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Binding Capacity (binding + capacity)
Kinds of Binding Capacity Selected AbstractsA Novel Approach to Magnetic Nanoadsorbents with High Binding Capacity for Bovine Serum AlbuminMACROMOLECULAR RAPID COMMUNICATIONS, Issue 3 2007Yabin Sun Abstract Magnetic nanoadsorbents using Fe3O4 nanoparticles as cores and poly(methyl acrylic acid) (PMAA) as ionic exchange groups were prepared through our novel approach. Two steps were involved in this approach: the first was to functionalize the magnetic nanoparticles (MNPs) with methacrylate double bonds via the combination of ligand exchange and condensation of methacryloxypropyltrimethoxysilane(MPS); the second was to graft PMAA chains onto the surface of MNPs through radical polymerization. The success of the various surface functionalization steps was ascertained using FTIR and XPS. The as-synthesized PMAA-coated MNPs were effective in binding bovine serum albumin (BSA) with a high capacity of 1,300 mg,·,g,1. [source] WHEN POSITIVELY CHARGED MILK PROTEINS CAN BIND TO DNAJOURNAL OF FOOD BIOCHEMISTRY, Issue 6 2002MAHMOUD SITOHY ABSTRACT The binding of three esterified milk proteins (,-lactoglobutin, ,-lactalbumin and ,-casein) to plasmid DNAs at pH 7.1 was followed by agarose-gel electrophoresis. Highly esterified ,-lactoglobulin and ,-lactalbumin samples showed DNA-binding capacities comparable to those exhibited by native basic proteins such as lysozymes and histones. All the studied esterified ,-casein samples failed to bind to DNA at the applied pH. Complete retardation of DNA migration on agarose gel was observed at a 1:1 ratio of protein basic groups (Lys + Arg) to DNA phosphate add groups in the case of highly esterified ,-lactoglobulin, esterified ,-lactalbumin and native basic proteins (lysozyme and histone). Binding capacity was dependent on the degree of esterification of the milk proteins. Hydrolysis of esterified milk proteins either suppressed or reduced their DNA-binding capacities according to the degree of hydrolysis and consequently to the average size of the resulting peptides. A prolonged peptic hydrolysis (25% degree of hydrolysis) completely suppressed DNA-binding capacity probably because of the small sizes of the resulting peptic peptides (< 1 kDa). Treatment with trypsin, which hydrolyzed the esterified proteins into relatively large peptide fragments, reduced the DNA-binding capacity to levels inversely proportional to the degree of hydrolysis. In the range of 2.7,12.3 kb, there was no influence of the DNA size on the binding of esterified milk proteins. The interactions DNA-esterified milk proteins did not depend on the DNA shape (circular or linear). Circular dichroism spectra of DNA in complex with methylated ,-lactoglobulin were markedly altered as compared to those obtained when DNA was in complex with native ,-lactoghbutin. [source] Effects of salts on protein,surface interactions: applications for column chromatographyJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 7 2007Kouhei Tsumoto Abstract Development of protein pharmaceuticals depends on the availability of high quality proteins. Various column chromatographies are used to purify proteins and characterize the purity and properties of the proteins. Most column chromatographies require salts, whether inorganic or organic, for binding, elution or simply better recovery and resolution. The salts modulate affinity of the proteins for particular columns and nonspecific protein,protein or protein,surface interactions, depending on the type and concentration of the salts, in both specific and nonspecific manners. Salts also affect the binding capacity of the column, which determines the size of the column to be used. Binding capacity, whether equilibrium or dynamic (under an approximation of a slow flow rate), depends on the binding constant, protein concentration and the number of the binding site on the column as well as nonspecific binding. This review attempts to summarize the mechanism of the salt effects on binding affinity and capacity for various column chromatographies and on nonspecific protein,protein or protein,surface interactions. Understanding such salt effects should also be useful in preventing nonspecific protein binding to various containers. © 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 1677,1690, 2007 [source] The rate of uptake of sex steroids from water by Tinca tinca is influenced by their affinity for sex steroid binding protein in plasmaJOURNAL OF FISH BIOLOGY, Issue 1 2005A. P. Scott Two experiments were carried out in which male and female tench Tinca tinca were placed in individual containers and tritiated steroids then added to the water. Water samples were collected over the next 6 or 7 h and the fish then sacrificed, bled and the gall bladder removed. Radioactivity was counted in all the samples. Over the course of the exposure period in the first experiment (7 h), radioactivity of 11-ketotestosterone (11-KT) in the water was depleted by 11%, 17,20,-dihydroxypregn-4-en-3-one (17,20ß-P) and 17,20,-dihydroxypregn-4-en-3-one (17,20,-P) by 28%, testosterone (T) by 56% and androstenedione (AD) by 68%. HPLC analysis of water samples at 3 h indicated that none of the steroids was extensively metabolized during the experiment. Females had a faster rate of uptake of AD than males. In the second experiment (6 h), radioactivity of cortisol in the water was depleted by 5%, 11-KT by 7%, 17-hydroxypregnen-4-ene (17-P) by 17%, 17,-oestradiol (E2) by 35%, T by 37% and AD by 44%. In both experiments, the amounts of radioactivity that were recovered from the gall bladder and plasma were positively correlated with the rate of disappearance of radioactivity from the water. The ability of the steroids to bind to sex steroid binding protein (SBP) of tench plasma was tested by incubating plasma with radioactive steroids and then separating bound and free with ice cold dextran-coated charcoal. When plasma at a final dilution of 1 : 60 (v/v) was incubated with 5 nM of each steroid, the percentage of radiolabel bound to SBP was: T 48% AD 44%, E2 30%, 17-P 17%, 11-KT 13·2%, 17,20,-P 10·3%, 17,20,-P 4·5% and cortisol 0%. Saturation analysis established dissociation constants (Kd; mean ± s.e.) of 3·4 ± 0·4, 2·2 ± 0·2, 4·0 ± 0·3. 