The effects of oxygen free radicals on the carbohydrate moiety of IgG

The CH2-linked glycoform of rheumatoid IgG is abnormal in having a reduced galactose content. This has been postulated to be a synthetic defect due to a decrease in the level of rheumatoid B cell galactosyltransferase. However, more recent work has indicated that agalactosylation may be common to chronic inflammatory diseases. In this work we have investigated the effect of oxygen free radicals (OFRs), which are generated by activated phagocytic cells at inflammatory sites, on the carbohydrate moiety of IgG. Radiolytically generated peroxy (ROO.) and hydroxyl radicals (OH.) but not superoxide anion radicals (O2.-) were found to destroy galactose on IgG. After OH. attack, this was associated with an increase in the availability of N-acetylglucosamine, possibly due to its presence as a terminal residue. These results suggest that the agalactosylation associated with chronic inflammation may not only be synthetic in nature, but may also be a consequence of post-synthetic degradation by OFRs.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Growth and regeneration of lichen thalli with the central portions artificially removed

The 12-month radial growth of Parmelia conspersa thalli with isidia or with apothecia and isidia was not influenced by removal of the thalli centres. When large thalli had their centres removed and the thallus perimeter was divided into fragments of about 1.0cm in diameter, growth of the fragments was less than the controls, but recovered to near control values after four or five months growth. These results suggest first, that fixed carbon for radial growth may be made in a narrow annulus at the perimeter and second, that there may be little transfer of fixed carbon between the annulus and the centre of the thallus ar around the annulus. Fragments of the centre and the perimeter regenerated growing points, suggesting that fragmentation may be an important method of vegetative reproduction in some lichens.

Does radial growth of the lichen Parmelia conspersa depend exclusively on growth processes at the lobe tip?

The objective of this study was to test the hypothesis that the radial growth of lobes of the lichen Parmelia conspersa depends largely on growth processes which occur at the lobe tip. First, individual lobes were removed from thalli and portions of the lobe removed to within various distances from the tip. Radial growth of the lobe was unaffected until less than 2 mm of the lobe tip remained. Second, the surfaces of individual lobes were painted with acrylic paint leaving different portions of the lobe exposed. Painting lobes to within 0.5 mm and 1 mm of the tip substantially reduced radial growth. Third, the levels of ribitol, arabitol and mannitol were measured in different regions behind the lobe tip on four occasions during 1994. The concentration of the three carbohydrates was greatest at the lobe tip and the levels declined linearly with distance from the tip. Fourth, painting one vertical half of the lobe tip did not affect radial growth but artificially bisecting the lobe tip with a scalpel reduced radial growth. Although transport of carbohydrate from other regions of the lobe cannot be ruled out, the results support the hypothesis that radial growth in P. conspersa depends largely on processes within a region approximately 2 mm behind the lobe tip.

Radial growth and carbohydrate levels in the lichen Parmelia conspersa on north and south facing rock surfaces

Parmelia conspersa (Ehrh. Ex Ach.)Ach. is a foliose lichen found more frequently on south facing compared with north facing rock surfaces in South Gwynedd, Wales, UK. The radial growth of thalli of P. conspersa from a north and a south facing rock surface was measured in situ at intervals of two months for 1 yr during 1990/1991. Mean annual radial growth rates were greater on the south compared with the north facing rock surface. In addition, the pattern of radial growth varied during the year with maximum growth recorded in the Feb/Mar. period especially on the south facing rock surface. The levels of ribitol, arabitol and mannitol were measured in individual lobes of P. conspersa collected from the same rock surfaces on 4 days (2 Jun; 7 July and 30 Nov. 1990 and 29 Mar. 1991) during 1990/1991. The total of the three carbohydrates varied between days; the levels of arbitol and ribitol being significantly lower in the 7 July sample on both north and south facing rock surfaces. In addition, the levels ribitol, arabitol and mannitol were higher on the south facing rock surface especially in the summer samples. The ratio of arabitol plus mannitol to ribitol and the mannitol/arabitol ratio varied more between days sampled than between north and south facing rock surfaces. The level of ribitol in individual thalli was positively correlated with arabitol on the north facing and with mannitol on the south facing slope. These results suggest that differences in the radial growth of P. conspersa thalli with aspect are more likely to reflect higher rates of photosynthesis on the south facing rock surface rather than large difference in the way carbohydrates were partitioned on the different surfaces. Lower radial growth rates may place P. conspersa at a competitive disadvantage on north facing rock surfaces.

