Chemical modification of papain for use in alkaline medium

Chemical modification is a useful method to recognize and modify functional determinants of enzymes. Papain, an endolytic cysteine protease (EC3.4.22.2) from Carica papaya latex has been chemically modified using different dicarboxylic anhydrides of citraconic, phthalic, maleic and succinic acids. These anhydrides reacted with five to six amino groups of the lysine residues in the enzyme, thereby changing the net charge of the enzyme from positive to negative. The resultant enzyme had its optimum pH shifted from 7 to 9 and change in temperature optima from 60 to 80 °C. The modified papain also had a higher thermostability. Stability of the modified papain was further increased by immobilization of the enzyme either by adsorption onto inert matrix or by entrapment in polysaccharide polymeric gels. Entrapment in starch gel showed better retention of enzyme activity. Incorporation of modified and immobilized enzymes to branded domestic detergent powders was found to have very good activity retention. The papain entrapped in starch gel showed better stability and activity retention than in other carbohydrate polymers when added to domestic detergent powders.     

Liquid modification of Baird-Parker's medium for the selective enrichment of Staphylococcus aureus

A liquid modification of Baird-Parker's medium is suggested for the detection of low (<20/g) numbers of Staphylococcus aureus. Model experiments showed that the medium had an acceptable level of selectivity and that it was non-inhibitory to injured cells. Practical evaluation demonstrated the advantage of selective enrichment procedures over both non-selective enrichment technique and direct plating methods.     

Oxidative modification and change of intramolecular hydrophobicity of hemoglobin A during incubation of human erythrocytes in Fenton's medium

It has been established by method of fluorescentive spectroscopy with using of zond FNA (N-phenilnaftilamine) that under incubation of human erythrocytes in Fenton's system (under intensive generation of oxygen active forms) the hydrophobicity of major hemoglobin fraction is lowered. The changes in the state of hemoglobin A intramolecular structure are correlated with formation of oxidative modification products. It has been shown that aldehydes and ketones of neutral character, which may be oxidative modification products of hydrophobic aminoacid residues, have the highest coefficient of correlation (r =-0.95).     

Cytoskeletal and DNA synthesis modification by concanavalin A in embryonic fibroblasts maintained in serum-free and serum-added medium

The administration of lectin concanavalin A (Con A) to in vitro cultures of chick embryo fibroblasts caused dose-dependent changes in cell shape, cytoskeleton network and DNA synthesis. After 6 hrs. even a low doses of Con A produced evident effects in serum-free cultures, whereas higher doses were required to cause alterations in cells cultured in serum-added medium. As the sugar competitor mannoside abolishes the effects, it would seem that the lectin acts by binding to transmembrane receptors and that the fibronectin present in the serum engages the receptors so that they are not available to Con A.     

A modification of the Pseudomonas selective medium, CFC, that allows differentiation between meat pseudomonads and Enterobacteriaceae

Certain members of the family Enterobacteriaceae developing on beef steaks packaged in modified atmospheres may grow on the medium CFC (Cephaloridine-Fucidin-Cetrimide), which is selective for pseudomonads. The addition of arginine (1% w/v) and the pH indicator phenol red (0.002% w/v) to this medium differentiated between the two groups. The pseudomonads produced ammonia from arginine, whereas Enterobacteriaceae generally did not use this substrate. The alkaline drift in pH in and around pseudomonad colonies gave a pink colouration with phenol red.     

Feeder-free culture of human embryonic stem cells in conditioned medium for efficient genetic modification

Realizing the potential of human embryonic stem cells (hESCs) in research and commercial applications requires generic protocols for culture, expansion and genetic modification that function between multiple lines. Here we describe a feeder-free hESC culture protocol that was tested in 13 independent hESC lines derived in five different laboratories. The procedure is based on Matrigel adaptation in mouse embryonic fibroblast conditioned medium (CM) followed by monolayer culture of hESC. When combined, these techniques provide a robust hESC culture platform, suitable for high-efficiency genetic modification via plasmid transfection (using lipofection or electroporation), siRNA knockdown and viral transduction. In contrast to other available protocols, it does not require optimization for individual lines. hESC transiently expressing ectopic genes are obtained within 9 d and stable transgenic lines within 3 weeks.     

A model of migration modification

Two subpopulations whose different sizes are in a constant ratio interact via migration. The fitness of the diploid organisms is determined by two alleles at a single locus and by the niche the organism is in. The rates of migration depend upon two neutral modifier genes at a second locus. The second modifying allele is introduced into an equilibrium where the first modifying allele is fixed, and where the other two alleles are already polymorphic. It is shown that the new migration modifier is selected for when it reduces migration. The similarity between this result and some recombination modifier models is noted.     

Conversion of Woodward's Reagent K in an aqueous medium: mathematical analysis applied to enzyme modification

A change in the optical density of Woodward's Reagent K solution at 340 nm has been shown. It is observed after the reagent has been dissolved in a weakly acidic medium. The optical density correlates with the reagent's ability to inhibit transketolase. A method for assay of the inhibitor concentration changes in the medium during enzyme modification is suggested.     

Homogeneous modification of sugarcane bagasse cellulose with succinic anhydride using a ionic liquid as reaction medium

The homogeneous chemical modification of sugarcane bagasse cellulose with succinic anhydride using 1-allyl-3-methylimidazolium chloride (AmimCl) ionic liquid as a reaction medium was studied. Parameters investigated included the molar ratio of succinic anhydride/anhydroglucose units in cellulose in a range from 2:1 to 14:1, reaction time (from 30 to 160min), and reaction temperature (between 60 and 110 degrees C). The succinylated cellulosic derivatives were prepared with a low degree of substitution (DS) ranging from 0.071 to 0.22. The results showed that the increase of reaction temperature, molar ratio of SA/AGU in cellulose, and reaction time led to an increase in DS of cellulose samples. The products were characterized by FT-IR and solid-state CP/MAS (13)C NMR spectroscopy, and thermal analysis. It was found that the crystallinity of the cellulose was completely disrupted in the ionic liquid system under the conditions given. The data also demonstrated that homogeneous modification of cellulose with succinic anhydride in AmimCl resulted in the production of cellulosic monoester. The thermal stability of the succinylated cellulose decreased upon chemical modification.     

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11mg (g-fresh cell weight)(-1), which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance.