Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in edible burdock (Arctium lappa L.)

<p>Abstract</p> <p>Background</p> <p>We have previously reported on the variation of total fructooligosaccharides (FOS), total inulooligosaccharides (IOS) and inulin in the roots of burdock stored at different temperatures. During storage at 0°C, an increase of FOS as a result of the hydrolysis of inulin was observed. Moreover, we suggested that an increase of IOS would likely be due to the synthesis of the IOS by fructosyltransfer from 1-kestose to accumulated fructose and elongated fructose oligomers which can act as acceptors for fructan:fructan 1-fructosyltransferase (1-FFT). However, enzymes such as inulinase or fructan 1-exohydorolase (1-FEH) involved in inulin degradation in burdock roots are still not known. Here, we report the isolation and functional analysis of a gene encoding burdock 1-FEH.</p> <p>Results</p> <p>A cDNA, named <it>aleh1</it>, was obtained by the RACE method following PCR with degenerate primers designed based on amino-acid sequences of FEHs from other plants. The <it>aleh1 </it>encoded a polypeptide of 581 amino acids. The relative molecular mass and isoelectric point (<it>pI</it>) of the deduced polypeptide were calculated to be 65,666 and 4.86. A recombinant protein of <it>aleh1 </it>was produced in <it>Pichia pastoris</it>, and was purified by ion exchange chromatography with DEAE-Sepharose CL-6B, hydrophobic chromatography with Toyopearl HW55S and gel filtration chromatography with Toyopearl HW55S. Purified recombinant protein showed hydrolyzing activity against β-2, 1 type fructans such as 1-kestose, nystose, fructosylnystose and inulin. On the other hand, sucrose, neokestose, 6-kestose and high DP levan were poor substrates.</p> <p>The purified recombinant protein released fructose from sugars extracted from burdock roots. These results indicated that <it>aleh1 </it>encoded 1-FEH.</p

Studies of the Electrical Parameters and Behavioral Effects of an EEG \u27\u27Seizure Discharge

Nemastrina monkeys with multiple electrodes implanted in the amygdaloid area of the brain were used to study the relationship of electrical seizures and overt behavior. The electrical seizures were produced by long duration electrical pulses administered through the implanted electrodes. There appears to be no relationship between this kind of electrical seizure and lever pressing performance. Relationships between depth EEG, depth impedance, DC potential and surface EEG are observed and as a consequence some ideas concerning the genesis of the electroencephalogram are put forth

Complete Genome Sequence of the Itraconazole Decreased Susceptible <i>Madurella fahalii</i> Type-Strain CBS 129176

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein

Complete Genome Sequence of the Itraconazole Decreased Susceptible Madurella fahalii Type-Strain CBS 129176

Madurella fahalii is a causative agent of the implantation mycosis mycetoma with decreased susceptibility to itraconazole, the preferred therapeutic drug to combat mycetoma. Here, we report the M. fahalii type-strain CBS 129176 genome assembly and annotation to identify a glutamic acid insert near the azole-binding pocket in the Cyp51A protein

Factors associated with physiotherapy provision in a population of elderly nursing home residents; a cross sectional study

BACKGROUND: Although physiotherapy (PT) plays an important role in improving activities of daily living (ADL functioning) and discharge rates, it is unclear how many nursing home residents receive treatment. Furthermore, there is a lack of insight into the determinants that influence the decision for treatment. In this study, we investigated how many nursing home residents receive PT. In addition, we analysed the factors that contribute to the variation in the provision of PT both between nursing homes and between residents. METHODS: A random sample of 600 elderly residents was taken from a random sample of 15 nursing homes. Residents had to be admitted for rehabilitation or for long-term care. Data were collected through interviews with the nursing home physician and the physiotherapist. Multilevel analysis was used to define the variation in the provision of PT and the factors that are associated with the question whether a resident receives PT or not. Furthermore the amount of PT provided was analysed and the factors that are associated with this. RESULTS: On average 69% of the residents received PT. The percentage of patients receiving treatment differed significantly across nursing homes, and especially the number of physiotherapists available, explained this difference between nursing homes. Residents admitted to a somatic ward for rehabilitation, and male residents in general, were most likely to receive PT. Residents who were treated by a physiotherapist received on average 55 minutes (sd 41) treatment a week. Residents admitted for rehabilitation received more PT a week, as were residents with a status after a total hip replacement. CONCLUSION: PT is most likely to be provided to residents on a somatic ward, recently admitted for rehabilitation to a nursing home, which has a relatively large number of physiotherapists. This suggests a potential under-use of PT for long-term residents with cognitive problems. It is recommended that physiotherapists reconsider which residents may benefit from treatment. This may require a shift in the focus of physiotherapists from 'recovery and discharge' to 'quality of life and well-being'

Linear viscoelastic behavior of aggregated colloidal dispersions

The viscoelastic behavior of a depletion-flocculated dispersion of colloidal spheres is investigated at different volume fractions of the spheres, using a controlled stress and a dynamic rheometer. Combining the results, we obtain the storage G′ and loss G′′ moduli over a frequency range of 0.02<ω<200rad/s. The measured G′ gradually increases with increasing frequency, while G′′ almost remains constant, indicating a broad spectrum of relaxation times. To describe and explain the observed behavior of the moduli as a function of frequency and volume fraction in terms of microscopic parameters, a microrheological model based on the fractal concept is proposed. Comparing experimental results with model calculations, we find a good agreement between the two, with physically plausible parameter values

The X-ray diffraction structure of a glycosyl hydrolase family 32 protein: 1-fructan exohydrolase IIa of<i>Cichorium intybus</i>

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Wheat genotypic variation in dynamic fluxes of WSC components in different stem segments under drought during grain filling

In wheat, stem water soluble carbohydrates (WSC), composed mainly of fructans, are the major carbon sources for grain filling during periods of decreasing photosynthesis or under drought stress after anthesis. Here, in a field drought experiment, WSC levels and associated enzyme activities were followed in different stem segments (peduncle, penultimate internode, lower parts of stem, and sheath) during grain filling. The focus was on two double haploid (DH) lines, DH 307 and DH 338, derived from a Westonia/Kauz cross, two drought-tolerant wheat varieties that follow different drought adaptation strategies during grain filling. The results showed that in irrigated plants, in the period between 20 and 30 days after anthesis (DAA), 70–80% of WSC were fructans. Before and after this period, the fructan proportion varied from 10 to 60%, depending on the location along the stem. Under drought, the fructan proportion changed, depending on genotype, and developmental stages. After anthesis, stem fructans accumulation occurred mainly in the peduncle and penultimate internode until 14 DAA in both DH lines, with clear genotypic variation in subsequent fructan degradation under drought. In DH 307 a significant reduction of fructans with a concomitant increase in fructose levels occurred earlier in the lower parts of the stem and the sheath, as compared to DH 338 or other stem segments in both lines. This was associated with an earlier increase of grain weight and thousand grain weight in DH 307. Spatiotemporal analysis of fructan dynamics and enzymatic activities in fructan metabolism revealed that several types of FEHs are involved in fructan remobilization to the grain under drought

An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases

Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne)

The main storage compounds in Lolium perenne are fructans with prevailing β(2–6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare