Increased throughput of proteomics analysis by multiplexing high-resolution tandem mass spectra.

High-resolution and high-accuracy Fourier transform mass spectrometry (FTMS) is becoming increasingly attractive due to its specificity. However, the speed of tandem FTMS analysis severely limits the competitive advantage of this approach relative to faster low-resolution quadrupole ion trap MS/MS instruments. Here we demonstrate an entirely FTMS-based analysis method with a 2.5-3.0-fold greater throughput than a conventional FT MS/MS approach. The method consists of accumulating together the MS/MS fragments ions from multiple precursors, with subsequent high-resolution analysis of the mixture. Following acquisition, the multiplexed spectrum is deconvoluted into individual MS/MS spectra which are then combined into a single concatenated file and submitted for peptide identification to a search engine. The method is tested both in silico using a database of MS/MS spectra as well as in situ using a modified LTQ Orbitrap mass spectrometer. The performance of the method in the experiment was consistent with theoretical expectations.

Effects of BRL 38227 on neurally-mediated responses in the guinea-pig isolated bronchus.

1. In guinea-pig isolated bronchus treated with indomethacin (2.8 microM), electrical field stimulation (EFS; 10 Hz, 0.5 ms, 60-70 V, for 10 s) evoked a tetrodotoxin (3 microM)-sensitive, biphasic contraction comprising a rapid, atropine (1 microM)-sensitive cholinergic response succeeded by a slowly developing, capsaicin (10 microM)-sensitive, non-adrenergic, non-cholinergic excitatory (NANCe) response. 2. BRL 38227 (0.3-3 microM), salmeterol (0.003-3 microM) and ketotifen (1.0-300 microM) each produced concentration-dependent inhibition of both NANCe and cholinergic responses to EFS in guinea-pig isolated bronchus. 3. Substance P (SP; 1 microM) and neurokinin A (NKA; 0.07 microM) produced contractions equivalent in magnitude to the NANCe response to EFS, which were inhibited by salmeterol (1 microM), but not by BRL 38227 (3 microM) or ketotifen (100 microM). 4. Acetylcholine (ACh; 6 microM) was equi-effective with the electrical activation of cholinergic neurones. BRL 38227 (3 microM) slightly inhibited responses to ACh (6 microM). Salmeterol (1 microM) and ketotifen (100 microM) markedly inhibited responses to ACh (6 microM). 5. In bronchial rings pre-contracted with ACh (100 microM), BRL 38227 (0.1-30 microM), salmeterol (0.001-3 microM) and ketotifen (0.1-100 microM) each produced concentration-dependent relaxation. Unlike ketotifen, BRL 38227 and salmeterol only partially (18.8 +/- 2.1% and 51.8 +/- 3.9% respectively) reversed the ACh-induced contraction. 6. The (+)-analogue of BRL 38227, BRL 38226 (0.3-100 microM), was without effect on responses to EFS and had no effect on the inhibition caused by BRL 38227. The K+-channel activators pinacidil (3.0-30 microM) and RP 52891 (3.0-30 microM) exerted similar inhibitory actions on responses to EFS as BRL 38227, but were less potent. Glibenclamide (0.1-1.O microM) and phentolamine (3 microM) antagonized the inhibitory effects of BRL 38227 on responses to EFS.7. It is concluded that BRL 38227 and ketotifen can inhibit NANCe neuroeffector transmission at concentrations exerting little or no inhibitory effects on responses to exogenously applied tachykinins.By contrast, in addition to suppressing NANCe responses to EFS, salmeterol also markedly inhibits responses to SP and NKA. At concentrations markedly suppressing cholinergic neuroeffector transmission, BRL 38227 has only minor effects on responses to exogenously-applied ACh. Salmeterol and ketotifen both depress responses to ACh within the concentration-range over which they inhibit cholinergic responses to EFS. The inhibitory effects of BRL 38227 on responses to EFS exhibit stereo-specificity and may involve the opening of a neuronal K+-channel. This K+-channel is glibenclamide-and phentolamine-sensitive and appears similar to the smooth muscle K+-channel which is modulated by BRL 38227.

The effects of epithelium removal on the actions of cholinomimetic drugs in opened segments and perfused tubular preparations of guinea-pig trachea.

