Leukotriene C4 synthase homo-oligomers detected in living cells by bioluminescence resonance energy transfer

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC(4) and its metabolites LTD(4) and LTE(4) stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC(4) is formed by addition of glutathione to LTA(4), catalyzed by the integral membrane protein, LTC(4) synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114-150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57-88. The functional significance of LTCS homo-oligomer formation is currently being investigated.     

Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.     

Identification of binding mechanisms in single molecule-DNA complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.     

Identification and characterization of adenosine 5'-tetraphosphate in human myocardial tissue

Endocrine functions of the human heart have been studied extensively. Only recently, nucleotidergic mechanisms have been studied in detail. Therefore, an isolation strategy was developed to isolate novel nucleotide compounds from human myocardium. The human myocardial tissue was fractionated by several chromatographic studies. A substance purified to homogeneity was identified as adenosine 5'-tetraphosphate (Ap(4)) by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), post-source decay MALDI MS, and enzymatic cleavage analysis. Furthermore, Ap(4) was also identified in ventricular specific granules. In the isolated perfused rat heart, Ap(4) elicited dose-dependent vasodilations. Vasodilator responses were abolished in the presence of the P(2Y1) receptor antagonist MRS 2179 (1 microm) or the NO synthase inhibitor N(G)-nitro-l-arginine methyl ester (50 microm). After removal of the endothelium by Triton X-100, Ap(4) induced dose-dependent vasoconstrictions. Inhibition of P(2X) receptors by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (30 microm) or desensitization of P(2X) receptors by alpha,beta-methylene ATP (alpha,beta-meATP, 1 microm) diminished these vasoconstrictor responses completely. In the present study Ap(4) has been isolated from human tissue. Ap(4) was shown to exist in human myocardial tissue and was identified in ventricular specific granules. In coronary vasculature the nucleotide exerted vasodilation via endothelial P(2Y1) receptors and vasoconstriction via P(2X) receptors on vascular smooth muscle cells. Ap(4) acts as an endogenous extracellular mediator and might contribute to the regulation of coronary perfusion.     

Effect of hypergravity on the Ca/Sr composition of developing otoliths of larval cichlid fish (Oreochromis mossambicus)

The amounts of calcium and strontium were measured by inductively coupled plasma mass spectrometry (ICP-MS) in saccular and utricular inner ear otoliths (sagittae and lapilli, respectively) of developing cichlid fish. These fish had been maintained for 22 days at 3-g hypergravity conditions within a centrifuge. During this time-span, the animals completed their ontogenetic development from hatch to the free-swimming stage. Neither the morphogenetic development nor the timely onset and gain of performance of the swimming behaviour was impaired by the experimental conditions. Experimental and control animals also did not differ concerning their size (total length). ICP-MS revealed that the otoliths contained significantly less calcium (in microg/otolith) after hyper-g exposure compared to parallelly raised 1-g control specimens (lapilli: 0.74+/-0.21 vs. 1.16+/-0.41; sagittae: 2.09+/-0.49 vs. 2.76+/-0.47). The content of strontium (in microg/otolith: lapilli: 0.0044+/-0.0023 vs. 0.0022+/-0.0013; sagittae: 0.0094+/-0.0026 vs. 0.0081+/-0.0016) and, consequently, the Sr/Ca ratio (Sr/Cax100) was increased (lapilli: 0.607+/-0.267 vs. 0.201+/-0.12; sagittae: 0.439+/-0.093 vs. 0.301+/-0.086). Since the calcium content can be taken as a proxy for otolith weight, and because parallelly undertaken morphometric investigations revealed smaller otoliths (maximum radius and surface area) due to hyper-g exposure, the results suggest that the growth of otoliths at hyper-g is slowed down. Since the concentration of trace elements incorporated into otoliths is likely based on the composition of the respective protein matrix, our findings suggest that the protein metabolism is affected by hypergravity.     

Site-directed mutagenesis of cysteine to threonine in Proteus vulgaris urease active site increases enzyme activity and stability

Cysteine-319 belongs to the flexible flap at the active site of Proteus vulgaris urease. Replacing this cysteine by threonine resulted in a 20-fold increase of specific activity. Temperature stability increased, susceptibility to inhibition by dipyridyl disulfide decreased, and pH optimum shifted from 8 to 6.9. K _m (35 to 12 mM) and V_max (47.4 to 1.8 mol min^–1) were substancially altered. Both variants of the enzyme were irreversibly inhibited by phenylmethanesulfonyl fluoride.     

Presenilin-dependent intramembrane proteolysis of CD44 leads to the liberation of its intracellular domain and the secretion of an Abeta-like peptide

Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.     

Dynamics of a mutualism in a multi-species context

Despite recent findings that mutualistic interactions between two species may be greatly affected by species external to the mutualism, the implications of such multi-species interactions for the population dynamics of the mutualists are virtually unexplored. In this paper, we ask how the mutualism between the shoot-base boring weevil Apion onopordi and the rust fungus Puccinia punctiformis is influenced by the dynamics of their shared host plant Cirsium arvense, and vice versa. In particular, we hypothesized that the distribution of the weevil's egg load between healthy and rust-infected thistles may regulate the abundance of the mutualists and their host plant. In contrast to our expectations we found that the dynamics of the mutualists are largely determined by the dynamics of their host. This is, to our knowledge, the first demonstration that the dynamics of a mutualism are driven by a third, non-mutualistic species.