6-Hydroxytoxol esters and a seco-dammarane derivative from Abrotanella forsterioides

Abrotanella forsterioides afrorded euparin, 6-hydroxytremetone and three 6-hydroxytoxol esters, one of them not being isolated previously. Furthermore a seco-triterpene was isolated. The tribal position of the genus Abrotanella in the Compositae is still an unsolved problem. Morphological investigations suggest that this genus should be transferred from the tribe Anthemideae to the Senecioneae [1,2]. So far two species have been studied chemically; one atforded ent-kaurane derivatives, while both contained euparin and hydroxytremetone [3].     

Neue furanoeremophilane und andere sesquiterpene aus vertretern der gattung Euryops

Investigation of a further 27 Euryops species affords, besides known compounds, 55 new furanoeremophilanes together with several other compounds. The configuration of six furanoeremophilanes earlier given as 10β-H has to be changed to 10α-H. Furthermore 9 new eremophilenes were isolated; the configurations of four compounds of this type isolated before have had to be changed. One of the eremophilenes is esterified with 3-methylpent-3(E)-enoic acid, not found before in nature. Two hemiacetals, which are probable precursors of the furanoerephilanes, and a corresponding lactone are also present. Furthermore two benzylic esters, cis-caryophyllene, a bicyclo-germacrene derivative, and a new oplopanone derivative have been found. The structures are elucidated mainly by spectroscopic methods together with some chemical transformations. From the chemotaxonomic viewpoint, the high concentration of very variable furanoeremophilanes may be important. The proposed differentiation into sections however can not be clearly correlated with the chemical constituents, though some taxonomic conclusions seem to be supported by the chemical evidence.     

From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.     

Diadenosine tetraphosphate (Ap4A) is compartmentalized in nuclei of mammalian cells

The intracellular compartmentation of Ap4A in various growth and cell-cycle stages in mammalian cells was studied by applying a non-aqueous extraction procedure for cell nuclei. In both slowly and in exponentially growing Ehrlich ascites tumour cells from random cultures, more than 75% of the whole cellular Ap4A content is localized in the nuclei. In G1 and early S-phase cells of synchronized baby hamster kidney (BHK) fibroblast cultures, approx. 90% of the intracellular Ap4A pool is confined to the nuclear compartment. In contrast, Ap4A is distributed to nearly equal amounts between cytoplasm and nuclei during mid-S phase. After transition through the S-phase, increasing proportions of Ap4A (78% 18 h and 96% 22 h after serum replenishing, respectively) are again localized in the nuclear compartment.     

Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Sister chromatid cohesion and recombination in meiosis

Sister chromatids are associated from their formation until their disjunction. Cohesion between sister chromatids is provided by protein complexes, of which some components are conserved across the kingdoms and between the mitotic and meiotic cell cycles. Sister chromatid cohesion is intimately linked to other aspects of chromosome behaviour and metabolism, in particular chromosome condensation, recombination and segregation. Recombination, sister chromatid cohesion and the relation between the two processes must be regulated differently in mitosis and meiosis. In meiosis, cohesion and recombination are modified in such a way that reciprocal exchange and reductional segregation of homologous chromosomes are ensured. Received: 11 October 1999; in revised form: 3 December 1999 / Accepted: 6 December 1999     

Reduced activity of juvenile hormone esterase in microsporidia-infected Lymantria dispar larvae

Infection of the fat body of Lymantria dispar (Lep.: Lymantriinae) larvae with the microsporidium Vairimorpha disparis has severe effects on juvenile hormone (JH) metabolism of the host. Beginning 8 days postinfection, activity of the JH degrading enzyme JH-esterase was significantly lower in the hemolymph of infected than uninfected larvae. Activity remained low as microsporidiosis progressed. JH titers were slightly elevated in infected larvae; the difference was not significant in most cases. This disturbance of JH metabolism may be due to generally impaired fat body functions and high demand for resources by the developing pathogen.     

