Biomedical subjects

P Neubauer

Publications and source records attributed to P Neubauer.

28 records · Page 2Linked to original sources

PubMed1996

Impact of plasmid presence and induction on cellular responses in fed batch cultures of Escherichia coli.

Fed batch cultivations of plasmid-free and recombinant Escherichia coli were employed in order to determine cellular responses and effects of plasmid presence and induction on the host cell physiology. While plasmid presence was shown to have minor influence on overall biomass yield, induction with 0.1 mM IPTG led to a marked reduction. The number of dividing cells, measured as colony forming ability, was influenced by plasmid presence and to a larger extent by induction. The latter caused a decline in the number of dividing cells to less than 10% of the population within 10 h. However, this cell segregation did not affect the specific rate of product formation, which was approximately constant throughout the cultivations. Analysis of the in vivo degradation rate of the product indicated that it was proteolytically stable. The cellular content of the stringent response signal substance, ppGpp, peaked immediately after transition from batch to fed batch mode to stabilise at a higher value than in the batch phase. When the specific growth rate declined below 0.06 h-1 an additional rise in ppGpp concentration was observed.

Cell Division↗

PubMed1995

Response of guanosine tetraphosphate to glucose fluctuations in fed-batch cultivations of Escherichia coli.

A rapid transient increase of guanosine 3'-diphosphate 5'-diphosphate (ppGpp) in Escherichia coli was found in response to short-term glucose fluctuations that may occur in large-scale fed-batch cultivations. The concentration of ppGpp was measured in laboratory-scale glucose limited fed-batch cultivations. Starvation zones were imitated by using an intermittent feeding scheme or a two-compartment reactor system. The cellular concentration of ppGpp per biomass increased from 80 nmol to 300-600 nmol per g cell dry weight within only 1 min after consumption of the residual glucose in dependence on the test system, which is much faster than earlier described in literature. Readdition of glucose caused immediate reduction of the ppGpp to the basic level which did not differ in cultivations with simulated starvation zones from control cultivations. Possible physiological consequences by an enhanced stringent response in cultivations with limited mass transfer have to be considered.

Biotechnology↗

PubMed1994

Efficient use of lactose for the lac promoter-controlled overexpression of the main antigenic protein of the foot and mouth disease virus in Escherichia coli under fed-batch fermentation conditions.

Derivatives of the lac promoter (tac, pac, rac) belong to the strongest bacterial promoters which are frequently used for the induced overexpression of foreign genes in Escherichia coli. However, their use in fermentation processes is strongly restricted because of the high cost of the inducer iso-propyl-beta-D-thiogalactopyranoside (IPTG). The aim of this work was to investigate the possibility of using lac-derived promoters in high cell density processes resulting in a high yield of the induced recombinant protein if glucose is the main carbon and energy source. Lactose is tested as inducer of the main antigenic coat protein (VP1) of the foot and mouth disease (FMD) virus in a T7-RNA polymerase expression system. It was shown that lactose is able to induce the expression of the recombinant gene to an amount of the VP1 protein corresponding to 20% of the total cell protein.

Antigens, Viral↗

PubMed1992

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells.

A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.

Alkaline Phosphatase↗

PubMed1992

Maximizing the expression of a recombinant gene in Escherichia coli by manipulation of induction time using lactose as inducer.

The use of isopropyl-beta-D-thiogalactoside (IPTG) for induction of the lac-promoter in small-scale cultivations is well established. However, for large-scale microbiological processes the cost of this inducer is a severe limitation. Here is described a method by which lactose is used as inducer of the lac promoter with the same efficiency as that of IPTG. It was found that after growth on glucose the time of the addition of lactose is important for the quality of induction. The resulting yield of the recombinant protein increased when lactose was added to the culture if the glucose concentration was rather low. By careful monitoring of the glucose level in the fermentation, using a biosensor, it was possible to add the inducer when the carbon source was nearly depleted. Using Escherichia coli BL21 (pET3), in which was cloned the main antigen coat protein of the foot and mouth disease virus, induction of the gene led to expression of the target protein at a level exceeding 20% of the total cell protein.

Aphthovirus↗

PubMed1991

Stringent control of replication of plasmids derived from coliphage lambda.

