Arabidopsis AtcwINV3 and 6 are not invertases but are fructan exohydrolases (FEHs) with different substrate specificities

The genome of Arabidopsis thaliana contains six putative cell-wall type invertase genes (AtcwINV1-6). Heterologous expression of AtcwINV1, 3 and 6 cDNAs in Pichia pastoris revealed that the enzymes encoded by AtcwINV3 and 6 did not show invertase activity. Instead, AtcwINV3 is a 6-FEH and AtcwINV6 is a fructan exohydrolase (FEH) that can degrade both inulin and levan-type fructans. For AtcwINV6 it is proposed to use the term (6&1) FEH. In contrast, AtcwINV1 is a typical invertase. FEH activity was also detected in crude extracts of different parts of Arabidopsis. To verify that the FEH activity of AtcwINV3 and 6 were not artefacts of the heterologous expression system, the protein corresponding to AtcwINV3 was isolated from whole Arabidopsis plants and indeed showed only 6-FEH activity and no invertase activity. Although no fructans can be detected in Arabidopsis plants, it is shown that kestoses (trimers) can be synthesized in crude leaf extracts. The putative physiological significance of FEH in so-called non-fructan plants is discussed.     

Increased breast density correlates with the proliferation-seeking radiotracer (99m)Tc(V)-DMSA uptake in florid epithelial hyperplasia and in mixed ductal carcinoma in situ with invasive ductal carcinoma but not in pure invasive ductal carcinoma or in mild epithelial hyperplasia

The purpose of this study was to assess the relationship of mammographic breast density (BD) and cell proliferation/focal adhesion kinase activation-seeking radiotracer technetium 99m pentavalent dimercaptosuccinic acid (99mTc(V)-DMSA) uptake in women with different breast histologies, that is, mild epithelial hyperplasia (MEH), florid epithelial hyperplasia (FEH), mixed ductal carcinoma in situ with invasive ductal carcinoma (DCIS + IDC), and pure IDC. Fifty-five women with histologically confirmed mammary pathologies were submitted preoperatively to mammography and 99mTc(V)-DMSA scintimammography. The percentage and intensity of 99mTc(V)-DMSA uptake and the percentage of BD were calculated by computer-assisted methods and compared (t-test) between the breast pathologies.?In breasts with increased BD, FEH and DCIS + IDC were found. On the contrary, pure IDC and MEH were identified in breasts with significantly lower BD values. In breasts with increased 99mTc(V)-DMSA area and intensity of uptake, FEH was the main lesion found compared to all other histologies. Linear regression analysis between BD and 99mTc(V)-DMSA uptake area and intensity revealed significant coefficients of correlation (r = .689, p < .001 and r = .582, p < .001, respectively). Increased BD correlates with the presence of FEH and mixed DCIS + IDC but not with pure IDC or MEH. Its close relationship to 99mTc(V)-DMSA, which also showed an affinity to FEH, indicates that stromal microenvironment may constitute a specific substrate leading to progression to different subtypes of cancerous lesions originating from different pathways.     

Fructosyl transferase and hydrolase activities in rhizophores and tuberous roots upon growth of Polymnia sonchifolia (Asteraceae)

The underground reserve organs of yacon (Polymnia sonchifolia Poep. Endl.), similarly to other economically important Asteraceae, accumulate more than 60%, on a DW basis, of inulin type β(2‐1) fructans, mainly oligomers (GF2–GF16). Although sucrose:sucrose 1‐fructosyl transferase (1‐SST), fructan:fructan 1‐fructosyl transferase (1‐FFT) and fructan 1‐exohydrolase (1‐FEH) were properly described and characterized from a number of plant species, detailed information about their activities in different organs during development are rather scarce in the literature. In the present work 1‐SST, 1‐FFT and 1‐FEH activities were measured monthly in rhizophores and tuberous roots of yacon plants during their complete growth cycle under field conditions. Results showed that 1‐SST activity in rhizophores was always higher than 1‐FFT activity and increased up to 8 months of cultivation, decreasing to initial values at the end of the growth period. In the tuberous roots 1‐SST activity was also higher than 1‐FFT but varied differently. The higher values were found at the beginning of tuberization (3‐month‐old plants) and at the flowering phase (7‐month‐old plants). Results also showed that synthesizing activities in yacon plants were always higher in rhizophores than in the tuberous roots, while hydrolysing activity predominated in the latter, mainly when 1‐kestose and nystose were used as substrates. 1‐FEH from yacon plants showed low efficiency when commercial inulin from Helianthus tuberosus was utilized as substrate. The analysis of the enzymatic activities performed during growth of yacon clearly indicated the most appropriate source organ and phase of development to obtain the highest enzymatic activities for purification purposes and for the production of fructo‐oligosaccharides (FOS). Furthermore, the results suggested that the relative levels of activities of 1‐SST, 1‐FFT and 1‐FEH could be involved in the chain length distribution of the fructan molecules found in rhizophores and in tuberous roots of this species.     

