An arabinogalactan from the skin of Opuntia ficus-indica prickly pear fruits

The cold-water extract from the skin of Opuntia ficus-indica fruits was fractionated by anion-exchange chromatography. The major fraction, which was purified by size exclusion chromatography, consisted of a polysaccharide composed of galactose and arabinose residues in the ratio 6.3:3.3, with traces of rhamnose, xylose and glucose, but no uronic acid. The results of methylation analysis, supported by (13)C NMR spectroscopy, indicated that this polysaccharide corresponded to an arabinogalactan having a backbone of (1-->4)-linked beta-D-galactopyranosyl residues with 39.5% of these units branched at O-3. The side-groups consisted either of single L-arabinofuranosyl units or L-arabinofuranosyl alpha-(1-->5)-linked disaccharides. This polysaccharide is thus an arabinogalactan that can be classified in the type I of the arabinogalactan family.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.     

Structure determination,apoptosis induction,and telomerase inhibition of CFP-2, a novel lichenin from Cladonia furcata

A great deal of experimental evidence has accumulated in the past several decades, suggesting that polysaccharides have wide bioactivities. Cladonia furcata polysaccharide, CFP-2, a water-soluble lichenin with a mean Mr 7.6 x 10(4), was first obtained by 0.25 M NaOH solution extraction, ethanol precipitation, DEAE-cellulose, and Sephadex G-200 column chromatography. Gas chromatography of acid hydrolyzate of CFP-2 suggested that it was composed of D-glucose, D-galactose, and D-mannose in the molar ratios of 8:1:1. Periodate oxidation, Smith degradation, IR, and NMR spectroscopy analysis revealed that CFP-2 had a backbone consisting of alpha-(1-->3) and alpha-(1-->4)-linked D-glucopyranosyl residues substituted at O-6 with beta-(1-->6)-linked D-galactopyranosyl residue and alpha-(1-->6)-linked D-mannopyranosyl residue. CFP-2 was able to reduce viability of cultured HL-60 and K562 cells. The antiproliferative properties of CFP-2 appeared to be attributable to its induction of apoptotic cell death as determined by ultrastructural change, internucleosomal DNA fragmentation, and increased proportion of the subdiploid cell population. To elucidate molecular events in the apoptosis, protein expressions of Bcl-2, Bax, Fas, and FasL were measured by Western blotting using specific antibodies in HL-60 cells. The level of Bcl-2 remained largely unchanged, but the Bax, Fas, and FasL expression showed up-regulation. Moreover, the telomerase activity analyzed by TRAP-ELISA assay in HL-60 cells treated with CFP-2 decreased as compared with the untreated control cells. These results suggest that CFP-2 could have a possible cancer therapeutic potential.     

Structural characterization and in vitro antitumor activity of a novel polysaccharide isolated from the fruiting bodies of Pleurotus ostreatus

A novel water-soluble polysaccharide (POPS-1) was obtained from the fruiting bodies of Pleurotus ostreatus by hot water extraction, ethanol precipitation, and fractionated by DEAE-cellulose ion exchange chromatography and sepharose CL-6B gel filtration chromatography using an ATKA explore 100 purifier. The structure characterization and antitumor activity of the POPS-1 were evaluated in this paper. According to GC analysis, HPGPC, FT-IR, partial acid hydrolysis, periodate oxidation and Smith degradation, methylation and GC-MS analysis, the results indicate POPS-1 (M(w)=31 kDa) was composed of Man; Gal; Glc with a molar ratio of 1:2.1:7.9, it had a backbone of beta-(1-->3)-linked glucose residues, which occasionally branches at O-6. The branches were composed of (1-->3)-linked Glc, (1-->4)-linked Gal, (1-->4)-linked Man, and terminated with Glc and Gal residues. Cytotoxicity assay showed POPS-1 presented significantly higher antitumor activity against Hela tumor cell in vitro, in a dose-dependent manner, and exhibited significantly lower cytotoxicity to human embryo kidney 293T cells than Hela tumor cells compared with 5-Fu. The results suggest POPS-1 may be considered as a potential candidate for developing a novel low toxicity antitumor agent.     

