Structure-Function Relations in the PI-PLC/GDPD Enzyme Superfamily

Phosphatidylinositol-specific phospholipases C (PI-PLC) are known to participate in many eukaryotic signal transduction pathways and act as virulence factors in lower organisms. Glycerophosphoryl diester phosphodiesterase (GDPD) enzymes are involved in phosphate homeostasis and phospholipid catabolism for energy production. Streptomyces antibioticus phosphatidylinositol-specific phospholipase C (SaPLC1) is a 38 kDa enzyme that displays characteristics of both enzyme superfamilies, representing an evolutionary link between these divergent enzyme classes. SaPLC1 also boasts a unique catalytic mechanism that involves a trans 1,6-cyclic inositol phosphate intermediate instead of the typical cis 1,2-cyclic inositol phosphate. The mechanism by which this occurs is still unclear. To attack this problem, we established a wide mutagenesis scan of the active site and measured activities of alanine mutants. A chemical rescue assay was developed to verify that the activity loss was due to the removal of the functional role of the mutated residue. 31P-NMR was employed in characterizing and quantifying intermediates in mutants that slowed the reaction sufficiently. We found that the H37A and H76A mutations support the hypothesis that these structurally conserved residues are also conserved in terms of their catalytic roles. H37 was found to be the general base (GB), while H76 plays the role of general acid (GA). K131 was identified as a semi-conserved key positive charge donor found at the entrance of the active site. By elucidating the SaPLC1 mechanism in relation to its active site architecture, we have increased our understanding of the structure-function relations that support catalysis in the PI-PLC/GDPD superfamily. These findings provide groundwork for in vivo studies of SaPLC1 function and its possible role in novel signaling or metabolism in Streptomyces.

Measurement of W+W- production in pp collisions at √s=7 TeV with the ATLAS detector and limits on anomalous WWZ and WWγ couplings

This paper presents a measurement of the W+W- production cross section in pp collisions at root s = 7 TeV. The leptonic decay channels are analyzed using data corresponding to an integrated luminosity of 4: 6 fb(-1) collected with the ATLAS detector at the Large Hadron Collider. The W+W- production cross section sigma(pp -> W+W- + X) is measured to be 51.9 +/- 2.0(stat) +/- 3.9(syst) +/- 2.0(lumi) pb, compatible with the Standard Model prediction of 44.7(-1.9)(+2.1) pb. A measurement of the normalized fiducial cross section as a function of the leading lepton transverse momentum is also presented. The reconstructed transverse momentum distribution of the leading lepton is used to extract limits on anomalous WWZ and WW gamma couplings.

Effects of prolonged endotoxemia on liver, skeletal muscle and kidney mitochondrial function

INTRODUCTION: Sepsis may impair mitochondrial utilization of oxygen. Since hepatic dysfunction is a hallmark of sepsis, we hypothesized that the liver is more susceptible to mitochondrial dysfunction than the peripheral tissues, such as the skeletal muscle. We studied the effect of prolonged endotoxin infusion on liver, muscle and kidney mitochondrial respiration and on hepatosplanchnic oxygen transport and microcirculation in pigs. METHODS: 20 anesthetized pigs were randomized to receive endotoxin or saline infusion for 24 hours. Muscle, liver and kidney mitochondrial respiration was assessed. Cardiac output (thermodilution), carotid, superior mesenteric and kidney arterial, portal venous (ultrasound Doppler) and microcirculatory blood flow (laser Doppler) were measured, and systemic and regional oxygen transport and lactate exchange were calculated. RESULTS: Endotoxin infusion induced hyperdynamic shock and impaired the glutamate- and succinate-dependent mitochondrial respiratory control ratio (RCR) in the liver (glutamate: endotoxemia: median [range] 2.8 [2.3-3.8] vs. controls: 5.3 [3.8-7.0]; p<0.001; succinate: endotoxemia: 2.9 [1.9-4.3] vs. controls: 3.9 [2.6-6.3] p=0.003). While the ADP:O ratio was reduced with both substrates, maximal ATP production was impaired only in the succinate-dependent respiration. Hepatic oxygen consumption and extraction, and liver surface laser Doppler blood flow remained unchanged. Glutamate-dependent respiration in the muscle and kidney was unaffected. CONCLUSIONS: Endotoxemia reduces the efficiency of hepatic but neither skeletal muscle nor kidney mitochondrial respiration, independent of regional and microcirculatory blood flow changes.

