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Biomedical subjects

A Lockhart

Publications and source records attributed to A Lockhart.

At least 55 records · Page 3Linked to original sources
PubMed1995

Kinesin and ncd bind through a single head to microtubules and compete for a shared MT binding site.

Kinesin and non claret disjunctional are closely related molecular motors that move in opposite directions along microtubules. We have used recombinant single-headed and double-headed constructs of both rat kinesin heavy chain and non claret disjunctional to investigate the interactions of these motor proteins with microtubules. At saturation the stoichiometry of binding for non claret disjunctional and kinesin to microtubules is one molecule (single or double-headed) per tubulin heterodimer. In the absence of added nucleotide, addition of increasing amounts of one motor results in the competitive displacement of the other motor from the microtubules. This effect is apparent also in the presence of the nucleotide analogue 5'-adenylimidodiphosphate, which tightens the binding of both kinesin and non claret disjunctional. Competition for binding sites occurs also under conditions of steady-state ATP turnover. We conclude that despite their opposite directionality, kinesin and non claret disjunctional compete for overlapping binding sites on the MT surface. Since the binding of the second head of a double-headed motor is sterically blocked, the data imply also that both kinesin and non claret disjunctional may translocate via a processive (alternating heads) mechanism with a minimum step size of approximately 8 nm.

Adenosine Triphosphate↗
PubMed1995

Effects of pretreatment with inhaled methoxamine on bronchial responses to histamine in asthmatic subjects.

The underlying mechanisms of bronchial obstruction in asthma are complex. Both bronchospasm and bronchial oedema are thought to play pivotal roles in asthma, but their respective importance in a given asthmatic individual is unknown. To address this question, we assessed the effects of pretreatment with inhaled methoxamine, a potent alpha 1-adrenoceptor agonist, on bronchial response to inhaled histamine in 10 asthmatic subjects. The study was conducted according to a double-blind, cross-over, randomized and placebo-controlled design. In each subject, dose-response curves for the effects on forced expiratory volume in one second (FEV1) of serially doubling doses of inhaled histamine were obtained on three different days, 15 min after pretreatment with either methoxamine (10 mg) or duplicated placebo. Histamine, first dose 100 micrograms (543 nmol), was delivered by a breath-activated dosimeter every 5 min. FEV1 was measured in triplicate after each dose and the largest value was retained. There was no difference in baseline and prechallenge FEV1 after placebo and methoxamine. Mean coefficient of variation of decrease in FEV1 induced by histamine on the two placebo days was 6.7 +/- 2%. On average, the bronchial responses to histamine were not modified by pretreatment with methoxamine as compared to placebo (delta FEV1 = 0.83 +/- 0.14 l on methoxamine versus 0.85 +/- 0.11 l and 0.86 +/- 0.13 l on the two placebo days).(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1995

The effect of intravenous phenylephrine on airway calibre in asthma.

Tracheobronchial vasoconstriction and subsequent reduction of airway wall thickness due to the alpha 1-agonist methoxamine, might be responsible for prevention of exercise-induced asthma, and reduction of bronchial hyperresponsiveness to methacholine increase in exercise performance in patients with impaired left ventricular function. Since bronchial wall oedema plays an important role in asthma, we have now investigated the bronchial response to the intravenously administered alpha 1-agonist, phenylephrine, in asthma of various severity. Increasing noncumulative intravenous phenylephrine doses (100 to 600 micrograms) were injected in 18 asthmatic subjects (three groups: mild asthma, mild asthma with recent acute attack, severe obstructive asthma) and in 11 control subjects. Changes in specific airways resistance (sRaw) on phenylephrine were linearly related to the dose administered in 16 out of 18 asthmatic subjects, and in only 3 out of 11 control subjects. In the asthmatic subjects, sRaw increased in 10 patients whose asthma was mild, or bronchial obstruction mild to moderate, and decreased in the remaining 8 asthmatic subjects with more severe disease or with a higher degree of bronchial obstruction. Changes in forced expiratory volume in one second (FEV1) were consistent with those of sRaw. In the five asthmatic subjects who underwent the protocol twice, results were reproducible. There was no difference in the responses of heart rate between the three groups of asthmatic subjects. It is likely that phenylephrine acts both via airway smooth muscle contraction, an effect which might predominate in mild asthma, and via mucosal vasoconstriction, which might overcome the effect on smooth muscle in more severe asthma with bronchial wall oedema.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Origins of reversed directionality in the ncd molecular motor.

