Problem based review: pleuritic chest pain.

Pleuritic pain, a sharp discomfort near the chest wall exacerbated by inspiration is associated with a number of pathologies. Pulmonary embolus and infection are two common causes but diagnosis can often be challenging, both for experienced physicians and trainees. The underlying anatomy and pathophysiology of such pain and the most common aetiologies are presented. Clinical symptoms and signs that may arise alongside pleuritic pain are then discussed, followed by an introduction to the diagnostic tools such as the Wells’ score and current guidelines that can help to select the most appropriate investigation(s). Management of pulmonary embolism and other common causes of pleuritic pain are also discussed and highlighted by a clinical vignette.

GABA-activated chloride channels in secretory nerve endings.

Neurotransmitters acting on presynaptic terminals regulate synaptic transmission and plasticity. Because of the difficulty of direct electrophysiological recording from small presynaptic terminals, little is known about the ion channels that mediate these actions or about the mechanisms by which transmitter secretion is altered. The patch-clamp technique is used to show that the predominant inhibitory presynaptic neurotransmitter, gamma-aminobutyric acid (GABA), activates a GABAA receptor and gates a chloride channel in the membranes of peptidergic nerve terminals of the posterior pituitary. The opening of a chloride channel by GABA weakly depolarizes the nerve terminal membrane and blocks action potentials. In this way, GABA limits secretion by retarding the spread of excitation into the terminal arborization.

Synaptotagmin modulation of fusion pore kinetics in regulated exocytosis of dense-core vesicles.

In the exocytosis of neurotransmitter, fusion pore opening represents the first instant of fluid contact between the vesicle lumen and extracellular space. The existence of the fusion pore has been established by electrical measurements, but its molecular composition is unknown. The possibility that synaptotagmin regulates fusion pores was investigated with amperometry to monitor exocytosis of single dense-core vesicles. Overexpression of synaptotagmin I prolonged the time from fusion pore opening to dilation, whereas synaptotagmin IV shortened this time. Both synaptotagmin isoforms reduced norepinephrine flux through open fusion pores. Thus, synaptotagmin interacts with fusion pores, possibly by associating with a core complex of membrane proteins and/or lipid.

5-HT3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line.

Serotonin (5-HT) induces a large inward current accompanied by a conductance increase when applied focally to either neurons of mouse hippocampal cultures or cells of the NG108-15 clonal cell line. In both systems, the response is blocked by ICS 205-930, curare, and metoclopramide, while 2-methyl-5-HT is an agonist. The actions of ICS 205-930 and 2-methyl-5-HT indicate that the response is mediated by the 5-HT3 receptor. In NG108-15 cells the response activated in as little as 35 ms. The rapidity of the response suggests a direct coupling between the 5-HT3 receptor and a channel, which are probably both part of a single membrane protein. In both cell types, prolonged application of 5-HT resulted in desensitization; the rates of desensitization were accelerated by the adenylate cyclase activator forskolin. The 5-HT3 receptor has much in common with the nicotinic receptor and is probably involved in rapid synaptic transmission in the mammalian brain. Since this response is modulated by manipulations that elevate intracellular cAMP levels, the central synapses in which this receptor operates may exhibit plasticity.

Adaptation of Ca(2+)-triggered exocytosis in presynaptic terminals.

Rapid increases in Ca2+ concentration, produced by photolysis of caged Ca2+, triggered exocytosis in squid nerve terminals. This exocytosis was transient in nature, decaying with a time constant of approximately 30 ms. The decay could not be explained by a decline in presynaptic Ca2+ concentration, depletion of synaptic vesicles, or desensitization of postsynaptic receptors. Experiments in which Ca2+ was increased either in a series of steps or continuously at different rates suggested that the decay is caused by adaptation of the exocytotic Ca2+ receptor to higher levels of Ca2+. This adjustable sensitivity to Ca2+ represents a novel property of the triggering mechanism that can be used to evaluate molecular models of exocytosis. Adaptation can limit the amount of transmitter released by a nerve terminal and permit the speed of a presynaptic Ca2+ rise to serve as a critical determinant of synaptic efficacy.

cGMP-mediated facilitation in nerve terminals by enhancement of the spike afterhyperpolarization.

cGMP has long been suspected to play a role in synaptic plasticity, but the inaccessibility of nerve terminals to electrical recording has impeded tests of this hypothesis. In posterior pituitary nerve terminals, nitric oxide enhanced Ca(2+)-activated K+ channel activity by activating guanylate cyclase and PKG. This enhancement occurred only at depolarized potentials, so the spike threshold remained unaltered but the afterhyperpolarization became larger. During spike trains, the enhanced afterhyperpolarization promoted Na+ channel recovery from inactivation, thus reducing action potential failures and allowing more Ca(2+) to enter. Activating guanylate cyclase, either with applied nitric oxide, or with physiological stimulation to activate nitric oxide synthase, increased action potential firing. Thus, the cGMP/nitric oxide cascade generates a short-term, use-dependent enhancement of release.

