A chemically defined medium for cephalosporin C production by Paecilomyces persicinus

A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours.     

Isolation and partial characterization of a D-amino acid oxidase active against cephalosporin C from the yeastTrigonopsis variabilis

Summary D-Amino acid oxidase has been isolated and purified in a simple procedure to homogeneity fromTrigonopsis variabilis. The enzyme was able to oxidatively deaminate cephalosporin C to keto adipic 7-aminocephalosporanic acid. The molecular weight of the native enzyme was estimated to be 86000. The enzyme was shown to contain two identical subunits with each subunit carrying probably one molecule of iron. The K_m values determined for the substrates cephalosporin C, D-phenylalanine, D-alanine, D-methionine and D-leucine were 13, 10, 76, 0.76 and 0.12 mM, respectively.Enzyme yield was shown to increase on growth of the organism in medium supplemented with DL-methionine.     

Repeated batch antibiotic fermentation by streptomycetes immobilized on cotton cloth

Summary Streptomyces cattleya and Streptomyces clavuligerus were found to form immobilized populations on cotton cloth. The resulting bacterial films were used as resident inocula in batch fermentations of thienamycin and cephalosporin C. During 20 cycles of 3-day batch fermentation, these antibiotics were produced at the constant yields.     

Changes in the lipid composition of Paecilomyces persicinus P-10 M1 during growth and cephalosporin C production

The lipid composition of the fungus Paecilomyces persicinus P-10 M1 was monitored daily for 6 days to detect any changes during growth and cephalosporin C production. Total lipid yields and cephalosporin C production were maximal after 72 h of incubation. Analysis of the total lipids revealed that the neutral lipid fraction was elevated at this time, whereas polar lipids were depressed. Phosphatidylethanolamine and phosphatidylcholine represented the major phospholipids detected. Phosphatidylethanolamine levels were descending when cephalosporin C was detected at its highest concentration. Increases in phosphatidylcholine levels paralleled those of cephalosporin C but reached a maximum at 48 h after the latter. Diphosphatidylglycerol, phosphatidic acid, and phosphatidylserine were also detected. Fatty acids present in the total lipid fraction ranged in carbon length from C12 to C24. The major acids were C16 (palmitic), C18:1 (oleic), and C18:2 (linoleic). All fatty acids exhibited minor variations in concentration during the 6-day period, and none displayed a direct correlation with cephalosporin C yields.     

Changes in the lipid composition of Paecilomyces persicinus P-10 M1 during growth and cephalosporin C production.

The lipid composition of the fungus Paecilomyces persicinus P-10 M1 was monitored daily for 6 days to detect any changes during growth and cephalosporin C production. Total lipid yields and cephalosporin C production were maximal after 72 h of incubation. Analysis of the total lipids revealed that the neutral lipid fraction was elevated at this time, whereas polar lipids were depressed. Phosphatidylethanolamine and phosphatidylcholine represented the major phospholipids detected. Phosphatidylethanolamine levels were descending when cephalosporin C was detected at its highest concentration. Increases in phosphatidylcholine levels paralleled those of cephalosporin C but reached a maximum at 48 h after the latter. Diphosphatidylglycerol, phosphatidic acid, and phosphatidylserine were also detected. Fatty acids present in the total lipid fraction ranged in carbon length from C12 to C24. The major acids were C16 (palmitic), C18:1 (oleic), and C18:2 (linoleic). All fatty acids exhibited minor variations in concentration during the 6-day period, and none displayed a direct correlation with cephalosporin C yields.     

Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea

A microsomal mixed function oxidase isolated from V. rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was dependent upon NADPH and oxygen and was associated with the 100,000 X g pellet which exhibited a characteristic reduced P-450-CO binding spectra. Light reversible inhibition by CO as well as differential sensitivity to other inhibitors established the hydroxylase as a cytochrome P-450 type. Cis-trans isomerase activity was not observed in this preparation. Both geraniol and nerol were shown to be hydroxylated almost exclusively at the C-10 methyl group.     

Optimum tissue culture conditions for selection of resistance to Phytophtora cinnamomi in pine callus tissue

The present experimentation compared the best nutrient medium, temperature, and growth hormones for callus induction and growth of various pine species from different seed sources with their effect on growth of Phytophthora cinnamomi. Callus tissues maintained on a modified Murashige and Skoog medium with 10^–5M 2,4-D at 26°C in the dark optimized the expression of differential resistance when inoculated with hyphae of P. cinnamomi. High concentration of 2,4-D (5×10^–5M) inhibited growth of P. cinnamomi.Abbreviations AL loblolly pine-Alabama - PL South Carolina - AS shortleaf pine-Alabama - CS Georgia - AV Virginia pine-Alabama     

Affinity chromatography purification of penicillinase of Bacillus licheniformis 749-C and its use to measure tuurnover of the cell bound enzyme

Affinity chromatography purification of small amounts of penicillinase using cephalosporin C covalently linked to Sepharose 4B has been used in examining the turnover of cell-bound penicillinase by B. licheniformis 749/C. Under conditions in which most of the nascent penicillinase is retained by the cells, turnover could be demonstrated (15% of the cell-bound enzyme in 2.5 hours), although most of the secreted enzyme is derived from newly-formed molecules.     

