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J E Bailey

Publications and source records attributed to J E Bailey.

At least 145 records · Page 8Linked to original sources
PubMed1991

Purification and aqueous two-phase partitioning properties of recombinant Vitreoscilla hemoglobin.

Soluble recombinant Vitreoscilla hemoglobin was purified from E. coli lysate by sequential two-phase extraction techniques. Extraction of lysate containing VHb in PEG/dextran gave a 3.6-fold increase in VHb purity in the PEG-rich phase via a size exclusion mechanism. Further extraction of the recovered PEG phase in PEG/sodium sulfate gave an additional 2.0-fold increase in purity in the PEG-rich phase due to an electrostatic mechanism. Final extraction of the PEG phase in PEG/magnesium sulfate gave an additional 1.3-fold increase in VHb purity in the magnesium sulfate-rich phase. The final yield from the extractive purification was 47% with purity of VHb estimated to be greater than 95%. Yields from the sulfate salt extractions are essentially quantitative due to the extreme partitioning behavior of VHb in these systems. VHb partition coefficients as large as 46 in PEG/sodium sulfate and as small as 0.06 in PEG/magnesium sulfate were observed. Similar small partition coefficients were obtained with PEG/manganese sulfate extractions. This dramatic effect of divalent cation content on the partition coefficient of VHb in PEG/sulfate salt systems was investigated by pH and magnesium ion titration experiments. Results show the effect to be largest and nearly constant for pH values greater than 6.0 and diminished at lower pH values. A model based on magnesium ion binding to negatively charged amino acids is shown to correlate with the data well. Based on model formulation and the partitioning behavior of contaminant proteins, the observed effect is expected to be applicable to other proteins.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Temperature and induction effects on the degradation rate of an abnormal beta-galactosidase in Escherichia coli.

Intracellular protein degradation was investigated using an unstable fragment of Escherichia coli beta-galactosidase, the CSH11 mutant, as a model protein. This abnormal protein was expressed from a single copy gene in the chromosome and is converted to a detectable degradable intermediate. The in vivo degradation rates of both beta-galactosidase fragments were measured using pulse-chase radioactive labeling techniques, and their intracellular concentrations were determined using alpha-complementation assays. In the physiological range of 30 to 37 degrees C, the apparent degradation rate constant for the CSH11 fragment follows Arrhenius behavior; while the intermediate's apparent degradation rate constant is nearly unchanged. However, above 37 degrees C the degradation rates of both fragments increase significantly. Analysis of the labeled intermediate's rate of change above 40 degrees C reveals that the CSH11 fragment is being degraded by a second pathway which does not produce the intermediate. When the induction level of the abnormal beta-galactosidase was varied the degradation rates of both fragments behaved similarly, but they unexpectedly decreased with increasing IPTG concentration. The two parallel degradation pathways for CSH11 apparently operated at only the lower IPTG levels. The measured degradation rates did not correlate directly with the intracellular concentration of abnormal proteins.

Escherichia coli↗
PubMed1991

Actinorhodin production by Streptomyces coelicolor and growth of Streptomyces lividans are improved by the expression of a bacterial hemoglobin.

Secondary metabolite production by Streptomyces is often highly sensitive to oxygen supply, which can be limiting in large-scale fermentations. In an attempt to improve oxygen utilization by the cells, we expressed a heterologous bacterial hemoglobin gene in Streptomyces coelicolor and Streptomyces lividans. Hemoglobin expression was demonstrated by immunoblot analysis and carbon monoxide binding activity. In batch fermentations run under reduced aeration, the expression of hemoglobin in S. coelicolor resulted in a ten-fold increase in specific yields of the aromatic polyketide, actinorhodin. Actinorhodin yields were also much less sensitive to aeration conditions in the hemoglobin-expressing strain. In addition, hemoglobin-expressing S. lividans cells grown under reduced aeration had higher final cell densities and exhibited greater oxygen consumption rates than non-expressing cells.

Anthraquinones↗
PubMed1990

Protein composition of Vitreoscilla hemoglobin inclusion bodies produced in Escherichia coli.

