Same but different - pseudo-pectin in the charophytic alga Chlorokybus atmophyticus.

All land-plant cell walls possess hemicelluloses, cellulose and anionic pectin. The walls of their cousins, the charophytic algae, exhibit some similarities to land plants' but also major differences. Charophyte 'pectins' are extractable by conventional land-plant methods, although they differ significantly in composition. Here, we explore 'pectins' of an early-diverging charophyte, Chlorokybus atmophyticus, characterising the anionic polysaccharides that may be comparable to 'pectins' in other streptophytes. Chlorokybus 'pectin' was anionic and upon acid hydrolysis gave GlcA, GalA and sulphate, plus neutral sugars (Ara≈Glc>Gal>Xyl); Rha was undetectable. Most Gal was the l-enantiomer. A relatively acid-resistant disaccharide was characterised as ß-d-GlcA-(1→4)-l-Gal. Two Chlorokybus 'pectin' fractions, separable by anion-exchange chromatography, had similar sugar compositions but different sulphate-ester contents. No sugars were released from Chlorokybus 'pectin' by several endo-hydrolases [(1,5)-α-l-arabinanase, (1,4)-ß-d-galactanase, (1,4)-ß-d-xylanase, endo-polygalacturonase] and exo-hydrolases [α- and ß-d-galactosidases, α-(1,6)-d-xylosidase]. 'Driselase', which hydrolyses most land-plant cell wall polysaccharides to mono- and disaccharides, released no sugars except traces of starch-derived Glc. Thus, the Ara, Gal, Xyl and GalA of Chlorokybus 'pectin' were not non-reducing termini with configurations familiar from land-plant polysaccharides (α-l-Araf, α- and ß-d-Galp, α- and ß-d-Xylp and α-d-GalpA), nor mid-chain residues of α-(1→5)-l-arabinan, ß-(1→4)-d-galactan, ß-(1→4)-d-xylan or α-(1→4)-d-galacturonan. In conclusion, Chlorokybus possesses anionic 'pectic' polysaccharides, possibly fulfilling pectic roles but differing fundamentally from land-plant pectin. Thus, the evolution of land-plant pectin since the last common ancestor of Chlorokybus and land plants is a long and meandering path involving loss of sulphate, most l-Gal and most d-GlcA; re-configuration of Ara, Xyl and GalA; and gain of Rha.

Fruit softening: evidence for pectate lyase action in vivo in date (Phoenix dactylifera) and rosaceous fruit cell walls.

BACKGROUND AND AIMS: The programmed softening occurring during fruit development requires scission of cell wall polysaccharides, especially pectin. Proposed mechanisms include the action of wall enzymes or hydroxyl radicals. Enzyme activities found in fruit extracts include pectate lyase (PL) and endo-polygalacturonase (EPG), which, in vitro, cleave de-esterified homogalacturonan in mid-chain by ß-elimination and hydrolysis, respectively. However, the important biological question of whether PL exhibits action in vivo had not been tested. METHODS: We developed a method for specifically and sensitively detecting in-vivo PL products, based on Driselase digestion of cell wall polysaccharides and detection of the characteristic unsaturated product of PL action. KEY RESULTS: In model in-vitro experiments, pectic homogalacturonan that had been partially cleaved by commercial PL was digested to completion with Driselase, releasing an unsaturated disaccharide ('ΔUA-GalA'), taken as diagnostic of PL action. ΔUA-GalA was separated from saturated oligogalacturonides (EPG products) by electrophoresis, then subjected to thin-layer chromatography (TLC), resolving ΔUA-GalA from higher homologues. The ΔUA-GalA was confirmed as 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→4)-d-galacturonic acid by NMR spectroscopy. Driselase digestion of cell walls from ripe fruits of date (Phoenix dactylifera), pear (Pyrus communis), rowan (Sorbus aucuparia) and apple (Malus pumila) yielded ΔUA-GalA, demonstrating that PL had been acting in vivo in these fruits prior to harvest. Date-derived ΔUA-GalA was verified by negative-mode mass spectrometry, including collision-induced dissociation (CID) fragmentation. The ΔUA-GalA:GalA ratio from ripe dates was roughly 1:20 (mol mol-1), indicating that approx. 5 % of the bonds in endogenous homogalacturonan had been cleaved by in-vivo PL action. CONCLUSIONS: The results provide the first demonstration that PL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Characterisation of the non-oxidative degradation pathway of dehydroascorbic acid in slightly acidic aqueous solution.

