Structural characterization of in vitro products

The physical and chemical techniques listed in Table 8-2 provide detailed structural information that can help understand some aspects related to the polymerization and crystallization of polysaccharides. They are complementary and it is actually not possible to have a complete structural characterization of a given polymer by using only one of these techniques. Other methods based on transmission electron microscopy examinations and involving cellulases coupled 

Amount of product required/ possibility of sample recovery 

Solubility (distinction between (l- 3)-P-D-glucan and cellulose however, this method is not completely reliable) 

Infrared spectroscopy (identification of linkage type/structure) 

Methylation (gas chromatography coupled to mass spectrometry) (identification of linkage type and estimation of the degree of polymerization if 100) C-NMR spectroscopy in DMSO (or NaOH) solution (structure) 

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The importance to achieve detailed structural characterization of in vitro products is illustrated by the application of the methods listed in Table 8-2 to the analysis of in vitro (1 3)-P-D-glucans synthesized under various conditions. For instance, it has been shown that for a given plant species the morphology and the stucture of the in vitro products are affected by the nature of the detergent used to extract the membrane-bound synthases (Lai Kee Him et al. 2001). Also, for a given detergent, enzymes from different plant species do not necessarily synthesize products that have the same morphology and structure (Lai Kee Him et al. 2001 Colombani et al. 2004). From these observations, it seems important... 

The development of highly sensitive proteomic methods for the characterization of membrane-bound proteins should facilitate the systematic sequencing of all the proteins that are present in fractions with high callose synthase activity. Also, the progress made on in vitro synthesis of cellulose is promising and proteomic analysis may also be used on fractions enriched in cellulose synthases in the near future. This approach combined with detailed structural characterization of in vitro products, immunochemical methods, molecular biology and gene... 

Lisa E. Cooper was born and raised in Renton, Washington. She received a B.S. in Biochemistry in 2004 from Western Washington University under the guidance of Professor Christopher J. A. Daley. Subsequently, she joined the laboratory of Professor Wilfred A. van der Donk at the University of Illinois at Urbana-Champaign, where she studied lantibiotic biosynthetic enzymes. After successfully engineering an in vitro production system for the novel two-component lantibiotic haloduracin, she characterized the structural features essential for the activity of this antibiotic. 

The characterization and crystal structure of bis(ferrocenyl)dioxolane 17, produced by the reaction of ferrocenecarbaldehyde and the 4,5-dimethyl-l,3,2-dioxaphosphole 16 (13JOM(739)40), and the synthesis, characterization, and in vitro antineoplastic activity evaluation of the diastereomeric ferrocene acetals 18 and 19 (13S1853) have been reported. Accounts on the synthesis and evaluation for anti-HIV-1 activity of a series of dioxolane pyrimidine nucleosides 20-22 (13T10884) and the synthesis of the natural product trichodermatide A 23 (13AGE3646) have been published. The crystal structure of asarinin 24, which was isolated from the shrub Zanthoxylum datum, has been reported (13AXCo87). 

It provides a direct structural characterization of the in vitro product, as opposed to biochemical techniques that rely on the use of radioactive substrate and a subsequent hydrolysis of the polysaccharides with specific hydrolases. In the case of P-glucans, Hquid C-NMR analysis allows distinction between (1—>3) and (1 4) linkages for products that are soluble in DMSO. For high molecular weight polymers that are insoluble in solvents commonly used for liquid C-NMR analysis, such as cellulose, solid-state NMR spectroscopy can be used for structural characterization. In addition, solid-state NMR spectroscopy provides information on the conformation of the glucan chains in a given preparation as well as structural details that cannot be obtained with Hquid NMR spectroscopy and methylation analysis. For instance, in the case of cellulose, solid-state NMR spectroscopy allows the determination of the proportions of the I and Ip allomorphs in the sample (Atalla and VanderHart 1984). 

Antioxidant capacities of common individual curcuminoids were determined in vitro by phosphomolybdenum and linoleic acid peroxidation methods. Antioxidant capacities expressed as ascorbic acid equivalents (pmol/g) were 3099 for curcumin, 2833 for demethoxycurcumin, and 2677 for bisdemethoxycurcumin at concentrations of 50 ppm. The same order of antioxidant activity (curcumin > demethoxycurcumin > bisdemethoxycurcumin) was observed when compared with BHT (buty-lated hydroxyl toluene) in linoleic peroxidation tests. The antioxidant activity of curcumin in the presence of ethyl linoleate was demonstrated and six reaction products were identified and structurally characterized. The mechanism proposed for this activity consisted of an oxidative coupling reaction at the 3 position of the curcumin with the lipid and a subsequent intramolecular Diels-Alder reaction. ... 

Increased use of liquid chromatography/mass spectrometry (lc/ms) for structural identification and trace analysis has become apparent. Thermo-spray lc/ms has been used to identify by-products in phenyl isocyanate precolumn derivatization reactions Liquid chromatography/thermospray mass spectrometric characterization of chemical adducts of DNA formed during in vitro reaction lias been proposed as an analytical technique to detect and identify those contaminants in aqueous environmental samples which have a propensity to be genotoxic, t.e.. to covalently bond to DNA. 

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