Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.)

Fructans, β2-1 and/or β2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively β2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed—as did the native enzyme from wheat—a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PC

Environmental factors influencing older adults\u27 walking for transportation: a study using walk-along interviews

<p>Abstract</p> <p>Background</p> <p>Current knowledge on the relationship between the physical environment and walking for transportation among older adults (≥ 65 years) is limited. Qualitative research can provide valuable information and inform further research. However, qualitative studies are scarce and fail to include neighborhood outings necessary to study participants’ experiences and perceptions while interacting with and interpreting the local social and physical environment. The current study sought to uncover the perceived environmental influences on Flemish older adults’ walking for transportation. To get detailed and context-sensitive environmental information, it used walk-along interviews.</p> <p>Methods</p> <p>Purposeful convenience sampling was used to recruit 57 older adults residing in urban or semi-urban areas. Walk-along interviews to and from a destination (e.g. a shop) located within a 15 minutes’ walk from the participants’ home were conducted. Content analysis was performed using NVivo 9 software (QSR International). An inductive approach was used to derive categories and subcategories from the data.</p> <p>Results</p> <p>Data were categorized in the following categories and subcategories: access to facilities (shops & services, public transit, connectivity), walking facilities (sidewalk quality, crossings, legibility, benches), traffic safety (busy traffic, behavior of other road users), familiarity, safety from crime (physical factors, other persons), social contacts, aesthetics (buildings, natural elements, noise & smell, openness, decay) and weather.</p> <p>Conclusions</p> <p>The findings indicate that to promote walking for transportation a neighborhood should provide good access to shops and services, well-maintained walking facilities, aesthetically appealing places, streets with little traffic and places for social interaction. In addition, the neighborhood environment should evoke feelings of familiarity and safety from crime. Future quantitative studies should investigate if (changes in) these environmental factors relate to (changes in) older adults’ walking for transportation.</p

Learning about pain from others: an observational learning account

Although direct experience and verbal instruction are important sources in the development of pain-related beliefs and behaviors, accumulating evidence indicates that observation of others in pain may be equally as important. Taking a contemporary view on learning as a starting point, we discuss available evidence on observational learning in the context of pain, highlight its importance for both development and management of chronic pain problems, and discuss potential moderators of observational learning effects. We argue that the capacity to understand and appreciate the experience of another person is fundamental to observational learning, including use of this information to establish the association between pain and antecedent or consequent stimuli. A main objective of this paper is to stimulate research on the role of learning about pain from others. Several lines for further research, including clinical applications, are delineated. Perspective: Based upon a contemporary view on learning, this focus article delineates how pain-related beliefs and behaviors may be learnt by observing others. It is discussed how further research on the acquisition of pain-related beliefs/behaviors might further our understanding of pain and disability. (PsycINFO Database Record (c) 2011 APA, all rights reserved) (journal abstract

Purification, cloning and functional characterization of a fructan 6-exohydrolase from wheat (Triticum aestivum L.)

Fructans, β2-1 and/or β2-6 linked polymers of fructose, are produced by fructosyltransferases (FTs) from sucrose. They are important storage carbohydrates in many plants. Fructan reserves, widely distributed in plants, are believed to be mobilized via fructan exohydrolases (FEHs). The purification, cloning, and functional characterization of a 6-FEH from wheat (Triticum aestivum L.) are reported here. It is the first FEH shown to hydrolyse exclusively β2-6 bonds found in a fructan-producing plant. The enzyme was purified to homogeneity using ammonium sulphate precipitation, ConA affinity-, ion exchange-, and size exclusion chromatography and yielded a single band of 70 kDa following SDS-PAGE. Sequence information obtained by mass spectrometry of in-gel trypsin digests demonstrated the presence of a single protein. Moreover, these unique peptide sequences, together with some ESTs coding for them, could be used in a RT-PCR based strategy to clone a 1.7 kb cDNA. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed—as did the native enzyme from wheat—a very high activity of the produced protein against bacterial levan, 6-kestose, and phlein whilst sucrose and inulin were not used as substrates. Therefore the enzyme is a genuine 6-FEH. In contrast to most FEHs from fructan-accumulating plants, this FEH is not inhibited by sucrose. The relative abundance of 6-FEH transcripts in various tissues of wheat was investigated using quantitative RT-PC

Fructan 1-Exohydrolases. β-(2,1)-Trimmers during Graminan Biosynthesis in Stems of Wheat? Purification, Characterization, Mass Mapping, and Cloning of Two Fructan 1-Exohydrolase Isoforms

Graminan-type fructans are temporarily stored in wheat (Triticum aestivum) stems. Two phases can be distinguished: a phase of fructan biosynthesis (green stems) followed by a breakdown phase (stems turning yellow). So far, no plant fructan exohydrolase enzymes have been cloned from a monocotyledonous species. Here, we report on the cloning, purification, and characterization of two fructan 1-exohydrolase cDNAs (1-FEH w1 and w2) from winter wheat stems. Similar to dicot plant 1-FEHs, they are derived from a special group within the cell wall-type invertases characterized by their low isoelectric points. The corresponding isoenzymes were purified to electrophoretic homogeneity, and their mass spectra were determined by quadrupole-time-of-flight mass spectrometry. Characterization of the purified enzymes revealed that inulin-type fructans [β-(2,1)] are much better substrates than levan-type fructans [β-(2,6)]. Although both enzymes are highly identical (98% identity), they showed different substrate specificity toward branched wheat stem fructans. Although 1-FEH activities were found to be considerably higher during the fructan breakdown phase, it was possible to purify substantial amounts of 1-FEH w2 from young, fructan biosynthesizing wheat stems, suggesting that this isoenzyme might play a role as a β-(2,1)-trimmer throughout the period of active graminan biosynthesis. In this way, the species and developmental stage-specific complex fructan patterns found in monocots might be determined by the relative proportions and specificities of both fructan biosynthetic and breakdown enzymes

The use of larvae of the black soldier fly as a high quality and sustainable protein source in broiler feed

The growing scarcity of protein sources for animal feed in the European Union has strongly increased prices in recent years. As a consequence, the industry is looking for alternative protein sources that can be used for animal feed. Several studies have proposed insects as a high quality, efficient and sustainable alternative protein source. A promising insect with a high protein content and short life-cycle is the black soldier fly (Hermetia illucens). In this project, we introduced larvae of the black soldier fly in feed for broiler chickens. To this end, the nutritional composition of larvae grown on super market waste was determined. Based on these data, broiler feed was formulated and subsequently the larvae were processed in broiler feed. All feeds were isoenergetic and isonitrogenous for the entire grow up (starter, grower and finisher). About a third of the soybean meal was replaced by the larvae. We evaluated the effect of this insect feed on broilers by assessing their growth performance, health status, meat quality and meat taste and compared the results with a control group that received conventional feed. No significant differences were observed in mean body weight, net carcass weight and feed conversion ratio between broilers that received conventional feed and insect feed. In addition, no significant difference was found between the taste of chicken breast from broilers which were grown on insect feed and grown on conventional feed.status: publishe