Although l-ascorbate (vitamin C) is an important biological antioxidant, its degradation pathways in vivo remain incompletely characterised. Ascorbate is oxidised to dehydroascorbic acid, which can be either hydrolysed to diketogulonate (DKG) or further oxidised. DKG can be further degraded, oxidatively or non-oxidatively. Here we characterise DKG products formed non-enzymically and non-oxidatively at 20 °C and at a slightly acidic pH typical of the plant apoplast. High-voltage electrophoresis revealed at least five products, including two novel CPLs (epimers of 2-carboxy-l-threo-pentonolactone), which slowly interconverted with CPA (2-carboxy-l-threo-pentonate). One of the two CPLs has an exceptionally low pKa. The CPL structures were supported by MS [(C6H7O7)-] and by 1H and 13C NMR spectroscopy. Xylonate and its lactone also appeared. Experiments with [1-14C]DKG showed that all five products (including the 5-carbon xylonate and its lactone) retained DKG's carbon-1; therefore, most xylonate arose by decarboxylation of CPLs or CPA, one of whose -COOH groups originates from C-2 or C-3 of DKG after a 'benzilic acid rearrangement'. Since CPLs appeared before CPA, a DKG lactone is probably the main species undergoing this rearrangement. CPA and CPL also form non-enzymically in vivo, where they may be useful to researchers as 'fingerprints', or to organisms as 'signals', indicating a non-oxidative, slightly acidic biological compartment.

Montbresides A-D: antibacterial p-coumaroyl esters of a new sucrose-based tetrasaccharide from Crocosmia × crocosmiiflora (montbretia) flowers.

Crocosmia × crocosmiiflora (montbretia) flowers yielded four esters (montbresides A-D) of a new sucrose-based tetrasaccharide, 3-O-ß-d-glucopyranosyl-4´-O-α-d-rhamnopyranosyl-sucrose [ß-d-Glc-(1 → 3)-α-d-Glc-(1↔2)-ß-d-Fru-(4 ← 1)-α-d-Rha]. All four possess O-p-coumaroyl residues on C-3 of fructose and C-4 of α-glucose, plus O-acetyl residues on C-2 and C-3 of rhamnose and C-6 of fructose. Montbresides A and B are additionally O-acetylated on C-1 of fructose. The p-coumaroyls are trans- in montbresides A and C and cis- in B and D. Elemental compositions were determined from MS data, and structures from 1D and 2D NMR spectra. Monosaccharide residues were identified from selective 1D TOCSY spectra and TLC, and acylation sites from 2D HMBC spectra. Enantiomers were distinguished by enzymic digestion. Montbretia flower extracts were cytotoxic against six human cancerous cell-lines, but purified montbresides lacked cytotoxicity. Each montbreside displayed antibacterial activity against Staphylococcus aureus (minimal inhibitory concentration ~6 µg/ml). Montbretia is a potential source of new cytotoxins and antibacterial agents.

The pectic disaccharides lepidimoic acid and ß-d-xylopyranosyl-(1→3)-d-galacturonic acid occur in cress-seed exudate but lack allelochemical activity.

BACKGROUND AND AIMS: Cress-seed (Lepidium sativum) exudate exerts an allelochemical effect, promoting excessive hypocotyl elongation and inhibiting root growth in neighbouring Amaranthus caudatus seedlings. We investigated acidic disaccharides present in cress-seed exudate, testing the proposal that the allelochemical is an oligosaccharin-lepidimoic acid (LMA; 4-deoxy-ß-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnose). METHODS: Cress-seed exudate was variously treated [heating, ethanolic precipitation, solvent partitioning, high-voltage paper electrophoresis and gel-permeation chromatography (GPC)], and the products were bioassayed for effects on dark-grown Amaranthus seedlings. Two acidic disaccharides, including LMA, were isolated and characterized by electrophoresis, thin-layer chromatography (TLC) and nuclear magnetic resonance (NMR) spectroscopy, and then bioassayed. KEY RESULTS: Cress-seed exudate contained low-Mr, hydrophilic, heat-stable material that strongly promoted Amaranthus hypocotyl elongation and inhibited root growth, but that separated from LMA on electrophoresis and GPC. Cress-seed exudate contained ∼250 µmLMA, whose TLC and electrophoretic mobilities, susceptibility to mild acid hydrolysis and NMR spectra are reported. A second acidic disaccharide, present at ∼120 µm, was similarly characterized, and shown to be ß-d-xylopyranosyl-(1→3)-d-galacturonic acid (Xyl→GalA), a repeat unit of xylogalacturonan. Purified LMA and Xyl→GalA when applied at 360 and 740 µm, respectively, only slightly promoted Amaranthus hypocotyl growth, but equally promoted root growth and thus had no effect on the hypocotyl:root ratio, unlike total cress-seed exudate. CONCLUSIONS: LMA is present in cress seeds, probably formed by rhamnogalacturonan lyase action on rhamnogalacturonan-I during seed development. Our results contradict the hypothesis that LMA is a cress allelochemical that appreciably perturbs the growth of potentially competing seedlings. Since LMA and Xyl→GalA slightly promoted both hypocotyl and root elongation, their effect could be nutritional. We conclude that rhamnogalacturonan-I and xylogalacturonan (pectin domains) are not sources of oligosaccharins with allelochemical activity, and the biological roles (if any) of the disaccharides derived from them are unknown. The main allelochemical principle in cress-seed exudate remains to be identified.

