Massive peatland carbon banks vulnerable to rising temperatures.

Peatlands contain one-third of the world's soil carbon (C). If destabilized, decomposition of this vast C bank could accelerate climate warming; however, the likelihood of this outcome remains unknown. Here, we examine peatland C stability through five years of whole-ecosystem warming and two years of elevated atmospheric carbon dioxide concentrations (eCO2). Warming exponentially increased methane (CH4) emissions and enhanced CH4 production rates throughout the entire soil profile; although surface CH4 production rates remain much greater than those at depth. Additionally, older deeper C sources played a larger role in decomposition following prolonged warming. Most troubling, decreases in CO2:CH4 ratios in gas production, porewater concentrations, and emissions, indicate that the peatland is becoming more methanogenic with warming. We observed limited evidence of eCO2 effects. Our results suggest that ecosystem responses are largely driven by surface peat, but that the vast C bank at depth in peatlands is responsive to prolonged warming.

Fine-root mortality rates in a temperate forest: estimates using radiocarbon data and numerical modeling.

* We used an inadvertent whole-ecosystem 14C label at a temperate forest in Oak Ridge, Tennessee, USA to develop a model (Radix1.0) of fine-root dynamics. Radix simulates two live-root pools, two dead-root pools, non-normally distributed root mortality turnover times, a stored carbon (C) pool, and seasonal growth and respiration patterns. * We applied Radix to analyze measurements from two root size classes (< 0.5 and 0.5-2.0 mm diameter) and three soil-depth increments (O horizon, 0-15 cm and 30-60 cm). * Predicted live-root turnover times were < 1 yr and approximately 10 yr for short- and long-lived pools, respectively. Dead-root pools had decomposition turnover times of approximately 2 yr and approximately 10 yr. Realistic characterization of C flows through fine roots requires a model with two live fine-root populations, two dead fine-root pools, and root respiration. These are the first fine-root turnover time estimates that take into account respiration, storage, seasonal growth patterns, and non-normal turnover time distributions. * The presence of a root population with decadal turnover times implies a lower amount of belowground net primary production used to grow fine-root tissue than is currently predicted by models with a single annual turnover pool.

Cleavage of DAP5 by coxsackievirus B3 2A protease facilitates viral replication and enhances apoptosis by altering translation of IRES-containing genes.

Cleavage of eukaryotic translation initiation factor 4G (eIF4G) by enterovirus proteases during infection leads to the shutoff of cellular cap-dependent translation, but does not affect the initiation of cap-independent translation of mRNAs containing an internal ribosome entry site (IRES). Death-associated protein 5 (DAP5), a structural homolog of eIF4G, is a translation initiation factor specific for IRES-containing mRNAs. Coxsackievirus B3 (CVB3) is a positive single-stranded RNA virus and a primary causal agent of human myocarditis. Its RNA genome harbors an IRES within the 5'-untranslated region and is translated by a cap-independent, IRES-driven mechanism. Previously, we have shown that DAP5 is cleaved during CVB3 infection. However, the protease responsible for cleavage, cleavage site and effects on the translation of target genes during CVB3 infection have not been investigated. In the present study, we demonstrated that viral protease 2A but not 3C is responsible for DAP5 cleavage, generating 45- and 52-kDa N- (DAP5-N) and C-terminal (DAP5-C) fragments, respectively. By site-directed mutagenesis, we found that DAP5 is cleaved at amino acid G434. Upon cleavage, DAP5-N largely translocated to the nucleus at the later time points of infection, whereas the DAP5-C largely remained in the cytoplasm. Overexpression of these DAP5 truncates demonstrated that DAP5-N retained the capability of initiating IRES-driven translation of apoptosis-associated p53, but not the prosurvival Bcl-2 (B-cell lymphoma 2) when compared with the full-length DAP5. Similarly, DAP5-N expression promoted CVB3 replication and progeny release; on the other hand, DAP5-C exerted a dominant-negative effect on cap-dependent translation. Taken together, viral protease 2A-mediated cleavage of DAP5 results in the production of two truncates that exert differential effects on protein translation of the IRES-containing genes, leading to enhanced host cell death.

