Alterations in carbohydrate metabolism of γ-irradiated cavendish banana

γ-Irradiation of preclimacteric banana resulted in a gradual increase in fructose content, which reached a maximum in 6 days. Although the catabolism of glucose-U-¹⁴C was less in irradiated banana, incorporation of label into fructose was high. Initial fructose accumulation in irradiated banana may be due to a shift in glucose utilization from the glycolytic to the pentose phosphate pathway. The ratio of resporatory CO₂ from glucose-6-¹⁴C and glucose-1-¹⁴C was halved in irradiated bananas indicating predominance of the pentose phosphate pathway. The radioactivity of fructose derived from glucose-6-¹⁴C was almost twice that from glucose-1-¹⁴C in irradiated bananas, whilst in control both fruit the labelled precursors yielded equal amounts. Studies on individual enzymes in these two pathways showed an increase in phosphorylase, phosphoglucomutase, glucose-6-phosphate dehydrogenase and fructose-6-phosphatase and a decrease in hexokinase in irradiated banana.     

Denitration of carbohydrate α-nitroepoxides by nucleophiles

Several nucleophiles were separately treated with methyl and phenyl 2,3-anhydro-4,6-O-benzylidene-3-deoxy-3-nitro-β-d-allopyranoside, to give 2-substituted aldos-3-ulose derivatives. In the latter case, the subsequent β-elimination of the aglyconic phenyl group always occurred to afford the corresponding glycal. Reaction mechanisms thereof are also discussed.     

Protein and carbohydrate moieties of a preparation of β-lactamase II

1. A crystalline preparation of beta-lactamase II has been separated into two moieties by gel filtration on a column of Sephadex G-100. 2. The first moiety consisted mainly of carbohydrate and showed virtually no beta-lactamase activity. 3. The second moiety was a protein of molecular weight 22500, which was enzymically active. 4. The protein moiety, like the original protein-carbohydrate complex, required Zn(2+) for beta-lactamase activity. It did not differ significantly from the complex in its behaviour to a number of cephalosporin substrates, but was less stable to heat than the complex. 5. About 30% of the total beta-lactamase activity was lost when the protein-carbohydrate complex was separated into the two moieties. This activity was regained when the protein and carbohydrate moieties were mixed, but the mixture did not show the heat stability of the original complex.     

Recognition roles of the carbohydrate glycotopes of human and bovine lactoferrins in lectin–N-glycan interactions

Background

Lactoferrin is an iron-binding protein belonging to the transferrin family. In addition to iron homeostasis, lactoferrin is also thought to have anti-microbial, anti-inflammatory, and anticancer activities. Previous studies showed that all lactoferrins are glycosylated in the human body, but the recognition roles of their carbohydrate glycotopes have not been well addressed.

Methods

The roles of human and bovine lactoferrins involved in lectin–N-glycan recognition processes were analyzed by enzyme-linked lectinosorbent assay with a panel of applied and microbial lectins.

Results and conclusions

Both native and asialo human/bovine lactoferrins reacted strongly with four Man-specific lectins — Concanavalia ensiformis agglutinin, Morniga M, Pisum sativum agglutinin, and Lens culinaris lectin. They also reacted well with PA-IIL, a LFuc>Man-specific lectin isolated from Pseudomonas aeruginosa. Both human and bovine lactoferrins also recognized a sialic acid specific lectin-Sambucus nigra agglutinin, but not their asialo products. Both native and asialo bovine lactoferrins, but not the human ones, exhibited strong binding with a GalNAc>Gal-specific lectin-Wisteria floribunda agglutinin. Human native lactoferrins and its asialo products bound well with four Gal>GalNAc-specific type-2 ribosome inactivating protein family lectins-ricin, abrin-a, Ricinus communis agglutinin 1, and Abrus precatorius agglutinin (APA), while the bovine ones reacted only with APA.

General significance

This study provides essential knowledge regarding the different roles of bioactive sites of lactoferrins in lectin–N-glycan recognition processes.     

Evolution of carbohydrate antigens--microbial forces shaping host glycomes?

Many glycans show remarkably discontinuous distribution across evolutionary lineages. These differences play major roles when organisms belonging to different lineages interact as host-pathogen or host-symbiont. Certain lineage-specific glycans have become important signals for multicellular host organisms, which use them as molecular signatures of their pathogens and symbionts through recognition by a toolkit of innate defense molecules. In turn, pathogens have evolved to exploit host lineage-specific glycans and are constantly shaping the glycomes of their hosts. These interactions take place in the face of numerous critical endogenous functions played by glycans within host organisms. Whether due to simple evolutionary divergence or adaptive changes under natural selection resulting from endogenous functional requirements, once different lineages elaborate on differential glycomes these mutual differences provide opportunities for host exploitation and/or pathogen defense between lineages. Such phylogenetic molecular recognition mechanisms will augment and likely contribute to the maintenance of lineage-specific differences in glycan repertoires.     

