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E T O'Brien

Publications and source records attributed to E T O'Brien.

At least 55 records · Page 3Linked to original sources
PubMed1991

Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap.

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubule of 60 microns/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437-1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.

Animals↗
PubMed1991

Reference values for the ambulatory blood pressure and the blood pressure measured at home: a population study.

In order to determine reference values for the ambulatory blood pressure, a population sample of 328 subjects, aged 20-81 years, who reported themselves to be in good health, was investigated. The ambulatory blood pressure was recorded over 24 h, taking measurements at 20 min intervals from 8 am to 10 pm, and at 45 min intervals from 10 pm to 8 am. Blood pressure was also measured by trained observers on each of two separate home visits (5 readings per visit). The ambulatory blood pressure in the 328 subjects averaged 118/71 mmHg over 24 h, 124/76 mmHg during the day (10 am-8 pm), and 108/62 mmHg at night (0 am-6 am). Blood pressure measured by an observer at the occasion of the second home visit was 4/5 mmHg lower (P less than 0.001) than the daytime ambulatory blood pressure. The 95th centiles for the daytime ambulatory pressures were 144/95 mmHg in 85 men below age 50; 154/90 mmHg in 74 men aged greater than or equal to 50 years; 132/85 mmHg in 96 women below age 50; and 151/91 mmHg in 73 women aged greater than or equal to 50 years. The 95th centiles for the nighttime pressures in these four sex-age groups were 124/79, 140/83, 121/70, and 132/72 mmHg, respectively.

Adult↗
PubMed1990

Effects of magnesium on the dynamic instability of individual microtubules.

We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.

Animals↗
PubMed1989

Assembly of pure tubulin in the absence of free GTP: effect of magnesium, glycerol, ATP, and the nonhydrolyzable GTP analogues.

We describe in vitro microtubule assembly that exhibits, in bulk solution, behavior consistent with the GTP cap model of dynamic instability. Microtubules assembled from pure tubulin in the absence of free nucleotides could undergo one cycle of assembly, but could not sustain an assembly plateau. After the initial peak of assembly was reached and bound E-site GTP hydrolyzed to GDP, the microtubules gradually disassembled. We studied buffer conditions that maximized this disassembly while still allowing robust assembly to take place. While both glycerol and glutamate increased the rate of initial assembly and then slowed disassembly, magnesium promoted initial assembly and, surprisingly, enhanced disassembly. After cooling, a second cycle of assembly was unsuccessful unless GTP or the hydrolyzable GTP analogue GMPCPOP was readded. The nonhydrolyzable GTP analogues GMPPNP and GMPPCP could not support the second assembly cycle in the absence of E-site GTP. Analysis using HPLC found no evidence that GMPPNP, GMPPCP, or ATP could bind to free tubulin, and these nucleotides did not compete with GTP for the E-site. We have, however, demonstrated that the nonhydrolyzable GTP analogues and ATP do have an important effect on microtubule assembly. GMPPNP, GMPPCP, and ATP could each enhance the rate of assembly and stabilize the plateau of assembled microtubules against disassembly, while not binding appreciably to free tubulin. We conclude that these nucleotides, as well as GTP itself, enhance assembly by binding to a site on microtubules that is not present on free, unpolymerized tubulin. We estimate the affinity (KD) of the polymeric site for nucleotide triphosphates to be approximately 10(-4)M.

Adenosine Triphosphate↗
PubMed1989

The efficacy and duration of action of sustained-release verapamil in essential hypertension.

The blood-pressure (BP)-lowering efficacy of sustained-release verapamil, using both clinic and ambulatory measurements, was assessed in patients with essential hypertension. In study 1, a between-patient comparison, we compared verapamil (n = 12) with propranolol (n = 10). Dosage of each agent was titrated to achieve optimal clinic BP control and this dose was maintained for the duration of the study. Both agents lowered clinic systolic and diastolic BP. Mean daily ambulatory BP was also reduced with propranolol by 23/8 mm Hg and with verapamil by 13/8 mm Hg. The mean percentage reduction of systolic BP was significantly greater with propranolol (p less than 0.01). In study 2 we assessed the duration of action of sustained-release verapamil (240 mg once daily) in 14 patients. Both clinic and mean ambulatory BP were significantly reduced by 17/12 and 16/8 mm Hg, respectively, and this reduction was maintained throughout the day. We conclude that this formulation of sustained-release verapamil is effective in lowering blood pressure in mild-to-moderate hypertension and that once-daily dosage with 240 mg maintains ambulatory BP reduction throughout the dosing interval.

Adult↗
PubMed1989

Selective inhibition of cytokinesis in sea urchin embryos by low concentrations of stypoldione, a marine natural product that reacts with sulfhydryl groups.

