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Biomedical subjects

J E Bailey

Publications and source records attributed to J E Bailey.

At least 127 records · Page 7Linked to original sources
PubMed1994

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↗
PubMed1994

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↗
PubMed1993

Construction and characterization of a novel cross-regulation system for regulating cloned gene expression in Escherichia coli.

A novel cross-regulation system employing two pairs of interacting promoter-repressor systems was constructed using the tac-lacI and lambda pL-cI promoter-operator-repressor systems. In particular, transcription of the cat gene and the fused cI gene is regulated by the tac promoter, while transcription of the lacI gene is controlled by the lambda pL promoter. In order to compare CAT production from this new system with a currently employed transcription control configuration, a control expression vector utilizing the constitutive repressor synthesis configuration was also constructed. In this construct, cat is under the control of the tac promoter, and lac repressor is provided from a single copy of the lacIq allele included in the plasmid. Induction results using different copy number vectors indicate that induced cat expression levels are at least twofold higher using the cross-regulation system which has very low basal expression. These results match well with previous mathematical modeling predictions indicating excellent control of basal expression and also higher cloned-gene expression post-induction over a broad range of copy numbers for a cross-regulation control configuration. Induction of the cross-regulation system both up-regulated the activation pathway and down-regulated the inhibition pathway, shifting the system steady-state from lac repressor expression to cat and cI expression. The control strategy presented here should be equally applicable to regulate transcription in diverse hosts.

Bacteriophage lambda↗
PubMed1993

Chemical reduction of FD&C yellow No. 5 to determine combined benzidine.

Data are presented suggesting the presence of the aromatic amine benzidine as a combined impurity in the regulated color additive FD&C Yellow No. 5. The benzidine exists as an azo-dye constituent that forms from free benzidine impurities introduced during the manufacture of the color additive. The presence of combined benzidine is ascertained by sodium dithionite reduction of the azo bonds in the commercial color additive. The resulting reduction products are extracted with chloroform, and the liberated benzidine is determined by high-performance liquid chromatography (HPLC). The levels of benzidine determined by HPLC exceed those levels of benzidine accounted for by direct determination of free aromatic amines in the unreduced color.

Benzidines↗
PubMed1993

Host-vector interactions in Escherichia coli.

Introduction of a DNA vector into E. coli for the purposes of cloned gene expression can perturb native cell functions at many levels. The presence of foreign DNA can alter regulation of chromosomal DNA replication, regulation of transcription of chromosomal genes, ribosome functions and RNA turnover, activities of regulatory proteins, chaperone and protease levels, membrane energetics and protein post-translational processing, as well as energy and intermediary metabolism of the cell. The combined effects of these interactions on the metabolic characteristics of the host-vector system have major implications for yields of cloned biotechnological products and interactions of genetically engineered organisms with their environment.

Biotechnology↗
PubMed1993

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↗
PubMed1993

A study of graduate education in physiological optics in the United States: 1938-1989.

For over 50 years, graduate programs in physiological optics (vision science) have been the primary source of supply to schools and colleges of optometry of Master of Science and Doctor of Philosophy degree recipients who teach and conduct research. To determine the supply of M.S. and Ph.D. degree recipients from 1938 through 1989 and the career pursuits of these recipients, a computerized data base was developed. A total of 456 degrees were awarded (287 M.S. and 169 Ph.D.). Among the M.S. recipients, 56 also completed a Ph.D. Although there was a steady increase in the number of degree recipients, the number who pursued optometric education as a career steadily declined, especially among those who received only a M.S. degree. Further analysis suggests that a loss of optometry faculty with graduate degrees, in particular those with Ph.D.'s, due to attrition will likely occur during this decade and become substantial during the first decade of the next century. Clearly, efforts need to be increased by schools and colleges of optometry in the very near future to foster graduate level study of physiological optics, and to encourage the pursuit of optometric education as a career.

Education, Graduate↗
PubMed1993

Overexpression of bacterial hemoglobin causes incorporation of pre-beta-lactamase into cytoplasmic inclusion bodies.