Flocculant and Chemical Properties of a Polysaccharide from Pullularia pullulans

An extracellular polysaccharide, PP-floc, was synthesized from glucose by Pullularia pullulans (or Aureobasidium pullulans) in a pilot plant batch fermentor containing 175 liters of culture medium. At 58 h of fermentation, the concentration of PP-floc was 1.03 g/100 ml, giving a 25.8% conversion of initial glucose to polysaccharide. The flocculant activity of the culture medium increased during the fermentation process and reached its maximum at 50 h of culture age. Less PP-floc (0.33 lb/ton of slimes [approximately 149.7 g/0.907 t]) was required to give the same flocculant activity as a synthetic polymer of acrylamide, Separan NP-10 (0.5 lb/ton of slimes [approximately 226.8 g/0.907 t]), at all temperatures from 25 to 100 C. The degree of inactivation of PP-floc and Separan NP-10 at elevated temperatures was almost identical, and they were completely inactivated at about the same temperature (80 C). PP-floc also gave better compaction of slimes than Separan NP-10 at all temperatures tested. PP-floc was soluble in water and its specific optical rotation was [alpha](D) (25) + 194 degrees in water (c, 0.4). PP-floc contained 83.3% carbohydrate, 3.2% protein, and 8.1% water. Glucose was found to be the principal sugar monomer with traces (>5%) of galactose and mannose present. Structural studies on the fractions of purified polysaccharide by methylation and by periodate oxidation techniques prove that PP-floc is linear and composed of alpha-(1 --> 4) and alpha-(1 --> 6) glucopyranosyl units in the approximate ratio of 2:1. The action of pullulanase on crude PP-floc suggested the ordered arrangement of two consecutive alpha-(1 --> 4)-linked glucopyranosyl units flanked by alpha-(1 --> 6)-linked glucopyranose residues.     

A study of fucoidan from the brown seaweed Chorda filum.

Fucoidan fractions from the brown seaweed Chorda filum were studied using solvolytic desulfation. Methylation analysis and NMR spectroscopy were applied for native and desulfated polysaccharides. Homofucan sulfate from C. filum was shown to contain poly-alpha-(1-->3)-fucopyranoside backbone with a high degree of branching, mainly of alpha-(1-->2)-linked single units. Some fucopyranose residues are sulfated at O-4 (mainly) and O-2 positions. Some alpha-(1-->3)-linked fucose residues were shown by NMR to be 2-O-acetylated. The 1H and 13C NMR spectra of desulfated, deacetylated fucan were completely assigned. The spectral data obtained correspond to a quasiregular polysaccharide structure with a branched hexasaccharide repeating unit. Other fucoidan fractions from C. filum have more complex carbohydrate composition and give rather complex methylation patterns. [formula: see text]     

(2-O-alpha-D-galactopyranosyl-4-O-methyl-alpha-D-glucurono)-D-xylan from Eucalyptus globulus Labill.

An unusual heteroxylan composed of galactosyl, 4-O-methyl-glucuronosyl and xylosyl residues with molar ratio 1:3:30 was isolated from the wood of Eucalyptus globulus Labill. The results of linkage analysis, supported by data of 1H, 2D 1H-1H COSY and 13C NMR spectroscopy, revealed that the polysaccharide is a (2-O-alpha-D-galactopyranosyl-4-O-methyl-alpha-D- glucurono)-D-xylan with a (1-->4)-linked beta-D-xylopyranosyl backbone branched at O-2 by short side chains composed of terminal 4-O-methyl-alpha-D-glucuronic acid and of 4-O-methyl-alpha-D-glucuronic acid substituted at O-2 with alpha-D-galactose.     

Structural characteristics of a bioactive polysaccharide from Sorghum arundinaceum

A polysaccharide, an alpha-D-glucan with an apparent molecular weight of 6.85 x 10(4), called PSa glucan, was isolated from fresh seeds of Sorghum arundinaceum by fractionation on Sephacryl S-300 HR and Sephadex G-25. Chemical and spectroscopic studies indicated that it has a highly branched glucan type structure composed of alpha-(1-->4) linked D-glucopyranose residues with (1-->3), (1-->6) branching points, and a significant amount of alpha-(1-->6) branching to alpha-(1-->3) linked D-glucopyranose residues. The anti-inflammatory activity of the polysaccharide was performed using the capillary permeability assay.     