9·0 ± 2·8 and 51·8 nM and binding capacities (Bmax) of 201 ± 29, 201 ± 33, 165 ± 3, 187 ± 15 and 13·4 nM for T, AD, E2,17-P and 17,20,-P respectively. The ability of steroids to displace tritiated T and AD from SBP was in the rank order AD > T > E2 > 17,20,P = 17,20,-P = 11-KT = 17-P > cortisol. Thus, the ability of tench plasma to bind certain steroids showed a relatively strong correlation with the ability of the fish to take up these steroids from water. Modelling of data for AD and 17,20,-P helped to show why and how plasma binding had a strong influence on the rate of uptake (and hence release) of the steroids. [source] Removal of Cadmium and Lead from Aqueous Solution by,Enterococcus faecium,StrainsJOURNAL OF FOOD SCIENCE, Issue 1 2010A. Topcu ABSTRACT:, Foods may be contaminated with heavy metals, which, even in small quantities, possess detrimental effects on human health. The aim of the present study was to investigate the uptake of cadmium or lead from an aqueous medium frequently found in foods, by 2,Enterococcus faecium,strains (E. faecium,EF031 and,E. faecium,M74). Also, the effects of the bacterial viability, incubation (contact) time, and pH on the binding capacities and binding stability were assessed. The results showed that both of the strains efficiently removed cadmium and lead. While EF031 removed 77.3% to 98.1% of cadmium and 66.9% to 98.9% of lead, M74 removed 53.5% to 91% of cadmium and 42.9% to 93.1% of lead throughout a 48 h incubation period at pH 5. It was found that, at 1 h, EF031 and M74 strains removed cadmium and lead, which was more than 60% of total removed cadmium and lead throughout the whole incubation period of 48 h. It suggests that the uptake of cadmium and lead by EF031 and M74 strains is a rapid process. The binding of both heavy metals increased with increasing pH of an aqueous medium and was the highest at pH 5. Also, the complexes formed between both heavy metals and bacterial cells were found to be stable. These findings indicate that,E. faecium,strains used in the study are able to bind the 2 heavy metals and may be used in the production of fermented functional foods, which will be healthy via its detoxification properties. [source] Differential and Age-Dependent Effects of Maternal Deprivation on the Hypothalamic-Pituitary-Adrenal Axis of Brown Norway Rats from Youth to SenescenceJOURNAL OF NEUROENDOCRINOLOGY, Issue 7 2001J. O. Workel Abstract In this study, the hypothesis was tested that infants deprived from maternal care show persistent changes in hypothalamic-pituitary-adrenal activity. For this purpose, we studied the effect of maternal deprivation in one cohort of the healthy ageing Brown Norway rat strain showing still more than 80% survival rate at 32 months of age. Three-day-old male Brown Norway rats were either maternally deprived for 24 h or remained with the dam. In 3, 12 and 30,32 months (young, adult, senescent) deprived rats and their nondeprived littermates (controls), we determined basal resting and stress-induced plasma adrenocorticotropic hormone (ACTH) and corticosterone as well as corticotropin releasing hormone (CRH) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus. Mineralocorticoid (MR) and glucocorticoid receptors (GR) in hippocampus and PVN were also assessed using in vitro cytosol binding and in situ hybridization. The effect of ageing per se showed that in the control nondeprived Brown Norway rats, basal corticosterone and ACTH concentrations did not change during life. However, with age, the corticosterone response to novelty stress became progressively attenuated, but prolonged, while there was an age-related increase in the ACTH response. CRH mRNA expression in PVN decreased with age. Hippocampal MR binding and MR mRNA expression in the dentate gyrus were reduced at senescence, as were the GR binding capacities in hippocampus and hypothalamus. Maternal deprivation did not affect survival rate, body weight, nor adrenal weight of the ageing Brown Norway rats. Basal corticosterone and ACTH levels were not affected by deprivation, except for a rise in basal corticosterone concentrations at 3 months. At this age, the corticosterone output in response to novelty was attenuated in the deprived rats. In contrast, a striking surge in novelty stress-induced corticosterone output occurred at midlife while, at senescence, the corticosterone and ACTH responses were attenuated again in the deprived animals, particularly after the more severe restraint stressor. CRH mRNA expression was reduced only during adulthood in the deprived animals. After maternal deprivation, the MR mRNA in dentate gyrus showed a transient midlife rise. GR binding in hypothalamus and hippocampus GR binding was reduced in young rats while, in the senescent deprived animals, a reduced GRmRNA expression was observed in PVN and hippocampal CA1. In conclusion, in the Brown Norway rat, ageing causes a progressive decline in corticosterone output after stress, which is paralleled at senescence by decreased MR and GR mRNA expression in hippocampus and hypothalamus. The long-term effects of maternal deprivation become manifest differently at different ages and depend on test conditions. The deprivation effect culminates in a midlife corticosterone surge and results at senescence in a strongly reduced corticosterone output. [source] Blunted Pituitary-Adrenocortical Stress Response in Adult Rats Following Neonatal Dexamethasone TreatmentJOURNAL OF NEUROENDOCRINOLOGY, Issue 10 2000K. Felszeghy Abstract Glucocorticoids have a prominent impact on the maturation of the stress-related neuroendocrine system and on the postnatal establishment of adaptive behaviour. The present study aimed at investigating the stress responsiveness of the hypothalamo-pituitary-adrenocortical (HPA) axis in young and adult rats after neonatal treatment with the synthetic glucocorticoid agonist, dexamethasone. Newborn male Wistar rats were injected s.c. with 1 µg/g dexamethasone on postnatal days 1, 3 and 5. Circulating adrenocorticotropic hormone (ACTH) and corticosterone concentrations were measured in the resting state and following a 30-min cold stress at the age of 10 days, as well as after a 30-min restraint stress at the age of 14 weeks. Also in adults, pituitary and adrenocortical hormone responsiveness was evaluated after i.v. administration of 2 µg/kg corticotropin releasing hormone (CRH). In addition, glucocorticoid (GR) and mineralocorticoid receptor (MR) binding capacities were assessed in the pituitaries of adult rats. The results showed that at day 10 basal ACTH concentration