Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase

To prevent diabetic complications derived from enhanced glucose flux via the polyol pathway the development of aldose reductase inhibitors (ARIs) has been established as a promising therapeutic concept. Here, we study the binding process of inhibitors to aldose reductase (ALR2) with respect to changes of the protonation inventory upon complex formation. Knowledge of such processes is a prerequisite to factorize the binding free energy into enthalpic and entropic contributions on an absolute scale. Our isothermal titration calorimetry (ITC) measurements suggest a proton uptake upon complex formation with carboxylate-type inhibitors. As the protonation event will contribute strongly to the enthalpic signal recorded during ITC experiments, knowledge about the proton-accepting and releasing functional groups of the system is of utmost importance. However, this is intricate to retrieve, if, as in the present case, both, binding site and ligand possess several titratable groups. Here, we present pKa calculations complemented by mutagenesis and thermodynamic measurements suggesting a tyrosine residue located in the catalytic site (Tyr48) as a likely candidate to act as proton acceptor upon inhibitor binding, as it occurs deprotonated to a remarkable extent if only the cofactor NADP+ is bound. We furthermore provide evidence that the protonation state and binding thermodynamics depend strongly on the oxidation state of the cofactor;s nicotinamide moiety. Binding thermodynamics of IDD 388, IDD 393, tolrestat, sorbinil, and fidarestat are discussed in the context of substituent effects.     

Clofibrate causes an upregulation of PPAR-{alpha} target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.     

Staphylococcus epidermidis polysaccharide intercellular adhesin activates complement

Staphylococcus epidermidis is a frequent cause of nosocomial infections. The central virulence factor of S. epidermidis is biofilm formation. Polysaccharide intercellular adhesin (PIA) constitutes the major biofilm matrix-component. PIA and biofilm have been implicated in S. epidermidis evasion of host immune defence. We examined the effects of S. epidermidis PIA on the inflammatory response with focus on complement activation. We used a human whole-blood ex vivo model of infection and compared the effects of a PIA-positive S. epidermidis strain (SE1457) and its PIA-negative isogenic mutant (M10). The independent effect of purified PIA on complement activation was investigated. In glucose-rich media, the mutant formed a proteinacious DNA-rich biofilm, whereas SE1457 formed a thick PIA-biofilm. In biofilm growth, SE1457 induced a stronger activation of the complement system compared with M10. We verified that purified PIA was independently responsible for a strong activation of the complement system. In contrast, M10 induced higher granulocyte activation by expression of CD11b and higher secretion of cytokines. We conclude that PIA has potent pro-inflammatory properties by activating the complement system. However, in a complex balance of the immune response, the decreased activation of granulocytes and cytokines by a PIA biofilm may limit host eradication of S. epidermidis.     

Belowground net primary productivity and biomass allocation of a grassland in Inner Mongolia is affected by grazing intensity

The root system of permanent grasslands is of outstanding importance for resource acquisition. Particularly under semi-arid conditions, the acquisition of water and nutrients is highly variable during the vegetation growth period and between years. Additionally, grazing is repeatedly disturbing the functional equilibrium between the root system and the transpiring leaf canopy. However, very few data is available considering grazing effects on belowground net primary productivity (BNPP) and root-shoot dry mass allocation in natural grassland systems. We hypothesise that grazing significantly reduces BNPP due to carbon reallocation to shoot growth. Root biomass and BNPP were estimated by soil coring in 2004, 2005 and 2006 and from ingrowth cores in 2005 and 2006 at one site which has been protected from grazing since 1979 (UG79), at one winter grazing (WG), and one heavily grazed (HG) site. BNPP was estimated from the summation of significant increments of total and live root biomass and from accumulated root biomass of ingrowth cores. Belowground biomass varied from 1,490–2,670 g m^?2 and was significantly lower under heavy grazing than at site UG79. Root turnover varied from 0.23 to 0.33 year^?1 and was not significantly different between sites. Heavy grazing significantly decreased live root biomass and BNPP compared to site UG79. Taking BNPP estimates from live root biomass dynamics and ingrowth cores as the most reliable values, the portion of dry mass allocated belowground relative to total net primary productivity (BNPP/NPP) varied between 0.50–0.66 and was reduced under heavy grazing in 2005, but not in 2006. The positive correlation between cumulative root length density of ingrowth cores and leaf dry matter suggests that the ingrowth core method is suitable for studying BNPP in this semi-arid steppe system. Grazing effects on BNPP and BNPP/NPP should be considered in regional carbon models and estimates of belowground nutrient cycling.     

