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P B Taylor

Publications and source records attributed to P B Taylor.

At least 19 recordsLinked to original sources
PubMed2000

Automated assay optimization with integrated statistics and smart robotics.

The transition from manual to robotic high throughput screening (HTS) in the last few years has made it feasible to screen hundreds of thousands of chemical entities against a biological target in less than a month. This rate of HTS has increased the visibility of bottlenecks, one of which is assay optimization. In many organizations, experimental methods are generated by therapeutic teams associated with specific targets and passed on to the HTS group. The resulting assays frequently need to be further optimized to withstand the rigors and time frames inherent in robotic handling. Issues such as protein aggregation, ligand instability, and cellular viability are common variables in the optimization process. The availability of robotics capable of performing rapid random access tasks has made it possible to design optimization experiments that would be either very difficult or impossible for a person to carry out. Our approach to reducing the assay optimization bottleneck has been to unify the highly specific fields of statistics, biochemistry, and robotics. The product of these endeavors is a process we have named automated assay optimization (AAO). This has enabled us to determine final optimized assay conditions, which are often a composite of variables that we would not have arrived at by examining each variable independently. We have applied this approach to both radioligand binding and enzymatic assays and have realized benefits in both time and performance that we would not have predicted a priori. The fully developed AAO process encompasses the ability to download information to a robot and have liquid handling methods automatically created. This evolution in smart robotics has proven to be an invaluable tool for maintaining high-quality data in the context of increasing HTS demands.

Automation↗
PubMed1996

Force-frequency response in isoproterenol-induced hypertrophied rat heart.

Rate-dependent force production was investigated using small trabecular muscle from control and hypertrophied rat cardiac muscle. Cardiac hypertrophy was induced by daily subcutaneous injection of isoproterenol (0.3 mg/kg body weight) for 12 days. The force-frequency relationship, for the control rat myocardium, is clearly biphasic. A stepped increase in stimulation frequency from 0.1 to 0.5 Hz results in a decrease in contractile force (negative phase). However, at higher stimulation frequency above 0.5 Hz, an increased contractile force is revealed (positive phase). Membrane action potential duration (APD50) was used to reflect sarcolemmal Ca2+ influx. The frequency-dependent increase in APD50 and the ability of nifedipine, a sarcolemmal L-type Ca2+ channel blocker, to eliminate the positive-force frequency response, indicate that sarcolemmal Ca2+ influx is important for force development at high stimulation frequency. Relative Ca2+ content of sarcoplasmic reticulum is estimated from rapid cooling contractures. The parallel change of rapid cooling contractures and twitch force suggests that the sarcoplasmic reticulum Ca2+ content alters with varying frequencies of stimulation. Isoproterenol-induced hypertrophied muscle shows a greater contractile force, increased nifedipine-sensitive force development and prolonged APD50 compared to controls. These data suggest a greater availability of intracellular Ca2+ to activate contraction in hypertrophied muscle, possibly by amplified Ca2+ influx via L-type channel.

Action Potentials↗
PubMed1996

Transformation of gypsy moth (Lymantria dispar) cell lines by infection with Glyptapanteles indiensis polydnavirus.

Glyptapanteles indiensis, a species of braconid parasitic wasp, infects its host Lymantria dispar (gypsy moth) with a polydnavirus (GiPDV) to suppress the host immune system during parasitization. Here it is shown that GiPDV can infect L. dispar cell lines and that a portion of the GiPDV genome is stably maintained in infected cells. Results of Southern hybridization analyses suggested that this portion of the GiPDV genome is integrated into the L. dispar cellular genome. This is the first report of an insect viral DNA molecule that can apparently integrate into lepidopteran insect cells.

Animals↗
PubMed1996

Altered contractile function in isoproterenol-induced hypertrophied rat heart.

OBJECTIVE: To study the calcium-dependent mechanisms contributing to altered contractile function in isoproterenol-induced ventricular hypertrophy of the rat heart. DESIGN: The force-interval relationship, systemic evaluation of changed contractile force and calcium sensitivity of the myofilaments were investigated using small trabecular muscle from hearts with isoproterenol-induced cardiac hypertrophy. METHODS: Cardiac hypertrophy was induced by daily subcutaneous injection of isoproterenol for 12 days. The calcium sensitivity of myofilaments was studied using non-ionic detergent (Triton-X-100)-skinned trabeculae. The contractile force was evaluated at various concentrations of extracellular calcium and muscle lengths. The force-interval relationship was used to reflect altered intracellular calcium handling. RESULTS: The isoproterenol-induced cardiac hypertrophy was associated with significantly enhanced contractile force at various concentrations of extracellular calcium and muscle lengths. Also, an amplified force-interval relationship in hypertrophied muscle at long rest intervals was found. However, this study revealed no change in calcium sensitivity of myofilaments. CONCLUSION: Altered intracellullar calcium handling contributes to enhanced contractile force in isoproterenol-induced cardiac hypertrophy.