Factors affecting cephalosporin C biosynthesis

Summary Caprolactam, butan-2-one, butan-2-ol, and butan-1, 3-diol were found to have a stimulating effect on the biosynthesis of cephalosporin C by a mutant strain ofCephalosporium. Caprolactam was shown to have a synergistic effect, when added to the fermentation medium together with each of the other three compounds.     

Cryopreservation of isolated mint shoot tips by vitrification

Shoot tips isolated from a mint clone, Mentha aquatica x M. spicata, were gradually exposed to a mixture containing 35% ethylene glycol, 1 M dimethylsulfoxide and 10% polyethylene glycol-8000 and then immersed into liquid nitrogen. Cooling and warming rates were approximately 4800°C/min and 9000°C/min respectively. Survival after liquid nitrogen treatment ranged from 31% to 75% among experiments. There was no obvious reason for this variation. In many cases the treated shoot tip directly developed into a shoot without any or with only slight callus formation.Abbreviations DSC differential scanning calorimetry - DMSO dimethylsulfoxide - EG ethylene glycol - PEG-8000 polyethylene glycol - MW avg. 8000 - LN liquid nitrogen - IBA indolebutyric acid - BA benzyladenine     

In vitro culture of pods from annual and perennial Medicago species

Because most interspecific Medicago embryos abort before they can be excised and cultured, our objective was to grow young pods in vitro. Various media were used to grow three-day-old pods of annuals [diploids, M. blancheana Boiss., M. disciformis DC., tetraploid M. scutellata (L.) Mill.] and perennials (diploid M. falcata L., tetraploid M. sativa L.).Few pods of perennial species grew to maturity on media containing modified Hoagland's plus 1% glucose or sucrose with or without 5% potato extract. Increasing sucrose to 6% increased the percentage of M. sativa pods that produced mature seeds. On DM (differentiation medium), the best medium, the percentage of pods producing viable seeds was: M. blancheana (82), M. disciformis (81), M. scutellata (48), M. sativa (63), M. falcata (15). DM plus 1 ppm indoleacetic or gibberellic acid did not enhance seed production.Abbreviations DM Differentiation medium - DMI DM plus 1 ppm indoleacetic acid - DMG DM plus 1 ppm gibberellic acid     

The cytotoxic T cell response to peptide analogs of the HLA-A*0201-restricted MUC1 signal sequence epitope,M1.2

The mucin MUC1 molecule is overexpressed on a variety of adenocarcinomas and is thus, a potential target for immunotherapy. Of the MUC1 peptides that bind to HLA-A*0201(A2), M1.2 (LLLLTVLTV) from the signal sequence appears to be the most immunogenic in humans. Here we have shown that large numbers (10⁹) of tetramer-binding M1.2-specific cytotoxic T lymphocytes (CTL) can be generated ex vivo from circulating precursors, derived from healthy adults. However, there was significant interpersonal variation in the level of co-stimulatory signal required. Tetramer-binding cells also required maturation in culture to become proficient killers of the HLA-A2⁺ MUC1⁺ MCF7 cell line, known to express a low number of endogenously processed M1.2. The functional avidity of M1.2-specific CTL, however, was low as compared to CTL specific for an HIV-1 epitope. Despite the low avidity, M1.2-specific CTL were polyfunctional, secreting multiple cytokines upon degranulation with antigen recognition. To identify potential agonist peptides that may be superior immunogens, an M1.2-specific CTL culture was used to scan a large nonameric combinatorial peptide library. Of 54 predicted peptides, 4 were “consensus” agonists because they were recognized by CTL from two other donors. Two agonists, p29 (LLPWTVLTV) and p15 (VLLWTVLTV), were equally stimulatory when loaded onto C1R target cells transfected with wild-type HLA-A2. Both agonists induced IL-2, TNF-α, IFN-γ, and degranulation with M1.2-specific CTL. In contrast, production of these cytokines, which are tightly regulated by specific activation through the T cell receptor, was restricted when the CTL were stimulated with peptides loaded onto C1R cells that were transfected with an HLA-A2 molecule bearing a mutation that abrogates binding to the CD8 co-receptor. Thus, activation by both M1.2 and its agonists was dependent upon CD8, showing that compensation by the co-receptor was necessary for the human T cell response to M1.2.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.     

