Selected ginsenosides of the protopanaxdiol series are novel positive allosteric modulators of P2X7 receptors.

BACKGROUND AND PURPOSE: The P2X7 receptor is an ATP-gated ion channel predominantly expressed in immune cells and plays a key role in inflammatory processes. Ginseng is a well-known Chinese herb with both pro- and anti-inflammatory properties and many of its actions have been ascribed to constituent ginsenosides. We screened a number of ginsenoside compounds for pharmacological activity at P2X7 receptors, that might contribute to the reported immunomodulatory actions of ginseng. EXPERIMENTAL APPROACH: We used several assays to measure responses of P2X7 receptors, ATP-mediated dye uptake, intracellular calcium measurement and whole-cell patch-clamp recordings. HEK-293 cells stably expressing human P2X7 receptors were used in addition to mouse macrophages endogenously expressing P2X7 receptors. KEY RESULTS: Four ginsenosides of the protopanaxdiol series, Rb1, Rh2, Rd and the metabolite compound K (CK) potentiated the dye uptake responses of P2X7 receptors, whereas other ginsenosides tested were ineffective (1-10 µM). The potentiation was rapid in onset, required a threshold concentration of ATP (>50 µM) and had an EC50 of 1.08 µM. CK markedly enhanced ATP-activated P2X7 currents, probably via an extracellular site of action. One of the consequences of this potentiation effect is a sustained rise in intracellular Ca(2+) that could account for the decrease in cell viability in mouse macrophages after a combination of 500 µM ATP and 10 µM CK that are non-toxic when applied alone. CONCLUSIONS AND IMPLICATIONS: This study identifies selected ginsenosides as novel potent allosteric modulators of P2X7 channels that may account for some of the reported immune modulatory actions of protopanaxdiol ginsenosides in vivo.

Therapeutic potential of Panax ginseng and ginsenosides in the treatment of chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major global health burden and will become the third largest cause of death in the world by 2030. It is currently believed that an exaggerated inflammatory response to inhaled irritants, in particular cigarette smoke, cause progressive airflow limitation. This inflammation, where macrophages, neutrophils and lymphocytes are prominent, leads to oxidative stress, emphysema, airways fibrosis and mucus hypersecretion. COPD responds poorly to current anti-inflammatory treatments including corticosteroids, which produce little or no benefit. Panax ginseng has a long history of use in Chinese medicine for respiratory conditions, including asthma and COPD. OBJECTIVES: In this perspective we consider the therapeutic potential of Panax ginseng for the treatment of COPD. RESULTS: Panax ginseng and its compounds, ginsenosides, have reported effects through multiple mechanisms but primarily have anti-inflammatory and anti-oxidative effects. Ginsenosides are functional ligands of glucocorticoid receptors and appear to inhibit kinase phosphorylation including MAPK and ERK1/2, NF-κB transcription factor induction/translocation, and DNA binding. They also inhibit pro-inflammatory mediators, TNF-α, IL-6, IL-8, ROS, and proteases such as MMP-9. Panax ginseng protects against oxidative stress by increasing anti-oxidative enzymes and reducing the production of oxidants. CONCLUSION: Given that Panax ginseng and ginsenosides appear to inhibit processes related to COPD pathogenesis, they represent an attractive therapeutic target for the treatment of COPD.

Atmospheric deposition of phosphorus to land and freshwater.