1. Isolated segments of guinea-pig trachea or perfused tracheal tubes were arranged for the recording of trachealis tension changes in Krebs solution containing indomethacin (2.8 microM). 2. In opened tracheal segments, epithelium removal caused modest (2-3 fold) potentiation of the effects of acetylcholine (ACh) and methacholine (MeCh) but failed to potentiate carbachol (CCh), bethanechol (BeCh), oxotremorine or KCl. 3. Pretreatment with ecothiopate potentiated effects of ACh and MeCh but not of CCh or BeCh. Removal of epithelium in ecothiopate-treated tissue potentiated effects of ACh and MeCh but not of CCh or BeCh. 4. Guinea-pig ileum challenged with ACh was used as a bioassay system for cholinesterase activity. Scrapings of tracheal epithelium did not hydrolyse ACh. 5. Histochemical staining revealed no fibres positive for acetylcholinesterase or pseudocholinesterase in the tracheal epithelium. However, the underlying tissues contained acetylcholinesterase-positive nerve fibres and the trachealis muscle itself stained positively for pseudocholinesterase activity. 6. Neither tetrodotoxin (3 microM) nor hexamethonium (500 microM) modified the ability of epithelium removal to potentiate ACh. 7. In perfused tracheal tubes where spasmogens were added to the luminal perfusate, epithelium removal potentiated effects of ACh (31 fold), CCh (10 fold), oxotremorine (2 fold) and KCl. 8. In perfused tracheal tubes where spasmogens were added to the Krebs solution superfusing the adventitial surface of the tissue, epithelium removal significantly reduced the potency of CCh, oxotremorine and KCl. 9. It is concluded that the selectivity and magnitude of the potentiation of cholinomimetics caused by epithelium removal depends on the route by which the cholinomimetic agent gains access to the trachealis muscle. The potentiation of acetic acid esters of choline seen in opened tracheal segments does not reflect the loss of epithelial cholinesterase activity and does not depend on the activity of nervous reflex arcs in the tracheal wall. The reduced potency of adventitially-applied cholinomimetics and KCl seen in epithelium-denuded tissue strongly suggests that the epithelium can moderate trachealis sensitivity to cholinomimetic agents not only by releasing epithelium-derived relaxing factor but also by acting as a barrier to drug diffusion.

Spasmogen action in guinea-pig isolated trachealis: involvement of membrane K+-channels and the consequences of K+-channel blockade.

1. Acetylcholine (ACh), histamine, prostaglandin E2 and potassium chloride (KCl) each evoked concentration-dependent spasm of guinea-pig isolated trachealis treated with indomethacin (2.8 microM). 2. Neither tetraethylammonium (TEA; 0.1-10 mM) nor procaine (0.1-10 mM) potentiated these spasmogens. Indeed, procaine (10 mM) depressed the log concentration-effect curves of all the spasmogens while TEA (1-10 mM) caused some depression of the log concentration-effect curve of prostaglandin E2. 3. Intracellular electrophysiological recording was performed in trachealis bathed by normal Krebs solution or by Krebs solution containing 2.8 microM indomethacin. In either medium the majority of trachealis cells exhibited spontaneous electrical slow waves while some cells were electrically quiescent. In either medium the spasmogenic effects of ACh (1 mM) and histamine (0.2 mM) were accompanied by depolarization and abolition of slow wave discharge. In many cases the record of membrane potential subsequently exhibited noise which incorporated fast, hyperpolarizing transients. 4. In the absence and presence of indomethacin, TEA (10 mM) and procaine (5 mM) markedly reduced the membrane noise and hyperpolarizing transients evoked by ACh or histamine without augmenting the evoked tension. 5. It is concluded that slow wave discharge does not depend on prostaglandin synthesis. The membrane noise and hyperpolarizing transients evoked by ACh and histamine represent the opening of membrane K+-channels. While such K+-channel opening may offset spasmogen-induced depolarization it does not moderate the evoked tension.

The spasmogenic action of oxytocin in the rat uterus--comparison with other agonists.