The head or motor domain of the ncd (non-claret disjunctional) molecular motor is 41% identical to that of kinesin, yet moves along microtubules in the opposite direction to kinesin. We show here that despite the reversed directionality of ncd, its kinetics in solution are homologous in key respects to those of kinesin. The rate limiting step, ADP release, occurs at 0.0033 s-1 at 100 mM NaCl and is accelerated approximately 1000-fold when the motor binds to microtubules. Other reaction steps are all very fast (> 0.1 s-1) compared with ADP release, and the motor is consequently paused in the ncd.ADP state until microtubule binding occurs (Kd = 2 microM), at which point ADP release is triggered and the motor locks onto the microtubule in a rigor-like state. These data identify close functional homology between the strong binding states of kinesin and ncd, and in view of this we discuss a possible mechanism for directional reversal, in which the strong binding states of ncd and kinesin are functionally identical, but the weak binding states are biased in opposite directions.

Adenosine Diphosphate↗
PubMed1994

The relationship between use of the maximum tolerated dose and study sensitivity for detecting rodent carcinogenicity.

The relationship between maximum tolerated dose (MTD) and study sensitivity for detecting rodent carcinogenicity was evaluated for 216 chemicals found to be carcinogens in laboratory animal studies conducted by the National Cancer Institute (NCI) and the National Toxicology Program (NTP). Approximately two-thirds of these rodent carcinogens would have been detected even without the top dose (estimated MTD), but in many of these studies, some site-specific carcinogenic effects would have been missed. Among the remaining one-third of the rodent carcinogens that required the top dose for statistical significance, approximately 80% had numerically elevated rates of the same site-specific tumors at lower doses as well. Only 13 of the NCI/NTP rodent carcinogens had increased tumor rates limited to the top dose for all sites of carcinogenicity. Alternatively, of the 838 site-specific carcinogenic effects observed in the NCI/NTP studies, 447 (53%) would have been detected even without the top dose. Of the remaining effects, 75% (294/391) showed numerically elevated site-specific tumor rates at lower doses. Our evaluation indicates that most carcinogenic effects observed at the top dose in rodent studies are also present (with reduced incidence that might or might not be statistically significant) at the lower doses typically employed (1/2MTD, 1/4MTD).

Animals↗
PubMed1994

A simple and rapid micromethod for genomic DNA extraction from jugal epithelial cells. Application to human lymphocyte antigen typing in one large family of atopic/asthmatic probands.

We describe a rapid and reliable micromethod for DNA isolation from buccal epithelial cells from the interior mouth mucosa. This convenient, noninvasive method could be applied to genetic typing in a small number of cells (about 2000 cells per cheek). We have shown that DNA released by this method is suitable for further amplification by polymerase chain reaction (PCR). Using this protocol, coupled with the PCR-RFLP (restriction fragment length polymorphism) method, we analyzed the allelic sequence diversity of the human lymphocyte antigen (HLA) class II genes in an extended family of 33 persons containing 14 asthmatic or atopic members. Six of eight DQA1 alleles, and 11 DQB1, 20 DPB1, and 10 DR haplotypes could be identified in a single DNA sample. Our results suggest that the DR53 group haplotype is frequently associated with allergic asthma and atopy. The micromethod described here may be useful in genetic epidemiology, especially in family studies involving small children.

Asthma↗
PubMed1994

Inhibition of substance P-induced microvascular leakage by inhaled methoxamine in rat airways.