was elevated while the cold stress-evoked ACTH response was attenuated in the dexamethasone-treated rats. As adults, treated rats showed a suppressed elevation of both ACTH and corticosterone plasma cncentrations in response to restraint, while basal hormonal concentrations were not altered. There was no difference in the magnitude of the CRH-induced elevation of ACTH and corticosterone concentrations initially; however, the dexamethasone-treated animals showed a prolonged secretion of both hormones. These animals also showed a selective decrease in pituitary GR binding capacity. Neonatal dexamethasone treatment strongly suppressed body weight gain, and adrenal and thymus weights in the early phase of postnatal development. By adulthood, the body and adrenal weights were normalized while thymus weight was greater than in controls. These findings indicate that neonatal dexamethasone treatment permanently alters HPA axis activity by reducing stress responses to cold and restraint probably through supra-pituitary actions, and by decreasing the effectiveness of feedback through a diminished GR binding in the pituitary. [source] Isolation of ATP from a yeast fermentation broth using a cryogel column at high flow velocitiesJOURNAL OF SEPARATION SCIENCE, JSS, Issue 22 2008Chen Yan Abstract This communication presents an effective method for isolating adenosine triphosphate (ATP) from a yeast fermentation broth using an anion-exchange supermacroporous cryogel column at high flow velocities. The breakthrough and elution behaviors of pure ATP in the cryogel bed were investigated at flow velocities of 2, 5, and 10 cm/min and the ATP binding capacities were determined. Then the ATP-containing yeast fermentation broth was employed as the test feedstock and various chromatographic runs were conducted to isolate ATP by the cryogel at different high flow velocities. The ATP samples obtained were analyzed quantitatively by HPLC. The results showed that even at a flow velocity of 5 or 10 cm/min, a product purity of 97.4 or 98.0% can be achieved, illustrating the potential of the present method for separation of high-purity ATP directly from fermentation feedstock at high flow velocities. [source] Purification of cell culture-derived modified vaccinia ankara virus by pseudo-affinity membrane adsorbers and hydrophobic interaction chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Michael W. Wolff Abstract A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated. The applied pseudo-affinity chromatography, based on reinforced sulfated cellulose and heparin-MA, was optimized in terms of dynamic binding capacities, virus yield and process productivity. HIC was introduced as a subsequent method to further reduce the DNA content. Therefore, two screens were undertaken. First, several HIC ligands were screened for different adsorption behavior between virus particles and DNA. Second, elution from pseudo-affinity MA and adsorption of virus particles onto the hydrophobic interaction matrix was explored by a series of buffers using different ammonium sulfate concentrations. Eventually, variations between different cultivation batches and buffer conditions were investigated. The most promising combination, a sulfated cellulose membrane adsorber with subsequent phenyl HIC resulted in overall virus particle recoveries ranging from 76% to 55% depending on the product batch and applied conditions. On average, 61% of the recovered virus particles were infective within all tested purification schemes and conditions. Final DNA content varied from 0.01% to 2.5% of the starting material and the level of contaminating protein was below 0.1%. Biotechnol. Bioeng. 2010;107: 312,320. © 2010 Wiley Periodicals, Inc. [source] Fractionation of ,-Lactoglobulin from whey by mixed matrix membrane ion exchange chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Syed M. Saufi Abstract Mixed matrix membranes (MMMs), which incorporate adsorptive particles during membrane casting, can be prepared simply and have performances that are competitive with other membrane chromatography materials. The application of MMM chromatography for fractionation of ,-Lactoglobulin from bovine whey is described in this article. MMM chromatography was prepared using ethylene vinyl alcohol polymer and lewatit anion exchange resin to form a flat sheet membrane. The membrane was characterized in terms of structure and its static and dynamic binding capacities were measured. The optimum binding for ,-Lactoglobulin was found to be at pH 6.0 using 20 mM sodium phosphate buffer. The MMM had a static binding capacity of 120 mg/g membrane (36 mg/mL membrane) and 90 mg/g membrane (27 mg/mL membrane) for ,-Lactoglobulin and ,-Lactalbumin, respectively. In batch fractionation of whey, the MMM showed selective binding towards ,-Lactoglobulin compared to other proteins. The dynamic binding capacity of ,-Lactoglobulin in whey solution was about 80 mg/g membrane (24 mg ,-Lac/mL of MMM), which is promising for whey fractionation using this technology. This is the first reported application of MMM chromatography to a dairy feed stream. Biotechnol. Bioeng. 2009;103: 138,147. © 2008 Wiley Periodicals, Inc. [source] An exclusion mechanism in ion exchange chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 5 2006C. Harinarayan Abstract Protein dynamic binding capacities on ion exchange resins are typically expected to decrease with increasing conductivity and decreasing protein charge. There are, however, conditions where capacity increases with increasing conductivity and decreasing protein charge. Capacity measurements on two different commercial ion exchange resins with three different monoclonal antibodies at various pH and conductivities exhibited two domains. In the first domain, the capacity unexpectedly increased with increasing conductivity and decreasing protein charge. The second domain exhibited traditional behavior. A mechanism to explain the first domain is postulated; proteins initially bind to the outer pore regions and electrostatically hinder subsequent protein transport. Such a mechanism is supported by protein capacity and confocal microscopy studies whose results suggest how knowledge of the two types of IEX behavior can be leveraged in optimizing resins and processes. © 2006 Wiley Periodicals, Inc. [source] No relationship between enzyme activity and structure of nucleotide binding site in sarcoplasmic reticulum