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Sustained plateau activity precedes and can generate ictal-like discharges in low-Cl(-) medium in slices from rat piriform cortex.

Interictal and ictal discharges represent two different forms of abnormal brain activity associated with epilepsy. Ictal discharges closely parallel seizure activity, but depending on the form of epilepsy, interictal discharges may or may not be correlated with the frequency, severity, and location of seizures. Recent voltage-imaging studies in slices of piriform cortex indicated that interictal-like discharges are generated in a two-stage process. The first stage consists of a sustained, low-amplitude depolarization (plateau activity) lasting the entire latent period prior to discharge onset. Plateau activity takes place at a site distinct from the site of discharge onset and serves to sustain and amplify activity initiated by an electrical stimulus. In the second stage a rapidly accelerating depolarization begins at the onset site and then spreads over a wide region. Here, we asked whether ictal-like discharges can be generated in a similar two-stage process. As with interictal-like activity, the first sign of an impending ictal-like discharge is a sustained depolarization with a plateau-like time course. The rapidly accelerating depolarization that signals the start of the actual discharge develops later at a separate onset site. As found previously with interictal-like discharges, local application of kynurenic acid to the plateau site blocked ictal-like discharges throughout the entire slice. However, in marked contrast to interictal-like activity, blockade of synaptic transmission at the onset site failed to block the ictal-like discharge. This indicates that interictal- and ictal-like discharges share a common pathway in the earliest stage of their generation and that their mechanisms subsequently diverge.

An estimate of the minimum amount of fluid lipid required for the growth of Escherichia coli.

The lipid phase transition of Escherichia coli was studied by high sensitivity differential scanning calorimetry. A temperature sensitive unsaturated fatty acid auxotroph was used to obtain lipids with subnormal unsaturated fatty acid contents. From these studies it was concluded that E. coli can grow nromally with as much as 20% of its membrane lipids in the ordered state but that if more than 55% of the lipids are ordered, growth ceases. Studies with wild-type cells show that the phase transition ends more than 10 degrees C below the growth temperature when the growth temperature is either 25 degrees C or 37 degrees C.

A Negative Hydraulic Message from Oxygen-Deficient Roots of Tomato Plants? (Influence of Soil Flooding on Leaf Water Potential, Leaf Expansion, and Synchrony between Stomatal Conductance and Root Hydraulic Conductivity).

Four to 10 h of soil flooding delayed and suppressed the normal daily increase in root hydraulic conductance (Lp) in tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants. The resulting short-term loss of synchrony between Lp and stomatal conductance decreased leaf water potential ([psi]L) relative to well-drained plants within 2 h. A decrease in [psi]L persisted for 8 h and was mirrored by decreased leaf thickness measured using linear displacement transducers. After 10 h of flooding, further closing of stomata and re-convergence of Lp in flooded and well-drained roots returned [psi]L to control values. In the second photoperiod, Lp in flooded plants exceeded that in well-drained plants in association with much increased Lp and decreased stomatal conductance. Pneumatic balancing pressure applied to roots of intact flooded plants to prevent temporary loss of [psi]L in the 1st d did not modify the patterns of stomatal closure or leaf expansion. Thus, the magnitude of the early negative hydraulic message was neither sufficient nor necessary to promote stomatal closure and inhibit leaf growth in flooded tomato plants. Chemical messages are presumed to be responsible for these early responses to soil flooding.

Export of Abscisic Acid, 1-Aminocyclopropane-1-Carboxylic Acid, Phosphate, and Nitrate from Roots to Shoots of Flooded Tomato Plants (Accounting for Effects of Xylem Sap Flow Rate on Concentration and Delivery).

