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Biomedical subjects

A Lockhart

Publications and source records attributed to A Lockhart.

At least 37 records · Page 2Linked to original sources
PubMed1997

[Ion transports across the respiratory epithelium].

The electrolytes and water transport across the respiratory epithelium have aroused particular interest for 20 years ago, with regard to the essential role they play in the regulation of the bronchial mucus composition and volume. The development of new in vitro and in vivo, techniques, allow better knowledge of these transport systems and their regulation. Transport involves two main ionic movements: chloride secretion and sodium absorption by the epithelial cells, associated with parallel water movements. In cystic fibrosis, the modification of bronchial mucus results from a defective protein-kinase dependent regulation of chloride secretion. This defect blocks water and chloride secretion by the respiratory epithelium causing dehydration of the mucus.

Body Water↗
PubMed1997

[Physiopathology of asthma: what role for leukotrienes?].

Leukotrienes are newly formed mediators derived from arachidonic acid that are released from membrane phospholipids upon stimulation of phospholipases. As a result of their various paracrine effects, these mediators-among which the most important are leukotrienes B4, C4, D4 and E4 (LTB4, LTC4, LTD4 and LTE4, respectively)-are likely to play a central role in bronchial obstruction in asthma. Indeed, LTC4 et LTD4 act on all three effectors of bronchial obstruction, namely bronchial smooth muscle, vessels and mucus secretory cells, whereas LTB4 is a potent chemotactic factor recruiting neutrophils and, in a lesser extent, eosinophils. Urinary excretion of LTE4 is also increased during acute asthma and after bronchial provocation tests with various agonists. Increased production of leucotrienes, however, does necessarily imply they are directly involved in asthma. This is, nevertheless, likely according to results obtained from numerous clinical trials assessing the therapeutical effects of competitive antagonists of LTC4 and LTD4 and 5-lipoxygenase (5-LO) inhibitors. Pretreatment with various LTC4 antagonists and 5-LO inhibitors reduces bronchial obstruction induced by exercise, hyperventilation, aspirin and allergens. However, it is still necessary to wait for results of clinical trials looking at the effects of these drugs in a large group of individuals studied over a long period, before one can assess the real therapeutical benefit of suppressing either the production or the action of leucotrienes in asthma.

Asthma↗
PubMed1997

[Occupational asthma caused by buckwheat flour].

Buckwheat flour, mainly used for pancakes, may induce asthma following inhalation and anaphylactic reactions following ingestion. These allergic reactions are mediated by specific IgE and may be confirmed by skin test and radio-allergo-sorbent test. The occupational asthma of a patient working in pancake restaurant was confirmed by specific challenge test with a computerised device to generate particles. A very small amount of buckwheat flour (10 micrograms) induced an immediate fall of the FEV1 to 56% of the initial value. No bronchial reaction was observed with lactose nor with wheat flour. Specific bronchial challenge identifies the allergen responsible for asthma, measures the level of sensitization and thus can prevent the occupational exposure.

Adult↗
PubMed1996

Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.

Kinesin and ncd motor proteins are homologous in sequence yet move in opposite directions along microtubules. We have previously shown that monomeric kinesin and ncd bind in the same orientation on equivalent sites relative to the ends of tubulin sheets of known polarity. We now report cryoelectron microscope images of 16-protofilament microtubules decorated with both single- and double-headed kinesin and double-headed ncd. Three-dimensional density maps and difference maps show that, in adenosine 5'-[beta,gamma-imido]triphosphate, both dimeric motors bind tightly to microtubules via one head, leaving the other free, though apparently in a fixed position. The attached heads of dimers bind to tubulin in the same way as single kinesin heads. The second heads are connected to the tops of the first but, whereas the second kinesin head is closely associated with the first, pairs of ncd heads are splayed apart. There is also a distinct difference in orientation: the second kinesin head is tilted toward the microtubule plus end, while the second head of ncd points toward the minus end.

Adenosine Diphosphate↗
PubMed1996

Microtubule minus ends can be labelled with a phage display antibody specific to alpha-tubulin.

To investigate the orientation of alpha- and beta-tubulin heterodimers within microtubules, we cloned a phage display antibody to alpha-tubulin. The N-terminal 100 residues of alpha-tubulin were bacterially expressed and used to select clones from a large repertoire of antibody-expressing phagemid particles. One clone reacted with the expressed alpha-tubulin N terminus and native tubulin dimer but not with the expressed beta-tubulin N terminus. Electron microscopy showed 30 nm gold beads coated with the antibody binding to one end of brain microtubules. The beads bound to the minus ends of axonemes but not to the brain tubulin extensions from their plus ends. In sliding motility assays with a plus end directed motor, beads were pushed ahead of the microtubules. Our results indicate that an N-terminal epitope of alpha-tubulin is exposed only at the minus ends of microtubules.