Sugar composition of the pectic polysaccharides of charophytes, the closest algal relatives of land-plants: presence of 3-O-methyl-D-galactose residues.

BACKGROUND AND AIMS: During evolution, plants have acquired and/or lost diverse sugar residues as cell-wall constituents. Of particular interest are primordial cell-wall features that existed, and in some cases abruptly changed, during the momentous step whereby land-plants arose from charophytic algal ancestors. METHODS: Polysaccharides were extracted from four charophyte orders [Chlorokybales (Chlorokybus atmophyticus), Klebsormidiales (Klebsormidium fluitans, K. subtile), Charales (Chara vulgaris, Nitella flexilis), Coleochaetales (Coleochaete scutata)] and an early-diverging land-plant (Anthoceros agrestis). 'Pectins' and 'hemicelluloses', operationally defined as extractable in oxalate (100 °C) and 6 m NaOH (37 °C), respectively, were acid- or Driselase-hydrolysed, and the monosaccharides analysed chromatographically. One unusual monosaccharide, 'U', was characterized by (1)H/(13)C-nuclear magnetic resonance spectroscopy and also enzymically. KEY RESULTS: 'U' was identified as 3-O-methyl-D-galactose (3-MeGal). All pectins, except in Klebsormidium, contained acid- and Driselase-releasable galacturonate, suggesting homogalacturonan. All pectins, without exception, released rhamnose and galactose on acid hydrolysis; however, only in 'higher' charophytes (Charales, Coleochaetales) and Anthoceros were these sugars also efficiently released by Driselase, suggesting rhamnogalacturonan-I. Pectins of 'higher' charophytes, especially Chara, contained little arabinose, instead possessing 3-MeGal. Anthoceros hemicelluloses were rich in glucose, xylose, galactose and arabinose (suggesting xyloglucan and arabinoxylan), none of which was consistently present in charophyte hemicelluloses. CONCLUSIONS: Homogalacturonan is an ancient streptophyte feature, albeit secondarily lost in Klebsormidium. When conquering the land, the first embryophytes already possessed rhamnogalacturonan-I. In contrast, charophyte and land-plant hemicelluloses differ substantially, indicating major changes during terrestrialization. The presence of 3-MeGal in charophytes and lycophytes but not in the 'intervening' bryophytes confirms that cell-wall chemistry changed drastically between major phylogenetic grades.

An unexpectedly lichenase-stable hexasaccharide from cereal, horsetail and lichen mixed-linkage ß-glucans (MLGs): implications for MLG subunit distribution.