The protein composition of inclusion bodies produced in recombinant Escherichia coli overproducing Vitreoscilla hemoglobin (VHb) was analyzed by one-dimensional and two-dimensional electrophoresis techniques. Results indicate the presence of two types of cytoplasmic aggregates of differing morphology in single bacterial cells. These aggregates also differ in their relative content of VHb and pre-beta-lactamase and are separable by differential centrifugation. Results further suggest that the cytoplasmic protein elongation factor Tu is integrated into VHb inclusion bodies. The presence of the outer membrane proteins OmpA and OmpF in inclusion body preparations is attributed to cell envelope contamination rather than specific involvement in inclusion bodies. The specificity of in vivo protein aggregation is discussed.

Animals↗
PubMed1990

An integrated modeling-experimental strategy for the analysis of metabolic pathways.

An inherent problem in studying the behavior of a metabolic pathway is the impossibility of developing a complete, detailed model that includes all the cellular processes that have an impact on the set of fluxes in such a pathway. Lacking this, one requires some means of modeling the interactions between a metabolic pathway and other cellular processes for the purpose of analyzing pathway characteristics within the cell (e.g., determining sensitivity coefficients for various steps in the pathway) with a minimal amount of time and effort. A general framework is developed for studying these issues in a rigorous manner. Using this framework, detailed knowledge about a metabolic pathway (i.e., a set of rate expressions for steps in the pathway) can be combined with the results from a relatively simple set of experiments in order to obtain estimates for the sensitivity of the pathway to enzyme activities, inhibition constants, and other parameters that determine the pathway's behavior, while accounting for the pathway's interaction with the rest of the cellular metabolism. A model system representing amino acid production is used to illustrate the problem and to provide results based on computational experiments. The modeling strategy described here should be useful in genetic design to improve pathway fluxes and metabolic network selectivity.

Amino Acids↗
PubMed1990

DNA distribution and respiratory activity of Spodoptera frugiperda populations infected with wild-type and recombinant Autographa californica nuclear polyhedrosis virus.

Spodoptera frugiperda cells were infected with a wild-type Autographa californica nuclear polyhedrosis virus and with a recombinant Autographa californica nuclear polyhedrosis virus. The recombinant virus was derived from the wild-type virus and produced beta-galactosidase instead of polyhedrin. The changes in cell size, cell growth, viability, DNA distribution, and respiratory activity were followed through the time course of the infection. The DNA content as measured by flow cytometry of infected cells increased to approximately 1.8 times the value of uninfected cells and the distributions of single-cell DNA content of the infected cells were strongly deformed. Early in the infection the respiratory activity passed through a maximum. The mitochondrial activity based on Rhodamine 123 labelling of cells infected with the recombinant virus, as determined by flow cytometry, also passed through a maximum at 24 h post infection while the mitochondrial activity of cells infected with the wild-type virus continued to increase. Evolution of single-cell mitochondrial activity was different in uninfected populations and in populations infected with wild-type and with recombinant virus. In all experiments performed, the recombinant virus influenced cell behavior and the measured parameters earlier than the wild-type virus. The influence of the multiplicity of infection was stronger for the wild-type virus than for the recombinant virus.

Animals↗
PubMed1990

Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter.

The oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.

Bacteria↗
PubMed1990

Expression of intracellular hemoglobin improves protein synthesis in oxygen-limited Escherichia coli.

We have previously cloned the Vitreoscilla hemoglobin gene (VHb) and expressed the protein in Escherichia coli in its active form. Under oxygen-limited conditions the presence of VHb improves protein synthesis as indicated by both total protein content and the activity of an enzyme expressed from a cloned gene present on a multicopy plasmid. Measurements of nitrogen utilization rates corroborate the observation of enhanced protein synthesis; however, the rates of carbon consumption and acid synthesis remain unchanged. This suggests that the net effect of VHb in E. coli is to improve the efficiency, rather than the kinetics, of oxygen-limited aerobic metabolism. We propose two possible models for the mechanism of action of VHb: the facilitated diffusion hypothesis and the intracellular redox effector hypothesis. These suggest other systems in which cloned VHb may enhance bioprocess productivity.

Bacterial Proteins↗
PubMed1990

Effect of information about waiting lists on referral patterns of general practitioners.

