Proteomic analysis of the site specificity of glycation and carboxymethylation of ribonuclease

Proteomic analysis using electrospray liquid chromatography-mass spectrometry (ESI-LC-MS) has been used to compare the sites of glycation (Amadori adduct formation) and carboxymethylation of RNase and to assess the role of the Amadori adduct in the formation of the advanced glycation end-product (AGE), N-is an element of-(carboxymethyl)lysine (CIVIL). RNase (13.7 mg/mL, 1 mM) was incubated with glucose (0.4 M) at 37 degreesC for 14 days in phosphate buffer (0.2 M, pH 7.4) under air. On the basis of ESI-LC-MS of tryptic peptides, the major sites of glycation of RNase were, in order, K41, K7, K1, and K37. Three of these, in order, K41, K7, and K37 were also the major sites of CIVIL formation. In other experiments, RNase was incubated under anaerobic conditions (1 mM DTPA, N-2 purged) to form Amadori-modified protein, which was then incubated under aerobic conditions to allow AGE formation. Again, the major sites of glycation were, in order, K41, K7, K1, and K37 and the major sites of carboxymethylation were K41, K7, and K37. RNase was also incubated with 1-5 mM glyoxal, substantially more than is formed by autoxidation of glucose under experimental conditions, but there was only trace modification of lysine residues, primarily at K41. We conclude the following: (1) that the primary route to formation of CIVIL is by autoxidation of Amadori adducts on protein, rather than by glyoxal generated on autoxidation of glucose; and (2) that carboxymethylation, like glycation, is a site-specific modification of protein affected by neighboring amino acids and bound ligands, such as phosphate or phosphorylated compounds. Even when the overall extent of protein modification is low, localization of a high proportion of the modifications at a few reactive sites might have important implications for understanding losses in protein functionality in aging and diabetes and also for the design of AGE inhibitors.

Endocannabinoid receptor antagonists: potential for obesity treatment

Obesity has been described as a global epidemic. Its increasing prevalence is matched by growing costs, not only to the health of the individual, but also to the medical services required to treat a range of obesity-related diseases. In most instances, obesity is a product of progressively less energetic lifestyles and the over-consumption of readily available, palatable, and highly caloric foods. Past decades have seen massive investment in the search for effective anti-obesity therapies, so far with limited success. An important part of the process of developing new pharmacologic treatments for obesity lies in improving our understanding of the psychologic and physiologic processes that govern appetite and bodyweight regulation. Recent discoveries concerning the endogenous cannabinoids are beginning to give greater insight into these processes. Current research indicates that endocannabinoids may be key to the appetitive and consummatory aspects of eating motivation, possibly mediating the craving for and enjoyment of the most desired, most fattening foods. Additionally, endocannabinoids appear to modulate central and peripheral processes associated with fat and glucose metabolism. Selective cannabinoid receptor antagonists have been shown to suppress the motivation to eat, and preferentially reduce the consumption of palatable, energy-dense foods. Additionally, these agents act to reduce adiposity through metabolic mechanisms that are independent of changes in food intake. Given the current state of evidence, we conclude that the endocannabinoids represent an exciting target for new anti-obesity therapies.

Low density lipoprotein undergoes oxidation within lysosomes in cells

The oxidised low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidised LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidised intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalised rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. The intracellular levels of oxysterols, measured by HPLC, increased during the chase incubation period, showing that LDL must have been oxidised inside the cells. Furthermore, we found that this oxidative modification was inhibited by lipid-soluble antioxidants, an iron chelator taken up by fluid-phase pinocytosis and the lysosomotropic drug chloroquine, which increases the pH of lysosomes. The results indicate that LDL oxidation can occur intracellularly, most probably within lysosomes.