Mixed-linkage (1→3),(1→4)-ß-d-glucan (MLG) is a biologically and technologically important hemicellulose, known to occur in three widely separated lineages: the Poales (including grasses and cereals), Equisetum (fern-allies), and some lichens e.g. Iceland moss (Cetraria islandica). Lichenase (E.C. 3.2.1.73) is widely assumed to hydrolyse all (1→4) bonds that immediately follow (1→3) bonds in MLG, generating predominantly the tetrasaccharide ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G4G4G3G; MLG4), the corresponding trisaccharide (G4G3G; MLG3), and sometimes also laminaribiose (G3G; MLG2). The ratio of the oligosaccharides produced characterises each polysaccharide. We report here that digestion of MLG from barley (Hordeum vulgare), Equisetum arvense and C. islandica by Bacillus subtilis lichenase also yields the unexpectedly stable hexasaccharide, ß-d-Glcp-(1→3)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→4)-ß-d-Glcp-(1→3)-d-Glc (G3G4G4G4G3G, i.e. MLG2-MLG4), identified by thin-layer chromatography, gel-permeation chromatography, HPLC (HPAEC), ß-glucosidase digestion, (1)H/(13)C-NMR spectroscopy and mass spectrometry. On HPLC, G3G4G4G4G3G is the major constituent of a peak previously ascribed solely to the nonasaccharide G4G4G4G4G4G4G4G3G. Because it was widely presumed that lichenase would cleave G3G4G4G4G3G to MLG2+MLG4, our data both redefine the substrate specificity of Bacillus lichenase and show previous attempts to characterise MLGs by HPLC of lichenase-digests to be flawed. MLG2 subunits are particularly underestimated; often reported as negligible, they are here shown to be an appreciable constituent of MLGs from all three lineages. We also show that there is no appreciable yield of water-soluble lichenase products with DP>9; potential identities of products previously labelled DP>9 are suggested. Finally, this discovery also provides a opportunity to investigate the spatial distribution of subunits along the MLG chain. We show that MLG2 subunits in barley and Cetraria MLG are not randomly distributed, but predominantly found at the non-reducing end of MLG4 subunits.

Characterisation of oligosaccharides from the chondroitin/dermatan sulphates: (1)H and (13)C NMR studies of oligosaccharides generated by nitrous acid depolymerisation.

The polymers chondroitin sulphate and dermatan sulphate have been fragmented by an anhydrous hydrazine/nitrous acid procedure. The resulting disaccharides from the polymer repeat sequences were reduced with NaBH(4) and purified by ion exchange chromatography. Whereas enzymatic depolymerisation leads to the loss of the distinction between glucuronic and iduronic acids of CS and DS in the resultant disaccharides, nitrous acid depolymerisation retains these structures. Complete (1)H and (13)C NMR data have been derived for the major components which were shown to have the structures: GlcA-(ß1→3)-anTal6S-ol (I) and L-IdoA-(α1→3)-anTal4S-ol (II), where anTal-ol represents (2,5)anhydro-D-talitol and 6S/4S represent O-ester sulphate groups at C-6/C-4 sites.

Impaired gluconeogenesis in a porcine model of paracetamol induced acute liver failure.

AIM: To investigate glucose homeostasis and in particular gluconeogenesis in a large animal model of acute liver failure (ALF). METHODS: Six pigs with paracetamol induced ALF under general anaesthesia were studied over 25 h. Plasma samples were withdrawn every five hours from a central vein. Three animals were used as controls and were maintained under anaesthesia only. Using (1)H NMR spectroscopy we identified most gluconeogenic amino acids along with lactate and pyruvate in the animal plasma samples. RESULTS: No significant changes were observed in the concentrations of the amino acids studied in the animals maintained under anaesthesia only. If we look at the ALF animals, we observed a statistically significant rise of lactate (P < 0.003) and pyruvate (P < 0.018) at the end of the experiments. We also observed statistically significant rises in the concentrations of alanine (P < 0.002), glycine (P < 0.005), threonine (P < 0.048), tyrosine (P < 0.000), phenylalanine (P < 0.000) and isoleucine (P < 0.01). Valine levels decreased significantly (P < 0.05). CONCLUSION: Our pig model of ALF is characterized by an altered gluconeogenetic capacity, an impaired tricarboxylic acid (TCA) cycle and a glycolytic state.

Lunacridine from Lunasia amara is a DNA intercalating topoisomerase II inhibitor.

An ethnobotanical survey of plants used to treat tropical ulcers in Papua New Guinea identified Lunasia amara as possessing anti-Staphylococcus aureus activity. Activity-guided fractionation of the aqueous bark extract resulted in the identification of the quinoline alkaloid lunacridine as the active principle. Lunacridine tends to cyslise at room temperature but the 2'-O-trifluoroacetyl derivative was found to be stable and therefore more suitable for biological assays. The compound exhibited a minimal inhibitory concentration (MIC) of 64 micro g/ml against Staphylococcus aureus NCTC 6571 and activity in the low micromolar range against HeLa and H226 cells; the latter showing signs of caspase-3/7 mediated apoptotic cell death. Experiments with drug resistant strains of Streptococcus pneumoniae suggested topoisomerase as a likely target for the drug in bacteria whilst decatenation assays with human topoisomerase II showed the compound to be a potent inhibitor of this isoform (IC(50)<5 micro M) thus explaining the drug's activity against human cell lines. Both lunacridine and 2'-O-trifluoroacetyl lunacridine exhibited mild DNA intercalation activity giving 50% decrease in ethidium DNA fluorescence at 0.22 and 0.6 mM, respectively, placing the drug amongst the DNA intercalating class of topoisomerase II inhibitors.