Actin Cytoskeleton↗
PubMed1995

Analytic requirements for the doubly labeled water method.

The doubly labeled water method is the first method that accurately measures total daily energy expenditure in free-living subjects over periods of days to weeks. Validations have indicated that the method can be performed with a coefficient of variation of between 3% and 5%. This precision, however, is dependent on the quality of the isotopic analyses. A recent interlaboratory comparison has indicated that there is a wide variation in the accuracy and precision with which deuterium and 18O enrichments are measured. This reduces the accuracy and precision with which a laboratory will perform the doubly labeled water method and in some cases may limit the application of this technique. Herein we review the analytical requirements for optimal use of the method and some of the potential sources of error in the stable isotope analysis.

Adult↗
PubMed1994

Kinetic and mutational analysis of human immunodeficiency virus type 1 reverse transcriptase inhibition by inophyllums, a novel class of non-nucleoside inhibitors.

Inophyllums are novel non-nucleoside inhibitors of human immunodeficiency virus (HIV) type 1 reverse transcriptase identified through an enzyme screening program and isolated from the plant Calophyllum inophyllum. The kinetics of reverse transcriptase inhibition by inophyllum B were characterized using recombinant purified enzyme, a heteropolymeric RNA template, and a scintillation proximity assay. Preincubation of inhibitor with the enzyme-template-primer complex for 11 min was required for maximal inhibition of reverse transcriptase to occur, suggesting that inophyllum B had a slow on-rate and that template-primer must bind to reverse transcriptase prior to inhibitor binding. Inhibition of reverse transcriptase by inophyllums was shown to be reversible. When thymidine triphosphate was the variable substrate, inophyllum B inhibited reverse transcriptase noncompetitively with a Ki of 42 nM. Enzyme inhibition with respect to template-primer was uncompetitive with a Ki of 26 nM. Reverse transcriptase enzymes containing point mutations in which tyrosine 181 was changed to either cysteine or isoleucine exhibited marginal resistance to inophyllums but were resistant to (+)-(5S)-4,5,6,7-tetrahydro-9-chloro-5-methyl-6- (3-methyl-2-butenyl)-imidazo[4,5,1-j,k][1,4]benzodiazepin-2-(1H)-t hione (TIBO R82913). A mutant enzyme in which tyrosine 188 was changed to leucine was cross-resistant to both inophyllum B and TIBO R82913, as was HIV type 2 reverse transcriptase. These studies suggest that inophyllum B and TIBO R82913 bind to distinct but overlapping sites. Inhibition of avian myeloblastosis virus reverse transcriptase and Moloney murine leukemia virus reverse transcriptase by inophyllum B was detectible, suggesting that these inhibitors may be more promiscuous than other previously described non-nucleoside inhibitors. Inophyllums were active against HIV type 1 in cell culture with IC50 values of approximately 1.5 microM. These studies imply that the inophyllums have a novel mechanism of interaction with reverse transcriptase and as such could conceivably play a role in combination therapy.

Antiviral Agents↗
PubMed1994

Low plasma ascorbate levels in patients with type 2 diabetes mellitus consuming adequate dietary vitamin C.

Low ascorbate concentrations in diabetes may be secondary to inadequate dietary vitamin C intake or may relate to the varied metabolic roles of the vitamin. To determine whether inadequate dietary intake is a factor we calculated daily vitamin C intakes using both a vitamin C questionnaire and a 4-day food diary in a group of 30 patients with Type 2 diabetes (mean age 68.8 +/- 6.9 yr, 17M/13F) and in 30 community controls (mean age 68.0 +/- 5.5 yr, 12M/18F)). Measures of plasma glucose, serum fructosamine, and plasma ascorbic and dehydroascorbic acid were obtained from 20 subjects in each group. There was no significant difference in daily vitamin C intake between the two groups using both methods: food diary, 61.4 +/- 28.3 (patients) vs 69.5 +/- 33.4 (controls) mg; questionnaire, 54.0 +/- 28.9 (patients) vs 65.0 +/- 30.9 (controls) mg. Vitamin C intake derived from both methods was significantly correlated (p < 0.001). Plasma ascorbate (30.4 +/- 19.1 mumol l-1) and dehydroascorbate (27.6 +/- 6.4 mumol l-1) levels were significantly lower in patients vs in controls (68.8 +/- 36.0 and 31.8 +/- 4.8 mumol l-1, respectively), p < 0.0001 and p < 0.01. Plasma ascorbate levels were significantly correlated with vitamin C intake derived from the food diary (p < 0.01) and questionnaire (p < 0.01) methods in the diabetic group only. Low ascorbate levels in diabetes appears to be a consequence of the disease itself and not due to inadequate dietary intake of vitamin C. A short vitamin C questionnaire is a convenient and reliable estimate of vitamin C intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗
PubMed1993

