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J W Dolan

Publications and source records attributed to J W Dolan.

54 records · Page 3Linked to original sources
PubMed1995

Characterization of a regulated form of phospholipase D in the yeast Saccharomyces cerevisiae.

Phospholipase D (PLD), which is present in bacterial, plant and animal cells, can serve as an important element of signal-transduction pathways. This study examined the potential role of this enzyme in the regulation of Saccharomyces cerevisiae. An assay in vitro using a fluorescent 1-acyl-2-alkyl glycerophosphocholine as substrate was used to assess PLD activity in yeast cell extracts. A neutral PLD activity is present in membranes prepared from both haploid and diploid yeast cells, as evidenced by the production of phosphatidic acid and phosphatidylbutanol in the presence of butanol. Alcohols, in addition to serving as substrates for transphosphatidylation, stimulate PLD activity. Increased PLD activity is detected in membranes when either haploid or diploid cells are incubated in the presence of a non-fermentable carbon source. Membrane PLD activity increases within 10 min after diploid cells are placed in a sporulation-inducing medium lacking nitrogen and containing a non-fermentable carbon source. The increased activity persists for 2-3 h, and then declines to control values. This response occurs in the presence of cycloheximide, an inhibitor of protein synthesis. These data indicate that PLD activity is present in yeast, and that activation of PLD is an early event in sporulation in this organism.

Adaptation, Physiological↗
PubMed1993

The effects on aqueous dynamics of PhXA41, a new prostaglandin F2 alpha analogue, after topical application in normal and ocular hypertensive human eyes.

OBJECTIVE: To study the effects of a topically applied prostaglandin F2 alpha analogue, PhXA41 (Latanoprost; 13,14-dihydro-17-phenyl-18,19,20-trinor-prostaglandin F2 alpha-isopropyl ester), on aqueous dynamics in the human eye. DESIGN: A randomized, double-masked study was carried out on 20 normal and 20 ocular hypertensive humans. One eye of each subject was treated with 0.006% PhXA41, while the contralateral eye received placebo twice daily for 5 days. MAIN OUTCOME: Compared with placebo, PhXA41 reduced intraocular pressure in both groups by approximately 20%. RESULTS: Tonographic facility of outflow was increased 24% in the normal group and 30% in the ocular hypertensive group; no changes were observed in the rates of aqueous humor flow in either group. The changes in tonographic facility were insufficient to fully explain the ocular hypotensive effect of the drug, suggesting that PhXA41 enhances outflow via the uveoscleral pathway. The suitability of fluorophotometry as a measure of flow was confirmed by three methods of comparing blood-aqueous barrier permeability: polarization of cameral fluorescence, intensity of backscattered light from the anterior chamber (flare), and cameral fluorescence after oral administration of fluorescein sodium. All of these measured parameters were normal, suggesting that this compound has no clinically significant effects on the blood-aqueous barrier or on the accuracy of fluorophotometry. PhXA41 was well tolerated in both groups. Only four of 40 subjects reported a transient foreign-body sensation, and only one of 40 subjects was observed to have greater than moderate conjunctival hyperemia. Most subjects had no symptoms and no measurable hyperemia. CONCLUSIONS: These results suggest that PhXA41 is a potentially useful ocular hypotensive agent that enhances the egress of aqueous humor via both major outflow pathways. The relative lack of ocular side effects in this study further suggests that this agent has promise for the treatment of chronic glaucoma.

Aqueous Humor↗
PubMed1992

Computer simulation for the prediction of separation as a function of pH for reversed-phase high-performance liquid chromatography. I. Accuracy of a theory-based model.

Computer simulation software (DryLab I/mp) is described for predicting high-performance liquid chromatographic separation as a function of changes in mobile phase pH. Three experimental runs with pH (only) varied are used to derive values of pKa plus capacity factors (k') for the ionized and non-ionized form of each ionizable solute. Various tests of the experimental data then allow classification of each solute as acidic, basic, neutral (including strong or weak acids or bases) and amphoteric. Experimental data are reported for the separation of several substituted anilines as a function of pH and solvent composition (%B). Experimental requirements for the accurate prediction of separation (ca. +/- 2-4% in alpha) as a function of pH are discussed. The reliability of the software is demonstrated for three different samples: mixtures of (a) substituted benzoic acids, (b) substituted anilines and (c) catecholamine-related compounds.

Aniline Compounds↗
PubMed1992

Computer simulation for the prediction of separation as a function of pH for reversed-phase high-performance liquid chromatography. II. Resolution as a function of simultaneous change in pH and solvent strength.

The optimization of reversed-phase high-performance liquid chromatographic separation by the simultaneous variation of pH and solvent strength (%B) was studied for acidic (substituted benzoic acids) and basic samples (substituted anilines). The combination of these two variables was expected to be more useful than either variable alone. This proved to be the case for the benzoic acid sample, but not for the aniline sample. Column plate numbers were also studied for each sample and as a function of pH. With the exception of one compound (3,5-dimethylaniline) in one particular pH range (3.0-4.5), plate numbers of 12,000-20,000 were observed for each sample.