Generation and contrasting gas-phase reactivity of 2-(2-alkenylpyrrol-1-yl)phenoxyl and thiophenoxyl radicals.

The pyrrolylacrylates 9 and 10 were synthesised and subjected to flash vacuum pyrolysis (FVP) at 650-700 degrees C to generate the radicals 11 and 18, respectively. The phenoxyl 11 underwent hydrogen capture to give a mixture of the phenol 12 and the pyrrolobenzoxazine 13 in low yields, which were also obtained by a Wittig reaction of the 2-formylpyrrole 14. The thiophenoxyl 18 gave a single major product in 41% yield which was identified as the pyrrolo[1,2-a]quinoline 17 by a sequence of NMR experiments. A mechanism for the formation of 17 by a rearrangement-sulfur extrusion sequence is proposed.

Characterization of oligosaccharides from the chondroitin/dermatan sulfates. 1H-NMR and 13C-NMR studies of reduced trisaccharides and hexasaccharides.

Chondroitin and dermatan sulfate (CS and DS) chains were isolated from bovine tracheal cartilage and pig intestinal mucosal preparations and fragmented by enzymatic methods. The oligosaccharides studied include a disaccharide and hexasaccharides from chondroitin ABC lyase digestion as well as trisaccharides already present in some commercial preparations. In addition, other trisaccharides were generated from tetrasaccharides by chemical removal of nonreducing terminal residues. Their structures were examined by high-field 1H and 13C NMR spectroscopy, after reduction using sodium borohydride. The main hexasaccharide isolated from pig intestinal mucosal DS was found to be fully 4-O-sulfated and have the structure: DeltaUA(beta1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S(beta1-4)L-IdoA(alpha1-3)GalNAc4S-ol, whereas one from bovine tracheal cartilage CS comprised only 6-O-sulfated residues and had the structure: DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. No oligosaccharide showed any uronic acid 2-sulfation. One novel disaccharide was examined and found to have the structure: GalNAc6S(beta1-4)GlcA-ol. The trisaccharides isolated from the CS/DS chains were found to have the structures: DeltaUA(beta1-3)GalNAc4S(beta1-4)GlcA-ol and DeltaUA(beta1-3)GalNAc6S(beta1-4)GlcA-ol. Such oligosaccharides were found in commercial CS/DS preparations and may derive from endogenous glucuronidase and other enzymatic activity. Chemically generated trisaccharides were confirmed as models of the CS/DS chain caps and included: GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc4S-ol and GalNAc6S(beta1-4)GlcA(beta1-3)GalNAc6S-ol. The full assignment of all signals in the NMR spectra are given, and these data permit the further characterization of CS/DS chains and their nonreducing capping structures.

Comparison of bioenergetic activity of primary porcine hepatocytes cultured in four different media.

Primary hepatocytes have extensively been used in biochemical, pharmacological, and physiological research. Recently, primary porcine hepatocytes have been regarded as the cells of choice for bioartificial liver support systems. The optimum culture medium for hepatocytes to be used in such devices has yet to be defined. In this study we investigated the effectiveness of four culture media in driving energy metabolism of primary porcine hepatocytes. The media selected were William's E medium, medium 1640, medium 199, and hepatocyte medium. Cells (3 x 10(10); viability 87 +/- 6%) were isolated from weanling piglets and seeded on 90-mm plates in the above media supplemented with antibiotics and hormones at a density of 8 x 10(6) viable cells per plate. Using 1H NMR spectroscopy we looked at indices of glycolysis, gluconeogenesis. ketogenesis, and ureagenesis on days 2, 4, and 6 of the experiments (n = 9). We also studied urea and albumin synthesis and total P450 content. The examined metabolic pathways of the hepatocytes were maintained by all media, although there were statistically significant differences between them. All media performed well in glycolysis, ureagenesis, and albumin synthesis. William's E medium and medium 199 outperformed the rest in gluconeogenesis. Medium 199 was best in ketogenesis. Overall, medium 199 was the best at driving energy metabolism from its constituent substrates and we think that it preferentially should be used in the culture of primary porcine hepatocytes.

