Initial characterization of ethyl(hydroxyethyl) cellulose using enzymic degradation and chromatographic methods

Two different ethyl(hydroxyethyl) cellulose (EHEC) samples were characterized by size-exclusion chromatography (SEC) with multiangle light scattering (MALS) detection and high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The aim of the study was to investigate the molar mass distribution and the heterogeneity of the substituent distribution, factors that are thought to affect the functional properties of EHEC. The presence of blocks of unsubstituted glucose units was studied by enzymic degradation of EHEC by two different endoglucanases from Trichoderma reesei. The SEC-MALS analysis of the hydrolysis products showed that both enzymes were strongly inhibited by the large number of substituents along the cellulose chain. However, as the weight-average molar mass was reduced from approximately 360,000 to 80,000 g/mol in one of the polymers and from 770,000 to 60,000 g/mol in the other polymer, it was suggested that both samples were composed of some unsubstituted regions where the enzymes got access to the glucosidic bonds. The amount of glucose released upon endoglucanase hydrolysis was determined by HPAEC-PAD, which gave information on the homogeneity of the substituent distribution. The production of unsubstituted glucose units indicated that one of the polymers had a more uneven distribution compared with the other. It was demonstrated that chemical characterization of EHEC is a complex task, which requires an analytical approach involving numerous different methods and techniques.     

Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.     

Freshwater for resilience: a shift in thinking

Humanity shapes freshwater flows and biosphere dynamics from a local to a global scale. Successful management of target resources in the short term tends to alienate the social and economic development process from its ultimate dependence on the life-supporting environment. Freshwater becomes transformed into a resource for optimal management in development, neglecting the multiple functions of freshwater in dynamic landscapes and its fundamental role as the bloodstream of the biosphere. The current tension of these differences in worldview is exemplified through the recent development of modern aquaculture contrasted with examples of catchment-based stewardship of freshwater flows in dynamic landscapes. In particular, the social and institutional dimension of catchment management is highlighted and features of social-ecological systems for resilience building are presented. It is concluded that this broader view of freshwater provides the foundation for hydrosolidarity.     

Tryptophan-tagged cutinase studied by steady state fluorescence for understanding of tag interactions in aqueous two-phase systems

Genetic engineering has been used to construct fusion proteins of Fusarium solani pisi cutinase and tryptophan-based tags, expressed in Saccharomyces cerevisiae, to increase the partitioning in aqueous two-phase systems. The separation systems were composed of thermoseparating polymers (random copolymers of ethylene oxide and propylene oxide, EOPO) and detergents (C(12)EO(n)). In this study, the fluorescence behaviour of the peptide-tagged protein, free peptide tag and tryptophan was investigated. The tryptophan-tagged proteins, cutinase-(WP)(4) and cutinase-TGGSGG-(WP)(4), showed emission spectra similar to the free peptides and tryptophan, indicating solvent exposure of the tag. The influence of polymers and detergents on the fluorescence of tagged proteins was examined. When peptides and tagged proteins were exposed to polymer, a slight blue shift of the emission maximum was observed. Larger blue shifts of the emission maximum were observed when C(12)EO(n) detergents were utilised. The results correlate with aqueous two-phase partitioning where addition of C(12)EO(n) detergents results in more extreme partitioning compared to systems containing only polymers. Dynamic light scattering (DLS) measurements of the EOPO copolymers were carried out, showing that the polymers did not aggregate at concentrations used in aqueous two-phase systems. Quenching of fluorescence with iodide for both proteins and peptide tags was studied. Plots according to the Stern-Volmer equation resulted in a linear fit, indicating exposed tryptophan residues for both free peptides and fusion proteins. The quenching constants were similar for both tagged protein and free peptide tag. The fluorescence results indicated that the tryptophan residues in the tag were exposed to the solvent and could interact with detergents and polymers in the two-phase systems.     

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.     

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.     

Coral reef disturbance and resilience in a human-dominated environment

Facing a human-dominated world, ecologists are now reconsidering the role of disturbance for coral reef ecosystem dynamics. Human activities alter the natural disturbance regimes of coral reefs by transforming pulse events into persistent disturbance or even chronic stress, by introducing new disturbance, or by suppressing or removing disturbance. Adding these alterations to natural disturbance regimes will probably result in unknown synergistic effects. Simultaneously, humans are altering the capacity of reefs to cope with disturbance (e.g. by habitat fragmentation and reduction of functional diversity), which further exacerbates the effects of altered disturbance regimes. A disturbance that previously triggered the renewal and development of reefs might, under such circumstances, become an obstacle to development. The implications of these changes for reef-associated human activities, such as fishing and tourism, can be substantial.     