Aniline Compounds↗
PubMed1992

Intravenous epinephrine stimulates aqueous formation in the human eye.

An experiment was done to determine if intravenously administered epinephrine would stimulate the rate of aqueous humor flow in sleeping human subjects. Twenty normal volunteers participated in a double-masked randomized placebo-controlled study. The rate of aqueous humor flow was measured by the rate of clearance of topically applied fluorescein. Measurements were made on two separate nights while the subjects slept, once during a placebo infusion and again during an intravenous infusion of epinephrine (rate, 1 microgram/hr; a rate calculated to be threefold the basal adrenal secretion in supine sedentary human subjects). Compared with placebo, epinephrine infusion had no effect on the blood pressure of sleeping subjects, increased the heart rate by 10%, and increased aqueous flow by 27%. Systolic blood pressure was 112 +/- 9 mmHg (mean +/- standard deviation) during placebo infusion and 114 +/- 13 during epinephrine infusion. The heart rate was 60 +/- 10 beats/min during placebo and 67 +/- 14 during epinephrine infusion (P = 0.011). Aqueous flow was 1.11 +/- 0.51 microliters/min during placebo and 1.42 +/- 0.52 during epinephrine infusion (P = 0.016). It was concluded that adrenal medullary secretion can stimulate aqueous formation and may explain in part the circadian rhythm of aqueous flow.

Adrenal Medulla↗
PubMed1991

Pheromone-dependent phosphorylation of the yeast STE12 protein correlates with transcriptional activation.

Haploid a and alpha cells of yeast respond to the pheromones alpha- and a-factor, respectively, by increasing the transcription of many genes whose products are essential for mating. The STE12 protein acts in this process by binding to the DNA sequence that mediates the increased transcription of pheromone-responsive genes. We show here that a hybrid protein containing STE12 fused to the DNA-binding domain of GAL4 can activate transcription of a reporter gene containing GAL4-binding sites but only after treatment of cells with pheromone. Thus, STE12 alone, when bound to DNA, is sufficient to mediate pheromone-induced transcription. By constructing hybrids of different STE12 regions with the GAL4 domain, we map the domain of STE12 necessary for this activation to the central third of the protein. Upon alpha-factor treatment, the hybrid of GAL4 with the complete STE12 sequence is rapidly phosphorylated, with kinetics consistent with the observed transcriptional induction of pheromone-responsive genes. The domain of STE12 necessary for this phosphorylation correlates with that involved in transcriptional activation. We propose that induction of pheromone-responsive genes is mediated by phosphorylation of STE12 to alter its activation function but not its DNA-binding ability.

Binding Sites↗
PubMed1990

Computer simulation (based on a linear-elution-strength approximation) as an aid for optimizing separations by programmed-temperature gas chromatography.

If the dependence of retention on temperature is specified for the various components of a sample in isothermal gas chromatography (GC), it is possible to predict retention, bandwidth, and resolution for programmed-temperature GC separations as a function of experimental conditions. The use of a linear-elution-strength (LES) approximation for isothermal retention allows these predictions to be carried out more easily and conveniently, in turn facilitating rapid simulations with a personal computer. This approach to GC method development appears promising, especially if segmented-temperature programs are used. The LES approximation also provides added insight into how different factors affect separation in programmed-temperature GC.

Chromatography, Gas↗
PubMed1990

Transcription termination factor rho has three distinct structural domains.

The domain structure of transcription termination factor rho was analyzed by partial trypsin cleavage and by photoaffinity labeling with ATP and oligo(C)5. A rho subunit consists of three distinct domains of nearly equal size that are connected by trypsin-sensitive linker segments. The amino-terminal domain binds to oligo(C)5 and has sequence segments with extended similarity to conserved elements in other RNA-binding proteins and a segment that has identities with another known cytidine nucleotide-binding protein. The middle domain has the ATP-binding site and has most of the sequence segments with similarity to conserved elements in other nucleoside triphosphate-binding proteins. The function of the third domain, the carboxyl-terminal domain, has not been identified. Both the amino- and carboxyl-terminal domains are relatively resistant to further trypsin treatment. The ATP-binding domain is also relatively resistant when it is linked to the amino-terminal, RNA-binding domain, but has not been detected as a separate digestion product. This result plus the finding that ADP and ATP inhibit the cleavage in the linker between the two domains indicate that those domains interact intimately in spite of their functional distinctions.

Adenosine Diphosphate↗
PubMed1990

Overproduction of the yeast STE12 protein leads to constitutive transcriptional induction.

