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.

Patterns of peroxidative ethane emission from submerged rice seedlings indicate that damage from reactive oxygen species takes place during submergence and is not necessarily a post-anoxic phenomenon.

Using ethane as a marker for peroxidative damage to membranes by reactive oxygen species (ROS) we examined the injury of rice seedlings during submergence in the dark. It is often expressed that membrane injury from ROS is a post-submergence phenomenon occurring when oxygen is re-introduced after submergence-induced anoxia. We found that ethane production, from rice seedlings submerged for 24-72 h, was stimulated to 4-37 nl gFW(-1), indicating underwater membrane peroxidation. When examined a week later the seedlings were damaged or had died. On de-submergence in air, ethane production rates rose sharply, but fell back to less than 0.1 nl gFW(-1) h(-1) after 2 h. We compared submergence-susceptible and submergence-tolerant cultivars, submergence starting in the morning (more damage) and in the afternoon (less damage) and investigated different submergence durations. The seedlings showed extensive fatality whenever total ethane emission exceeded about 15 nl gFW(-1). Smaller amounts of ethane emission were linked to less extensive injury to leaves. Partial oxygen shortage (O(2) levels <1%) imposed for 2 h in gas phase mixtures also stimulated ethane production. In contrast, seedlings under anaerobic gas phase conditions produced no ethane until re-aerated: then a small peak was observed followed by a low, steady ethane production. We conclude that damage during submergence is not associated with extensive anoxia. Instead, injury is linked to membrane peroxidation in seedlings that are partially oxygen deficient while submerged. On return to air, further peroxidation is suppressed within about 2 h indicating effective control of ROS production not evident during submergence itself.

Physiological and metabolic adaptations of Potamogeton pectinatus L. tubers support rapid elongation of stem tissue in the absence of oxygen.

Tubers of Potamogeton pectinatus L., an aquatic pondweed, over-winter in the anoxic sediments of rivers, lakes and marshes. Growth of the pre-formed shoot that emerges from the tuber is remarkably tolerant to anoxia, with elongation of the stem occurring faster when oxygen is absent. This response, which allows the shoot to reach oxygenated waters, occurs despite a 69-81% reduction in the rate of ATP production, and it is underpinned by several physiological and metabolic adaptations that contribute to efficient energy usage. First, extension of the pre-formed shoot is the result of cell expansion, without the accumulation of new cellular material. Secondly, after over-wintering, the tuber and pre-formed shoot have the enzymes necessary for a rapid fermentative response at the onset of growth under anoxia. Thirdly, the incorporation of [(35)S]methionine into protein is greatly reduced under anoxia. The majority of the anoxically synthesized proteins differ from those in aerobically grown tissue, implying an extensive redirection of protein synthesis under anoxia. Finally, anoxia-induced cytoplasmic acidosis is prevented to an unprecedented degree. The adaptations of this anoxia-tolerant plant tissue emphasize the importance of the mechanisms that balance ATP production and consumption in the absence of oxygen.

Response and adaptation by plants to flooding stress.

Stress on plants imposed by flooding of the soil and deeper submergence constitutes one of the major abiotic constraints on growth, species' distribution and agricultural productivity. Flooding stress is also a strong driver of adaptive evolution. This has resulted in a wide range of biochemical, molecular and morphological adaptations that sanction growth and reproductive success under episodic or permanently flooded conditions that are highly damaging to the majority of plant species. However, even seemingly poorly adapted species possess some short-term resilience that is important for overall success of these plants in various habitats. The papers contained in this Special Issue address these topics and emphasize molecular, biochemical and developmental processes that impact on flooding tolerance. Most of the articles are based on lectures given to the 8th Conference of the International Society for Plant Anaerobiosis (ISPA), held at the University of Western Australia, Perth, 20-24 September, 2004. Reviews and research papers are presented from the leading laboratories currently working on plant responses to flooding stress.