1. The effect of the inhaled alpha-adrenoceptor agonist, methoxamine (MTX), was studied on experimental airway oedema induced by injection of substance P (SP) in the rat. Sprague-Dawley rats (300-350 g) were anaesthetized with sodium thiopentone, tracheotomized and artificially ventilated. 2. MTX or its vehicle was administered by inhalation. Airway resistance and blood pressure were monitored continuously. Evans Blue dye (EB, 20 mg kg-1) was injected through a jugular catheter 1 min before SP (14.8 nmol kg-1). Airways were dissected out, weighed and placed in formamide for EB extraction and determination by spectrophotometry. 3. EB extravasation induced by SP was significantly reduced in distal intraparenchymal bronchi by inhaled MTX at doses of 50 micrograms kg-1 (58 +/- 9 vs 96 +/- 9 ng EB mg-1 tissue after vehicle, P < 0.001) and 100 micrograms kg-1 (69 +/- 11 vs 137 +/- 26 ng EB mg-1 tissue after vehicle, P < 0.01). Inhaled MTX by itself (100 micrograms kg-1) increased blood pressure: 172 +/- 6 vs 132 +/- 10 mmHg baseline (P < 0.02), but neither induced extravasation nor increased airway resistance. 4. In another set of experiments without SP, MTX was administered intravenously 1 min after EB. At 100 micrograms kg-1, i.v. MTX increased blood pressure to a similar extent as inhaled MTX (180 vs 147 mmHg baseline, P < 0.01), increased airway resistance and caused leakage of plasma proteins in distal intraparenchymal bronchi (79 +/- 7 vs 47 +/- 1 ng EB mg-1 tissue, P < 0.02). 5 Similarly, after sequential i.v. injections of doubling doses of MTX (50-800 microg kg-1), a marked EB extravasation was found in the airways. This was abrogated by pretreatment with prazosin (100 microg kg-1)but not with propranolol (2 mg kg-1).6 These results suggest that microvascular leakage and airway oedema induced by i.v. MTX may be linked to an increase in pressure in the pulmonary circulation, resulting from vasoconstriction of the pulmonary vasculature and acute cardiac dysfunction due to systemic hypertension.7 Our results with inhaled MTX show that direct deposition of MTX at the bronchial vasculature induces a reduction in SP-induced microvascular leakage in rat airways and that inhaled MTX does not share the untoward effect of i.v. MTX inducing airway oedema.

Administration, Inhalation↗
PubMed1994

HLA class II alleles in isocyanate-induced asthma.

Studying genetic factors that control human immune responsiveness may further our understanding of specific types of asthma in which the role of immune factors is uncertain to date. HLA Class II gene products are involved in the control of immune responses. Therefore, we investigated whether HLA Class II genetic markers contribute to susceptibility or resistance to isocyanate-induced asthma (IAA) in exposed workers. We collected venous blood samples from two groups of unrelated white adults: (1) patients with isocyanate-induced asthma documented by a positive inhalation challenge; and (2) exposed individuals with no history of IAA. The second exon of DQA1, DQB1, DPB1, and DRB genes was selectively amplified by the polymerase chain reaction (PCR) method. HLA typing was carried out by the PCR-RFLP method, which allowed discrimination of most HLA DQA1, DQB1, DPB1, and DRB alleles. No significant difference was found in the distribution of DPB1 alleles between patients and control subjects. Allele DQB1*0503 and allelic combination DQB1*0201/0301 were associated with susceptibility to the disease. Conversely, allele DQB1*0501 and the DQA1*0101-DQB1*0501-DR1 haplotype conferred significant protection to exposed healthy control subjects. Our results are consistent with the hypothesis that immune mechanisms are involved in isocyanate-induced asthma and that specific genetic factors may increase or decrease the risk of developing IAA in exposed workers.

Adult↗
PubMed1994

Effect of furosemide on prostaglandin synthesis by human nasal and bronchial epithelial cells in culture.

Inhaled furosemide protects asthmatic subjects against bronchial obstruction caused by indirect provocants. We have attempted to correlate the protective effect of furosemide with its ability to alter prostaglandin (PG) synthesis by the airway epithelium. Human epithelial cells from nasal polyps and bronchi were cultured in DME-Ham's F12 medium with 10% fetal calf serum. Confluent cells (days 6 through 8) were incubated for 30 min in fresh medium, and the PGs in the supernatant were measured by radioimmunoassay. Spontaneous output (ng.ml-1.mg-1 cell protein) was as follows (mean +/- SEM): PGE2 = 7.74 +/- 2.10 (n = 12), PGF2 alpha = 1.66 +/- 0.12 (n = 15), 6-keto-PGF1 alpha = 4.32 +/- 1.37 (n = 11), PGD2 = 0.73 +/- 0.16 (n = 11) for bronchial cells and PGE2 = 7.24 +/- 0.80 (n = 32), PGF2 alpha = 1.38 +/- 0.12 (n = 17), 6-keto-PGF1 alpha = 6.79 +/- 2.50 (n = 15), PGD2 = 0.42 +/- 0.07 (n = 17) for nasal cells. Incubation with arachidonic acid (25 micrograms/ml) for 30 min significantly increased the amounts of the four PGs. Incubation with furosemide (10(-4) M) for 30 min caused a marked reduction in both basal and arachidonic acid-stimulated production of PGE2 and PGF2 alpha but did not reduce production of 6-keto-PGF1 alpha and PGD2. Incubation with bumetanide (10(-4) M) for 30 min did not modify the PGE2 synthesis by nasal epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1994

[Is there a genetic component in bronchial hyperreactivity in the human species?].

