Biomedical subjects

J E Bailey

Publications and source records attributed to J E Bailey.

At least 55 records · Page 3Linked to original sources

PubMed2000

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells.

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.

Alkaline Phosphatase↗

PubMed2000

Analysis of growth rate effects on productivity of recombinant Escherichia coli populations using molecular mechanism models. Reprinted from Biotechnology and Bioengineering, Vol. 26, Issue 1, Pages 66-73 (1984).

The influence of growth rate on Escherichia coli plasmid content and expression of a cloned-gene product has been described by a mathematical model based upon the molecular mechanism of lambdadv plasmid replication and known relationships between growth rate and transcription and translation activities of the host cell. The model simulates correctly decreases in plasmid content with increasing growth rate as observed experimentally for pBR322, NR1, R1, and Col E1 plasmids. A maximum with respect to growth rate in intracellular product accumulation is indicated by the model, as is a transient overshoot in product concentration following a shift from smaller to larger growth rate. Available data, although very limited, show the same trends. These results, obtained without parameter or kinetic form adjustments or manipulation, clearly illustrate the advantages of kinetic descriptions of recombinant systems based upon the pertinent molecular mechanisms.

Escherichia coli↗

PubMed2000

Altered regulation of pyruvate kinase or co-overexpression of phosphofructokinase increases glycolytic fluxes in resting Escherichia coli.

Glycolytic fluxes in resting Escherichia coli were enhanced by overexpression of heterologous pyruvate kinases (Pyk) from Bacillus stearothermophilus and Zymomonas mobilis, but not homologous Pyk. Compared to the control, an increase of 10% in specific glucose consumption and of 15% in specific ethanol production rates was found in anaerobic resting cells, expressing the heterologous Pyks, that were harvested from exponentially growing aerobic cultures. A further increase in glycolytic flux was achieved by simultaneous overexpression of E. coli phosphofructokinase (Pfk) and Pyk with specific glucose consumption and ethanol production rates of 25% and 35% greater, respectively, than the control. Fluxes to lactate were not significantly affected, contrary to previous observations with resting cells harvested from anaerobically growing cultures. To correlate the physiology of resting cells with the physiology of cells prior to harvest, we determined the relevant growth parameters from aerobic and anaerobic precultures. We conclude that glycolytic fluxes in E. coli with submaximal (aerobic) metabolic activity can be increased by overexpression of pyruvate kinases which do not require allosteric activation or co-overexpression with Pfk. However, such improvements require more extensive engineering in E. coli with near maximal (anaerobic) metabolic activity.

Escherichia coli↗

PubMed2000

Co-overexpression of RspAB improves recombinant protein production in Escherichia coli.

The Escherichia coli mutant CWML2 was previously reported to exhibit improved physiological characteristics, including recombinant protein production. Here we investigate the molecular basis of this phenotype by comparing the cellular level of three RNA polymerase sigma subunits by immunoblot analysis. While the level of housekeeping sigma(D) was similar in parent and mutant, the levels of the flagella synthesis regulator sigma(F) and the stationary phase regulator sigma(S) were higher in the mutant strain, indicating a different motility and stationary phase phenotype. Evidence for this conclusion was provided by the significantly higher motility of CWML2, compared to its parent. Based on these results, we hypothesized that alterations in ppGpp regulation via a homoserine lactone-dependent mechanism may be relevant for the mutant phenotype. Indeed, transcription of the rspAB operon, which was previously described to be involved in the degradation of homoserine lactone, was found to be deregulated in CWML2 in a plasmid-based reporter protein assay. By overexpression of the E. coli rspAB operon, we could partly mimic the mutant phenotype and demonstrate that co-overexpression of RspAB is a pertinent metabolic engineering strategy to improve recombinant protein production.

Blotting, Western↗

PubMed2000

Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae.

Yeast vacuoles are highly dynamic and flexible organelles. In a previous paper, we have shown that subtle, often unrecognised amino acid limitations lead to much lower final cell densities in cultures of different commonly used auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the number and morphology of vacuoles, and that these changes are correlated with the cell cycle in batch cultures in a similar way as is known from synchronized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for sample preparation that so far have been barely successful in yeast. Cells of leucine-limited cultures had single, large vacuoles with a hexagonal tonoplast pattern and were partially arrested in G1 phase. To relieve leucine-limitation, additional leucine was supplied extracellularly via the medium or intracellularly via enhanced leucine biosynthesis due to plasmid-based expression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast pattern and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and cell cycle distribution present a note of caution for morphological and cell cycle studies in yeast.