Alternative methodologies - stretching the in vitro box

Current gas-based in vitro evaluation systems are extremely powerful research techniques. However they have the potential to generate a great deal more than simple fermentation dynamics. Details from four experiments are presented in which adaptation, and novel application, of an in vitro system allowed widely differing objectives to be examined. In the first two studies, complement methodologies were utilised. In such assays, an activity or outcome is inferred through the occurrence of a secondary event rather than by direct observation. Using an N-deficient incubation medium, the increase in starch fermentation, when supplemented with individual amino acids (i.e., known level of N) relative to that of urea (i.e., known quantity and N availability), provided an estimate of their microbial utilisation. Due to the low level of response observed with some arnino acids (notably methionine and lysine), it was concluded, that they may not need to be offered in a rumen-inert form to escape rumen microbial degradation. In another experiment, the extent to which degradation of plant cell wall components was inhibited by lipid supplementation was evaluated using fermentation gas release profiles of washed hay. The different responses due to lipid source and level of inclusion suggested that the degree of rumen protection required to ameliorate this depression was supplement dependent. That in vitro inocula differ in their microbial composition is of little interest per se, as long as the outcome is the same (i.e., that similar substrates are degraded at comparable rates and end-product release is equivalent). However where a microbial population is deficient in a particular activity, increasing the level of inoculation will have no benefit. Estimates of hydrolytic activity were obtained by examining fermentation kinetics of specific substrates. A number of studies identified a fundamental difference between rumen fluid and faecal inocula, with the latter having a lower fibrolytic activity, which could not be completely attributed to microbial numbers. The majority of forage maize is offered as an ensiled feed, however most of the information on which decisions such as choice of variety, crop management and harvesting date are made is based on fresh crop measurements. As such, an attempt was made to estimate ensiled maize quality from an in vitro analysis of the fresh crop. Fermentation profiles and chemical analysis confirmed changes in crop composition over the growing season, and loss of labile carbohydrates during ensiling. In addition, examination of degradation residues allowed metabolizable energy (ME) contents to be estimated. Due to difficulties associated with starch analysis, the observation that this parameter could be predicted by difference (together with an assumed degradability), allowed an estimate of ensiled maize ME to be developed from fresh material. In addition, the contribution of the main carbohydrates towards ME showed the importance of delaying harvest until maximum starch content has been achieved. (c) 2005 Elsevier B.V. All rights reserved.

Low density lipoprotein undergoes oxidation within lysosomes in cells

The oxidized low density lipoprotein (LDL) hypothesis of atherosclerosis proposes that LDL undergoes oxidation in the interstitial fluid of the arterial wall. We have shown that aggregated (vortexed) nonoxidized LDL was taken up by J774 mouse macrophages and human monocyte-derived macrophages and oxidized intracellularly, as assessed by the microscopic detection of ceroid, an advanced lipid oxidation product. Confocal microscopy showed that the ceroid was located in the lysosomes. To confirm these findings, J774 macrophages were incubated with acetylated LDL, which is internalized rapidly to lysosomes, and then incubated (chase incubation) in the absence of any LDL. The intracellular levels of oxysterols, measured by HPLC, increased during the chase incubation period, showing that LDL must have been oxidized inside the cells. Furthermore, we found that this oxidative modification was inhibited by lipid-soluble antioxidants, an iron chelator taken up by fluid-phase pinocytosis and the lysosomotropic drug chloroquine, which increases the pH of lysosomes. The results indicate that LDL oxidation can occur intracellularly, most probably within lysosomes.

An investigation of the germylene addition reaction, GeH2+C2H2: Time-resolved gas-phase kinetic studies and quantum chemical calculations of the reaction energy surface

Time resolved studies of germylene, GeH2, generated by laser flash photolysis of 3,4-dimethylgermacyclopentene-3, have been carried out to obtain rate constants for its bimolecular reaction with acetylene, C2H2. The reaction was studied in the gas-phase over the pressure range 1-100 Tort, with SF6 as bath gas, at 5 temperatures in the range 297-553 K. The reaction showed a very slight pressure dependence at higher temperatures. The high pressure rate constants (obtained by extrapolation at the three higher temperatures) gave the Arrhenius equation: log(k(infinity)/cm(3) molecule(-1) s(-1)) (-10.94 +/- 0.05) + (6.10 +/- 0.36 kJ mol(-1))/RTln10. These Arrhenius parameters are consistent with a fast reaction occurring at approximately 30% of the collision rate at 298 K. Quantum chemical calculations (both DFT and ab initio G2//B3LYP and G2//QCISD) of the GeC2H4 potential energy surface (PES), show that GeH2 + C2H2 react initially to form germirene which can isomerise to vinylgermylene with a relatively low barrier. RRKM modelling, based on a loose association transition state, but assuming vinylgermylene is the end product (used in combination with a weak collisional deactivation model) predicts a strong pressure dependence using the calculated energies, in conflict with the experimental evidence. The detailed GeC2H4 PES shows considerable complexity with ten other accessible stable minima (B3LYP level), the three most stable of which are all germylenes. Routes through this complex surface were examined in detail. The only product combination which appears capable of satisfying the (P-3) + C2H4.C2H4 was confirmed as a product by GC observed lack of a strong pressure dependence is Ge(P-3) + C2H4. C2H4 was confirmed as a product by GC analysis. Although the formation of these products are shown to be possible by singlet-triplet curve crossing during dissociation of 1-germiranylidene (1-germacyclopropylidene), it seems more likely (on thermochernical grounds) that the triplet biradical, (GeCH2CH2.)-Ge-., is the immediate product precursor. Comparisons are made with the reaction of SiH2 with C2H2.