Kinetics of ethanol and acetaldehyde release suggest a role for acetaldehyde production in tolerance of rice seedlings to micro-aerobic conditions.

BACKGROUND AND AIMS: This paper examines the basis of the greater tolerance of an indica rice cultivar FR13A to complete submergence compared with relatively intolerant japonica rice CT6241. We study whether this superior tolerance is related to its greater tolerance to O2 shortage and to an ability to run a more favourable rate of alcoholic fermentation during and after O2 deprivation. METHODS Fermentation products were analysed using sensitive laser-based photoacoustics at high time resolution to establish patterns and rates of ethanol and acetaldehyde emission by intact rice seedlings exposed to micro-aerobic (0.05-0.5 % O2) or zero O2 supply, and also during their return to air. Oxygen and CO2 emission or uptake was also quantified. KEY RESULTS: In the dark, no acetaldehyde and ethanol emission was observed until external O2 concentration in a gas phase decreased to

De-submergence-induced ethylene production in Rumex palustris: regulation and ecophysiological significance.

Rumex palustris responds to total submergence by increasing the elongation rate of young petioles. This favours survival by shortening the duration of submergence. Underwater elongation is stimulated by ethylene entrapped within the plant by surrounding water. However, abnormally fast extension rates were found to be maintained even when leaf tips emerged above the floodwater. This fast post-submergence growth was linked to a promotion of ethylene production that is presumed to compensate for losses brought about by ventilation. Three sources of ACC contributed to post-submergence ethylene production in R. palustris: (i) ACC that had accumulated in the roots during submergence and was transported in xylem sap to the shoot when stomata re-opened and transpiration resumed, (ii) ACC that had accumulated in the shoot during the preceding period of submergence and (iii) ACC produced de novo in the shoot following de-submergence. This new production of ethylene was associated with increased expression of an ACC synthase gene (RP-ACS1) and an ACC oxidase gene (RP-ACO1), increased ACC synthase activity and a doubling of ACC oxidase activity, measured in vitro. Out of seven species of Rumex examined, a de-submergence upsurge in ethylene production was seen only in shoots of those that had the ability to elongate fast when submerged.

Dynamic aspects of alcoholic fermentation of rice seedlings in response to anaerobiosis and to complete submergence: relationship to submergence tolerance.

Rice plants are severely damaged by complete submergence. This is a problem in rice farming and could be the result, in part, of tissue anoxia imposed by a reduced availability of oxygen. To investigate this possibility we monitored alcoholic fermentation products as markers for tissue anaerobiosis using sensitive laser-based spectroscopy able to sense ethanol and acetaldehyde down to 3 nl l(-1) and 0.1 nl l(-1), respectively. Acetaldehyde emission began within 0.5 h of imposing an oxygen-free gas phase environment followed closely by ethanol. As treatment progressed, ethanol output increased and came to exceed acetaldehyde emission as this stabilized considerably after approx. 3 h. On re-entry of air, a sharp post-anaerobic peak of acetaldehyde production was observed. This was found to be diagnostic of a preceding anoxic episode of 0.5 h or more. When anaerobiosis was lengthened by up to 14 h, the size of the post-anaerobic acetaldehyde outburst increased. After de-submergence from oxygen-free water, a similarly strong but slower post-anaerobic acetaldehyde upsurge was seen, which was accompanied by an increase in ethanol emission. Light almost, but not completely, eliminated fermentation in anaerobic surroundings and also the post-anaerobic or post-submergence peaks in acetaldehyde production. All photosynthetically generated oxygen was consumed within the plant. There was no substantial difference in acetaldehyde and ethanol output between FR13A and the less submergence-tolerant line CT6241 under any submergence treatment. In some circumstances, submergence damaged CT6241 more than FR13A even in the absence of vigorous fermentation. We conclude that oxygen deprivation may not always determine the extent of damage caused to rice plants by submergence under natural conditions.

Interpretation and optimization of absorbance and fluorescence signals from voltage-sensitive dyes.