Animals↗

PubMed2000

Streptomycetes in micro-cultures: growth, production of secondary metabolites, and storage and retrieval in the 96-well format.

Mycelium-forming Streptomyces strains were grown in one milliliter liquid micro-cultures in square deep-well microtiter plates. Growth was evaluated with respect to biomass formation and production of secondary metabolites which were found to be very similar in the micro-cultures, bioreactor, and shake flask cultivations, respectively. Despite repetitive sampling and extensive growth on the walls of the wells, no cross contamination occurred. Furthermore, we successfully employed cold storage at -20 degrees C of spore suspensions (in the 96-well format), directly prepared from cultures grown on agar in the microtitre plate. Cultures were retrieved by replicating aliquots from the frozen spore suspensions.

Bacteriological Techniques↗

PubMed2000

A temperature-regulated replicon-based DNA expression system.

We present a temperature-regulated, alphavirus replicon-based DNA expression system. The system is regulated by a viral temperature-sensitive RNA-dependent RNA replicase, creating a temperature-dependent RNA amplification loop. Because of this positive feedback, the system exhibits both low background and high inducibility. We observed 700-fold induction in transiently transfected cells, and over 104-fold induction in stably transfected cells. The high stringency of inducibility allowed the generation of stable cell lines expressing a highly toxic protein upon temperature shift. These data suggest that the present expression system could simplify bioprocess engineering strategies, especially in situations where the cloned protein has detrimental effects on host cell metabolism.

Alphavirus↗

PubMed2000

A mathematical model of caspase function in apoptosis.

Caspases (cysteine-containing aspartate-specific proteases) are at the core of the cell's suicide machinery. These enzymes, once activated, dismantle the cell by selectively cleaving key proteins after aspartate residues. The events culminating in caspase activation are the subject of intense study because of their role in cancer, and neurodegenerative and autoimmune disorders. Here we present a mechanistic mathematical model, formulated on the basis of newly emerging information, describing key elements of receptor-mediated and stress-induced caspase activation. We have used mass-conservation principles in conjunction with kinetic rate laws to formulate ordinary differential equations that describe the temporal evolution of caspase activation. Qualitative strategies for the prevention of caspase activation are simulated and compared with experimental data. We show that model predictions are consistent with available information. Thus, the model could aid in better understanding caspase activation and identifying therapeutic approaches promoting or retarding apoptotic cell death.

Apoptosis↗

PubMed2000

Streptogramin-based gene regulation systems for mammalian cells.

Here we describe repressible (PipOFF) as well as inducible (PipON) systems for regulated gene expression in mammalian cells, based on the repressor Pip (pristinamycin-induced protein), which is encoded by the streptogramin resistance operon of Streptomyces coelicolor. Expression of genes placed under control of these systems was responsive to clinically approved antibiotics belonging to the streptogramin group (pristinamycin, virginiamycin, and Synercid). The versatility of these systems was demonstrated by streptogramin-regulated expression of mouse erythropoietin (EPO), human placental secreted alkaline phosphatase (SEAP), or green fluorescent protein (GFP) in diverse cell lines (BHK, CHO, HeLa, and mouse myoblasts). Analysis of isogenic constructs in CHO cells demonstrated the PipOFF system gave lower background and higher induction ratios than the widely used tetracycline-repressible (TetOFF) expression systems. The streptogramin-based expression technology was functionally compatible with the TetOFF system, thus enabling the selective use of different antibiotics to independently control two different gene activities in the same cell.

Alkaline Phosphatase↗

PubMed2000

Preoptometry and optometry school grade point average and optometry admissions test scores as predictors of performance on the national board of examiners in optometry part I (basic science) examination.

PURPOSE: To evaluate preoptometry and optometry school grade point averages and Optometry Admission Test (OAT) scores as predictors of performance on the National Board of Examiners in Optometry NBEO Part I (Basic Science) (NBEOPI) examination. METHODS: Simple and multiple correlation coefficients were computed from data obtained from a sample of three consecutive classes of optometry students (1995-1997; n = 278) at Southern California College of Optometry. RESULTS: The GPA after year two of optometry school was the highest correlation (r = 0.75) among all predictor variables; the average of all scores on the OAT was the highest correlation among preoptometry predictor variables (r = 0.46). Stepwise regression analysis indicated a combination of the optometry GPA, the OAT Academic Average, and the GPA in certain optometry curricular tracks resulted in an improved correlation (multiple r = 0.81). Predicted NBEOPI scores were computed from the regression equation and then analyzed by receiver operating characteristic (roc) and statistic of agreement (kappa) methods. From this analysis, we identified the predicted score that maximized identification of true and false NBEOPI failures (71% and 10%, respectively). Cross validation of this result on a separate class of optometry students resulted in a slightly lower correlation between actual and predicted NBEOPI scores (r = 0.77) but showed the criterion-predicted score to be somewhat lax. CONCLUSIONS: The optometry school GPA after 2 years is a reasonably good predictor of performance on the full NBEOPI examination, but the prediction is enhanced by adding the Academic Average OAT score. However, predicting performance in certain subject areas of the NBEOPI examination, for example Psychology and Ocular/Visual Biology, was rather insubstantial. Nevertheless, predicting NBEOPI performance from the best combination of year two optometry GPAs and preoptometry variables is better than has been shown in previous studies predicting optometry GPA from the best combination of preoptometry variables.

