Characterization of a solid state reaction product from a lyophilized formulation of a cyclic heptapeptide. A novel example of an excipient-induced oxidation.

PURPOSE: To elucidate the structure of a degradation product arising from a lyophilized formulation of a cyclic heptapeptide, and to provide a mechanism to account for its formation. METHODS: Preparative HPLC was used to isolate the degradate in quantities sufficient for structural studies. A structure assignment was made on the basis of the compounds spectroscopic properties (UV, MS, NMR) and the results of amino acid analysis. RESULTS: The degradate was identified as a benzaldehyde derivative arising from the oxidative deamination of an aminomethyl phenylalanine moiety. The extent of formation of this product is influenced by the amount of mannitol used as an excipient in the formulation. A mechanism is proposed whereby reducing sugar impurities in mannitol act as an oxidizing agent via the intermediacy of Schiff base adducts which subsequently undergo tautomerization and hydrolysis. CONCLUSIONS: Reducing sugar impurities in mannitol are responsible for the oxidative degradation of the peptide via a mechanism that involves Schiff base intermediates. This mechanism may be a potential route of degradation of other arylmethyl amines in mannitol-based formulations.

Gas chromatographic/mass spectrometric analysis of methyl methanesulphonate and ethyl methanesulphonate in the bismesylate salt of DPI 201-106, a positive inotropic agent for the treatment of heart failure.

A gas chromatographic/mass spectrometric procedure using single-ion monitoring and repetitive scanning was developed to characterize and determine methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS) in the free base and bismesylate salt of DPI 201-106, a positive inotropic agent used in the treatment of heart failure. Mass spectral fragmentations, leading to product ions, are rationalized and mechanisms of potential rearrangement pathways are described. The apparent levels of MMS and EMS, as measured against the internal standard n-propyl methanesulphonate, were found to be 0.51 and 1.31 micrograms per gram of bismesylate salt, respectively. The presence of these alkylating agents in the free base was not observed.

Field and genetic studies testing optimal outcrossing in Agave schottii, a long-lived clonal plant.

In thisstudy we combine field experiments, designed to test the predictions of optimal outcrossing theory in Agave schottii, with molecular genetic studies, using RAPD (random amplified polymorphic DNA), polymerase chain reaction to assess the underlying genetic hypothesis of optimal outcrossing theory. Initially, 48 "females" of A. schottii were hand-pollinated with pollen collected from 1 m, 10 m, 100 m, and 2500 m distances. Each female received all four distance treatments. Additionally, a subset of the focal females and their pollen donors were used in an analysis of genetic similarity across the four distances. Results of hand-pollinations showed that crosses of 1 m had significantly lower seed set than 10 m and 100 m crosses. Crosses of 2500 m had intermediate seed set. Combined relative fitness was significantly lower for 1 m crosses compared to 10 m crosses, while 100 m and 2500 m crosses were intermediate. Thus, A. schottii experiences inbreeding depression and a trend toward outbreeding depression. Genetic analyses showed a similar pattern: individuals 1 m apart had on average higher genetic similarity (proportion of bands shared) than individuals separated by greater distances, with a trend toward lower genetic similarity for plants located 2500 m distant. The observed spatial genetic patterns are likely maintained by the combined effects of clonal reproduction, clone longevity, limited seed dispersal and the substantial number of inbred progeny produced, counteracting distant allele transfer which tends to reduce population genetic structure. The correspondence between our ecological and genetic results indicates that RAPD markers are useful tools for assessing ecological phenomena.

Hepatic metabolism of spironolactone. Production of 3-hydroxy-thiomethyl metabolites.

