Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates.

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography-tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 µM RDX within 4 h. Nitroso-intermediates hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0-96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.

Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6-trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 µg mL(-1) RDX were transferred each time the RDX concentration decreased to 5 µg mL(-1) or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound.

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding.

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.

Isolation and characterization of the major glycosphingolipids from the liver of the rainbow trout (Oncorhynchus mykiss): identification of an abundant source of 9-O-acetyl GD3.

The carbohydrate structures of the major glycosphingolipids from the liver of the rainbow trout Oncorhynchus mykiss have been examined. We have isolated and identified four major neutral (glucosylceramide, galactosylceramide, lactosylceramide, and globoside) and five acidic (sulfatide, GM3, GM2, GD1a, and 9-O-Acetyl GD3) glycosphingolipids from trout liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, fast atom bombardment mass spectrometry, and specific monoclonal antibodies. Significantly, the relatively scarce ganglioside 9-O-acetyl GD3 was found to comprise approximately 23% of the total ganglioside content of normal rainbow trout liver. 9-O-Acetyl GD3 is, however, abundant in human melanoma and as such, trout liver may be a suitable source of this antigen.

NMR study of the possible interaction in solution of angiotensin II with a peptide encoded by angiotensin II complementary RNA.

The potential binding of angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) (AII) to a peptide encoded by its complementary RNA (Lys-Gly-Val-Asp-Val-Tyr-Ala-Val) (IIA) has been studied by monitoring the 1H NMR spectrum of IIA in aqueous phosphate or Tris.HCl buffer (2H2O) as it is titrated with AII. For molar ratios of AII/IIA ranging from 0.2 to 1.8, the NMR spectra are unchanged as compared to the spectra of the isolated peptides. Based on these findings, the Kd for the putative biomolecular complex of the two peptides under these conditions is calculated to be greater than 10(-4) M. This result does not support the suggestion of Elton et al. [Elton, T. S., Dion, L.D., Bost, K. L., Oparil, S. & Blalock, J. E. (1988) Proc. Natl. Acad. Sci. USA 85, 2518-2522] that AII and IIA engage in high-affinity binding (Kd approximately 5 x 10(-8) M) with each other.

Isolation and characterization of four major neutral glycosphingolipids from the liver of the English sole (Parophrys vetulus). Presence of a novel branched lacto-ganglio-iso-globo hybrid structure.

We have previously reported on carbohydrate structures of the major acidic glycosphingolipids from the liver of the English sole, Parophrys vetulus (Ostrander, G. K., Levery, S. B., Hakomori, S., and Holmes, E. H. (1988) J. Biol. Chem. 263, 3103-3110). We have now isolated four major neutral glycosphingolipids from English sole liver. They have been characterized by 1H nuclear magnetic resonance spectroscopy, methylation analysis, and by fast atom bombardment-mass spectrometry. In addition to neutral glycosphingolipids with known structures (CMH, lactosylceramide, and Gg3), a major polar neutral glycosphingolipid was isolated and characterized as having the following novel structure: (formula; see text) The compound represents a novel hybrid of neolacto-, ganglio-, and iso-globo-series glycosphingolipid structures, a combination not previously encountered. Furthermore, the linkage Fuc alpha 1----3GalNac has not been previously reported for a glycosphingolipid. The relationship of these structural elements to known glycoconjugates is discussed.

Synthesis and characterization of lyso-GM3 (II3Neu5Ac Lactosyl sphingosine), de-N-acetyl-GM3 (II3NeuNH2 lactosyl Cer), and related compounds.

Various GM3 derivatives which are present in A431 cells have different effects on the activity of the EGF receptor kinase. In order to systematically study these effects, the following GM3 derivatives have been synthesized: de-N-acetyl-GM3 (D1), de-N-acetyl-lyso-GM3 (D2), lyso-GM3 (D3), de-N-acetyl-GM3 with N-acetylsphingosine (D4), and GM3 with N-acetylsphingosine (D3). A crucial step for the preparation of D1 is the use of mild alkaline conditions of hydrolysis under which the N-acetyl group of sialic acid is preferentially hydrolyzed. For the preparation of D3, conditions which allowed preferential N-acetylation of the amino group of the neuraminic acid moiety were devised, i.e., D2 was incorporated in a dipalmitoyl-phosphatidylcholine (dpPC) liposome in which the sphingosine moiety was protected and the amino group of neuraminic acid was N-acetylated with acetate and a water-soluble catalyst, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (DEC). When an aqueous micellar solution of D2 was treated with acetic anhydride and sodium hydrogencarbonate, N-acetylation occurred at the amino groups of both neuraminosyl and sphingosyl residues, yielding D5. The structures of these derivatives were verified by 1H-n.m.r. spectroscopy and mass spectrometry.

2D NMR studies of aminoglycoside antibiotics. Use of relayed coherence transfer for 1H resonance assignment and in situ structure elucidation of amikacin derivatives in reaction mixtures.

Phase-sensitive 2D 1H/1H COSY spectra can be used to identify the structures of individual pure specimens of the aminoglycoside antibiotic amikacin and its N-hemisuccinyl derivatives. However, even at 500 MHz the 2D chemical shift dispersion does not allow for unambiguous assignment of all cross-peaks. By use of 2D relayed coherence transfer experiments (RELAY) optimized to detect two-step 1H/1H scalar interactions in which one of the J-values is small, sufficient additional correlations can be obtained from the frequency-isolated resonances to allow facile tracing of all scalar connectivities. Complete assignments of the 1H NMR spectra of amikacin, its 6'-N-hemisuccinamide, and a novel bis(acylate) [gamma-N-(p-vinylbenzoyl)amikacin 6'-N-hemisuccinamide] were obtained for aqueous media. The NMR spectrum of amikacin free base was also assigned in dimethyl sulfoxide solution. The RELAY experiment can be extended to the analysis of reaction mixtures, which allows for the identification and resonance assignment of regioisomeric amikacin haptens in the mixture state. All of the N-monohemisuccinyl isomers of amikacin have been identified in reaction mixtures through the RELAY experiment. The relative reactivities of the amino functions of amikacin toward acylating agents were found to be 6'-N greater than 3-N equal to or greater than 3"-N equal to or greater than gamma-N. However, this reactivity order is altered after the initial acylation event.

Tropical intussusception in adults.

In contrast with the incidence in Britain and the United States, about 50% of intussusceptions in Sri Lanka occur in adults, the maximum age incidence being in the fourth decade. Seventy-six cases of intussusception in adults are analysed in this paper, 62 of which were of the caecocolic type, which though only rarely described in the West, have also been frequently reported from other tropical countries. The clinical picture was characteristic, and a mass was palpable in 90% of the patients, facilitating diagnosis without ancillary investigations. On the basis of the histological examination of resected specimens it is concluded that amoebic granulomatous formation in the dependent "diverticulum" of the caecum is the predisposing cause of the caecocolic intussusception, accounting for the chronicity of a large number of cases. Irrespective of the duration of the illness, gangrene did not occur in any of the cases if this type, although resection was occasionally required on account of irreducibility. In view of the proliferation of fibrous tissue in the wall of the caecum, complete evagination of the intussusception can only be achieved by surgical exploration and manipulation, the results of which are excellent.