We compiled published and newly-obtained data on the directly-measured atmospheric deposition of total phosphorus (TP), filtered total phosphorus (FTP), and inorganic phosphorus (PO4-P) to open land, lakes, and marine coasts. The resulting global data base includes data for c. 250 sites, covering the period 1954 to 2012. Most (82%) of the measurement locations are in Europe and North America, with 44 in Africa, Asia, Oceania, and South-Central America. The deposition rates are log-normally distributed, and for the whole data set the geometric mean deposition rates are 0.027, 0.019 and 0.14 g m(-2) a(-1) for TP, FTP and PO4-P respectively. At smaller scales there is little systematic spatial variation, except for high deposition rates at some sites in Germany, likely due to local agricultural sources. In cases for which PO4-P was determined as well as one of the other forms of P, strong parallels between logarithmic values were found. Based on the directly-measured deposition rates to land, and published estimates of P deposition to the oceans, we estimate a total annual transfer of P to and from the atmosphere of 3.7 Tg. However, much of the phosphorus in larger particles (principally primary biological aerosol particles) is probably redeposited near to its origin, so that long-range transport, important for tropical forests, large areas of peatland and the oceans, mainly involves fine dust from deserts and soils, as described by the simulations of Mahowald et al. (Global Biogeochemical Cycles 22, GB4026, 2008). We suggest that local release to the atmosphere and subsequent deposition bring about a pseudo-diffusive redistribution of P in the landscape, with P-poor ecosystems, for example ombrotrophic peatlands and oligotrophic lakes, gaining at the expense of P-rich ones. Simple calculations suggest that atmospheric transport could bring about significant local redistribution of P among terrestrial ecosystems. Although most atmospherically transported P is natural in origin, local transfers from fertilised farmland to P-poor ecosystems may be significant, and this requires further research.

Controls on soil solution nitrogen along an altitudinal gradient in the Scottish uplands.

Nitrogen (N) deposition continues to threaten upland ecosystems, contributing to acidification, eutrophication and biodiversity loss. We present results from a monitoring study aimed at investigating the fate of this deposited N within a pristine catchment in the Cairngorm Mountains (Scotland). Six sites were established along an elevation gradient (486-908 m) spanning the key habitats of temperate maritime uplands. Bulk deposition chemistry, soil carbon content, soil solution chemistry, soil temperature and soil moisture content were monitored over a 5 year period. Results were used to assess spatial variability in soil solution N and to investigate the factors and processes driving this variability. Highest soil solution inorganic N concentrations were found in the alpine soils at the top of the hillslope. Soil carbon stock, soil solution dissolved organic carbon (DOC) and factors representing site hydrology were the best predictors of NO(3)(-) concentration, with highest concentrations at low productivity sites with low DOC and freely-draining soils. These factors act as proxies for changing net biological uptake and soil/water contact time, and therefore support the hypothesis that spatial variations in soil solution NO(3)(-) are controlled by habitat N retention capacity. Soil percent carbon was a better predictor of soil solution inorganic N concentration than mass of soil carbon. NH(4)(+) was less affected by soil hydrology than NO(3)(-) and showed the effects of net mineralization inputs, particularly at Racomitrium heath and peaty sites. Soil solution dissolved organic N concentration was strongly related to both DOC and temperature, with a stronger temperature effect at more productive sites. Due to the spatial heterogeneity in N leaching potential, a fine-scale approach to assessing surface water vulnerability to N leaching is recommended over the broad scale, critical loads approach currently in use, particularly for sensitive areas.

The present is the key to the past, but what does the future hold for the recovery of surface waters from acidification?

Analogue matching was used to identify close modern water quality analogues for a set of 59 acid-sensitive lakes in the Galloway region of south-west Scotland. Modern analogues were identified that closely matched the pre-disturbance conditions of these lakes using simulated water quality parameters from the MAGIC (Model of Acidification of Groundwater in Catchments) model for key years from 1860 to 2100. The lakes were matched with hydrochemical samples from a large spatial data set in the UK. For the majority of the 59 lakes, several close modern analogues were identified from the training set for specified years. The close modern analogues for the reference year (1860) were predominantly located in north-west Scotland, an area of low acid deposition and high-status water quality. A clear recovery in the regional surface water acid neutralising capacity (ANC) was simulated by MAGIC in 2015 compared to the situation in 1970 at the height of acid emissions. Predicted trends in surface water chemistry from present day to 2015 indicate some improvement in water quality with c. 23 +/- 0.97% recovery towards pre-acidification (1860) ANC for the region. Output from the MAGIC model was used with the analogue technique to investigate the combined influence of future changes in deposition and climate on biogeochemical processes and water quality at the Round Loch of Glenhead (RLGH). Our results demonstrate that pre-acidification restoration targets will not be achieved by simply reducing acid deposition, and climate change will further confound the beneficial effects of deposition reductions. Results for 2015 and beyond show that modern analogues for these periods were predominantly concentrated in North Wales, with some in north-west Scotland, Galloway and the Lake District. Evidence from model simulations and modern analogues indicate that more stringent measures to further reduce acid deposition and combat climate change in the future are necessary if the majority of lakes in the Galloway region are to be restored to their pre-acidification target chemistry. The identified analogues for selected periods may be used to study wider ecological conditions to better define reference conditions and future recovery trajectories. That modern analogues were identified for the simulated chemistry in 2100 at RLGH suggests that unprecedented chemical conditions are unlikely to be observed as a result of future climate change.

