Surface plasmon resonance imaging for real-time, label-free analysis of protein interactions with carbohydrate microarrays

Plant lectin recognition of glycans was evaluated by SPR imaging using a model array of N-biotinylated aminoethyl glycosides of β-d-glucose (negative control), α-d-mannose (conA-responsive), β-d-galactose (RCA₁₂₀-responsive) and N-acetyl-β-d-glucosamine (WGA-responsive) printed onto neutravidin-coated gold chips. Selective recognition of the cognate ligand was observed when RCA₁₂₀ was passed over the array surface. Limited or no binding was observed for the non-cognate ligands. SPR imaging of an array of 40 sialylated and unsialylated glycans established the binding preference of hSiglec7 for α2-8-linked disialic acid structures over α2-6-sialyl-LacNAcs, which in turn were recognized and bound with greater affinity than α2-3-sialyl-LacNAcs. Affinity binding data could be obtained with as little as 10–20 μg of lectin per experiment. The SPR imaging technique was also able to establish selective binding to the preferred glycan ligand when analyzing crude culture supernatant containing 10–20 μg of recombinant hSiglec7-Fc. Our results show that SPR imaging provides results that are in agreement with those obtained from fluorescence based carbohydrate arrays but with the added advantage of label-free analysis.     

Metabolic characterisation of a novel vanillylmandelate-degrading bacterium

A newly isolated gram-negative bacterium, possibly Brevundimonas diminuta, utilised d,l-vanillylmandelate (d,l-VMA) as a sole carbon and energy source. The organism converted d,l-VMA to vanillylglyoxylate using a soluble NAD-dependent dehydrogenase specific for d-VMA and a dye-linked, membrane-associated l-VMA dehydrogenase. Vanillylglyoxylate was further metabolised by decarboxylation, dehydrogenation and demethylation to protocatechuate. A 4,5-dioxygenase cleaved protocatechuate to 2-hydroxy-4-carboxymuconic semialdehyde. Partially purified d-VMA dehydrogenase exhibited optimal activity at 30° C and pH 9.5 and had an apparent K _m for d-VMA of 470 μM. Although induced by several substituted mandelates, the enzyme had a narrow substrate specificity range with virtually no activity towards d-mandelate. Such properties render the enzyme of potential use in both diagnostic and biosynthetic applications. Received: 23 January 1996 / Accepted: 9 April 1996     

Influence of carbohydrates and polyols on l-lactic acid production and fatty acid formation by Rhizopus arrhizus

Growth and l-lactic acid production on 24 different carbohydrates and polyols (glycerol, mannitol and sorbitol) by Rhizopus arrhizus CCM 8109 were determined. The d- but not the l-forms of xylose, fructose, galactose, mannose, glucose, cellobiose, maltose and sucrose and partially hydrolysed starch were converted to l-lactic acid. Changes in lipid formation and fatty acid composition were detected in biomass grown on the different sugars. In the presence of polyols, growth and considerable production of lipids were observed with little or no lactate production. Invertase was mainly associated with the mycelium during growth on sucrose, whereas glucoamylase and -amylase were produced extracellularly during growth on starch.The authors are with the Department of Biochemical Technology, Faculty of Chemical Technology, Slovak Polytechnical University, Radlinského 9, SK-812 37 Bratislava, Slovak Republic     

Nutritional requirements ofXanthomonas phaseoli var.fuscans

As found by Starr (1946),l-glutamic acid is necessary for the growth ofXanthomonas phaseoli var.fuscans. According to our results, the growth is stimulated byl-asparagine in the presence ofl-glutamic acid;l-asparagine itself, however, does not serve as a source of carbon and nitrogen.Xanthomonas phaseoli var.fuscans grew well in a medium containing tryptone. Some peptides of the acidic fraction isolated from tryptone affected the growth as much as tryptone itself. Vitamins and plant growth substances did not affect the growth of the bacteria; proteins appeared to be a poor carbon and nitrogen source. On substituting glucose in a glutamic acid-containing medium with another saccharide, the growth of the bacteria was found equal or better in media containing mannose, sucrose, fructose, maltose or starch. The bacteria grew less satisfactorily in media containing galactose and cellobiose as compared with media containing glucose.     