The first events of lambda plasmid replication in vivo, which probably regulate this process, are the transcriptional activation of the origin of replication by RNA polymerase and the binding of the initiator protein, lambda O, to this nucleotide sequence. The lambda O protein is known for its rapid proteolytic degradation; hence amino acid starvation of Escherichia coli should result in inhibition of lambda plasmid replication caused by inhibition of protein synthesis. However, contrary to this prediction, we found that lambda plasmid replication, as measured by the increase in plasmid content per bacterial mass, proceeds for hours in an amino acid-starved, relaxed mutant, whereas it is inhibited in its wild-type stringent partner. lambda plasmid replication in amino acid-starved, relaxed cells reveals absolute lambda O dependence and is not inhibited by chloramphenicol at 200 micrograms/ml. This process also occurs in wild-type cells treated with chloramphenicol. We conclude that lambda plasmid replication is under stringent control, probably as a result of the action of ppGpp, the indirect product of the relA gene, on RNA polymerase. The problem of stability of the lambda O initiator protein is discussed.

Bacteriophage lambda↗

PubMed1991

[Unusual course of acute leukemia with massive CNS involvement].

The article presents the case of a 37-year-old patient with acute leukemia. Four days before the appearance of acute symptoms, the patient manifested unspecific prodromes, with headache, coughing, and tiredness. Within 18 hours, he then presented the complete picture of acute leukemia, with multiple brain hemorrhages, diffused leukemic infiltration of all organs, and the typical alterations in blood picture. Eighteen hours after the first specific symptoms, the patient died of therapy-resistant cerebral pressure.

Acute Disease↗

PubMed1990

Amplification of pBR322 plasmid DNA in Escherichia coli relA strains during batch and fed-batch fermentation.

Fermenter studies under batch and fed-batch conditions were carried out to test the possibility of plasmid pBR322 production in large amounts by using E. coli relA strains. High amplification rates of pBR322 plasmid DNA were observed in E. coli CP79 (relA) and E. coli CP143 (relA) in both batch and fed-batch cultivation after exhaustion of the amino acid arginine. The concentrations of plasmid DNA per unit of biomass were nearly the same in batch and in fed-batch fermentations of E. coli CP79 and E. coli CP143. Therefore, the significantly higher biomass concentration of the two strains after fed-batch fermentation gave a dramatic increase in the yield of plasmid DNA per litre of medium in comparison to the batch process. The results support the suggestion that E. coli relA strains are suitable hosts for production of large amounts of ColE1-derived plasmids for recombinant DNA research.

DNA, Bacterial↗

PubMedDate not supplied

Growth rate related concentration changes of the starvation response regulators sigmaS and ppGpp in glucose-limited fed-batch and continuous cultures of Escherichia coli.

The entry of Escherichia coli into glucose starvation is connected to the induction of both the stringent response and the general stress response. The concentration of the corresponding response regulators ppGpp and sigmaS (RpoS, KatF) was investigated during glucose-limited growth at different growth rates and with different cultivation strategies, including fed-batch fermentation, chemostat, and a continuous-cultivation technique with computer-controlled decrease of the feeding rate (A-stat). This study confirms the model of the in vivo effect of ppGpp on sigmaS by the direct measurement of ppGpp and sigmaS levels. The response of both regulators was clearly shown in situations where a nutrient shift occurs. A correlation in the concentration of both regulators was also shown under steady-state conditions. However, during transients with slow continuous changes of the nutrient availability, this concerted reaction of ppGpp and sigmaS is less apparent, indicating the specific importance of these regulators for the adaptation of the cells to fast changes of environmental parameters.

Bacterial Proteins↗

PubMedDate not supplied

Evaluation of a province-wide physiotherapy monitoring service in an arthritis control program.

We evaluated our rheumatoid arthritis patient follow-up service in British Columbia in a 1 yr prospective study. The follow-up by 45 specially trained Arthritis Society physiotherapists involved contact of 1,701 patients; 1,081 were assessed and 440 were judged to need therapy or physician services; 279 required further physiotherapy, 191 had functional problems needing occupational therapy and 108 had medical problems requiring return to the family doctor. Within 2 months 72% of patients requiring physiotherapy and 84% of patients requiring occupational therapy had received their therapy. The study underlines the value of physiotherapy follow-up monitoring of rheumatoid patients.

Arthritis, Rheumatoid↗