Interactive effects of nitrogen deposition and fire on plant and soil chemistry in an alpine heathland.

The response of alpine heathland vegetation and soil chemistry to N additions of 0, 10, 20 and 50 kg N ha(-1) year(-1) in combination with simulated accidental fire (+/-) was monitored over a 5-year period. N addition caused rapid and significant increases in plant tissue N content and N:P and N:K of Calluna vulgaris, suggesting increasing phosphorus and potassium limitation of growth. Soil C:N declined significantly with N addition, indicating N saturation and increasing likelihood of N leakage. Fire further decreased soil C:N and reduced potential for sequestration of additional N. This study shows that alpine heathlands, which occupy the headwaters of many rivers, have limited potential to retain deposited N and may rapidly become N saturated, leaking N into downstream communities and surface waters.

Modelling the effect of climate change on recovery of acidified freshwaters: relative sensitivity of individual processes in the MAGIC model.

The MAGIC model was used to evaluate the relative sensitivity of several possible climate-induced effects on the recovery of soil and surface water from acidification. A common protocol was used at 14 intensively studied sites in Europe and eastern North America. The results show that several of the factors are of only minor importance (increase in pCO(2) in soil air and runoff, for example), several are important at only a few sites (seasalts at near-coastal sites, for example) and several are important at nearly all sites (increased concentrations of organic acids in soil solution and runoff, for example). In addition changes in forest growth and decomposition of soil organic matter are important at forested sites and sites at risk of nitrogen saturation. The trials suggest that in future modelling of recovery from acidification should take into account possible concurrent climate changes and focus specially on the climate-induced changes in organic acids and nitrogen retention.

Nitrate leaching as a confounding factor in chemical recovery from acidification in UK upland waters.

Over the period 1988-2002, data from 18 of the 22 lakes and streams in the UK Acid Waters Monitoring Network (AWMN) show clear trends of declining excess sulphate concentrations in response to reductions in sulphur deposition, but fewer trends in increasing pH or alkalinity. There has been no significant decline in the deposition of total nitrogen over the same period, and no sites show a trend in nitrate concentration. Peak nitrate concentrations have already surpassed excess sulphate on occasion in half of the AWMN sites. Furthermore, current understanding of terrestrial N saturation processes suggests that nitrate leaching from soils may increase, even under a constant N deposition load. Best-case projections indicate that nitrate will overtake sulphate as the major excess acid anion in many sites within 10 years, while worst-case predictions with steady-state models suggest that in the longer-term, nitrate could become the dominant excess acid anion in most of the UK.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), TRAIL receptors, and the soluble receptor osteoprotegerin in human gestational membranes and amniotic fluid during pregnancy and labor at term and preterm.