Stability of peatland carbon to rising temperatures.

Peatlands contain one-third of soil carbon (C), mostly buried in deep, saturated anoxic zones (catotelm). The response of catotelm C to climate forcing is uncertain, because prior experiments have focused on surface warming. We show that deep peat heating of a 2 m-thick peat column results in an exponential increase in CH4 emissions. However, this response is due solely to surface processes and not degradation of catotelm peat. Incubations show that only the top 20-30 cm of peat from experimental plots have higher CH4 production rates at elevated temperatures. Radiocarbon analyses demonstrate that CH4 and CO2 are produced primarily from decomposition of surface-derived modern photosynthate, not catotelm C. There are no differences in microbial abundances, dissolved organic matter concentrations or degradative enzyme activities among treatments. These results suggest that although surface peat will respond to increasing temperature, the large reservoir of catotelm C is stable under current anoxic conditions.

Effect of pertussis toxin on the inhibition of secretory activity by prostaglandin E2, somatostatin, epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate in parietal cells from rat stomach.

Rat parietal cells were incubated for 2 h with pertussis toxin (100 ng/ml) which ADP-ribosylates and inactivates guanine nucleotide regulatory proteins (G proteins) of the 'Gi-like' family. The effect of this pretreatment on the action of inhibitors of parietal cell acid secretion was investigated by using the accumulation of the weak base aminopyrine as an index of secretory activity. The inhibitory actions of near maximally effective concentrations of prostaglandin E2 (PGE2), somatostatin and epidermal growth factor (EGF) on histamine-stimulated aminopyrine accumulation were reduced by 83%, 72% and 70%, respectively, by preincubation with pertussis toxin. By contrast, the inhibitory action of a near maximally effective concentration of 12-O-tetradecanoylphorbol 13-acetate on histamine-stimulated aminopyrine accumulation was reduced by only 12%. It is concluded that G-proteins are involved in the inhibitory actions of PGE2, somatostatin and EGF on parietal cells. However, since the inhibitory actions of PGE2 and EGF can be distinguished by the blockade of the action of EGF, but not that of PGE2, by 3-isobutyl-1-methylxanthine, it is possible that PGE2 and EGF either activate the same G-protein in different ways or work through different G-proteins.

Nitric oxide synthase and arginase in cells isolated from the rat gastric mucosa.

Nitric oxide (NO) synthase activity, which converts arginine to citrulline and NO, is present in homogenates of rat gastric mucosal cells. The aims of this study were to identify the form of NO synthase expressed in gastric cells isolated from fed rats, and to investigate the metabolism of arginine by suspensions of intact mucosal cells. Antibodies directed against the neuronal form of NO synthase recognised a protein of 160 kDa on immunoblots of extracts of gastric cells, and stained isolated cells of approx. 8 microm in diameter. NO synthase was enriched in a cell fraction which banded at high-density in a Percoll gradient, and was inhibited (IC50) by N(G)-nitro-L-arginine (0.8 microM), N(G)-monomethyl-L-arginine (12.6 microM), L-canavanine (147 microM), trifluoperazine (140 microM) and by phosphorylation involving protein kinase C. Intact gastric cells converted exogenous arginine to ornithine and citrulline. Arginase was present in the cells, and was predominantly responsible for arginine metabolism because formation of ornithine and citrulline was reduced by the arginase inhibitors, N(G)-hydroxy-L-arginine and L-ornithine, but not by NO synthase inhibitors such as N(G)-nitro-L-arginine. In conclusion, NO synthase that resembles the neuronal isoform is present in gastric mucosal cells, but a pathway involving arginase seems to be largely responsible for citrulline formation from exogenous arginine in intact mucosal cells.

Inhibitory effect of 12-O-tetradecanoylphorbol 13-acetate on acid secretion by rat stomach in vivo.