Reclassification of Bacteroides putredinis (Weinberg et al., 1937) in a New Genus Alistipes gen. nov., as Alistipes putredinis comb. nov., and description of Alistipes finegoldii sp nov., from human sources

During studies on the bacteriology of appendicitis in children, we often isolated from inflamed and non-inflamed tissue samples, an unusual bile-resistant pigment-producing strictly anaerobic gram-negative rod. Phenotypically this organism resembles members of Bacteroides fragilis group of species, as it is resistant to bile and exhibits a special-potency-disk pattern (resistance to vancomycin, kanamycin and colistin) typical for the B. fragilis group. However, the production of brown pigment on media containing haemolysed blood and a cellular fatty acid composition dominated by iso-C15:0, suggests that the organism most closely resembles species of the genus Porphyromonas. However, the unidentified organism differs from porphyromonads by being bile-resistant and by not producing butyrate as a metabolic end-product. Comparative 16S ribosomal RNA gene sequencing studies show the unidentified organism represents a distinct sub-line, associated with but distinct from, the miss-classified species Bacteroides putredinis. The clustering of the unidentified bacterium with Bacteroides putredinis was statistically significant, but they displayed >4% sequence divergence with each other. Chromosomal DNA-DNA pairing studies further confirmed the separateness of the unidentified bacterium and Bacteroides putredinis. Based on phenotypic and phylogenetic considerations, it is proposed that Bacteroides putredinis and the unidentified bacterium from human sources be classified in a new genus Alistipes, as Alistipes putredinis comb. nov. and Alistipes finegoldii sp. nov., respectively. The type strain of Alistipes finegoldii is CCUG 46020(T) (= AHN2437(T)).

Cetobacterium somerae sp nov from human feces and emended description of the genus Cetobacterium

Phenotypic and phylogenetic studies were performed on four isolates of an unidentified gram-negative, microaerotolerant, non-spore-forming, rod-shaped bacterium isolated from the feces of children. The unknown organism was bile resistant and produced acetic acid as the major end product of metabolism of peptides and carbohydrates. It possessed a low DNA G + C content of 31 mol %. Comparative 16S rRNA gene sequencing demonstrated that the four isolates were phylogenetically identical (100% 16S rRNA sequence similarity) and represent a hitherto unknown sub-line within the genus Cetobacterium. The novel bacterium displayed approximately 5% sequence divergence with Cetobacterium ceti, and can be readily distinguished from the latter by physiological and biochemical criteria. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown fecal bacterium be classified in the genus Cetobacterium, as Cetobacterium somerae sp. nov. The proposed type strain of Cetobacterium somerae is WAL 14325(T) (ATCC BAA-474(T) = CCUG 46254T).

Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease

Accumulation of advanced glycation end-products (AGEs) on proteins is associated with the development of diabetic complications. Although the overall extent of modification of protein by AGEs is limited, localization of these modifications at a few critical sites might have a significant effect on protein structure and function. In the present study, we describe the sites of modification of RNase by glyoxal under physiological conditions. Arg(39) and Arg(85), which are closest to the active site of the enzyme, were identified as the primary sites of formation of the glyoxal-derived dihydroxyimidazolidine and hydroimidazolone adducts. Lower amounts of modification were detected at Arg(10), while Arg(33) appeared to be unmodified. We conclude that dihydroxyimidazolidine adducts are the primary products of modification of protein by glyoxal, that Arg(39) and Arg(85) are the primary sites of modification of RNase by glyoxal, and that modification of arginine residues during Maillard reactions of proteins is a highly selective process.