Continous annular chromatography for the separation of carbohydrate mixtures

The continuous separation of beet molasses resulting in a sucrose rich product and a non-sugar waste product was carried out using a rotating annular chromatograph. The annulus was 12 mm wide and 1.4 m long and was packed with a sodium charged 5.5% cross-linked polystyrene ion exchange resin. Separation was achieved by the simultaneous mechanisms of ion exclusion, size exclusion and partition chromatography. The entire packed bed was slowly rotated while beet molasses was fed continuously through a stationary feed nozzle to the top of the bed. Each molasses constituent having a different relative affinity for the packing and the deionised water mobile phase describes a characteristic helical path as it progresses from the stationary feed point to the bottom of the rotating bed. Each solute then elutes from the annulus at a different angular distance from the feed and separation of the multicomponent mixture is thereby achieved. When a 35% w/w sucrose beet molasses feed was used the throughput achievable was 45.1 kg sucrose m~3 resin h"1. In addition to beet molasses separation other carbohydrate mixtures were separated. In particular the separation of glucose and fructose by Ligand exchange chromatography on a calcium charged ion exchange bed was carried out. The effects of flowrates, concentration, rotation rate, temperature and particle size on resolution and dilution of constituents in the mixtures to be separated were studied. A small test rig was designed and built to determine the cause of liquid maldistribution around the annulus. The problem was caused by the porous bed support media becoming clogged with fines being introduced by eluent flows and off the resin. An outer ring was constructed to house the bed support which could be quickly replaced with the onset of maldistribution. The computer simulation of the operation of the rotating annular chromatograph has been carried out successfully.

The production of fructose from carbohydrate feedstocks

A review of the general chromatographic theory and of continuous chromatographic techniques has been carried out. Three methods of inversion of sucrose to glucose and fructose in beet molasses were explored. These methods were the inversion of sucrose using the enzyme invertase, by the use of hydrochloric acid and the use of the resin Amberlite IR118 in the H+ form. The preferred method on economic and purity considerations was by the use of the enzyme invertase. The continuous chromatographic separation of inverted beet molasses resulting in a fructose rich product and a product containing glucose and other non-sugars was carried out using a semi-continuous counter-current chromatographic refiner (SCCR6), consisting of ten 10.8cm x 75cm long stainless steel columns packed with a calcium charged 8% cross-linked polystyrene resin Zerolit SRC 14. Based on the literature this is the first time such a continuous separation has been attempted. It was found that the cations present in beet molasses displaced the calcium ions from the resin resulting in poor separation of the glucose and fructose. Three methods of maintaining the calcium form of the resin during the continuous operation of the equipment were established. Passing a solution of calcium nitrate through the purge column for half a switch period was found to be most effective as there was no contamination of the main fructose rich product and the product concentrations were increased by 50%. When a 53% total solids (53 Brix) molasses feedstock was used, the throughput was 34.13kg sugar solids per m3 of resin per hour. Product purities of 97% fructose in fructose rich (FRP) and 96% glucose in the glucose rich (GRP) products were obtained with product concentrations of 10.93 %w/w for the FRP and 10.07 %w/w for the GRP. The effects of flowrates, temperature and background sugar concentration on the distribution coefficients of fructose, glucose, betaine and an ionic component of beet molasses were evaluated and general relationships derived. The computer simulation of inverted beet molasses separations on an SCCR system has been carried out successfully.

The chromatographic separation of carbohydrate mixtures

The separation performance of a semicontinuous counter-current chromatographic refiner (SCCR7), consisting of twelve 5.4 cm id x 75cm long columns packed with calcium charged cross-linked polysytrene resin (KORELA VO7C), was optimised. An industrial barley syrup was used containing 42% fructose, 52% glucose and 6% maltose and oligosaccharides. The effects of temperature, flow rates and concentration on the distribution coefficients were evaluated and quantified by deriving general relationships. The effects of flow rates, feed composition and concentration on the separation performance of the SCCR7 were identified and general relationships between them and the switch time, which was found to be the controlling parameter, were developed. Fructose rich (FRP) and glucose rich (GRP) product purities of 99.9% were obtained at 18.6% w/v feed concentrations. When a 66% w/v feed concentration was used and product splitting technique was employed, the throughput was 32.1 kg sugar solids/m3 resin/hr. The GRP contained less than 4.5% fructose, the FRP was over 95% pure, and the respective concentrations were 22.56 and 11.29% w/v. Over 94% of the glucose and 95.78% of the fructose in the feed were recovered in the GRP and FRP respectively. By recycling the dilute product split fractions, the GRP and FRP concentrations were increased to 25.4 and 12.96% w/v; the FRP was 90.2% pure and the GRP contained 6.69% w/v fructose. A theoretical link between batch and semicontinuous chromatographic equipments has been determined. A computer simulation was developed predicting successfully the purging concentration profiles at `pseudo-equilibrium', and also certain system design parameters. An important further aspect of the work has been to study the behaviour of chromatographic bioreactor-separators. Such batch systems of 5.4cm id and lengths varying between 30 and 230cm, were used to investigate the effect of scaling up on the conversion of sucrose into dextran and fructose in the presence of the dextransucrase enzyme. Conversions of over 80% were achieved at 4 hr sucrose residence times. The crude dextransucrase was purified using centrifugation, ultrafiltration and cross-flow microfiltration techniques. Better enzyme stability was obtained by first separating the non-solid impurities using cross-flow microfiltration, and then removing the cells from the enzyme immediately before use by continuous centrifugation.