C-terminal region amino acid substitutions contribute to catalytic differences between murine class alpha glutathione transferases mGSTA1-1 and mGSTA2-2 toward anti-diol epoxide isomers of benzo[c]phenanthrene

The molecular basis for catalytic differences between structurally closely related murine class alpha glutathione (GSH) transferases mGSTA1-1 and mGSTA2-2 in the GSH conjugation of anti-diol epoxide isomers of benzo[c]phenanthrene (anti-B[c]PDE) was investigated. GSH conjugation of both (-)- and (+)-enantiomers of anti-B[c]PDE was observed in the presence of mGSTA1-1 (60 and 40% GSH conjugation, respectively), whereas mGSTA2-2 exhibited a preference for the (-)-anti-isomer (>97%). In addition, the specific activity of mGSTA2-2 toward the (-)-anti-B[c]PDE isomer was relatively higher than that of mGSTA1-1. The amino acid sequences of mGSTA1-1 and mGSTA2-2 differ at 10 positions that are distributed in three sections. Section I contains amino acid residues in positions 65 and 95; section II contains residues in positions 157, 162, and 169, and section III contains residues in positions 207, 213, 218, 221, and 222. Enzyme activity measurements with chimeras of mGSTA1-1 and mGSTA2-2 revealed that amino acid substitutions in section III account for their differential enantioselectivity and catalytic activity toward anti-B[c]PDE. Site-directed mutagenesis of amino acid residues in section III of mGSTA2-2 with corresponding residues of mGSTA1-1 followed by activity measurements of the wild type and mutated enzymes indicates that leucine 207 and phenylalanine 221 may be critical for the high catalytic activity of mGSTA2-2 toward (-)-anti-B[c]PDE. Molecular modeling studies demonstrated that the active site of mGSTA1-1 accommodates both enantiomers of anti-B[c]PDE, whereas the (-)-anti-isomer interacts more favorably with active site residues in mGSTA2-2. The results of this study clearly indicate that amino acid substitutions in the C-terminal region contribute to catalytic differences between mGSTA1-1 and mGSTA2-2 with respect to anti-B[c]PDE.     

Does the antibacterial activity of silver nanoparticles depend on the shape of the nanoparticle? A study of the Gram-negative bacterium Escherichia coli

In this work we investigated the antibacterial properties of differently shaped silver nanoparticles against the gram-negative bacterium Escherichia coli, both in liquid systems and on agar plates. Energy-filtering transmission electron microscopy images revealed considerable changes in the cell membranes upon treatment, resulting in cell death. Truncated triangular silver nanoplates with a {111} lattice plane as the basal plane displayed the strongest biocidal action, compared with spherical and rod-shaped nanoparticles and with Ag(+) (in the form of AgNO(3)). It is proposed that nanoscale size and the presence of a {111} plane combine to promote this biocidal property. To our knowledge, this is the first comparative study on the bactericidal properties of silver nanoparticles of different shapes, and our results demonstrate that silver nanoparticles undergo a shape-dependent interaction with the gram-negative organism E. coli.     

Purification and properties of cis-aconitic decarboxylase

Summary Lyophilized and stored in a deep-freeze, the mycelial material was found to retain cis-aconitic decarboxylase activity unimpaired at the end of 2 months. Mycelia could be stored also in the frozen condition but after squeezing hard to remove as much of adherent water as possible. Extracts with maximum cis-aconitic decarboxylase activity were obtained when the frozen or better the lyophilized mycelia of Aspergillus terreus were ground in a mortar with phosphate buffer using pyrex glass powder as abrasive. Cis-aconitic decarboxylase was purified 25-fold by fractionation with ammonium sulfate, starting from extracts of the mycelia in phosphate buffer. The purified enzyme was considerably more stable than the crude extracts to storage and dialysis. The optimum pH was 5.8 using 0.2 m phosphate buffer; Km value was 5×10^-3 m at pH 5.8 and 37°C. EDTA and 8-hydroxyquinoline activated the enzyme; all metals tested inhibited the enzyme, Zn⁺⁺ and Cu⁺⁺ leading to complete inactivation. Fluoride, arsenite and azide also inhibited the enzyme activity.     

MsrA Efflux Pump Inhibitory Activity of Piper cubeba L.f. and its Phytoconstituents against Staphylococcus aureus RN4220

MsrA, an efflux pump belonging to ATP‐binding cassette (ABC) transporter family that conferred resistance to macrolides, was detected in Staphylococcus aureus strains. Herein, we report the isolation of phytoconstituents from Piper cubeba fruit methanol extract and investigated their efflux pump inhibitory potential against S. aureus MsrA pump. Four isolated compounds, viz. pellitorine, sesamin, piperic acid and tetrahydropiperine studied in combination with erythromycin in S. aureus RN4220, exhibited 2–8‐fold reduction in minimum inhibitory concentration (MIC) of erythromycin. Pellitorine and sesamin decreased MIC of erythromycin by 8‐fold. The real‐time fluorometry‐based efflux and accumulation studies of ethidium bromide (EtBr) on S. aureus RN4220 in the presence of these compounds showed reduced efflux and enhanced uptake, thus indicating inhibition of the efflux pump. Pellitorine showed significant post‐antibiotic effect of erythromycin. The results revealed that the primary mechanism of action of these compounds involves steady ATP production impairment.     

Doubled Haploid Production Using an Improved Anther Culture Protocol for Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Sorghum [Sorghum bicolor (L.) Moench] can benefit from accelerated breeding and release of improved varieties through doubled haploid technology. The technology has been used in speeding up the breeding of other major cereals such as wheat, maize and rice, for which generally widely applied optimised protocols exist. A reproducible protocol for the crop, that can overcome genotype dependency and other species-specific challenges such as phenolic exudation is however lacking. This study aimed at sorghum doubled haploids production thereby contributing to the development of an improved protocol. From the 28 hybrid genotypes, both F₁ registered- and experimental hybrids involved, this study successfully produced haploids from five genotypes and subsequently, four confirmed doubled-haploid lines on W14mf medium or its modification with 1.0 gl⁻¹ L-proline, 1.0 gl⁻¹ L-asparagine and 1.0 gl⁻¹ KH₂PO₄. Medium 190-2Cu was used for regeneration and rooting, which occurred successfully, if the calli were transferred on to it less than 7 days after induction, and temperature was maintained at 25˚C under light condition. Genotype dependency was not wholly overcome; however, sorghum’s high tillering ability and abiotic stress tolerance were observed to contribute to attainment of haploid plantlets. Spontaneous diploids producing seeds at rates of upto 80.5% were obtained, therefore eliminating the need for colchicine duplication.