activity of a CsA-like, ether-extractable component, associated with good clinical outcome in vivo. Since the in vitro immunosuppressive CsA metabolites, M-17 and M-1, are erythrocyte-bound, the immunosuppressive activity demonstrated in patient serum suggests that other immunosuppressive metabolites need exist. To test this hypothesis and obtain CsA metabolites for study, ether-extracted bile from tritiated and nonradioactive CsA-treated pigs was processed by novel high performance liquid and thin-layer chromatography (HPLC and HPTLC) techniques. Initial MLC screening of potential metabolites revealed a component, designated M-E, to have immunosuppressive activity. Pig bile-derived M-E was characterized as a CsA metabolite, by radioactive CsA tracer studies, by 56% crossreactivity in CsA radioimmunoassay, and by mass spectrometric (MS) analysis. MS revealed a CsA ring structure, hydroxylated at a site other than at amino acid one. M-E was different than M-1 and M-17, as demonstrated by different retention properties for each metabolite, using HPTLC and a novel rhodamine B/ $\alpha$-cyclodextrin stain, and using HPLC, performed by Sandoz, that revealed M-E to be different than previously characterized metabolites. The immunosuppressive activity of M-E was quantified by determination of mean metabolite potency ratio in human MLC assays, which was found to be 0.79 $\pm$ 0.23 (CsA, 1.0). Similar to parent drug, M-E revealed inter-individual differences in its immunosuppressive activity. M-E demonstrates inhibition of IL-2 production by concanavalin A stimulated C3H mouse spleen cells, similar to CsA, as determined with an IL-2 dependent mouse cytotoxic T-cell line. ^

Role of FbpA and FbpB in the pathogenesis and mycolyltransferase activity of Mycobacterium tuberculosis

Mycobacterium tuberculosis infects more people worldwide each year than any other single organism. The Antigen 85 Complex, a family of fibronectin-binding proteins (Fbps) found in several species of mycobacteria and possibly involved in host interaction, is considered among the putative virulence factors of M. tuberculosis. These proteins are implicated in the production of trehalose dimycolate (TDM) and arabinogalactan-mycolate (AG-M), two prominent components of the mycobacterium cell wall and potent modulators of the immune system during infection. For these reasons, the principal members of the complex, FbpA and FbpB, were the focus of these studies. The genes encoding these proteins, fbpA and fbpB, were each disrupted by insertion of a kanamycin resistance cassette in a pathogenic strain of M. tuberculosis, H37Rv. Neither mutation affected growth in routine broth culture. Thin layer chromatography analysis of TDM and AG-M showed no difference in content between the parent strain H37Rv and the FbpA- and FbpB-deficient mutants grown under two different culture conditions. However, metabolic radiolabeling of the strains showed that the production of TDM (but not its precursor TMM) was delayed in the FbpA- and FbpB-deficient mutants compared to the parent H37Rv. During this same labeling period, FbpA-deficient mutant LAa1 failed to produce AG-M and in the FpbB-deficient mutant LAb1 production was decreased. In macrophage tissue culture assay, LAa1 failed to multiply when bacteria in early log phase were used to infect monolayers while LAb1 grew like the parent strain. The growth deficiency of LAa1 as well as the deficiencies in TDM and AG-M production were restored by complementing LAa1 with a functional fbpA gene. These results suggest that the FbpA and FbpB proteins are involved in synthesis of TDM (but not its precursor TMM) as well as AG-M. Other members of the complex appear to compensate for defects in synthesis caused by mutation of single genes in the complex over time. Mutation of the FbpA gene causes greater in vivo effect than mutation of the FbpB gene despite very similar deficiencies in the rate of production of mycolate containing molecules on the cell surface. ^

Association of pellicle growth morphological characteristics and clinical presentation of Mycobacterium tuberculosis isolates