Ca2+ -ATPase from short-term stimulated rat muscleACTA PHYSIOLOGICA, Issue 4 2009T. Mishima Abstract Aim:, We examined whether structural alterations to the adenine nucleotide binding site (ANBS) within sarcoplasmic (endo) reticulum Ca2+ -ATPase (SERCA) would account for contraction-induced changes in the catalytic activity of the enzyme as assessed in vitro. Methods:, Repetitive contractions were induced in rat gastrocnemius by electrical nerve stimulation. Measurements of sarcoplasmic reticulum properties were performed on control and stimulated muscles immediately after or at 30 min after the cessation of 5-min stimulation. In order to examine the properties at the ANBS, the binding capacity of SERCA to fluorescence isothiocyanate (FITC), a competitive inhibitor at the ANBS, was analysed in microsomes. Results:, Short-term electrical stimulation evoked a 23.9% and 32.6% decrease (P < 0.05) in SERCA activity and in the FITC binding capacity, respectively, in the superficial region of the muscle. Whereas SERCA activity reverted to normal levels during 30-min recovery, a restoration of the FITC binding capacity did not occur. Conclusion:, The discordant changes between the enzyme activity and the FITC binding suggest that, at least during recovery after exercise, changes in SERCA activity may not correlate closely with structural alterations to the ANBS within the enzyme. [source] Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemiaCYTOMETRY, Issue 6 2007Eva D. Rossmann Abstract Background: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors. Methods: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITEÔ (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC). Results: The ABCs of CD4 and CD20 antigens estimated with QSC (ABCQSC) were higher than those assigned with QB (ABCQB) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABCQ-MESF) were ,15% higher than ABCQSC, whereas ABCQ-MESF was ,49% higher than ABCQB. Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down-regulation of CD3 antigen on T cells from patients with CLL. Conclusions: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi-center clinical studies. © 2007 Clinical Cytometry Society [source] Serum Iron and Matrix Metalloproteinase-9 Variations in Limbs Affected by Chronic Venous Disease and Venous Leg UlcersDERMATOLOGIC SURGERY, Issue 6 2005Paolo Zamboni MD Background. Severe chronic venous disease (CVD) is characterized by both dermal hemosiderin accumulation and matrix metalloproteinase (MMP) hyperactivation. The iron-driven pathway is one of the recognized mechanisms of MMP hyperactivation. Objective. To investigate the potential consequences of leg hemosiderin deposits on both iron metabolism and activation of MMPs. Methods. We contemporaneously assessed the following in the serum of the arm and ankle veins of 30 patients (C4,6) with CVD and 14 normal subjects: ferritin, transferrin, iron, percentage of transferrin iron binding capacity (%TIBC), and MMP-9. Optical microscopy examinations with Perls' staining of chronic wounds were also performed. Results. Histology consistently revealed iron deposits. Serum ferritin, iron, and %TIBC were significantly increased in the legs affected by severe CVD compared with the arm of the same subjects or the controls. In addition, iron and %TIBC were significantly elevated in the legs of ulcer patients. The rate of activation of MMP-9 was significantly elevated in CVD. Conclusions. The increased iron deposition in legs affected by CVD seems to be more instable in ulcer patients, leading to iron release in the serum of the affected leg. Our data suggest the iron-driven pathway as a further mechanism for MMP hyperexpression leading to tissue lesion. [source] Serotonin and dopamine transporter binding in children with autism determined by SPECTDEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 8 2008Ismo Makkonen MD Disturbances in the serotonergic system have been recognized in autism. To investigate the association between serotonin and dopamine transporters and autism, we studied 15 children (14 males, one female; mean age 8y 8mo [SD 3y 10mo]) with autism and 10 non-autistic comparison children (five males, five females; mean age 9y 10mo [SD 2y 8mo]) using single-photon emission computed tomography (SPECT) with [123I] nor-,-CIT. The children, with autism were studied during light sedation. They showed reduced serotonin transporter (SERT) binding capacity in the medial frontal cortex, midbrain, and temporal lobe areas. However, after correction due to the estimated effect of sedation, the difference remained significant only in the medial frontal cortex area (p=0.002). In the individuals with autism dopamine transporter (DAT) binding did not differ from that of the comparison group. The results indicate that SERT binding capacity is disturbed in autism. The reduction is more evident in adolescence than in earlier childhood. The low SERT binding reported here and the low serotonin synthesis capacity shown elsewhere may indicate maturation of a lesser number of serotonergic nerve terminals in individuals with autism. [source] An Electroanalytical Investigation on the Redox Properties of Calcium Antagonist DihydropyridinesELECTROANALYSIS, Issue 10 2003Rosanna Toniolo Abstract The antioxidant capacity of some calcium antagonists and one calcium agonist 1,4-dihydropyridines (DHPs) was evaluated by a competitive kinetic procedure. With the exception of Amlodipine, all the calcium antagonist DHPs display an unambiguous antioxidant capacity, while for the calcium agonist DHP (Bay K 8644) no measurable reactivity towards peroxyl radicals could be detected. The finding was corroborated by an electroanalytical investigation of the redox properties of DHPs compounds to get an insight about both the thermodynamic constraints of their oxidation process and reaction pattern. The oxidation potentials decrease with both antioxidant capacity and increasing basic character, thus suggesting the relevance of the electron density on the DHP ring. For all the compounds investigated, the overall oxidation process takes place through a primary one-electron step accompanied by a fast proton release and the formation of a neutral radical undergoing a second much easier one-electron step. The protonated form of the parent pyridine derivative is thus generated as the final product. This pattern is relevant for the antioxidant effect, since the radical intermediate