Biomedical subjects

J W Dolan

Publications and source records attributed to J W Dolan.

At least 37 records · Page 2Linked to original sources

PubMed2000

Reversed-phase separation of achiral isomers by varying temperature and either gradient time or solvent strength.

The separation of several isomer pairs of widely varied structure was studied as a function of changes in temperature, gradient time, mobile phase pH, column type ("monomeric" vs. "polymeric" C18-silica) and organic solvent (methanol vs. acetonitrile). General conclusions are drawn which may prove useful in future attempts at the separation of these and other achiral isomers.

Solvents↗

PubMed2000

Computer simulation for the convenient optimization of isocratic reversed-phase liquid chromatographic separations by varying temperature and mobile phase strength.

Software is described which allows the rapid development of separations by means of isocratic reversed-phase liquid chromatography (RP-LC), based on the optimization of column temperature (T) and mobile phase strength (%B). For a given sample, four initial experiments are carried out at two different temperatures, using either isocratic or (better) gradient elution. If isocratic experiments are chosen for computer simulation, it is necessary to select appropriate values of %B for these initial runs. Literature data for solute retention as a function of T are reviewed, as a basis for estimating suitable values of %B at the two values of T selected. The use of optimized values of T and %B led to acceptable separations for three representative samples. The prediction of isocratic separation on the basis of initial gradient experiments is more convenient than the use of initial isocratic experiments, but less reliable. When gradient experiments are used, one additional isocratic experiment can improve the accuracy of such predictions by a "reflection" procedure. The latter approach was confirmed for predictions of both isocratic and gradient separation from initial gradient experiments.

Chromatography, Liquid↗

PubMed2000

Control of column temperature in reversed-phase liquid chromatography.

When separations by reversed-phase liquid chromatography (RP-LC) are carried out at temperatures other than ambient, resulting retention times and bandwidths can depend on the equipment used. As a result, an RP-LC separation that is adequate when carried out on one LC system may prove inadequate when the separation is repeated on a second system. In the present study, various temperature-related problems which can result in a failure of method transfer for non-ambient RP-LC methods were examined. Means for correcting for such effects and thereby ensuring method transferability are described.

Chromatography, Liquid↗

PubMed2000

Computer simulation for the simultaneous optimization of any two variables and any chromatographic procedure.

Computer software that allows the simulation of any chromatographic separation as a function of simultaneous changes in any one or two variables that can affect sample separation order (selectivity) is described. For one example, an application is described for the simultaneous variation of the mobile phase pH and gradient time in reversed-phase liquid chromatography. The accuracy of such predictions is examined for a sample mixture of 17 substituted benzoic acids and anilines, and requirements for an acceptable predictive accuracy are summarized. In a second example, the separation of three peptides by capillary electrophoresis is optimized.

Benzoates↗

PubMed2000

Undecylenic acid inhibits morphogenesis of Candida albicans.

Resilient liners are frequently used to treat denture stomatitis, a condition often associated with Candida albicans infections. Of 10 liners tested, 2 were found to inhibit the switch from the yeast form to hyphae and a third was found to stimulate this switch. The inhibitor was determined to be undecylenic acid.

Candida albicans↗

PubMed2000

Carotid cavernous fistula associated with persistent primitive trigeminal artery.

A patient with diplopia had a carotid cavernous fistula associated with a persistent primitive trigeminal artery that was seen with angiography. Balloon occlusion of the carotid cavernous fistula resulted in flow stasis of the persistent primitive trigeminal artery and resolution of the symptoms. Persistent primitive trigeminal artery may be associated with a carotid cavernous fistula.

Aged↗

PubMed1999

Endogenous mutations in human uncoupling protein 3 alter its functional properties.

Human uncoupling protein (UCP3) is a mitochondrial transmembrane carrier that uncouples oxidative phosphorylation and is a candidate gene for obesity. Expression of native human UCP3 mutations in yeast showed complete loss (R70W), significant reduction (R143X), or no effect (V102I and IVS6+1G > A) on the uncoupling activity of UCP3. It is concluded that certain mutations in UCP3 alter its functional impact on membrane potential (deltaphi), possibly conferring susceptibility to develop metabolic diseases.

Carrier Proteins↗

PubMed1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations.