Biochemical prognostic markers of outcome in non-paracetamol-induced fulminant hepatic failure.

BACKGROUND: Fulminant hepatic failure (FHF) is associated with major metabolic disturbances, the onset and severity of which can predict clinical outcome. This study uses admission blood samples to identify early biochemical markers of clinical outcome in patients with non-paracetamol-induced FHF. PATIENTS AND METHODS: Fifty-nine patients admitted to the Scottish Liver Transplant Unit with non-paracetamol-induced FHF were studied. Plasma samples were collected at a median of 5.4 hr after admission to our unit and analyzed using conventional laboratory tests and nuclear magnetic resonance spectroscopy. RESULTS: A total of 19 patients underwent transplantation, 15 patients died without undergoing transplantation, and 25 patients survived with medical management alone. There were significantly lower levels of lactate, alanine, valine, and bilirubin and significantly higher levels of pyruvate and albumin in patients who survived spontaneously compared with the other two groups. By use of multiple logistic regression analysis, an equation was devised that best predicted clinical outcome: 0.5x(albumin [g/L])-2x(lactate [mmol/L])-36x(valine [mmol/L])-38x(pyruvate [mmol/L]). Values of less than 2 were associated with poor clinical outcome and had a positive predictive value of 91%, a negative predictive value of 86%, a sensitivity of 94%, and a specificity of 86% for death or transplantation. This algorithm can be applied on admission, thus expediting decision-making. CONCLUSION: We identified biochemical markers that may be useful in predicting outcome in patients with non-paracetamol-induced FHF and should be evaluated further in a different patient population.

alpha-D-Glucuronosyl-(1-->3)-L-galactose, an unusual disaccharide from polysaccharides of the hornwort Anthoceros caucasicus.

Acid hydrolysis of cell wall-rich material from thalli of the hornwort Anthoceros caucasicus yielded substantial amounts of an unusual disaccharide (1). Hydrolysis of 1 yielded only GlcA, Gal and unhydrolysed 1. Compound 1 was identified as alpha-D-GlcpA-(1-->3)-L-Gal by 1H and 13C NMR spectroscopic analysis and by the susceptibility of its monosaccharide units to phosphorylation by enantiomer-specific kinases. Compound 1 was not detected in acid hydrolysates of other land plants including mosses, leafy and thalloid liverworts, lycopodiophytes and euphyllophytes; it was also absent from charophytes. The Anthoceros polysaccharide that yields 1 was partially extractable in cold aqueous buffer (pH 4.7) and Na(2)CO(3), but not in EDTA or NaOH, suggesting that it was not a typical pectin or hemicellulose. The yield of 1 from various polysaccharide fractions correlated with the yields of Xyl, suggesting a previously unreported polymer containing D-GlcA, L-Gal and Xyl. The existence of a unique polysaccharide in an evolutionarily isolated plant (Anthoceros) supports the view that major steps in plant phylogeny were accompanied by significant changes in cell wall composition.

The effect of hypothermia on primary porcine hepatocyte metabolism monitored by (1H) nuclear magnetic resonance spectroscopy.

OBJECTIVE: The aim of our study was to use (1H) nuclear magnetic resonance (NMR) spectroscopy as a tool to assess metabolic functions of hepatocytes and to monitor major metabolic pathways of these cells during culture following hypothermic preservation. METHODS: After isolation, 2 x 10(7) primary porcine hepatocytes were preserved at 4 degrees C in supplemented Leibovitz L-15 medium for 48 h. Viability was assessed at isolation, 24 and 48 h. At isolation and at 48 h cells were plated and cultured with serum free supplemented Williams E medium. 1H NMR spectroscopy was used to assess indices of glucose metabolism, ammonia clearance indices and ketone bodies precursors at 48 h post-plating. Peak integration was applied with sodium 3-(trimethylsilyl-2,2,3,3-2H4)-1-propionate as an internal standard to obtain quantitative results. RESULTS: Results were obtained from six isolations. Viability was 78.1 +/- 1.2% at isolation, 69 +/- 3.4% at 24 h and 58.9 +/- 3.8% at 48 h of hypothermia. Plating efficiency was 87 +/- 4% for freshly isolated cells and 33.6 +/- 7.6% for hypothermically preserved cells. Glucose consumption was comparable in both groups. Hypothermically preserved cells consumed more threonine, produced more pyruvate and alanine but less lactate. They also produced less acetate and consumed less tyrosine. Glutamate and glutamine concentrations were similar under both conditions. CONCLUSION: 1H NMR spectroscopy is a valid method for assessing metabolic pathways of cultured primary porcine hepatocytes. Although hypothermically preserved cells had a reduced plating efficiency, they were still metabolically active. Thus, hypothermia can be used as a temporary preservation technique for primary porcine hepatocytes.

