Evidence for trans-trans and cis-cis farnesyl pyrophosphate synthesis in Gossipium hirsutum

A protein fraction capable of catalysing the formation of all four geometrical isomers of farnesyl pyrophosphate has been isolated from cotton roots. Using neryl pyrophosphate and isopentenyl pyrophosphate as substrates the product was found to be cis-cis farnesyl pyrophosphate and possibly trans-cis farnesyl pyrophosphate. Geranyl pyrophosphate and isopentenyl pyrophosphate as substrates yielded trans-trans and possible cis-trans farnesyl pyrophosphate. During purification of the active protein fraction, the ratio of utilization of geranyl pyrophosphate and neryl pyrophosphate did not remain constant, indicating that two enzymes may be involved, one specific for cis C₁₀-substrate and the other for trans C₁₀-substrate.     

Rapid assay for detecting gyrA and parC mutations associated with fluoroquinolone resistance in Enterobacteriaceae

We developed a PCR-RFLP assay to detect mutations in the quinolone-resistance determining regions of gyrA and parC associated with fluoroquinolone resistance in Enterobacteriaceae. The assay detected mutations associated with reduced susceptibility to fluoroquinolones and may therefore serve as a specific, rapid, inexpensive, and simple testing alternative.     

Interconversion of trans, trans and cis, trans farnesol by enzymes from Andrographis

When trans, trans-farnesol [4,8,12-¹⁴C₃,1-³H₂] is isomerized to cis, trans-farnesol by soluble enzymes from Andrographis paniculata tissue cultures, 50% of the tritium label is lost. The same loss is observed when isomerization occurs in the opposite direction. This is in accordance with the proposed mechanism for isomerization via aldehydes.     

Storage glucan biosynthesis in Cyanidium, Chlorella and Prototheca: evidence for endosymbiosis?

The storage glucans of Chlorella pyrenoidosa and Prototheca zopfii are identical and consist of a linear polyglucan akin to amylose and a branched amylopectin component. The branched glucans of these algae differ markedly from that formed by the hot-springs alga, Cyanidium caldarium. The more highly branched Cyanidium glucan appears to be formed by branching glucosyltransferases which are different from those of the other two algae. The relevance of the data to the possibility of Cyanidium being a Prototheca-like Chlorella that has acquired symbiotic Cyanobacteria as chloroplasts is discussed.     

Dinuclear ruthenium sawhorse-type complexes containing bridging ligands with ferrocenyl substituents in endo/endo, endo/exo and exo/exo orientations

The dinuclear ruthenium complexes Ru₂(CO)₄(OOCC₅H₄FeC₅H₅)₂L₂ (L = NC₅H₅: 1, L = PPh₃: 2) have been synthesized from Ru₃(CO)₁₂, ferrocene carboxylic acid and pyridine or triphenylphosphine, respectively. The single-crystal X-ray structure analysis reveals for 1 and 2 a Ru₂(CO)₄ sawhorse backbone with the two ferrocenyl substituents of the two carboxylato bridges being endo/exo with respect to each other in the solid state. With the new pyridine derivative NC₅H₄OOCC₅H₄FeC₅H₅ (4-ferrocenoyl pyridine) (3) as axial ligand, the complex Ru₂(CO)₄(OOCC₅H₄FeC₅H₅)₂(NC₅H₄OOCC₅H₄FeC₅H₅)₂ (4) was obtained, the single crystal X-ray structure analysis showing an exo/exo orientation of the two carboxylato bridges in the solid state. The endo/endo orientation is found in the solid-state structure of Ru₂(CO)₄(HNOCC₅H₄FeC₅H₅)₂(PPh₃)₂ (5), the two OCNH bridges being transoïd with respect to each other; this complex is accessible from Ru₃(CO)₁₂, ferrocenamide and triphenylphosphine.     