The separation of samples that contain more than 15 to 20 analytes (n > 15-20) is typically difficult and usually requires gradient elution. We have examined the reversed-phase liquid chromatographic separation of 24 samples with 8 < or = n < or = 48 as a function of temperature T and gradient time tG. The required peak capacity was determined for each sample, after selecting T and tG for optimum selectivity and maximum sample resolution. Comparison of these results with estimates of the maximum possible peak capacity in reversed-phase gradient elution was used to quantify the maximum value of n for some required sample resolution (when T and tG have been optimized). These results were also compared with literature studies of similar isocratic separations as a function of ternary-solvent mobile phase composition, where the proportions of methanol (MeOH), tetrahydrofuran (THF) and water were varied simultaneously. This in turn provides information on the relative effectiveness of these two different method development procedures (optimization of T and tG vs. % MeOH and % THF) for changing selectivity and achieving maximum resolution.

Chromatography, Liquid↗

PubMed1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time III. Improving the accuracy of computer simulation.

Previous studies have shown that four experimental runs, where both temperature T and gradient time tG are varied, can be used for the reliable prediction of separation as a function of these two variables (two-dimensional optimization). Computer simulation (e.g., DryLab) can then be used to predict "optimized" conditions for maximum sample resolution using either isocratic or gradient elution. Samples that contain a large number of components (e.g., n>15-20) present a greater challenge. Resolution for these more complex samples is often quite sensitive to small changes in T or tG in turn requiring greater accuracy in predictions that result from computer simulation. In the present study of several samples, we have examined computer simulation errors that can arise from inexact expressions for retention time as a function of T, tG or isocratic %B. Resulting conclusions are applicable to both complex and simpler samples, in either one- or two-dimensional optimization. Means to anticipate and minimize the impact of these predictive errors are examined.

Chromatography, Liquid↗

PubMed1999

Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time II. Two-run assay procedures.

By optimizing column temperature T and gradient time tG, complex samples can often be separated by means of reversed-phase high-performance liquid chromatography (RP-LC). Conclusions reached in Part I suggest that the complete separation of such samples will be difficult, however, when more than 15-20 components are present in the sample. An alternative approach is to carry out two separations with different conditions (T, tG) in each run. The combination of results from these two runs then allows the total analysis of the sample, providing that every sample component is adequately resolved in one run or the other. Examples of this approach, carried out by means of computer simulation, are shown here for several samples of varying complexity. Also considered is the ability of a single separation where T and tG are optimized to enable the separation and analysis of one or more individual sample components from complex mixtures (e.g., drugs in animal plasma), including the resolution of isomeric compounds from each other.

Chromatography, Liquid↗

PubMed1998

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development. I. Application to 14 different samples using computer simulation.

The optimized reversed-phase HPLC separation of 14 different samples is reported, based on simultaneous changes in temperature and gradient steepness. Four experimental runs are required for each sample, following which preferred conditions can be predicted using computer simulation software (DryLab). The overall accuracy and effectiveness of this method development approach is discussed, with particular attention to the use of resolution maps provided by the software. These maps are useful for maximizing resolution for the total sample, for optimizing the separation of a smaller number of selected sample compounds, and as an initial step in the separation of more demanding samples.

Adrenal Cortex Hormones↗

PubMed1998

Simultaneous variation of temperature and gradient steepness for reversed-phase high-performance liquid chromatography method development. II. The use of further changes in conditions.

The preceding paper (Part I) suggests that simply optimizing temperature and gradient steepness will often provide an adequate reversed-phase HPLC separation. In some cases, however, this procedure will prove unsuccessful, and then further method-development experiments (involving change in other separation conditions) will be required. One strategy is to change a variable other than temperature or gradient steepness, followed by re-optimization of the latter two variables. The present paper examines the application of this approach with the aid of computer simulation to several samples.

Acetonitriles↗

PubMed1998

Maintaining fixed band spacing when changing column dimensions in gradient elution.

In gradient elution separations, it may be required to change either column length (to increase resolution or shorten run time) or column diameter (for an increase in sensitivity or for preparative separations). In either of these changes of column dimensions, it is usually desired to maintain the same relative band spacing (selectivity), so as to increase resolution in proportion to (column plate number)1/2 when increasing column length, or to maintain constant resolution when changing column diameter. A general rule for avoiding changes in band spacing in these situations is to maintain the quantity [(gradient time) x (flow-rate)/(column volume)] constant, while holding the initial and final gradient mobile phase compositions (%B) fixed. This rule is only valid as long as the equipment hold-up volume (dwell volume) is negligible, or if all sample components are strongly retained at the start of the gradient. When neither of the latter conditions apply, then significant changes in band spacing may result when changing column size. Rules are presented for recognizing this potential problem for a given sample/HPLC-equipment combination, and adjustments in separation conditions that can avoid this problem are discussed. Changes in band spacing as a result of change in column size are of special concern when developing procedures for preparative chromatography under gradient conditions.