The effect of 12-O-tetradecanoylphorbol 13-acetate (TPA) on acid secretion by rat stomach in vivo has been investigated to establish whether a previously determined inhibitory effect of TPA on aminopyrine accumulation by isolated parietal cells genuinely reflected a reduced acid secretion. Perfusion of 1 microM TPA through the lumen of the stomach had little effect on basal or carbachol-stimulated secretion, but inhibited secretion induced by histamine or pentagastrin. 4 alpha-Phorbol 12,13-didecanoate did not affect pentagastrin-stimulated secretion. It is concluded that TPA exerts a specific inhibitory effect on acid secretion in vivo, which parallels that found on aminopyrine accumulation in isolated parietal cells.

Opposite effects of flurbiprofen and the nitroxybutyl ester of flurbiprofen on apoptosis in cultured guinea-pig gastric mucous cells.

The nitric oxide (NO)-donating nitroxybutyl ester of flurbiprofen (NO-flurbiprofen), shows reduced gastro-intestinal toxicity relative to flurbiprofen. NO may exert either pro- or anti-apoptotic effects, while non-steroidal anti-inflammatory drugs may induce apoptosis. The aim of the present work was therefore to compare the effects of flurbiprofen and NO-flurbiprofen on apoptosis in guinea-pig gastric mucous cells. Apoptotic activity was assessed by assay of caspase activity and from the fragmentation and condensation of nuclei. Incubation with flurbiprofen for 24 h produced a concentration-dependent induction of apoptosis in cells attached to the culture plate (caspase 3-like activity increased by 257% at 500 microM), while NO-flurbiprofen inhibited basal apoptosis (caspase 3-like activity decreased by 71% at 500 microM). Caspase activity and nuclear fragmentation were substantially increased in cells that had spontaneously detached from the culture plate. NO-flurbiprofen inhibited caspase activity (55% at 500 microM) but not nuclear fragmentation in these detached cells. NO flurbiprofen inhibited the activation of apoptosis by 25 microM C(6)-ceramide in cells attached to the culture plate. Inhibition of caspase activity by NO-flurbiprofen was detectable after 6 h of incubation with intact cells, but by contrast with the NO-donor S-nitrosyl-N-acetyl-penicillamine, was not demonstrable with cell homogenates. Activation of caspase 3-like activity by flurbiprofen was slow (>6 h incubation needed) and was inhibited by cycloheximide. The presence of a nitroxybutyl ester moiety on flurbiprofen prevents the pro-apoptotic activity of the parent compound and may contribute to the reduced gastro-intestinal toxicity of NO-flurbiprofen.

Sensitivity of stomatal and canopy conductance to elevated CO2 concentration - interacting variables and perspectives of scale.

• The hydrological response of forests to rising CO2 is a critical biotic feedback in the study of global climate change. Few studies, however, have investigated this highly dynamic response at relevant temporal and spatial scales. • A combination of leaf and whole-tree measurements and stand-level extrapolations were used to assess how stomatal conductance, canopy transpiration and conductance, and evapotranspiration might be affected by future, higher CO2 concentrations. • Midday measurements of stomatal conductance for leaves sampled in a 12-yr-old sweetgum (Liquidambar styraciflua) stand exposed to free-air CO2 enrichment were up to 44% lower at elevated than at ambient CO2 concentrations, whereas canopy conductance, averaged over the growing season, was only 14% lower in stands exposed to CO2 enrichment. The magnitude of this response was dependent on vapor pressure deficit and soil water potential. Annual estimates of evapotranspiration showed relatively small reductions due to atmospheric CO2 enrichment. • These data illustrate that the hydrological response of a closed-canopy plantation to elevated CO2 depends on the temporal and spatial scale of observation. They emphasize the importance of interacting variables and confirm that integration of measurements over space and time reduce what, at the leaf level, might otherwise appear to be a large and significant response.

Interactions between drought and elevated CO2 on growth and gas exchange of seedlings of three deciduous tree species.

