Colorimetric measurement of dermatan sulphate

The periodate-Schiff reaction has been adapted for the measurement of dermatan sulphate. The method is specific for this glycosaminoglycan, provided that glycogen and glycoproteins are removed. Measurements of dermatan sulphate present in the urine of patients affected by various mucopolysaccharidoses indicate a good agreement between the values obtained with enzymic methods and those obtained with the colorimetric method described.     

Quantification of dissimilatory (bi)sulphite reductase gene expression in Desulfobacterium autotrophicum using real-time RT-PCR

We developed a real-time RT-PCR method for the quantification of dissimilatory (bi)sulphite reductase (DSR) mRNA in Desulfobacterium autotrophicum cells. The amount of DSR mRNA was determined relative to the amount of 16S rRNA at different growth conditions during transition from exponential to stationary phase: sulphate respiration with lactate, thiosulphate respiration with lactate, sulphate respiration with H2 and pyruvate fermentation. The dsr gene was expressed constitutively, although DSR mRNA content per-cell varied under different growth conditions. The maximum DSR mRNA per-cell content was 2.0 to 4.1-fold higher during sulphate or thiosulphate respiration than during pyruvate fermentation. After transfer of a pyruvate-fermenting culture into sulphate-rich medium, upregulation of the DSR mRNA content was observed. Irrespective of the mode of metabolism the per-cell DSR mRNA content changed significantly during growth (up to 310-fold from the early to the late exponential phase during respiration with thiosulphate). The maximum DSR mRNA per-cell contents correlated with cell-specific sulphate reduction rates for all experiments. Environmental applications for the quantification of DSR mRNA are discussed.     

The mechanisms of inhibition of Desulfovibrio and Desulfotomaculum species by selenate and molybdate

The effects of two specific inhibitors of sulphate-reducing bacteria, selenate and molybdate, have been studied by measurement of sulphate uptake and by measurement of the effects of these compounds on growth of the bacteria on a variety of media. The results obtained with selenate are consistent with the view that it acts as a competitive inhibitor of sulphate transport. The results obtained with molybdate show that there is more than one site of action and suggest that the primary effect is also on sulphate transport.     

A sensitive GLC-method for component sugars andO-glycosidic linkage monosaccharides of cartilage proteoglycans

A method is described for the measurement ofN-acetylgalactosamine,N-acetylglucosamine, galactose, mannose and xylose present in the different carbohydrate chains of cartilage proteoglycans (PG). Bovine articular cartilage PG samples corresponding to the minimum of 1 nmol of each monosaccharide were reproducibly quantified following hydrolysis with 2 M HCl and derivatization into alditol acetates. An on-column injection mode and an OV-1701 fused silica capillary column were used for chromatography.Alkaline borohydride treatment of the PG was exploited to reduce the acid labile xylose in the base of the chondroitin sulphate chain into more stable xylitol, allowing the assay of chondroitin sulfate chain length as anN-acetylgalactosamine/xylose ratio. A novel procedure is described for the measurement of the galactosaminitol evolving from the protein linkage of oligosaccharides and of keratan sulphate.     

Evaluation of methods to assess adequacy of potential soil S supply to crops

Eight methods were evaluated for assessment of the likely soil S-supply to a crop and in particular to identify likely deficiencies in this supply. Of the methods, only sulphate production from incubated soil amended both with a S-containing amino acid and elemental S correlated with field response of grass yield to S-fertilizer applications. Tissue S-concentrations in plants grown for a short-term bioassay also correlated with field S-fertilizer response. Neither sulphate extracted by 3 different salt extractions, nor sulphate production from unamended incubated soil, nor soil arylsulphatase activity, correlated with field fertilizer response.     

Coupling of glycosaminoglycans to agarose beads (Sepharose 4B)

1. Heparin, heparan sulphate, chondroitin sulphate and dermatan sulphate were covalently attached to beads of agarose activated by cyanogen bromide. The bond is probably mediated by the amino group of a serine or peptide residue at the reducing end of the polysaccharide chain. 2. The uptake of glycosaminoglycan during the coupling procedure is about 0.9mg/ml of wet gel. However, direct analysis of washed and freeze-dried gels reveals that only about one-third of this amount is firmly attached to the gel. 3. The use of the gels for polysaccharidase analyses is exemplified by a hyaluronidase assay. Further applications, e.g. interaction studies and preparative purposes, are discussed.     