Time-resolved gas-phase kinetic study of the germylene addition reaction, CH3C CCH3

Time-resolved kinetic studies of the reaction of germylene, GeH2, generated by laser. ash photolysis of 3,4-dimethyl-1-germacyclopent-3-ene, have been carried out to obtain rate constants for its bimolecular reaction with 2-butyne, CH3C CCH3. The reaction was studied in the gas phase over the pressure range 1-100 Torr in SF6 bath gas, at five temperatures in the range 300-556 K. The second order rate constants obtained by extrapolation to the high pressure limits at each temperature, fitted the Arrhenius equation: log(k(infinity)/cm(3) molecule(-1) s(-1)) = (-10.46 +/- 10.06) + (5.16 +/- 10.47) kJ mol(-1)/ RT ln 10 Calculations of the energy surface of the GeC4H8 reaction system were carried out employing the additivity principle, by combining previous quantum chemical calculations of related reaction systems. These support formation of 1,2-dimethylvinylgermylene (rather than 2,3-dimethylgermirene) as the end product. RRKM calculations of the pressure dependence of the reaction are in reasonable agreement with this finding. The reactions of GeH2 with C2H2 and with CH3CRCCH3 are compared and contrasted.

The gas-phase reaction between silylene and 2-butyne: kinetics, isotope studies, pressure dependence studies and quantum chemical calculations

Time-resolved kinetic studies of the reactions of silylene, SiH2, and dideutero-silylene, SiD2, generated by laser. ash photolysis of phenylsilane and phenylsilane-d(3), respectively, have been carried out to obtain rate coefficients for their bimolecular reactions with 2-butyne, CH3C CCH3. The reactions were studied in the gas phase over the pressure range 1-100 Torr in SF6 bath gas at five temperatures in the range 294-612 K. The second-order rate coefficients, obtained by extrapolation to the high pressure limits at each temperature, fitted the Arrhenius equations where the error limits are single standard deviations: log(k(H)(infinity)/cm(3) molecule(-1) s(-1)) = (-9.67 +/- 0.04) + (1.71 +/- 0.33) kJ mol(-1)/RTln10 log(k(D)(infinity)/cm(3) molecule(-1) s(-1)) = (-9.65 +/- 0.01) + (1.92 +/- 0.13) kJ mol(-1)/RTln10 Additionally, pressure-dependent rate coefficients for the reaction of SiH2 with 2-butyne in the presence of He (1-100 Torr) were obtained at 301, 429 and 613 K. Quantum chemical (ab initio) calculations of the SiC4H8 reaction system at the G3 level support the formation of 2,3-dimethylsilirene [cyclo-SiH2C(CH3)=C(CH3)-] as the sole end product. However, reversible formation of 2,3-dimethylvinylsilylene [CH3CH=C(CH3)SiH] is also an important process. The calculations also indicate the probable involvement of several other intermediates, and possible products. RRKM calculations are in reasonable agreement with the pressure dependences at an enthalpy value for 2,3-dimethylsilirene fairly close to that suggested by the ab initio calculations. The experimental isotope effects deviate significantly from those predicted by RRKM theory. The differences can be explained by an isotopic scrambling mechanism, involving H - D exchange between the hydrogens of the methyl groups and the D-atoms in the ring in 2,3-dimethylsilirene-1,1-d(2). A detailed mechanism involving several intermediate species, which is consistent with the G3 energy surface, is proposed to account for this.

The role of transfer in language variation and change: Evidence from contact varieties of French

In this Special Issue, the focus is on contact-induced language variation and change in situations of societal bilingualism that involve long-term contact between French and another language. As is well known, when two or more languages are spoken by groups of speakers in the same geographical area, over time, features from one language can be transferred to the other language, especially when the languages in question are unequal in terms of prestige, institutional support and demographic factors. The process that leads to the adoption of such features in the contact languages is generally known as INTERFERENCE or TRANSFER, and these terms are also used to describe the features in question (i.e. the end product of the process of transfer). In this issue we prefer to use the term TRANSFER over the use of the notion INTERFERENCE, as the former has fewer negative connotations than the latter.