Isolation and structural study of polysaccharides from campion Silene vulgaris

Silenan SV, a pectic polysaccharide, was isolated from the aerial part of Silene vulgaris (Moench) Garke (Oberna behen (L.) Ikonn.), widespread through the European North of Russia. The polysaccharide was found to contain residues of galacturonic acid (63%), arabinose, galactose, and rhamnose as the main constituents. The results of a partial acidic hydrolysis, pectinase digestion, and NMR studies of silenan SV indicated that its molecule contains a linear alpha-1,4-D-galacturonan backbone and ramified regions. The core of the ramified regions is composed of residues of alpha-1,4-D-galacturonic acid along with 2-substituted alpha-rhamnopyranose residues. The NMR data showed that the silenan SV side chains are composed of the blocks built from the terminal alpha-1,5-linked arabinofuranose and beta-1,4-linked galactopyranose residues; these most likely are the side chains of rhamnogalacturonan, characteristic of other pectic polysaccharides. The nonreducing ends of these side chains contain alpha-arabinofuranose residues.     

A fucoidan fraction from Ascophyllum nodosum.

A fucoidan fraction was purified from the brown alga Ascophyllum nodosum. The polysaccharide contained L-fucose and sulfate as the only constituents. Combination of methylation analysis, Smith degradation, FTIR and NMR spectroscopy on the native and the de-sulfated polymers demonstrated that the fucoidan consisted of a highly branched core region with primarily alpha-(1-->3)-linked fucosyl residues and a few alpha-(1-->4) linkages. Branch points were at position 2 of the -->3-linked internal residues. The side chains consisted of single and multi-unit fucosyl residues. The combined analytical data suggested also a complex sulfation pattern with substitution principally at position 2 and/or position 4. Such diversity in the structural features of this fucoidan may be of importance for its various biological properties.     

Structural analysis of a pectic polysaccharide from the leaves of Diospyros kaki

A pectic polysaccharide DL-2A with a molar mass of 8.5 x 10(5), was obtained from the boiling water extract of Diospyros kaki leaves. It had [alpha]20D -21.8 degrees (c 0.22, H2O) and consisted of rhamnose, arabinose, galactose, xylose and galacturonic acid units in the molar ratio of 0.4:3.4:2.4:1.0:0.8, along with traces of glucuronic acid. About 16.7% of galacturonic acid existed as the methyl ester. A combination of linkage analyses, periodate oxidation, partial acid hydrolysis, selective lithium-degraded reaction, ESIMS, 1H- and 13C- NMR spectral analyses revealed its structural features. It was found that DL-2A possessed an alpha-(1-->4)-galacturonan backbone with some insertions of alpha-1,2-Rhap residues. The side-chains of arabino-3,6-galactan were attached to the backbone via O-4 of Rhap residues and O-3 of GalAp residues, while 4-linked xylose residues (forming short linear chains) were directly linked to O-4 of rhamnose residues, not as part of the xylogalacturonan. These novel structural features enlarge the knowledge on the fine structure of pectic substances in the plant kingdom.     

Enzymic degradation of the mycobacterial O-methyl-D-glucose polysaccharide by a Rhizopus-mold alpha amylase, an enzyme active on 6-O-methyl-amylo-oligosaccharides

An enzyme activity that catalyzes hydrolysis of an alpha-(1----4)-linked 6-O-methyl-D-glucan was detected in, and purified from, Rhizopus oryzae mold. The enzyme acts like an alpha amylase and digests unmodified amylo-oligosaccharides 10 to 15 times as fast as it does the 6-O-methyl and 6-deoxy derivatives. When the limit product obtained by digesting the mycobacterial O-methyl-D-glucose polysaccharide with pancreatic alpha amylase and Aspergillus glucoamylase was further digested with the Rhizopus alpha amylase, di-, tri-, and tetra-saccharide fragments composed of alpha-(1----4)-linked 6-O-methyl-D-glucose were released. The rest of the molecule was recovered as oligosaccharides terminated by two, or three, alpha-(1----4)-linked 6-O-methyl-D-glucose residues.     

Structures of two O-chain polysaccharides of Citrobacter gillenii O9a,9b lipopolysaccharide. A new homopolymer of 4-amino-4,6-dideoxy-D-mannose (perosamine).