The inophyllums, novel inhibitors of HIV-1 reverse transcriptase isolated from the Malaysian tree, Calophyllum inophyllum Linn.

As part of a search for novel inhibitors of HIV-1 reverse transcriptase, the acetone extract of the giant African snail, Achatina fulica, was shown to be active. Fractionation of the extract yielded inophyllums A, B, C, and E and calophyllolide (1a, 2a, 3a, 3b, and 6), previously isolated from Calophyllum inophyllum Linn., a known source of nutrition for A. fulica. From a methanol/methylene chloride extract of C. inophyllum, the same natural products in considerably greater yield were isolated in addition to a novel enantiomer of soulattrolide (4), inophyllum P (2b), and two other novel compounds, inophyllums G-1 (7) and G-2 (8). The absolute stereochemistry of inophyllum A (1a) was determined to be 10(R), 11(S), 12(S) from a single-crystal X-ray analysis of its 4-bromobenzoate derivative, and the relative stereochemistries of the other inophyllums isolated from C. inophyllum were established by a comparison of their 1H NMR NOE values and coupling constants to those of inophyllum A (1a). Inophyllums B and P (2a and 2b) inhibited HIV reverse transcriptase with IC50 values of 38 and 130 nM, respectively, and both were active against HIV-1 in cell culture (IC50 of 1.4 and 1.6 microM). Closely related inophyllums A, C, D, and E, including calophyllic acids, were significantly less active or totally inactive, indicating certain structural requirements in the chromanol ring. Altogether, 11 compounds of the inophyllum class were isolated from C. inophyllum and are described together with the SAR of these novel anti-HIV compounds.

Acetylation↗
PubMed1992

A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates.

Various steps involved in the preparation of partially methylated alditol acetates (PMAAs) from glycoprotein-derived carbohydrates were improved to obtain the derivatives in a rapid manner with excellent yields. Carbohydrates were permethylated in dimethyl sulfoxide (DMSO), using a fine suspension of sodium hydroxide and methyl iodide (CH3I). The fine suspension of NaOH was prepared conveniently from commercially available 50% aqueous NaOH in DMSO by sonication and washing the precipitate with DMSO. Methylation of ovalbumin and fetuin glycopeptides using the fine suspension of NaOH and CH3I was complete within 5 min, and the methylation reaction did not generate any nonsugar artifacts. Methylated carbohydrates without any purification were hydrolyzed in a mixture of volatile organic acids, which permitted rapid removal of the acids from samples by evaporation. Acetylation of partially methylated alditols with acetic anhydride for 2-4 h at ambient temperature using 4-N,N'-dimethylaminopyridine as a catalyst and the reaction was free from generating nonsugar reaction artifacts. The reaction time course for methylation, hydrolysis, and acetylation was determined to obtain optimum reaction conditions for preparation of the PMAAs. The procedure facilitated rapid identification and quantitation of PMAAs due to diminished reaction artifacts and the quality of the chromatogram depended only on the purity of starting material and the reagents used for the methylation analysis. Utility of these simple methods for rapid methylation analysis was demonstrated in the characterization of oligosaccharides isolated in small amounts using a carbohydrate analyzer.

Acetates↗
PubMed1992

Myocardial stretch alters twitch characteristics and Ca2+ loading of sarcoplasmic reticulum in rat ventricular muscle.

