Biosynthesis of glyantrypine by Aspergillus clavatus

The biosynthesis of glyantrypine from radiolabelled amino acid precursors has been shown experimentally to involve anthranilic acid, tryptophan and glycine. Low values for percentage incorporation of radiolabel into glyantrypine were partly influenced by a complex array of other novel alkaloids shown by the radiolabelling experiments to be related to glyantrypine. Interpretation of radiolabel incorporation from [14C-carboxyl]-anthranilic acid into microbial metabolites seen to contain an anthranilyl moiety in various biosynthetic arrangements is discussed. The possibility of diversion of anthranilic acid from the kynurenine pathway to glyantrypine biosynthesis is recognised.     

Purification of a class C A-type beta-lactamase from a derepressed strain of Enterobacter cloacae. Comparison of the wild-type and mutant enzyme with those from strains P99, 208 and GN7471.

Enterobacter cloacae strain 5822 expresses low levels of a class C beta-lactamase which can be induced 100-fold by imipenem. Mutants that constitutively express high levels of beta-lactamase can be selected on aztreonam or cefotaxime. The beta-lactamase from one such mutant (5822M2) has been purified to homogeneity and compared on the basis of subunit Mr, pI, substrate specificity, inhibitor sensitivity and immunological cross-reactivity with the enzyme from strains P99, GN7471 and 208, which have been studied previously. The enzyme from strain 5822M2 is clearly related to these other forms and is of the A-type according to the criteria of Seeberg, Tolxdorff-Neutzling & Wiedemann [Antimicrob. Agents Chemother. (1983) 23, 918-925]. The enzyme from the wild-type strain (5822) is shown to be identical to that found in the depressed strain (5822M2), indicating that the mutation is in a regulatory gene. A detailed analysis of the kinetics of the enzyme from strain 5822M2 shows that all of the beta-lactams studied are substrates and that a mechanism involving the formation of an acyl-enzyme is probably applicable in every case. The substrates however can clearly be grouped into two classes, i.e. 'good' substrates with kcat. values of 80-1200 s-1 and 'poor' substrates/good inhibitors with kcat. values of 0.009-0.00007 s-1. The permeability barrier to aztreonam is 4-fold less in the derepressed strain when compared with the wild-type strain. This is associated with significant changes in the expression of outer membrane porins. The observed resistance in the derepressed mutant appears to be linked to the elevated levels of beta-lactamase (3000-fold) rather than to the modest changes in the permeability barrier.     

Nephrotoxicity of Penicillium aurantiogriseum and P. commune from an endemic nephropathy area of Yugoslavia

Seven out of nine Penicillium isolates from mouldy maize in Yugoslavia have been differentiated into the adjacent species P. aurantiogriseum and P. commune. Nephrotoxicity of cultured mycelia in the rat has been demonstrated for all isolates of both species and was correlated usefully, though indirectly, with the production of benzodiazepine secondary metabolites, notably auranthine. Shredded wheat (22 g) moulded by an example of each species and fed to a rat over 4 days elicited renal pathology in the P₃ segment of proximal tubules, involving frequent pyknosis and extensive mitosis typical of this as yet uncharacterised toxin. The effect was attributed in P. aurantiogriseum at least partly to the spores. Prominent pathology was elicited by only lg of spores given over 4 days.     

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.     

The effects of delipidation on the major antigenic determinant of purified human intestinal mucin

To investigate whether the antigenicity of purified human intestinal mucin was dependent on the presence of associated lipid, native mucin (purified by equilibrium density gradient centrifugation in CsCl (twice) and gel filtration on Sepharose 2B) was extracted five times with organic solvents to remove any noncovalently bound lipid and, subsequently, treated with hydroxylamine to release any covalently bound fatty acids. The first organic extract contained cholesterol, phosphatidylethanolamine, and phosphatidylserine, with lesser amounts of phosphatidylcholine, triglycerides, fatty acids, sphingomyelin, and glycolipids. In total, this noncovalently bound lipid amounted to less than 5% by weight of the native mucin preparation. Further organic extracts were free of lipid. Removal of noncovalently bound lipid had essentially no effect on mucin antigenicity, as assessed by radioimmunoassay. Treatment of the delipidated mucin with hydroxylamine caused no detectable changes in mucin antigenicity or composition and the release of covalently (ester) bound fatty acids could not be demonstrated. We therefore conclude that although purified human intestinal mucin contains small amounts of noncovalently bound lipid this lipid is not involved in mucin antigenicity.     

Studies on Claviceps purpurea (Fr.) Tul. parasitic on Phragmites communis Trin

Isolates from C. purpurea sclerotia occurring naturally on Phragmites communis usually sporulated vigorously on the culture medium employed, and their failure to produce alkaloid in vitro was associated with a thin white growth form. Such isolates also failed to produce sclerotia on the host plants tested. A variant having a plectenchymatic morphology in vitro and producing a thick pigmented non-sporulating growth form yielded alkaloid (up to approximately 300μg/ml mainly δ8–9 and δ9–10 lysergic acids and chanoclavine) in surface or submerged culture and developed typical ergot sclerotia (containing 0·2-0·4% alkaloid, mainly ergotoxine and ergotamine) in vivo. Improved alkaloid yield in vitro was obtained from a strain reselected after passing through a parasitic phase. Aetiological aspects of the P. communis ergot are discussed.     

Evidence for two forms of ligandin (YaYa dimers of glutathione S-transferase) in rat liver and kidney.

CM-cellulose chromatography of rat liver and kidney cytosol at pH6 reveals the presence of a second Ya-subunit dimer of glutathione S-transferase (GST-F) in addition to the recently described GST-YaYa (GST-L; our nomenclature) [Hayes & Clarkson (1982) Biochem. J. 207, 459-470]. The two forms are structurally similar (by the criteria of CNBr- and Staphylococcus-V8-proteinase-cleavage peptide maps), and both are sensitive to inhibition by haemin. However, their kinetic parameters with 1-chloro-2,4-dinitrobenzene are quite distinct, and they show differential inducibility by phenobarbitone. These results suggest a similar heterogeneity in Ya-subunits to that previously described for Yb-subunits of glutathione S-transferase and indicate that significant gene duplication may have occurred in these multifunctional intracellular binding proteins.     

Biosynthesis of radiolabeled verruculogen by Penicillium simplicissimum.

In surface culture of Penicillium simplicissimum, verruculogen was shown to be biosynthesized from the intact carbon skeletons of tryptophan and proline, isoprenoid derivatives of mevalonic acid, and a methyl group donated by methionine. Selected radiolabeled precursors (1 mCi) pulse-fed at the optimum stage of fermentation yielded verruculogen (specific activity, 5.89 X 10(2) microCi mmol-1) labeled in the prolyl and isoprenyl regions of the molecule and suitable for metabolic studies.