We have studied TNF-related apoptosis-inducing ligand (TRAIL) and its membrane-bound (R1-R4) and soluble receptors [osteoprotegerin (OPG)] in gestational membranes to assess their significance in preterm parturition and premature rupture of membranes (PROM). TRAIL was detected by ELISA in extracts of term choriodecidual (but not amnion) tissues and explant-conditioned media. Concentrations of OPG (determined using ELISA) in gestational membranes were 20- to 50-fold greater than those of TRAIL. Median OPG concentrations in amniotic fluid (AF) at 15-17 wk gestation were similar to those at term before and during labor, whereas levels in pregnancies sampled preterm were significantly elevated. OPG levels in AF from women with preterm PROM were similar to those from women in preterm labor. In contrast, in pooled AF samples (n = 23-33), TRAIL concentrations at term with and without labor were elevated compared with samples from preterm deliveries. TRAIL-R3 and -R4 decoy receptors were detected in term amnion and choriodecidual extracts by immunoblotting and were localized by immunohistochemistry to amnion epithelial cells and chorionic trophoblasts. TRAIL (100 ng/ml) had little or no effect on amnion or choriodecidual cell viability or apoptosis, although these tissues responded to TNF-alpha with increased prostaglandin E(2) production. Our findings suggest that OPG is abundant in gestational membranes and, in concert with TRAIL decoy receptors, may protect resident cells of the fetal membranes against the proapoptotic effects of TRAIL and other related ligands during pregnancy.

Cytokines, prostaglandins and parturition--a review.

The elaboration of cytokines, chemokines and immunomodulatory proteins in the placenta and gestational membranes has been extensively investigated in the context of both normal and abnormal pregnancy and delivery. Patterns of expression of cytokines in the foetal membranes and decidua suggest that inflammatory activation occurs modestly with term labour, but much more robustly in preterm delivery, particularly in the presence of intrauterine infection. Enhanced chemokine expression, particularly evident in deliveries with an infected amniotic cavity, is presumably responsible for recruiting infiltrating leukocytes into the membranes thereby amplifying the inflammatory process and hastening membrane rupture and delivery. Anti-inflammatory cytokines suppress inflammatory reactions in the placenta, but under some circumstances may act in a pro-inflammatory fashion in the membranes. Intracellular signalling by cytokines is modulated by proteins such as SOCS (Silencer Of Cytokine Signalling)-1, -2 and -3. Changes in the abundance of these proteins occur with term labour, implicating them as modulators of cytokine actions around the time of parturition. Prostaglandins, released by the membranes in response to stretch and the actions of pro-inflammatory cytokines, act not only upon the myometrium and cervix, but may also exert paracrine/autocrine effects on cell viability and matrix protein integrity. The localization and regulation of prostanoid isomerases, responsible for converting PGH(2) (derived from prostaglandin H synthase-1 and -2) to bioactive prostanoids, are being studied in these tissues, particularly in the context of cytokine interactions. Although the gestational tissues are known to be sources of PGD(2), PGJ(2) and its derivatives, the regulation of production of these prostaglandins has yet to be studied in any detail and their actions, which may include apoptosis and suppression of inflammation, remain poorly defined. A more complete understanding of these aspects of cytokine-prostaglandin interactions in pregnancy and parturition will, no doubt, unfold as current studies come to fruition.

Water quality in the Scottish uplands: a hydrological perspective on catchment hydrochemistry.

Land above 300 m covers approximately 75% of the surface of Scotland and most of the nation's major river systems have their headwaters in this upland environment. The hydrological characteristics of the uplands exert an important influence on the hydrochemistry of both headwater streams and downstream river systems. Thus, many of the spatial and temporal patterns in the chemical quality of surface waters are mediated by hydrological processes that route precipitation through upland catchments. These hydrological pathways also have an important influence on how the hydrochemistry of upland streams is responding to increasing pressures from environmental changes at the global and regional scales. At the present time, atmospheric deposition remains an issue in many parts of the Scottish uplands, where critical loads of acidity are exceeded, particularly in areas affected by increasing N deposition. Moreover, climatic change forecasts predict increasingly wetter, warmer and more seasonal conditions, which may modify the hydrochemical regimes of many river systems, particularly those with a strong snowmelt component. On a more localised scale, land management practices, including felling of commercial forests, expansion of native woodlands, agricultural decline and moorland management all have implications for the freshwater environment. Moreover, increasing public access to upland areas for a range of recreational activities have implications for water quality. Understanding the hydrology of the uplands, through integrated field and modelling studies, particularly of the hydrological pathways that regulate chemical transfers to streamwaters, will remain an important research frontier for the foreseeable future.