Mild acid degradation of the lipopolysaccharide of Citro- bacter gillenii O9a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4- acetamido-4,6-dideoxy-d-mannose (d-Rha4NAc, N-acetyl-d-perosamine). PS was studied by methylation analysis and (1)H-NMR and (13)C-NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and H-detected (1)H,(13)C heteronuclear correlation experiments. It was found that PS includes two structurally different polysaccharides: an alpha1-->2-linked homopolymer of N-acetyl-d-perosamine [-->2)-alpha-d-Rhap4NAc-(1-->, PS2] and a polysaccharide composed of tetrasaccharide repeating units (PS1) with the following structure: -->3)-alpha-d-Rhap4NAc-(1-->2)-alpha-d-Rhap4NAc-(1-->2)-alpha-d-Rhap4NAc-(1-->3)-alpha-d-Rhap4 N Ac2Ac-(1--> where the degree of O-acetylation of a 3-substituted Rha4NAc residue at position 2 is approximately 70%. PS could be fractionated into PS1 and PS2 by gel-permeation chromatography on TSK HW-50S. Matrix-assisted laser desorption ionization MS data indicate sequential chain elongation of both PS1 and PS2 by a single sugar unit, with O-acetylation in PS1 beginning at a certain chain length. Anti-(C. gillenii O9a,9b) serum reacted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a structurally related O-chain polysaccharide were reactive.     

Some structural features of a new sulphated heteropolysaccharide from Padina pavonia

An acid-extractable, water-soluble, polysaccharide sulphate, isolated from Padina pavonia, comprised variable proportions of glucuronic acid, galactose, glucose, mannose, xylose, and fucose in addition to a protein moiety. Partial acid hydrolysis and autohydrolysis of the free acid polysaccharide yielded several oligosaccharides. Evidence from periodate oxidation studies indicated that the inner polysaccharide portion is composed of (1 → 4)-linked β-D-glucuronic acid, (1 → 4)-linked β-D-mannose and (1 → 4)-linked β-D-glucose residues. The heteropolymeric partially sulphated exterior portion is attached to the inner part and comprises various ratios of (1 → 4)-linked β-D-galactose, β-D-galactose-3-sulphate residues, (1 → 4)-linked β-D-glucose residues, (1 → 2)-linked α-L-fucose 4-sulphate residues and (1 → 3)-linked β-D-xylose residues.     

Structural studies of a heteroxylan from Plantago major L. seeds by partial hydrolysis, HPAEC-PAD, methylation and GC-MS, ESMS and ESMS/MS.

The seed mucilage from Plantago major L. contains acidic heteroxylan polysaccharides. For further structural analysis, oligosaccharides were generated by partial acid hydrolysis and then isolated by high-pH anion-exchange chromatography (HPAEC). Each HPAEC fraction was shown by ESMS to contain one major oligosaccharide and several minor components. Partial structures of the oligosaccharides were determined using GC-MS, ESMS and ES tandem mass spectrometry (ESMS/MS). A (1-->4)-linked xylan trisaccharide and (1-->3)-linked xylan oligosaccharides with DP 6-11 suggested that the backbone of the heteroxylan polysaccharide consisted of blocks of (1-->4)-linked and (1-->3)-linked Xylp residues. A (1-->2)-linked Xylp disaccharide and a branched tetrasaccharide were also found, revealing that single Xylp residues are linked to the O-2 of some of the (1-->4)-linked Xylp residues in the backbone. In addition, our results confirm the presence of side chains consisting of the disaccharide GlcpA-(1-->3)-Araf.     

Structure of a heteroxylan of gum exudate of the palm Scheelea phalerata (uricuri)

