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Biomedical subjects

J E Bailey

Publications and source records attributed to J E Bailey.

At least 19 recordsLinked to original sources

The effects of clonidine on cardiovascular responses to standing in healthy volunteers.

This study assessed the effects of clonidine on blood pressure (BP) and heart rate responses to active standing, recorded continuously using a Finapres monitor. Ten subjects were given a placebo infusion over 1 h, followed by clonidine hydrochloride 1.5 micrograms/kg over 2 h. During placebo and at 1, 3 and 19 h following the clonidine infusion, heart rate and blood pressure were recorded during the second half of supine rest for 10 min, active standing and quiet standing for 7 min. Clonidine did not alter the size of immediate drop in BP on standing, although the nadir was lower. BP recovery was impaired, with a loss of the usual BP overshoot in most subjects and with delays in reaching supine levels of diastolic BP (6.1 versus 9.6 s; p < 0.01) and systolic BP (8.1 versus 12.3 s; p < 0.05). The compensatory initial heart rate rise was significantly increased from 47 to 53 beats/min (p < 0.05), although the peak rate reached was reduced from 114 to 104 beats/min (p < 0.05). These results demonstrate that impairment of central sympathetic vasomotor drive leads to a delay in BP recovery and loss of initial BP overshoot immediately after standing, together with impaired maintenance of early steady-state BP.

Adult

Sponge-like electrophoresis media: mechanically strong materials compatible with organic solvents, polymer solutions and two-dimensional electrophoresis.

A new range of sponge-like media, called "electrophoresis sponges", is presented. They differ from electrophoresis gels primarily in that they are mechanically stronger, providing a permanent structure of directly measurable pore size dimensions. The new media are similar to capillary electrophoresis in terms of pore size range, they are mechanically strong with directly definable walls, and are compatible with polymer solutions. The sponges differ from capillary electrophoresis in that they provide large numbers of channels, with a corresponding high load capacity for simultaneous runs in multiple channels and they are compatible directly with multi-dimensional separations, such as high resolution two-dimensional electrophoresis. Furthermore, they can be molded (or cut) to any shape and retain that shape, they can be handled more easily than gels, they can be reused if necessary, they can be distributed in the same format between labs easily, and they can be stored indefinitely. Chemically, they can be hydrophilic or hydrophobic, with capability ranging from inert to reactive surfaces. Pore sizes can range from the sub-nanometer to 100 micron scale. Results with various hydrophobic sponges are reported for the carrier ampholyte-based isoelectric focusing of proteins. Broad and narrow pH gradients are established in the sponges that are more linear than those achieved with polyacrylamide gels. One- and two-dimensional electrophoresis of proteins has been achieved, for example with high resolution of the charge isomers of the haptoglobin beta chain, using sponge-based isoelectric focusing. Isoelectric focusing is about threefold faster in the tested sponges than in equivalent polyacrylamide gels. This improved speed is probably related to the larger sponge pores.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Gel, Two-Dimensional

A pharmacodynamic study of the alpha 2-adrenergic receptor antagonist ethoxyidazoxan in healthy volunteers.

Ethoxyidazoxan, a potent and highly selective alpha 2-adrenergic receptor antagonist, was administered intravenously to six healthy male volunteers in a double-blind, placebo-controlled, dose-rising design. Doses of 6 micrograms/kg and 8 micrograms/kg infused intravenously over 30 minutes produced significant elevations of plasma norepinephrine and body temperature and inhibited norepinephrine-induced platelet aggregation. Central activity was shown by reversal of the slowing of saccadic eye movements and an increase in saccade peak velocity at the higher dose. Modest increases in blood pressure were produced without change in heart rate. Ethoxyidazoxan was well tolerated, with slight changes in subjective alertness and sedation.

Adrenergic alpha-Antagonists

Photoreceptor orientation in iris coloboma.

PURPOSE: Investigate photoreceptor orientation and alignment in an observer with a simple coloboma of the iris and no retinal involvement. METHOD: Using a psychophysical procedure, extensive measurements of the Stiles-Crawford functions of the first kind (SCE I) were obtained from this observer. RESULTS: In this observer, the usual center-of-the-exit-pupil-of-the-eye alignment tendency of photoreceptors (inferred from the peaks of the SCE I functions) is absent. The inferred orientation of photoreceptors at numerous retinal loci tested lies outside the pupil, opposite the direction of the downwardly displaced pupil center. CONCLUSIONS: These results add support to previous suggestions that nonphototropic forces such as retinal tractional forces should be considered as an integral part of the mechanism(s) subserving human photoreceptor alignment in both normal and anomalous observers.