Stypoldione is a marine natural product that inhibits cells division in marine embryos and in mammalian cell cultures. The mechanism responsible for the ability of the compound to inhibit cell division is not known. The compound was found in early studies to inhibit polymerization of tubulin into microtubules in vitro, which lead to the suggestion that inhibition of microtubule polymerization in cells might be responsible for the ability of the compound to inhibit cell division. More recently, stypoldione was found to react covalently with the sulfhydryl groups of a number of proteins including tubulin and with sulfhydryl groups of peptides and small molecules. Thus, stypoldione could potentially react with a large number of cellular targets. In the present study, we have examined the effects of stypoldione on the organization of microtubules and chromatin in cells, in relation to the ability of the compound to inhibit cell division. We used indirect immunofluorescence light microscopy of fixed and stained sea urchin embryos during the first and second divisions after fertilization, with stains specific for tubulin and DNA. We found that stypoldione exerted qualitatively different effects on cell division and microtubule organization and function at different concentrations. At the lowest effective concentrations, 5-10 microM, stypoldione selectively inhibited cytokinesis. Mitotic division occurred normally, usually with no discernible perturbation of microtubule organization or function, and cells became multinuclear. At somewhat higher concentrations, 20-40 microM, stypoldione blocked embryos before streak stage of the first division and, although microtubules were present, their organization was perturbed and they often formed unusual "spiral aster" arrays. At 80 microM and above, microtubules in blocked cells were largely absent. Thus, stypoldione uncouples cytokinesis from mitosis at the lowest effective concentrations and, although it can disrupt microtubules at relatively higher concentrations, it inhibits cell division at the lowest effective concentrations by a selective action on cytokinesis through a mechanism that does not appear to involve disassembly of microtubules.

Animals↗
PubMed1988

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies.

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.

Animals↗
PubMed1988

Isolated systolic hypertension: data from the European Working Party on High Blood Pressure in the Elderly.

The European Working Party on High Blood Pressure in the Elderly study (EWPHE) was a placebo-controlled, double-blind, inter-patient assessment of diuretic treatment in hypertensive patients aged 60 years or more. Of the 840 patients included in the study 247 had isolated systolic hypertension (systolic blood pressure greater than or equal to 160 and diastolic blood pressure less than or equal to 95 mmHg). In those (n = 120) randomized to active treatment (diazide-hydrochlorothiazide with triamterene +/- methyldopa) blood pressure after 3 years was, on average, 19/8 mmHg lower than in the placebo group (n = 119) and after 5 years the difference was 9/7 mmHg. Data on mortality and morbidity were insufficient for firm conclusions to be drawn. The data are presented here only to communicate the trends observed and to provide information that may be useful in the design of future trials. The trends observed for cardiac mortality, terminating non-fatal events (including severe heart failure) and combined fatal and non-fatal cardiovascular events follow a similar pattern to that observed in the trial overall, in that active treatment appeared to confer benefit. However, in the case of isolated systolic hypertension none of these differences between active and placebo treatment achieved statistical significance. We conclude that, given the epidemiological data incriminating systolic hypertension as a risk factor and the data presented here, a rigorous assessment of the value of treating isolated systolic hypertension is justified.

Aged↗
PubMed1988

The antihypertensive efficacy of verapamil in the elderly evaluated by ambulatory blood pressure measurement.

To assess the efficacy, tolerability and pharmacokinetics of verapamil in the elderly, ten patients with blood pressure greater than 160/90 mmHg were studied in a randomized double-blind placebo-controlled cross-over trial. Nine patients aged 75 (+/- 4.9) years completed the study. After titration, doses of verapamil varying from 40 to 120 mg (40 mg in four, 80 mg in one and 120 mg in four patients) twice daily for six weeks were taken. Mean (+/- SEM) clinic lying blood pressure was reduced on verapamil from 187 +/- 6.8/100 +/- 4.1 to 167 +/- 4.6/86 +/- 3.1 mmHg, [P less than 0.001). Mean ambulatory blood pressure was reduced from 174 +/- 1.4/95 +/- 1.0 to 169 +/- 1.3/90 +/- 0.8 mmHg, (P less than 0.01). Lying heart rate was significantly reduced but glomerular filtration rate, renal blood flow and mental function, were not altered by treatment. The mean plasma half-life of verapamil was 6.9 +/- 1.1 hours. Side effects were minimal. We conclude that verapamil is an effective blood pressure lowering agent in the elderly.

Aged↗
PubMed1987

GTP hydrolysis during microtubule assembly.

The GTP cap model of dynamic instability [Mitchison, T., & Kirschner, M.W. (1984) Nature (London) 312, 237] postulates that a GTP cap at the end of most microtubules stabilizes the polymer and allows continuing assembly of GTP-tubulin subunits while microtubules without a cap rapidly disassemble. This attractive explanation for observed microtubule behavior is based on the suggestion that hydrolysis of GTP is not coupled to assembly but rather takes place as a first-order reaction after a subunit is assembled onto a polymer end. Carlier and Pantaloni [Carlier, M., & Pantaloni, D. (1981) Biochemistry 20, 1918] reported a lag of hydrolysis behind microtubule assembly and a first-order rate constant for hydrolysis (kh) of 0.25/min. A lag has not been demonstrated by other investigators, and a kh value that specifies such a slow rate of hydrolysis is difficult to reconcile with reported steady-state microtubule growth rates and frequencies of disassembly. We have looked for a lag using tubulin free of microtubule-associated protein at concentrations of 18.5-74 microM, assembly with and without glycerol, and two independent assays of GTP hydrolysis. No lag was observed under any of the conditions employed, with initial rates of hydrolysis increasing in proportion to rates of assembly. If hydrolysis is uncoupled from assembly, we estimate that kh must be at least 2.5/min and could be much greater, a result that we argue may be advantageous to the GTP cap model. We also describe a preliminary model of assembly coupled to hydrolysis that specifies formation and loss of a GTP cap, thus allowing dynamic instability.