Chromatography, High Pressure Liquid↗

PubMed1997

Characterization of a sphingomyelinase activity in Saccharomyces cerevisiae.

Sphingomyelinases (SMase), which hydrolyze sphingolipids to yield ceramide, participate in signal transduction pathways in mammalian cells. Although yeast express many homologs of mammalian signaling proteins, SMase activity had not been previously demonstrated in yeast. In this study, we used an in vitro assay to characterize yeast SMase activity. Activity was detected in yeast membranes at both acid and neutral pH. The enzyme exhibited a requirement for magnesium or manganese, and was sensitive to detergents. The pI of the enzyme was approximately 5.9. SMase was separable from phospholipase D (PLD) activity, and was expressed at normal levels in yeast lacking expression of PLD1. While sphingosine and phytosphingosine inhibited growth, other sphingolipid metabolites had no effect on yeast growth. Intact yeast generate ceramide from exogenous sphingomyelin. These studies demonstrate that yeast express a membrane-localized neutral SMase activity.

Animals↗

PubMed1996

Combined use of temperature and solvent strength in reversed-phase gradient elution. I. Predicting separation as a function of temperature and gradient conditions.

It has been shown previously that computer simulation based on two initial experiments can predict separation in reversed-phase gradient elution as a function of gradient conditions (gradient steepness, gradient range and gradient shape) and column conditions (column length, flow-rate and particle size). The present study extends this capability for changes in temperature. Four initial experiments (two different gradient times, two different temperatures) provide input data that allow predictions of separation as a function of temperature as well as gradient and column conditions. A semi-empirical relationship, tR = a + bT, is able to relate gradient retention time tR to column temperature T (other conditions constant). The accuracy of this approach has been evaluated for 102 solutes and a variety of experimental conditions, including the use of five different HPLC instruments (four different models).

Chromatography, High Pressure Liquid↗

PubMed1996

Characterization of Saccharomyces cerevisiae deficient in expression of phospholipase D.

A gene encoding phospholipase D (PLD) in Saccharomyces cerevisiae was identified. The 195 kDa product of PLD1 has 24% overall sequence identity with a plant PLD. Expression of yeast PLD activity was eliminated by one-step gene disruption. Yeast haploids lacking PLD activity were deficient in growth on non-fermentable carbon sources. Diploids lacking expression of PLD1 were unable to sporulate.

Amino Acid Sequence↗

PubMed1996

Novel aspects of pheromone-induced cell-cycle arrest in yeast.

Saccharomyces cerevisiae responds to pheromones by arresting cell-cycle progression at the G1/S boundary. This arrest has been shown to require the activation by phosphorylation of Far1, a cyclin-dependent kinase inhibitor. Arrest resembling pheromone-induced arrest can be triggered by high levels of Ste12, the pheromone-responsive transcriptional activator. In the present study, cell-cycle arrest induced by high levels of Ste12 is characterized. This arrest occurs predominately at the G1/S boundary and is independent of Far1 and upstream components of the pheromone response pathway. Since Ste12 is a transcriptional activator, it is proposed that cell-cycle arrest in response to high levels of Ste12 is due to transcription of a Ste12-dependent arrest factor regulated primarily, but not necessarily exclusively, at the level of transcription. The data presented here suggest that a novel component has a role in arrest in response to pheromone.

Biological Factors↗

PubMed1995

A role for the Gal11 protein in pheromone-induced transcription in Saccharomyces cerevisiae.

The product of the Saccharomyces cerevisiae GAL11 gene, Gal11p, is required for the proper expression of a wide range of genes including many mating-type-specific genes. In this study Gal11p is shown to have a role in the induction of transcription by pheromone. Basal transcription of an a-specific gene is unaffected by loss of Gal11p but pheromone treatment fails to increase transcription. A haploid-specific gene is shown to retain pheromone-inducibility in the absence of Gal11p but the extent of induction is drastically reduced. Evidence is also presented that suggests the existence of strain differences that can alter the phenotype of gal11 mutants.

Base Sequence↗