is much more prone to be oxidized than to be reduced, thus fully preventing the propagation of the oxidative chain reaction. In the case of calcium antagonist DHPs, the above release of protons complicates the overall oxidation process by introducing a parasitic side reaction where a coupling between protons and the starting species takes place. This DHP self-protonation subtracts part of the original species from the electrode process because the parent cationic species is no longer electroactive. Conversely, the calcium agonist DHP, which is more difficult to be oxidized, turned out to be such a weak base as to be unable to undergo the self-protonation reaction. The combined effect of oxidation potentials and proton binding capacity of DHPs is a key element for the redox transition, which could support their antioxidant effect and should be considered to some extent in accounting for the calcium antagonist vs calcium agonist effect. [source] Biological significance of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa),ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 3 2006Martina G. Vijver Abstract Metal ions in excess of metabolic requirements are potentially toxic and must be removed from the vicinity of important biological molecules to protect organisms from adverse effects. Correspondingly, metals are sequestrated in various forms, defining the accumulation pattern and the magnitude of steady-state levels reached. To investigate the subcellular fractions over which Ca, Mg, Fe, Cu, Zn, Cd, Pb, Ni, and As are distributed, earthworms (Aporrectodea caliginosa) collected from the field were analyzed by isolating metal-rich granules and tissue fragments from intracellular microsomal and cytosolic fractions (i.e., heat-stable proteins and heat-denatured proteins). The fractions showed metal-specific binding capacity. Cadmium was mainly retrieved from the protein fractions. Copper was equally distributed over the protein fraction and the fraction comprising tissue fragments, cell membranes, and intact cells. Zinc, Ca, Mg, and As were mainly found in this fraction as well. Lead, Fe, and Ni were mainly isolated from the granular fraction. To study accumulation kinetics in the different fractions, three experiments were conducted in which earthworms were exposed to metal-spiked soil and a soil contaminated by anthropogenic inputs and, indigenous earthworms were exposed to field soils. Although kinetics showed variation, linear uptake and steady-state types of accumulation patterns could be understood according to subcellular compartmentalization. For risk assessment purposes, subcellular distribution of metals might allow for a more precise estimate of effects than total body burden. Identification of subcellular partitioning appears useful in determining the biological significance of steady-state levels reached in animals. [source] Hormetic effects of gamma radiation on the stress axis of natural populations of meadow voles (Microtus pennsylvanicus)ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 2 2005Rudy Boonstra Abstract We tested the hypothesis that low doses of gamma radiation have beneficial, hormetic effects on the stress axis (the hypothalamic-pituitary-adrenocortical axis) of free-ranging meadow vole populations (Microtus pennsylvanicus). Voles were exposed to chronic gamma radiation from a 137Cs field irradiator. In isolated populations, voles received one of three treatments over a four-year period: Controls (0.19,0.42 ,Gy/h , levels that were 2,5X above background levels [0.1 ,Gy/h] and live-trapped in all years , 1982,1985), low doses (22.6 ,Gy/h , 50,200X background, live-trapped from November 1982,April 1985), or high doses (3,840 ,Gy/h , 40,000X background, live-trapped from November 1983,April 1985). Voles exposed to a low dose had levels of free and total corticosterone that were significantly higher than those in the control or high-dose groups. Differences in response to radiation between the sexes were apparent for maximum corticosterone-binding capacity, with females exposed to low doses having higher binding capacity than control or high-dose females, whereas males exposed to low doses had lower binding capacity than control or high-dose males. Low-dose voles had higher counts of neutrophils than either the controls or high-dose voles; hematocrit was greater in the controls than in irradiated voles. These results indicate that voles display a hormetic response to radiation, wherein low doses of an otherwise harmful agent produce a beneficial effect. The stimulation of the stress axis resulting in the increased secretion of glucocorticoids, which may protect against the excessive actions of the immune and inflammatory responses, may be a key mechanism producing this effect. [source] Cadmium uptake by earthworms as related to the availability in the soil and the intestineENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2001Leonard A. Oste Abstract The free metal concentration in the soil solution is often considered a key parameter for metal uptake by and toxicity to soft-bodied soil organisms. The equilibrium partitioning theory, which assumes a relationship between the contaminant concentration in pore water and the contaminant concentration in the body tissue, can be used to describe uptake by earthworms. This theory has proved useful for organic chemicals, but its applicability is less clear for metals. In this study, the Cd concentration in soil pore water (pw) was varied by increasing the soil pH by the addition of lime (Ca(OH)2) and by adding manganese oxide (MnO2), which has a high metal binding capacity. Both lime (0.135% w/w) and MnO2 (1% w/w) decreased [Cd2+]pw by a factor of 25, while CdWorm was reduced only by a factor of 1.3 in lime-treated soils and 2.5 in MnO2 -treated soils. Cadmium uptake was weakly related to the free metal concentration (R2adj = 0.66). Adding pH as an explanatory variable increased R2adj to 0.89, indicating that Cd uptake from pore water is pH dependent, which might be attributed to competition of protons and Cd at the surface of the earthworm body. However, previous earthworm experiments in reconstituted groundwater showed a conspicuously smaller pH dependency of Cd uptake. The differences in metal uptake between earthworms in lime- and MnO2 -treated soils are therefore more likely to reflect the predominance of pH-independent intestinal uptake of Cd. Equilibrating the soil