We determined whether root stress alters the output of physiologically active messages passing from roots to shoots in the transpiration stream. Concentrations were not good measures of output. This was because changes in volume flow of xylem sap caused either by sampling procedures or by effects of root stress on rates of whole-plant transpiration modified concentrations simply by dilution. Thus, delivery rate (concentration x sap flow rate) was preferred to concentration as a measure of solute output from roots. To demonstrate these points, 1-aminocyclopropane-1-carboxylic acid (ACC), abscisic acid, phosphate, nitrate, and pH were measured in xylem sap of flooded and well-drained tomato (Lycopersicon esculentum Mill., cv Ailsa Craig) plants expressed at various rates from pressurized detopped roots. Concentrations decreased as sap flow rates were increased. However, dilution of solutes was often less than proportional to flow, especially in flooded plants. Thus, sap flowing through detopped roots at whole-plant transpiration rates was used to estimate solute delivery rates in intact plants. On this basis, delivery of ACC from roots to shoots was 3.1-fold greater in plants flooded for 24 h than in well-drained plants, and delivery of phosphate was 2.3-fold greater. Delivery rates of abscisic acid and nitrate in flooded plants were only 11 and 7%, respectively, of those in well-drained plants.

Increased 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activity in Shoots of Flooded Tomato Plants Raises Ethylene Production to Physiologically Active Levels.

Soil flooding increased 1-aminocyclopropane-1-carboxylic (ACC) acid oxidase activity in petioles of wild-type tomato (Lycopersicon esculentum L.) plants within 6 to 12 h in association with faster rates of ethylene production. Petioles of flooded plants transformed with an antisense construct to one isoform of an ACC oxidase gene (ACO1) produced less ethylene and had lower ACC oxidase activity than those of the wild type. Flooding promoted epinastic curvature but did so less strongly in plants transformed with the antisense construct than in the wild type. Exogenous ethylene, supplied to well-drained plants, also promoted epinastic curvature, but transformed and wild-type plants responded similarly. Flooding increased the specific delivery (flux) of ACC to the shoots (picomoles per second per square meter of leaf) in xylem sap flowing from the roots. The amounts were similar in both transformed and wild-type plants. These observations demonstrate that changes in ACC oxidase activity in shoot tissue resulting from either soil flooding or introducing ACC oxidase antisense constructs can influence rates of ethylene production to a physiologically significant extent. They also implicate systemic root to shoot signals in regulating the activity of ACC oxidase in the shoot.

Potamogeton pectinatus Is Constitutively Incapable of Synthesizing Ethylene and Lacks 1-Aminocyclopropane-1-Carboxylic Acid Oxidase.

A highly sensitive laser-driven photoacoustic detector responsive to [less than or equal to]2.1 nmol m-3 ethylene (50 parts per trillion [v/v]) was used for ethylene analysis. Dark-grown plants of Potamogeton pectinatus L. growing from small tubers made no ethylene. Exposure of shoots to white light, wounding, submergence in water followed by desubmergence, partial oxygen shortage, indole acetic acid, or carbon dioxide failed to induce ethylene production, although clear effects were observed in Pisum sativum L. Some ethylene was released after applying high concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC; 10 mol m-3) to P. pectinatus, but the amount was trivial compared with that released by P. sativum. More endogenous ACC was found in P. pectinatus than in P. sativum. Considerable ACC oxidase activity was present in tissue extracts of P. sativum. However, no ACC oxidase activity was found in P. pectinatus, indicating that this is where ethylene production is arrested.

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap.

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.

Stable expression of insect GABA receptors in insect cell lines. Promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines.

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect gamma-aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selected marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chlorine ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the generic transformation of non-Drosophilid insects.

Expression of a Drosophila GABA receptor in a baculovirus insect cell system. Functional expression of insecticide susceptible and resistant GABA receptors from the cyclodiene resistance gene Rdl.

Recombinant baculoviruses containing two alternative splice forms of the Drosophila Rdl GABA receptor gene were constructed. Spodoptera frugiperda (Sf21) cells infected with either splice form expressed a transcript of expected size (2.5 kb). Western blotting of cell membrane extracts and immunoprecipitation experiments with an anti-Rdl antiserum recognized a protein of the expected size of approximately 65 kDa. Whole cell patch clamp analysis of cells infected with either splice form revealed functional expression of GABA gated chloride ion channels which were blocked by application of 1 microM picrotoxinin. Following replacement of alanine 302 with a serine, a mutation associated with resistance to picrotoxinin and cyclodiene insecticides, mutant channels showed similar levels of insensitivity to picrotoxinin (approximately 100-fold) as those observed in recordings from cultured Drosophila neurons. The significance of the expression of an insect GABA receptor in an insect cell line and the similarity of the results from these functional expression studies to recordings from cultured neurons is discussed.

Total lipid and fatty acid composition of milk from women with and without insulin-dependent diabetes mellitus.

