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     

On-line parameter and state estimation of continuous cultivation by extended Kalman filter

Summary The on-line estimation of biomass concentration and of three variable parameters of the non-linear model of continuous cultivation by an extended Kalman filter is demonstrated. Yeast growth in aerobic conditions on an ethanol substrate is represented by an unstructured non-linear stochastic t-variant dynamic model. The filter algorithm uses easily accessible data concerning the input substrate concentration, its concentration in the fermentor and dilution rate, and estimates the biomass concentration, maximum specific growth rate, saturation constant and substrate yield coefficient. The microorganismCandida utilis, strain Vratimov, was cultivated on the ethanol substrate. The filter results obtained with the real data from one cultivation experiment are presented. The practical possibility of using this method for on-line estimation of biomass concentration, which is difficult to measure, is discussed.Nomenclature D dilution rate (h^-1) - DO₂ dissolved oxygen concentration (%) - E identity matrix - F Jacobi matrix of the deterministic part of the system equations g - g continuousn-vector non-linear real function - h m-vector non-linear real function - K Kalman filter gain matrix - K _S saturation constant (kgm^-3) - K_S expectation of the saturation constant estimate - M Jacobi matrix of the deterministic part of the measurement equations h - P(t₀) co-variance matrix of the initial values of the state - P(t_k/t_k) c-variance matrix of the error in (t _k|t _k) - P(t_k+1/t_k) co-variance matrix of the error in (t _k+1|t _k - Q co-variance matrix of the state noise - R co-variance matrix of the output noise - S substrate concentration (kgm^-3) - S _i input substrate concentration - t time - t _k discrete time instant with indexk=0, 1, 2,... - u(t) input vector - v(t_k) measurement (output) noise sequence - w(t) n-vector white Gaussian random process - x(t₀) initial state of the system - (t₀) expectation of the initial state values - x(t) n-dimensional state vector - x(t_k) state vector at the time instantt _k - (t_k|t_k) expectation of the state estimate at timet _k when measurements are known to the timet _k - (t_k+1|t_k) expectation of the state prediction - X biomass concentration (kgm^-3) - expectation of the biomass concentration estimate - y(t_k) m-dimensional output vector at the time instantt _k - Y _XIS substrate yield coefficient - _X|S expectation of the substrate yield coefficient estimate - specific growth rate (h^-1) - _M maximum specific growth rate (h^-1) - expectation of the maximum specific growth rate estimate - state transition matrix     

Carnivora: primary structure of the hemoglobins from ratel (Mellivora capensis)

The erythrocytes of adult ratel contain two hemoglobin components, with two alpha- and one beta-chains. In this paper, their complete amino acid sequences are presented. The two alpha-chains differ in one residue at position 34 (Ala----Val) only. The primary structure of the chains was determined by sequencing the N-terminal regions (45 steps) and the tryptic peptides after their isolation from the digests by reversed-phase high-performance liquid chromatography. The alignment of these peptides was deduced from homology with other carnivora globins. The alpha-chains show 21 and the beta-chains 11 exchanges compared with human globin chains. In the alpha-chains, one heme- and two alpha 1/beta 1 contacts are exchanged. In the beta-chains there are three exchanges which involve one alpha 1/beta 1-, one alpha 1/beta 2- and one heme-contact. Between the ratel hemoglobin and those of carnivora a high degree of homology was found.     

Enzymatic resynthesis of the "reactive site" bond in the modified aprotinin derivatives [seco-15/16]aprotinin and [Di-seco-15/16,39/40]aprotinin

On incubation of [di-seco-15/16,39/40]aprotinin with human plasmin, porcine pancreatic kallikrein or bovine or porcine trypsin in neutral or slightly alkaline solutions [seco-39/40]aprotinin is slowly formed with enzymatic resynthesis of the reactive-site bond 15/16. With chymotrypsin, however, further degradation of [di-seco-15/16,39/40]aprotinin takes place without enzymatic resynthesis. The apparent rate constants for the synthesis of [seco-39/40]aprotinin with kallikrein and trypsin have been determined and indicate that the bond-forming reaction is 10-200-fold slower with [di-seco-15/16,39/40]aprotinin than with [seco-15/16]aprotinin. The newly formed [seco-39/40]aprotinin has similar kinetic constants for the complexation with its cognate enzymes as aprotinin, indicating that any distortion of the secondary binding region due to cleavage of the Arg39-Ala40 bond does not seriously influence binding and affinities.     