with a solution of 0.01 M CaCl2 and 0.1 M triethanolamine (buffered at pH 7.2), simulating the conditions prevailing in the worm intestine, yielded free Cd concentrations that were closely (R2adj = 0.83) and linearly related to the Cd concentration in the earthworm tissue. [source] Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4+ T-cell proliferation and IFN-, production in response to myelin basic protein and myelin oligodendrocyte glycoproteinEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2010Christopher T. Harp Abstract Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-, secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4+ T-cell proliferation and IFN-, secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4+ T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells. [source] Photopolymerizable Hydrogels Made from Polymer-Conjugated Albumin for Affinity-Based Drug Delivery,ADVANCED ENGINEERING MATERIALS, Issue 1-2 2010Liat Oss-Ronen As a drug delivery vehicle, biodegradable albumin hydrogels can combine the high binding capacity of albumin with the structural stability of a polymeric hydrogel network to enable controlled release of small molecules based on both binding affinity and physical interactions. In the present study, we report on the development of a hybrid hydrogel composed of albumin conjugated to poly(ethylene glycol) (PEG) for drug delivery applications where controlled release is accomplished using the natural affinity of the drugs to the serum albumin. Bovine serum albumin was conjugated to PEG-diacrylate having a molecular weight of 1.5, 4, or 10,kDa to form a PEGylated albumin macromolecule (mono-PEGylated or multi-PEGylated). Biodegradable hydrogels were formed from the PEGylated albumin using photopolymerization. Two model drugs, Warfarin and Naproxen, were used for equilibrium dialysis and release experiments from the hydrogels, both having relatively low molecular weights and a known high affinity for albumin. Equilibrium dialysis experiments showed that multi-PEGylation of albumin significantly decreased the drug affinity to the protein compared to non-PEGylated controls, irrespective of the PEG molecular weight. However, the results from drug release experiments showed that mono-PEGylation of albumin did not change its natural affinity to the drug. Comparing the release profiles with a Fickian diffusion model provided strong evidence that hydrogels containing mono-PEGylated albumin exhibited sub-diffusive drug release properties based on the affinity of the drug to the tethered protein. [source] Zinc ion adsorption on montmorillonite,Al hydroxide polymer systemsEUROPEAN JOURNAL OF SOIL SCIENCE, Issue 2 2003R. P. T. Janssen Summary Clay,Al hydroxide polymers (CAlHO) can bind heavy metals effectively and may play an important role in the adsorption behaviour and metal binding capacity of soils. We studied the dependence of Al loading and pH on the adsorption of Zn on Na-saturated montmorillonite,Al hydroxide polymer systems. The available binding sites on Al hydroxide polymers (AlHO) had a very strong affinity for Zn ions. Zinc binding on the clay surface became important when the binding sites on the AlHO were nearly all occupied. The pH had a very strong effect on the Zn binding. At pH 6.6 much more Zn could be adsorbed to the AlHO than at pH 5.0. The effect of the Al:clay ratio on Zn binding was influenced by pH. At pH 6.6, Zn binding to the AlHO, expressed per mole AlHO, was independent of the Al:clay ratio, whereas at pH 5.0 this relation was dependent. This is related to the constant charge of the AlHO at pH 6.6, whereas at pH 5.0 the charge decreases with increasing Al:clay ratio. If clay,Al hydroxide polymers are present in the soil their Zn binding to the AlHO will strongly influence the availability of the Zn. [source] Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activitiesEXPERIMENTAL DERMATOLOGY, Issue 8 2010Annica Hedberg Please cite this paper as: Deposition of chromatin-IgG complexes in skin of nephritic MRL-lpr/lpr mice is associated with increased local matrix metalloprotease activities. Experimental Dermatology 2010; 19: e265,e274. Abstract:, Chromatin-IgG complexes appear as electron dense structures (EDS) in glomerular basement membranes in lupus nephritis. Here, we present results of comparative analyses of the composition of EDS in murine lupus dermatitis and nephritis. One focus was to perform an analytical approach to understand why such complex structures bind skin basement membrane components. Transcription of skin membrane-encoding genes was analysed to see if expression of such genes was increased, eventually indicating that binding capacity of immune complexes increased when dermatitis developed. Variations in matrix metalloprotease 2 (MMP2), MMP9 and Dnase1 mRNA levels and enzymatic activities were correlated with circulatory chromatin-IgG complexes and deposition in skin. We also examined if glomerular deposits of EDS predicted similar deposits in skin of (NZB × NZW)F1 or MRL-lpr/lpr mice, as we observed chromatin-IgG complexes in capillary lumina in skin and glomeruli in both strains. EDS consisting of chromatin fragments and IgG were found sub-epidermally in skin with LE-like lesions of end-stage nephritic MRL-lpr/lpr mice. Dermal MMP-encoding genes were up-regulated during disease progression, and gelatinolytic activity was increased in affected skin. Dnase1 mRNA level and total nuclease activity remained stable in skin during the disease, in contrast to progressive loss of renal Dnase1 mRNA and total renal nuclease activity during development of nephritis. Loss of renal Dnase1 may explain release of chromatin fragments, while increased MMP activity may disrupt membranes making them accessible for chromatin fragment-IgG complexes. Circulatory chromatin-IgG complexes, and up-regulated intradermal MMP activity may be crucial for deposition of immune complexes in skin of lupus-prone mice. [source] Physicochemical properties and distinct DNA binding capacity of the repressor of temperate Staphylococcus aureus phage ,11FEBS JOURNAL, Issue 7 2009Tridib Ganguly The repressor protein and cognate operator DNA of any temperate Staphylococcus aureus phage have not been investigated in depth, despite having the potential to enrich the molecular biology of the staphylococcal system. In the present study, using the extremely pure repressor of temperate Staphylococcus aureus phage ,11 (CI), we demonstrate that CI is composed of ,-helix and ,-sheet to a substantial extent at room temperature, possesses two domains, unfolds at temperatures above 39 °C and binds to two sites in the ,11 cI - cro intergenic region with variable affinity. The above CI binding sites harbor two homologous 15 bp inverted repeats (O1 and O2), which are spaced 18 bp apart. Several guanine bases located in and around O1 and O2 demonstrate interaction with CI, indicating that these 15 bp sites are used as operators for repressor binding. CI interacted with O1 and O2 in a cooperative manner and was found to bind to operator DNA as a homodimer. Interestingly, CI did not show appreciable binding to another homologous 15 bp site (O3) that was located in the same primary immunity region as O1 and O2. Taken together, these results suggest that ,11 CI and the ,11 CI,operator complex resemble significantly those of the lambdoid phages at the structural level. The mode of action of ,11 CI, however, may be distinct from that of the repressor proteins of , and related phages. [source] Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalisFEBS JOURNAL, Issue 22 2001Mari Yotsu-Yamashita Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 ± 0.97 nmol·mg,1 protein and a Kd of 14.6 ± 0.33 nm for [3H]saxitoxin in equilibrium binding assays. [3H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 µm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish. [source] Coupling of endothelin receptors to the ERK/MAP kinase pathway,FEBS JOURNAL, Issue 20 2001Roles of palmitoylation Endothelins are potent mitogens that stimulate extracellular signal-regulated kinases (ERK/MAP kinases) through their cognate G-protein-coupled receptors, ETA and ETB. To address the role of post-translational ET receptor modifications such as acylation on ERK activation and to identify relevant downstream effectors coupling the ET receptor to the ERK signaling cascades we have constructed a panel of palmitoylation-deficient ET receptor mutants with differential G, protein binding capacity. Endothelin-1 stimulation of wild-type ETA or ETB induced a fivefold to sixfold increase in ERK in COS-7 and CHO cells whereas full-length nonpalmitoylated ETA and ETB mutants failed to stimulate ERK. A truncated ETB lacking the C-terminal tail domain including putative phosphorylation and arrestin binding site(s) but retaining the critical palmitoylation site(s) was still able to fully stimulate ERK activation. Using mutated ET receptors with selective G-protein-coupling we found that endothelin-induced stimulation of G,q, but not of G,i or G,s, is essential for endothelin-mediated ERK activation. Inhibition of protein kinases A and C or epidermal growth factor receptor kinase failed to prevent ETA - and ETB -mediated ERK activation whereas blockage of phospholipase C-, completely abrogated endothelin-promoted ERK activation through ETA and ETB in recombinant COS-7 and native C6 cells. Complex formation of Ca2+ or inhibition of Src family tyrosine kinases prevented ET-1-induced ERK-2 activation in C6-cells. Our results indicate that endothelin-promoted ERK/MAPK activation criticially depends on palmitoylation but not on phosphorylation of ET receptors, and that the G,q/phospholipase C-,/Ca2+/Src signaling cascade is necessary for efficient coupling of ET receptors to the ERK/MAPK pathway. [source] Preparation and Characteristics of Esculin-Imprinted PolymersHELVETICA CHIMICA ACTA, Issue 6 2007Guo-Song Wang Abstract Four molecularly imprinted polymers (MIPs) were prepared in MeOH with esculin (=6,7-dihydroxycoumarin 6-(, - D -glucopyranoside)=6-(, - D -glucopyranosyloxy)-7-hydroxy-2H -1-benzopyran-2-one) as the imprinted molecule, methacrylic acid (=2-methylprop-2-enoic acid; MAA), acrylamide (=prop-2-enamide; AM), 4-vinylpyridine (=4-ethenylpyridine; 4-VP), or 2-vinylpyridine (=2-ethenylpyridine; 2-VP) as the functional monomer, respectively, as well as ethylene glycol dimethacrylate (=2-methylprop-2-enoic acid ethane-1,2-diyl ester; EGDMA) as the cross-linking agent. The interaction between the template and the functional monomers was investigated by fluorescence and UV spectrophotometry, respectively, which revealed the presence of esculin/monomer complexes in the stoichiometric ratio 1,:,2 in the pre-polymerization mixture. The resultant polymers were studied in equilibrium binding experiments to evaluate the recognition ability and the binding capacity towards esculin. The results showed that MIP1, prepared with MAA as the functional monomer, exhibited advantageous characteristics of high binding capacity, optimal imprinting effect, and good selectivity towards esculin. The Scatchard analysis indicated that there are two types of binding sites in MIP1, and its binding parameters including the apparent maximum numbers of binding sites and the dissociation constants were calculated. Finally, by packing an SPE column (SPE=solid-phase extraction) with MIP1, the esculin was separated and enriched successfully by this sorbent from samples of Cortex fraxini, and the average recovery was up to 74.7%. [source] Direct and indirect manipulation of the MEK-ERK pathway regulates the formation of a pericellular HA-dependent matrix by chick articular surface cells without modifying CD44 expresssionINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Edward R. Bastow Introduction Recent evidence suggests that hyaluronan (HA) facilitates the mechano-dependent joint cavity-forming process through the elaboration and retention of a HA-rich pericellular matrix in the developing joint interzone (IZ). The presumptive joint IZ phenotype shows a capacity to bind and synthesize HA and also exhibits elevated activated ERK, prior to synovial joint cavity formation (Lamb et al. 2001; Edwards et al. 1994; Dowthwaite et al. 1998). We have found that immobilization, which induces embryonic joint fusion with loss of the joint IZ phenotype, also reduces ERK activity levels in the IZ. As the signalling events regulating the synthesis and binding of HA have yet to be determined, we hypothesize that ERK activation plays a pivotal role in determining the presumptive joint IZ phenotype through HA synthetic and binding capacity. Materials and methods Chick articular surface (AS) cells were harvested from proximal tibiotarsal joints of embryos by collagenase digestion. Pericellular coat formation was assessed using the erythrocyte exclusion assay and