Structural and functional studies of the nicotinic acetylcholine receptor by solid-state NMR

Over the last seven years, solid-state NMR has been widely employed to study structural and functional aspects of the nicotinic acetylcholine receptor. These studies have provided detailed structural information relating to both the ligand binding site and the transmembrane domain of the receptor. Studies of the ligand binding domain have elucidated the nature and the orientation of the pharmacophores responsible for the binding of the agonist acetylcholine within the agonist binding site. Analyses of small transmembrane fragments derived from the nicotinic acetylcholine receptor have also revealed the secondary structure and the orientation of these transmembrane domains. These experiments have expanded our understanding of the channels structural properties and are providing an insight into how they might be modulated by the surrounding lipid environment. In this article we review the advances in solid-state NMR applied to the nicotinic acetylcholine receptor and compare the results with recent electron diffraction and X-ray crystallographic studies.Presented at the Biophysical Society Meeting on Ion channels – from structure to disease held in May 2003, Rennes, France     

Application of sterilised human amnion for reconstruction of the ocular surface

BACKGROUND: Despite improved surgical techniques and the use of new medication, healing of corneal and conjunctival defects cannot always be achieved. In this connection the clinical use of human amnion, produced by different techniques, has represented a successful alternative for the past ten years. The purpose of the present investigation was the development of a clinically secure, therapeutically efficient, and easy-to-handle (transport, storage, application) allogenic amnion transplant. PATIENTS AND METHODS: A new method for an amnion preparation, which contains a sterilisation process in peracetic acid and a drying process in a laminar flow cabinet, was developed as an alternative to previous techniques described in the literature. Amnion transplantation was used to treat 41 patients, 36 of them with corneal ulcer. Further indications for amnion transplantation were symblepharon, descemetocele, as well as dehiscence of conjunctiva after cerclage. RESULTS: Seventy-three percent of cases showed postoperative improvement evidenced by constant vision, while 15 percent showed decreased vision. CONCLUSIONS: The study confirms the observations of previous investigators who consider amnion transplantation an efficient therapeutic method for a multitude of eye diseases. The new method described in this report, guarantees patients' safety by using a validated new sterilisation process against infections that can be transmitted by human tissue. At present this method constitutes the only process available in Germany, and is approved by the Federal Institute for Drugs and Medical Products (BfArM) for the manufacture of human amnion transplants as a finished medical product.     

The redox protein construction kit: pre-last universal common ancestor evolution of energy-conserving enzymes

Genome analyses and the resolution of three-dimensional structures have provided evidence in recent years for hitherto unexpected family relationships between redox proteins of very diverse enzymes involved in bioenergetic electron transport. Many of these enzymes appear in fact to be constructed from only a limited set of building blocks. Phylogenetic analysis of selected units from this "redox enzyme construction kit" indicates an origin for several prominent bioenergetic enzymes that is very early, lying before the divergence of Bacteria and Archaea. Possible scenarios for the early evolution of selected complexes are proposed based on the obtained tree topologies.     

Leishmania-induced inhibition of macrophage antigen presentation analyzed at the single-cell level

A number of studies have previously examined the capacity of intracellular Leishmania parasites to modulate the capacity of macrophages to process and present Ags to MHC class II-restricted CD4(+) T cells. However, the bulk culture approaches used for assessing T cell activation make interpretation of some of these studies difficult. To gain a more precise understanding of the interaction between Leishmania-infected macrophages and effector T cells, we have analyzed various parameters of T cell activation in individual macrophage-T cell conjugates. Leishmania-infected macrophages efficiently stimulate Ag-independent as well as Ag-dependent, TCR-mediated capping of cortical F-actin in DO.11 T cells. However, infected macrophages are less efficient at promoting the sustained TCR signaling necessary for reorientation of the T cell microtubule organizing center and for IFN-gamma production. A reduced ability to activate these T cell responses was not due to altered levels of surface-expressed MHC class II-peptide complexes. This study represents the first direct single-cell analysis of the impact of intracellular infection on the interaction of macrophages with T cells and serves to emphasize the subtle influence Leishmania has on APC function.     