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5 degrees C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly beta-2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81% in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.     

Factors affecting fructosyltransferases and fructan exohydrolase activities in <Emphasis Type="Italic">Agave tequilana</Emphasis> Weber var. azul

There is great interest in the fructosyltransferases (FTFs) involved in fructan metabolism and agents affecting their activity. Agaves accumulate fructans, fructose polymers linked by glycosidic β(2–1) and β(2–6) bonds in linear or branched configurations. In plants, fructans provide protection under stress conditions. The sucrose:sucrose 1-fructosyltransferase (1-SST), fructan:fructan 1-fructosyltransferase (1-FFT), fructan:fructan 6G-fructosyltransferase (6G-FFT), and fructan exohydrolase (FEH) activities were analyzed in micropropagated Agave tequilana plants in the absence and presence of HgCl₂, AgNO₃, MgCl_2, sodium deoxycholate (DNa), and sodium dodecyl sulfate (SDS). Kestose, nystose and neokestose were synthesized by the respective FTFs. HgCl₂ and AgNO₃ inhibited all FTFs, mainly up to 90 % in 1-SST and 1-FFT. DNa increased 1-SST (32 %) and 1-FFT (45 %) activities, and SDS increased 6G-FFT activity by 96 %. Finally, AgNO₃ inhibited FEH activity by 78 %. Our results might be relevant on the regulation of FTFs in agave and other crops, for instance by the increment the fructans synthesis in stressed plants.     

Effects of nitrogen deficiency on accumulation of fructan and fructan metabolizing enzyme activities in sink and source leaves of barley (Hordeum vulgare)

Seedlings of barley ( Hordeum vulgare L. cv. Agneta) were grown hydroponically under continuous light, constant temperature and relative humidity. During the first two weeks, the relative growth rate (RGR) was kept at 25% by limiting only the supply of nitrogen. The cultures were then transferred to nitrogen-free media and the amounts of fructan, starch, sucrose, glucose and fructose in sink and source leaves were measured at 0, 12, 24, 48, 72, 120 and 156 h. The activities of two key enzymes in fructan metabolism, sucrose:sucrose fructosyltransferase (SST), fructan exohydrolase (FEH), as well as acid invertase were also measured in the two types of leaves.
The fructan and starch levels in both sink and source leaves increased during nitrogen deficiency. The highest increase in starch was 200% of the control while for fmctans a 700% increase was recorded. The activity of SST increased parallel to fructan accumulation in sink leaves. However the FEH activity was constant and not affected by nitrogen deficiency. The invertase activity both in sink and source leaves was reduced by nitrogen deficiency. More fructans as well as sucrose and fructose accumulated in source leaves compared to sink leaves both before and after nitrogen starvation. The results show that fructan is the major carbohydrate reserve accumulating under nitrogen deficiency both in sink and source leaves in barley plants. The induction of fructan accumulation in sink leaves caused by nitrogen deficiency is intimately connected with the regulation of SST     

Structure prediction and molecular simulation of gases diffusion pathways in hydrogenase

Although hydrogen is considered to be one of the most promising future energy sources and the technical aspects involved in using it have advanced considerably, the future supply of hydrogen from renewable sources is still unsolved. The [Fe]- hydrogenase enzymes are highly efficient H(2) catalysts found in ecologically and phylogenetically diverse microorganisms, including the photosynthetic green alga, Chlamydomonas reinhardtii. While these enzymes can occur in several forms, H(2) catalysis takes place at a unique [FeS] prosthetic group or H-cluster, located at the active site. 3D structure of the protein hydA1 hydrogenase from Chlamydomonas reinhardtti was predicted using the MODELER 8v2 software. Conserved region was depicted from the NCBI CDD Search. Template selection was done on the basis NCBI BLAST results. For single template 1FEH was used and for multiple templates 1FEH and 1HFE were used. The result of the Homology modeling was verified by uploading the file to SAVS server. On the basis of the SAVS result 3D structure predicted using single template was chosen for performing molecular simulation. For performing molecular simulation three strategies were used. First the molecular simulation of the protein was performed in solvated box containing bulk water. Then 100 H(2) molecules were randomly inserted in the solvated box and two simulations of 50 and 100 ps were performed. Similarly 100 O(2) molecules were randomly placed in the solvated box and again 50 and 100 ps simulation were performed. Energy minimization was performed before each simulation was performed. Conformations were saved after each simulation. Analysis of the gas diffusion was done on the basis of RMSD, Radius of Gyration and no. of gas molecule/ps plot.     