Voltage-sensitive dyes produce absorbance and fluorescence changes that can be used to image voltage. The present study develops a systematic approach to the optimization of these signals. A mathematical analysis assesses the dye optical density ( OD) that optimizes the signal-to-noise ratio in absorbance and fluorescence measurements. The signal-to-noise ratio is maximal for a dye OD of 2 (natural logarithm) in absorbance and ~1 in fluorescence. The fluorescence result is approximate because, in contrast to absorbance, the optimal dye OD varies with the amount of scattering and intrinsic absorbance of the tissue. The signal-to-noise ratio of absorbance is higher in thick preparations such as brain slices; fluorescence is superior in thin preparations such as cell culture. The optimal OD for absorbance and fluorescence, as well as the superiority of absorbance, were confirmed experimentally on hippocampal slices. This analysis also provided insight into the interpretation of signals normalized to resting light intensities. With both absorbance and fluorescence, the normalized signal (Delta I/I) varies with OD, and does not reflect the change in dye absorbance. In absorbance this problem is remedied by dividing Delta I/I by the dye OD to obtain the absorbance change. For fluorescence a correction is possible, but is more complicated. Because this analysis indicates that high levels of stain optimize the signal-to-noise, dyes were tested for pharmacological actions and phototoxicity. The absorbance dye RH155 was found to have pharmacological action at high staining levels. The fluorescent dye RH414 was phototoxic. Adverse effects could not be detected with the absorbance dye RH482.

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.

Epileptiform discharges with in-vivo-like features in slices of rat piriform cortex with longitudinal association fibers.

Brain slices serve as useful models for the investigation of epilepsy. However, the preparation of brain slices disrupts circuitry and severs axons, thus complicating efforts to relate epileptiform activity in vitro to seizure activity in vivo. This issue is relevant to studies in transverse slices of the piriform cortex (PC), the preparation of which disrupts extensive rostrocaudal fiber systems. In these slices, epileptiform discharges propagate slowly and in a wavelike manner, whereas such discharges in vivo propagate more rapidly and jump abruptly between layers. The objective of the present study was to identify fiber systems responsible for these differences. PC slices were prepared by cutting along three different nearly orthogonal planes (transverse, parasagittal, and longitudinal), and epileptiform discharges were imaged with a voltage-sensitive fluorescent dye. Interictal-like epileptiform activity was enabled by either a kindling-like induction process or disinhibition with bicuculline. The pattern of discharge onset was very similar in slices cut in different planes. As described previously in transverse PC slices, discharges were initiated in the endopiriform nucleus (En) and adjoining regions in a two-stage process, starting with low-amplitude "plateau activity" at one site and leading to an accelerating depolarization and discharge onset at another nearby site. The similar pattern of onset in slices of various orientations indicates that the local circuitry and neuronal properties in and around the En, rather than long-range fibers, assume dominant roles in the initiation of epileptiform activity. Subtle variations in the onset site indicate that interneurons can fine tune the site of discharge onset. In contrast to the mode of onset, discharge propagation showed striking variations. In longitudinal slices, where rostrocaudal association fibers are best preserved, discharge propagation resembled in vivo seizure activity in the following respects: propagation was as rapid as in vivo and about two to three times faster than in other slices; discharges jumped abruptly between the En and PC; and discharges had large amplitudes in superficial layers of the PC. Cuts in longitudinal slices that partially separated the PC from the En eliminated these unique features. These results help clarify why epileptiform activity differs between in vitro and in vivo experiments and suggest that rostrocaudal pyramidal cell association fibers play a major role in the propagation of discharges in the intact brain. The longitudinal PC slice, which best preserves these fibers, is ideally suited for the study their role.

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.

Fundamental Ca2+ signaling mechanisms in mouse dendritic cells: CRAC is the major Ca2+ entry pathway.