Coloboma

Effect of biosynthetic manipulation of heme on insolubility of Vitreoscilla hemoglobin in Escherichia coli.

Vitreoscilla hemoglobin (VHb) is accumulated at high levels in both soluble and insoluble forms when expressed from its native promoter on a pUC19-derived plasmid in Escherichia coli. Examination by atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy revealed that the insoluble form uniformly lacks the heme prosthetic group (apoVHb). The purified soluble form contains heme (holoVHb) and is spectroscopically indistinguishable from holoVHb produced by Vitreoscilla cells. This observation suggested that a relationship may exist between the insolubility of apoVHb and biosynthesis of heme. To examine this possibility, a series of experiments were conducted to chemically and genetically manipulate the formation and conversion of 5-aminolevulinic acid (ALA), a key intermediate in heme biosynthesis. Chemical perturbations involved supplementing the growth medium with the intermediate ALA and the competitive inhibitor levulinic acid which freely cross the cell barrier. Genetic manipulations involved amplifying the gene dosage for the enzymes ALA synthase and ALA dehydratase. Results from both levulinic acid and ALA supplementations indicate that the level of soluble holoVHb correlates with the heme level but that the level of insoluble apoVHb does not. The ratio of soluble to insoluble VHb also does not correlate with the level of total VHb accumulated. The effect of amplifying ALA synthase and ALA dehydratase gene dosage is complex and may involve secondary factors. Results indicate that the rate-limiting step of heme biosynthesis in cells overproducing VHb does not lie at ALA synthesis, as it reportedly does in wild-type E. coli (S. Hino and A. Ishida, Enzyme 16:42-49, 1973).

Bacterial Proteins

Characterization of inclusion bodies in recombinant Escherichia coli producing high levels of porcine somatotropin.

The protein composition of inclusion bodies (IBs) formed in recombinant Escherichia coli producing high levels of porcine somatotropin (pST) was analyzed by one- and two-dimensional protein gel electrophoresis. Recombinant pST is exclusively recovered from the insoluble cell fraction. Results indicate that, in addition to the main species of pST, subspecies with different isoelectric points and degradative fragments are contained within IBs. The presence of outer membrane proteins in IB fractions results from coprecipitation of cell debris during IB preparation and not from specific in vivo or in vitro interaction of these proteins with IBs. Cells producing pST contain up to three IBs located in the cytoplasm. The implication of high level gene expression on the uniformity of the desired product is discussed.

Animals

Proteolytic response to the expression of an abnormal beta-galactosidase in Escherichia coli.

Because induction of proteolytic activity and stress-response proteins can significantly affect expression levels in recombinant Escherichia coli, the influence of low-level expression of a mutant beta-galactosidase was investigated. A single copy of the well-characterized CSH11 mutant of the lacZ gene was integrated into the chromosome. Induction of expression of the mutant beta-galactosidase caused a measurable increase in ATP-dependent intracellular proteolytic activity but resulted in no significant change in ATP-independent proteolytic activity. Growth at temperatures above 40 degrees C resulted in a significant decrease in the level of ATP-independent proteolytic activity compared to growth at 37 degrees C, and the ATP-dependent activity increased 2.5-fold from 30 to 42 degrees C. Synthesis of stress-response proteins was evident in two-dimensional gel electrophoresis analysis of proteins in the strain expressing the abnormal beta-galactosidase at 37 degrees C, but no such response was evident when mutant beta-galactosidase expression was induced at 30 degrees C. In separate experiments, stress proteins were overexpressed by inducing expression of the htpR gene on a plasmid. Resulting increases in stress-protein levels correlated with an increase in ATP-dependent proteolytic activity with no significant change in the intracellular ATP-independent proteolytic activity. These data suggest that even very low levels of abnormal protein can substantially influence protease levels and stress response in E. coli. These responses were reduced by induction at lower temperatures.

Adenosine Triphosphate

Protein compositional analysis of inclusion bodies produced in recombinant Escherichia coli.

Culture conditions favouring the simultaneous formation of soluble protein and inclusion bodies (IBs) were chosen for producing the cytoplasmic protein beta-galactosidase or the periplasmic protein TEM-beta-lactamase. Soluble and insoluble cell fractions of Escherichia coli producing either beta-galactosidase or TEM-beta-lactamase were analyzed by one- and two-dimensional gel electrophoresis and subsequent silver staining or immunodetection of the recombinant protein. The results show that truncated fragments of the recombinant protein were not present in the soluble cell fraction but accumulate in the IB fraction. The presence of other cellular, non-plasmid-encoded proteins in IB preparations such as the outer membrane proteins OmpF, OmpC, and OmpA or the ribosomal subunit proteins L7/L12 was attributed to co-precipitation of cell-debris-associated components. Protein-folding enzymes were not detected in IB preparations. The specificity of in-vivo protein association in the formation of IBs and its implication on protein purification is discussed.