Interactions between elevated atmospheric CO2 and drought on growth and gas exchange of American sycamore (Platanus occidentalis L.), sweetgum (Liquidambar styraciflua L.) and sugar maple (Acer saccharum Marsh.) were investigated using 1-yr-old seedlings, planted in 8 1 pots and grown in four open-top chambers, containing either ambient air or ambient air enriched with 300 µmol mol-1 CO2 . Two soil moisture regimes were included within each chamber: a 'well-watered' treatment with plants watered daily and a 'drought' treatment in which plants were subjected to a series of drought cycles. Duration and depth of the drought cycles were determined by soil matric potential. Mean soil water potential at rewatering for the water-stressed seedlings under ambient CO2 for sugar maple, sweetgum and sycamore was -0.5, -0.7 and -1.8 MPa, respectively, compared with > -0.1 MPa for all well-watered plants. Elevated CO2 increased relative growth rate of well-watered sugar maple by 181%, resulting in a 4.3-fold increase in total plant dry weight after 81 d, compared with 1.4 and 1.6-fold increases for sweetgum and sycamore, respectively, after 69 d. Although elevated CO2 increased net CO2 assimilation rate of sugar maple by 115%, there was a 10-fold increase in leaf area which contributed to the growth response. Although drought did not eliminate a growth response of sugar maple to elevated CO2 it greatly reduced the elevated CO2 -induced enhancement of relative growth rate. In contrast, relative growth rates of sweetgum and sycamore were not significantly increased by elevated CO2 . Drought, under elevated CO2 , reduced leaf area of all three species to a greater extent than it reduced net CO2 assimilation rate. The response ranged from no effect in sugar maple to a 40 % reduction in sycamore, with sweetgum exhibiting an intermediate response. Results indicate that drought may alter the growth response, gas exchange and water relations of tree species growing in an elevated CO2 atmosphere. Under high nutrient and water availability, sugar maple may benefit the most (of the three species studied) from a CO2 - enriched atmosphere, but productivity gains will be limited if frequent drought is prevalent.

Inhibition by Ro 31-8220 of acid secretory activity induced by carbachol indicates a stimulatory role for protein kinase C in the action of muscarinic agonists on isolated rat parietal cells.

The bisindolylmaleimide Ro 31-8220 is a selective inhibitor of protein kinase C. This compound was used to investigate the involvement of protein kinase C in the stimulation of gastric acid secretion by the muscarinic cholinergic receptor on rat isolated parietal cells. The accumulation of the weak base aminopyrine by both crude and enriched preparations of parietal cells was used as an index of secretory activity. Ro 31-8220 antagonized (IC50, 1.0 microM) the effect of the activator of protein kinase C,12-O-tetradecanoylphorbol 13-acetate (TPA), on histamine-stimulated aminopyrine accumulation. Ro 31-8220 (0.1-2.14 microM) inhibited the aminopyrine response to 0.1 mM carbachol (IC50, 0.78 microM; 49% inhibition at 2.14 microM Ro 31-8220) and shifted the dose-response curve for the effect of carbachol concentration of aminopyrine accumulation downwards and to the right. No inhibition of aminopyrine accumulation induced by histamine was found with Ro 31-8220 (0.1-2.14 microM). In a preparation containing > 80% parietal cells incubated with 0.1 mM carbachol, 2.14 microM Ro 31-8220 inhibited aminopyrine accumulation by 43%, but had no effect on the increase in intracellular Ca2+ which was measured by using the fluorescent probe FURA-2. In conclusion, Ro 31-8220 (0.1-2.14 microM) produced a selective reduction in secretory activity in parietal cells by inhibition of protein kinase C. The predominant role of protein kinase C in parietal cells activated with carbachol is not to cause feedback inhibition of the response but to facilitate stimulation of secretory activity.

Induction of heat shock protein 72 by a nitric oxide donor in guinea-pig gastric mucosal cells.