Trehalose dimycolate (TDM) is a mycobacterial glycolipid that is released from the surface of virulent M. tuberculosis. We evaluated the rate of growth, colony characteristics and production of TDM by Mycobacterium tuberculosis strains isolated from different clinical sites. Since detergent removes TDM from organisms, we analyzed growth rate and colony morphology of 79 primary clinical isolates grown as pellicles on the surface of detergent free Middlebrook 7H9 media. The genotype of each had been previously characterized. TDM production was measured by thin layer chromatography on 32 of these isolates. We found that strains isolated from pulmonary sites produced large amounts of TDM, grew rapidly as thin spreading pellicles, showed early cording (<1 week) and climbed the sides of the dish. In contrast, the extrapulmonary isolates (lymph node and bone marrow) produced less TDM (p<0.01), grew as discrete patches with little tendency to spread or climb the walls (p<0.02). The Beijing pulmonary (BP) isolates produced more TDM than non Beijing pulmonary isolates. The largest differences were observed in Beijing strains. The Beijing pulmonary isolates produced more TDM and grew faster than the Beijing extrapulmonary isolates (p<0.01). This was true even when the pulmonary and extrapulmonary isolates were derived from the same clade. These growth characteristics were consistently observed only on the first passage after primary isolation. This suggests that the differences in growth rate and TDM production observed reflect differences in gene expression patterns of pulmonary and extrapulmonary infections, that Mycobacterium tuberculosis in the lung grows more rapidly and produces more TDM than it does in extrapulmonary sites. This provides new opportunities to investigate gene expression of Mycobacterium tuberculosis in human.^

Mutagenicity study of the urinary metabolites of 2-aminonaphthalene

The mutagenicity study of the urinary metabolites of 2-aminonaphthalene was conducted to determine whether differences in metabolism between different acetylator phenotypes could account for a proposed mechanism of bladder carcinogenesis. This required the use of fast and slow acetylator rabbits with phenotypic similarities to humans. In the absence of available slow acetylators, it was necessary to inhibit fast acetylators. The proposed mechanism was that slow acetylators were at greater potential risk of bladder carcinogenesis due to low rates of acetylation, a detoxification mechanism for certain aromatic amines. The alternate metabolic pathway will be hydroxylation. The fast acetylators were proposed to exhibit lower risk of bladder carcinogenicity as a result of higher acetylation rates and less mutagenic metabolites.^ This hypothesis was approached by determining from in vitro mutagenicity assays with Salmonella typhimurium strains TA98 and TA100 whether different metabolites were mutagenic. The acetylation rate of each rabbit and a suitable method of acetylation inhibition were determined through oral exposure to dapsone and the acetylation inhibitor, K-p-aminosalicylic acid. Residues of dapsone and its acetylated metabolite were extracted from blood samples and analyzed by ultra-violet spectrometry using standard curves for each metabolite. The urine samples were concentrated on XAD-2 resin and analyzed both as whole urine concentrates and as isolated metabolites from spots on high performance thin layer chromatography plates. The major isolated spots were identified and quantified through extraction and analysis by high performance liquid chromatography when possible.^ Acetylation rate determination and inhibition were successfully demonstrated in rabbits. Significant mutagenicity was noted for several critical metabolites. None of the mutagenic metabolites were detected in higher concentration in the inhibited acetylators and thus, no clear relationship of metabolite concentration to bladder carcinogenesis was evident for the compounds analyzed. There was some evidence that the inhibitor may have affected critical enzyme systems other than acetylation alone. This would account for the lower concentrations of mutagenic hydroxylated compounds observed. ^

Composition of organic matter from samples of massive sulfides, bottom sediments, and seawater collected within the Logachev and Broken Spur hydrothermal fields, Mid-Atlantic Ridge

Lipid components of hydrothermal deposits from the unusual field at 14°45'N MAR and from the typical field at 29°N MAR were studied. For the first time mixed nature of organic matter (OM) from hydrothermal sulfide deposits was established with use of biochemical, gas chromatographic, and molecular methods of studies. In composition of OM lipids of phytoplankton, those of chemosynthesis bacteria and non-biogenic synthesis lipids were determined. Specific conditions of localization of sulfide deposits originated from ''black smokers'' (reducing conditions, absence of free oxygen, presence of reduced sulfur preventing OM from decomposition) let biogenic material, including bacterial one, be preserved in sulfide deposits. The hydrothermal system at 14°45'N MAR is characterized by geological, geochemical and thermodynamic conditions allowing abiogenic synthesis of methane and petroleum hydrocarbons. For sulfide deposits at 29°N and other active hydrothermal fields known at MAR, abiogenic synthesis of hydrocarbons occurs in lower scales.

Organic geochemistry of lower Cretaceous sediments at DSDP Hole 77-535

At Site 535, the four lithologic units of Cretaceous age are controlled by two types of sedimentologic facies: (1) the massive light-colored limestones or marly limestones in which the total organic carbon (TOC) content is low and the organic matter more or less oxidized and (2) laminated dark facies in which the TOC content is higher and associated with a well-preserved organic matter of Type II origin. Very little typical Type III organic matter occurs in the whole series from late Berriasian to Aptian and Cenomanian. Fluctuations from oxidizing to reducing environments of deposition are proposed to account for the variations in properties of the Type II organic matter between the different facies. Dark laminated layers are good but immature potential source rocks: petroleum potential is often higher than 2 kg HC/t of rock.