The expression of Vitreoscilla hemoglobin (VHb) in Escherichia coli JM101 (pRED2) causes the incorporation of the TEM beta-lactamase precursor into cytoplasmic inclusion bodies (IBs). Less pre-beta-lactamase is translocated and processed to its mature, periplasmic form in the strain coexpressing VHb than in the control strain E. coli JM101(pUC19) not expressing VHb. When cells are grown in a special fed-batch procedure, the formation of cytoplasmic IBs consisting of pre-beta-lactamase is also inducible in the control strain. Comparative microscopic and compositional analyses of IBs generated in E. coli JM101(pUC19) and JM101(pRED2) under identical growth conditions strongly suggest that pre-beta-lactamase and VHb coaggregate into common IBs in E. coli JM101 (pRED2).

Escherichia coli↗
PubMed1993

Reflex cardiovascular effects of continuous prostacyclin administration into an isolated in situ lung in the dog.

Bolus administration of prostacyclin (PGI2), an inflammatory product produced by the arachidonic acid cyclooxygenase pathway, into the pulmonary circulation is known to stimulate pulmonary C-fibers and to produce transient reflex cardiovascular depression. We used an isolated perfused in situ left lung preparation to examine the reflex cardiovascular effects of continuous administration of PGI2 into the isolated pulmonary circulation of dogs. PGI2 was infused for 30 min (5 micrograms.kg-1 x min-1) into the innervated isolated left pulmonary circulation in six dogs and into the denervated isolated circulation in seven dogs. Mean arterial pressure and cardiac output were significantly decreased from baseline values during the final 20 min of PGI2 infusion. There were no significant changes in maximum rate of change of left ventricular pressure during systole and in heart rate. All variables returned to baseline levels within 15 min of discontinuation of PGI2 infusion. PGI2 infusion produced no significant measured hemodynamic changes in the denervated group. Control experiments demonstrated that the observed response was not affected by duration of the preparation, lung denervation, or infusion vehicle. These findings suggest that continuous infusion of PGI2 into the isolated pulmonary circulation produces sustained cardiovascular depression via a vagal reflex mechanism that is probably mediated by pulmonary C-fibers.

Animals↗
PubMed1993

Mechanical precision in two-dimensional electrophoresis can improve protein spot positional reproducibility.

Current methods for high resolution two-dimensional electrophoresis (2DE) of proteins are capable of separating over 5000 protein spots in one procedure. Running and analysing such 2DE gels requires skilled technical work. However, the variable reproducibility of spot positions means that, even under the best circumstances, one gel cannot be overlain directly on another for precise comparison. Therefore, new and improved technologies that enhance gel-to-gel reproducibility are required. To this end, we have designed and built a research instrument to test whether a precise mechanical device could improve the gel-to-gel reproducibility by reducing the amount of distortion and positional variation between the first and second dimension gels. Other causes of poor reproducibility, including sample type and preparation, gel matrices and running conditions were not varied in order to limit this study to the mechanical variations inherent in current 2DE systems. We found that the sample standard deviation of pooled data for measured protein spot-to-spot distances in the prototype device was 1.3 mm as compared to 4.3 mm in a conventional 2DE system. These improvements support the possibility that greater automation of the multistep 2DE process will enhance reproducibility. This approach seems justified in order to achieve significantly better matching between gels and between results from different laboratories.

Cerebrospinal Fluid Proteins↗
PubMed1993

Optics of phakic, pseudophakic, and aphakic eyes: effects on the Stiles-Crawford (SCE I) function.

In many experimental situations one has to work with suitable optical corrections for ocular elements which have been eliminated (lens in aphakia, for example). Analysis of the optics of the total system (eye plus correction and other optical elements) shows substantial changes in the positions of the cardinal points. These changes are meaningful and must be taken into account in subsequent analysis. We discuss the pertinent optical considerations and show as an example the results of measurements of the Stiles-Crawford effect of the first kind in an aphakic subject with spectacle and contact lens correction.

Aphakia↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

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↗