Gastric mucosal cells may be exposed to exogenous nitric oxide (NO) from a variety of sources. The response of primary cultures of guinea-pig gastric mucosal cells to the NO donor S-nitroso-N-acetyl-penicillamine was therefore investigated. Exposure to S-nitroso-N-acetyl-penicillamine for 8 h caused a concentration-dependent induction of heat shock protein 72 (HSP 72). Induction was inhibited by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and by blockade of transcription with actinomycin D. Induction of HSP 72 by S-nitroso-N-acetyl-penicillamine was enhanced by diethyl maleate which decreased the intracellular reduced thiol content. By contrast, HSP 72 formation after heat shock was associated with an elevation of reduced thiol. Incubation with S-nitroso-N-acetyl-penicillamine for 18 h increased detachment of cells from the culture plate. The effect of S-nitroso-N-acetyl-penicillamine on detachment was exacerbated by the presence of actinomycin D. In conclusion, exogenous NO induces HSP 72 in guinea-pig gastric mucosal cells and this response may in part protect the cells from the deleterious effects of NO.

Stimulation by carbachol of mucus gel thickness in rat stomach involves nitric oxide.

Instillation of carbachol (150 micrograms/kg) into the gastric lumen in vivo increased the thickness of the mucus gel layer. Intravenous administration of the inhibitor of nitric oxide (NO) synthase, NG-nitro-L- arginine methyl ester (L-NAME, 0.4-5 mg/kg) dose-dependently reduced the stimulation by carbachol, the half-maximal inhibitory dose being 0.57 mg/kg. This effect of L-NAME was abolished by administration of L-arginine but not by D-arginine (100 mg/kg i.v.). By contrast L-NAME (5 mg/kg) did not reduce the stimulatory effect of intraluminal 16,16-dimethyl prostaglandin E2 (50 micrograms/kg) on mucus gel thickness. These results implicate NO in the cholinergic activation of gastric mucus secretion.

Nitric oxide donors increase mucus gel thickness in rat stomach.

Instillation of the nitric oxide (NO) generator isosorbide dinitrate (0.1-1 mM) into the rat gastric lumen in vivo produced a dose-related increase in mucus gel thickness that was prevented by coadministration of oxyhaemoglobin (10 microM). Isosorbide dinitrate did not induce epithelial cell damage. S-Nitroso-N-acetyl-penicillamine (0.3 mM) and dibutyryl cyclic GMP (1 mM) also increased mucus thickness. These findings, along with the presence of NO synthase in the gastric mucosa, imply a role for NO in vivo in mediation of gastric mucus release.

Lipopolysaccharide induces Ca(2+)-independent nitric oxide synthase activity in rat gastric mucosal cells.

Ca(2+)-independent nitric oxide synthase was detected in gastric mucosal cells isolated from rats injected 4 h previously with Escherichia coli lipopolysaccharide (3 mg/kg i.v.). Induced nitric oxide synthase was located in an elutriated cell fraction of intermediate size which contained epithelial cells, but was absent from the parietal cell fraction. Administration of dexamethasone (2 mg/kg i.p.) 1 h before lipopolysaccharide inhibited the appearance of Ca(2+)-independent nitric oxide synthase, and prevented the observed reduction in cell viability (trypan blue exclusion). Ca(2+)-independent nitric oxide synthase activity can thus be induced in certain cells of the gastric mucosa, and may contribute to gastric pathologies where there is activation of the immune system.

Effect of nitric oxide on apoptotic activity in the rat gastrointestinal tract.

The effect of nitric oxide (NO) on apoptosis in the gastrointestinal mucosa was investigated. Experiments involved long-term exposure of rat gastric mucosal cells in vitro to exogenous NO delivered from the NO, donor S-nitroso-N-acetyl-penicillamine, and the effect of intravenous administration of lipopolysaccharide in vivo, in the presence and absence of the selective inhibitor of inducible NO synthase N-(3-(aminomethyl)benzyl) acetamidine (1400 W). S-nitroso-N-acetyl-penicillamine produced a dose-related inhibition of caspase 3-like activity and DNA fragmentation in isolated gastric mucosal cells. Caspase 3-like activity and DNA fragmentation in gastric, ileal and colonic mucosa were increased both 5 and 24 h after injection of lipopolysaccharide (3 mg/kg, i.v.) to rats in vivo. Administration of 1400 W (5 mg/kg, i.v.) immediately after lipopolysaccharide enhanced caspase 3-like activity and DNA fragmentation above that found with lipopolysaccharide alone. In conclusion, data obtained both in vitro and in vivo suggest that NO exerts an anti-apoptotic effect on rat gastrointestinal mucosal cells.