Animals↗
PubMed1996

Weak and strong states of kinesin and ncd.

Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.003 micro M, K(ncd)d approximately 0.24 micro M), whilst the ADp state was the weakest binding (K(kin)d approximately 11.32 micro M, K(ncd)d approximately 12.02 micro M). In ATP, the motor. ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (K(kin)d approximately 4.23 micro M, K(ncd)d approximately 2.34 micro M), consistent with a tight-binding motor. ATP-like state being enriched. In the presence of non-hydrolysable analogue beta,gamma-imidoadenosine 5'-triphosphate the binding is still tighter (K(kin)d approximately <0.27 micro M, K(ncd)d approximately 0.21 micro M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesis and ncd switch from weak to strong binding via ADP release, and back again via ADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.

Animals↗
PubMed1996

Kinetics and motility of the Eg5 microtubule motor.

We have investigated the kinetic properties of the slow plus end directed microtubule (MT) motor Eg5. The recombinantly expressed fusion protein E437GST, containing residues 12-437 of Eg5 fused to the N-terminus of glutathione S-transferase (GST), is dimeric and motile, translocating MTs at an average speed of 0.063 (+/-0.01) micrometers(-1). The kinetics of ATP turnover by E437GST were investigated using the fluorescent ATP analogue methylanthraniloyl-ATP (mantATP). In the absence of MTs, mantADP release from E437GST is slow (0.006 s(-1) in 50 mM NaCl) and rate-limiting. MTs accelerate this kinetic step approximately 850-fold to a maximal rate of 4.94 s(-1). Under these conditions, the steady-state rate of mantATP turnover was 1.92 s(-1), indicating that MT-activated mantADP release accounts for at least 40% of the total cycle time of the motor and is probably rate-limiting. This step is around 10-fold slower in Eg5 than in kinesin, consistent with it limiting the rate of physical stepping in both Eg5 and kinesin. The dissociation constants of the motor in the presence of various nucleotides were determined using MT pelleting assays. ADP stabilizes the weakest bound state of the motor, while ATP, ATP gamma S, AMPPNP, and apyrase all induce a shift toward tighter binding states. Overall, the data indicate that Eg5 displays strong kinetic homologies with the two other well-characterized MT motors, kinesin and non claret disjunctional, suggesting that all kinesin superfamily motors may share the same basic mechanochemistry.

Adenosine Diphosphate↗
PubMed1996

Changes in the testes following vasectomy in the rat.

Six months and more after unilateral vasectomy in the rat, ipsilateral (same side) and bilateral testicular atrophy are common; this paper investigates their etiology. Ipsilateral atrophy is associated with the presence of a sperm granuloma in the head of the corresponding epididymis and appears to be pressure-mediated. That sperm granulomas at the epididymal head fail to relieve intraluminal pressure in the epididymal duct of the rat was supported by a study of the effects of ligation of that region. No histological evidence was found to indicate that bilateral atrophy was the result of an immune response. A radiological study does not support the idea that compression or displacement of testicular blood vessels by sperm granulomas is responsible for bilateral atrophy.

Animals↗
PubMed1996

Phenotypic and quantitative changes in mast cells after syngeneic unilateral lung transplantation in the rat.

1. Lung transplantation causes a total interruption of the inneration and vascularization within the transplanted organ, followed by repair processes. This is frequently associated with bronchial hyper-responsiveness. A common feature of tissue repair is an increase in the number of mast cells. Three phenotypically distinct mast cell subsets, with respect to their protease content, have been identified in rat lung, and it is probable that mast cells of differing protease phenotype fulfil different functions. 2. We have compared the number, protease phenotype and distribution of mast cells in left lung from transplanted and control Lewis rats 1 month after syngeneic unilateral left lung transplantation, without interference of inflammation, graft rejection or of any treatment. Connective and mucosal-type mast cell phenotypes were characterized using antibodies directed against their specific rat mast cell proteases, RMCPI and RMCPII, respectively. 3. After transplantation, RMCPI and RMCPII tissue concentrations increased by 172% and 239%, respectively, compared with controls (13.1 +/- 1.2 and 5.6 +/- 1.0 micrograms/g). 4. Localization of mast cell phenotypes was studied by immunohistochemistry after double immunostaining. The number of mast cells increased after transplantation: the increase in the number of RMCPI-immunoreactive mast cells (RMCPI+) was significant around bronchioles and arterioles, around large vessels and in the pleura. The number of RMCPII+ mast cells also significantly increased around bronchioles and arterioles, as well as in the smooth muscle layer of large airways. Some mast cells stained for the presence of both RMCPI and RMCPII, supporting the existence of co-expressing phenotype in rat lung. The number of mast cells of the RMCPI+/II+ phenotype significantly increased around bronchioles and arterioles and in the pleura. Moreover, the distribution of the mast cell phenotypes was modified in the different areas after transplantation. 5. This indicates a local differentiation/maturation of mast cells after transplantation.

