The characterization of two biliary glutathione conjugates of amsacrine using liquid secondary ion mass spectrometry.

An additional biliary glutathione (GSH) conjugate of the anilinoacridine anti-tumour agent amsacrine (4'-(9-acridinylamino)methanesulphon-m-anisidide, NSC 249992) has been identified in bile collected from male Wistar rats by cannulation of the common bile duct and from male BDF1 mice by removal of the gall bladder after treatment with amsacrine. The presence of this conjugate, at the 6'-position of the anilino ring, has been confirmed by liquid secondary ion (LSI) mass spectrometric analysis of selected biliary metabolites separated by high-performance liquid chromatography. The two major metabolites each gave a daughter ion spectrum which was diagnostic for either 5'- or 6'-GSH conjugation. This pattern was confirmed by comparison with LSI mass spectral data obtained from authentic chemical standards formed on reaction of the quinone diimine derivative of amsacrine with methanethiol or mercaptoethanol.

Host recognition and the study of a chemical basis for attraction by cuckoo bumble bees (Hymenoptera: Apidae).

Species ofPsithyrus (Hymenoptera; Apidae) are obligate bumble bee social parasites. In this study, females ofP. vestalis andP. ashtoni were presented with pentane extracts prepared from different body parts of queens of their respective host species,Bombus terrestris andB. terricola. Parasites of both species were capable of distinguishing host bees from other bumble bee species using chemical cues contained within extracts. Among extracts of several body parts presented to parasites, the abdomen produced the greatest behavioral response, with Dufour's gland and terminal tergal segments eliciting the greatest response among abdominal regions. Extracts of these two body parts obtained fromB. terrestris queens shared a number of compounds, identified by GC-MS. Among the identified compounds are a number that have been reported to be of importance in bee sociochemistry.

Differences in the metabolism of the antitumour agents amsacrine and its derivative CI-921 in rat and mouse.

1. Rats and mice were treated with the antitumour agent CI-921 (I), and parent compound amsacrine, with all biliary metabolites being analysed. 2. In both rat and mouse the major biliary metabolites of amsacrine are the 5'- and 6'-glutathione (GSH) conjugates, with no C9-GSH conjugate being detected. 3. 5'- and 6'-GSH conjugates of I are also formed in both species. However, two additional products were detected and their structures confirmed by liquid secondary ion mass spectrometry and 1H-n.m.r. spectrometry, and by comparison with synthetic standards. 4. Additional metabolites of I are the C9-GSH conjugate and the 4-hydroxymethyl derivative which, either as the aglycone or glucuronide, is the predominant product in rat bile (comprising 56% of the dose eliminated over 3.5 h). 5. Relative amounts of the C9-GSH conjugate to the 5'- and 6'-GSH conjugates to the 4-hydroxymethyl derivatives, were 24:65:10 in mouse bile and 2:8:90 in rat bile. 6. These differences indicate first, likely enzyme involvement in the formation of the C9-GSH conjugate of I, and second, in comparison with amsacrine, alternative pathways which may decrease formation of the reactive quinone diimine intermediate of I and consequent hepatotoxicity.

Cytosol mediated metabolism of the experimental antitumor agent acridine carboxamide to the 9-acridone derivative.

The acridine antitumor agent N-[2'-(dimethylamino)ethyl]acridine-4-carboxamide (AC; NSC 601316; acridine carboxamide) is oxidized efficiently in vitro by rat and mouse hepatic cytosolic fractions. Under these conditions the oxidase activity has an apparent Km of 11 microM towards AC. A single product is formed which has been identified as the corresponding 9(10H)-acridone carboxamide by 1H-NMR and mass spectrometry. Inhibition with menadione and amsacrine, but not allopurinol, indicates that this reaction is most likely to be catalysed by aldehyde oxidase (EC 1.2.3.1). Several AC analogues with modifications to the side chain (the N-oxide, N-monomethyl-, and amino-derivatives) are also metabolized to the equivalent acridone product but the 7-hydroxylated and 4-carboxylic acid acridine derivatives are not.

Bovine ceroid-lipofuscinosis (Batten's disease): the major component stored is the DCCD-reactive proteolipid, subunit C, of mitochondrial ATP synthase.