Partitioning the variation within the acid neutralizing capacity of surface waters in Scotland in relation to land cover, soil and atmospheric depositional factors.

A method of decomposing the variation in the acid neutralizing capacity (ANC) of surface waters in Scotland is described. Using national datasets, a series of variables relating to 703 catchments across Scotland is divided into three components representing (i) land cover, (ii) soil and (iii) atmospheric deposition/altitude. Redundancy analysis (RDA) and (partial) redundancy analysis are used to quantify the amount of variation in ANC uniquely attributable to each of these components, independent of the effects of the others. The variation accounted for by covarying combinations of these components is also determined. Approximately 55% of the total variation in ANC across the 703 sites is explained by the variables representing catchment characteristics and atmospheric deposition. Of this, 8.5%, 2.4% and 6.9% are uniquely attributable to the land cover, soil and deposition/altitude components, respectively. A further 38% of ANC variation is associated with the covariation between components, with 18% accounted for by the combination of all three. Approximately 45% of the variation in ANC remains unexplained. The results reflect the integrated nature of catchment processes and demonstrate, for these data, that it is a combination of land cover, soil and deposition and altitude factors which most explain variation in freshwater ANC level. The approach offers a tool with which to assess the sensitivity of surface waters to acid deposition at a regional scale and provides a way of identifying regional differences in catchment response to acid loading.

Interaction of nitrogen deposition and land use on soil and water quality in Scotland: issues of spatial variability and scale.

Over large areas of the Scottish uplands anthropogenic sulfur (S) deposition is declining in response to stringent national and European controls on S emissions. At the same time, however, the relative contribution of nitrogenous (N) compounds to the total anthropogenic deposition loading has increased. To investigate the significance of N deposition on the potential acidification of surface waters, national, regional, and catchment databases were developed to assess the relationships between N deposition, soil C/N ratios, land use and surface water NO3 concentrations. National classification schemes for land use and soils were used as only limited empirical data are available at such large spatial scales. Data were screened to eliminate areas where N inputs are dominated by non-atmospheric sources. From these screened datasets, it was apparent that areas with the highest risk of N leaching were situated predominantly in the upland areas of south-west and west Scotland (areas with low soil C/N ratios). At the regional scale, surface-water NO3 concentration in afforested catchments was negatively correlated with soil C/N ratios below 20. This relationship was not evident in moorland catchments, where NO3 leaching was strongly related to N deposition and the loch/catchment ratio, rather than the soil C/N ratio. Temporal trends of regional water quality highlighted as increasing loch NO3 concentrations between 1988 and 1996-1997, presumably reflecting an increase in N deposition, enhanced leaching losses from the terrestrial component of the catchment, or altered in-lake processes. The hydrochemical records for two catchments in NE Scotland (Lochnagar and Allt a Mharcaidh) highlight the importance of within catchment process in controlling the nitrogen response observed in surface waters. The potential mechanisms through which vegetation and soils may modify incoming deposition are discussed.

15-deoxy-delta12,14-prostaglandin J2-induced apoptosis in amnion-like WISH cells.

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.

Glial cell line derived neurotrophic factor (GDNF) regulates VR1 and substance P in cultured sensory neurons.

Many nociceptive dorsal root ganglion (DRG) sensory neurons express the high affinity nerve growth factor (NGF) receptor TRKA, and respond to NGF. However, many do not express TRKA but are thought to respond to glial cell-derived neurotrophic factor (GDNF) and related molecules. We therefore cultured DRG neurons in the presence of GDNF, and looked at the expression of substance P and of the capsaicin receptor, VR1, two nociceptive properties already known to be NGF regulated. Using several different approaches we demonstrated that GDNF produced clear increases in expression of both properties, comparable in magnitude to increases seen with NGF. Following axotomy, aberrant expression of substance P in A fibres may be involved in the generation of neuropathic pain. Factors regulating substance P and other properties in the absence of retrogradely transported NGF may therefore be of significance in neuropathic pain states.