Whether a hereditary, genetic component of human non allergenic bronchial hyperresponsiveness exists in man is controversial. Therefore, the "Dutch hypothesis" that bronchial hyperresponsiveness is a risk factor of asthma and chronic bronchitis has not been validated by hard data, although it was put forward more than two decades ago. In some inbred strains of mice and rats, bronchial hyperresponsiveness to acetylcholine or serotonin is transmitted as a Mendelian monogenic trait or a polygenic one. Genetic studies of human bronchial hyperresponsiveness are difficult, if only because it is an ill-defined phenotype, and because acquired, inflammation-related bronchial hyperresponsiveness is at the forefront in airway diseases such as asthma. Studies of concordance in mono- and dizygotic twins, of distribution in the general population, and of familial inheritance suggest transmission from generation to generation of human bronchial hyperresponsiveness. This only applies to bronchial response to cholinergic agents because of the small number of studies done with other bronchial challenges. Furthermore, there is no agreement on the mode of transmission of this specific type of bronchial hyperresponsiveness. One hopes that combining genetic epidemiology with molecular genetics in the forthcoming years will result in a better understanding of genetic risk factors of human bronchial hyperresponsiveness.

Asthma↗
PubMed1994

[Bronchial hyperreactivity in 1994, a cheerful quinquagenarian. Conclusions].

A review is made of the principal themes that are discussed in this supplement of the Revue des Maladies Respiratoires. Three important questions on bronchial hyperresponsiveness, a key feature of asthma, are raised: 1) the clinical usefulness of its assessment, mostly in the atypical forms of asthma and in occupational asthma and, less so, to estimate the severity of asthma; data derived from the Bayesian analysis lead to the conclusion that it is a test which is better for excluding rather than for confirming asthma; in an epidemiological setting, the assessment of bronchial responsiveness should be carried out to assess both host factors and the effect of environmental exposure; 2) the method of assessment which should take into account the stimulus, be it pharmacological or not, the physiological index used to assess the response and the mode of expression of results; 3) finally, the mechanisms of bronchial responsiveness are probably multiple as examined in different chapters of this supplement.

Asthma↗
PubMed1994

[Bronchial hyperreactivity. Definitions and history].

The various definitions that are proposed for bronchial hyper-responsiveness (BH) are discussed. The semantic problems related to these definitions are presented: 1) the translation of bronchial hyperresponsiveness into French can vary; 2) BH is not synonymous of abnormal contraction of airway smooth muscle; 3) there is also some confusion of BH with the clinical status of asthma, which is excessive. The authors also review the difficulties related to the purely operational character of the definition itself: 1) BH has a continuous distribution in a general population; 2) BH can be expressed by various indices; 3) stimuli to reveal BH are various; 4) there are various types of BH related to the causal conditions; 5) there are different sites of BH within the airways. The authors also review the historical background of BH with some emphasis on the contribution of French-speaking research workers.

Animals↗
PubMed1993

Structural and functional domains of the Drosophila ncd microtubule motor protein.

Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein that is required for proper chromosome distribution in Drosophila. Despite its sequence similarity to kinesin heavy chain, ncd translocates with the opposite polarity as kinesin, toward microtubule minus ends. We have expressed different regions of the protein in bacteria and analyzed the proteins for function. Results indicate that ncd consists of three domains: a basic, proline-rich N-terminal "tail," a central alpha-helical coiled-coil stalk, and a C-terminal motor domain. The ncd N terminus proteins bundle microtubules in motility assays and show ATP-independent binding to microtubules in solution. Truncated proteins, lacking the tail but containing the predicted motor domain and differing lengths of the stalk, did not support microtubule gliding in in vitro assays but showed microtubule-stimulated MgATPase activity in solution. Addition of a nonspecific N terminus to two of the truncated proteins restored directional gliding and rotation of microtubules in motility assays, demonstrating that these properties map to the predicted mechanochemical domain of ncd. Physical properties of the C terminus proteins indicate that the stalk region is important for dimerization and that the ncd protein probably exists and functions as a dimer.