First evidence on induced topological changes in supercoiled DNA by an aluminium <Emphasis Type="SmallCaps">d</Emphasis>-aspartate complex

Aluminium is a debatable and suspected etiological factor in neurodegenerative disorders. Aluminium–amino acid complexes also play an important role in the complex biology of the metal. Recent reports indicate the presence of d-aspartate and d-glutamate in aging brain, human breast tumors, core amyloid plaques and neurofibrillary tangles of Alzheimer's brain. This stereoinversion from the l- to the d-enantiomer is enhanced by Al. Further, the observation that Al is localized in the chromatin region encouraged the present study of the interaction of Al–amino acid complexes with DNA. This study used circular dichroism of supercoiled DNA and showed that Al–d-Asp caused a native B-DNA to C-DNA conformational change, while Al–l-Asp, Al–l-Glu and Al–d-Glu did not alter the native B-DNA conformation. This differential DNA binding property of Al–amino acid complexes is assigned to the stereoisomerism and chirality of the complexes. Interestingly, polyamines like spermine further induced an asymmetric condensation of the "limit C-motif" induced by Al–d-Asp to a -DNA. The results were supported by computer modeling, gel studies and ethidium bromide binding. We also propose a mechanism of Al–d-Asp binding and its ability to modulate DNA topology.     

<Emphasis Type="SmallCaps">d</Emphasis>-Galacturonic acid catabolism in microorganisms and its biotechnological relevance

d-Galacturonic acid is the main constituent of pectin, a naturally abundant compound. Pectin-rich residues accumulate when sugar is extracted from sugar beet or juices are produced from citrus fruits. It is a cheap raw material but currently mainly used as animal feed. Pectin has the potential to be an important raw material for biotechnological conversions to fuels or chemicals. In this paper, we review the microbial pathways for the catabolism of d-galacturonic acid that would be relevant for the microbial conversion to useful products.     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

The impact of supplemental carbon sources on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> growth,chlorophyll content and anthocyanin accumulation

This research explores the impacts of a broad range of supplemental carbon sources on growth and development of Arabidopsis thaliana. Parameters measured include dark-germinated hypocotyl length, light-germinated root growth, rosette growth, chlorophyll concentration and anthocyanin content. Treatment sugars include sucrose, maltose, d-glucose, d-fructose, l-arabinose, l-fucose, d-galactose, d-mannose, l-rhamnose and d-xylose each supplied at 4, 20 or 100 mM. This comparison of the effect of different carbon sources on multiple parameters and under identical conditions showed that every carbon source had unique qualitative and quantitative effects on Arabidopsis growth and development. Root growth was particularly sensitive to supplemental carbon source. Growth on 100 mM sucrose, maltose, glucose or xylose stimulated root growth by ~100%. Growth on arabinose, fucose, galactose, mannose or rhamnose inhibited root growth by 50% or more. Several sugars that strongly inhibited root growth had either no effect (galactose and fucose) or a positive effect (arabinose) on hypocotyl elongation and rosette growth. Rhamnose was the only carbon source that inhibited hypocotyl elongation across all concentrations. Sucrose, maltose, glucose, fructose, arabinose or xylose stimulated rosette growth by ~50%. Chlorophyll content was strongly reduced by mannose while sucrose, glucose, galactose and rhamnose caused smaller reductions. Anthocyanin accumulation was strongly induced by both galactose and mannose. Only mannose impacted all parameters across all concentrations. Based on these data it can be concluded that the effect of each carbon source on Arabidopsis growth and development is specific in terms of both magnitude and the parameters impacted.     

Carbon sources,growth, sclerotium formation and carbohydrate composition of Sclerotinia sclerotiorum

