Molecular BioSystems

doi: 10.1039/c004973ppmid: N/A

doi: 10.1039/c004974npmid: N/A

Brnjic, Slavica; Olofsson, Maria Hägg; Havelka, Aleksandra Mandic; Linder, Stig

doi: 10.1039/b920805dpmid: 20567760

Calcium (Ca2+) is used as a signaling molecule to regulate many cellular processes. Calcium signaling generally involves transient elevations of the concentration of free Ca2+ in the cytosol. More pronounced and sustained elevations of intracellular Ca2+ concentrations are observed during apoptosis (programmed cell death). These Ca2+ elevations have been shown to lead to the activation of proteases (calpains) and to changes in protein phosphorylation. Recent evidence, using chemical biology, has raised the possibility that calcium signaling is involved in sustained JNK activation during late phases of apoptosis. For at least some stimuli, calcium release leads to activation of calmodulin kinase II (CaMKII), apoptosis signaling kinase 1 (ASK1) and JNK. Calcium signaling may help to orchestrate the apoptotic response during the execution phase.

Espitia, Clara; Servín-González, Luis; Mancilla, Raúl

doi: 10.1039/b916394hpmid: 20567761

Glycosylation is a common post-translational modification of surface exposed proteins and lipids present in all kingdoms of life. Information derived from bacterial genome sequencing, together with proteomic and genomic analysis has allowed the identification of the enzymatic glycosylation machinery. Among prokaryotes, O-mannosylation of proteins has been found in the actinomycetes and resembles protein O-mannosylation in fungi and higher eukaryotes. In this review we summarize the main features of the biosynthetic pathway of O-mannosylation in prokaryotes with special emphasis on the actinomycetes, as well as the biological role of the glycosylated target proteins.

Quinn, Amy M.; Allali-Hassani, Abdellah; Vedadi, Masoud; Simeonov, Anton

doi: 10.1039/b921912apmid: 20567762

Methylation of lysine residues, catalyzed by histone methyltransferase (HMT) enzymes, is one of many modifications of core histone proteins that regulate transcription and chromatin structure. G9a is the predominant HMT in mammalian euchromatin and recent data suggest that it is required to perpetuate a malignant phenotype in cancer cells and is implicated in metastasis, supporting this HMT as a therapeutic target for cancer and other diseases associated with epigenetic regulation. Of the methods currently used to measure methyltransferase activity, many involve a separation step or utilize coupling enzymes complicating implementation and data interpretation. Here we describe a homogeneous assay to measure G9a HMT activity using the chemiluminescence-based AlphaScreen immunoassay technology. Methylation of biotinylated-histone peptide is measured through specific antibody-based detection, in conjunction with streptavidin-coated donor and secondary antibody-coated acceptor beads. The method is particularly well suited for detection of inhibitors acting by the desired histone peptide competitive mechanism and is applicable to testing other HMTs, demonstrated here with the G9a homolog EHMT1, also known as GLP.

Murakami, Tatsuya; Tsuchida, Kunihiro; Hashida, Mitsuru; Imahori, Hiroshi

doi: 10.1039/c001442gpmid: 20567763

A phospholipid–protein hybrid nanostructure was found to reveal various hydrodynamic diameters dependent on the amount of the drugs incorporated, which could be over 10 times larger than that of the starting nanostructure. This observation was likely related to the thermal stabilization of the nanostructure during incorporation of the drugs by using the increased amount of phospholipids for the preparation. Furthermore, hydrophobicity or molecular weight of drugs is at least needed for size control.

Darius, Aw Kang Lie; Ling, Neo Jia; Mahesh, Uttamchandani

doi: 10.1039/c001923bpmid: 20567764

The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Most reports on its use have however been constrained to proof-of-concept demonstrations using synthetic oligonucleotides. This is due to the inherent complexities of the assay, and the multiple components involved. Herein, we have overcome several of these challenges and have successfully integrated asymmetric PCR with the visual detection step, allowing enriched targets from complex samples to be conveniently detected. This workflow would enable the DNAzyme system to be applied for point-of-care DNA detection applications. We have established the platform herein as a simple and robust method to determine the presence of target DNA sequences post-PCR (as required for pathogen detection), without the need for gels or other apparatus. Nanomolar concentrations of amplified targets were detected using the workflow established, enabling the detection of salmonella and mycobacterium targets through a color change reaction, observable just by eye.