Phytoplankton biomass, chlorophyll concentration, and variability of lipid content in Calanus euxinus in the Black Sea in June 1991

During field studies relationships between chlorophyll concentrations, phytoplankton biomass (total, individual sizes and species) and level of accumulation of total lipids, wax esters, triacylglycerols, and phospholipids in C. euxinus (copepodites V and females) were studied. These relationships allowed to display not only simple trophic relations between isolated parts of the C. euxinus population and phytoplankton, but also selective role of individual algae species in forming lipid reserves too. Besides it was found that geographical variability of chlorophyll concentration and phytoplankton biomass correlates closely only with those lipid fractions (wax esters and phospholipids) of C. euxinus, which accumulated and kept in a body for a fairly long time. No correlation was found between phytoplankton and for rapidly metabolized triacylglycerols, which have to be utilized within few hours.

Composition of particulate lipids in the intermediate zone between the Kara and Laptev Seas

Chemical group composition of particulate lipids from the intermediate zone between the Kara and Laptev Seas is studied by thin-layer chromatography with flame ionization detection (IATROSCAN TH-10). Hydrocarbons and complex polar lipids similar to those found in the previously studied southeastern area of the Kara Sea are basic components of particulate lipids. High content of triglycerides in the upper layers of the water mass north of the Severnaya Zemlya Islands is a characteristic feature of group composition of particulate lipids. Distribution of triglycerides correlates with localization of the ice cover boundary and complies with process of phytoplankton blooming in the ice edge zone. Distribution of lipid concentration depends on water stratification in the intermediate zone between the Kara and Laptev Seas.

Biometry, and fatty acid and fatty alcohol composition of the Arctic ctenophore Mertensia ovum from Kongsfjorden

Lipids of the Arctic ctenophore Mertensia ovum, collected from Kongsfjorden (Svalbard) in 2001, were analysed to investigate seasonal variability and fate of dietary lipids. Total lipids, lipid classes and fatty acid and alcohol compositions were determined in animals, which were selected according to age-group and season. Changes in lipids of age-group 0 animals were followed during growth from spring to autumn. Total lipids increased from May to September. Lipids as percentage of dry mass were lowest in August indicating their use for reproduction. Higher values occurred in September, which may be due to lipid storage for overwintering. Wax esters were the major lipid class accounting for about 50% of total lipids in age-group 0 animals from July and August. Phospholipids were the second largest lipid fraction with up to 46% in this age-group. The principal fatty acids of M. ovum from all age-groups were 22:6(n-3), 20:5(n-3) and 16:0. Wax ester fatty alcohols were dominated by 22:1(n-11) and 20:1(n-9) followed by moderate proportions of 16:0. The unique feature of M. ovum lipids was the high amount of free fatty alcohols originating probably from the dietary wax esters. In May, free alcohols exhibited the highest mean proportion with 14.6% in age-group 0 animals. We present the first data describing a detailed free fatty alcohol composition in zooplankton. This composition was very different from the alcohol composition of M. ovum wax esters because of the predominance of the long-chain monounsaturated 22:1 (n-11) alcohol accounting for almost 100% of total free alcohols in some samples. The detailed lipid composition clearly reflected feeding of M. ovum on the herbivorous calanoid species, Calanus glacialis and C. finmarchicus, the abundant members of the zooplankton community in Kongsfjorden. Other copepod species or prey items seem to be less important for M. ovum.

Concentrations of particulate organic carbon in the Kara Sea and in the Obskaya Guba (Ob River estuary) in September 1993

Contents and distribution of particulate lipids were studied by thin-layer chromatography technique with flame ionization detection (Iatroscan TH-10) along the transect from the Ob River towards the Kara Sea. Lipid contents range from 18.4 to 266 µg/l with, average 84.97 µg/l, which comprises from 4.06 to 58.32 % of total particulate organic matter. Principal constituents of particulate lipids are hydrocarbons (32.14 % of total lipids on the average), polar compounds (29.85 %), wax and sterol esters (13.04 %), and mono- and diglycerides (12.52 %). Secondary components are presented by fatty acid esters (5.14 %), free fatty acids (4.56 %), triglycerides (2.32 %), and sterols (1.04 %). Specific composition of particulate lipids along the Ob River - Kara Sea transect is formed under strong impact of river run-off. Particulate lipid composition reflects differences between processes of organic matter transformation in estuarine and marine parts of the transect, as well as peculiarities of species composition of Arctic living organisms.