Spironolactone (SP) is used clinically as a renal aldosterone antagonist and as an antiandrogen. It is known that the drug is extensively metabolized and that metabolites mediate its therapeutic actions, but hepatic metabolism of SP has not been comprehensively investigated. Hepatic disposition may also be important in the toxicity of SP, because the parent compound prevents the hepatocarcinogenic effects of its metabolite, canrenone (CAN). Using a combination of in vivo and in vitro approaches, we studied the metabolism of SP by guinea pig livers. The major compounds detected in livers in vivo following SP treatment were the known metabolites, 7 alpha-thiomethyl-spirolactone (TM) and CAN, and a previously uncharacterized compound whose mass spectral and UV absorption characteristics suggested that it was an A-ring-reduced derivative of TM. In vitro incubation of liver homogenates with SP also resulted in the formation of the unknown metabolite. A combination of MS and NMR spectroscopy was used to identify unequivocally the unknown metabolites as 3 alpha-hydroxy-TM. Another metabolite produced in vitro was identified as 3 beta-hydroxy-TM. It is possible that these two new metabolites of SP contribute to the pharmacological actions of the drug. In addition, production of 3 alpha-hydroxy-TM suggests a mechanism to account for the prevention of CAN-induced carcinogenicity by SP. TM may block the conversion of CAN to mutagenic 3-hydroxy-CAN metabolites by serving as a competitive substrate for hepatic 3-keto reductases.

Identification of spironolactone metabolites in plasma and target organs of guinea pigs.

Spironolactone (SL) is a renal aldosterone antagonist that is used clinically in the treatment of hypertension and congestive heart failure. Among the side effects of the drug are degradation of cytochrome P-450 and inhibition of steroidogenesis in the testes. It has long been recognized that the effects of SL are mediated by metabolites of the drug, but questions remain about the identities of the active metabolites. Because tissue metabolites of SL had not previously been investigated, experiments were done to determine the identities of metabolites in target organs after SL administration to guinea pigs. Metabolites were identified by HPLC and MS. The major plasma metabolite was 7 alpha-thiomethyl-SL (TM) with smaller amounts of canrenone (CAN) and 7 alpha-thio-SL (TH) also present. In kidneys, TM also was the principal metabolite, but CAN was the only other compound consistently found. By contrast, in testes, substantial amounts of SL and TH were present in addition to TM and CAN. It is possible that local metabolism of SL contributes to the differences in metabolite profiles between plasma and target organs. Data also suggest that TM is principally responsible for the renal antimineralocorticoid effects of SL and support the purported role of TH in the degradation of testicular cytochrome P-450.

Pulsed field gel electrophoresis reveals chromosome length differences between strains of Cladosporium fulvum (syn. Fulvia fulva).

Methods are described for the electrophoretic separation of chromosome-sized DNA molecules from the fungal tomato pathogen Cladosporium fulvum (syn. Fulvia fulva). Using a hexagonal electrode array and switching times of 75 min at 45 V for 14 days, nine bands could be resolved. By comparison with co-electrophoresed Aspergillus nidulans chromosomal DNA (which was resolved into seven bands), the sizes of the C. fulvum bands are estimated to be between 1.9 Mb and 5.4 Mb. The two largest bands are believed to be doublets, giving a minimum genome size of 44 Mb. Cloned probes for the ribosomal DNA repeat, an anonymous single copy fragment and a newly discovered retrotransposon were hybridized to blots of the pulsed field gels, demonstrating the use of this technique for genomic mapping. Most strains of C. fulvum had an identical pattern of bands. Two strains exhibited two polymorphisms which could be due to a translocation.

An evolutionary relationship between the ColE5-099 and the ColE9-J plasmids revealed by nucleotide sequencing.

The nucleotide sequence of a 1124 bp fragment of the ColE5-099 plasmid which encodes colicin E5 immunity, a lys gene involved in colicin release from the host cell, and the 3' end of the colicin E5 structural gene has been determined. Open reading frames corresponding to the three genes have been located by analogy with similar sequences from other E colicin plasmids. The location of these open reading frames corresponds with the position of the genes as determined by subcloning and transposon mutagenesis. The amino acid sequence of the carboxy-terminal 107 amino acid residues of the colicin E5 gene shows no homology with any other E colicin, suggesting a different mode of action in killing sensitive cells. A comparison of the nucleotide sequence of this region of the ColE5-099 plasmid with that of the equivalent region of the ColE9-J plasmid suggests a close evolutionary relationship between these two plasmids.

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Electron-paramagnetic-resonance studies on the molybdenum centre.