Facilitatory effect of Ca2+ on the noradrenaline-evoked cation current in rabbit portal vein smooth muscle cells.

1. The facilitatory effect of external calcium ions (Ca2+o) on the alpha1-adrenoceptor-activated non-selective cation current (Icat) was investigated in rabbit portal vein cells using noise and voltage-jump relaxation analysis of the whole-cell macroscopic current. 2. Micromolar concentrations of Ca2+o potentiated the peak amplitude of Icat at a holding potential (Vh) of -50 mV. The effective [Ca2+]o which produced a 50% potentiation (EC50) was 3 microM. 3. From noise analysis the estimated single channel conductance (gamma) was approximately 23 pS with [Ca2+]o between 3 and 100 microM, whereas in < 10 nM or 1 microM Ca2+o gamma was approximately 10 pS. 4. The spectral density function of Icat at negative potentials could be described by the sum of two Lorentzians in every [Ca2+]o examined. The time constant of the lower frequency Lorentzian component (tau1) was about 11 ms in < 10 nM Ca2+o and was about 45 ms in micromolar concentrations of Ca2+o (1-100 microM). In contrast, the time constant of the higher frequency component (tau2) was similar in < 10 nM Ca2+o and 100 microM Ca2+o (between 1 and 2 ms). 5. The lower frequency Lorentzian component was responsible for about half the total current variance in < 10 nM Ca2+o whereas in micromolar concentrations of Ca2+o it was responsible for most of the measured current variance. 6. In voltage-jump experiments, on stepping the voltage from -50 to +50 mV the instantaneous current was followed by an exponential decline of Icat. Stepping back to -30 mV produced an exponential inward relaxation (Irelax,-30 mV) leading to an increase in the steady-state amplitude of Icat in micromolar concentrations of Ca2+o, but this relaxation was not observed in < 10 nM Ca2+o. The relative amplitude of Irelax,-30 mV increased in an [Ca2+]o-dependent manner (EC50 was 2 microM) although the time constant of this relaxation (taurelax,-30 mV) remained unchanged (about 60 ms between 2 and 100 microM Ca2+o). 7. The data suggest that Ca2+o produces marked changes in the kinetics and single channel conductance of cation channels, which may account for the facilitatory effect of micromolar concentrations of Ca2+o on the peak amplitude of Icat.

Capsaicin sensitivity is associated with the expression of the vanilloid (capsaicin) receptor (VR1) mRNA in adult rat sensory ganglia.

A vanilloid receptor (VR1) has recently been cloned and shown to be a target for capsaicin, the excitotoxic component of capsicum peppers (Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D. and Julius, D., Nature, 389 (1997) 816-824). The effects of capsaicin appear to be selective for a subset of sensory neurones which includes polymodal nociceptors. The present study describes the distribution of VR1 mRNA, together with measurements of capsaicin sensitivity, in sensory nerve ganglia of different embryological origins and a single sympathetic ganglion, the superior cervical ganglion (SCG). In situ hybridisation revealed the expression of VR1 mRNA in small-to-medium-sized neurones of the dorsal root, trigeminal and vagal ganglia. No hybridisation signal was observed in the SCG neurones. This pattern of expression correlated with capsaicin sensitivity measured by whole-cell voltage clamp where, in similar sized cells, over 80% of neurones from dorsal root and vagal ganglia were capsaicin sensitive, but all SCG neurones were insensitive to capsaicin.

Contrasting expression of KAL in cell-free systems: 5' UTR and coding region structural effects on translation.

