Solution phase parallel synthesis and evaluation of MAPK inhibitory activities of close structural analogues of a Ras pathway modulator

A solution phase parallel synthesis approach was undertaken to rapidly explore the structure-activity relationship of an inhibitor of the Ras/Raf protein interaction identified from a small molecule compound library. Evaluation of the MAPK pathway signaling inhibitory activity of the synthesized analogues as well as their antiproliferative activity and ability to inhibit soft agar growth were performed.     

Cognitive neuroscience: acting on numbers

The parietal cortex is a central part of the brain's system for representing numbers and magnitudes. Activity in the parietal cortex might reflect number representation or actions made in response to the numbers.     

Functional stability of the aristaless gene in appendage tip formation during evolution

The appendages of an insect are subdivided into distinct segments or podomeres. Many genes responsible for the regionalization of the growing limb into subdomains have been isolated from Drosophila. So far, only one gene is known in the leg that is solely required for specifying the distal-most pattern element—the pretarsal claw. In Drosophila, the gene aristaless is expressed in the centre of the antennal and leg imaginal disc that represents the most distal position of appendages, and in a proximal region. When Drosophila aristaless function is impaired, antennae and legs develop without their distal-most structures—the arista and the claw. We describe here the analysis of aristaless in the beetle Tribolium—an insect that shows a different, more ancestral mode of appendage formation than Drosophila. In Tribolium, appendages grow out continuously during embryogenesis, and no imaginal discs are formed. Tribolium aristaless (Tc-al) expression starts midway during appendage elongation, and is seen in a distal and a proximal position of head and trunk appendages. At the end of embryogenesis, Tc-al is seen in four expression domains in the leg, in the dorsal epidermis, and ventrally in every segment in lateral groups of cells, presumably the histoblasts. Like in the Drosophila adult, Tc-al is required in the larva for the formation of the most distal structures of the leg and the antenna as revealed by RNAi experiments. We conclude that aristaless is evolutionarily robust, meaning that it has retained its expressional and functional characteristics, although a heterochronic change of the process of appendage elongation took place towards the evolution of the highly derived diptera.Edited by D. Tautz     

Bioelectrical impedance assay to monitor changes in cell shape during apoptosis

Apoptosis is a strictly regulated and genetically encoded cell 'suicide' that may be triggered by cytokines, depletion of growth factors or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell-cell contacts. We applied a non-invasive electrochemical technique referred to as electric cell-substrate impedance sensing (ECIS) in order to monitor the apoptosis-induced changes in cell shape in an integral and quantitative fashion with a time resolution in the order of minutes. In ECIS the cells are grown directly on the surface of small gold-film electrodes (d = 2 mm). From readings of the electrical impedance of the cell-covered electrode, performed with non-invasive, low amplitude sensing voltages, it is possible to deduce alterations in cell-cell and cell-substrate contacts. To improve the sensitivity of this impedance assay we used endothelial cells derived from cerebral micro-vessels as cellular model systems since these are well known to express electrically tight intercellular junctions. Apoptosis was induced by cycloheximide (CHX) and verified by biochemical and cytological assays. The time course of cell shape changes was followed with unprecedented time resolution by impedance readings at 1 kHz and correlated with biochemical parameters. From impedance readings along a broad frequency range of 1-10(6) Hz we could assign the observed impedance changes to alterations on the subcellular level. We observed that disassembly of barrier-forming tight junctions precedes changes in cell-substrate contacts and correlates strongly with the time course of protease activation.     