Summary Sclerotinia sclerotiorum (Lib.) D By. was grown in stationary liquid mineral-salts medium, pH 4.3, containing various carbon sources and the weight of mycelia and sclerotia was determined at regular intervals. When grown on various glucose concentrations (0–24 g of C/l), more sclerotia were produced at 8–12 g of C/l. Sclerotia were not usually formed in shake cultures. The ability of the fungus to use other carbon sources for growth and sclerotium formation was tested at 12 g of C/l in the stationary mineral-salts medium. The highest weights of mycelia and sclerotia occurred with raffinose, sucrose, maltose, lactose, d-mannose, d-glucose, d-fructose or l-arabinose. Good growth but decreased sclerotium production were found on cellobiose and d-xylose. Reduced or poor growth, a long lag period and few or no sclerotia occurred on trehalose, melibiose, l-sorbose, l-rhamnose, d-ribose, d-arabinose, l-xylose or 8 polyols. No growth was observed with erythritol or i-inositol. A combination of glucose plus trehalose or polyols resulted in increased growth and the formation of sclerotia. Organic acids supported little or no growth and no sclerotia were produced. Generally culture filtrates which supported growth and formation of sclerotia became acid (about pH 3.5). The pH of the culture filtrate usually increased slowly during the growth period when the fungus grew poorly and no sclerotia were formed. The alcoholsoluble sugars and polyols present in culture filtrates, mycelia and sclerotia were determined by paper and thin-layer chromatography. Regardless of the carbon source, mannitol was usually present in culture filtrates. The occurrence of other compounds in the filtrates depended on the carbon source. Trehalose, mannitol and usually small quantities of glucose or fructose were present in mycelia and sclerotia from all carbon sources. Galactitol or pentitols occurred in mycelia and sclerotia when the fungus grew on galactose and oligosaccharides containing galactose or the corresponding pentose, sugars. Acid hydrolyzates of the alcohol-insoluble fraction of mycelia or sclerotia contained glucose, smaller amounts of galactose and mannose and traces of ribose and rhamnose.     

Effect of Resveratrol on Gliotransmitter Levels and p38 Activities in Cultured Astrocytes

Accumulating evidence suggests that resveratrol may have beneficial effects against traumatic brain injury. However, its effect on the regulation of extracellular levels of gliotransmitter and on the activation of p38 MAPK in astrocytes is still unknown. We have examined whether resveratrol regulates extracellular levels of gliotransmitter as well as the activation of p38 MAPK in cultured astrocytes before and after stretch injury. The extracellular levels of glutamate, d-/l-serine and d-serine were apparently reduced by 100 μM resveratrol in control astrocyte cultures. The dramatic increase of glutamate and d-serine release induced by stretch injury was also clearly inhibited by resveratrol. Resveratrol mediates this response by reduction of release through inhibition of extracellular calcium influx and increment of gliotransmitter uptake through enhancement of amino acid transporter expressed in the membrane of astrocyte. In addition, resveratrol definitely reduced the activation of p38 MAPK in cultured astrocytes following stretch injury. AMPA receptor is involved in the activation of p38 following injury. Conversely, the levels of glutamine and glycine were not obviously affected by resveratrol before and after injury. Intracellular levels of glutamate and d-serine are not apparently changed by stretch injury. Collectively, our data suggest that resveratrol might play an important role in protection of the nervous system after injury by decreasing the extracellular levels of gliotransmitter and inhibiting activation of p38 MAPK following injury.     

Synthesis and cleavage of octulose bisphosphates with liver and muscle aldolases

Rabbit muscle aldolase was used to synthesize d-glycero-d-altro-octulose 1,8-bisphosphate and d-glycero-d-ido-octulose 1,8-bisphosphate. The products, isolated by ion-exchange chromatography, were characterized with the cysteine-sulfuric acid reaction and shown to be 90–95% pure by analysis for organic phosphorus and for dihydroxyacetone phosphate formed on cleavage with aldolase. The kinetic constants for synthesis and cleavage of these octulose bisphosphates with muscle and liver aldolases were determined. In the direction of cleavage both octulose bisphosphates were excellent substrates for liver aldolase, comparable to fructose 1,6-bisphosphate with respect to both V and K_m. With muscle aldolase the rate of cleavage was 1–5% of that with fructose bisphosphate and comparable to that with fructose 1-phosphate. In the direction of synthesis, ribose 5-phosphate was a better substrate than arabinose 5-phosphate for both the liver and muscle enzymes, although for both pentose phosphates the values of K_m fell in the range between 5 and 25 mm. It is concluded that reactions catalyzed by aldolase might account for the reported presence of these eight-carbon sugar phosphate esters in liver and in red cells.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Losses and biogeochemical cycling of soil organic nitrogen with prolonged arable cropping in the South African Highveld – evidence from - and -amino acids