Escherichia coli

Structural characteristics of an abnormal protein influencing its proteolytic susceptibility.

Structural properties of two similar beta-galactosidase fragments were investigated to determine how they influence the fragments' degradation rate in Escherichia coli. Both fragments resulting from a C-terminal nonsense mutation in lacZ, the CSH11 polypeptide and its 90 kDa degradative intermediate, exist predominantly as monomer subunits instead of in the tetrameric form characteristic of the native enzyme. However, both fragments appear to produce trace amounts of dimers and tetramers. The tetramer and higher molecular weight aggregates formed by the wild-type subunit confer greater protection for the enzyme's N-terminal auto-alpha polypeptide than does the monomer state of the beta-galactosidase fragments. The thermally induced aggregation of both beta-galactosidase fragments correlates with their sensitivity to alpha-chymotrypsin. The relatively low thermal stability of the 90 kDa degradative intermediate appears to be the cause of the significant increase in its proteolytic susceptibility at moderately high temperatures.

Blotting, Western

Cysteine to serine substitutions in basic fibroblast growth factor: effect on inclusion body formation and proteolytic susceptibility during in vitro refolding.

We have investigated the effect of cysteine to serine substitutions in human basic fibroblast growth factor (bFGF) on the formation of inclusion bodies in Escherichia coli. Using a temperature-sensitive expression, system, about 30% of human bFGF, which contains four cysteines at positions 26, 70, 88, and 93, is deposited into inclusion bodies. A single mutation at position 88 and a double mutation at positions 70 and 88 do not greatly alter the partition of bFGF into soluble and insoluble cell fractions. However, a single substitution of cysteine 70 by serine decreases the fraction of soluble bFGF significantly. When cysteines 26 and 93 (conserved among related growth factors) are replaced by serines, no soluble bFGF is formed in E. coli. Cysteine to serine substitutions also affect proteolytic susceptibility of bFGF during in vitro refolding from crude inclusion bodies. About 60% of human bFGF is lost to proteolytic degradation during in vitro refolding. Replacement of cysteines by serines increases the total recovery of bFGF, although more aggregates are formed during refolding. Ser-88-bFGF was expressed at the highest level, gave the highest soluble fraction in vivo, and exhibited the greatest fractional recovery and was recovered with the largest insoluble fraction after in vitro refolding. Thermal stability experiments at 42 degrees C and 70 degrees C revealed that cysteine to serine substitutions did not cause aggregation of the folded protein in vitro.

Base Sequence

Toward a science of metabolic engineering.

Application of recombinant DNA methods to restructure metabolic networks can improve production of metabolite and protein products by altering pathway distributions and rates. Recruitment of heterologous proteins enables extension of existing pathways to obtain new chemical products, alter posttranslational protein processing, and degrade recalcitrant wastes. Although some of the experimental and mathematical tools required for rational metabolic engineering are available, complex cellular responses to genetic perturbations can complicate predictive design.

Amino Acids

Secondary structure perturbations in salt-induced protein precipitates.

The secondary structure implications of precipitation induced by a chaotropic salt, KSCN, and a structure stabilizing salt, Na2SO4, were studied for twelve different proteins. alpha-helix and beta-sheet content of precipitate and native structures were estimated from the analysis of amide I band Raman spectra. A statistical analysis of the estimated perturbations in the secondary structure contents indicated that the most significant event is the formation of beta-sheet structures with a concomitant loss of alpha-helix on precipitation with KSCN. The conformational changes for each protein were also analyzed with respect to elements of primary, secondary and tertiary structure existing in the native protein; primary structure was quantified by the fractions of hydrophobic and charged amino acids, secondary structure by x-ray estimates of alpha-helix and beta-sheet contents of native proteins and tertiary structure by the dipole moment and solvent-accessible surface area. For the KSCN precipitates, factors affecting beta-sheet content included the fraction of charged amino acids in the primary sequence and the surface area. Changes in alpha-helix content were influenced by the initial helical content and the dipole moment. The enhanced beta-sheet contents of precipitates observed in this work parallel protein structural changes occurring in other aggregative phenomena.

Chemical Precipitation

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

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

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

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

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