Mutations in chicory FEH genes are statistically associated with enhanced resistance to post-harvest inulin depolymerization

Key message

Nucleotidic polymorphisms were identified in fructan exohydrolases genes which are statistically associated with enhanced susceptibility to post-harvest inulin depolymerization.

Abstract

Industrial chicory (Cichorium intybus L.) root is the main commercial source of inulin, a linear fructose polymer used as dietary fiber. Post-harvest, inulin is depolymerized into fructose which drastically increases processing cost. To identify genetic variations associated with enhanced susceptibility to post-harvest inulin depolymerization and related free sugars content increase, we used a candidate-gene approach focused on inulin and sucrose synthesis and degradation genes, all members of the family 32 of glycoside hydrolases (GH32). Polymorphism in these genes was first investigated by carrying out EcoTILLING on two groups of chicory breeding lines exhibiting contrasted response to post-harvest inulin depolymerization. This allowed the identification of polymorphisms significantly associated with depolymerization in three fructan exohydrolase genes (FEH). This association was confirmed on a wider panel of 116 unrelated families in which the FEH polymorphism explained 35 % of the post-harvest variance for inulin content, 36 % of variance for sucrose content, 18 % for inulin degree of polymerization, 23 % for free fructose content and 22 % for free glucose content. These polymorphisms were associated with significant post-harvest changes of inulin content, inulin chain length and free sugars content.     

Induction of focal epithelial hyperplasia in tongue of young bk6-E6/E7 HPV16 transgenic mice

Squamous cell carcinoma (SCC) of the oral cavity is one of the most common neoplasms in the world. During the past 2 decades, the role of high-risk human papilloma virus (HR-HPV) has been studied and the data supporting HPV as a one of the causative agents in the development and progression of a sub-set of head and neck squamous cell carcinomas (HNSCC) has accumulated. In order to investigate the role of HR-HPV oncogene expression in early epithelial alterations in vivo, we produced transgenic mice expressing HPV16 early region genes from the promoter of the bovine keratin 6 gene (Tg[bK6-E6/E7]). In this article, we demonstrate that E6/E7 transgene was abundantly expressed and cellular proliferation was increased in the middle tongue epithelia of transgenic mice, and that in the same region young (27 weeks old) Tg[bK6-E6/E7] mice spontaneously developed histological alterations, mainly focal epithelial hyperplasia (FEH).     

Activities of fructan- and sucrose-metabolizing enzymes in wheat stems subjected to water stress during grain filling

This study investigated if a controlled water deficit during grain filling of wheat (Triticum aestivum L.) could accelerate grain filling by facilitating the remobilization of carbon reserves in the stem through regulating the enzymes involved in fructan and sucrose metabolism. Two high lodging-resistant wheat cultivars were grown in pots and treated with either a normal (NN) or high amount of nitrogen (HN) at heading time. Plants were either well-watered (WW) or water-stressed (WS) from 9 days post anthesis until maturity. Leaf water potentials markedly decreased at midday as a result of water stress but completely recovered by early morning. Photosynthetic rate and zeatin + zeatin riboside concentrations in the flag leaves declined faster in WS plants than in WW plants, and they decreased more slowly with HN than with NN when soil water potential was the same, indicating that the water deficit enhanced, whereas HN delayed, senescence. Water stress, both at NN and HN, facilitated the reduction in concentration of total nonstructural carbohydrates (NSC) and fructans in the stems but increased the sucrose level there, promoted the re-allocation of pre-fixed ¹⁴C from the stems to grains, shortened the grain-filling period, and accelerated the grain-filling rate. Grain weight and grain yield were increased under the controlled water deficit when HN was applied. Fructan exohydrolase (FEH; EC 3.2.1.80) and sucrose phosphate synthase (SPS; EC 2.4.1.14) activities were substantially enhanced by water stress and positively correlated with the total NSC and fructan remobilization from the stems. Acid invertase (EC 3.2.1.26) activity was also enhanced by the water stress and associated with the change in fructan concentration, but not correlated with the total NSC remobilization and ¹⁴C increase in the grains. Sucrose:sucrose fructosyltransferase (EC 2.4.1.99) activity was inhibited by the water stress and negatively correlated with the remobilization of carbon reserves. Sucrose synthase (EC 2.4.1.13) activity in the stems decreased sharply during grain filling and showed no significant difference between WW and WS treatments. Abscisic acid (ABA) concentration in the stem was remarkably enhanced by water stress and significantly correlated with SPS and FEH activities. Application of ABA to WW plants yielded similar results to those for WS plants. The results suggest that the increased remobilization of carbon reserves by water stress is attributable to the enhanced FEH and SPS activities in wheat stems, and that ABA plays a vital role in the regulation of the key enzymes involved in fructan and sucrose metabolism.Abbreviations ABA Abscisic acid - DAS Days after sowing - DPA Days post anthesis - ESC Ethanol-soluble carbohydrate - FEH Fructan exohydrolase - HN High amount of nitrogen - INV Invertase - NN Normal amount of nitrogen - NSC Nonstructural carbohydrate - _leaf Leaf water potential - _soil Soil water potential - Pr Photosynthetic rate - SPS Sucrose phosphate synthase - SS Sucrose synthase - SST Sucrose:sucrose fructosyltransferase - V_limit Limiting substrate - V_max Saturated substrate - WS Water stressed - WSC Water-soluble carbohydrate - WW Well watered - Z Zeatin - ZR Zeatin riboside     