Coupling ensemble weather predictions based on TIGGE database with Grid-Xinanjiang model for flood forecast

The incorporation of numerical weather predictions (NWP) into a flood forecasting system can increase forecast lead times from a few hours to a few days. A single NWP forecast from a single forecast centre, however, is insufficient as it involves considerable non-predictable uncertainties and lead to a high number of false alarms. The availability of global ensemble numerical weather prediction systems through the THORPEX Interactive Grand Global Ensemble' (TIGGE) offers a new opportunity for flood forecast. The Grid-Xinanjiang distributed hydrological model, which is based on the Xinanjiang model theory and the topographical information of each grid cell extracted from the Digital Elevation Model (DEM), is coupled with ensemble weather predictions based on the TIGGE database (CMC, CMA, ECWMF, UKMO, NCEP) for flood forecast. This paper presents a case study using the coupled flood forecasting model on the Xixian catchment (a drainage area of 8826 km2) located in Henan province, China. A probabilistic discharge is provided as the end product of flood forecast. Results show that the association of the Grid-Xinanjiang model and the TIGGE database gives a promising tool for an early warning of flood events several days ahead.

Tracking the uncertainty in flood alerts driven by grand ensemble weather predictions

The incorporation of numerical weather predictions (NWP) into a flood warning system can increase forecast lead times from a few hours to a few days. A single NWP forecast from a single forecast centre, however, is insufficient as it involves considerable non-predictable uncertainties and can lead to a high number of false or missed warnings. Weather forecasts using multiple NWPs from various weather centres implemented on catchment hydrology can provide significantly improved early flood warning. The availability of global ensemble weather prediction systems through the ‘THORPEX Interactive Grand Global Ensemble’ (TIGGE) offers a new opportunity for the development of state-of-the-art early flood forecasting systems. This paper presents a case study using the TIGGE database for flood warning on a meso-scale catchment (4062 km2) located in the Midlands region of England. For the first time, a research attempt is made to set up a coupled atmospheric-hydrologic-hydraulic cascade system driven by the TIGGE ensemble forecasts. A probabilistic discharge and flood inundation forecast is provided as the end product to study the potential benefits of using the TIGGE database. The study shows that precipitation input uncertainties dominate and propagate through the cascade chain. The current NWPs fall short of representing the spatial precipitation variability on such a comparatively small catchment, which indicates need to improve NWPs resolution and/or disaggregating techniques to narrow down the spatial gap between meteorology and hydrology. The spread of discharge forecasts varies from centre to centre, but it is generally large and implies a significant level of uncertainties. Nevertheless, the results show the TIGGE database is a promising tool to forecast flood inundation, comparable with that driven by raingauge observation.

The effect of organic materials on the mobility and toxicity of metals in contaminated soils

Organic materials such as compost are often proposed as suitable materials for the remediation of contaminated brownfield sites intended for soft end-use. In addition to vitalising the soil, they are also believed to immobilise metals thereby breaking contaminant-receptor pathways and reducing the ecotoxicity of the contaminants. However, some research has demonstrated contradictory effects between composts on metal immobilisation. In the present study, four different composts and a liming product containing organic matter (LimeX70) were tested to examine both their metal retention and toxicity reduction capabilities on three different metal contaminated soils. Leaching tests, a plant growth test with Greek cress (Lepidium sativum), an earthworm (Eisenia fetida) survival and condition test and a bacterial toxicity test using Vibrio fischeri were carried out. The leaching test results showed that spent mushroom compost caused an increase in metal concentration in the leachates, while LimeX70 caused a decrease. The variation in behaviour between different amendments for each soil was high, so a generic conclusion could not be drawn. Toxicity tests showed significant reduction of metal bioavailability and toxicity for Greek cress, earthworms and bacteria. The results also suggest that more research should be undertaken to understand the mechanisms involved in metal complexation using different types of organic matter, in order to optimise the use of organic materials like compost for soil remediation. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.