Studies on themodulation of immunoglobulin production by prostaglandins

The present study investigated the effect of prostaglandins (PG) on the in vitro production of polyclonal IgG and IgM by pokeweed mitogen- stimulated normal human peripheral mononuclear cells. Concentrations of PGE₁ and PGA₁ in excess of 10⁻⁶M were suppressive. PGE₂ and PGs of the F series were less effective and significant suppression was seen in concentrations greater than 10⁻³M. Indomethacin added to cell cultures did not enhance Ig production. This discrepancy between physiologic PG concentrations and the very large pharmacologic concentration necessary to suppress Ig synthesis in vitro makes the physiologic role of PG in the modulation of Ig synthesis questionable.     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.     

Fusion of Agrobacterium and E. coli spheroplasts with Nicotiana tabacum protoplasts — Direct gene transfer from microorganism to higher plant

Spheroplasts of Agrobacterium tumefaciens strains and E. coli were fused with protoplasts of Nicotiana tabacum. Fusion products were cultured in the presence of antibiotics to eliminate remaining bacterial spheroplasts. On hormone free medium, tobacco protoplasts treated with wild type Agrobacterium-strains formed colonies with an average frequency of 10^–4. Opine synthesis was detected in the tissues. Some calli derived from protoplasts treated with A. tumefaciens C58C1pRi15834 formed typical hairy roots. Kanamycin resistant calli were obtained after fusion with A. tumefaciens containing pLGVTi23 neo (frequency=10^–3). Fusion of E. coli spheroplasts containing a virulent pTiB6S3::RP4 co-integrate with tobacco protoplasts yielded two hormone independent growing calli producing octopine out of 10⁵ microcalli.Abbreviations PEG Polyethylene glycol - PVA Polyvinyl alcohol     

Metabolism of valproic acid by hepatic microsomal cytochrome P-450

Incubation of valproic acid with rat liver microsomes led to the formation of 3-, 4- and 5-hydroxy-valproic acid. The latter two metabolites, which have been characterized previously from in vivo studies, may be regarded as products of fatty acid ω-1 and ω hydroxylation, respectively. 3-Hydroxy-valproic acid, however, had been thought to derive from the β-oxidation pathway in mitochondria. Conversion of valproic acid to all three metabolites in microsomes required NADPH (NADH was less effective), utilized molecular oxygen, was suppressed by inhibitors of cytochrome P-450 and was stimulated (notably at C-3 and C-4) by phenobarbital pretreatment of the rats. It is concluded that rat liver microsomal cytochrome P-450 catalyzes ω-2 hydroxylation of valproic acid, a reaction not detected previously with fatty acids in mammalian systems, and that the product, 3-hydroxyvalproic acid, should not be used to assess in vivo metabolism of valproate via the β-oxidation pathway.     

Effect of cinnamoyl putrescines on in vitro cell multiplication and differentiation of tobacco explants

Hydroxycinnamoyl putrescines promote the cell multiplication of leaf discs of a tobacco mutant, RMB₇, cultivatedin vitro on the Murashige and Skoog medium. This mutant never accumulates these molecules during its development and does not enter in floweirng. Maximal effect is obtained at 2.5·10^–4M. The same molecules inhibit bud formation ofNicotiana tabacum var. Xanthi nc, at 5·10^–5 M but promote callus formation. From 10^–4 M to 5·10^–3 M they strongly inhibit cell multiplication and bud formation without toxic effect. Their possible role in plant metabolism is discussed.Abbreviations IAA indole 3-acetic-acid - BA benzyladenine     

Effect of AgNO3 and aminoethoxyvinylglycine on in vitro shoot and root organogenesis from seedling explants of recalcitrant Brassica genotypes

The presence of 1–10 M aminoethoxyvinylglycine (AVG) or 5–30 M AgNO₃ markedly enhanced shoot regeneration from cotyledon and hypocotyl cultures of eight recalcitrant Brassica campestris and B. juncea genotypes tested. Expiants of B. campestris ssp. chinensis and ssp. parachinensis grown with a high AVG concentration (20 M), regenerated poorly. All cytokinins tested were equally effective in promoting shoot formation, except that kinetin was inhibitory to shoot regeneration from hypocotyls of B. campestris ssp. pekinensis (cv. Wong Bok). Both AgNO₃ and AVG had no effect on percent rooting and number of roots per rooted cutting of Wong Bok, White Sun and Leaf Heading, but AgNO₃ was inhibitory to rooting of India Mustard. However, root elongation of all cuttings was markedly inhibited by AVG at concentrations of 5 and 10 M.Abbreviations AVG aminoethoxyvinylglycine - BA benzyladenine - IBA indole-3-butyric acid - 2ip 6-{ie195-01}-{ie195-02}-dimethylallylamino purine - MS Murashige and Skoog - NAA naphthaleneacetic acid