Although Ca(2+)-signaling processes are thought to underlie many dendritic cell (DC) functions, the Ca(2+) entry pathways are unknown. Therefore, we investigated Ca(2+)-signaling in mouse myeloid DC using Ca(2+) imaging and electrophysiological techniques. Neither Ca(2+) currents nor changes in intracellular Ca(2+) were detected following membrane depolarization, ruling out the presence of functional voltage-dependent Ca(2+) channels. ATP, a purinergic receptor ligand, and 1-4 dihydropyridines, previously suggested to activate a plasma membrane Ca(2+) channel in human myeloid DC, both elicited Ca(2+) rises in murine DC. However, in this study these responses were found to be due to mobilization from intracellular stores rather than by Ca(2+) entry. In contrast, Ca(2+) influx was activated by depletion of intracellular Ca(2+) stores with thapsigargin, or inositol trisphosphate. This Ca(2+) influx was enhanced by membrane hyperpolarization, inhibited by SKF 96365, and exhibited a cation permeability similar to the Ca(2+) release-activated Ca(2+) channel (CRAC) found in T lymphocytes. Furthermore, ATP, a putative DC chemotactic and maturation factor, induced a delayed Ca(2+) entry with a voltage dependence similar to CRAC. Moreover, the level of phenotypic DC maturation was correlated with the extracellular Ca(2+) concentration and enhanced by thapsigargin treatment. These results suggest that CRAC is a major pathway for Ca(2+) entry in mouse myeloid DC and support the proposal that CRAC participates in DC maturation and migration.

Characterisation of programmed cell death during aerenchyma formation induced by ethylene or hypoxia in roots of maize (Zea mays L.).

Aerenchyma is a tissue type characterised by prominent intercellular spaces which enhance flooding tolerance in some plant species by facilitating gas diffusion between roots and the aerial environment. Aerenchyma in maize roots forms by collapse and death of some of the cortical cells in a process that can be promoted by imposing oxygen shortage or by ethylene treatment. Maize roots grown hydroponically in 3% oxygen, 1 microl x l(-1) ethylene or 21% oxygen (control) were analysed by a combination of light and electron microscopy. Use of in-situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) suggested internucleosomal cleavage of DNA. However, chromatin condensation detectable by electron microscopy was preceded by cytoplasmic changes including plasma membrane invagination and the formation of vesicles, in contrast to mammalian apoptosis in which chromatin condensation is the first detectable event. Later, cellular condensation, condensation of chromatin and the presence of intact organelles surrounded by membrane resembling apoptotic bodies were observed. All these events were complete before cell wall degradation was apparent. Therefore, aerenchyma formation initiated by hypoxia or ethylene appears to be a form of programmed cell death that shows characteristics in part resembling both apoptosis and cytoplasmic cell death in animal cells.

Whole-cell voltage clamp recording.

This unit describes the use of whole-cell voltage clamping to study voltage-gated channels. Stepwise changes in voltage produced by this technique cause channels to interconvert between different states, and these transitions are monitored as changes in membrane current. The time course of this redistribution of states contains a great deal of information about the mechanism of channel gating. Furthermore, the voltage clamp can be used to activate different populations of channels selectively. In this way, a specific channel targeted by biological or pharmacological manipulations can often be identified and studied in detail. This technique is also readily adapted to the study of ligand-gated channels, synaptic potentials, and exocytosis.

Single-channel recording.

This unit provides detailed descriptions for the steps of patch excision, data acquisition, and data analysis, and elaborates upon the relevant issues discussed in other units from Chapter 3. It includes discussions of the instrumentation for single-channel recording and the key concepts necessary for the interpretation of single-channel data.

Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release.

Anoxia tolerance in the aquatic monocot Potamogeton pectinatus absence of oxygen stimulates elongation in association with an unusually large pasteur effect.