Formate dehydrogenase from Pseudomonas aeruginosa contains molybdenum, a [4Fe-4S] cluster and cytochrome b. This paper reports the detection of molybdenum as Mo(V) by e.p.r. spectroscopy. In order to generate Mo(V) signals, addition of amounts of excess formate varying between 10- and 50-fold over enzyme, followed by 200-fold excess of sodium dithionite, were used. Two Mo(V) species were observed. One, the major component, has g1 = 2.012, g2 = 1.985 and g3 = 1.968, appeared at low concentrations of formate and increased linearly in intensity with increasing concentrations of formate up to 25-fold excess over the enzyme. At higher formate concentration this signal disappeared. The appearance and disappearance of this Mo(V) signal seems to parallel the state of reduction of the [4Fe-4S] clusters. A second, minor, Mo(V) species with g-values g1 = 1.996, g2 = 1.981 and g3 = 1.941 appears at a constant level during the formate-dithionite titration. No evidence has been obtained for nuclear hyperfine coupling to protons. The major Mo(V) species has unusual e.p.r. signals compared with other molybdenum-containing enzymes, except for that observed in the formate dehydrogenase from Methanobacterium formicicum [Barber, Siegel, Schauer, May & Ferry (1983) J. Biol. Chem. 258, 10839-10845]. The present work suggests that the enzyme is acting as a CO2 reductase, with dithionite as an electron donor to a [4Fe-4S] cluster, which in turn donates electrons to molybdenum, producing a Mo(V) species with CO2 bound to the metal.

Electron-paramagnetic-resonance and magnetic-circular-dichroism studies on the formate dehydrogenase-nitrate reductase particle from Pseudomonas aeruginosa.

The membrane-bound respiratory particle complex of Pseudomonas aeruginosa, which reduces nitrate to nitrite using formate as the electron donor, was prepared and characterized by e.p.r. and low-temperature magnetic c.d. (m.c.d.) spectroscopy. The particle complex has two enzymic components, namely nitrate reductase (NiR) and formate dehydrogenase (FDH), which are multi-centred proteins containing molybdenum, iron-sulphur clusters and cytochrome. By using results from work on the purified extracted enzymes NiR and FDH to aid in the assignment, it has been possible to observe spectroscopically all the components of the electron-transfer chain in the intact particle. This led to a proposal for the organization of the metal components of the FDH-NiR chain. Molybdenum ions are at opposite ends of the chain and interact with, respectively, the formate-CO2 couple and the nitrate-nitrite couple. The molybdenum ion at the low-potential end of the chain passes electrons to cytochrome b of FDH, a bishistidine-co-ordinated haem with unusual steric restraint at the iron. The next component is a [4Fe-4S] cluster. This comprises all the components of FDH. Electrons are passed to the molybdenum of NiR via a number, probably two, of [4Fe-4S] clusters. No evidence has been found in this work for the presence of a quinone to mediate electron transfer between FDH and NiR. Cytochrome c appears to be able to feed electrons into the chain at the level of one of the [4Fe-4S] centres of NiR.

Purification and properties of formate dehydrogenase from Pseudomonas aeruginosa. Characterization of haem and iron-sulphur centres by magnetic-circular-dichroism and electron-paramagnetic-resonance spectroscopy.