The carbohydrate structures ofβ-galactosidase from human liver

Acid -galactosidase (EC3.2.1.23) was obtained from human liver in a pure monomeric state (M_r63 000). The carbohydrate content of the enzyme was established to be, 9% by weight; mannose,N-acetylglucosamine, galactose andN-acetylneuraminic acid were found to be the constituent monosaccharides. The carbohydrate structures of the enzyme were studied at the glycopeptide level by employing 500 MHz¹H-NMR spectroscopy, carbohydrate composition analysis and methylation analysis involving GLCMS. Based upon the intensities of relevant signals in the¹H-NMR spectrum, approximately 60% of the chains were found to be of theN-acetyllactosamine type, having the structure The rest appeared to be of the oligomannoside type (Man₅-₆GlcNAc₂Asn). The carbohydrate composition and methylation analysis results sustained these findings, although the calculation of the distribution based upon these techniques indicated a somewhat lower percentage ofN-acetyllactosamine type chains. There are approximately three oligosaccharide chains per molecule. These findings offer an explanation for the abnormal distribution of -galactosidase in tissues and cultured fibroblasts of patients with I-cell disease.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid -l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kD_a and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHz¹H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 31. While the oligomannoside-type chains show some heterogeneity in size (Man_5–8GlcNAc₂), theN-acetyllactosaminetype chains are exclusively bi-(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of -l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, -N-acetylhexosaminidase, -l-fucosidase and -galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

The oncofetal Thomsen–Friedenreich carbohydrate antigen in cancer progression

The oncofetal Thomsen–Friedenreich carbohydrate antigen (Galβ1-3GalNAcα1-Ser/Thr TF or T antigen) is a pan-carcinoma antigen highly expressed by about 90% of all human carcinomas. Its broad expression and high specificity in cancer have attracted many investigations into its potential use in cancer diagnosis and immunotherapy. Over the past few years increasing evidence suggests that the increased TF occurrence in cancer cells may be functionally important in cancer progression by allowing increased interaction/communication of the cells with endogenous carbohydrate-binding proteins (lectins), particularly the members of the galactoside-binding galectin family. This review focuses on the recent progress in understanding of the regulation and functional significance of increased TF occurrence in cancer progression and metastasis.     

Quantification of lignin–carbohydrate linkages with high-resolution NMR spectroscopy

A quantitative approach to characterize lignin–carbohydrate complex (LCC) linkages using a combination of quantitative ¹³C NMR and HSQC 2D NMR techniques has been developed. Crude milled wood lignin (MWLc), LCC extracted from MWLc with acetic acid (LCC-AcOH) and cellulolytic enzyme lignin (CEL) preparations were isolated from loblolly pine (Pinus taeda) and white birch (Betula pendula) woods and characterized using this methodology on a routine 300 MHz NMR spectrometer and on a 950 MHz spectrometer equipped with a cryogenic probe. Structural variations in the pine and birch LCC preparations of different types (MWL, CEL and LCC-AcOH) were elucidated. The use of the high field NMR spectrometer equipped with the cryogenic probe resulted in a remarkable improvement in the resolution of the LCC signals and, therefore, is of primary importance for an accurate quantification of LCC linkages. The preparations investigated showed the presence of different amounts of benzyl ether, γ-ester and phenyl glycoside LCC bonds. Benzyl ester moieties were not detected. Pine LCC-AcOH and birch MWLc preparations were preferable for the analysis of phenyl glycoside and ester LCC linkages in pine and birch, correspondingly, whereas CEL preparations were the best to study benzyl ether LCC structures. The data obtained indicate that pinewood contains higher amounts of benzyl ether LCC linkages, but lower amounts of phenyl glycoside and γ-ester LCC moieties as compared to birch wood.     

Variation in carbohydrate source–sink relations of forest and treeline white spruce in southern, interior and northern Alaska

Two opposing hypotheses have been presented to explain reduced tree growth at the treeline, compared with growth in lower elevation or lower latitude forests: the carbon source and sink limitation hypotheses. The former states that treeline trees have an unfavorable carbon balance and cannot support growth of the magnitude observed at lower elevations or latitudes, while the latter argues that treeline trees have an adequate carbon supply, but that cold temperatures directly limit growth. In this study, we examined the relative importance of source and sink limitation in forest and treeline white spruce (Picea glauca) in three mountain ranges from southern to northern Alaska. We related seasonal changes in needle nonstructural carbohydrate (NSC) content with branch extension growth, an approach we argue is more powerful than using needle NSC concentration. Branch extension growth in the southernmost Chugach Mountains was much greater than in the White Mountains and the Brooks Range. Trees in the Chugach Mountains showed a greater seasonal decline in needle NSC content than trees in the other mountain ranges, and the seasonal change in NSC was correlated with site-level branch growth across mountain ranges. There was no evidence of a consistent difference in branch growth between the forest and treeline sites, which differ in elevation by approximately 100 m. Our results point to a continuum between source and sink limitation of growth, with high-elevation trees in northern and interior Alaska showing greater evidence of sink limitation, and those in southern Alaska showing greater potential for source limitation.     