OBJECTIVE: The aim was to determine the influence of diastolic muscle length on force development and timing parameters of cardiac muscle twitch contraction and to determine whether a length dependency exists for the calcium loading capacity of the sarcoplasmic reticulum. METHODS: Right ventricular papillary muscles and trabeculae were isolated from hearts of female Wistar rats weighing 220-280 g. Papillary muscles were stretched to diastolic lengths of 90, 95, and 100% Lmax and paced at 1.0 Hz. Individual twitch profiles were characterised by their peak force and the maximum rate (dF/dt) of the positive and negative force changes. Intrinsic timing was identified through waveform analysis that divided the twitch profile into time domains for the ascending limb (T0-T1; T1-T2) and the descending limb (T2-T3; T3-T4). Each domain was compared at three muscle lengths. The sarcoplasmic reticular calcium content at short (1.88 microns) and long (2.11 microns) sarcomere lengths was characterised by rapid cooling contractures after 1 s and 60 s of diastolic rest. RESULTS: Peak developed force and the maximum rate of positive and negative force development decreased as diastolic muscle length was reduced from Lmax to 90% Lmax. The intrinsic timing for the segment that reflects the relaxation phase of the twitch (T1-T4) was shortened as muscle length was reduced. The time domain that reflects the combined effects of calcium release and the early phase of contraction (T0-T1) was insensitive to diastolic muscle length. The fractional release of sarcoplasmic reticular calcium at different muscle lengths was approximately 32-35% of the total sarcoplasmic reticulum calcium pool. CONCLUSIONS: The data on the intrinsic timing of the twitch characteristics coupled with rapid cooling contracture analysis suggests a fractional calcium release that is approximately 32-35% of the total sarcoplasmic reticular capacity at either long or short muscle lengths. However, the loading capacity of the sarcoplasmic reticulum is greater when the muscle operates at a shorter diastolic length. This can be interpreted as meaning that diastolic muscle length differentially influences sarcoplasmic reticular calcium storage and release processes.

Animals↗
PubMed1991

Quantitative determination of phenyl isothiocyanate-derivatized amino sugars and amino sugar alcohols by high-performance liquid chromatography.

Simple and rapid methods for the preparation of phenylthiocarbamyl (PTC) derivatives of amino sugars and amino sugar alcohols and their quantitative determination with high sensitivity (less than 10 pmol) by C18 reversed-phase high-performance liquid chromatography are described. Rapid sample preparation of the phenyl isothiocyanate (PITC)-derivatized amino sugars and amino sugar alcohols was achieved by a simple extraction of the reaction mixture with chloroform to remove the excess PITC and its adducts. Baseline separation of the PTC derivatives of amino sugars and amino sugar alcohols was obtained within 30 min, using a simple solvent system consisting of 0.2% each of n-butylamine, phosphoric acid, and tetrahydrofuran. The mobile phase containing n-butylamine, in conjunction with a C18 stationary phase, mimics the conditions for the separation of carbohydrates on an amino-bonded column. GlcNH2 and GalNH2 derived from the initial protein-sugar linkages were also separated from the amino acids for quantitative estimation of sugar chains in glycoproteins. Amino sugar alcohols gave single reaction products with PITC while the reaction with amino sugars was accompanied by the formation of secondary products. Apparently the secondary products were formed in an acid-catalyzed intramolecular cyclization of the PTC-hexosamines involving the aldehyde functional group. Conditions were developed to stop the transformations and maintain the stability of PTC derivatives for their convenient determination by HPLC.

Amino Sugars↗
PubMed1991

Rapid characterization of asparagine-linked oligosaccharides isolated from glycoproteins using a carbohydrate analyzer.

Chromatographic methods were developed for the separation and characterization of acidic (sialylated) and neutral (asialo-complex and high-mannose) oligosaccharides released from glycoproteins with peptide N-glycosidase F. endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H using a carbohydrate analyzer (Dionex BioLC). All the carbohydrate separations were carried out on a polymeric pellicular anion-exchange column HPIC-AS6/CarboPac PA-1 (Dionex) using only two eluants namely, 0.5 M NaOH and 3% acetic acid/NaOH pH 5.5, which were mixed with water to generate various gradients. Developed conditions for quantitative detection of carbohydrates with pulsed amperometry were necessary to obtain steady baselines at 0.1-0.3 microA output with suitable sensitivity (less than 5 pmol) in separations employing a variety of acidic and alkaline sodium acetate gradients. Oligosaccharides released from heat-denatured and trypsin-treated glycoproteins were purified initially from large-scale digestion (greater than 0.1 g) by extraction of peptide material into phenol/chloroform and finally by ion-exchange chromatography of the acqueous phase. Oligosaccharides isolated from the peptide N-glycosidase digests of bovine fetuin, human transferrin and alpha 1-acid glycoprotein gave multiple peaks in each charge group in separations based on the charge content at pH 5.5. Alkaline sodium acetate gradients were developed to obtain oligosaccharide maps of the glycoproteins within 60 min, in which separated oligosaccharides eluted in the order of neutral, mono-, di-, tri- and tetra-sialylated species based on both charge, size and structure. Baseline separations were obtained with neutral oligosaccharide types but mixtures of high-mannose and complex types were poorly resolved. The high-mannose peaks were eliminated specifically from complex oligosaccharides by digesting with alpha-mannosidase. Treatment with beta-galactosidase, beta-N-acetylglucosaminidase and alpha-mannosidase resulted in a decrease of the oligosaccharide elution times corresponding to the number of sugar residues lost, the profile of changes was highly reproducible. In contrast, treatment with alpha-L-fucosidase, endo-beta-N-acetylglucosaminidase F and endo-beta-N-acetylglucosaminidase H resulted in an increase in their corresponding oligosaccharide retention times similar to the presence of an additional sugar residue. Conditions developed for separation of the reduced oligosaccharides and also a mixture of monosaccharide to oligosaccharide containing about 15 sugar residues within 30 min were useful in determining the effect of endo- and exo-glycosidases on porcine thyroglobulin oligosaccharides. Changes in elution time of the oligosaccharides following specific glycosidase digestions combined with methylation analysis provided a rapid and sensitive tool for confirmation of the carbohydrate primary structures present in thyroglobulin.