The purification of formate dehydrogenase (FDH) from Pseudomonas aeruginosa after anaerobic growth on nitrate-containing medium was carried out. The separation of the FDH enzyme from nitrate reductase (NiR), which are found together in a particle fraction and constitute the short respiratory chain of this bacterium, has been followed by optical, magnetic c.d. (m.c.d.) and e.p.r. spectroscopy. These techniques have allowed the haem, iron-sulphur clusters and molybdenum components to be detected and, in part, their nature to be determined. Attempts to extract FDH anaerobically in the absence of sodium dithionite led to loss of activity. Addition of sodium dithionite maintained the activity of the enzyme, even after subsequent exposure to air, in an assay involving formate reduction with Nitro Blue Tetrazolium as reductant. Three preparations of FDH have been examined spectroscopically. The preparations vary in the amount of contaminating nitrate reductase, the amount of cytochrome c present and the concentration of oxidized [3Fe-4S] cluster. Optical spectra and low-temperature m.c.d. spectroscopy show the loss of a cytochrome-containing protohaem IX co-ordinated by methionine and histidine as NiR is separated from the preparation. In its purest state FDH contains one molecule of cytochrome co-ordinated by two histidine ligands in the oxidized state. This cytochrome has an e.p.r. spectrum with gz = 3.77, the band having the unusual ramp shape characteristic of highly anisotropic low-spin ferric haem. It also shows a charge-transfer band of high intensity in the m.c.d. spectrum at 1545 nm. It has recently been shown [Gadsby & Thomson (1986) FEBS Lett. 197, 253-257] that these spectroscopic properties are diagnostic of a bishistidine co-ordinated haem with steric constraint of the axial ligands. The e.p.r. and m.c.d. spectra of the reduced state of FDH reveal the presence of an iron-sulphur cluster of the [4Fe-4S]+ type. The g-values are 2.044, 1.943 and 1.903. An iron-sulphur cluster of the class [3Fe-4S], detected by e.p.r. spectroscopy in the oxidized state and by low-temperature m.c.d. spectroscopy in the reduced state, is purified away with the NiR. Finally, an e.p.r. signal at g = 2.0 with a narrow bandwidth which persists to 80 K is observed in the purest preparation of FDH. This may arise from an organic radical species.

A new mutation for multiple drug resistance and modified plasma membrane ATPase activity in Schizosaccharomyces pombe.

The mutant JV66 was selected from the wild type strain of S. pombe 972h- ade7-413 by its ability to grow on solid rich medium containing 200 micrograms Dio-9/ml. The single nuclear mutation, designated pma1 gives resistance towards diguanidines and several other positively charged compounds. The pma1 mutation also decreases plasma membrane ATPase activity and confers resistance of ATPase to vanadate. The pma1 locus is localized on chromosome I at 5.3 map units from cyh1-C7 and at about 20.7 map units from the centromere. This new mutation is genetically and phenotypically different from the mutation cyh3 and cyh4 previously described (Johnston and Coddington 1983).

Acid phosphatase deficient mutants of Schizosaccharomyces pombe are defective in tyrosine uptake.

The uptake of tyrosine and arginine into wild type and acid phosphatase deficient mutants (pho 1) of Schizosaccharomyces pombe was investigated. All 11 pho 1-alleles tested exhibited a reduced tyrosine uptake and impaired uptake cosegregated with the lack of acid phosphatase activity. Kinetic analyses using wild type cells grown in high phosphate medium (acid phosphatase repressed) and low phosphate medium (acid phosphatase derepressed) showed staturation kinetics for tyrosine with a KM of about 2 x 10(-4) M for both media and a V of about 5 nmol min-1 mg-1 and 2 nmol min-1 mg-1 for derepressed and repressed cells respectively. The pho 1-118 strain completely lacked this saturable uptake system for tyrosine. Preliminary evidence suggests that tyrosine uptake may be via a general amino acid permease system and we conclude that mutations in the structural gene of acid phosphatase which abolish enzyme activity lead to a loss of this uptake system. In contrast to tyrosine, arginine uptake seems not to be significantly affected either by different acid phosphatase levels in wild type cells or by the pho 1-118 mutation.

Drug resistance in the fission yeast Schizosaccharomyces pombe: pleiotropic mutations affecting the oleic acid and sterol composition of cell membranes.

The whole cell lipid and sterol content of the drug resistant strains cyh1, cyh3 and cyh4 was compared with that of wild type by thin layer and gas liquid chromatography and by UV spectrophotometric analysis. The cyh3 and cyh4 strains had a decreased content of the unsaturated 18:1 fatty acid oleic acid, a decreased content of ergosterol and an increased content of 24,28 dehydroergosterol with respect to wild type. The cyh1 strain, however, only showed a decreased content of ergosterol and an increased content of 24,28 dehydro-ergosterol when compared to wild type.

Multiple drug resistance in the fission yeast Schizosaccharomyces pombe: Correlation between drug and amino acid uptake and membrane ATPase activities.