Azospirillum brasilense colonisation of wheat roots and the role of lectin–carbohydrate interactions in bacterial adsorption and root-hair deformation

The dynamics of adsorption of the nitrogen-fixing soil bacteria Azospirillum brasilense 75 and 80 (isolated from soil samples collected in Saratov Oblast, southern Russia) and A. brasilense Sp245 to the roots of seedlings of common spring wheat was studied in relation to inoculum size, period of incubation with the roots and bacterial-growth phase. The number of root-attached cells increased with increasing size of inoculum and time of contact. The saturation of root-surface adsorption was observed by 24 h of co-incubation for A. brasilense 75, by 6 h for A. brasilense 80, and by 3 h for A. brasilense Sp245. The firmness of bacterial–root attachment increased after extended co-incubation. Differences in the adsorption kinetics of the azospirilla were found that were associated with bacterial-growth phases. Azospirilla attached to the roots of their host cultivar more actively than they did to the roots of a non-host cultivar. Adsorption was partially inhibited when the roots were treated with N-acetyl-D-glucosamine. Maximal inhibition occurred after a 3-h exposure of the roots to the bacteria. Root-hair deformation induced with polysaccharide-containing complexes from the Azospirillum capsular material was inhibited by N-acetyl-D-glucosamine and chitotriose, specific haptens of wheat germ agglutinin. A possible mechanism of the mutual influence of bacteria and plants may involve key roles of wheat germ agglutinin, present on the roots, and the polysaccharide-containing components of the Azospirillum capsule.     

Determination of the structure of the carbohydrate chains of acid α-glucosidase from human placenta

Acid α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) from human placenta (70 and 76 kDa) was found to contain 4 N-glycosidic carbohydrate chains per molecule. Sugar analysis of purified enzyme revealed the presence of mannose, N-acetylglucosamine and fucose at a molar ratio of 5.0:2.0:0.6. In addition, trace amounts of galactose and N-acetylneuraminic acid were detected. The sugar chains were liberated from the polypeptides by the hydrazinolysis procedure and subsequently fractionated by gel filtration and HPLC. Purified compounds were investigated by 500-MHz ¹H-NMR spectroscopy. Oligomannoside-type chains of intermediate size, e.g., Man₅GlcNAcGlcNAc-ol and Man₇GlcNAcGlcNAc-ol, and N-type chains of smaller size e.g., Man_2–3GlcNAc[Fuc]_0–1GlcNAc-ol, were demonstrated to be present at a ratio of 2:3. In addition, a small amount of sialylated N-acetyllactosamine-type chains has been found. The possible biosynthetic route of the fucose-containing small-size chains is discussed.     

Seasonal influences on carbohydrate metabolism in the CAM bromeliad Aechmea ��Maya��: consequences for carbohydrate partitioning and growth

Background and Aims

Photosynthetic plasticity in response to a range of environmental factors that include [CO₂], water availability, light intensity and temperature, is ubiquitous among plants with crassulacean acid metabolism (CAM). The present study examined how seasonal changes in light availability, as experienced by greenhouse CAM crops in northern latitude regions, influence diel carboxylation patterns and impact on carbon gain and seasonal accumulation of biomass.

Methods

In the CAM bromeliad Aechmea ‘Maya’ integrated measurements of leaf gas exchange, diel metabolite dynamics (e.g. malate, soluble sugars and starch) and biomass accumulation were made four times a year, i.e. in winter, spring, summer and autumn.

Key Results

During the brighter seasons (spring and summer) daytime Phases II and IV were dominated by C₄ carboxylation, whilst the higher diurnal uptake in the autumn and winter was characterized by equal contributions of both Rubisco and PEPC. As a consequence, net CO₂ uptake showed a significant depression at the end of the day in the darker months when supplementary illumination was turned off. Remarkable seasonal consistency was found in the amount of storage reserves available for nocturnal carboxylation, a consequence of predominantly daytime export of carbohydrate in spring and summer whilst nocturnal export was the major sink for carbohydrate in autumn and winter.