Milk lipid and fatty acids were measured from women with insulin-dependent diabetes mellitus (IDDM), a comparison group of women without diabetes, and reference women. Milk samples were collected 80 min after breakfast at 2, 3, 7, 14, 42, and 84 d postpartum (pp). Total lipid and medium-chain fatty acids (MCFAs) in the group with IDDM were similar to or greater than those of control and reference groups at all times and were within normal, reported ranges. Milk long-chain polyunsaturated fatty acids (LC-PUFAs) were lower in women with IDDM from 14 to 84 d pp. Lower milk LC-PUFAs may be a result of altered fatty acid metabolism in women with IDDM.

Increased plasma noradrenaline during severe sodium restriction does not stimulate platelet release in essential hypertension.

Seventeen 50-year old hypertensive men, previously untreated with blood pressure 157 +/- 4/110 +/- 2 mmHg (means +/- SE) were given a low sodium diet for 2 weeks. During the second week, the diet was supplemented with potassium. The urinary Na+/K+ excretion ratio changed from 2:1 to 1:4 and 1:11, respectively. Sympathetic noradrenergic tone increased considerably during the first week. Thus, venous plasma noradrenaline increased from 254 +/- 22 to 347 +/- 28 pg/ml (p less than 0.001) and arterial concentration from 253 +/- 36 to 317 +/- 42 pg/ml (n = 10, p less than 0.001). No significant change was observed in sympathetic adrenal tone as reflected by normal plasma adrenaline in venous (42 +/- 5 vs 43 +/- 6 pg/ml, ns) or arterial blood (71 +/- 10 vs 82 +/- 15 pg/ml, n = 10, ns) or in venous plasma concentration of the blood platelet release product beta-thromboglobulin (BTG) (50 +/- 8 vs 43 +/- 5 ng/ml, ns). During the second week sympathetic noradrenergic tone remained highly significantly elevated compared to baseline but still no change in plasma adrenaline or plasma BTG was found. Thus, whereas sodium depletion did increase plasma noradrenaline concentration markedly in these hypertensive men, no change in adrenaline concentration was observed, and blood platelet release reaction was unchanged. Plasma noradrenaline within the physiological concentration range does not seem to serve as a regulator of in vivo platelet function.

Neuroactive steroids modulate GABAA receptors in peptidergic nerve terminals.

Neuroactive steroid modulation of GABAA receptors was investigated in the peptidergic nerve terminals of the posterior pituitary using patch clamp techniques. In common with GABAA receptors in cell bodies, the nerve terminal GABAA receptor was potentiated by the synthetic steroid alphaxalone and by physiological concentrations of the progesterone metabolite allopregnanolone. Both of these agents enhanced Cl- currents elicited by GABA. Estradiol-17 beta had a weak inhibitory effect on GABA responses of nerve terminals, but only at high concentrations. The potentiating action was manifest as an increase in the probability of channel opening, with no effect on the rate of desensitization of the GABAA receptor. Neuroactive steroids enhanced GABA-gated Cl- channel activity in cell-free membrane patches, thus demonstrating a membrane delimited response. These results indicated that with regard to allosteric modulation by neuroactive steroids, the nerve terminal GABAA receptor is similar to the GABAA receptors of nerve cell bodies and endocrine cells. Neuroactive steroids are thus capable of altering the chemosensitivity of nerve terminal membranes by enhancing GABA inhibition at this location. The neuroactive steroid sensitivity of nerve terminal GABAA receptors provides a pathway by which gonadal steroid derivatives could regulate peptide secretion from neurosecretory neurons. Such a pathway could participate in the coordination of neuropeptide secretion during complex neuroendocrine functions. With specific regard to the neurohypophysis, neuroactive steroid-induced changes in the sensitivity of nerve terminal GABAA receptors could play a role in the initiation of oxytocin secretion during the transition between pregnancy and parturition.

Imaging epileptiform discharges in slices of piriform cortex with voltage-sensitive fluorescent dyes.

Voltage imaging techniques were used to investigate epileptiform discharges in brain slices containing piriform cortex (PC). These experiments pinpointed the site of discharge onset in the endopiriform nucleus (En). Under some conditions, discharge onset also occurred simultaneously in adjoining neocortex. With slightly suprathreshold electrical stimulation, discharge generation was a two-stage process in which onset was preceded by a sustained spatially localized depolarization denoted as plateau activity. Plateau activity was seen away from the onset site, in a border region between En and layer III of PC. A similar two-stage sequence was seen for slices taken from a variety of planes, using two different interictal models as well as an ictal model. Plateau activity was found to be necessary for the generation of both kinds of discharge. Synaptic transmission at the site of onset was found to be required for the generation of interictal-like discharges, but ictal-like discharges were different in that they could still be generated when synaptic transmission at this site was impaired. These studies identify specialized regions with potentially important roles in epileptogenesis and help to elucidate the neuronal circuitry that can produce epileptiform activity.