Kanlaya, Rattiyaporn; Pattanakitsakul, Sa-nga; Sinchaikul, Supachok; Chen, Shui-Tein; Thongboonkerd, Visith

doi: 10.1039/b923864fpmid: 20567765

Our previous study using expression proteomics demonstrated that many proteins, particularly five forms of heterogeneous nuclear ribonucleoproteins (hnRNPs), were up-regulated in human endothelial cells upon dengue virus infection. To address functional significance of these proteins in response to dengue virus infection, we performed a functional proteomics study to identify hnRNPs-interacting proteins in the infected EA.hy926 cells. Immunoprecipitation followed by 2-D PAGE and mass spectrometric analyses revealed 18 and 13 interacting partners of hnRNP C1/C2 and hnRNP K, respectively. Interestingly, vimentin was a common partner for both hnRNP C1/C2 and K. The interaction between vimentin and these hnRNPs was confirmed by reciprocal immunoprecipitation followed by Western blot analysis and also by double immunofluorescence staining. Disruption of vimentin intermediate filament by acrylamide not only dissociated these complexes but also reduced nuclear hnRNPs expression, whereas cytosolic hnRNPs expression was unchanged. We also demonstrated that vimentin was strongly associated with dengue non-structural protein 1 (NS1). Disruption of vimentin intermediate filament not only dissociated this complex but also reduced dengue NS1 expression, as well as viral replication and release. Our data report for the first time that vimentin interacts with hnRNPs and dengue NS1, and plays a crucial role in replication and release of dengue virus.

Ausländer, Simon; Ketzer, Patrick; Hartig, Jörg S.

doi: 10.1039/b923076apmid: 20567766

The possibility to externally control gene expression is of fundamental importance in both basic and applied life sciences. Although there are some techniques available to regulate expression in mammalian cells, they rely on the presence of ligand-sensing transcription factors, making it necessary to generate cell lines or organisms that stably express these regulatory factors. In recent years, mechanisms relying on direct RNA-ligand interactions for controlling gene expression have been both discovered in nature and engineered artificially. Among the latter, RNA switches relying on catalytically active RNA have been described. In principle, ligand-dependent triggering of mRNA self-cleavage should be a universal mechanism in order to control gene expression in a variety of organisms. Nevertheless, no examples of such aptazymes acting as RNA-based switches have been reported so far in mammalian cells. Here we present the theophylline-induced activation of an mRNA-based hammerhead ribozyme, resulting in an off-switch of gene expression. Starting from an artificial aptazyme switch reported to function in bacteria, we identified and optimized important parameters such as artificial start codons and the communicating sequence connecting ribozyme and aptamer, resulting in an RNA switch that allows for controlling transgenic expression in mammalian cells without the need to express a corresponding ligand-sensing transcription factor.

Ahmad, Mark; Taylor, Charles R.; Pink, David; Burton, Kerry; Eastwood, Daniel; Bending, Gary D.; Bugg, Timothy D. H.

doi: 10.1039/b908966gpmid: 20567767

Two spectrophotometric assays have been developed to monitor breakdown of the lignin component of plant lignocellulose: a continuous fluorescent assay involving fluorescently modified lignin, and a UV-vis assay involving chemically nitrated lignin. These assays have been used to analyse lignin degradation activity in bacterial and fungal lignin degraders, and to identify additional soil bacteria that show activity for lignin degradation. Two soil bacteria known to act as aromatic degraders, Pseudomonas putida and Rhodococcus sp. RHA1, consistently showed activity in these assays, and these strains were shown in a small scale experiment to breakdown lignocellulose, producing a number of monocyclic phenolic products. Using milled wood lignin prepared from wheat straw, pine, and miscanthus, some bacterial lignin degraders were found to show specificity for lignin type. These assays could be used to identify novel lignin degraders for breakdown of plant lignocellulose.