Elongation by stems of overwintered tubers of Potamogeton pectinatus (L.) is strongly promoted over several days by oxygen-free conditions. Characteristics of the respiration underpinning this unusual response were examined. Anaerobic plants produced ethanol and CO(2) in approximately equimolar amounts, indicating that glycolysis coupled to alcoholic fermentation was the principal CO(2)-producing respiratory pathway. Rates of CO(2) evolution by aerobic and anaerobic whole plants (shoot and tuber) were similar, suggesting a rate of glycolysis three times that of aerobic plants, i.e. a strong Pasteur effect. In the shoot alone, anaerobic CO(2) production was twice the aerobic rate indicating a 6-fold increase in the rate of glycolysis in this tissue. Anoxic stems contained more sucrose at a stronger concentration than slower-growing aerobic stems or anaerobic leaves, demonstrating that sugar supply to the site of most rapid growth exceeded demand in the absence of oxygen. Concentrations of potentially toxic acetaldehyde in the external medium were small (approximately 0.2 mol m(-3)) during anoxia and on return to aerated conditions. Lactic acid was undetectable under anaerobic conditions and in vivo (31)P-NMR analysis of shoots revealed a cytoplasmic acidification of only

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.

Induction of persistent sodium current by exogenous and endogenous nitric oxide.

Most voltage-gated Na(+) channels inactivate almost completely at depolarized membrane potentials, but in some cells a residual Na(+) current is seen that is resistant to inactivation. This persistent Na(+) current can have a profound impact on the electrical behavior of excitable cells, and the regulation of this property could have important biological consequences. However, the biological signaling mechanisms that regulate the persistence of Na(+) channels are not well understood. This study showed that in nerve terminals and ventricular myocytes nitric oxide (NO) reduced the inactivation of Na(+) current. This effect was independent of cGMP, was blocked by N-ethylmaleimide, and could be elicited in cell-free outside-out patches. Thus, a reactive nitrogen species acts directly on the channel or closely associated protein. Persistent Na(+) current could also be induced by endogenous NO generated enzymatically by NO synthase (NOS). Application of ionomycin to raise the intracellular Ca(2+) concentration in myocytes activated NOS. The NO produced in response to ionomycin was detected with an NO-sensitive fluorescent dye. Persistent Na(+) current was enhanced by the same treatment, and NOS inhibitors abolished both the elevation of NO and the induction of persistent Na(+) current. These experiments show that NO is a potential endogenous regulator of persistent Na(+) current under physiological and pathophysiological conditions.

Characteristics of plateau activity during the latent period prior to epileptiform discharges in slices from rat piriform cortex.

The deep piriform region has an unusually high seizure susceptibility. Voltage imaging previously located the sites of epileptiform discharge onset in slices of rat piriform cortex and revealed the spatiotemporal pattern of development of two types of electrical activity during the latent period prior to discharge onset. A ramplike depolarization (onset activity) appears at the site of discharge onset. Onset activity is preceded by a sustained low-amplitude depolarization (plateau activity) at another site, which shows little if any overlap with the site of onset. Because synaptic blockade at either of these two sites blocks discharges, it was proposed that both forms of latent period activity are necessary for the generation of epileptiform discharges and that the onset and plateau sites work together in the amplification of electrical activity. The capacity for amplification was examined here by studying subthreshold responses in slices of piriform cortex using two different in vitro models of epilepsy. Under some conditions electrically evoked responses showed a nonlinear dependence on stimulus current, suggesting amplification by strong polysynaptic excitatory responses. The sites of plateau and onset activity were mapped for different in vitro models of epilepsy and different sites of stimulation. These experiments showed that the site of plateau activity expanded into deep layers of neighboring neocortex in parallel with expansions of the onset site into neocortex. These results provide further evidence that interactions between the sites of onset and plateau activity play an important role in the initiation of epileptiform discharges. The site of plateau activity showed little variation with different stimulation sites in the piriform cortex, but when stimulation was applied in the endopiriform nucleus (in the sites of onset of plateau activity), plateau activity had a lower amplitude and became distributed over a much wider area. These results indicate that in the initiation of epileptiform discharges, the location of the circuit that generates plateau activity is not rigidly defined but can exhibit flexibility.