Animals↗
PubMed1996

Role of NO in the pulmonary artery hyporeactivity to phenylephrine in experimental biliary cirrhosis.

The aim of this study was to see whether increased activity of nitric oxide (NO) might account for decreased pulmonary vascular tone seen in the hyperdynamic circulation of cirrhosis. We compared the pulmonary vascular reactivity of isolated pulmonary arteries (PA) from control rats (n = 10), and rats with biliary cirrhosis (n = 10) induced by chronic bile duct ligation (4 weeks). The responses of PA rings to cumulative concentrations of phenylephrine, acetylcholine, and sodium nitroprusside were studied, and also the effects of inhibition of synthesis of NO by the L-arginine analogue, N omega-nitro-L-arginine (L-NOARG) in PA rings challenged with cumulative concentrations of phenylephrine and acetylcholine. The contractile response to phenylephrine was significantly reduced in cirrhotic PA rings as compared with controls. Pretreatment with L-NOARG (10(-4) M) significantly increased the contractile response to phenylephrine in PA rings from cirrhotic rats but not in control PA rings. Furthermore, L-NOARG restored the response to phenylephrine in cirrhotic PA rings back to normal. There was no difference in the relaxation of PA rings from both groups in response to acetylcholine and sodium nitroprusside. We conclude that in vitro pulmonary artery ring hyporeactivity to phenylephrine results from increased nitric oxide production in the pulmonary circulation of cirrhotic rats and might account for the hepatopulmonary syndrome.

Acetylcholine↗
PubMed1996

Exhaled nitric oxide during acute changes of airways calibre in asthma.

It has been shown that endogenous nitric oxide (NO), measured in exhaled air, is increased in asthmatic subjects and after allergen challenge in sensitized animals. NO is also a paracrine molecule with some, though weak, bronchodilator effects. However, whether the amount of endogenous NO that originates in the lungs can reflect the degree of bronchial tone and airways calibre in asthmatic subjects has not yet been investigated. The aim of this study was, therefore, to determine whether NO production could be modified by acute changes of airways calibre in mild, nonatopic, asthmatic subjects. NO output was measured in the exhaled air of 14 steroid-free asthmatics, 8 steroid-treated asthmatics and 21 control subjects. In seven steroid-free asthmatics, exhaled NO was measured after methacholine challenge, and then after salbutamol-induced bronchial dilatation. Exhaled tidal breathing was collected for 30 s and NO in the exhaled air was measured with a chemiluminescence analyser. Both NO concentration and its output were significantly higher in the steroid-free asthmatic patients (15.6 +/- 1.5 parts per billion (ppb) and 6.3 +/- 0.7 nmol.min-1, respectively) as compared with the control subjects (8.9 +/- 1.0 ppb and 3.5 +/- 0.3 nmol.min-1, respectively; p < 0.001 for both) and with the steroid-treated asthmatic patients (11.3 +/- 3.3 ppb and 3.7 +/- 0.9 nmol.min-1, respectively; p < 0.05 for both). Neither methacholine-induced bronchial obstruction nor salbutamol-induced bronchial dilatation caused a significant change in exhaled NO. We conclude that NO production is higher in steroid-free than in steroid-treated asthmatics and in control subjects. Furthermore, NO production is not affected by acute pharmacologically-induced changes of airways calibre in asthmatic subjects. Our results suggest that NO production is a marker of airways inflammation rather than an endogenous modulator of bronchial tone in asthma.

Adult↗
PubMed1996

[Measurement of nasal transepithelial potential difference: a diagnostic test for cystic fibrosis].

Measurement of nasal transepithelial potential difference allows the exploration of transepithelial ionic transports in vivo. Cystic fibrosis is an interesting indication of this test. Indeed, this disease is characterized by a chloride and water secretion deficit across respiratory epithelium. We have measured nasal potential in 8 healthy volunteers. Measurements were repeated 3 times a day, during 3 days for each subject. The reproducibility of the data was analysed with factorial variance model. The mean nasal potential in the healthy volunteers group and in 10 patients with cystic fibrosis was compared. In the cystic fibrosis group, the nasal potential was measured 3 times with a 2 mn-interval between the measurements. No significant variation of the nasal potential values was found from day to day or in the same day from one measurement to another. Mean value was -19 +/- 3.5 mv in normal subjects and -42.6 +/- 5.1 mv in cystic fibrosis patients. We conclude that nasal potential measurement is an easy and reproducible test that might be a complementary tool routinely used along with the classical tests in the diagnosis of cystic fibrosis.