Animals↗
PubMed1991

Structural classification of carbohydrates in glycoproteins by mass spectrometry and high-performance anion-exchange chromatography.

A general strategy has been developed for determining the structural class (oligomannose, hybrid, complex), branching types (biantennary, triantennary, etc.), and molecular microheterogeneity of N-linked oligosaccharides at specific attachment sites in glycoproteins. This methodology combines mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection to take advantage of their high sensitivity and the capability for analysis of complex mixtures of oligosaccharides. Glycopeptides are identified and isolated by comparative HPLC mapping of proteolytic digests of the protein prior to, and after, enzymatic release of carbohydrates. Oligosaccharides are enzymatically released from each isolated glycopeptide, and the attachment site peptide is identified by fast atom bombardment mass spectrometry (FAB-MS) of the mixture. Part of each reaction mixture is then permethylated and analyzed by FAB-MS to identify the composition and molecular heterogeneity of the carbohydrate moiety. Fragment ions in the FAB mass spectra are useful for detecting specific structural features such as polylactosamine units and bisecting N-acetylhexosamine residues, and for locating inner-core deoxyhexose residues. Methylation analysis of these fractions provides the linkages of monomers. Based on the FAB-MS and methylation analysis data, the structural classes of carbohydrates at each attachment site can be proposed. The remaining portions of released carbohydrates from specific attachment sites are preoperatively fractionated by high-performance anion-exchange chromatography, permethylated, and analyzed by FAB-MS. These analyses yield the charge state and composition of each peak in the chromatographic map, and provide semiquantitative information regarding the relative amounts of each molecular species. Analytically useful data may be obtained with as little as 10 pmol of derivatized carbohydrate, and fmol sensitivity has been achieved. The combined carbohydrate mapping and structural fingerprinting procedures are illustrated for a recombinant form of the CD4 receptor glycoprotein.

Amino Acid Sequence↗
PubMed1991

Inotropic interventions and myocardial force-interval relation: a quantitative approach.

OBJECTIVES: To analyze inotropic influence on the early and late phases of cardiac sarcoplasmic reticulum calcium loading. DESIGN: Papillary muscles with parallel edges and no evidence of tissue branching were selected from the heart. Only muscle preparations that maintained stable passive diastolic and developed forces were used for analysis. Muscles were stretched to their maximum length and stimulated at 0.2 Hz. The early and late phases of sarcoplasmic reticulum calcium loading were evaluated quantitatively by mathematical fitting of the force-interval relation. Increasing the extracellular calcium or decreasing the extracellular sodium was used to increase the inotropic state. ANIMALS: Right ventricular papillary muscles were isolated from female Wistar rats weighing 200 to 220 g. Electrical stimulation and data acquisition were controlled through a microcomputer. MAIN RESULTS: Increasing the extracellular calcium concentration from 0.5 to 1.0 mM produced a 90% increase in developed tension. This was accounted for by a 41% increase in the early phase of sarcoplasmic reticulum loading and a 29% increase in the late phase. A 20% reduction in the extracellular sodium concentration increased contractile force 100% and shifted the force-interval curve to the left. This was accounted for through an increase in both early and late phases of sarcoplasmic reticulum loading. CONCLUSION: These results are consistent with the current model of excitation-contraction coupling and clearly indicate that various positive inotropic interventions have selective effects on each process of the force-interval relation that cooperatively interact with each other. Mathematical fitting of data clearly improves the quantitative aspect of the force-interval response.

Animals↗