In situ detection of expression of thegus reporter gene in transgenic plants: ten years of blue genes

Among the methods now available to localize the sites of gene expression in plant materials, reporter genes based on thegus (uidA) gene ofEscherichia coli, which encodes a -glucuronidase (E.C. 3.2.1.31; GUS), have been the most widely used during the last ten years. The apparent simplicity of the histochemical GUS assay has been a major factor in the increase in articles usinggus genes. However, over the last four years, there have been occasional reports expressing doubts concerning the specificity of the observed localizations based on discrepancies between results obtained with GUS histochemistry and immunocytochemistry and/orin situ hybridization. This brief review compares the results obtained with immunocytochemistry with those obtained with various GUS substrates for histochemical studies. Certain sources of artefact are discussed, as are the limits that should be imposed on interpretation of GUS histochemistry results at the organ, tissue and cell levels.     

Antibacterial Activity of Long-Chain Primary Alcohols from <Emphasis Type="Italic">Solena amplexicaulis</Emphasis> Leaves

Extraction, thin layer chromatography and gas chromatography–mass spectrometry of Solena amplexicaulis (Lam.) Gandhi, commonly known as creeping cucumber, (Cucurbitaceae) leaves revealed 21 long-chain primary alcohols, and 100 g leaves indicated presence of 3651.59 ± 327.18 SE µg long-chain primary alcohols. 1-Heptadecanol and 1-triacontanol were the predominant and least abundant primary alcohols, representing for 780.44 ± 42.59 and 3.28 ± 0.55 SE μg, respectively. Antibacterial property of the complete synthetic blend (0.1%), comparable to long-chain alcohols as detected by GC-FID of 100 g S. amplexicaulis leaf extracts was evaluated on the pathogenic bacteria Salmonella gallinarum by agar well diffusion method, and exhibited 20.4, 26.7 and 38.2 mm zone of inhibition at 25, 50 and 100 μl doses, respectively. One hundred µl dose of 6 individual pure synthetic compounds, 1-tridecanol, 1-pentadecanol, 1-heptadecanol, 1-nonadecanol, 1-eicosanol and 1-tricosanol comparable to the amounts present in 0.1% solution of pure isolated alcohols from S. amplexicaulis leaves displayed 16.2, 17.7, 18.6, 22.8, 15.8 and 14.5 mm zone of inhibition against this bacterium, respectively. Hundred µl dose from a synthetic blend of above 6 compounds (comparable to the proportions as present in 0.1% solution of pure isolated alcohols from 100 g S. amplexicaulis leaves) exhibited 38.1 mm zone of inhibition against this bacterium. Furthermore, 100 μl dose from a mixture (1:1) comprising of chloramphenicol (1 µg/ml) and a synthetic blend of above 6 compounds displayed 38.8 mm inhibition zone against S. gallinarum, and hence, this combination might be used against this pathogenic bacteria.     

A synthetic S6 segment derived from KvAP channel self-assembles, permeabilizes lipid vesicles, and exhibits ion channel activity in bilayer lipid membrane

KvAP is a voltage-gated tetrameric K(+) channel with six transmembrane (S1-S6) segments in each monomer from the archaeon Aeropyrum pernix. The objective of the present investigation was to understand the plausible role of the S6 segment, which has been proposed to form the inner lining of the pore, in the membrane assembly and functional properties of KvAP channel. For this purpose, a 22-residue peptide, corresponding to the S6 transmembrane segment of KvAP (amino acids 218-239), and a scrambled peptide (S6-SCR) with rearrangement of only hydrophobic amino acids but without changing its composition were synthesized and characterized structurally and functionally. Although both peptides bound to the negatively charged phosphatidylcholine/phosphatidylglycerol model membrane with comparable affinity, significant differences were observed between these peptides in their localization, self-assembly, and aggregation properties onto this membrane. S6-SCR also exhibited reduced helical structures in SDS micelles and phosphatidylcholine/phosphatidylglycerol lipid vesicles as compared with the S6 peptide. Furthermore, the S6 peptide showed significant membrane-permeabilizing capability as evidenced by the release of calcein from the calcein-entrapped lipid vesicles, whereas S6-SCR showed much weaker efficacy. Interestingly, although the S6 peptide showed ion channel activity in the bilayer lipid membrane, despite having the same amino acid composition, S6-SCR was significantly inactive. The results demonstrated sequence-specific structural and functional properties of the S6 wild type peptide. The selected S6 segment is probably an important structural element that could play an important role in the membrane interaction, membrane assembly, and functional property of the KvAP channel.     

Effects of tyrosine kinase and phosphatase inhibitors on mitosis progression in synchronized tobacco BY-2 cells

To test whether reversible tubulin phosphorylation plays any role in the process of plant mitosis the effects of inhibitors of tyrosine kinases, herbimycin A, genistein and tyrphostin AG 18, and of an inhibitor of tyrosine phosphatases, sodium orthovanadate, on microtubule organization and mitosis progression in a synchronized BY-2 culture has been investigated. It was found that treatment with inhibitors of tyrosine kinases of BY-2 cells at the G₂/M transition did not lead to visible disturbances of mitotic microtubule structures, while it did reduce the frequency of their appearance. We assume that a decreased tyrosine phosphorylation level could alter the microtubule dynamic instability parameters during interphase/prophase transition. All types of tyrosine kinase inhibitors used caused a prophase delay: herbimycin A and genistein for 2 h, and tyrphostin AG18 for 1 h. Thereafter the peak of mitosis was displaced for 1 h by herbimycin A or genistein exposure, but after tyrphostin AG18 treatment the timing of the mitosis-peak was comparable to that in control cells. Enhancement of tyrosine phosphorylation induced by the tyrosine phosphatase inhibitor resulted in the opposite effect on BY-2 mitosis transition. Culture treatment with sodium orthovanadate during 1 h resulted in an accelerated start of the prophase and did not lead to the alteration in time of the mitotic index peak formation, as compared to control cells. We suppose that the reversible tyrosine phosphorylation can be involved in the regulation of interphase to M phase transition possible through regulation of microtubule dynamics in plant cells.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

New spectrophotometric methods for the determination of nifedipine in pharmaceutical formulations

Two simple, sensitive and economical spectrophotometric methods were developed for the determination of nifedipine in pharmaceutical formulations. Method A is based on the reaction of the nitro group of the drug with potassium hydroxide in dimethyl sulphoxide (DMSO) medium to form a coloured product, which absorbs maximally at 430 nm. Method B uses oxidation of the drug with ammonium molybdate and subsequently reduced molybdenum blue is measured at 830 nm. Beer's law is obeyed in the concentration range of 5.0-50.0 and 2.5-45.0 microg ml(-1) with methods A and B, respectively. Both methods have been successfully applied for the assay of the drug in pharmaceutical formulations. No interference was observed from common pharmaceutical adjuvants. The reliability and the performance of the proposed methods are established by point and interval hypothesis tests and through recovery studies.