The ceroid-lipofuscinoses (Batten's disease) are a group of recessively inherited lysosomal storage diseases of children and animals in which there is intracellular accumulation of a fluorescent lipopigment in a wide variety of cells. Lipopigment bodies isolated from pancreas, liver, kidney and brain tissue from a heifer affected with ceroid-lipofuscinosis contained between 55 and 62% protein. A dominant component comigrated on LDS-PAGE with the major low molecular weight protein stored in ovine ceroid-lipofuscinosis. It was identified by amino acid sequence and mass spectroscopy as the full subunit c of mitochondrial ATP synthase, normally found only in the inner mitochondrial membrane, where it is estimated to account for 2-4% of the membrane protein. In pancreatic lipopigment it accounted for at least 40% of the total lipopigment mass and this storage was considered specific to the disease. No other mitochondrial proteins were found in storage bodies. These results are similar to those found in studies on the ovine and the late infantile and juvenile human forms of the disease. It is concluded that bovine ceroid-lipofuscinosis is also a proteolipid proteinosis in which subunit c of mitochondrial ATP synthase is specifically stored in lysosome derived organelles.

Functional antithrombin-III variant (41 Pro----Leu) identified by liquid secondary ion mass spectrometry.

A genetic variant of antithrombin with impaired heparin cofactor activity was identified in 4 members of a French family. Both the variant and normal antithrombin component were purified by affinity chromatography on heparin Sepharose. Reverse phase peptide mapping revealed a single altered peak when tryptic digests of both antithrombins were compared. After further purification of the aberrant peptide, amino acid analysis indicated a substitution of 41 Leu----Pro (antithrombin Basel). This result was confirmed by liquid secondary ion mass spectrometry which gave a measured mass of 816.4655 Da for the new peptide compared to a calculated mass of 800.3579 Da for the normal peptide and 816.4579 for the Leu----Pro substitution.

The sequence of the major protein stored in ovine ceroid lipofuscinosis is identical with that of the dicyclohexylcarbodiimide-reactive proteolipid of mitochondrial ATP synthase.

The ceroid lipofuscinoses are a group of neurodegenerative lysosomal storage diseases of children and animals that are recessively inherited. In diseased individuals fluorescent storage bodies accumulate in a wide variety of cells, including neurons. Previous studies of these bodies isolated from tissues of affected sheep confirmed that the storage occurs in lysosomes, and showed that the storage body is mostly made of a single protein with an apparent molecular mass of 3500 Da with an N-terminal amino acid sequence that is the same as residues 1-40 of the c-subunit (or dicyclohexylcarbodi-imide-reactive proteolipid) of mitochondrial ATP synthase. In the present work we have shown by direct analysis that the stored protein is identical in sequence with the entire c-subunit of mitochondrial ATP synthase, a very hydrophobic protein of 75 amino acid residues. As far as can be detected by the Edman degradation, the stored protein appears not to have been subject to any post-translational modification other than the correct removal of the mitochondrial import sequences that have been shown in other experiments to be present at the N-terminal of its two different precursors. No other protein accumulates in the storage bodies to any significant extent. Taken with studies of the cDNAs for the c-subunit in normal and diseased sheep, these results indicate that the material that is stored in lysosomes of diseased animals has probably entered mitochondria and has been subjected to the proteolytic processing that is associated with mitochondrial import. This implies that the defect that leads to the lysosomal accumulation concerns the degradative pathway of the c-subunit of ATP synthase. An alternative, but less likely, hypothesis is that for some unknown reason the precursors of subunit c are being directly mis-targeted to lysosomes, where they become processed to yield a protein identical with the protein that is normally found in the mitochondrial ATP synthase assembly, and which then accumulates.

Molecular characterization of antithrombin Barcelona-2: 47 arginine to cysteine.

The molecular characterization of antithrombin Barcelona-2 is reported. The abnormal antithrombin was isolated from plasma by chromatography on heparin-Sepharose at pH 6.0, and ion exchange on DEAE-Sephadex at pH 8.6 and 6.0. The tryptic peptides were mapped by reverse-phase HPLC and amino acid sequencing and mass spectrometry showed arginine-47 to be replaced by cysteine. The affinity of Barcelona-2 for heparin is dramatically decreased. The new cysteine does not form a mixed disulphide with DTNB, implying it is present as a disulphide with some other available thiol molecule such as cysteine. This extra bulk at position 47 accounts for the low heparin affinity compared with two other mutations (Rouen-1 47 His; Rouen-2 47 Ser) at this residue. These results confirm the view that Arg-47 is an important residue in heparin binding. No dimers of Barcelona-2 were observed suggesting that steric hindrance of the new cysteine at residue 47 prevents dimerisation.

Successful autografting in chronic myeloid leukaemia after maintenance of marrow in culture.

