A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor.

A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.

Identification and characterization of a zinc metalloprotease associated with invasion by the fish pathogen Vibrio anguillarum.

An invasiveness-defective mutant of the fish-pathogenic bacterium Vibrio anguillarum was isolated. Compared with the wild type, this mutant had a 1,000-fold higher 50% lethal dose after immersion infection of rainbow trout, Oncorhynchus mykiss, while after intraperitoneal infection, the mutant had only a 10-fold higher 50% lethal dose. In addition, the mutant showed a lower level of protease activity. Two forms of the protease (Pa and Pb) were found after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of nonheated samples. Pa was found predominantly in protease preparations of the wild type, while Pb was the predominant form in the mutant. Conversion of Pb to Pa was observed in protease preparations after incubation at 4 degrees C. Characterization of the protease showed that it was an elastolytic enzyme which required Zn2+ for activity and Ca2+ for stability. The molecular mass of the protease was 36 kilodaltons. N-terminal amino acid sequence analysis of the protease of V. anguillarum revealed homology to the elastase of Pseudomonas aeruginosa and the protease of Legionella pneumophila.

Proteolytic modification of tissue plasminogen activator: importance of the N-terminal part of the catalytically active B-chain for enzymatic activity.

Native one-chain tissue plasminogen activator (t-PA) was rapidly converted to the two-chain form by trypsin-Sepharose cleavage. This caused an increase in the amidolytic activity on low molecular weight peptide substrates, while plasminogen activation in the presence of fibrin markedly decreased. Cleavage sites were identified by N-terminal sequence analysis of reduced and carboxymethylated peptides. In the B-chain, the expected cleavage at Arg278-Ile279 was identified. Furthermore, a specific cleavage site was found at Arg302-Ser303, 24 amino acids from the N-terminus of the B-chain. The peptide released by this cleavage (designated B1-24) remained associated with the activator molecule by strong noncovalent interactions but could be dissociated under denaturing conditions (4 mol/L of guanidine hydrochloride), leading to a 20-fold decrease in amidolytic activity. Addition of purified B1-24 peptide to t-PA treated in this manner restored the activity in a concentration-dependent way. In contrast to trypsin, cleavage of the single-chain t-PA molecule with endoproteinase Lys-C generated a two-chain form of the activator, without simultaneous increase in the amidolytic activity. By sequence analysis, a major cleavage was identified at Lys280-Gly281, two residues into the B-chain. Together, the results presented provide additional information on the one-chain to two-chain conversion of t-PA and the role of the free N-terminus of the B-chain.

Proteolytically induced variations in the enzymatic properties of tissue plasminogen activator. Activations, inactivations and reactivations.

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50,000/52,000 and 17,000/20,000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420-421 and 423-424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420-421 and 423-424, convert the active one-chain activator into an 'inactive' zymogen, which is again 'activated' by plasmin cleavage.

Fibrinolysis mediated by tissue plasminogen activator. Disclosure of a kinetic transition.

The rate of 'Glu'-plasminogen activation by tissue plasminogen activator was repeatedly determined during a fibrinolytic process. The process was found to proceed via two distinct phases. The kinetics of each phase obeyed Michaelis-Menten equation: First phase; kcat about 0.17 s-1 and Km about 1 microM, second phase; kcat about 0.13 s-1 and Km about 0.06 microM. Practically identical results were obtained with one-chain as with two-chain tissue plasminogen activator. Transition from first to second phase occurred when the system had been exposed to a certain degree of plasmin digestion. Electrophoretic analysis demonstrated time correlation between the appearance of minimally degraded fibrin (X-fragments) and the transition. No such correlation was found between transition and conversion of 'Glu'-plasminogen to 'Lys'-plasminogen. The effect can result in an acceleration (up to 13-fold) of the fibrinolytic process once a slight degradation of the fibrin has taken place. In vivo, the effect described may constitute a mechanism that protects a fibrin clot from premature lysis.

Comparison studies of chlorazepate administered as a divided daily dose and as a single dose at night.

The residual effects of dipotassium chlorazepate administered as either a single daily dose of 20 mg at bedtime or a divided daily dose (5 + 5 + 10 mg) were studied in a placebo-controlled, double-blind trial comprising 12 out-patients. The following tests were used to determine changes in perceptual wakefulness, performance ability, fine motor skills, and coordination: critical flicker fusion test, car driving in a simulator, and the "bead and needle tests." In addition, the patients underwent a clinical assessment and also filled out a self-rating scale for judging factors related to the tests. No significant differences were found between the dosage schedules or between the active medication and the placebo. The clinical results were not dependent on the dosage schedule.

Clinical and experimental comparison of diazepam, chlorazepate and placebo.

A double blind clinical evaluation of chlorazepate (dipotassium-7-chloro-5-phenyl-2,2-dihydroxy-3-carboxy-1,2-dihydro-2H-1,4-benzodiazepine), diazepam and placebo on a patient group of young students, mean age 25 years, is reported. Both drugs were significantly better than placebo and according to global assessment chlorazepate was superior to diazepam. An analysis of the main target symptom revealed better effects of chlorazepate on the following items: anxiety, feeling of muscular tension and gastro-intestinal disturbances. With respect to irritability and sleep disturbances both drugs were found to be equally effective. In patients' self-ratings chlorazepate was considered superior to diazepam in giving more alertness and less drowsiness during day time. The results are discussed with reference to EEG-studies and pharmaco-kinetic properties of chlorazepate and diazepam. Performance tests in simulated car-driving by healthy volunteers did not demonstrate any significant difference as compared with placebo. The psychophysiological eeffects, however, are more pronounced after diazepam than after chlorazepate medication.