Generation of bioactive peptides by biological libraries

Biological libraries are powerful tools for discovery of new ligands as well as for identification of cellular interaction partners. Since the first development of the first biological libraries in form of phage displays, numerous biological libraries have been developed. For the development of new ligands, the usage of synthetic oligonucleotides is the method of choice. Generation of random oligonucleotides has been refined and various strategies for random oligonucleotide design were developed. We trace the progress and design of new strategies for the generation of random oligonucleotides, and include a look at arising diversity biases. On the other hand, genomic libraries are widely employed for investigation of cellular protein-protein interactions and targeted search of proteomic binding partners. Expression of random peptides and proteins in a linear form or integrated in a scaffold can be facilitated both in vitro and in vivo. A typical in vitro system, ribosome display, provides the largest available library size. In vivo methods comprise smaller libraries, the size of which depends on their transformation efficiency. Libraries in different hosts such as phage, bacteria, yeast, insect cells, mammalian cells exhibit higher biosynthetic capabilities. The latest library systems are compared and their strengths and limitations are reviewed.     

Aquaporins and unloading of phloem-imported water in coats of developing bean seeds

Nutrients are imported into developing legume seeds by mass flow through the phloem, and reach developing embryos following secretion from their symplasmically isolated coats. To sustain homeostasis of seed coat water relations, phloem-delivered nutrients and water must exit seed coats at rates commensurate with those of import through the phloem. In this context, coats of developing French bean seeds were screened for expression of aquaporin genes resulting in cloning PvPIP1;1, PvPIP2;2 and PvPIP2;3. These genes were differentially expressed in all vegetative organs, but exhibited their strongest expression in seed coats. In seed coats, expression was localized to cells of the nutrient-unloading pathway. Transport properties of the PvPIPs were characterized by expression in Xenopus oocytes. Only PvPIP2;3 showed significant water channel activity (Pos = 150-200 microm s(-1)) even when the plasma membrane intrinsic proteins (PIPs) were co-expressed in various combinations. Permeability increases to glycerol, methylamine and urea were not detected in oocytes expressing PvPIPs. Transport active aquaporins in native plasma membranes of seed coats were demonstrated by measuring rates of osmotic shrinkage of membrane vesicles in the presence and absence of mercuric chloride and silver nitrate. The functional significance of aquaporins in nutrient and water transport in developing seeds is discussed.     

Complex nitrogen cycling in the sponge Geodia barretti

Marine sponges constitute major parts of coral reefs and deep‐water communities. They often harbour high amounts of phylogenetically and physiologically diverse microbes, which are so far poorly characterized. Many of these sponges regulate their internal oxygen concentration by modulating their ventilation behaviour providing a suitable habitat for both aerobic and anaerobic microbes. In the present study, both aerobic (nitrification) and anaerobic (denitrification, anammox) microbial processes of the nitrogen cycle were quantified in the sponge Geodia barretti and possible involved microbes were identified by molecular techniques. Nitrification rates of 566 nmol N cm^?3 sponge day^?1 were obtained when monitoring the production of nitrite and nitrate. In support of this finding, ammonia‐oxidizing Archaea (crenarchaeotes) were found by amplification of the amoA gene, and nitrite‐oxidizing bacteria of the genus Nitrospira were detected based on rRNA gene analyses. Incubation experiments with stable isotopes (¹⁵NO₃^‐ and ¹⁵NH₄⁺) revealed denitrification and anaerobic ammonium oxidation (anammox) rates of 92 nmol N cm^?3 sponge day^?1 and 3 nmol N cm^?3 sponge day^?1 respectively. Accordingly, sequences closely related to ‘Candidatus Scalindua sorokinii’ and ‘Candidatus Scalindua brodae’ were detected in 16S rRNA gene libraries. The amplification of the nirS gene revealed the presence of denitrifiers, likely belonging to the Betaproteobacteria. This is the first proof of anammox and denitrification in the same animal host, and the first proof of anammox and denitrification in sponges. The close and complex interactions of aerobic, anaerobic, autotrophic and heterotrophic microbial processes are fuelled by metabolic waste products of the sponge host, and enable efficient utilization and recirculation of nutrients within the sponge–microbe system. Since denitrification and anammox remove inorganic nitrogen from the environment, sponges may function as so far unrecognized nitrogen sinks in the ocean. In certain marine environments with high sponge cover, sponge‐mediated nitrogen mineralization processes might even be more important than sediment processes.