We have previously shown that Philadelphia chromosome (Ph1)-positive cells rapidly disappear when marrow from patients with chronic myeloid leukaemia (CML) is cultured under conditions that maintain normal haematopoiesis for many weeks. The ability of marrow maintained in culture for 10 days to serve as an autograft has now been tested in three patients treated with intensive chemoradiotherapy. Two weeks after transplantation, marrow samples from all patients showed trilineage haematopoiesis. Neutrophil counts greater than 1.0 x 10(9)/l were achieved in all patients within 4 weeks, and platelet counts greater than 20 x 10(9)/l were achieved in two patients within 5 weeks. During this period of haematopoietic recovery, marrow cells were exclusively Ph1-negative in two patients and predominantly so in the third. These results suggest that engraftment can occur from Ph1-negative haematopoietic stem cells selected by maintenance of autologous CML marrow in culture for 10 days. Thus, the feasibility of using this approach to allow intensive and potentially curative therapy for CML has been established.

Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules.

Two nodule-specific Rhizobium loti compounds were identified in Lotus tenuis and Lotus pedunculatus nodules induced by strain NZP2037. One, a silver nitrate-positive cation called rhizolotine, has been characterized as the riboside of a novel alpha-hydroxyimino acid containing a 1,4,5,6-tetrahydropyrimidine ring (G. J. Shaw, R. D. Wilson, G. A. Lane, L. D. Kennedy, D. B. Scott, and G. J. Gainsford, J. Chem. Soc. Chem. Commun., p. 180-181, 1986), and the other, yellow-1, stains yellow with ninhydrin. Both compounds were degraded by R. loti NZP2037 but not by strains of Rhizobium meliloti, Rhizobium trifolii, or Agrobacterium tumefaciens. Under the conditions tested neither compound was able to serve as a sole source of C or N for growth of R. loti NZP2037. Rhizolotine and yellow-1 were found in nodules from a range of different legumes inoculated with NZP2037, suggesting that the Rhizobium and not the host plant determines their synthesis. Neither compound was found in nodulelike structures of L. pedunculatus induced by transposon Tn5-induced noninfectious (Inf-) mutants of NZP2037 or in similar structures induced by a transconjugant of NZP2037 containing the symbiotic (Sym) cointegrate plasmid pPN1 of R. trifolii. Both compounds were also absent in the ineffective nodules induced by the bacterial-release-negative (Bar-) mutant, strain PN239. However, both compounds were present in nodules induced by the fixation-negative (Fix-) mutant PN235 and in Fix+ nodules formed by a plasmid-cured derivative of NZP2037. These results would suggest that infection and bacterial release from the infection thread are necessary for nodule (symbiotic) synthesis of these compounds.

The monounsaturated acyl- and alkyl- moieties of wax esters and their distribution in commercial orange roughy (Hoplostethus atlanticus) oil.

Wax esters were isolated from commercial orange roughy (Hoplostethus atlanticus) oil by column chromatography and fractionated by argentation thin layer chromatography. Following transesterification, the resultant fatty acid methyl esters and fatty alcohols were analyzed by gas chromatography. Both acyl- and alkyl-moieties were mainly of the monoene structure within the 16:1-22:1 range. After derivatization, the positions of the double bonds of even numbered fatty acid and fatty alcohol isomers were located by chromatography-mass spectrometry and compared. Results of these positional analyses indicate that the primary desaturation reactions takes place in the delta 9 position of pre-existing (C14 to C24) acyl chains. It is proposed that acyl components from 18:1 are subjected to chain elongation to form a mixture of 24:1 isomers as the final product. Apart from the 24:1 acyl moiety of the wax esters, in which the double bond was almost exclusively in the delta 15 position, de novo biosynthetic reactions on acids and alcohols appear to yield related acyl- and alkyl-moieties of resynthesized wax esters.

Anthophora Bees: Unusual Glycerides from Maternal Dufour's Glands Serve as Larval Food and Cell Lining.

The Dufour's gland of Anthophora abrupta, a solitary bee, secretes a complex mixture of liquid triglycerides containing one long-chain and two shortchain fatty acids. This is applied inside the earthen brood cells and added to the provision, where it is converted, perhaps by enzymes from the bee's saliva or gut, to solid diglycerides that are later eaten by the bee larvae. This use of Dufour's gland secretion as food and its nutritive function are reminiscent of the royal jelly secreted by honey bees.

Mass spectra of some specifically deuterated tryptamines.

The mass spectra of the four tryptamine derivatives, N-acetyl-5-methoxytryptamine (melatonin), N-acetyl-5-hydroxytryptamine (N-acetyl-serotonin), N,N-dimethyl-5-hydroxtryptamine (bufotenine) and N,N-dimethyl-5-methoxytryptamine (O-methylbufotenine), with specifically labeled [D4] aminoethyl sidechains have been measured. Comparison of these spectra with those of the unlabeled compounds enable the major fragmentations of the compounds to be defined.

The synthesis of alpha, alpha, beta, beta-d4-serotonin.

Alpha, alpha, beta, beta-d4-Serotonin (94% d4, 6% d3) has been synthesized for use as an internal standard in mass spectrometric determinations of serotonin in biological systems.