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Biomedical subjects

J E Bailey

Publications and source records attributed to J E Bailey.

At least 163 records · Page 9Linked to original sources
PubMed1989

Effect of iodine-treated water on the performance of broiler chickens reared under various stocking densities.

A total of 544 day-old sexed broiler chicks were used to investigate the effects of 2 ppm iodine-treated water on body weight and feed efficiency when the chicks were reared at 900, 720, 540, and 360-cm2 stocking densities. Chicks were fed a corn-soybean meal diet calculated to contain 3,212 kcal ME/kg and 21.58% CP. Iodine added to the drinking water at the rate of 2 ppm improved growth significantly after 4 wk of age. Feed efficiency was not significantly affected by the addition of iodine to the drinking water. Maximum effects on growth were obtained when the broilers were housed at stocking densities of 720 and 540 cm2/bird. A stocking density of 360 cm2 appeared to provide a crowding stress severe enough to negate the beneficial effects of adding iodine to the drinking water.

Animals↗
PubMed1989

A new oxygen-regulated promoter for the expression of proteins in Escherichia coli.

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.

Bacterial Proteins↗
PubMed1988

A mathematical model of recombinational amplification of the 2 mu plasmid in the yeast Saccharomyces cerevisiae.

A mathematical model of 2 mu plasmid recombinational amplification in Saccharomyces cerevisiae has been developed, based on mechanisms of 2 mu recombination and replication presented in the literature. A probabilistic description reveals the limits inherent in the recombinational mode of plasmid amplification. These limits correspond well with values calculated from reported results. In the model, copy number control is effected by the constitutive expression of a repressor of recombinase expression. Estimation of the model parameters is accomplished via a set of heuristic rules which restrict the feasible parameter space considerably. It is demonstrated that many parameter sets arbitrarily chosen from the feasible parameter space reproduce the observed characteristics of 2 mu plasmid amplification: rapid correction of downward copy number deviations, with a lack of strict control of steady-state copy number.

DNA Replication↗
PubMed1988

Heterologous expression of a bacterial haemoglobin improves the growth properties of recombinant Escherichia coli.

Rational design of novel as well as improved cellular biocatalysts by genetic manipulation of cellular metabolism has recently attracted considerable interest. A wide range of bacteria have been genetically modified by integrating new enzymatic functions into their metabolic network. A central problem in the aerobic growth of any cell culture is the maintenance of dissolved oxygen (DO) concentrations above growth-limiting levels especially in high cell-density fermentations which are usually of a fed-batch type. The optimal rate of nutrient addition (and consequently the productivity) is ultimately limited by the rate at which cells can aerobically catabolize the carbon source without generating growth-inhibitory metabolites such as lactate and acetate. All approaches thus far have concentrated on improving the oxygen mass transfer rates by manipulating various environmental parameters. We have isolated the gene for a haemoglobin-like molecule, expressed by the aerobic bacterium Vitreoscilla in poorly-oxygenated environments, and expressed it in Escherichia coli. The recombinant cells contain enhanced haem as well as active haemoglobin, and they grow faster and to considerably greater cell densities than comparable plasmid-containing cells which do not express haemoglobin. This haemoglobin increases the rate of oxygen use, especially when dissolved oxygen is less than 5% of air saturation.

Aerobiosis↗
PubMed1988

A single-cell assay of beta-galactosidase activity in Saccharomyces cerevisiae.

A novel assay of single-cell exogenous beta-galactosidase activity in Saccharomyces cerevisiae has been developed. Intracellular fluorescence due to the hydrolysis of resorufin-beta-D-galactopyranoside attains a steady state between production of resorufin and its subsequent leakage from the cell. The cells are permeabilized with Triton X-100, and the assay is performed at 0 degrees C. These conditions were chosen to minimize intercellular fluorescence communication. Free resorufin in the extracellular space is bound by bovine serum albumin to prevent its uptake by cells. Two regimes of fluorescence accumulation are observed, one limited by the rate of diffusion of substrate into the cell, and one limited by the rate of enzymatic cleavage of the substrate. A quantitative correlation between fluorescence and beta-galactosidase activity is obtained under optimized assay conditions.

Cell Separation↗
PubMed1988

The Vitreoscilla hemoglobin gene: molecular cloning, nucleotide sequence and genetic expression in Escherichia coli.