In a two year trial general practitioners in the West Midlands were provided with extended waiting time information for hospital consultation and treatment in general surgery. Selected general practitioners were sent monthly bulletins on comparative times to wait for both outpatient appointment and inpatient treatment throughout the region. Their referrals to consultant general surgeons were monitored, alongside those of a matched control group not receiving such information. Differences were found between the two groups which indicate the willingness of general practitioners to change their referral practice when adequate information is available. In addition, patients referred to their local hospital had longer waiting times than patients matched for clinical condition and district of origin who were sent to hospitals where it was indicated that a shorter wait might be expected.

Appointments and Schedules↗
PubMed1990

Development of an instrument for the management of computer user attitudes in hospitals.

Measuring and managing user attitudes toward various aspects of computer systems is an important part of making those systems effective. In this paper, results of an effort to adapt and test a technique for measuring user satisfaction in hospitals are presented. These results include extensive empirical tests of the technique. Comparison norms for hospital computer user satisfaction are also presented and interpreted. Analysis software for using the technique as a management tool is presented. Finally, experiences using the concept are presented.

Attitude to Computers↗
PubMed1989

Evidence for partial export of Vitreoscilla hemoglobin into the periplasmic space in Escherichia coli. Implications for protein function.

The Vitreoscilla hemoglobin protein has been implicated in earlier studies to serve a globin-like function under oxygen-limited growth conditions. Evidence is presented using fractionation as well as proteinase K accessibility techniques to prove that a considerable amount of this protein is localized in the periplasmic space of the cell. Genetic evidence points towards the existence of information within the N-terminal domain of the protein that plays a role in the process of protein export. However, this sequence is not cleaved in the process of translocation. Analysis of the primary structure of this region reveals several unusual features. Instead of positively charged residues at its amino terminus, it has a negative charge. The overall hydrophobicity of the central region of this sequence is significantly lower than in typical leader peptides due to the presence of a charged residue. In keeping with the likelihood that such an export signal may not be very efficient, a substantial fraction of the total cellular hemoglobin can also be detected in the cytoplasm. Heme is incorporated in both cytoplasmic and periplasmic globin as indicated by the ability of protein from both fractions to bind carbon monoxide. The secretion of this protein into the periplasm raises questions concerning the physiological significance of its localization. Dimensional analysis of a model based on the facilitated diffusion hypothesis, which was initially proposed to account for the effects of eukaryotic globins on oxygen transport, suggests that periplasmic globin can support an additional oxygen flux to the respiratory apparatus that may be physiologically significant.

Amino Acid Sequence↗
PubMed1989

Application of flow cytometric measurement of surface IgG in kinetic analysis of monoclonal antibody synthesis and secretion by murine hybridoma cells.

The kinetics of antibody synthesis and secretion from a murine hybridoma cell line were studied using measurements of total cell-associated IgG, surface IgG, and IgG secreted into the medium. Kinetic analysis of IgG secretion demonstrates approximately constant secretion rate per viable cell over the entire batch cultivation. A correlation was observed (r2 = 0.74) between mean surface immunofluorescence and the total cell-associated IgG determined by ELISA of detergent-extracted cell lysates. No correlation was found between specific secretion rate and mean surface IgG level estimated by immunofluorescence flow cytometry measurements. Material balances on cellular IgG demonstrated that about 7% of the antibody which was synthesized during exponential batch growth was not released to the growth medium. Distributions of single-cell surface antibody content showed two subpopulations, one with very low surface IgG. The fraction of the population with low surface IgG increased throughout a batch cultivation.