cell-coat area ratios determined. ERK activity was modulated by transient transfection of GFP constructs of constitutively active (CA-) or dominant negative (DN-) forms of MEK, the direct upstream regulator of ERK or by treatment with the MEK inhibitor PD98059 (50 µm). ERK activation was monitored by immunochemistry. CD44 expression and ERK activation in PD98059-treated cells were monitored by immunoblotting and medium HA concentrations by ELISA. Results AS cells form large pericellular coats that are lost following hyaluronidase treatment and thus dependent upon HA for their construction. Treatment with PD98059 significantly reduced pericellular coat formation after 6 h. In parallel, we confirmed that PD98059 diminished active ERK expression without modifying overall levels of ERK, suggesting that the elaboration of large HA-pericellular coats is dependent upon MEK's activation of ERK. Western blot analysis of PD98059-treated cells showed that loss of pericellular coats was not, however, associated with any decreased levels of the cell surface HA receptor CD44. Although treatment with PD98059 did not change medium HA concentration after short times of exposure, at times (up to 6 h) during which coat loss was evident, prolonged treatment over 24 h significantly decreased medium HA concentration. Consistent with a role for ERK in pericellular coat formation, transfection with DN-MEK diminished, while CA-MEK increased, both active ERK expression and coat formation efficiency. We also found that, commensurate with this modification in coat forming efficiency, cells expressing DN-MEK exhibited a significant reduction in labelling of free HA on the cell surface. Discussion These studies extend our recent work to indicate that: (i) direct modulation of ERK activation by transfection with its endogenous upstream regulator modifies cell surface-associated HA (ii) PD98059-induced blockade of ERK activation restricts medium HA release and (iii) ERK-mediated changes in pericellular coat elaboration are independent of changes in cellular CD44 expression. These findings suggest an intimate relationship between ERK activation and the formation/retention of HA-rich pericellular matrices in vitro and highlight the role for ERK activation in regulating joint line-related differentiation. [source] ORIGINAL ARTICLE: Pro-hepcidin and iron metabolism parameters in multi-time blood donorsINTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY, Issue 5 2010J. BOINSKA Summary A high number of blood donations may cause iron depletion. The pathophysiology behind this process may involve hepcidin, a recently discovered peptide that acts by inhibiting iron absorption and promoting iron retention in reticuloendothelial macrophages. The aim of this study was to determine serum pro-hepcidin levels and iron metabolism parameters in multi-time blood donors. The study group consisted of 132 multi-time male blood donors and 25 healthy male volunteers (nondonors). Complete blood cell count and iron status including serum iron, ferritin, soluble transferrin receptor (sTfR), total iron binding capacity (TIBC), unsaturated iron binding capacity (UIBC), erythropoietin and pro-hepcidin (ELISA) were assessed. In blood donors, ferritin level drops markedly in relation to donation frequency (P < 0.001). In contrast, TIBC and UIBC levels increase progressively corresponding to annual donation frequency. Pro-hepcidin concentration increases significantly with the number of donations per year (P = 0.0290). In blood donors having donated blood with the highest frequency per year, pro-hepcidin levels were positively correlated with haemoglobin (R = 0.31, P < 0.05) and negatively with sTfR (R = ,0.31, P < 0.05). Pro-hepcidin levels increase in relation to blood donation frequency per year. Longitudinal studies focusing on changes in serum hepcidin levels are required to address the question whether hepcidin may contribute to iron metabolism disturbances in multi-times blood donors. [source] Oxygen Controls the Phosphorus Release from Lake Sediments , a Long-Lasting Paradigm in LimnologyINTERNATIONAL REVIEW OF HYDROBIOLOGY, Issue 4-5 2008Michael Hupfer Abstract The pioneer works of Einsele, Mortimer, and Ohle on the linking between phosphorus (P) and iron (Fe) cycles seven decades ago created the theoretical basis for a long-standing paradigm among limnologists i.e., ,oxygen controls the P release from sediments'. While many empirical studies as well as strong correlations between oxygen depletion and P release seem to support this paradigm, various field observations, laboratory experiments, and repeated failures of hypolimnetic oxygenation measures cast doubt on its universal validity. The temporal existence of a thin oxidized sediment surface-layer could affect only fluctuations of the temporary P pool at the sediment surface but not the long-term P retention. On longer time scales P release is the imbalance between P sedimentation and P binding capacity of anoxic sediment layers. The P retention of lake sediments strongly depends on sediment characteristics and land use of the catchment. The presence of redox-insensitive P-binding systems such as Al(OH)3 and unreducible Fe(III) minerals can enhance the P retention and completely prevent P release even in case of anoxic conditions. Alternative release mechanisms such as a dissolution of calcium-bound P and decomposition of organic P under both, aerobic and anaerobic conditions, are often more important than the redox driven Fe-coupled P cycle. Additionally, bacteria affect P cycling not only by altering the redox conditions but also by releasing P during mineralization of organic matter and by accumulation and release of bacterial P. Since microbial processes consume oxygen and liberate P it is difficult to distinguish whether oxygen depletion is the result or the cause of P release. Nowadays, the old paradigm is discarded and a paradigm shift takes place. Sedimentary P exchange ought to be considered as a complex process which is mainly determined by the amount and species of settled P as well as their subsequent diagenetic transformation in the sediment. The classical paradigm is only valid in special cases since reality is much more complex than suggested by that paradigm. Everything should be made simple as possible, but not simpler! Albert Einstein (© 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] | ||||||||||||||||||||||||||||||||||||||||