Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Nitrogen assimilation in opaque and normal sorghum during grain development

Activity of key nitrogen assimilating enzymes was studied in developing grains of high-lysine opaque sorghum P-721 and normal sorghum CSV-5. The higher percentage of protein in opaque sorghum was mainly due to lower starch content since protein per grain was less than in CSV-5. During grain development, albufn and globulin decreased while prolafne and glutelin increased. Prolafne content in CSV-5 was higher than in opaque sorghum. Average nitrate reductase activity in flag and long leaf were similar in both the varieties. The nitrate reductase activity decreased during grain development. Glutamate dehydrogenase activity was higher during early development and lower at later stages in opaque sorghum than in CSV-5. Glutamate oxaloacetate transaminase activity was higher and glutamine synthetase lower in opaque sorghum than in CSV-5 grains during development. Glutamate synthase activity was higher in opaque sorghum up to day 20 and lower thereafter than in CSV-5. It is suggested that reduced activities of glutamine synthetase as well as glutamate synthase in opaque sorghum as compared to CSV-5 during later stages of development may restrict protein accumulation in the former.     

A rapid method for the purification of D-amino acid oxidase of Trigonopsis variabilis by hydrophobic chromatography

Summary A relatively simple method has been described for the rapid purification of D-amino acid oxidase from Trigonopsis variabilis by hydrophobic chromatography on Phenyl-Sepharose CL-4B and negative adsorption on DEAE-cellulose. The purified enzyme had a specific activity of 22–24 units at 25°C and exhibited three bands on enzymatic staining.     

Conformational changes of plasma fibronectin detected upon adsorption to solid substrates: a spin-label study

Changes in local environment of the free sulfhydryl groups in plasma fibronectin upon adsorption of the protein to polystyrene beads have been examined by electron spin resonance (ESR) spin-label spectroscopy. The two free sulfhydryl groups per subunit of plasma fibronectin were modified chemically with an [15N, 2H]maleimide spin-label. For soluble fibronectin, both free sulfhydryl groups are shown to be in confined environments as evidenced from the labeled protein exhibiting a strongly immobilized ESR spectrum as described previously using [14N, 1H]maleimide spin-labels [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. When the labeled protein was adsorbed to the beads, half of the strongly immobilized component was found to convert into a weakly immobilized component, a result indicating that one of the two labeled sites becomes exposed and exhibit a fast tumbling motion. Experiments conducted using various spin-labeled fibronectin fragments suggest that the newly exposed labeled site is located between the DNA-binding and the cell-binding regions of the molecule. The data obtained indicate that, upon adsorption to polystyrene beads, plasma fibronectin undergoes a conformational change through which the buried free sulfhydryl group near the cell-binding region of the molecule is exposed. This observation may have important implications regarding the expression of cell adhesive properties of the fibronectin molecule.     

Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase

The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.     

Neurotoxic mechanisms of triclosan: The antimicrobial agent emerging as a toxicant

Several scientific studies have suggested a link between increased exposure to pollutants and a rise in the number of neurodegenerative disorders of unknown origin. Notably, triclosan (an antimicrobial agent) is used in concentrations ranging from 0.3% to 1% in various consumer products. Recent studies have also highlighted triclosan as an emerging toxic pollutant due to its increasing global use. However, a definitive link is missing to associate the rising use of triclosan and the growing number of neurodegenerative disorders or neurotoxicity. In this article, we present systematic scientific evidence which are otherwise scattered to suggest that triclosan can indeed induce neurotoxic effects, especially in vertebrate organisms including humans. Mechanistically, triclosan affected important developmental and differentiation genes, structural genes, genes for signaling receptors and genes for neurotransmitter controlling enzymes. Triclosan-induced oxidative stress impacting cellular proteins and homeostasis which triggers apoptosis. Though the scientific evidence collated in this article unequivocally indicates that triclosan can cause neurotoxicity, further epidemiological studies may be needed to confirm the effects on humans.