Cyh3 and cyh4, multiple drug resistant strains of Schizosaccharomyces pombe, show a much reduced uptake of trichodermin, chloramphenicol, cycloheximide, L-lysine, glycine, L-threonine, L-glutamine, L-arginine and L-glutamic acid when compared to wild type. The plasma membrane and mitochondrial ATPase activities of these mutants are also greatly reduced. Since the uptake of such compounds is likely to be driven by a proton electrochemical gradient set up by the membrane ATPase it is suggested that the primary effect of these mutations is at the level of the membrane ATPase. Another drug resistant strain, cyh1, which is resistant only to high levels of cycloheximide, shows increased uptake of trichodermin, L-lysine, glycine, L-threonine, L-glutamine when compared to wild type. The plasma membrane and mitochondrial ATPases of cyh1 are considerably greater than those of wild type. It has been shown previously that cyh1 possesses an altered 60S ribosonal subunit protein when compared to wild type and this makes it resistant to cycloheximide. There is no obvious explanation as to how this change could lead to the alterations in drug and amino acid uptake and in ATPase activities observed.

Multiple drug resistance in the fission yeast Schizosaccharomyces pombe: evidence for the existence of pleiotropic mutations affecting dependent transport systems.

The uptake of L-tyrosine into wild type and antibiotic resistant strains of Schizosaccharomyces pombe requires an energy source, is initially linear with respect to time, is inhibited by 2,4-dinitrophenol and sodium azide and is saturable. However the initial uptake rates and the amount of L-tyrosine accummulated by antibiotic resistant strains are much less than wild type. Comparison of the kinetic constants of uptake shows that mutant strains have a reduced maximum velocity of uptake compared to wild type and a larger Km. Since the three mutant strains possess a permeability barrier to L-tyrosine as well as being drug resistant this is an indication that antibiotic resistance may be caused by a decrease in plasma membrane permeability.

Characterisation of ribosomes from drug resistant strains of Schizosaccharomyces pombe in a poly U directed cell free protein synthesising system.

Mutants of Schizosaccharomyces pombe were isolated as resistant either to trichodermin or to anisomycin. Growth tests showed that the majority of mutants isolated were cross resistant to both drugs and also to cycloheximide. A limited genetic analysis showed that mutants at least four loci, tri3, tri4, ani1 and ani2, had this phenotype as was also the case for mutants at three cycloheximide resistant loci, cyh2, cyh3 and cyh4 reported previously (Ibrahim and Coddington, 1976). Allelism tests showed that the tri3, ani2 and cyh4 strains were allelic. A mutant at another trichodermin resistant locus, tri5, was cross resistant to anisomycin but sensitive to cycloheximide. Ribosomes from wild type and selected strains were analysed in a poly U directed cell free protein synthesising system. Three strains, cyh1-C7, ani1-F1 and tri-N15 (probably a tri5 allele) possessed ribosomes which were more resistant than the wild type to the drugs used in their isolation. In each case the site of the resistance was in the 60S subunit. Ribosomes from the cyh2, cyh3 and cyh4 strains were as sensitive to cycloheximide as those from wild type.

Genetical studies on revertants to sensitivity from a cycloheximide resistant strain of Schizosaccharomyces pombe.

Six UV induced cycloheximide-sensitive revertants were isolated from the cyh1-C7 strain of Schizosaccharomyces pombe which is resistant to cycloheximide. In all cases reversion to sensitivity was due to a forward mutation in a second suppressor gene. Genetical analysis showed that at least two genes, designated scr1 and scr2 (scr=suppression of cycloheximide resistance) were involved. Both scr1 and scr2 suppressed the resistance of six independently isolated alleles at the cyh1 locus. They had no effect on two known nonsense mutations in the ade7 locus. The cyh1-C7 strain has an altered 60S ribosomal protein which can be detected by two-dimensional polyacrylamide gel electrophoresis. In two suppressed strains, cyh1-C7 scr1 and cyh1-C7 scr2, the original altered protein was present. However no further ribosomal protein differences were observed which could be correlated with the presence of the scr genes. Both scr mutations conferred cold sensitivity on the organism indicating that they were of the missense type. Hence it seems certain that scr1 and scr2 are not mutations in tRNA genes leading to either nonsense or missense suppression. There is however no direct evidence that they code for ribosomal proteins and exert their effect on cyh1-C7 at the ribosomal level.