Function of liver activation-regulated chemokine/CC chemokine ligand 20 is differently affected by cathepsin B and cathepsin D processing

Chemokine processing by proteases is emerging as an important regulatory mechanism of leukocyte functions and possibly also of cancer progression. We screened a large panel of chemokines for degradation by cathepsins B and D, two proteases involved in tumor progression. Among the few substrates processed by both proteases, we focused on CCL20, the unique chemokine ligand of CCR6 that is expressed on immature dendritic cells and subtypes of memory lymphocytes. Analysis of the cleavage sites demonstrate that cathepsin B specifically cleaves off four C-terminally located amino acids and generates a CCL20(1-66) isoform with full functional activity. By contrast, cathepsin D totally inactivates the chemotactic potency of CCL20 by generating CCL20(1-55), CCL20(1-52), and a 12-aa C-terminal peptide CCL20(59-70). Proteolytic cleavage of CCL20 occurs also with chemokine bound to glycosaminoglycans. In addition, we characterized human melanoma cells as a novel CCL20 source and as cathepsin producers. CCL20 production was up-regulated by IL-1alpha and TNF-alpha in all cell lines tested, and in human metastatic melanoma cells. Whereas cathepsin D is secreted in the extracellular milieu, cathepsin B activity is confined to cytosol and cellular membranes. Our studies suggest that CCL20 processing in the extracellular environment of melanoma cells is exclusively mediated by cathepsin D. Thus, we propose a model where cathepsin D inactivates CCL20 and possibly prevents the establishment of an effective antitumoral immune response in melanomas.

Coronary collateral function long after drug-eluting stent implantation

OBJECTIVES: This study was designed to compare coronary collateral function in patients after bare-metal stent (BMS) or drug-eluting stent (DES) implantation. BACKGROUND: Drug-eluting stents have an inhibitory effect on the production of cytokines, chemotactic proteins, and growth factors, and may therefore negatively affect coronary collateral growth. METHODS: A total of 120 patients with long-term stable coronary artery disease (CAD) after stent implantation were included. Both the BMS group and the DES group comprised 60 patients matched for in-stent stenosis severity of the vessel undergoing collateral flow index (CFI) measurement at follow-up and for the duration of follow-up. The primary end point of the investigation was invasively determined coronary collateral function 6 months after stent implantation. Collateral function was assessed by simultaneous aortic, coronary wedge, and central venous pressure measurements (yielding CFI) and by intracoronary electrocardiogram during balloon occlusion. RESULTS: There were no differences between the groups regarding age, gender, body mass index, frequency of cardiovascular risk factors, use of cardiovascular drugs, severity of CAD, or site of coronary artery stenoses. Despite equal in-stent stenosis severity (46 +/- 34% and 45 +/- 36%) and equal follow-up duration (6.2 +/- 10 months and 6.5 +/- 5.4 months), CFI was diminished in the DES versus BMS group (0.154 +/- 0.097 vs. 0.224 +/- 0.142; p = 0.0049), and the rate of collaterals insufficient to prevent ischemia during occlusion (intracoronary electrocardiographic ST-segment elevation > or =0.1 mV) was higher with 50 of 60 patients in the DES group and 33 of 60 patients in the BMS group (p = 0.001). CONCLUSIONS: Collateral function long after coronary stenting is impaired with DES (sirolimus and paclitaxel) when compared with BMS. Considering the protective nature of collateral vessels, this could lead to more serious cardiac events in the presence of an abrupt coronary occlusion.

C-reactive protein production in term human placental tissue

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Planting and production of switchgrass (Panicum virgatum L.) as a bioenergy crop in Michigan's Upper Peninsula

Switchgrass (Panicum virgatum L.) is a perennial grass holding great promise as a biofuel resource. While Michigan’s Upper Peninsula has an appropriate land base and climatic conditions, there is little research exploring the possibilities of switchgrass production. The overall objectives of this research were to investigate switchgrass establishment in the northern edge of its distribution through: investigating the effects of competition on the germination and establishment of switchgrass through the developmental and competitive characteristics of Cave-in-Rock switchgrass and large crabgrass (Digitaria sanguinalis L.) in Michigan’s Upper Peninsula; and, determining the optimum planting depths and timing for switchgrass in Michigan’s Upper Peninsula. For the competition study, a randomized complete block design was installed June 2009 at two locations in Michigan’s Upper Peninsula. Four treatments (0, 1, 4, and 8 plants/m2) of crabgrass were planted with one switchgrass plant. There was a significant difference between switchgrass biomass produced in year one, as a function of crabgrass weed pressure. There was no significant difference between the switchgrass biomass produced in year two versus previous crabgrass weed pressure. There is a significant difference between switchgrass biomass produced in year one and two. For the depth and timing study, a completely randomized design was installed at two locations in Michigan’s Upper Peninsula on seven planting dates (three fall 2009, and four spring 2010); 25 seeds were planted 2 cm apart along 0.5 m rows at depths of: 0.6 cm, 1.3 cm, and 1.9 cm. Emergence and biomass yields were compared by planting date, and depths. A greenhouse seeding experiment was established using the same planting depths and parameters as the field study. The number of seedlings was tallied daily for 30 days. There was a significant difference in survivorship between the fall and spring planting dates, with the spring being more successful. Of the four spring planting dates, there was a significant difference between May and June in emergence and biomass yield. June planting dates had the most percent emergence and total survivorship. There is no significant difference between planting switchgrass at depths of 0.6 cm, 1.3 cm, and 1.9 cm. In conclusion, switchgrass showed no signs of a legacy effect of competition from year one, on biomass production. Overall, an antagonistic effect on switchgrass biomass yield during the establishment period has been observed as a result of increasing competing weed pressure. When planting switchgrass in Michigan’s Upper Peninsula, it should be done in the spring, within the first two weeks of June, at any depth ranging from 0.6 cm to 1.9 cm.