Cloning and functional analysis of chicory root fructan1‐exohydrolase I (1‐FEH I): a vacuolar enzyme derivedfrom a cell‐wall invertase ancestor? Mass fingerprint of the 1‐FEH I enzyme

This paper describes the cloning and functional analysis of chicory (Cichorium intybus L.) fructan 1-exohydrolase I cDNA (1-FEH I). To our knowledge it is the first plant FEH cloned. Full-length cDNA was obtained by a combination of RT-PCR, 5' and 3' RACE using primers based on N-terminal and conserved amino acid sequences. Electrophoretically purified 1-FEH I enzyme was further analyzed by in-gel trypsin digestion followed by matrix-assisted laser desorption ionization and electrospray time-of-flight tandem mass spectrometry. Functionality of the cDNA was demonstrated by heterologous expression in potato tubers. 1-FEH I takes a new, distinct position in the phylogenetic tree of plant glycosyl hydrolases being more homologous to cell-wall invertases (44-53%) than to vacuolar invertases (38-41%) and fructosyl transferases (33-38%). The 1-FEH I enzyme could not be purified from the apoplastic fluid at significantly higher levels than can be explained by cellular leakage. These and other data suggest a vacuolar localization for 1-FEH I. Also, the pI of the enzyme (6.5) is lower than expected from a typical cell-wall invertase. Unlike plant fructosyl transferases that are believed to have evolved from a vacuolar invertase, 1-FEH I might have evolved from a cell-wall invertase-like ancestor gene that later obtained a vacuolar targeting signal. 1-FEH I mRNA quantities increase in the roots throughout autumn, and especially when roots are stored at low temperature.     

Roles of the fructans from leaf sheaths and from the elongating leaf bases in the regrowth following defoliation of Lolium perenne L

The study of carbohydrate metabolism in perennial ryegrass (Lolium perenne L. cv. Bravo) during the first 48 h of regrowth showed that fructans from elongating leaf bases were hydrolysed first whereas fructans in mature leaf sheaths were degraded only after a lag of 1.5 h. In elongating leaf bases, the decline in fructan content occurred not only in the differentiation zone (30–60 mm from the leaf base), but also in the growth zone. Unlike other soluble carbohydrates, the net deposition rate of fructose remained positive and even rose during the first day following defoliation. The activity of fructan exohydrolase (FEH; EC 3.2.1.80) was maximal in the differentiation zone before defoliation and increased in all segments, but peaked in the growth zone after defoliation. These data strongly indicate that fructans stored in the leaf growth zone were hydrolysed and recycled in that zone to sustain the refoliation immediately after defoliation. Despite the depletion of carbohydrates, leaves of defoliated plants elongated at a significantly higher rate than those of undefoliated plants, during the first 10 h of regrowth. This can be partly attributed to the transient increase in water and nitrate deposition rate. The results are discussed in relation to defoliation tolerance. Received: 16 June 2000 / Accepted: 17 October 2000     

Spinal muscular atrophy: a deficiency in a ubiquitous protein; a motor neuron-specific disease

Spinal muscular atrophy (SMA) is a neurodegenerative disease in humans and the most common genetic cause of infant mortality. The disease results in motor neuron loss and skeletal muscle atrophy. Despite a range of disease phenotypes, SMA is caused by mutations in a single gene, the Survival of Motor Neuron 1 (SMN1) gene. Recent advances have shed light on functions of the protein product of this gene and the pathophysiology of the disease, yet, fundamental questions remain. This review attempts to highlight some of the recent advances made in the understanding of the disease and how loss of the ubiquitously expressed survival of motor neurons (SMN) protein results in the SMA phenotype. Answers to some of the questions raised may ultimately result in a viable treatment for SMA.