Applying dimorphic yeasts as model organisms to study mycelial growth: part 2. Use of mathematical simulations to identify different construction principles in yeast colonies

The dimorphic yeasts Candida boidinii and Yarrowia lipolytica were applied as model organisms to study mycelial growth. A mathematical model of hybrid cellular automaton type was developed to analyze the impact of different biological assumptions on the predicted development of filamentous yeast colonies. The one-dimensional model described discrete cells and continuous distribution of nutrients. The simulation algorithm accounted for proliferation of cells, diffusion of nutrient, as well as biomass decay and recycling inside the mycelium. Simulations reproduced the spatio-temporal development of C. boidinii colonies when a diffusion-limited growth algorithm based on the growth of pseudohyphal cells was applied. Development of Y. lipolytica colonies could only be reproduced when proliferation was restricted to the colony boundary, and cell decay and biomass recycling were incorporated into the model. The results suggested that cytoplasm, which served as the secondary nutrient resource, had to be translocated inside the hyphal network.     

Changes in the microbial community in a forest soil amended with aluminium in situ

Considerable knowledge exists about the effect of aluminium (Al) on root vitality, but whether elevated levels of Al affect soil microorganisms is largely unknown. We thus compared soils from Al-treated and control plots of a field experiment with respect to microbial and chemical parameters, as well as root growth and vitality. The field experiment was established in a 50-year-old Norway spruce (Picea abies L.) stand where no Al or low concentrations of Al had been added every 7–10 days during the growth season for 7 years. Analysis of soil solutions collected using zero tension lysimeters and porous suction cups showed that Al treatment lead to increased concentrations of Al, Ca and Mg and lower pH and [Ca + Mg + K/Al] molar ratio. Corresponding soil analyses showed that soil pH remained unaffected (pH 3.8), that exchangeable Al increased, while exchangeable Ca and Mg decreased due to the Al treatment. Root in-growth into cores placed in the upper 20 cm of the soil during three growth seasons was not affected by Al additions, neither was nutrient concentration or mortality of these roots. The biomass of some taxonomic groups of soil microorganisms, analyzed using specific membrane components (phospholipid fatty acids; PLFAs), was clearly affected by the imposed Al treatment, both in the organic soil horizon and in the underlying mineral soil. Microbial community structure in both horizons was also clearly modified by the Al treatment. Shifts in PLFA trans/cis ratios indicative of short term physiological stress were not observed. Yet, aluminium stress was indicated both by changes in community structure and in ratios of single PLFAs for treated/untreated plots. Thus, soil microorganisms were more sensitive indicators of subtle chemical changes in soil than chemical composition and vitality of roots.     

Effects of common atopy-associated amino acid substitutions in the IL-4 receptor alpha chain on IL-4 induced phenotypes

The human IL-4 receptor alpha chain gene (IL4R) is highly polymorphic and controversial reports have been published with respect to the association of different single nucleotide polymorphisms (SNPs) with atopy markers. Here we analyzed the functional and associational relevance of common IL4R coding SNPs. Transfection of B cell lines expressing the IL-4R variant V75+R576 did not result in enhanced IL-4 induced CD23 expression compared to cell lines expressing the wild type IL-4R alpha chain. Transfection of the IL-4R variant P503 into a murine T cell line did not influence IL-4 induced T-cell proliferation compared to wild type constructs. Analysis of six IL4R coding SNPs (I75V, E400A, C431R, S436L, S503P, Q576R) and common haplotypes (frequency 0.05%) in blood donors (n=300) did not indicate a significant association with elevated serum IgE level. Moreover, the most informative IL4R coding SNPs (I75V, C431R, Q576R) and related two- and three-point haplotypes (frequency 0.05%) were analyzed in a second, extended group of blood donors (n=689). Again, no significant association with elevated serum IgE was detectable. We conclude that common coding SNPs in the IL4R gene are unlikely to contribute significantly to increased IgE levels and variations outside the coding region may influence atopy susceptibility.     

C2 can do it, too

In this issue of Cell, report that the C2 domain of the serine/threonine protein kinase Cdelta is a phosphotyrosine binding domain and present the crystal structure of this C2 domain bound to a peptide containing phosphotyrosine. Prior to this work, C2 domains were thought to bind only to phospholipids or to unphosphorylated proteins, and the SH2 and PTB domains were the only signaling domains known to recognize phosphotyrosine. This new role for the C2 domain links phosphotyrosine recognition directly to serine/threonine kinase activity and reveals an unexpected mechanism for crosstalk between distinct signaling pathways.