On the design and efficacy assessment of self‐assembling peptide‐based hydrogel‐glycosaminoglycan mixtures for potential repair of early stage cartilage degeneration

Peptide‐based hydrogels are of interest for their potential use in regenerative medicine. Combining these hydrogels with materials that may enhance their physical and biological properties, such as glycosaminoglycans, has the potential to extend their range of biomedical applications, for example in the repair of early cartilage degeneration. The aim of this study was to combine three self‐assembling peptides (P₁₁‐4, P₁₁‐8, and P₁₁‐12) with chondroitin sulphate at two molar ratios of 1:16 and 1:64 in 130 and 230 mM Na⁺ salt concentrations. The study investigates the effects of mixing self‐assembling peptide and glycosaminoglycan on the physical and mechanical properties at 37°C. Peptide alone, chondroitin sulphate alone, and peptide in combination with chondroitin sulphate were analysed using Fourier transform infrared spectroscopy to determine the β‐sheet percentage, transmission electron microscopy to determine the fibril morphology, and rheology to determine the elastic and viscous modulus of the materials. All of the variables (peptide, salt concentration, and chondroitin sulphate molar ratio) had an effect on the mechanical properties, β‐sheet formation, and fibril morphology of the hydrogels. P₁₁‐4 and P₁₁‐8‐chondroitin sulphate mixtures, at both molar ratios, were shown to have a high β‐sheet percentage, dense entangled fibrillar networks, as well as high mechanical stiffness in both (130 and 230 mM) Na⁺ salt solutions when compared with the P₁₁‐12/chondroitin sulphate mixtures. These peptide/chondroitin sulphate hydrogels show promise for biomedical applications in glycosaminoglycan depleted tissues.     

Effects of ammonium-N on development and infectivity of the take-all fungus

Effects of applications of a mixture of ammonium sulphate and mono-ammonium phosphate and of ammonium nitrate on the incidence of take-all disease of wheat (caused by Gaeumannomyces graminis var. tritici) and on subsequent inoculum levels were studied in field and glasshouse experiments. In a field experiment in Western Australia, on a sandy soil at pH 5·4, nitrogen applications had no detectable effect on disease severity at anthesis, but ammonium sulphate treatment increased the number of propagules of the pathogen in the soil. In a pot experiment, in which seed was sown in the field experiment soils, disease was greater in soil from plots treated with ammonium sulphate and least in soil from the nil-nitrogen plots, reflecting the respective inoculum levels in the field plot. However, treatment of the soils of lower inoculum with ammonium sulphate and ammonium nitrate during this pot experiment decreased disease. A second pot experiment confirmed the effectiveness of ammonium sulphate and ammonium nitrate in reducing take-all at lower inoculum levels, and their ineffectiveness at higher inoculum levels.     

Preliminary research on body composition measurement using X-ray phase contrast imaging

Body composition measurement is of cardinal significance for medical and clinical applications. Currently, the dual-energy X-ray absorptiometry (DEXA) technique is widely applied for this measurement. In this study, we present a novel measurement method using the absorption and phase information obtained simultaneously from the X-ray grating-based interferometer (XGI). Rather than requiring two projection data sets with different X-ray energy spectra, with the proposed method, both the areal densities of the bone and the surrounding soft tissue can be acquired utilizing one projection data set. By using a human body phantom constructed to validate the proposed method, experimental results have shown that the compositions can be calculated with an improved accuracy comparing to the dual energy method, especially for the soft tissue measurement. Since the proposed method can be easily implemented on current XGI setup, it will greatly extend the applications of the XGI, and meanwhile has the potential to be an alternative to DEXA for human body composition measurement.     

The extraction of plasma 3-hydroxy-17-oxo steroid sulphates and the measurement of the constituent dehydroepiandrosterone sulphate and androsterone sulphate

1. A simple method for the extraction of 17-oxo steroid sulphates of plasma is described; glucosiduronates and orthophosphates are extracted, but to a smaller extent. 2. Four methods of analyses of the extracts are given and are relatively simple. Three of these are specific for steroid sulphates and two measure the sulphate conjugates directly. 3. Values for dehydroepiandrosterone sulphate and androsterone sulphate concentrations of normal and pathological plasmas are given.     