We know little about the mechanisms that cause rapid losses in the soil organic N pool during cropping. As the analysis of amino acid enantiomers can provide insight into both the fate of microbial N and the ageing of cells in the environment, we used this technique as a tool to examine how the pool of protein-bound N in subtropical Plinthosols responds to increasing duration of arable cropping. The samples comprised bulk soils (0–20 cm) and clay fractions from each of three agro-ecosystems in semiarid South Africa; the sites have been cropped for periods varying from 0 to 98 years. The amino acid enantiomers contributed 34% to the total N content. With increasing number of years a piece of land has been cropped, the amino acid concentrations declined bi-exponentially to about 30% of their initial level in the native grasslands. Changes of the remaining soil protein-N pool were indicated by alterations in the d-content of individual amino acids. As the years of arable cropping increased, the proportions of d-alanine and d-glutamic acid increased relative to the respective l-enantiomers. This was attributed to an accumulation of N in residues of bacterial cell walls. In contrast, the d/l-ratios of leucine and aspartic acid declined in the long-term cultivated plots, probably reflecting losses of old amino acid-N reserves at the most degraded arable land.     

Die Proteinstruktur der Defektmutante PM 2 des Tabakmosaikvirus

Summary The protein structure of the TMV mutant PM2 has been studied. PM2 produces a non-functioning protein unable to aggregate into rodlike particles (Siegel et al., 1962). The two amino acid replacements in PM2-protein are localized in position 28 (threonineisoleucine), and position 95 (glutamic acidaspartic acid). This finding is discussed with relation to the spatial structure of the virus.

---

---

Herrn ProfessorG. Melchers zum 60. Geburtstag gewidmet.     

L-Arabinose is not a gratuitous inducer of α-galactosidase from Saccharomyces carlsbergensis

L-Arabinose has been described as a gratuitous inducer of the yeast -galactosidase. This has been found to be an artefact resulting from galactose contamination of commercial samples of L-arabinose. The inactivation produced on UDP-glucose 4-epimerase by the pentose does not amplify the inducer activity of contaminating D-galactose.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii

The ability to convert d-galactose into d-tagatose was compared among a number of bacterial l-arabinose isomerases (araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis–Menten constants of the enzyme determined with l-arabinose, d-galactose and d-fucose also indicated that this enzyme is an unusual, versatile l-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of d-tagatose at 65 °C. Starting from a 30% solution of d-galactose, the yield of d-tagatose was 42% and no sugars other than d-tagatose and d-galactose were detected. Direct conversion of lactose to d-tagatose in a single reactor was demonstrated using a thermostable -galactosidase together with the thermostable l-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.     

Pseudomonas aeruginosa Biofilms in Disease

Analysis of the composition of the marine-dissolved organic matter has highlighted the importance of d-amino acids, whose origin is attributed mainly to the remains of bacterial peptidoglycan released as a result of grazing or viral lysis. However, very few studies have focused on the active release of d-amino acids by bacteria. With this purpose, we measured the concentration of dissolved amino acids in both enantiomeric forms with two levels of complexity: axenic cultures of Vibrio furnissii and Vibrio alginolyticus and microcosms created from marine microbial assemblages (Biscay Bay, Cantabrian Sea) with and without heterotrophic nanoflagellates (HNFs). Axenic cultures showed that only d-Ala was significantly released and accumulated in the medium up to a concentration of 120 nM, probably as a consequence of the rearrangement of peptidoglycan. The marine microbial assemblages showed that only two d-amino acids significantly accumulated in the environment, d-Ala and d-aspartic acid (Asp), in both the absence and presence of HNFs. The d/l ratio increased during the incubation and reached maximum values of 3.0 to 4.3 for Ala and 0.4 to 10.6 for Asp and correlated with prokaryotic and HNF abundance as well as the rate of prokaryotic thymidine and leucine incorporation. Prokaryotes preferentially consumed l-amino acids, but the relative uptake rates of d-Ala significantly increased in the growth phase. These results demonstrate that bacteria can release and consume d-amino acids at high rates during growth, even in the absence of viruses and grazers, highlighting the importance of bacteria as producers of dissolved organic matter (DOM) in the sea.     

The taxonomic significance of the oxidation of carbon compounds by different strains ofMicrococcus luteus

The oxidation of 17 carbon compounds by 13 strains ofMicrococcus luteus was studied. It was shown that all strains oxidized Na-acetate, Na-lactate, glycerol, glucose, galactose, sucrose, maltose and fructose. The oxidation of mannitol, sorbitol, xylose, ribose, rhamnose, starch, lactose and arabinose was variable. Dulcitol was not oxidized at all. We have shown that the species, considered byKocur andMartinec (1962) to be identical withM. luteus, possess the same oxidation pattern.