A molecular dynamics investigation of mono and dimeric states of the outer membrane enzyme OMPLA

OMPLA is a phospholipase found in the outer membranes of many Gram-negative bacteria. Enzyme activation requires calcium-induced dimerisation plus bilayer perturbation. As the conformation of OMPLA in the different crystal forms (monomer versus dimer; with/without bound Ca(2+)) is remarkably similar we have used multi-nanosecond molecular dynamics (MD) simulations to probe possible differences in conformational dynamics that may be related to enzyme activation. Simulations of calcium-free monomeric OMPLA, of the Ca(2+)-bound dimer, and of the Ca(2+)-bound dimer with a substrate analogue covalently linked to the active site serine have been performed, all with the protein embedded in a phospholipid (POPC) bilayer. All simulations were stable, but differences in the dynamic behaviour of the protein between the various states were observed. In particular, the stability of the active site and the hydrophobic substrate-binding cleft varied. Dimeric OMPLA is less flexible than monomeric OMPLA, especially around the active site. In the absence of bound substrate analogue, the hydrophobic substrate-binding cleft of dimeric OMPLA collapses. A model is proposed whereby the increased stability of the active site in dimeric OMPLA is a consequence of the local ordering of water around the nearby calcium ion. The observed collapse of the substrate-binding cleft may explain the experimentally observed occurrence of multiple dimer conformations of OMPLA, one of which is fully active while the other shows significantly reduced activity.     

The naphthoquinol oxidizing cytochrome bc1 complex of the hyperthermophilic knallgasbacterium Aquifex aeolicus: properties and phylogenetic relationships

Phylogenetic analysis of constituent proteins of Rieske/cytochrome b complexes [Schütz et al. (2000) J. Mol. Biol. 300, 663-675] indicated that the respective enzyme from the hyperthermophile Aquifex (A.) aeolicus is closely related to proteobacterial counterparts, in disagreement with positioning of its parent species on small subunit rRNA trees. An assessment of the details and possible reasons for this discrepancy necessitates a thorough understanding of the biochemical and biophysical properties of the enzyme in addition to the bioinformatic data. The cytochrome bc(1) complex from A. aeolicus, which is part of the "Knallgasreaction" pathway, was therefore studied in membranes and in detergent-solubilized, isolated complex. Hemes b(L) (E(m,7) = -190 mV; g(z)= 3.7), b(H) (E(m,7) = -60 mV; g(z )= 3.45), and c(1) (E(m,7) = +160 mV; g(z )= 3.55) were identified by EPR and optical spectroscopy in combination with electrochemical methods. Two electrochemically distinct (E(m,7) = +95 mV; E(m,7) = +210 mV) Rieske centers were detected in membranes, and the +210 mV species was shown to correspond to the Rieske center of the cyt bc(1) complex. The gene coding for this latter Rieske protein was heterologously expressed in Escherichia coli, and the resulting protein was characterized in detail. The pool quinone of A. aeolicus was determined to be naphthoquinone. The redox poises of the individual electron-transfer steps are compared to those of other Rieske/cyt b complexes. The Aquifex enzyme was found to represent the only extant naphthoquinol oxidizing true cyt bc(1) complex described so far. An improved scenario for the phylogenetic positioning of the Aquifex cyt bc(1) complex is proposed.     

Benzyl-functionalized cycloSal-d4T monophosphates

Synthetic routes to benzyl-functionalized cycloSal-d4T monophosphates (7CH2X-cycloSal-d4TMP) have been developed. Their hydrolytic behavior in basic aqueous solution (pH = 7.3) was studied and their hydrolysis half-lives were determined. It turned out that two different degradation pathways are leading to different products: beside the formation of the expected d4TMP and a styrene type derivative, a phenyl-d4T-phosphodiester was obtained as well. The product distribution was specified.     

What cell lineages tell us about the evolution of spiralia remains to be seen

Abstract.— Cell-lineage trees may contain information about spiralian phylogeny, as proposed by Guralnick and Lind-berg (2001). Here we discuss this possibility further and conclude that the cell-division pattern must be known in greater detail and the coding methods refined before a possible phylogenetic signal can be identified.     

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.