Adenosine Triphosphatases↗
PubMed1993

Locally deposited but not inhaled frusemide reduces nasal potential difference in healthy subjects.

Previous publications suggest that prolonged inhalation of frusemide (F) does not cause a fall in the nasal transepithelial potential difference (PD) whereas locally deposited F does. In an attempt to reconcile these observations, we have measured the effect of inhalation through the nose and local deposition of F, amiloride (A), bumetanide (B) and salbutamol (S) on nasal PD in 7 healthy male volunteers in a randomised, double blind study. Solutions of drugs ranging from 10(-6) M to 10(-3) M (3 x 10(-8) M to 3 x 10(-5) M for B) in phosphate buffered saline 0.5 ml (PBS) were sequentially deposited in both nostrils, and nasal PD was measured 5 min after each dose. In 10 further volunteers, 10(-2) M solutions of A, F and S (3 x 10(-4) M for B) 5 ml were nebulised through the nose for 15 min, when nasal PD was measured. Resting PD was similar in the left and right nostrils averaging -17.1 mV (lumen negative). Placebo, inhaled of deposited B and S, and inhaled F did not change nasal PD. Topically deposited F significantly lowered PDmax in a dose-dependent manner [10(-4) M, -12% from baseline; 10(-3) M, -24%] as did the more potent A [10(-5) M, -19%; 10(-4) M, -31%; 10(-3) M, -47%]. Nebulised A (10(-2) M) had the same effect on nasal PD as deposited A (10(-4) M). The effects of locally deposited F and A (10(-3) M) on nasal PD were additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Inhalation↗
PubMed1993

Muscarinic receptors after syngeneic unilateral lung transplantation.

We have characterized the muscarinic cholinergic receptor population by binding assay in left and right lung membranes from syngeneic Lewis rats one month after unilateral left lung transplantation and from controls. The density of muscarinic receptors measured by [3H](-)QNB binding was similar in left and right lung membranes one month after unilateral left lung transplantation (Bmax = 28.2 +/- 2.2 and 29.0 +/- 1.4 fmol/mg protein, respectively) and was similar between transplanted and control rats. Binding affinity also was similar on both sides (Kd = 28.5 +/- 9.5 and 32.3 +/- 2.7 pM, respectively). Binding parameters (Ki) of four selective muscarinic antagonists determined from competitive binding experiments of 0.5 nM [3H](-)QNB showed an order of potency: atropine > 4-DAMP > AF-DX 116 BS > pirenzepine both in transplanted and in control lungs. There was no significant difference in Ki values between left transplanted and right non transplanted lungs (1.8 +/- 0.1 and 1.9 +/- 0.1 nM; 4.3 +/- 0.5 and 3.5 +/- 0.5 nM; 80.9 +/- 13.8 and 78.9 +/- 18.8 nM; 480.4 +/- 40.9 and 481.7 +/- 78.2 nM, respectively for atropine, 4-DAMP, AF-DX 116 BS and pirenzepine). Values were also similar between transplanted and control lungs. These observations suggest a similar population of muscarinic receptors in the transplanted and the non transplanted lung in the Lewis rat. This result is in accordance with the similar physiological effect of exogenous acetylcholine that we previously observed in transplanted and non transplanted bronchi.

Animals↗
PubMed1993

Modelling of passive expiration in patients with adult respiratory distress syndrome.

The time-course of volume change during passive expiration preceded by an end-inspiratory hold was studied with a biexponential model in six adult respiratory distress syndrome (ARDS) patients. We measured the initial volumes and time constants of the fast (tau 1), and the slow (tau 2) compartments of expiration, as well as the static elastance of the respiratory system. The results were compared to those of 11 normal subjects. We observed that: 1) the biexponential model fitted closely the volume decay; 2) the fast compartment was responsible for 81 +/- 7% (ARDS) versus 84 +/- 10% (controls) of the total volume exhaled, with tau 1 = 0.35 +/- 0.11 s (ARDS) versus 0.50 +/- 0.22 s (controls); 3) the slow compartment contributed only 19 +/- 6% (ARDS) versus 16 +/- 7% (controls), with tau 2 = 4.67 +/- 2.38 s (ARDS) versus 3.27 +/- 1.54 s (controls); and 4) static elastance was higher in ARDS patients. The findings could be explained in terms of a four parameter viscoelastic model of the respiratory system.

Adolescent↗