Haploid a and alpha cells of the yeast Saccharomyces cerevisiae respond to the pheromones alpha- and a-factor, respectively, by increasing transcription of inducible genes, arresting cell division, and forming cell-surface projections. These responses are dependent on the activity of several genes, including STE12, whose product binds to the pheromone response element located within the regulatory DNA sequences of inducible genes. We assayed the effects of overproducing the STE12 protein in both STE+ cells, as well as ste2, ste7, and ste11 mutant cells. We find that overproduction leads to increased transcription of pheromone-inducible genes and is able to suppress the mating defect of the ste mutants. These results suggest that one effect of pheromone treatment may be to increase the ability of the STE12 protein to activate transcription. In addition, we observed that cells cannot tolerate very high levels of the STE12 protein, and many arrest in G1 with a large size and morphological changes. Thus, constitutively high-level transcription of pheromone-inducible genes causes cells to display some features similar to treatment with pheromone.

Fungal Proteins↗
PubMed1989

The yeast STE12 protein binds to the DNA sequence mediating pheromone induction.

The STE12 gene product of Saccharomyces cerevisiae is required for the transcription of two sets of cell-type-specific genes: the a-specific genes (active only in a cells) and the alpha-specific genes (active only in alpha cells). We show that radiolabeled STE12 protein, prepared by in vitro transcription and translation, is capable of forming complexes with unlabeled DNA fragments from two a-specific genes. Wild-type yeast, but not a ste12 mutant, produce a factor that forms complexes with labeled DNA from these same two genes. We use assays with yeast extracts to localize the binding site for the STE12-dependent activity. This site corresponds to the sequence identified as the pheromone induction element, which is responsible for increased transcription of genes when cells are exposed to alpha-factor or a-factor. Thus the STE12 protein may be an ultimate effector in the signal transduction pathway triggered by pheromone.

Amino Acid Sequence↗
PubMed1981

Liquid-chromatographic analysis for common tricyclic antidepressant drugs and their metabolites in serum or plasma with the technicon "FAST-LC" system.

We describe a single procedure for assay of seven tricyclic antidepressant drugs and metabolites in serum or plasma: protriptyline, nortriptyline, amitriptyline, desmethyldoxepin, doxepin, desipramine, and imipramine. With the Technicon "FAST-LC" system, samples are aspirated directly into the unit and pretreated via double extraction; the concentration of each drug is then determined by "high-performance" liquid chromatography. Final chromatograms are monitored at 205 nm, at analysis rates of 7.5 samples/h. Concentration and absorbance are linearly related for each drug from 0 to 1400 micrograms/L. Day-to-day CVs averaged 5 to 6% for each drug, and there is good correlation of FAST-LC values with those obtained by gas-chromatographic methods. Total sample volume is 750 microliters.

Amitriptyline↗
PubMed1980

On-line liquid-chromatographic analysis for drugs in serum with the Technicon "FAST-LC" system: performance data for theophylline and for four commonly used anticonvulsants and their metabolites.

We describe a new instrument for use in assay of therpeutic drugs in serum by "high-performance" liquid chromatography, the "FAST-LC" system (Technicon). Serum samples are aspirated directly into the unit, extracted with solvent, and the evaporated and redissolved extract is injected onto a chromatographic column. We illustrate the performance of the system by assays in serum for theophylline and four anticonvulsants (primidone, phenobarbital, phenytoin, and carbamazepine) plus two of their active metabolites (phenylethylmalonamide and carbamazepine epoxide). For theophylline, final chromatograms are monitored at 270 nm, at analysis rates of 10/h. Concentration and absorbance are linearly related from 0 to 130 mg of theophylline per liter. For the anticonvulsants, chromatograms are monitored at 200 nm, at analysis rates of 7.5/h. The six individual determinations are each linear beyond the therapeutic range. For both drug panels, day-to-day CV's were 4 to 6%. Results correlate well with those by enzyme immunoassay. A total sample volume of 150 microL is required.

Anticonvulsants↗
PubMed1978

Continuous-flow automated HPLC analysis of fat-soluble vitamins in tablets.

A prototype automated system involving continuous-flow analysis and high performance liquid chromatography (CFA/HPLC) has been developed for the analysis of fat-soluble vitamins in individual pharmaceutical tablets. The novel features are the front-end coupling of CFA to HPLC, injection of hexane solutions on reversed phase columns, separation/quantitation of vitamins A, D2 and E within single chromatographic runs for a wide variety of tablets, and a dynamic range sufficient to accommodate the 1000-fold higher levels of vitamins a and E over D2 in the same tablets. The analysis rate is 10 samples per hour, the precision better than 6% for all three vitamins, and the recovery is 70-90% of that obtained by the standard AOAC method. Although the system is a prototype, it already greatly outperforms current manual analyses which are time consuming, tedious, and demanding in terms of the level of skill and experience of the experimenter. Included in this work are some retention comparisons of commercial columns.

Autoanalysis↗