Animals↗
PubMed1987

Treatment of elderly hypertensives: results and implications of recent trials.

The results of recent trials on the value of antihypertensive treatment are reviewed with special reference to their implications for the elderly. In the European Working Party on High Blood Pressure in the Elderly (EWPHE) trial, there were fewer cardiac deaths on active treatment than in the Australian National Blood Pressure Study (ANBPS) and the Medical Research Council (MRC) trial in which the patients were not elderly and the benefits with treatment were attributable to fewer strokes. An explanation for this difference may be that the combination of hypertension and myocardial infarction is especially lethal in the elderly. In the EWPHE trial there was a significant reduction in nonfatal strokes but not in fatal strokes. The benefits from reducing the incidence of nonfatal stroke in this population merit consideration. As propranolol was less effective than bendrofluazide in lowering blood pressure with increasing age in the MRC trial, and as the incidence of diuretic-induced side effects in the EWPHE trial was high it seems appropriate to study alternate antihypertensive drugs in the elderly.

Aged↗
PubMed1986

Mechanism of action of the marine natural product stypoldione: evidence for reaction with sulfhydryl groups.

Stypoldione, a marine natural product that possesses an o-quinone functional group, has been shown to inhibit a variety of biological processes including cell division. We found that stypoldione binds covalently to sulfhydryl groups of thiol-containing compounds via addition of sulfur to the C-4' position of the quinone ring. We examined the ability of stypoldione to add to sulfhydryl groups of a number of thiol-containing substances, including glutathione, thiophenol, beta-mercaptoethanol, and the protein tubulin. We suggest that the biological actions of stypoldione may be caused by the addition of this compound to thiol groups of biological molecules.

Cell Division↗
PubMed1984

Acute fractures and dislocations of the carpus.

In this article, the diagnosis and treatment of acute carpal fractures and dislocations are discussed. The techniques of securing and maintaining proper reduction are emphasized. The management of the complications of carpal injuries is also discussed.

Adult↗
PubMed1984

The efficacy of indapamide in hypertensive patients failing to respond to a beta-blocker alone.

A double-blind, placebo-controlled, cross-over study was carried out to evaluate the efficacy and safety of 2.5 mg indapamide in 24 hypertensive patients failing to respond to oxprenolol alone. An additional 6 patients were assessed by ambulatory blood pressure recordings over a 15-hour period with a Remler M2,000 semi-automatic sphygmomanometer. On average, indapamide reduced supine blood pressure by 18.5/10 mmHg and standing blood pressure by 19.6/8.9 mmHg. The ambulatory recordings carried out in 6 patients detected a fall in diastolic pressure not observed using clinic readings in these 6 patients, suggesting that this is a more sensitive method of detecting antihypertensive effect. These responses were not associated with significant changes in heart rate or body weight and there was no significant postural fall in blood pressure. No serious side-effects were reported. Changes in serum potassium, chloride and urate similar to those seen with diuretics were observed. These results suggest that indapamide is a useful and safe adjunct to beta-adrenoceptor blocking therapy for uncontrolled hypertension.

Adult↗
PubMed1983

Inhibition of bovine brain microtubule assembly in vitro by stypoldione.

Stypolidione, an orthoquinone derived from the brown seaweed Stypopodium zonale, inhibited the polymerization of three-cycle-purified bovine brain microtubule protein in vitro in a concentration-dependent manner. Fifty per cent inhibition of the extent of polymerization beginning under initiating conditions occurred at a stypoldione concentration of approximately 25 microM, and 50% inhibition of tubulin addition to the assembly ends of microtubules at steady state occurred at a concentration of approximately 8 microM. Only slight structural abnormalities could be detected by negative stain electron microscopy in some of the microtubules that did assemble in the presence of the drug, and no aberrant structural forms of microtubule protein were detected. Stypoldione inhibited the binding of [3H]colchicine to tubulin, with 50% inhibition of colchicine binding activity occurring at a stypoldione concentration of 12-15 microM. Inhibition of colchicine binding activity appeared noncompetitive and was at least partially reversible, suggesting that stypoldione and colchicine bind at separate sites. By assuming that the inhibition constant for the ability of stypoldione to prevent the binding of colchicine to tubulin was equivalent to the dissociation constant for the binding of stypoldione to tubulin, we calculated that approximately 62% of the tubulin present free in solution under initiating conditions and 35-37% of the soluble tubulin under steady-state conditions was complexed with stypoldione when polymerization was inhibited by 50%. These data are consistent with a mechanism in which stypoldione interacts with soluble tubulin and inactivates the tubulin so that it is unable to add to microtubule ends, although a colchicine-like mechanism involving an action of stypoldione at microtubule ends has not been eliminated.

Animals↗