Screen of Bacillus thuringiensis toxins for transgenic rice to control Sesamia inferens and Chilo suppressalis

Transgenic rice to control stem borer damage is under development in China. To assess the potential of Bacillus thuringiensis (Bt) transgenes in stem borer control, the toxicity of five Bt protoxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ba and Cry1Ca) against two rice stem borers, Sesamia inferens (pink stem borer) and Chilo suppressalis (striped stem borer), was evaluated in the laboratory by feeding neonate larvae on artificial diets containing Bt protoxins. The results indicated that Cry1Ca exhibited the highest level of toxicity to both stem borers, with an LC₅₀ of 0.24 and 0.30 μg/g for C. suppressalis and S. inferens, respectively. However, S. inferens was 4-fold lower in susceptibility to Cry1Aa, and 6- and 47-fold less susceptible to Cry1Ab and Cry1Ba, respectively, compared to C. suppressalis. To evaluate interactions among Bt protoxins in stem borer larvae, toxicity assays were performed with mixtures of Cry1Aa/Cry1Ab, Cry1Aa/Cry1Ca, Cry1Ac/Cry1Ca, Cry1Ac/Cry1Ba, Cry1Ab/Cry1Ac, Cry1Ab/Cry1Ba, and Cry1Ab/Cry1Ca at 1:1 (w/w) ratios. All protoxin mixtures demonstrated significant synergistic toxicity activity against C. suppressalis, with values of 1.6- to 11-fold higher toxicity than the theoretical additive effect. Surprisingly, all but one of the Bt protoxin mixtures were antagonistic in toxicity to S. inferens. In mortality-time response experiments, S. inferens demonstrated increased tolerance to Cry1Ab and Cry1Ac compared to C. suppressalis when treated with low or high protoxin concentrations. The data indicate the utility of Cry1Ca protoxin and a Cry1Ac/Cry1Ca mixture to control both stem borer populations.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

Nitrogen-containing non-steroidal secondary metabolites of Solanum, Cyphomandra, Lycianthes and Margaranthus

Of the 49 species of Solanum studied, cuscohygrine has been detected in 25, solamine and related amines in 17 and solamine-derived amides in 16. Five species of Cyphomandra examined all contained both amines and amides. From roots of Margaranthus solanaceus cuscohygrine has been isolated which probably occurs, too, in roots of Lycianthes rantonnettii. The distribution of these compounds throughout the taxa could be of chemotaxonomic value.     

Leishmania (Viannia) panamensis: An in vitro assay using the expression of GFP for screening of antileishmanial drug

Promastigotes of Leishmania (Viannia) panamensis were successfully transfected with p6.5-egfp to express green fluorescent protein. The transfectants remained infective to macrophages, providing an in vitro model for screening antileishmanial drugs. This was demonstrated by flow cytometry of macrophage-associated GFP after exposure of infected cultures to known antileishmanial drugs, i.e. amphotericin B and glucantime^®. Fluorescence of GFP diminished progressively from infected cells with increasing drug concentrations used in both cases. The availability of this fluorescent assay for infection of macrophages by L. (V.) panamensis facilitates drug discovery program for the Viannia species, which differ significantly from those of the Leishmania subgenus.     

Iridoid glucosides in Griselinia, Aralidium and Toricellia

From three of five investigated species of Griselinia a new iridoid glucoside, griselinoside, was isolated. It was found to be present also in foliage of Aralidium pinnatifidum and Toricellia angulata, accompanied in the former by aralidioside another novel iridoid glucoside. The structures and absolute configurations of the two iridoids were elucidated by NMR spectroscopy and chemical conversions. From G. littoralis and T. angulata the glucosides magnolioside and syringoside respectively were isolated. ¹³C NMR spectra are given for thirteen iridoid derivatives.     

Fluorescence in situ hybridization for intracellular localization of nifH mRNA

Few reports on in situ mRNA detection in bacteria have been published, even though a major aim in environmental microbiology is to link function/activity to the identity of the organisms. This study reports a reliable approach for the in situ detection of nifH mRNA using fluorescence hybridization based on a previously described protocol for pmoA. nifH codes for a dinitrogenase reductase, a key enzyme in dinitrogen fixation. nifH mRNA was hybridized with a digoxigenin-labelled polynucleotide probe. The hybrid was detected with an anti-DIG-antibody labelled with horseradish peroxidase. Subsequently, the signal was amplified by catalyzed reporter deposition (CARD) with fluorochrome-labelled tyramides. Furthermore, the imaged organisms were identified using standard fluorescence in situ hybridization of rRNA. Thus, the approach enabled us specifically to link in situ the information from the dinitrogen fixation activity of an organism to its identity. Unexpectedly, the signals derived from nifH mRNA hybridization showed a distinct uneven pattern within the cells. This indicated that the method used could even give insights about the localization of the detected mRNA within the cell, which is a potential use of mRNA fluorescence in situ hybridization (FISH) that has not been reported up to now for bacterial cells.     

