Polysaccharide Contaminants Research Articles

High throughput DNA extraction of legume root nodules for rhizobial metagenomics

A novel combination of tissue homogenization, cell lysis, and DNA purification techniques was developed for isolating total DNA from whole legume root nodules on an automated robotic system. Silica dehydrated root nodules from soy bean, Glycine max cv. Tara, and mung bean, Vigna radiata cv. Crystal, were homogenized in 96-well plates and were enzymatically and chemically lysed before being loaded into the AutoGenprep 965 for automated phenol–chloroform based DNA isolation. The resulting DNAs were of relatively high concentration, low fragmentation, and free of phenolic and polysaccharide contaminants. In contrast to manual methods, this adaptation of the Autogen Prep automated DNA extraction instrument allows for 100’s to 1000’s of samples to be prepared in a fraction of the time and cost.

Minimizing Environmental and Economic Risks With a Proppant-Sized Solid-Scale-Inhibitor Additive in the Bakken Formation

SummaryWithin the past decade, hydraulic fracturing has proved to enhance the efficiency and economics of recovering oil and natural gas from shale formations. In a previous paper (SPE-134414-MS, Gupta et al. 2010), a summary of treatment results from placing a solid scale inhibitor into formations by means of the fracturing process for more than 5 years and in more than 1,500 wells was discussed fully; however, the practice of hydraulic fracturing has come under scrutiny because of concerns about environmental impact, health, and safety. Therefore, a novel, biodegradable scale inhibitor with an outstanding ecotoxicity profile for fresh water incorporated into a solid matrix was recently developed and deployed in North Dakota. For one operator with 150 Bakken producing wells in this area, severe carbonate-mineral scaling in the pump and production tubing has been observed in 22 of these producing wells, which led to well failure (Cenegy et al. 2011), whereas the results from more than 290 Bakken wells now fractured with the new solid-inhibitor additives indicate no reported scale failures to date.This paper provides a detailed description of the first deployment of this environmentally preferred proppant-sized solid-scale-inhibitor additive under severe scaling conditions in the field. In addition, an analytical method developed to track the residual of this additive in the produced fluid containing polysaccharide contaminants is also discussed.

Rapid isolation of DNA from Dioscorea species suitable for PCR, restriction digestion and pathogen screening

The methods employed for DNA extraction from many plants is difficult because of the metabolites that interfere with DNA isolation procedures. We have developed a reliable and efficient method for isolating genomic DNA free from polysaccharide, polyphenols and protein contaminants from Dioscorea spp. The method involves inactivation of contaminant proteins by using CTAB/Proteinase K and precipitation of polysaccharides in the presence of high concentration of salt. The purity of genomic DNA was confirmed by A260/280 and A260/230 ratios calculated from the spectrophotometric readings and further by restriction analysis of the isolated DNA using restriction enzymes Eco RI. The total genomic DNA extracted by the new protocol was used for polymerase chain reaction amplification, RAPD analysis, restriction digestion and pathogen screening. The new protocol can be successfully used for both small- and large-scale preparation of genomic DNA from different tissues of Dioscorea spp. The quarantine of seed tubers and use of pathogen-free tubers for planting is a prerequisite for integrated disease management strategy. The protocol can be used for the isolation of genomic DNA from other crop plants too.

Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

Structural variation and rheological properties of water-extractable arabinoxylans from six Greek wheat cultivars

Aqueous extractions (at 25°C) of flours from six Greek wheat cultivars that differed in their bread-making quality, followed by porcine pancreas α-amylase digestion to remove starch contaminants and precipitation with ammonium sulphate (saturation at 95%), yielded arabinoxylan isolates with notable differences in molecular weight, ranging from 146,500 to 397,000, and in arabinose-to-xylose ratios (Ara/Xyl, varying between 0.57 and 0.71). The arabinoxylan isolates contained only small amounts of proteins and were relatively free of other polysaccharide contaminants. Generally a very high ratio of trans to cis-ferulic acid content was detected for all samples. The genotype also had an impact on the molecular characteristics of these polysaccharides. 1H NMR spectroscopy revealed variations in the un-substituted (61.1–67.9%) and di- (19.4–28.1%) and mono-substituted xylose residues (10.0–16.6%). Using 2D NMR spectroscopy, it was feasible to identify the spin sequence of individual monosaccharide residues and elucidate their structure, despite the extensive overlapping of 1H and 13C resonances. Differences in the physicochemical properties of the arabinoxylans, such as the critical concentration values (c∗ and c∗∗), viscosity, shear-thinning behaviour, as well as the gelling ability, could not be fully explained by differences in molecular size and structure of the isolated polysaccharides.

