Physiological control and process kinetics of clavine alkaloid production byClaviceps purpurea

Summary Kinetic parameters of production of clavine alkaloids were evaluated in twoClaviceps purpurea strains. Mutagenesis brought about enhanced resistance of the biosynthetic system towards alkaloids. Addition of glucose into the fermentation medium altered the zero order kinetics of production to activation-inhibition kinetics. The glucose treatment allowed performance of both elymoclavine-inhibitionless and clavine alkaloid-decompositionless fermentations if a combination of fermentation and separation units in a closed loop was used.Nomenlacture k ₁ rate constant of agroclavine synthesis (mg Agro · mg Elymo/l·g DW·day for stage 1, mg Agro/g DW·day for stage 2) - k ₂ parameter describing inhibition of agroclavine formation rate by elymoclavine (mg Elymo/l) - k ₃ specific rate of agroclavine decay (l/g DW·day) - k ₄ maximal specific rate of elymoclavine synthesis (stage 1, 1/g DW·day, stage 2, mg Elymo/g DW·day) - k ₄ ^– maximal specific rate of elymoclavine synthesis in stage 1 (inhibition-activation mechanism) (mg Elymo/g DW·day) - k ₅ physiological constant describing the elymoclavine decay rate (l²/g DW·day·mg Elymo) - k ₅ ^– physiological constant describing the activation of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₆ physiological constant describing the repression of elymoclavine biosynthesis by elymoclavine (mg Elymo/l) - k ₇ maximal specific growth rate (1/day) - k ₈ specific rate of biomass decay (l/g DW·day) - A agroclavine concentration (mg/l) - E elymoclavine concentration (mg/l) - r _A specific rate of agroclavine biosynthesis (mg Agro/g DW·day) - r _E specific rate of elymoclavine biosynthesis (mg Elymo/g DW·day) - r _i specific rate of alkaloid biosynthesis (mg alkaloid/g DW·day) - X dry biomass concentration (g/l) - specific growth rate (1/day) Abbreviations Agro agroclavine - Elymo elymoclavine - Chano chanoclavine - DW dry weight of biomass     

Irreversible Binding and Recovery of the Norepinephrine Uptake System Using an Alkylating Derivative of Norepinephrine

The effects of bromoacetylaminomenthylnorepinephrine (BAAN) on the sodium-dependent, high-affinity norepinephrine (NE) uptake system in rat brain synaptosomes and CNS neuronal cultures were investigated. BAAN inhibited [3H]NE uptake into synaptosomes in a dose- and time-dependent manner (IC50, 6.5 microM). Pretreatment of cortical synaptosomes or neuronal cells with BAAN alone, followed by washing to remove free drug, reduced the Vmax but did not alter the Km value for [3H]NE uptake. The BAAN-induced reduction in Vmax was attenuated by concurrent pretreatment with desipramine and blocked by the reaction of BAAN with dithiothreitol or cysteine. In contrast, BAAN was 19-fold less potent at inhibiting [3H]dopamine uptake in striatal synaptosomes, and no change in the Vmax or Km value for [3H]dopamine uptake was observed after a pretreatment with BAAN followed by washing. Furthermore, the irreversible beta-antagonist, bromoacetylalprenololmentane, was equipotent to BAAN for inhibiting [3H]NE uptake into cortical synaptosomes, but did not alter the Vmax or Km for [3H]NE after pretreatment. In neuronal cultures, BAAN inhibited sodium-dependent uptake of [3H]NE (IC50, 5.6 microM) with no effect on sodium-independent uptake. After pretreatment of cultures with 30 microM BAAN followed by washing, there was a 74% decrease in the Vmax for [3H]NE uptake. Following a 24-h lag period, uptake recovered to the control level within 48 h; however, recovery was completely blocked by cycloheximide. The data indicate that BAAN irreversibly binds to the [3H]NE uptake system in both CNS synaptosomes and neuronal cultures and may be a useful probe for studying the turnover of the [3H]NE uptake system.     

A comparison of the drying of chymotrypsin bound to bead cellulose by lyophilisation and in air at 25 to 70°C

Summary The effect was studied of the method of drying chymotrypsin attached to bead cellulose on its chemical and physical characteristics. These characteristics are not deteriorated when replacing the commonly used lyophilisation by fluid drying in the air stream. The preparations saved essentially the same proteolytic activity as well as stability even when increasing the drying air temperature to 70°C.     