Vitreoscilla hemoglobin is involved in oxygen metabolism of this bacterium, possibly in an unusual role for a microbe. We have isolated the Vitreoscilla hemoglobin structural gene from a pUC19 genomic library using mixed oligodeoxy-nucleotide probes based on the reported amino acid sequence of the protein. The gene is expressed in Escherichia coli from its natural promoter as a major cellular protein. The nucleotide sequence, which is in complete agreement with the known amino acid sequence of the protein, suggests the existence of promoter and ribosome binding sites with a high degree of homology to consensus E. coli upstream sequences. In the case of at least some amino acids, a codon usage bias can be detected which is different from the biased codon usage pattern in E. coli. The downstream sequence exhibits homology with the 3' end sequences of several plant leghemoglobin genes. E. coli cells expressing the gene contain greater than fivefold more heme than controls.

Amino Acid Sequence↗
PubMed1987

Determination of p-cresidine in FD&C red no. 40 by the diazotization and coupling procedure followed by reversed-phase high-performance liquid chromatography.

The free aromatic amine p-cresidine is determined at low ppb (10(9] levels in the regulated color additive FD&C Red No. 40. The determination involves chloroform extraction of p-cresidine and other unsulfonated impurities from the color additive, followed by diazotization of the aromatic amines and coupling with the disodium salt of 3-hydroxy-2,7-naphthalenedisulfonic acid (R-salt). The coupling products are then determined by reversed-phase high-performance liquid chromatography. Calibration is performed in the presence of the color additive by using the external standard method. The identity of the p-cresidine coupling product is confirmed by obtaining a UV-VIS spectrum of the eluting solute. The presence of aniline in commercial batches is also confirmed in this manner by using 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid (pyrazolone-T) as an alternative coupling agent.

Aniline Compounds↗
PubMed1986

Flow cytometry analysis of recombinant Saccharomyces cerevisiae populations.

A new fluorescent stain has been developed for detecting cloned beta-galactosidase activity in individual cells of Saccharomyces cerevisiae by flow cytometry. The staining reaction is based on enzymatic cleavage of alpha-naphthol-beta-D-galactopyranoside by intracellular beta-galactosidase and trapping of the liberated naphthol by hexazoniumpararosaniline yielding a fluorescent, insoluble end product. This stain, in connection with an appropriate host strain, has been applied for detecting plasmids encoding inducible beta-galactosidase in unstable recombinant cell populations carrying plasmids with different origins of replication. The method enables rapid determination of the fraction of plasmid-containing cells as well as quantitation of intracellular beta-galactosidase content by kinetic enzyme assay. Inducibility of the marker enzyme is important for maintaining correlation between enzyme and gene content.

Flow Cytometry↗
PubMed1986

Studies of host-plasmid interactions in recombinant microorganisms.

Plasmid genes redirect some components of cellular metabolism into synthesis of plasmid gene products and additional plasmids. The stoichiometric and kinetic implications of these host-plasmid interactions have been investigated theoretically and experimentally. Using known pathway energetics, maximum theoretical yield factors based on ATP, glucose, and O2 have been estimated for recombinant Escherichia coli and compared with corresponding estimates for host cells alone, indicating major changes in carbon and energetic stoichiometry in recombinant cells in cases of high cloned gene expression. The influence of the number of plasmids in recombinant E. coli has been experimentally characterized using a series of pMB1 derivatives stably propagated at copy numbers from 12 to 408. Recombinant cell growth rate declines monotonically as plasmid content increases as does efficiency of plasmid gene expression. A detailed metabolically structured single-cell model for E. coli has successfully simulated these trends. Interrelationships among number of plasmids per cell, induction of expression of a plasmid gene, and recombinant population growth rate have been experimentally delineated for Saccharomyces cerevisiae containing plasmid pLGSD5 and derivatives in which the 2-micron origin of replication has been replaced by a cloned ARS1 sequence or its deletion fragments. The CEN4 centromere sequence has been included in some of these plasmids to provide more regular segregation. Specific growth rate of these recombinant yeasts exhibits a maximum as a function of plasmid content, an effect attributed to the interplay between beneficial effects of the plasmid in selective medium and parasitic effects on metabolism at larger plasmid content or with more plasmid gene expression activity. The yeast strains investigated exhibit substantial segregational instability that was characterized using a rapid-flow cytometry measurement based upon single-cell deletion of E. coli beta-galactosidase activity in recombinant cells.

DNA, Recombinant↗
PubMed1986

Performance and clinical utility of a commercially available 'C-terminal' PTH assay.

The performance and clinical utility of a 'C-terminal' parathyroid hormone (PTH) radioimmunoassay (Dac-Cel, Wellcome Diagnostics) is described. Parathyroid hormone, as measured by the Dac-Cel method, is stable in whole blood samples for at least 24 h. 84% of patients with hypercalcaemia due to primary hyperparathyroidism have values above the upper limit seen in normocalcaemic subjects (0.5 micrograms/L), with detectable serum PTH demonstrable in the remaining 16%. In patients with hypocalcaemia due to hypoparathyroidism serum PTH was undetectable in 73% and 'inappropriately' low in the remainder. In 50% of patients with malignancy-associated hypercalcaemia serum PTH was undetectable, but was above 0.5 micrograms/L in 13%. Increased PTH concentrations were invariably found in patients with renal failure. The Dac-Cel method is a reliable and robust technique for measurement of PTH and in conjunction with determination of calcium facilitates the diagnosis of primary parathyroid disorders. Caution is required in the interpretation of PTH measurements in patients with renal failure; the significance of detectable PTH in some patients with malignancy-associated hypercalcaemia is not clear.