Relative pathogenicity of nuclear polyhedrosis viruses from Heliothis punctigera and Heliothis zea for larvae of Heliothis armigera and Heliothis punctigera

Laboratory bioassays indicated that the potency of a nuclear polyhedrosis virus from Heliothis zea derived from a commercial American formulation was similar to that of a naturally occurring nuclear polyhedrosis virus from H. punctigera in Australia. Both viruses exhibited high virulence for neonate larvae of H. armigera and H. punctigera, the major pest species in this genus in Australia. Hence evaluation of the virus in Australia can proceed employing virus from either H. punctigera or H. zea.     

Cortinarius sect. Brunnei (Basidiomycota, Agaricales) in North Europe

The section Brunnei was extensively studied based on material from North Europe. To stabilise the nomenclature we studied the relevant types of taxa included in this section. Phylogenetic relationships and species limits were investigated using rDNA ITS sequences and the results were compared with the morphological data. We recognised 11 species: Cortinarius brunneus, C. clarobrunneus comb. nov., C. coleoptera, C. ectypus, C. gentilis, C. glandicolor (neotypified), C. pseudorubricosus, and four species described as new C. caesiobrunneus, C. albogaudis, C. carabus, and C. cicindela. They are described here and their taxonomy, ecology, distribution, and relationships are discussed. In addition, a key to species of the section Brunnei is provided. A total of 77 new sequences of 11 species are published including nine type sequences. Also the taxonomic assignments of sequences in the public databases belonging to the section Brunnei are revised.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.     

Cloning of a gene required for tryptophan biosynthesis from Leptospira biflexa serovar patoc into Escherichia coli

A clone bank, consisting of approx. 8100 colonies, has been created for the spirochete Leptospira biflexa serovar patoc in Escherichia coli using pBR322 as the vector. One of these clones contains the genetic information needed to complement a defect in the trpE gene of E. coli. The information resides on a 20.5-kb plasmid designated pYCl, which carries a 16-kb insert consisting of three HindIII fragments. It does not complement defects in other genes needed for the biosynthesis of tryptophan in E. coli.     

Lavandula angustifolia essential oil as a novel and promising natural candidate for tick (Rhipicephalus (Boophilus) annulatus) control

Lavandula angustifolia is a well known herbal medicine with a variety of useful properties, including its acaricidal effect. This experiment was carried out to study the bioacaricidal activity of L. angustifolia essential oil (EO) against engorged Rhipicephalus (Boophilus) annulatus (Acari; Ixodidae) females. For this purpose six serial concentrations (0.5, 1.0, 2.0, 4.0, 6.0 and 8.0% w/v) of L. angustifolia EO were used. There was considerable mortality in concentrations more than 4.0% although there were no differences between 6.0 and 8.0% in all measured criteria. The mortality rate 24 h after inoculation was 73.26 and 100% in groups treated with 4.0 and 8.0% EO, respectively. Lavender EO also reduced tick egg weight in a concentration-dependent manner. The amount of eggs produced varied from 0.12 g (at 0.5% EO) to 0.00 g (at 8.0% EO) but did not differ statistically from the control. L. angustifolia EO caused 100% failure in egg laying at 6.0 and 8.0% whereas this value in the control group was zero. A positive correlation between L. angustifolia EO concentration and tick control, assessed by relative mortality rate and egg-laying weight, was observed by the EO LC/EC₅₀, which, when calculated using the Probit test, was 2.76-fold higher than the control. Lavender is a promising acaricidal against R. (B.) annulatusin vitro.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

Establishment of a cell line from Chrysodeixis chalcites permissive for Chrysodeixis chalcites and Trichoplusia ni nucleopolyhedrovirus

A new cell line was established from the embryos of the insect Chrysodeixis chalcites (Lepidoptera, Noctuidae, Plusiinae). The cell line contains several morphologically different cell types and was distinguished from three other lepidopteran cell lines propagated in the laboratory by DNA amplification fingerprinting. The cultured cells, which we officially named WU-CcE-1 cells, were permissive for infection by C. chalcites nucleopolyhedrovirus (ChchNPV) and large numbers of occlusion bodies were produced that retained their infectivity for C. chalcites larvae. The CcE-1 cells were also permissive for Trichoplusia ni single nucleopolyhedrovirus (TnSNPV). ChchNPV could be passaged in these cells for at least four passages indicating that budded virus production was supported. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa armigera (Hear) NPV both induced apoptosis in these cells. The results obtained indicate that the CcE-1 cell line will be a useful tool in the study of both ChchNPV and TnSNPV.