Cancer vaccine design: a novel bacterial adjuvant for peptide-specific CTL induction

The recent identification of tumor Ags as potential vaccines has prompted the search for efficient adjuvants and delivery systems, especially in the case of peptide-based vaccination protocols. Here, we investigated the adjuvant potential of the recombinant 40-kDa outer membrane protein of Klebsiella pneumoniae (P40) for specific CTL induction. We studied the CTL response induced in HLA-A*0201/K(b) transgenic mice immunized with peptides derived from two melanoma-associated differentiation Ags, the HLA-A*0201-restricted decapeptide Melan-A(26--35) substituted at position 2 and the K(b)-restricted tyrosinase-related protein 2(181--188) T cell epitope. We found that both peptides are able to generate a specific CTL response when mixed with the protein in the absence of conventional adjuvant. This CTL response is a function of the amount of P40 used for immunization. Moreover, the CTL response generated against the tyrosinase-related protein 2(181-188) peptide in presence of P40 is associated with tumor protection in two different experimental models and is independent of the presence of CD4(+) T lymphocytes. Thus, the recombinant bacterial protein P40 functions as a potent immunological adjuvant for specific CTL induction.     

Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice

We have isolated and characterised the promoter of the mouse Scnn1a (alpha ENaC) gene. Using transient transfections of serial deletion mutants into Scnn1a-expressing cells, we demonstrate that 1.56 kb of 5' upstream sequence is required for cell-specific expression and corticosteroid-mediated regulation. These 5' sequences are not sufficient to drive expression of a lacZ reporter gene or a rat Scnn1a cDNA in transgenic mice, where they failed to rescue Scnn1a deficiency.     

Caspase inhibitors

Caspases are the key effector molecules of the physiological death process known as apoptosis, although some are involved in activation of cytokines, rather than cell death. They exist in most of our cells as inactive precursors (zymogens) that kill the cell once activated. Caspases can be controlled in two ways. The processing and activation of a caspase can be regulated by molecules such as FADD, APAF-1, Bcl-2 family members, FLIP and IAPs. Active caspases can be controlled by a variety of inhibitors that directly interact with the protease. This review describes the later direct caspase inhibitors that have been identified, products of both viral and cellular genes, and artificial caspase inhibitors that have been developed both as research tools and as pharmaceutical agents to inhibit cell death in vivo.     

Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus

Rhodobacter capsulatus contains two PII-like proteins, GlnB and GlnK, which play central roles in controlling the synthesis and activity of nitrogenase in response to ammonium availability. Here we used the yeast two-hybrid system to probe interactions between these PII-like proteins and proteins known to be involved in regulating nitrogen fixation. Analysis of defined protein pairs demonstrated the following interactions: GlnB-NtrB, GlnB-NifA1, GlnB-NifA2, GlnB-DraT, GlnK-NifA1, GlnK-NifA2, and GlnK-DraT. These results corroborate earlier genetic data and in addition show that PII-dependent ammonium regulation of nitrogen fixation in R. capsulatus does not require additional proteins, like NifL in Klebsiella pneumoniae. In addition, we found interactions for the protein pairs GlnB-GlnB, GlnB-GlnK, NifA1-NifA1, NifA2-NifA2, and NifA1-NifA2, suggesting that fine tuning of the nitrogen fixation process in R. capsulatus may involve the formation of GlnB-GlnK heterotrimers as well as NifA1-NifA2 heterodimers. In order to identify new proteins that interact with GlnB and GlnK, we constructed an R. capsulatus genomic library for use in yeast two-hybrid studies. Screening of this library identified the ATP-dependent helicase PcrA as a new putative protein that interacts with GlnB and the Ras-like protein Era as a new protein that interacts with GlnK.     

Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium

Myxobacteria are gram-negative bacteria which are most noted for their ability to form fruiting bodies upon starvation. Within the last two decades, they increasingly gained attention as producers of natural products with biological activity. Here, recent and future biotechnological research on certain key myxobacteria and on their ability to produce natural products is reviewed with the focus on the production of myxovirescin, soraphen and epothilone. Aspects of product improvement and yield as well as statistics regarding secondary metabolite formation are discussed. Future research will deal with the exploitation of the biosynthetic potential of the myxobacteria, for example via the isolation of new myxobacterial species with different physiological properties. Additionally, the genetic potential of myxobacteria to form natural products can be exploited by the identification and activation of biosynthetic gene clusters. These can be found frequently within their genomes, which is shown by the analysis of the unfinished genomes of Myxococcus xanthus and Sorangium cellulosum. The current status of the S. cellulosum functional genome project with model strain So ce56 is discussed.