A low concentration (0.2 nM) of oxytocin induced phasic tension development in the isolated uterus of the day-22 pregnant rat. Tonic spasm was also induced by higher concentrations of oxytocin (2 and 20 nM). Spasmogenic responses to bradykinin and potassium chloride (KCl) also contained phasic and tonic components while acetylcholine induced tonic spasm only. The phasic component of the responses to oxytocin and to bradykinin and both components of the response to KCl were inhibited by (+)-cis diltiazem (0.1 and 1 microM). The tonic component of the responses to oxytocin and to bradykinin and the responses to acetylcholine were only reduced by (+)-cis diltiazem at concentrations greater than 10 microM. (-)-cis Diltiazem was less potent than (+)-cis diltiazem as an inhibitor of calcium (Ca2+)-induced spasm in a depolarizing medium and of the phasic spasms induced by oxytocin. The two isomers were of similar potency as inhibitors of oxytocin-induced tonic spasm. Spasmogenic responses to oxytocin, bradykinin, acetylcholine and KCl were decreased when uteri were bathed in media which were Ca2+-free or of low Na+ content. However, there was no correlation between the rank order of sensitivity of the four spasmogens to the changed media and to their inhibition by (+)-cis diltiazem. Oxytocin (0.2 nM) increased the frequency, duration and amplitude of spike activity, measured by extracellular electrical recording, in parallel with enhancement of phasic tension development. With higher concentrations of oxytocin (2 and 20 nM) spike firing was initially continuous but often subsequently ceased despite the associated tonic contracture. After incubation in (+)-cis diltiazem (10 microM), oxytocin (0.2, 2 and 20 nM) produced graded tonic spasm without spike activity. Oxytocin (0.2 nM) produced a small increase in 45Ca2+ influx into myometrium as assessed by the 'lanthanum method'. Higher concentrations of oxytocin (2 and 20 nM) did not increase 45Ca2+ influx. It is concluded that the phasic component of the response of the uterus to oxytocin and bradykinin is associated with Ca2+ influx via voltage-dependent Ca2+ channels. The tonic component is due to another mechanism(s) which does not appear to involve Ca2+ influx. All of the spasmogenic response to KCl can be explained by Ca2+ influx through voltage-dependent Ca2+ channels. These channels do not appear to be involved in the spasmogenic response to acetylcholine.

Faculty practice: how it enhances teaching.

This article is a presentation of the two authors' views on faculty practice and how both have operationalized the practice option. Both describe how the practice option has positively affected their teaching and their students' learning in a university baccalaureate nursing program located in the midwestern United States. The authors are full-time, master's prepared faculty members who have been in the faculty practice track for 2 years. Both teach in the junior year of a 4-year baccalaureate program. The first author (D.M.G.) is a staff nurse who works in a community hospital and serves on the hospital's clinical nursing research committee. The second author (C.R.S.) is a clinical research nurse coordinator who performs and manages pharmaceutical trials at multiple inpatient and outpatient locations. In this article, the authors examine faculty practice in general and the various models currently used. In addition, they explain their individual academic positions, the duties and responsibilities accompanying each of their practice roles, and the integration of those practice experiences into their clinical and classroom learning.

Social support: gender differences in multiple sclerosis spousal caregivers.

The purpose of this study was to investigate gender differences in social support of spousal caregivers of persons with multiple sclerosis (MS). The sample consisted of 37 male and 28 female caregivers of individuals with MS. It was found that female caregivers scored significantly higher than males on the total number of resources available, perceived social support and the perceived availability of friends and self-help groups. There was a positive relationship between caregiver-perceived social support and the ability of the mate to perform intimate functions. Caregiver-perceived social support was also found to be positively correlated with the caregiver's level of commitment to the spousal relationship.

Effects of levcromakalim and RP52891 on NANCe nerve-mediated changes in pulmonary dynamics evoked by vagal stimulation in the guinea-pig.

The effects of the potassium channel activators (KCA) levcromakalim and RP52891 on NANCe nerve-mediated changes in pulmonary dynamics were investigated in the anaesthetized guinea-pig, using a newly-developed respiratory dynamics computer. Levcromakalim (0.025-0.2 mg/kg i.v.) and RP52891 (0.05-0.5 mg/kg i.v.) caused dose-dependent inhibition of NANCe nerve-mediated increases in airways resistance (RAW) and decreases in dynamic compliance (Cdyn). These effects of the KCAs persisted for at least 1 h. Unlike NANCe nerve-mediated responses, equivalent challenges with exogenously-administered substance P (SP; 10-25 micrograms/kg i.v.) and neurokinin A (NKA; 0.5-2.0 micrograms/kg i.v.) tended to produce progressively increasing responses but this effect was not statistically significant. Levcromakalim (0.2 mg/kg i.v.) and RP52891 (0.5 mg/kg i.v.) did not significantly decrease responses to exogenously-administered SP, although NKA-induced bronchoconstriction was attenuated. Glibenclamide (25 mg/kg i.v.) partially reversed the NANCe-inhibitory effects of levcromakalim (0.1 mg/kg i.v.) and RP52891 (0.25 mg/kg i.v.) and fully reversed their hypotensive effects. We have shown that levcromakalim and RP52891 inhibit bronchoconstrictor responses to NANCe nerve stimulation. This involves the opening of a glibenclamide-sensitive K(+)-channel and may represent effects at a pre-junctional site on NANCe neurones to reduce transmitter release.