Patterns of methyl and O-acetyl esterification in spinach pectins: new complexity.

Driselase-digestion of cell walls from suspension-cultures of spinach (Spinacia oleracea L.), followed by anion-exchange chromatography, gel-permeation chromatography, preparative paper chromatography and preparative paper electrophoresis, yielded ten uronic acid-containing products in addition to free galacturonic acid (GalA). These included 4-O-methylglucuronic acid, alpha-L-rhamnopyranosyl-(1-->4)-D-glucuronic acid and several oligosaccharides containing GalA residues. The structures were unambiguously determined by a combination of 1- and 2-dimensional NMR spectroscopic techniques. Five of the six homogalacturonan-derived oligosaccharides purified contained 3-O-acetyl-GalA residues; however, methyl-esterified GalA residues occurred adjacent to both 2-O-acetyl-GalA and 3-O-acetyl-GalA residues. An acetylated, rhamnogalacturonan-I-derived oligosaccharide that was purified also contained 3-O-acetyl-GalA residues. Taken together with published data, our findings indicate considerable diversity in the patterns of pectin esterification. The implications for the action of pectin esterases are discussed.

Design of ET(B) receptor agonists: NMR spectroscopic and conformational studies of ET7-21[Leu7, Aib11, Cys(Acm)15].

In a previous report we have shown that the endothelin-B receptor-selective linear endothelin peptide, ET-1[Cys (Acm)1,15, Ala3, Leu7, Aib11], folds into an alpha-helical conformation in a methanol-d3/water co-solvent [Hewage et al. (1998) FEBS Lett., 425, 234-238]. To study the requirements for the structure-activity relationships, truncated analogues of this peptide were subjected to further studies. Here we report the solution conformation of ET7-21[Leu7, Aib11, Cys(Acm)15], in a methanol-d3/water co-solvent at pH 3.6, by NMR spectroscopic and molecular modelling studies. Further truncation of this short peptide results in it displaying poor agonist activity. The modelled structure shows that the peptide folds into an alpha-helical conformation between residues Lys9-His16, whereas the C-terminus prefers no fixed conformation. This truncated linear endothelin analogue is pivotal for designing endothelin-B receptor agonists.

1H NMR spectroscopy as a tool to evaluate key metabolic functions of primary porcine hepatocytes after cryopreservation.

Proton NMR spectroscopy of biological fluids has produced interesting results lately. We used the technique to investigate the effects of cryopreservation on primary porcine hepatocytes as successful cryopreservation of primary porcine hepatocytes is of importance to the development of bioartificial liver support systems. After isolation 10(8) hepatocytes were cryopreserved for 1 week in Williams E/10% DMSO, either by quick freezing (-5 to -30 degrees C/min), slow freezing (-0.3 to -3 degrees C/min) or stepwise freezing protocols on cell suspensions and confluent cell plates. Plating efficiency was assessed by percentage LDH release. Metabolic functions of cryopreserved hepatocytes at 24 h post-thawing were compared with those of fresh hepatocyte cultures at 48 h. 1H nuclear magnetic resonance spectroscopy of the culture medium post-incubation, using the presaturation technique, assessed the following: glucose metabolism, transamination and glutamine synthesis and succinate synthesis. Freshly isolated cells had a viability of 82 +/- 4.3% and a plating efficiency of 87 +/- 3.8%. All cryopreservation protocols resulted in significantly reduced viability and plating efficiency. No significant differences were observed between different cryopreservation media or protocols. When comparing cryopreserved with freshly isolated cells, we observed that metabolism of acetyl-CoA precursors was significantly impaired in cryopreserved cells. Lactate and pyruvate production was also significantly less, although glucose consumption was similar. No differences were observed in gluconeogenic amino acid metabolism, transamination and urea synthesis. 1H NMR spectroscopy can be used to provide information about metabolic activity and functions of cultured primary cells.