Isolation of highly multidrug-resistant P388 cells from drug-sensitive P388/S cells by flow cytometric cell sorting

To investigate the spontaneous frequency of occurrence of stable multidrug-resistant cells in a population of drug-sensitive cells, we exposed drug sensitive P388/S cells to daunorubicin (dnr) for 1 h, then used fluorescence-activated cell sorting based on intracellular dnr fluorescence to isolate cells within P388/S having different intracellular content of drug. One of the sort windows chosen (low dnr content sort window) isolated only P388/S cells with intracellular drug content equal to or less than that of the known multidrug-resistant subline P388/adr. This sort window constituted approximately 3% of P388/S cells with lowest dnr content. By such a procedure we were able, on one of seven attempts, to isolate and cultivate stable, highly multidrug-resistant cells (comparable to that of P388/adr) from the P388/S cells obtained from the low dnr-content sort window. Net growth of cells in culture was observed 15-20 days after sorting, indicating that of the P388/S cells collected from the low dnr-content sort window, very few were actually highly drug-resistant. On no occasion could resistant cells be cultivated from cells sorted from P388/S with higher dnr content, as would be expected if mutation to a multidrug-resistant phenotype had occurred as a result of exposure to drug. The resistant cells isolated from P388/S by sorting (called P388/LoSort) displayed low intracellular accumulation of dnr that was enhanced by verapamil, were cross-resistant to vincristine and actinomycin-D, and distinct from P388/S, possessed a 150- to 160-kD membrane species identified by Vinca alkaloid photoaffinity labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Plasmid inverted repeats provide for duplication or precise excision of DNA inserts

A plasmid containing inverted repeats is constructed in Bacillus subtilis. Insertion of DNA fragments into the plasmid inverted repeats results either in the precise excision of the insert or in its duplication in the opposite inverted repeat. These rearrangements are due to the presence of inverted repeats only. Two recombination events are possibly responsible for these phenomena. During the first step of the recombination two plasmid monomers form a dimer molecule. During the second step the intramolecular recombination between the direct repeats in the dimer structure leads to the formation of two rearranged plasmid monomers devoid of insertion or containing two DNA inserts. Proposed dimeric intermediate is unstable in B. subtilis. However, it is isolated from Escherichia coli recA, cells. Plasmids containing the inverted repeats can serve as a model to study plasmid recombination in B. subtilis cells.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Interaction between glucose utilization and left ventricular heart function in type I-diabetics

The aim of the present study was to check whether equal, therapeutically relevant, positively inotropic doses of different adrenergic agents elicit equal inotropic and metabolic effects in 6 type I-diabetics as in 6 matched nondiabetic subjects. The effects of increasing doses of norepinephrine (NE)- and orciprenaline (0.12, 0.20, 0.33 microgram/kg min) on heart function (systolic time interval, heart rate, blood pressure) and on serum fatty acid (NEFA), glucose, lactate, pyruvate and insulin concentrations were recorded. In the therapeutic dose range, NE, and orciprenaline elicited in diabetics without clinical signs of any cardiovascular disease a diminished myocardial inotropic response (20-40%), less marked vascular effects (vasoconstriction, vasodilatation), but greater metabolic changes in right atrial blood (NEFA, pyruvate, lactate) compared to matched controls (p less than 0.05). The smaller increase of cardiac performance in diabetics to exogenous catecholamines cannot be explained by sympathetic cardiac denervation, since chronotropic beta 1-beta 2-stimulation with orciprenaline provoked nearly equal dose-dependent changes in diabetics and controls. It is suggested that the smaller positive inotropic effect during NE and orciprenaline infusion in type I-diabetics is a result first of all of alterations in myocardial energy turnover in diabetes due to reduced myocardial glucose utilization. It seems necessary to secure continuous myocardial glucose utilization and subnormal NEFA concentrations in the serum during the therapeutic application of inotropic adrenergic agents in severe cardiac failure and cardiogenic shock in diabetics.     

Restriction map of permuted DNA of Erwinia carotovora temperate bacteriophage 59

The cyclic permutation and terminal redundancy were found in the genomes of Erwinia carotovora temperate bacteriophage 59 by electron microscopic studies. The headful mechanism for bacteriophage DNA cleavage and packaging during the phage morphogenesis was confirmed by the restriction analysis technique. Restriction map of the bacteriophage 59 DNA was constructed for restriction endonucleases BamHI, Bg1II, Eco31, Sa1I, SmaI, EcoRI.