Biosynthesis of penicillin N and cephalosporin C: Antibiotic production and other features of the metabolism of a Cephalosporium sp

1. The production of penicillin N and cephalosporin C by two mutants of a Cephalosporium sp. has been studied with cultures grown in a chemically defined medium and with suspensions of washed mycelium in water or a buffered salt solution. 2. Antibiotic synthesis began at an early stage of growth and its rate per unit weight of mycelium appeared to pass its maximum as morphological changes were occurring in young hyphae. This rate subsequently declined, but rapid production could continue after net growth had ceased. 3. In a series of shake-flask fermentations in the growth medium, increases in the yield of penicillin N above the mean were correlated with much smaller increases in the yield of cephalosporin C and vice versa. 4. In suspensions of washed mycelium, moderate decreases in the efficiency of aeration increased the yield of penicillin N and decreased that of cephalosporin C. A similar result normally followed the addition of methionine to the suspension fluid, and in both cases there was usually an increase in the yield of the two antibiotics combined. 5. The apparent intracellular concentrations of the antibiotics were much lower than those attained extracellularly and also much lower than those of most of the amino acids in the intracellular pool. No detectable amount of [(14)C]penicillin N added to the extracellular fluid was found to enter the mycelium. 6. Very small amounts of peptide material whose behaviour was similar to that of the sulphonic acid of delta-(alpha-amino-adipoyl)cysteinylvaline on paper electrophoresis at pH1.8 were found in extracts of the mycelium that had been oxidized with performic acid. 6-Aminopenicillanic acid and 7-aminocephalosporanic acid were not detected. 7. Ultrasonic treatment of the mycelium resulted in rapid fragmentation of mycelial chains, rupture of many individual cells, and the liberation of amino acids and other substances into the medium. 8. Ultrasonically treated preparations synthesized penicillin N and cephalosporin C rapidly after a lag of 12hr. Antibiotic synthesis was accompanied by the growth of hyphae from swollen mycelial fragments and by the re-establishment of permeability barriers resulting in the uptake of amino acids from the medium.     

Differential Effect of Polyamines on T4 Morphogenesis

The 5-fluorouracil (5 FU) technique for the phenotypic reversion of amber mutants was used to demonstrate that under certain circumstances, in the presence of putrescine or spermidine, early mutants have an enhanced response to 5 FU, whereas late mutants have a delayed response. Bacteria infected by T4D wild-type bacteriophage did not produce phage in the presence of high putrescine concentrations. Pulse treatments with putrescine showed that the production of lysozyme depends on a putrescine-sensitive process that begins immediately after infection at 26 C and ends at 36 min or even later. The addition of putrescine at any time during the critical period between 0 and 36 min led to a corresponding delay in lysozyme synthesis after the inhibitor was removed. Intracellular phage maturation was delayed by the addition of 100 mumoles of putrescine per ml. Early enzymes were not affected by the diamine, but the level of phage deoxyribonucleic acid was considerably decreased by the inhibitor. The putrescine-sensitive process that affects the timing of maturation is suggested to be the natural process controlling the T4 "clock."     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Lysosomal aryl sulphatase in the liver

Summary Lysosomal aryl sulphatase activity in rat liver is demonstrated by a modification of the existing processes of fixation, incubation and processing. The choice and concentration of the fixative, duration of fixation and thickness of liver slices are found to be important factors in maintaining the levels of enzyme activity. Reliable and reproducible results are obtained by fixing thin liver slices (1 mm) for 18–24 h, in 2% glutaraldehyde buffered to pH 7.4 by 0.1M cacodylate buffer and incubating sections inHopsu et al. (1967) medium using (160 mg) nitrocatechol sulphate as substrate. Aryl sulphatase activity is localised in discrete pericanalicular granules recognised as lysosomes, which stain less intensely than acid phosphatase by the lead method.Supported by a grant from the Nuffield Foundation.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Relation between optical configuration and immunogenicity of synthetic polypeptides