Conclusions

Throughout the different seasons Aechmea ‘Maya’ showed considerable plasticity in the timing and magnitude of C₃ and C₄ carboxylation processes over the diel cycle. Under low PPFD (i.e. winter and autumn) it appears that there was a constraint on the amount of carbohydrate exported during the day in order to maintain a consistent pool of transient carbohydrate reserves. This gave remarkable seasonal consistency in the amount of storage reserves available at night, thereby optimizing biomass gain throughout the year. The data have important practical consequences for horticultural productivity of CAM plants and suggest a scenario for reconciling carbohydrate partitioning between competing sinks of nocturnal acidification and export for growth.Key words: Aechmea ‘Maya’, seasonal, CAM, bromeliad, carbon budgets, gas exchange, metabolite dynamics, PEPC, photoperiod extension, PPFD, photosynthetic plasticity, Rubisco     

Structural assessment of β-glucuronidase carbohydrate chains by lectin affinity chromatography

Rat liver -glucuronidase was studied by sequential lectin affinity chromatography. -Glucuronidase glycopeptides were obtained by extensive Pronase digestion followed byN-[¹⁴C]acetylation and desialylation by neuraminidase treatment. According to the distribution of the radioactivity in the various fractions obtained by chromatography on different lectins, and on the assumption that all glycopeptides were acetylated to the same specific radioactivity, a relative distribution of glycan structure types is proposed. The presence of complex biantennary and oligomannose type glycans (56.8% and 42.7%, respectively) was indicated by Concanavalin A-Sepharose chromatography.Ulex europaeus agglutinin-agarose chromatography revealed the presence of (1-3) linked fucose in some of the complex biantennary type glycans (16.6% of the total glycopeptides). Wheat germ agglutinin chromatography indicated that the minority (0.5%) were hybrid or poly (N-acetyllactosamine) type glycans. Furthermore, the absence of O-glycans, tri-, tetra- and bisected biantennary type glycans was demonstrated by analysis of Concanavalin A-Sepharose unbound fraction by chromatography on immobilized soybean agglutinin,Ricinus communis agglutinin andPhaseolus vulgaris erythroagglutinin.     

The effect of hypoxia on carbohydrate metabolism in flounder (Platichthys flesus L.)—I. Utilization of glycogen and accumulation of glycolytic end products in various tissues

• 1.1. Resting oxygen consumption at 10°C did not change from normoxia (150 mm Hg) down to an oxygen tension of 55 mm Hg for the flounder, Platichtys flesus.
• 2.2. Flounders exposed to hypoxia showed increased levels of blood glucose and lactate, dependent on the degree of hypoxia.
• 3.3. Due to hypoxia glycogen was depleted in the liver and swimming muscle but in the heart there was no significant change.
• 4.4. Liver glucose increased after 7 hr of hypoxia. Heart and muscle glucose did not change but the absolute glucose concentration in the heart was five times higher than in the muscle.
• 5.5. There is a transient accumulation of lactate in heart, liver and kidney after 7 hr of hypoxia while lactate accumulation in the swimming muscle is significant only after 21 hr of hypoxia.
• 6.6. Succinate only accumulated in the liver while alanine accumulated in muscle, heart and liver.

     

Differential carbohydrate metabolism conducts morphogenesis in embryogenic callus of Hevea brasiliensis (Müll. Arg.)

Somatic embryogenesis in Hevea is stimulated when the embryogenesis induction medium contains maltose, rather than glucose, fructose, or sucrose, in equimolarity (Blanc et al., 1999). Kinetic analyses were carried out on various physiological and biochemical indicators over the 8 weeks that the induction phase then expression of somatic embryogenesis can take. Embryogenesis induction in the presence of glucose, fructose or sucrose revealed strong callus growth in the first 3-4 weeks, associated with a high intra- and extracellular hexose content, a high starch content and a substantial decline in protein synthesis. In the presence of maltose, callus growth was slow and only half that seen with sucrose. This morphogenetic behaviour is associated with a drop in endogenous hexose and starch contents, and an increase in protein synthesis in the first three weeks of culture. The induction of embryogenesis in the presence of maltose was uniform and twice as fast as with sucrose supply. At the end of culture, peroxidase activity, antioxidant and membrane protein contents increased in these calluses; these characteristics may be associated with somatic embryo organization and with the maintenance of effective membrane integrity within a nutrient environment that has become limiting. These new results tally with data in the literature on the roles of sugars, and provide some precise information with regard to the 'carbohydrate deficit' hypothesis usually put forward to explain maltose action. An analysis of these results led to the hypothesis that regulation of endogenous hexose contents at a low level, through slow maltose hydrolysis, was a key element of the biochemical signal leading this callus towards somatic embryogenesis.