Certification↗

PubMed2000

Effect of eltenac in horses with induced endotoxaemia.

Ten horses were used in a crossover study to evaluate the effectiveness of eltenac against endotoxaemia. Eltenac (0.5 mg/kg bwt) or saline control was given i.v. then 15 min later, intravenous infusion of endotoxin was begun and continued for 120 min (total dose 100 ng/kg bwt). Horses were monitored for heart and respiratory rates, pulmonary and carotid arterial pressure and core body temperature. Blood was sampled at intervals for measurement of haematological variables and plasma concentrations of lactate, prostanoid metabolites, tumour necrosis factor (TNF) and stress hormones. In comparison with saline-treatment, use of eltenac significantly protected against endotoxin-induced changes in respiratory rate, core temperature, systemic arterial blood pressure (SAP), pulmonary arterial pressure, PCV, and plasma protein, 6-keto prostaglandin F1 alpha, thromboxane B2, epinephrine, and cortisol concentrations. Despite statistical effect of eltenac on SAP, values in both treatment groups remained well above baseline throughout the evaluation period. Significant protective effect of eltenac was not found for heart rate, white blood cell count, plasma lactate concentration or TNF activity. On the basis of these results, it is expected that use of eltenac will provide clinical benefit in horses with naturally occurring endotoxaemia.

Aniline Compounds↗

PubMed2000

Expression of Alcaligenes eutrophus flavohemoprotein and engineered Vitreoscilla hemoglobin-reductase fusion protein for improved hypoxic growth of Escherichia coli.

Expression of the vhb gene encoding hemoglobin from Vitreoscilla sp. (VHb) in several organisms has been shown to improve microaerobic cell growth and enhance oxygen-dependent product formation. The amino-terminal hemoglobin domain of the flavohemoprotein (FHP) of the gram-negative hydrogen-oxidizing bacterium Alcaligenes eutrophus has 51% sequence homology with VHb. However, like other flavohemoglobins and unlike VHb, FHP possesses a second (carboxy-terminal) domain with NAD(P)H and flavin adenine dinucleotide (FAD) reductase activities. To examine whether the carboxy-terminal redox-active site of flavohemoproteins can be used to improve the positive effects of VHb in microaerobic Escherichia coli cells, we fused sequences encoding NAD(P)H, FAD, or NAD(P)H-FAD reductase activities of A. eutrophus in frame after the vhb gene. Similarly, the gene for FHP was modified, and expression cassettes encoding amino-terminal hemoglobin (FHPg), FHPg-FAD, FHPg-NAD, or FHP activities were constructed. Biochemically active heme proteins were produced from all of these constructions in Escherichia coli, as indicated by their ability to scavenge carbon monoxide. The presence of FHP or of VHb-FAD-NAD reductase increased the final cell density of transformed wild-type E. coli cells approximately 50 and 75%, respectively, for hypoxic fed-batch culture relative to the control synthesizing VHb. Approximately the same final optical densities were achieved with the E. coli strains expressing FHPg and VHb. The presence of VHb-FAD or FHPg-FAD increased the final cell density slightly relative to the VHb-expressing control under the same cultivation conditions. The expression of VHb-NAD or FHPg-NAD fusion proteins reduced the final cell densities approximately 20% relative to the VHb-expressing control. The VHb-FAD-NAD reductase-expressing strain was also able to synthesize 2.3-fold more recombinant beta-lactamase relative to the VHb-expressing control.

Aerobiosis↗

PubMed2000

Sleep and daytime sleepiness the next day following single night-time dose of fluvoxamine, dothiepin and placebo in normal volunteers.