Effect of the air pollution component ammonium sulphate on the VAM infection rate of three heathland species

Three heathand species, Antennaria dioica, Arnica montana and Hieracium pilosella, were artificially rained with ammonium sulphate solutions at increasing concentrations in a greenhouse experiment. The same species were also artificially rained with increasing ammonium sulphate solutions under field conditions. Dry weights of the plants in the field experiments did not change with increasing ammonium sulphate applications. Nor did the dry weights of plants in the greenhouse experiments change with increasing ammonium sulphate concentrations, except for Arnica montana, which showed an increase in dry weight. VAM infection percentage of Antennaria dioica increased in both the greenhouse and the field experiment. The results of the field experiment show that VAM infection rates are reduced after two years of artificial rain in the plant species Arnica montana, which grows naturally under nutrient poor conditions and is presently declining in its natural habitat in the Netherlands. In the greenhouse experiment, VAM infection of Arnica montana did not change with increasing ammonium sulphate concentrations. VAM infection rates of Hieracium pilosella, which presently is not declining, did not change with increasing ammonium sulphate concentrations.     

Microbial heparin/heparan sulphate lyases: potential and applications

Heparin/heparan sulphate glycosaminoglycans (HSGAGs) are composed of linear chains of 20–100 disaccharide units of N-acetylated d-glucosamine α (1–4) linked to glucuronic acid. HSGAGs are widely distributed on the cell surface and extracellular cell matrix of virtually every mammalian cell type and play critical role in regulating numerous functions of blood vessel wall, blood coagulation, inflammation response and cell differentiation. These glycosaminoglycans present in this extracellular environment very significantly influence the blood coagulation system and cardiovascular functions. Recent studies have investigated the mechanism by which cancer causes thrombosis and emphasizes the importance of the coagulation system in angiogenesis and tumour metastasis. Heparan sulphate/heparin lyases or heparinases are a class of enzymes that are capable of specifically cleaving the (1–4) glycosidic linkages in heparin and heparan sulphate to generate biologically active oligosaccharides with substantially significant and distinct clinical, pharmaceutical and prophylactic/therapeutic applications. Bioavailability and pharmacokinetic behaviour and characteristics of these oligosaccharides vary significantly depending on the origin/nature of the substrate (heparin or heparan sulphate-like glycosaminoglycans), the source of enzyme and method of preparation. Various microorganisms are reported/patented to produce these enzymes with different properties. Heparinases are commercially used for the depolymerization of unfractionated heparin to produce low molecular weight heparins (LMWHs), an effective anticoagulant. Individual LMWHs are chemically different and unique and thus cannot be interchanged therapeutically. Heparinases and LMWHs are reported to control angiogenesis and metastasis also. This review catalogues the degradation of HSGAGs by microbial heparin/heparan sulphate lyases and their potential either specific to the enzymes or with the dual role for generation of oligosaccharides for a new generation of compounds, as shown by various laboratory or clinical studies.     

Vitamin A and the biosynthesis of sulphated mucopolysaccharides. Experiments with rats and cultured neoplastic mast cells

1. The uptake and incorporation of [(35)S]sulphate into mucopolysaccharides by colon and duodenum in vitro are unaffected by the vitamin A status of the animals. 2. Uptake and incorporation in vivo are unaffected at 4hr. after injection of [(35)S]sulphate, but at later times are decreased in some tissues of vitamin A-deficient animals. 3. The rate of removal of (35)S from blood, its rate of appearance in urine, the plasma concentration of sulphate and the uronic acid content of several tissues are not significantly altered in vitamin A deficiency. 4. These results, and direct measurement of (35)S in mucopolysaccharides at various times after injection of [(35)S]sulphate, suggest that the synthesis of mucopolysaccharides is unaffected but that their turnover is increased in vitamin A deficiency. 5. Neither the growth rate of, nor the incorporation of [(35)S]sulphate into heparin by, P815Y and HC cultured neoplastic mast cells is decreased when the horse serum necessary for growth is treated with ultraviolet light or is replaced by serum from vitamin A-deficient rats. 6. The addition of citral is no more toxic to growth rate or to incorporation of (35)S than is the addition of vitamin A itself. 7. It is concluded that neoplastic mast cells in culture do not require vitamin A for growth or for the synthesis of heparin. 8. None of these results is compatible with the view that vitamin A or a derivative is directly involved in the biosynthesis of sulphated mucopolysaccharides.     

A non-enzymic procedure for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)ethylene glycol (MHPG sulphate) in human urine using stable isotope dilution and gas chromatography—mass spectrometry

A method is described for the quantitative analysis of (3-methoxy-4-sulphoxyphenyl)-ethylene glycol (MHPG sulphate) in human urine, based on selected ion monitoring gas chromatography—mass spectrometry and using a specifically deuterium-labelled analogue of MHPG sulphate as internal standard. The procedure involves extraction of the urine sample on Amberlite XAD-2, followed by isolation of MHPG sulphate by column chromatography on Sephadex LH-20. Cleavage of the sulphate conjugate and formation of the MHPG tris(trifluoroscetate) derivative are carried out in a one-step reaction, without recourse to enzymic hydrolysis.     