The immune geography of IgA induction and function

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.

Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Three-year cost analysis of function-centred versus pain-centred inpatient rehabilitation in patients with chronic non-specific low back pain

OBJECTIVE: To compare costs of function- and pain-centred inpatient treatment in patients with chronic low back pain over 3 years of follow-up. DESIGN: Cost analysis of a randomized controlled trial. PATIENTS: A total of 174 patients with chronic low back pain were randomized to function- or pain-centred inpatient treatment. METHODS: Data on direct and indirect costs were gathered by questionnaires sent to patients, health insurance providers, employers, and the Swiss Disability Insurance Company. RESULTS: There was a non-significant difference in total medical costs after 3 years' follow-up. Total costs were 77,305 Euros in the function-centred inpatient treatment group and 83,085 Euros in the pain-centred inpatient treatment group. Likewise, indirect costs after 3 years from lost work days were non-significantly lower in the function-centred in-patient treatment group (6354 Euros; 95% confidence interval -20,892, 8392) and direct medical costs were non-significantly higher in the function-centred inpatient treatment group (574 Euros; 95% confidence interval -862, 2011). CONCLUSION: The total costs of function-centred and pain-centred inpatient treatment were similar over the whole 3-year follow-up.

Direct and indirect associations between plant species richness and productivity in grasslands: regional differences preclude simple generalization of productivity-biodiversity relationships

Plant species richness of permanent grasslands has often been found to be significantly associated with productivity. Concentrations of nutrients in biomass can give further insight into these productivity- plant species richness relationships, e.g. by reflecting land use or soil characteristics. However, the consistency of such relationships across different regions has rarely been taken into account, which might significantly compromise our potential for generalization. We recorded plant species richness and measured above-ground biomass and concentrations of nutrients in biomass in 295 grasslands in three regions in Germany that differ in soil and climatic conditions. Structural equation modelling revealed that nutrient concentrations were mostly indirectly associated with plant species richness via biomass production. However, negative associations between the concentrations of different nutrients and biomass and plant species richness differed considerably among regions. While in two regions, more than 40% of the variation in plant species richness could be attributed to variation in biomass, K, P, and to some degree also N concentrations, in the third region only 15% of the variation could be explained in this way. Generally, highest plant species richness was recorded in grasslands where N and P were co-limiting plant growth, in contrast to N or K (co-) limitation. But again, this pattern was not recorded in the third region. While for two regions land-use intensity and especially the application of fertilizers are suggested to be the main drivers causing the observed negative associations with productivity, in the third region the little variance accounted for, low species richness and weak relationships implied that former intensive grassland management, ongoing mineralization of peat and fluctuating water levels in fen grasslands have overruled effects of current land-use intensity and productivity. Finally, we conclude that regional replication is of major importance for studies seeking general insights into productivity-diversity relationships.

Thermal 2-loop master spectral function at finite momentum

When considering NLO corrections to thermal particle production in the “relativistic” regime, in which the invariant mass squared of the produced particle is K2 ~ (πT)2, then the production rate can be expressed as a sum of a few universal “master” spectral functions. Taking the most complicated 2-loop master as an example, a general strategy for obtaining a convergent 2-dimensional integral representation is suggested. The analysis applies both to bosonic and fermionic statistics, and shows that for this master the non-relativistic approximation is only accurate for K2 ~(8πT)2, whereas the zero-momentum approximation works surprisingly well. Once the simpler masters have been similarly resolved, NLO results for quantities such as the right-handed neutrino production rate from a Standard Model plasma or the dilepton production rate from a QCD plasma can be assembled for K2 ~ (πT)2.