Animals↗
PubMed1989

Structure-function relationships in the inorganic salt-induced precipitation of alpha-chymotrypsin.

alpha-Chymotrypsin (alpha CT) was used as a model protein to study the effects of salt-induced precipitation on protein conformation. Process parameters investigated included the type and amount of salt used to induce precipitation. The salts studied included Na2SO4, NaCl, NaBr, KBr and KSCN. Precipitate secondary structure content was examined via laser Raman spectroscopy. Conventional and saturation transfer electron paramagnetic resonance spectroscopy were employed to probe the tertiary structure of the active site in spin-labelled alpha CT precipitates. As the molal surface tension increment of the inducing salt increased, the beta-sheet content increased and the alpha-helix content decreased. There was no significant variation in secondary structure with the amount of salt used. The fraction of precipitate that recovered activity on redissolution was correlated with the change in secondary structure content. Spin-labelled precipitate spectra indicated that the active site remains unaltered during precipitation. Molecular modelling was employed to investigate how physical property of alpha CT were affected by these types of conformational change. Estimated physical property changes could not account entirely for observed deviations from current equilibrium theory for salt-induced precipitation. The spectroscopic observations were also combined with activity/solubility results to propose a mechanism for the salt-induced precipitation of globular proteins.

Animals↗
PubMed1989

Isolation, characterization and determination of trace organic impurities in FD&C red no. 40.

The unsulfonated aromatic amine 4-nitro-p-cresidine (2-methoxy-5-methyl-4-nitrobenzenamine) was identified as an impurity in the regulated color additive FD&C Red. No. 40. The compound was isolated from the water-soluble color by extraction with chloroform, followed by transfer of the free amines to acid solution and subsequent separation by reversed-phase high-performance liquid chromatography. The 4-nitro-p-cresidine was collected and then identified by gas chromatography-mass spectrometry. The levels of 4-nitro-p-cresidine as well as p-cresidine and aniline were determined in commercial batches of FD&C Red. No. 40.

Aniline Compounds↗
PubMed1989

Characterization of the oxygen-dependent promoter of the Vitreoscilla hemoglobin gene in Escherichia coli.

The gene coding for the Vitreoscilla hemoglobin (VHb) molecule has been cloned and functionally expressed in Escherichia coli. By using a plasmid-encoded gene as well as single-copy integrants, the oxygen-dependent VHb gene (VHb) promoter was shown to be functional in E. coli. The promoter was maximally induced under microaerobic conditions (dissolved oxygen levels of less than 2% air saturation). Direct analysis of mRNA levels as well as the use of gene fusions with lacZ showed that oxygen-dependent regulation occurred at the level of transcription. Transcriptional activity decreased substantially under anaerobic conditions, suggesting the presence of a regulatory mechanism that is maximally induced under hypoxic but not completely anaerobic conditions in E. coli. Primer extension analysis was used to identify the existence of two overlapping promoters within a 150-base-pair region upstream of the structural VHb gene. The oxygen-dependent activity of both promoters was qualitatively similar, suggesting the existence of a common mechanism by which available oxygen concentrations influence expression from the two promoters. Analysis of promoter activity in crp and cya mutants showed that both cyclic AMP and catabolite activator protein were required for full activity of the promoter. The VHb promoter contained a region of significant homology to the catabolite activator protein-binding site near the E. coli lac promoter.

Aerobiosis↗
PubMed1989

DNA polymerase I is required for premeiotic DNA replication and sporulation but not for X-ray repair in Saccharomyces cerevisiae.

We have used a set of seven temperature-sensitive mutants in the DNA polymerase I gene of Saccharomyces cerevisiae to investigate the role of DNA polymerase I in various aspects of DNA synthesis in vivo. Previously, we showed that DNA polymerase I is required for mitotic DNA replication. Here we extend our studies to several stages of meiosis and repair of X-ray-induced damage. We find that sporulation is blocked in all of the DNA polymerase temperature-sensitive mutants and that premeiotic DNA replication does not occur. Commitment to meiotic recombination is only 2% of wild-type levels. Thus, DNA polymerase I is essential for these steps. However, repair of X-ray-induced single-strand breaks is not defective in the DNA polymerase temperature-sensitive mutants, and DNA polymerase I is therefore not essential for repair of such lesions. These results suggest that DNA polymerase II or III or both, the two other nuclear yeast DNA polymerases for which roles have not yet been established, carry out repair in the absence of DNA polymerase I, but that DNA polymerase II and III cannot compensate for loss of DNA polymerase I in meiotic replication and recombination. These results do not, however, rule out essential roles for DNA polymerase II or III or both in addition to that for DNA polymerase I.

DNA Polymerase I↗