Identification and removal of colanic acid from plasmid DNA preparations: implications for gene therapy

Polysaccharide contaminants in plasmid DNA, including current good manufacturing practices (cGMP) clinical preparations, must be removed to provide the greatest safety and efficacy for use in gene therapy and other clinical applications. We developed assays and methods for the detection and removal of these polysaccharides, our Super Clean DNA (SC-DNA) process, and have shown that these contaminants in plasmid DNA preparations are responsible for toxicity observed post-injection in animals. Furthermore, these contaminants limit the efficacy of low and high doses of plasmid DNA administered by numerous delivery routes. In particular, colanic acid (CA) that is mainly long-chained, branched and has high molecular weight (MW) is most refractory when complexed to cationic delivery vehicles and injected intravenously (IV). Because CA is often extremely large and tightly intertwined with DNA, it must be degraded, in order, to be effectively removed. We have produced a recombinant, truncated colanic acid degrading enzyme (CAE) that successfully accomplishes this task. Initially, we isolated a newly identified CAE from a bacteriophage that required truncation for proper folding while retaining its full enzymatic activity during production. Any plasmid DNA preparation can be digested with CAE and further purified, providing a critical advance to non-viral gene therapy.

The Tainted Heparin Story: An Update

Recently, certain batches of heparin have been associated with an acute, rapid onset of serious side-effects indicative of allergic-type reactions. These reports generated significant concern regarding the possible presence of a dangerous contaminant within heparin and highlighted the need to re-assess the purity criteria of heparin preparations for clinical use. Given the nature of the array of all possible contaminants, traditional screening tests cannot safely differentiate between contaminated and uncontaminated heparin preparations. Mono- and bi-dimensional NMR spectroscopy are powerful techniques that are able to detect and quantify a wide variety of potential sulfated polysaccharide contaminants. As such, these techniques are powerful tools for the analysis and assessment of heparin preparations.

Prostaglandin E release from human monocytes treated with lipopolysaccharides isolated from Bacteroides intermedius and Salmonella typhimurium: potentiation by gamma interferon

The purpose of this investigation was to examine gamma interferon potentiation of lipopolysaccharide (LPS) responses in human monocytes by using phenol-water-extracted (unfractionated) and highly purified LPS preparations isolated from Bacteroides intermedius and Salmonella typhimurium. Phenol-water-extracted LPS preparations from these bacteria were further purified by chromatography over Sepharose-CL-4B. LPS enrichment in pooled column fractions was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation of hydroxy-fatty acid and 2-keto-3-deoxyoctulosonic acid content, protein contamination, and anthrone-reactive material. Monocyte stimulation by LPS, measured as prostaglandin E (PGE) release, was assessed with and without gamma interferon treatment. Cells were either treated simultaneously with gamma interferon and LPS or pretreated with gamma interferon prior to LPS stimulation. PGE release from counterflow-isolated monocytes was quantitated during the 0- to 24-h and 24- to 48-h culture intervals. Contrary to previous results from this laboratory, phenol-water-extracted LPS preparations from B. intermedius and S. typhimurium were similar in their capacities to stimulate PGE release from monocytes. Molecular sieve chromatography was found to remove substantial amounts of high-molecular-weight polysaccharide contaminants only from the B. intermedius LPS but did not significantly alter the potency of either B. intermedius or S. typhimurium LPS. Gamma interferon cotreatment did not potentiate the release of PGE with any of the LPS preparations tested. However, 24-h pretreatment of monocytes with gamma interferon followed by a 24-h exposure to LPS resulted in significant potentiation of PGE release over LPS alone. In addition, B. intermedium preparations were approximately threefold more potent than similarly prepared LPS isolates from S. typhimurium following gamma interferon pretreatment. These results indicate that gamma interferon can selectively potentiate the effects of B. intermedius LPS in human monocyte isolates.

Purification of DNA by affinity chromatography: Removal of polysaccharide contaminants

DNA, as isolated, is often contaminated with polysaccharidelike components. To overcome this difficulty a simple and rapid procedure has been developed based on the principle of affinity chromatography. Deproteinized DNA fractions are introduced onto a column of concanavalin A linked to Sepharose. Glycogen and starchlike polysaccharide contaminants are effectively bound to the carbohydrate exchange resin while nucleic acids pass through (recovery,95%). The column can then be regenerated for further use. DNA samples from a variety of organisms have been successfully freed of polysaccharide contaminants by this method.