Selected subspecies names from Richter,Plantae Europeae,Vol. 1, 1890

The bookK. Richter, Plantae europeae, Vol. 1, 1890, is analyzed from the viewpoint of nomenclature of subspecies names. 264 subspecies names interesting from the standpoint of contemporary taxonomy and nomenclature are selected from the total number of 840 subspecies names for a commented list. Seventeen important cases are dealt with separately in greater detail. Eleven new nomenclatural combinations (two for species, nine for subspecies) are proposed in the Appendix. Types for two generic names (Acorellus Palla andJuncellus C. B. Clarke) are proposed. A new term “parabasionym” is applied for names establishing the priority of infraspecific epithets in the rank in question. The need to respect the newly accepted principle of priority of autonymic subspecies names at proposing new nomenclatural combinations is stressed. Examples ofRichter’s subspecies names having priority, subspecies names with necessary changes in their authorship and names having a possible importance as parabasionyms are enumerated in separate lists.     

Sterol composition of a δ5,7-sterol-rich strain ofSaccharomyces cerevisiae during batch growth

Sterol composition was examined during batch growth on complex media containing ethanol, molasses or glucose as the carbon source. The molasses-grown cells exhibited a balanced sterol composition throughout growth, maintaining the proportion of ergosterol to 24:28-dehydroergosterol equal to 1.4. The negative effect of glucose on sterol synthesis manifested itself by decreasing the accumulation of 24:28-dehydroergosterol and total sterols but not of ergosterol. Using ethanol as the sole carbon source, a large amount of 24:28-dehydroergosterol accumulated, partly at the expense of other sterols. The gradual addition of nitrogen source during growth significantly decreased the accumulation of ergosterol, 24:28-dehydroergosterol and of total sterols. A general scheme of regulation of sterol synthesis in baker's yeast is presented.     

Saprophytic production of Clavine alkaloids and activity of Hydroxymethylglutaryl-CoA reductase

In submerged Claviceps cultures the activity of hydroxymethylglutaryl-CoA reductase preceded the increase of alkaloid production and of sterol content. During the first alkaloid phase, cell mevalonate was involved in the biosynthesis of both alkaloids and steroids. In the second production phase, it was predominantly used for alkaloid synthesis. Hydroxymethylglutaryl-CoA reductase appears to be a suitable target for physiological manipulation to increase clavine alkaloid yields.     

Possible mechanism of the allosteric activation of cAMP receptor protein

Secondary structure of cAMP receptor protein of E. coli was predicted and compared to its crystal structure in the complex with cAMP solved by McKay and Steitz. The two conformations coincide in the DNA binding domain but strikingly differ in the other domain which binds cAMP and causes protein dimerization. The comparison indicates that cAMP destabilizes a very long helix instead of which sheets are formed creating a hydrophobic pocket where cAMP binds. Consequently, the helix-sheets isomerization and a resulting change in the relative monomer disposition in the dimer appears to be the origin of cAMP-induced allosteric activation of the protein. Extremely long helices were also predicted in the regions of the regulatory subunit of cAMP-dependent protein kinase from bovine cardiac muscle where cAMP binds. It is thus likely that the proposed mechanism of the effect of cAMP on protein structure has wider implications.     

Phosphoenolpyruvate-dependent phosphorylation of a 55-kDa protein of Streptococcus faecalis catalyzed by the phosphotransferase system

Abstract A protein with an M _r of 55000 was isolated from glucose-grown Streptococcus faecalis cells. The protein becomes phosphorylated in a phosphoenolpyruvate-dependent reaction catalyzed by enzyme I and HPr of the bacterial phosphotransferase system. It did not stimulate phosphoenolpyruvate-dependent glucose phosphorylation. Several sugars were tested for their ability to dephosphorylate the phosphorylated protein in the presence of membrane fragments. Even though some of the sugars were able to dephosphorylate phospho-HPr quickly, the factor III-like 55-kDa protein remained phosphorylated. We therefore assumed that this protein is not involved in any sugar uptake reaction but that it exerts a regulatory function in Gram-positive bacteria comparable to the function of factor III specific for glucose in Escherichia coli .