Improved purification of α-L-rhamnosidase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> naringinase

To achieve an efficient separation and purification of α-L-rhamnosidase (Rha) from naringinase (Nar) that was prepared from a fermented broth of Aspergillus niger, improved experimental methods were developed with aid of a chemical dithiothreitol (DTT) and a novel HPLC method. The addition of DTT did not negatively affect the purification of Rha and Nar, but greatly simplified the purification steps due to its strong capability of separating the Rha from Nar. The novel HPLC method enabled simultaneous measurement and differentiation of the Rha and the Nar with the naringin as the substrate. These improvements resulted in an efficient purification of the homogenous Rha with an estimated molecular weight of 87 kDa. Otherwise, Rha could not be extracted with enough purity even by the combination of sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The modified methods have ensured efficient purification of the Rha from A. niger naringinase. This study provides a powerful and simple procedure to separate and purify the Rha of Nar, which will facilitate further more in-depth studies of these enzymes, as well as their industrial applications.     

An improved sulphur assay applied to a problem of isethionate metabolism in squid axon and other nerves

An assay has ken developed for total sulphur which is based on a wet oxidation and measurement with a spectrophotofluorometer of light scattering by barium sulphate. The method has been adapted to the measurement of isethionate in squid nerve and blood, in other cephalopod nerve, and in the nerve tissue of other species including mammals. A correlation has been found between isethionate contents and the activity in the same tissues of one kind of DFP-hydrolysing enzyme, the highest levels of both being in squid nerve. Squid nerve also took up cysteine rapidly and metabolized it predominantly to hypotaurine but not to isethionate. We speculate that a hypotaurine derivative is a reserve form of isethionate, and that the so-called DFPase is involved in the release of hypotaurine and its metabolism to isethionate as needed.     

The molecular-weight distribution of glycosaminoglycans

1. A rapid and sensitive method for the accurate estimation of the molecular-weight distribution of keratan sulphate and chondroitin sulphate isolated from adult bovine nasal septum and intervertebral disc is described. The method utilizes gel chromatography of reductively labelled glycosaminoglycan and end-group estimation of number-average molecular weight for each fraction across the peak of eluted glycosaminoglycan. 2. Chain-length distribution data obtained by this procedure are used to evaluate mechanisms of chondroitin sulphate biosynthesis.     

Simulation of pregnancy levels of plasma oestrone sulphate by infusion in the non-pregnant mare: A preliminary study

Oestrone sulphate was infused intravenously in saline solution into two, non-pregnant pony mares in repeated trials to simulate levels of the oestrogen found in plasma after midpregnancy in mares. Rates of infusion of oestrone sulphate ranged from 32 to 231 mg of oestrone equivalent per hour. Blood samples were taken from the opposite jugular vein for measurement of oestrone sulphate by radioimmunoassay. Plasma levels rose rapidly from the start of each infusion of the oestrogen and remained elevated over the hour-long periods, with concentrations as oestrone ranging from about 175 to 700 ng/ml. During each subsequent infusion with saline alone the oestrogen levels in plasma fell within 1 h to about 25% of the preceding values. Results were similar for each animal and simulation of plasma levels of oestrone sulphate in pregnancy was achieved.     

Multisensor Data Fusion for High Quality Data Analysis and Processing in Measurement and Instrumentation

Multisensor data fusion （MDF） is an emerging technology to fuse data from multiple sensors in order to make a more accurate estimation of the environment through measurement and detection. Applications of MDF cross a wide spectrum in military and civilian areas. With the rapid evolution of computers and the proliferation of micro-mechanical/electrical systems sensors, the utilization of MDF is being popularized in research and applications. This paper focuses on application of MDF for high quality data analysis and processing in measurement and instrumentation. A practical, general data fusion scheme was established on the basis of feature extraction and merge of data from multiple sensors. This scheme integrates artificial neural networks for high performance pattern recognition. A number of successful applications in areas of NDI （Non-Destructive Inspection） corrosion detection, food quality and safety characterization, and precision agriculture are described and discussed in order to motivate new applications in these or other areas. This paper gives an overall picture of using the MDF method to increase the accuracy of data analysis and processing in measurement and instrumentation in different areas of applications.