Wine lees are the residual precipitate formed during wine fermentation, storage, fi ltration or centrifugation (1). The (poly)phenolic compounds in lees determine the colour, intensity and organoleptic properties of wine. Through biochemical processes in lees, enzymatic activitiy induces the transformation of phenols to high value--added polyphenolic compounds with in vivo physiological activity, such as gallic acid or ellagic acid, catechin, caff eic acid, resveratrol and others (2)(3)(4)(5) SummaryThe study examines the potential of wine industry by-product, the lees, as a rich mixture of natural polyphenols, and its physiological potential to reduce postprandial metabolic and oxidative stress caused by a cholesterol-rich diet in in vivo model. Chemical analysis of wine lees showed that their total solid content was 94.2 %. Wine lees contained total phenols, total nonfl avonoids and total fl avonoids expressed in mg of gallic acid equivalents per 100 g of dry mass: 2316.6±37.9, 1332.5±51.1 and 984.1±28.2, respectively. The content of total anthocyanins expressed in mg of cyanidin-3-glucoside equivalents per 100 g of dry mass was 383.1±21.6. Antioxidant capacity of wine lees determined by the DPPH and FRAP methods and expressed in mM of Trolox equivalents per 100 g was 259.8±1.8 and 45.7±1.05, respectively. The experiment lasted 60 days using C57BL/6 mice divided in four groups: group 1 was fed normal diet and used as control, group 2 was fed normal diet with added wine lees, group 3 was fed high-cholesterol diet (HCD), i.e. normal diet with the addition of sunfl ower oil, and group 4 was fed HCD with wine lees. HCD increased serum total cholesterol (TC) by 2.3-fold, triacylglycerol (TAG) by 1.5-fold, low-density lipoprotein (LDL) by 3.5-fold and liver malondialdehyde (MDA) by 50 %, and reduced liver superoxide dismutase (SOD) by 50 %, catalase (CAT) by 30 % and glutathione (GSH) by 17.5 % compared to control. Conversely, treatment with HCD and wine lees reduced TC and LDL up to 1.4 times more than with HCD only, with depletion of lipid peroxidation (MDA) and restoration of SOD and CAT activities in liver, approximating values of the control. HDL levels were unaff ected in any group. Serum transaminase activity showed no hepatotoxic properties in the treatment with lees alone. In the proposed model, wine lees as a rich polyphenol source could be a basis for functional food products without alcohol.

The ability of aminoacyl‐tRNA synthetases to distinguish between similar amino acids is crucial for accurate translation of the genetic code. Saccharomyces cerevisiae seryl‐tRNA synthetase (SerRS) employs tRNA‐dependent recognition of its cognate amino acid serine [Lenhard, B., Filipic, S., Landeka, I., Skrtic, I., Söll, D. & Weygand‐Durasevic, I. (1997) J. Biol. Chem.272, 1136–1141]. Here we show that dimeric SerRS enzyme complexed with one molecule of tRNASer is more specific and more efficient in catalyzing seryl‐adenylate formation than the apoenzyme alone. Sequence‐specific tRNA–protein interactions enhance discrimination of the amino acid substrate by yeast SerRS and diminish the misactivation of the structurally similar noncognate threonine. This may proceed via a tRNA‐induced conformational change in the enzyme's active site. The 3′‐terminal adenosine of tRNASer is not important in effecting the rearrangement of the serine binding site. Our results do not provide an indication for a readjustment of ATP binding in a tRNA‐assisted manner. The stoichiometric analyses of the complexes between the enzyme and tRNASer revealed that two cognate tRNA molecules can be bound to dimeric SerRS, however, with very different affinities.

The active site of class II aminoacyl-tRNA synthetases contains the motif 2 loop, which is involved in binding of ATP, amino acid, and the acceptor end of tRNA. In order to characterize the active site of Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS), we performed in vitro mutagenesis of the portion of the SES1 gene encoding the motif 2 loop. Substitutions of amino acids conserved in the motif 2 loop of seryl-tRNA synthetases from other sources led to loss of complementation of a yeast SES1 null allele strain by the mutant yeast SES1 genes. Steady-state kinetic analyses of the purified mutant SerRS proteins revealed elevated K m values for serine and ATP, accompanied by decreases in k cat (as expected for replacement of residues involved in aminoacyl-adenylate formation). The differences in the affinities for serine and ATP, in the absence and presence of tRNA are consistent with the proposed conformational changes induced by positioning the 3-end of tRNA into the active site, as observed recently in structural studies of Thermus thermophilus SerRS (Cusack, S., Yaremchuk, A., and Tukalo, M. (1996) EMBO J. 15, 2834 -2842). The crystal structure of this moderately homologous prokaryotic counterpart of the yeast enzyme allowed us to produce a model of the yeast SerRS structure and to place the mutations in a structural context. In conjunction with structural data for T. thermophilus SerRS, the kinetic data presented here suggest that yeast seryl-tRNA synthetase displays tRNA-dependent amino acid recognition.The formation of aminoacyl-tRNA, catalyzed by aminoacyltRNA synthetases, is a crucial step in maintaining the fidelity of protein biosynthesis. This family of enzymes can be partitioned into two classes of 10 enzymes each, based on conserved sequences (1) and structural motifs (2). All members of class I contain a common loop with the signature sequence KMSKS (3) and a region of homology with the HIGH peptide (4) as part of a Rossmann dinucleotide binding fold of parallel ␤-sheets (5). Class II synthetases have a different topology of dinucleotide binding based on antiparallel ␤-sheets (2, 6, 7). The three common signature motifs of class II synthetases are found in this domain. Motif 1 forms part of the conserved inter-subunit interface of homodimeric (6, 8) and heteromeric (9) synthetases. Motifs 2 and 3 contain many of the active-site residues important for ATP, amino acid, and tRNA acceptor stem recognition (10 -15). The elucidation of the crucial role of sequence motifs in substrate binding have resulted from the solution of several crystal structures of enzymes and enzyme-substrate complexes from both class I and class II (16) and numerous biochemical studies involving mutant synthetases (17-22).The evolution of tRNA recognition systems has recently gained much attention (23-27). The primary structure of several prokaryotic and eukaryotic seryl-tRNA synthetases (Ref.23; see also the legends to Fig. 1 and 3), including the enzyme that probably functions in yeast mitochondria (28), have been determi...