The chaperone-like activity of a small heat shock protein is lost after sulfoxidation of conserved methionines in a surface-exposed amphipathic alpha-helix

The small heat shock proteins (sHsps) possess a chaperone-like activity which prevents aggregation of other proteins during transient heat or oxidative stress. The sHsps bind, onto their surface, molten globule forms of other proteins, thereby keeping them in a refolding competent state. In Hsp21, a chloroplast-located sHsp in all higher plants, there is a highly conserved region forming an amphipathic alpha-helix with several methionines on the hydrophobic side according to secondary structure prediction. This paper describes how sulfoxidation of the methionines in this amphipathic alpha-helix caused conformational changes and a reduction in the Hsp21 oligomer size, and a complete loss of the chaperone-like activity. Concomitantly, there was a loss of an outer-surface located alpha-helix as determined by limited proteolysis and circular dichroism spectroscopy. The present data indicate that the methionine-rich amphipathic alpha-helix, a motif of unknown physiological significance which evolved during the land plant evolution, is crucial for binding of substrate proteins and has rendered the chaperone-like activity of Hsp21 very dependent on the chloroplast redox state.     

Cause-Specific Cardiovascular Risk Associated with Nonsteroidal Anti-Inflammatory Drugs among Myocardial Infarction Patients - A Nationwide Study

Background

Non steroidal anti-inflammatory drugs (NSAIDs) increase mortality and morbidity after myocardial infarction (MI). We examined cause-specific mortality and morbidity associated with NSAIDs in a nationwide cohort of MI patients.

Methods and Results

By individual-level linkage of nationwide registries of hospitalization and drug dispensing from pharmacies in Denmark, patients aged >30 years admitted with first-time MI during 1997–2009 and their subsequent NSAID use were identified. The risk of three cardiovascular specific endpoints: cardiovascular death, the composite of coronary death and nonfatal MI, and the composite of fatal and nonfatal stroke, associated with NSAID use was analyzed by Cox proportional hazard analyses. Of 97,698 patients included 44.0% received NSAIDs during follow-up. Overall use of NSAIDs was associated with an increased risk of cardiovascular death (hazard ratio [HR] 1.42, 95% confidence interval [CI] 1.36–1.49). In particular use of the nonselective NSAID diclofenac and the selective cyclooxygenase-2 inhibitor rofecoxib was associated with increased risk of cardiovascular death (HR 1.96 [1.79–2.15] and HR1.66 [1.44–1.91], respectively) with a dose dependent increase in risk. Use of ibuprofen was associated with increased risk of cardiovascular death (HR 1.34[1.26–1.44]), whereas naproxen was associated with the lowest risk of (e.g., HR 1.27[1.01–1.59].

Conclusion

Use of individual NSAIDs is associated with different cause-specific cardiovascular risk and in particular rofecoxib and diclofenac were associated with increased cardiovascular morbidity and mortality. These results support caution with use of all NSAIDs in patients with prior MI.     

Characterization of chemical substitution of hydroxypropyl cellulose using enzymatic degradation

The distribution of substituents along the polymer backbone will have a strong influence on the properties of modified cellulose. Endoglucanases were used to degrade a series of hydroxypropyl cellulose (HPC) derivatives with a high degree of substitution. The HPCs were characterized with cloud-point analysis prior to degradation. The extent of enzymatic degradation was determined with size-exclusion chromatography with online multi-angle light scattering and refractive index detection and also with high-pH anion exchange chromatography with pulsed amperometric detection. To further characterize the formed products, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was employed for analysis of short-chained oligosaccharides. The different endoglucanases showed varying degradation capability depending on structure of the active site. The highly substituted HPCs had different susceptibility to degradation by the endoglucanases. The results show a difference in substituent distribution between HPCs, which would explain the differing cloud-point behaviors. Increased number of regions with low substitution could be correlated with lower polymer cloud point. The study shows the usefulness of enzymatic degradation to study the distribution of substituents in soluble biopolymer derivates.