Adult↗
PubMed1985

Determination of the lower sulfonated subsidiary colors in FD&C Yellow No. 6 by reversed-phase high-performance liquid chromatography.

Data are presented for the determination of 5-(phenylazo)-6-hydroxynaphthalene-2-sulfonic acid (ANSC) and 4-[(2-hydroxynaphthalene-1-yl)azo]benzenesulfonic acid (BNSC) in FD&C Yellow No. 6 by reversed-phase high-performance liquid chromatography (RP-HPLC). The method employs gradient elution with a water-tetrahydrofuran solvent system buffered with ammonium acetate and quantitation in the UV (254 nm) and visible (490 nm) regions. The external calibration method was used with twelve data points for each analyte. Each analysis was performed in duplicate. In analyses of commercial FD&C Yellow No. 6, ANSC was found at levels ranging from none to 0.52, and BNSC was found at levels ranging from none to 0.73%. The identities of the analytes were confirmed by using a rapid-scan diodearray spectrophotometer to obtain the electronic absorption spectra during RP-HPLC analysis.

Azo Compounds↗
PubMed1985

Determination of unsulfonated aromatic amines in FD&C Yellow No. 6 by the diazotization and coupling procedure followed by reversed-phase high-performance liquid chromatography.

Data are presented for the determination of parts-per-billion (10(9)) levels of aniline, benzidine, 4-aminobiphenyl (4-ABP) and 4-aminoazobenzene in the regulated color additive FD&C Yellow No. 6. The determination involves chloroform extraction of the amines from the color additive, followed by diazotization and coupling with the disodium salt of 3-hydroxy-2,7-naphthalenedisulfonic acid (R-salt). The coupling products are then analyzed by reversed-phase high-performance liquid chromatography. An interference discovered during the determination of 4-ABP required the use of 4,5-dihydro-5-oxo-1-(4-sulfophenyl)-1H-pyrazole-3-carboxylic acid (pyrazolone-T) as an alternative coupling agent. The identity of each coupling product is confirmed by obtaining a UV-visible spectrum of the eluting solute. The liquid chromatograph is calibrated in the presence of the color additive by using the external standard method.

Amines↗
PubMed1985

Determination of 1,3-diphenyltriazene and azobenzene in D&C Red No. 33 by solvent extraction and reversed-phase high-performance liquid chromatography.

Data are presented for the determination of trace levels of 1,3-diphenyltriazene (DPT) and azobenzene (AB) in D&C Red No. 33. The contaminants are extracted with chloroform from an aqueous solution of the color, and the chloroform is removed under vacuum. The residue is dissolved in acetonitrile and the solution is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with detection at 365 nm. DPT and AB were determined at levels of less than or equal to 439 ppb and less than or equal to 2.2 ppm, respectively, in samples of commercial D&C Red No. 33. Method precision for the DPT determination (five analyses) was 6.8% relative standard deviation. The UV-Vis spectra of the HPLC analytes, obtained by using a rapid-scan diode-array spectrophotometer, were used to confirm the identity of the DPT and AB responses produced during analysis of the D&C Red No. 33 extracts. The method is also useful for the separation of cis- and trans-AB.

Aniline Compounds↗
PubMed1985

Effect of ARS1 mutations on chromosome stability in Saccharomyces cerevisiae.

We have used a set of deletion mutations in the ARS1 element of Saccharomyces cerevisiae to measure their effect on chromosome stability. This work establishes the previously proposed existence of three domains in ARS1. Domain C, which we have previously inferred, but not proved, to be a part of ARS1, is now established. In addition, we show that increasingly large deletions of the domain have increasingly large effects, which was not realized before. Furthermore, we have provided the first positive evidence for the central importance of a 14-base-pair core sequence containing the ARS consensus element by showing that it has the ability to act as a replicator on a plasmid containing no other ARS1 flanking sequence. The method of analyzing plasmid stability used in our study employs a novel and sensitive flow cytometry assay for beta-galactosidase. We discuss ways in which flow cytometry, based on this assay, could be generalized beyond its particular application in this work to studying other aspects of the cell biology of yeast and higher cells. The actual flow cytometry method will be described in detail elsewhere.

Chromosomes↗