To explore the effects of sedating and non-sedating antidepressants, we conducted a placebo-controlled, double-blind cross-over study in 12 normal subjects of the effects of a single night-time dose of fluvoxamine 100 mg, dothiepin 100 mg or placebo on night-time sleep recorded at home, and sleepiness and performance the following day. Night-time sleep was altered significantly by both drugs, with main effects on rapid eye movement (REM) sleep and sleep continuity. Dothiepin increased total sleep time, REM latency and stage 2 sleep and decreased arousals, wake after sleep onset and stage 1, whereas fluvoxamine decreased total sleep time and REM time and increased wake after sleep onset. Sleep latencies in daytime naps were significantly shorter for dothiepin and longer for fluvoxamine, showing that subjects were more sleepy when taking dothiepin. Electroencephalograms (EEG) performed during performance tasks failed to distinguish significantly between drugs. There were no significant differences between groups on our measures of tracking performance or reaction time; however, these tasks were designed primarily to provide a standard setting in which to monitor continuous EEG, and were unsuitable to detect sleepiness effects themselves. Saccadic eye movement velocity, acceleration and deceleration showed small non-significant changes after both drugs. Mood self ratings showed no significant differences among the groups. Subjective measures of night-time sleep reflected the objective measures of sleep continuity, and the items for difficulty and speed of wakening in the morning were significantly higher (i.e. more difficulty and slower) in the dothiepin group. The home-recorded sleep findings after fluvoxamine in this study were very similar to sleep laboratory studies with other antidepressant drugs, thus providing more validation of the home recording method.

Adult↗

PubMed2000

Adverse effects of increased body weight on quantitative measures of mammographic image quality.

OBJECTIVE: The purpose of our study was to show that compressed breast thickness on mammograms in overweight and obese women exceeds the thickness in normal-weight women and that increased thickness results in image degradation. SUBJECTS AND METHODS: Three hundred consecutive routine mammograms were reviewed. Patients were categorized according to body mass index. Compression thickness, compressive force, kilovoltage, and milliampere-seconds were recorded. Geometric unsharpness and contrast degradation were calculated for each body mass index category. RESULTS: Body mass index categories were lean (3%), normal (36%), overweight (36%), and obese (25%). Body mass index was directly correlated with compressed thickness. In the mediolateral oblique view, the mean thickness of the obese category exceeded normal thickness by 18 mm (p < 0.01), corresponding to a 32% increase in geometric unsharpness. Mean obese thickness exceeded lean thickness by 33 mm (p < 0.01), corresponding to a 79% increase in unsharpness. Similar trends were observed for the craniocaudal view. In the mediolateral oblique projection, there was an increase of 1.0 kVp (p < 0.01) for obese compared with normal and 1.7 kVp (p < 0.01) between lean and obese, corresponding, respectively, to a 16% and a 25% decrease in image contrast because of scatter and kilovoltage changes. Milliampere-seconds increased by 47% on the mediolateral oblique images in the obese category compared with normal body mass index. CONCLUSION: An increased body mass index was associated with greater compressed breast thickness, resulting in increased geometric unsharpness, decreased image contrast, and greater potential for motion unsharpness.

Adult↗

PubMed2000

Butorphanol/xylazine/ketamine immobilization of free-ranging Baird's tapirs in Costa Rica.

Cardiopulmonary effects and the utility of a butorphanol/xylazine/ketamine combination were evaluated during twenty immobilizations of sixteen Baird's tapirs (Tapirus bairdii) between March 1996 and January of 1998 in Corcovado National Park (Costa Rica). The animals were attracted to a bait site and darted from tree platforms. The tapirs were estimated to weigh between 200 to 300 kg. Actual weights of three tapirs taken at later dates fell within the estimated range. A butorphanol, 48+/-1.84 (x +/- SE) mg/animal IM, and xylazine, 101+/-2.72 mg/animal IM, combination was used to immobilize the animals. In some instances, ketamine was used either IM or IV at 187+/-40.86 mg/animal to prolong the immobilization period in addition to the butorphanol/xylazine combination. Naltrexone was used IM to reverse butorphanol at 257+/-16.19 mg/animal. Either yohimbine, 34+/-0.61 or tolazoline at 12+/-10.27 mg/animal, was used to reverse xylazine. The mean time from dart impact to first visible effect was 4.63+/-0.50 min (x +/- SE). Mean time to sternal recumbency was 12.21+/-1.08 min. Mean time the tapirs were immobilized was 45.63+/-3.6 min. Mean time to return to sternal recumbency and standing in animals that received yohimbine and naltrexone was 3.16+/-1.06 and 5.33+/-1.45 min, respectively. Mean time to return to sternal recumbency and standing in animals that received tolazoline and naltrexone was 1.57+/-0.39 and 3.14+/-0.51 min, respectively. Cardiopulmonary parameters including heart rate, respiratory rate, body temperature, electrocardiogram, percent oxygen satoration, and indirect blood pressure were recorded. Arterial blood gas analysis was performed on four animals. A mild degree of hypoxemia was evidenced by low arterial oxygen saturations. Five of 14 (36%) animals measured had oxygen saturations below 90%. Bradycardia (heart rates <45 BPM) was an expected finding in 11 (55%) immobilizations. Induction, recovery and muscle relaxation of each immobilization was graded. Premature arousal, which occurred in six (30%) animals, was the only problem associated with the immobilizations. Butorphanol/xylazine is a recommended protocol for immobilization of calm, free-ranging tapirs lasting less than 30 min. Supplemental intravenous administration of ketamine is recommended for longer procedures. Nasal insufflation of oxygen is recommended.