Enteroviruses, endoscopy and infection control: an applied study.

Decontamination methods for medical equipment are based largely on bacterial studies yet enteroviruses are more resistant to disinfection than most vegetative bacteria and other viruses. To study the elimination of enteroviruses from endoscopes, poliovirus was aspirated into the suction-biopsy channels of five gastroscopes. Endoscopes were cleaned in detergent and disinfected in 2% alkaline glutaraldehyde. Contamination was measured before and after cleaning and after various periods of disinfection by irrigating the channels with viral medium and quantifying surviving virus by plaque assay. The effectiveness of glutaraldehyde against cell-free and cell-associated polio virus, dried to a surface in a protein coagulum, was also studied. Cleaning reduced virus by a mean of 4.6 log10 plaque forming units (pfu) ml-1. Samples were virus-free after 2 min disinfection. Virus dried on surfaces was inactivated in 1 min by 2% and 1% glutaraldehyde, with a reduction of > 6 log10 pfu ml-1. Thus, cleaning was effective against heavy viral contamination while glutaraldehyde rapidly inactivated poliovirus even when dried to a surface in serum.

Viral transmission and fibreoptic endoscopy.

Fibreoptic endoscopes have been responsible for outbreaks of infection with bacteria although viral transmission has been reported only once. The emergence of human immunodeficiency virus (HIV) has prompted a review of infection control practices in endoscopy units because of the theoretical possibility that HIV might be transmitted at endoscopy. Recent studies have shown that bronchoscopes and gastroscopes used on AIDS patients become contaminated with HIV genetic material although cleaning equipment in detergent removes all traces of the virus. Thorough precleaning has been shown to eliminate even high titres of HIV from endoscopes and 2% alkaline glutaraldehyde has been found to inactivate the virus rapidly even if the virus is dried in serum to a surface. These findings support the British Society of Gastroenterology recommendations for the cleaning and disinfection of endoscopic equipment and demonstrate that a uniform policy of infection control is practicable in endoscopy units.

A study of glutaraldehyde disinfection of fibreoptic bronchoscopes experimentally contaminated with Mycobacterium tuberculosis.

Mycobacteria are difficult to inactivate, and concern about the spread of tuberculosis at bronchoscopy has a major influence on infection control practices. Recommendations from the UK Department of Health are based largely on in-vitro mycobactericidal assays which do not take into account the particular conditions encountered in endoscopy units. In this applied study cleaning and disinfection methods were examined using five bronchoscopes that were heavily contaminated with a recent isolate of Mycobacterium tuberculosis in sputum. Cleaning reduced contamination by a mean 3.5 log(10) colony forming units (cfu) per ml; all bronchoscopes were free of detectable mycobacteria after 10 min in 2% alkaline glutaraldehyde (AG). It is recommended that all bronchoscopes be thoroughly pre-cleaned and disinfected in 2% AG for 20 min as part of a uniform policy of infection control.

The rise of Clostridium difficile: the effect of length of stay, patient age and antibiotic use.

Hospitals in the UK have recently seen a marked increase in C. difficile for reasons which are unclear. Reduced standards of hygiene, increasingly elderly patients, greater cephalosporin use and longer hospital stay have been suggested. We retrospectively studied all cases of C. difficile diarrhoea at Princess Margaret Hospital, Swindon, over two years. Cephalosporins, patient age and LOS appeared unrelated to the rise in C. difficile; penicillins and macrolides were related. Our policy of using amoxycillin and clarithromycin for community-acquired pneumonia coincided with this study and may explain the observed rise in C. difficile.