Adult↗
PubMed1996

Evidence of a strong, positive association between atopy and the HLA class II alleles DR4 and DR7.

BACKGROUND: Atopy, with or without associated asthma, provides a useful model for evaluating the genetic factors that control human immune responsiveness. HLA class II gene products are involved in the control of immune responses. OBJECTIVES: We investigated whether susceptibility or resistance to the disease was associated with HLA class II genes. METHODS: Blood samples were obtained from two groups of unrelated European-born white adults: 56 atopic patients (52 of them with asthma) and 39 healthy controls with no personal or familial history of asthma or atopy. Genomic DNA was extracted from peripheral blood lymphocytes. The exons of DQA1, DQB1, DRB and DPB1 genes were selectively amplified by the polymerase chain reaction (PCR) method. Genotyping was carried out by digestion of the amplified DNA products with allele-specific endonucleases (PCR-RFLP), which can recognize allelic variations in the polymorphic exon. RESULTS: We found no significant differences in the frequency of DPB1 alleles between patients and controls. HLA class II DR4 and DR7 alleles were present in 39.2% of the patients and in 2.5% of the healthy subjects (Pc*2 < or = 3.9 10(-3)). Conversely, DQA1*0103 and DQB1*0502 alleles were more frequent in the control subjects. These results confirm a previous study of an extended pedigree, which showed that DR4 and DR7 alleles were absent in all healthy members of the family and were frequently observed in atopic and/or in asthmatic subjects. CONCLUSION: We observed that HLA-DR 4 and DR7 alleles are significantly implicated in their susceptibility to the disease and suggest that this susceptibility is more related to atopy than to specific responses to allergens. According to previous studies, we could also submit that in atopic patients with asthma, DR4 alleles at the least, could be more closely associated with atopy than with asthma per se. Conversely, we suggest that some allelic DQA1 and DQB1 sequences might confer protection against the disease.

Adult↗
PubMed1995

ADP release is the rate-limiting step of the MT activated ATPase of non-claret disjunctional and kinesin.

The motor protein non-claret disjunctional (ncd) moves towards the minus ends of microtubules (MTs), whereas its close relative kinesin moves in the opposite direction towards the plus ends of MTs. The mechanisms of movement and directional reversal for these motor proteins are unknown. Here we report the rate constants for MT activated ADP release from a recombinant double-headed ncd protein, GST-MC5, and a recombinant double-headed kinesin protein, K delta 401, measured using the fluorescent nucleotide analogues methylanthranilyol ATP (mantATP) and mantADP. Comparison of the maximal MT activated mantADP release rates for these proteins with their maximal MT activated mantATP turnover rates indicates that ADP release is the rate-limiting step for ATP turnover for both ncd and kinesin. This data supports the view that directional reversal may result from structural rather than chemical kinetic differences in the way the motors interact with MTs.

Adenosine Diphosphate↗
PubMed1995

Nucleotide-dependent angular change in kinesin motor domain bound to tubulin.

Kinesin is a 'motor' molecule, consisting of two head domains, an alpha-helical coiled coil rod, and a tail part that binds to its cargo. When expressed in a bacterial system, the head domain is functional, and can bind to microtubules with the stoichiometry of one head per tubulin dimer. Kinesin moves along microtubules by means of a cyclic process of nucleotide binding, hydrolysis and product release. We have used negative-stain electron microscopy and image analysis to study the structures of microtubules and tubulin sheets decorated with the motor domain (head) of kinesin in three states: in the presence of an unhydrolysable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP); without nucleotides; and with adenosine 5'-diphosphate (ADP). A single kinesin head bound to a microtubule has a pear-shaped structure, with the broader end towards the 'plus' end of the microtubule under all conditions; the reverse motor, ncd, is similarly oriented. Three-dimensional maps reveal that kinesin heads have a spike that is assumed to form the attachment to the tail of a complete kinesin molecule. This spike is perpendicular to the microtubule axis in the presence of ADP, but points towards the plus end (approximately 45 degrees) in the presence of AMP-PNP or absence of nucleotides. Our results provide direct evidence for a conformational change of the kinesin motor domain during the ATPase cycle.

Adenylyl Imidodiphosphate↗