1. Three random linear copolymers composed of two or three of the amino acids d-tyrosine, d-glutamic acid, d-alanine and d-lysine, and a branched multichain copolymer with a poly-d-lysine backbone and polymeric side chains of d-tyrosine and d-glutamic acid, were found to be non-antigenic in rabbits, by precipitin and passive cutaneous anaphylaxis, and in guinea pigs, by delayed hypersensitivity tests. The corresponding four copolymers of l-amino acids were shown to be antigenic by all the three criteria. 2. No immunological cross-reactions were observed between the polypeptides composed of d-amino acids and the corresponding l-amino acid copolymers. 3. Similarly, an azobenzenearsonic acid conjugate of poly-d-tyrosine was shown to be non-antigenic in guinea pigs, in contrast with an analogous conjugate of poly-l-tyrosine. Animals sensitized with the conjugate of poly-l-tyrosine did not exhibit delayed skin reactions, when cross-tested with the d-conjugate. 4. A linear polymer composed of d-tyrosine, l-glutamic acid and l-alanine was found to be immunogenic and to cross-react with the corresponding polymer composed exclusively of d-amino acids.     

A possible role for PAF in allergen-induced late responses: modification by a selective antagonist

We determined whether platelet-activating factor (PAF) plays a role in allergen-induced airway responses by studying the effects of a selective PAF antagonist WEB-2086 on antigen-induced early and late airway responses in allergic sheep. In seven sheep, inhaled Ascaris suum produced significant early (282%) and late (176%) increases in specific lung resistance (sRL). WEB-2086 (1 mg/kg iv) given 20 min before antigen challenge did not affect the early response, but the peak late increase in sRL was only 37% over base line (P less than 0.05 vs. control). To study the mechanism by which PAF contributes to antigen-induced responses, we evaluated the effects of pharmacological probes on PAF-induced bronchoconstriction. Inhaled PAF (dose range 75-700 micrograms) caused reproducible (r = 0.781, P less than 0.05) increases in sRL in eight sheep. The PAF-induced bronchoconstriction was blocked by WEB-2086 (1 mg/kg iv) and by the leukotriene antagonist FPL-55712 (30 mg by aerosol); however, neither the cyclooxygenase blocker indomethacin (2 mg/kg iv) nor the histamine H1-antagonist chlorpheniramine (2 mg/kg iv) blocked the PAF response. WEB-2086, however, did not block bronchoconstriction induced by aerosol leukotriene D4, indicating that PAF acts indirectly through leukotrienes. Finally, we determined whether PAF could induce late airway responses. Inhaled PAF produced an immediate increase in sRL in all seven sheep tested, but late airway responses were observed in only three of the seven sheep.(ABSTRACT TRUNCATED AT 250 WORDS)     

A PAF antagonist blocks antigen-induced airway hyperresponsiveness and inflammation in sheep

We studied the effects of WEB-2086, a specific antagonist of platelet-activating factor (PAF), on the development of antigen-induced airway hyperresponsiveness and inflammation in sheep (n = 8). For these studies, airway responsiveness was determined from slopes of carbachol dose-response curves (DRC) performed at base line (prechallenge) and 2 h after Ascaris suum antigen challenges in the following three protocols: 1) antigen challenge alone (control trial), 2) WEB-2086 (1 mg/kg iv) given 30 min before antigen challenge (WEB pretreatment), and 3) WEB-2086 given 2 h after antigen challenge, immediately before the postchallenge DRC (WEB posttreatment). Airway inflammation was assessed by bronchoalveolar lavage (BAL) before antigen challenge and after the postchallenge DRC for each trial. A. suum challenge resulted in acute increases in specific lung resistance that were not different among the three trials. Antigen challenge (control trial) caused a 93% increase (P less than 0.05) in the slope of the carbachol DRC when compared with the prechallenge value. WEB pretreatment (1 mg/kg) reduced (P less than 0.05) this antigen-induced hyperresponsiveness, whereas pretreatment with a 3-mg/kg dose completely prevented it. WEB posttreatment was ineffective in blocking this hyperresponsiveness. BAL neutrophils increased after antigen challenge in the control trial and when WEB-2086 was given after antigen challenge (P less than 0.05). Pretreatment with WEB-2086 (1 or 3 mg/kg) prevented this neutrophilia. This study provides indirect evidence for antigen-induced PAF release in vivo and for a role of endogenous PAF in the modulation of airway responsiveness and airway inflammation after antigen-induced bronchoconstriction in sheep.