The polysaccharide isolated from the gum exudate of palm Scheelea phalerata (SPN) was water-insoluble and composed of Fuc, Ara, Xyl, and uronic acid moieties in a 5:34:54:7 molar ratio: 12% of phenolics were also present. A soluble polysaccharide (SPNa) was obtained after alkaline treatment, which contained Fuc, Ara, Xyl and uronic acid in a 7:44:42:7 molar ratio, with only 2% phenolics. SPNa had an M(W) approximately 1.04 x 10(5) g mol(-1) and was almost monodisperse (M(W)/M(N) : 1.25 +/-0.22). It had a branched structure with side chains of 2-O-substituted Xylp (approximately 8%) and 3-O-substituted Araf (12%) units, and a large proportion of nonreducing end-units of Araf (15%), Fucp (10%), Xylp (4%), and Arap (6%). The (1 --> 4)-linked beta-Xylp main-chain units were 3-O- (9%), 2-O- (13%), and 2,3-di-O- (13%) substituted. Its (13)C NMR spectrum contained at least 9 C-1 signals, those at delta 108.6 and 107.7 arising from alpha-Araf units. Others were present at delta 175.4 from C-6 of alpha-GlcpA and delta 15.6 from C-6 of Fucp units. The main chain of SPNa was confirmed by analysis of a Smith-degraded polysaccharide (SPDS): methylation analysis provided a 2,3-Me(2)-Xyl (65%) derivative and its (13)C NMR spectrum showed five main signals typical of a (1 --> 4)-linked beta-Xylp units. Methylation analysis of a carboxy-reduced polysaccharide (SPN-CR) revealed a 2,3,4,6-Me(4)-Glc derivative (4%) arising from nonreducing end-units of GlcpA. Alpha-GlcpA-(1 --> 2)-alphabeta-Xy1p and alpha-GlcpA-(1 --> 2)-beta-Xylp-(1 --> 4)-alphabeta-Xylp were obtained via partial acid hydrolysis of SPN, showing the structure of side-chain substituents on O-2 of the main-chain units.     

Substrate specificity of the alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The alpha-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1-->5)-alpha-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [alpha-(1-->5)-linked] of the arabinan backbone rather than the arabinosyl side chain [alpha-(1-->3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1-->5)-Araf, T-Araf, (1-->3, 5)-Araf and (1-->3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1-->5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [alpha-(1-->5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the alpha-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this alpha-L-arabinofuranosidase can only hydrolyse alpha-L-arabinofuranosyl residues of arabinan.     

Structural studies of a mucilage from Abroma augusta root bark

The structure of an acidic polysaccharide isolated from Abroma augusta root bark was determined by sugar and methylation analyses and high resolution ¹H- and ¹³C-NMR spectroscopy. The main chain of the polysaccharide was composed of 1,2-linked - -rhamnopyranose and 1,4- or 1,3-linked - -galacturonic acid residues. The terminal β- -glucuronic acid residue was attached to the 3- and/or 4-position of the - -galacturonic acid residue.     

An extracellular polysaccharide produced by Zoogloea ramigera 115

A weakly acidic polysaccharide was purified from the extracellular zoogloeal matrix produced by Zoogloeal ramigera 115. The purified polysaccharide was homogeneous as judged by sedimentation analysis, and the average molecular weight was estimated to be about 10(5) by gel permeation chromatography of the fully methylated preparation. The polysaccharide was composed of D-glucose, D-galactose and pyruvic acid in an approximate molar ratio 11:3:1.5. On the basis of methylation, periodate oxidation, Smith degradation and partial hydrolysis, the following highly branched structure was deduced for the polysaccharide: a long chain mainly consisting of beta 1 leads to 4-linked glucose residues branching at the C-3 or C-6 position of galactose residues which are present in beta 1 leads to 4 or beta 1 leads to 3 linkages as the minor component of the long chain; pyruvic acid residues, the sole acidic component, are linked to the nonreducing end and/or 1,3-linked glucose residues through 4,6-ketal linkages. The purified polysaccharide was not readily soluble in water and had a high affinity for several metallic ions (e.g, 0.25 mumol Fe3+/mg, and 0.17 mumol Fe2+ mg). Upon addition of metallic ions (1 mM) to a gelatinous aqueous solution of the polysaccharide (K+ form, 0.125%), more than 80% of it immediately coprecipitated out with them.     

Structural studies of an acidic polysaccharide from Ocimum basilicum seeds

An acidic polysaccharide isolated from the seeds of Ocimum basilicum by DEAE-cellulose fractionation was ～92% pure, having an associated glucan impurity (～8%). The polysaccharide is composed of d-xylose, l-arabinose, l-rhamnose, and d-galacturonic acid in the molar ratios 15:9:7:12, together with traces or galactose and glucose. Methylation analysis indicated that the polysaccharide contained a (1→4)-linked xylan backbone carrying branch-points at C-2 and C-3 of the xylosyl residues, and revealed the structural features of the side chains. Periodateoxidation and Smith-degradation studies support the results of methylation analysis.