We investigated the expression of two different X-linked Kallmann (KAL) gene cDNAs in two different cell-free systems using rabbit reticulocyte lysate: (system A) transcription/translation coupled and (system B) noncoupled. System A yielded a single band of 76 kDa corresponding to anosmin-1, the expected full-length gene product, and upon addition of canine microsomal membranes produced a 85-kDa glycosylated form. System B did not produce any detectable protein band despite the expression of a beta-galactosidase-positive control gene. The first 179 bases of the coding sequence are 74% GC-rich and showed the potential to form imperfect hairpin structures, which in part may explain the translation inhibition of KAL in system B. This has further led us to speculate that coupling transcription to translation may either be preventing translating-inhibiting hairpin formation or be compensating for the lack of certain tissue-specific proteins in reticulocyte lysate that are essential in overcoming inhibitory hairpins during translation. Substitution of the 5'-UTR with an encephalomyocarditis virus internal ribosomal entry site (EMCV IRES) sequence resulted paradoxically in a lower yield of anosmin-1, suggesting that elements in the 5'UTR may be necessary for maintaining a "normal" level of expression. The use of KAL and luciferase reporters (containing different 5'UTRs) demonstrated that the native KAL 5' UTR is not involved in translational efficiency. However, this sequence may influence faithful translation initiation. Theoretical RNA conformation data imply that effective EMCV IRES usage with KAL may require favorable pairing between the IRES and unidentified sequences within the 5' coding region of the gene. This work provides a foundation both for the investigation of KAL regulation and for the characterization of its function.

Will reduced sulphur emissions under the Second Sulphur Protocol lead to recovery of acid sensitive sites in UK?

A conceptual model of the combined effects of acid deposition and land-use, Model of Acidification of Groundwater In Catchments (MAGIC), was applied to 21 upland sites in the UK Acid Waters Monitoring Network (AWMN) to assess the likely future recovery in response to the latest international agreements controlling anthropogenic sulphur emissions throughout Europe. Future estimates of sulphur deposition were generated by the Hull Acid Rain Model (HARM), based on the agreed reductions outlined in the Second Sulphur Protocol. The results indicate only a limited degree of recovery in surface-water chemistry at all sites over the next 50 years; moreover, a continuing decline in soil base status is predicted to occur at 70% of sites, resulting in longer term reacidification of surface-water at 38% of sites. However, compared with a 'business as usual' scenario the recovery is pronounced, although acidified sites will require further reductions in acidic deposition if recovery to pre-industrial chemical conditions are to be achieved. Furthermore, land-use scenarios at afforested sites suggest that replanting of felled forest will lead to a further increase in acidification. This strengthens the argument that plantation forestry should be avoided in areas considered geologically sensitive to acidic deposition.

Modulation of Ca(2+)-activated Cl- currents in rabbit portal vein smooth muscle by an inhibitor of mitochondrial Ca2+ uptake.

1. The effects of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an inhibitor of mitochondrial Ca2+ uptake, was investigated on the properties of Ca(2+)-activated chloride currents (ICl(Ca)) in rabbit portal vein smooth muscle cells using the perforated patch whole-cell voltage-clamp technique to ascertain whether this Ca2+ uptake process influences the time course of the subsarcolemmal Ca2+ signal that activates ICl(Ca). 2. In cells bathed in either physiological calcium (2 mM Cao2+) or high calcium (10 mM Cao2+) external solutions, application of CCCP (1-2 microM) evoked an inward current and prolonged the exponential decay time constant (tau) of Ca(2+)-activated Cl- 'tail' currents (Itail) evoked by Ca2+ influx through voltage-dependent calcium channels (VDCCs). The effect of CCCP on tau was greater in cells where the amplitude of Itail was relatively large and, in different cells, the effect of CCCP on tau was positively correlated with the amplitude of Itail. 3. CCCP abolished spontaneously occurring transient Ca(2+)-activated Cl- currents (STICs), but did not alter their time course before complete block. 4. Thapsigargin and cyclopiazonic acid (inhibitors of the sarcoplasmic Ca(2+)-ATPase) inhibited STICs, but did not affect the decay of Itail or STICs. 5. In conclusion, when Ca2+ enters the cell through VDCCs, the time course of the consequent Ca2+ signal in the subsarcolemmal domain containing Ca(2+)-activated chloride channels appears to be regulated by Ca2+ uptake into mitochondria. In contrast, inhibition of Ca2+ uptake by the sarcoplasmic reticulum ATPase does not seem to influence the time course of ICl(Ca).