Adrenergic alpha-Agonists↗

PubMed1999

A mathematical description of regulation of the G1-S transition of the mammalian cell cycle.

A mathematical model of regulation of the G1-S transition of the mammalian cell cycle has been formulated to organize available experimental molecular-level information in a systematic quantitative framework and to evaluate the ability of this manifestation of current knowledge to calculate correctly experimentally observed phenotypes. This model includes nine components and includes cyclin-cdk complexes, a pocket protein (pRb), a transcription factor (E2F-1), and a cyclin-cdk complex inhibitor. Simulation of the model equations yields stable oscillatory solutions corresponding to cell proliferation and asymptotically stable solutions corresponding to cell cycle arrest (quiescence). Bifurcation analysis of the system suggests changes in the intracellular concentrations of either E2F or cyclin E can activate cell proliferation and that co-overexpression of these molecules can prevent cell proliferation. Further analysis suggests that the amount of inhibitor necessary to prevent cell proliferation is independent of the concentrations of cyclin E and E2F and depends only on the equilibrium ratio between the bound and unbound forms of the inhibitor to the complex.

Animals↗

PubMed1999

Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells.

The glycosylation patterns of recombinant therapeutic glycoproteins can be engineered by overexpression of glycosyltransferases in the host cells used for glycoprotein production. Most prior glycosylation engineering experiments have involved constitutive expression of cloned glycosyltransferases. Here we use tetracycline-regulated expression of two glycosyltransferases, N-acetylglucosaminlytransferases III and V (GnTIII and GnTV) to manipulate glycoform biosynthesis in Chinese hamster ovary (CHO) cells and to study the effect of glycosyltransferase overexpression on this host. The amount of GnTIII and GnTV in these cells, and the glycosylation patterns of several cellular glycoproteins, could be controlled simply by manipulating the concentration of tetracycline in the culture medium. Using this system, it was found that overexpression of either GnTIII or GnTV to high levels led to growth inhibition and was toxic to the cells, indicating that this may be a general feature of glycosyltransferase overexpression. This phenomenon has not been reported previously, probably due to the widespread use of constitutive promoters, and should be taken into account when designing vectors for glycosylation engineering. The growth inhibition effect sets an upper limit to the level of glycosyltransferase overexpression, and may thereby also limit the maximum extent of in vivo modification of poorly accessible glycosylation sites. Also, such inhibition implies a bound on constitutive glycosyltransferase expression which can be cloned.

Animals↗

PubMed1999

A novel autoregulated proliferation-controlled production process using recombinant CHO cells.

Controlled proliferation bioprocesses have shown great enhancement of heterologous protein production. This novel technology has been implemented here using a multicistronic expression unit encoding the product gene and a cytostatic cell-cycle-arresting gene (p27) under control of a single tetracycline-repressible (tet(off)) promoter. The strict genetic linkage of both genes allows the dissection of the production process into a nonproductive growth phase (dicistronic expression unit repressed) followed by a proliferation-inhibited production phase (dicistronic expression unit induced) when the cells have reached an optimal cell density. Based on rapid degradation of the external repressible agents tetracycline (tet) and doxycycline (dox) in the cell culture medium, we developed a self-regulated process for transition from the growth phase to the production phase in a fashion that is dependent only on the starting cell population and the initial concentration of the tetracyclines. With this process, no change in medium is required to accomplish the transition from growth to production phase. The two-phase bioprocess achieved here by tet switch-controlled proliferation is reliable and allows a growth-arrested production phase of at least 7 days, during which cells remain in a well-defined, highly viable physiological state and show enhanced heterologous protein production. This Tet(SWITCH) process is readily adaptable to a variety of industrial processes designed for production of difficult-to-express protein pharmaceuticals.

Animals↗