Cosmogenic production rates and recoil loss effects in micrometeorites and interplanetary dust particles

We present a purely physical model to determine cosmogenic production rates for noble gases and radionuclides in micrometeorites (MMs) and interplanetary dust particles (IDPs) by solar cosmic-rays (SCR) and galactic cosmic-rays (GCR) fully considering recoil loss effects. Our model is based on various nuclear model codes to calculate recoil cross sections, recoil ranges, and finally the percentages of the cosmogenic nuclides that are lost as a function of grain size, chemical composition of the grain, and the spectral distribution of the projectiles. The main advantage of our new model compared with earlier approaches is that we consider the entire SCR particle spectrum up to 240 MeV and not only single energy points. Recoil losses for GCR-produced nuclides are assumed to be equal to recoil losses for SCR-produced nuclides. Combining the model predictions with Poynting-Robertson orbital lifetimes, we calculate cosmic-ray exposure ages for recently studied MMs, cosmic spherules, and IDPs. The ages for MMs and the cosmic-spherule are in the range <2.2–233 Ma, which corresponds, according to the Poynting-Robertson drag, to orbital distances in the range 4.0–34 AU. For two IDPs, we determine exposure ages of longer than 900 Ma, which corresponds to orbital distances larger than 150 AU. The orbital distance in the range 4–6 AU for one MM and the cosmic spherule indicate an origin either in the asteroid belt or release from comets coming either from the Kuiper Belt or the Oort Cloud. Three of the studied MMs have orbital distances in the range 23–34 AU, clearly indicating a cometary origin, either from short-period comets from the Kuiper Belt or from the Oort Cloud. The two IDPs have orbital distances of more than 150 AU, indicating an origin from Oort Cloud comets.

Calibration of cosmogenic noble gas production in ordinary chondrites based on 36 Cl- 36 Ar ages. Part 1: Refined produced rates for cosmogenic 21 Ne and 38 Ar

We measured the concentrations and isotopic compositions of He, Ne, and Ar in bulk samples and metal separates of 14 ordinary chondrite falls with long exposure ages and high metamorphic grades. In addition, we measured concentrations of the cosmogenic radionuclides 10Be, 26Al, and 36Cl in metal separates and in the nonmagnetic fractions of the selected meteorites. Using cosmogenic 36Cl and 36Ar measured in the metal separates, we determined 36Cl-36Ar cosmic-ray exposure (CRE) ages, which are shielding-independent and therefore particularly reliable. Using the cosmogenic noble gases and radionuclides, we are able to decipher the CRE history for the studied objects. Based on the correlation 3He/21Ne versus 22Ne/21Ne, we demonstrate that, among the meteorites studied, only one suffered significant diffusive losses (about 35%). The data confirm that the linear correlation 3He/21Ne versus 22Ne/21Ne breaks down at high shielding. Using 36Cl-36Ar exposure ages and measured noble gas concentrations, we determine 21Ne and 38Ar production rates as a function of 22Ne/21Ne. The new data agree with recent model calculations for the relationship between 21Ne and 38Ar production rates and the 22Ne/21Ne ratio, which does not always provide unique shielding information. Based on the model calculations, we determine a new correlation line for 21Ne and 38Ar production rates as a function of the shielding indicator 22Ne/21Ne for H, L, and LL chondrites with preatmospheric radii less than about 65 cm. We also calculated the 10Be/21Ne and 26Al/21Ne production rate ratios for the investigated samples, which show good agreement with recent model calculations.

Measurements of Production Properties of K0S mesons and Lambda hyperons in Proton-Carbon Interactions at 31 GeV/c

Spectra of K0S mesons and Λ hyperons were measured in p+C interactions at 31 GeV/c with the large acceptance NA61/SHINE spectrometer at the CERN SPS. The data were collected with an isotropic graphite target with a thickness of 4% of a nuclear interaction length. Interaction cross sections, charged pion spectra, and charged kaon spectra were previously measured using the same data set. Results on K0S and Λ production in p+C interactions serve as reference for the understanding of the enhancement of strangeness production in nucleus-nucleus collisions. Moreover, they provide important input for the improvement of neutrino flux predictions for the T2K long baseline neutrino oscillation experiment in Japan. Inclusive production cross sections for K0S and Λ are presented as a function of laboratory momentum in intervals of the laboratory polar angle covering the range from 0 up to 240 mrad. The results are compared with predictions of several hadron production models. The K0S mean multiplicity in production processes and the inclusive cross section for K0S production were measured and amount to 0.127 ± 0.005 (stat) ± 0.022 (sys) and 29.0 ± 1.6 (stat) ± 5.0 (sys) mb, respectively.