A neuropeptide was isolated from a frog brain extract by HPLC purification and characterized by mass spectrometry. This 26-aa neuropeptide, which belongs to the RFamide peptide family, was designated 26RFa, and its primary structure was established as VGTALGSLAEELNGYNRKKGGFSFRF-NH 2. Research in databases revealed the presence of sequences homologous to frog 26RFa in the human genome and in rat ESTs. On the basis of this sequence information, the cDNAs encoding the human and rat 26RFa precursors were cloned. The two preproteins show a similar organization, with the 26RFa sequence located in the C-terminal region of the precursor. Human preprotein (prepro)-26RFa encodes an additional putative RFamide peptide that is not found in the rat precursor. The primary structures of human, rat, and frog 26RFa exhibit Ϸ80% identity, and the C-terminal octapeptide has been fully conserved from amphibians to mammals. In situ hybridization histochemistry revealed that, in the rat brain, the 26RFa gene is exclusively expressed in the ventromedial hypothalamic nucleus and in the lateral hypothalamic area. 26RFa induced a dosedependent stimulation in cAMP production by rat pituitary cells in vitro and markedly increased food intake in mice. The conservation of the primary structure of 26RFa during vertebrate evolution, the discrete localization of the mRNA encoding its precursor in hypothalamic nuclei involved in the control of feeding behavior, and the observation that 26RFa possesses orexigenic properties indicate that this neuropeptide may play important biological functions.

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 [␤-(2,1)] are much better substrates than levan-type fructans [␤-(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 ␤-(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.Starch is the most prominent storage carbohydrate in plants, but about 15% of flowering plant species use fructan (a Fru polymer) as a storage compound (Hendry, 1993). Inulin-type fructan consists of linear ␤-(2,1)-linked fructofuranosyl units and occur mainly in dicotyledonous species. Levan consists of linear ␤-(2,6)-linked fructofuranosyl units, but more complex and branched fructan types (graminan, inulin neoseries, and levan neoseries) are common in monocotyledonous species (Vijn and Smeekens, 1999;Pavis et al., 2001b) Next to their obvious role as reserve compounds, fructan might have other functions in plants like stress protectants (drought and cold) or osmoregulators (Vergauwen et al., 2000; Hincha et al., 2002, and refs. therein). Unlike starch, fructans are water soluble and are believed to be stored in the vacuole , although the exclusive vacuolar localization has been questioned .Although the metabolism of inulin has become clear in dicotyledonous species and the respective biosynthetic and breakdown enzymes have been cloned (Edelman and Jefford, 1968;Van den Ende and Van Laere, 1996a; van der Meer et al., 1998;Hellwege et al., 2000;Van den Ende et al., 2000, fructan metabolism in monocots is not yet completely unraveled. So far, four different fructosyltransferases, each with their own specificity, are believed to be involved in monocot fructan biosynthesis. In addition to inulin biosynthesis by Suc:Suc 1-fructosyl tr...

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 nonfructan plants is discussed.

Food ingredients commonly undergo heat treatment. Meat, in particular, is typically consumed after some form of heating, such as boiling or roasting. Heating of meat can introduce a wide range of structural changes in its proteinaceous components. At the 3-dimensional structural level, meat proteins may denature and form aggregates upon heating. At the molecular level, primary structure (amino acid residue) alterations reported in cooked meat include protein carbonylation, modification of aromatic residues, and the formation of Maillard reaction products. Identification of these modifications is essential for determining the mechanism of thermal processing of meat and allowing better control of the nutritional and functional properties of products. This article reviews and summarizes the current state of understanding of protein modifications at the molecular level in commonly consumed mammalian food. In addition, relevant case studies relating to characterization of heat-induced amino acid residue-level modifications in other biological materials such as milk and wool are discussed to provide complementary insights.

The cloning of two highly homologous chicory (Cichorium intybus var. foliosum cv Flash) fructan 1-exohydrolase cDNAs (1-FEH IIa and 1-FEH IIb) is described. Both isoenzymes could be purified from forced chicory roots as well as from the etiolated "Belgian endive" leaves where the 1-FEH IIa isoform is present in higher concentrations. Full-length cDNAs were obtained by a combination of reverse transcriptase-polymerase chain reaction (PCR), PCR and 5Ј-and 3Ј-rapid amplification of cDNA ends using primers based on N-terminal and conserved amino acid sequences. 1-FEH IIa and 1-FEH IIb cDNA-derived amino acid sequences are most homologous to a new group of plant glycosyl hydrolases harboring cell wall-type enzymes with acid isoelectric points. Unlike the observed expression profiles of chicory 1-FEH I, northern analysis revealed that 1-FEH II is expressed when young chicory plants are defoliated, suggesting that this enzyme can be induced at any developmental stage when large energy supplies are necessary (regrowth after defoliation).

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SummaryAbout 15% of¯owering plant species synthesize fructans. Fructans serve mainly as reserve carbohydrates and are subject to breakdown by plant fructan exohydrolases (FEHs), among which 1-FEHs (inulinases) and 6-FEHs (levanases) can be differentiated. This paper describes the unexpected ®nding that 6-FEHs also occur in plants that do not synthesize fructans. The puri®cation, characterization, cloning and functional analysis of sugar beet (Beta vulgaris L.) 6-FEH are described. Enzyme activity measurements during sugar beet development suggest a constitutive expression of the gene in sugar beet roots. Classical enzyme puri®ca-tion followed by in-gel trypsin digestion and mass spectrometry (quadruple-time-of-¯ight mass spectrometry (Q-TOF) MS) led to peptide sequence information used in subsequent RT-PCR based cloning. Levantype fructans (b-2,6) are the best substrates for the enzyme, while inulin-type fructans (b-2,1) and sucrose are poorly or not degraded. Sugar beet 6-FEH is more related to cell wall invertases than to vacuolar invertases and has a low iso-electric point (pI ), clearly different from typical high pI cell wall invertases. Poor sequence homology to bacterial or fungal FEHs makes an endophytic origin highly unlikely. The functionality of the 6-FEH cDNA was further demonstrated by heterologous expression in Pichia pastoris. As fructans are absent in sugar beet, the role of 6-FEH in planta is not obvious. Like chitinases and bglucanases hydrolysing cell-surface components of fungal plant pathogens, a straightforward working hypothesis for further research might be that plant 6-FEHs participate in hydrolysis (or prevent the formation) of levan-containing slime surrounding endophytic or phytopathogenic bacteria.

Summary• Cereals accumulate graminan-type fructans which are subject to stress-related degradation by fructan 1-exohydrolases (1-FEHs) and fructan 6-exohydrolases (6-FEHs). To find new FEH genes related to freezing tolerance, a cold-hardened wheat crown cDNA library was screened.• Here we report the cloning, purification and characterization of two novel 6-kestosidase (6-KEH) isoenzymes from wheat crowns ( Triticum aestivum ). Functional characterization in Pichia pastoris confirmed the extreme substrate selectivity for the fructan trisaccharide 6-kestose.• Northern blotting showed that 6-KEH transcripts were constantly detected at the same level from autumn to winter in crown but not in leaf tissues. Apoplastic fluid isolations and activity measurements strongly suggest that 6-KEH is localized in the apoplast.• It is proposed that 6-KEHs, together with other FEHs, might be involved in the breakdown of apoplastic fructans which may fulfil a role as membrane protectors under stress. Alternatively, a role in signalling processes, or in the degradation of exogenous 6-kestose from bacterial origin, cannot be excluded.