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Biomedical subjects

A Engel

Publications and source records attributed to A Engel.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis.

A gene encoding a AAA ATPase was discovered in the 5' region of the second operon of 20 S proteasome subunits in the nocardioform actinomycete Rhodococcus erythropolis NI86/21. The gene was cloned and expressed in Escherichia coli. The protein, ARC (AAA ATPase forming Ring-shaped Complexes), is a divergent member of the AAA family. The deduced product of the arc gene is 591 residues long (66 kDa). The purified protein possesses a low, N-ethylmaleimide-sensitive ATPase activity and forms rings of six subunits, arranged symmetrically around a central opening or cavity. Two-dimensional crystals grown on lipid monolayers yielded images of the ATPase molecules in "end-on" orientation at 1.9 nm resolution.

Amino Acid Sequence↗
PubMed1998

Evaluating atomic models of F-actin with an undecagold-tagged phalloidin derivative.

We have prepared an undecagold-tagged phalloidin derivative to determine this mushroom toxin's binding site and orientation within the F-actin filament by scanning transmission electron microscopy (STEM) and 3-D helical reconstruction. Remarkably, when stoichiometrically bound to F-actin, the undecagold moiety of the derivative could be directly visualized by STEM along the two half-staggered long-pitch helical strands of single filaments. Most importantly, the structural data obtained when combined with various biochemical constraints enabled us to critically evaluate two distinct atomic models of the F-actin filament (i.e. the Holmes-Lorenz versus the Schutt-Lindberg model). Taken together, our data are in excellent agreement with the Holmes-Lorenz model.

Actins↗
PubMed1998

Surface analysis of the photosystem I complex by electron and atomic force microscopy.

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e' or were at the sites of putative alpha-helix-connecting loops estimated from the 4 A map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

Amino Acid Sequence↗
PubMed1998

Progress on the structure and function of aquaporin 1.

Life exists in water as universal solvent, and cells need to deal with its influx and efflux. Nature has accomplished the almost impossible, creating membrane channels with both a high flux and a high specificity for water. The first water channel was discovered in red blood cell membranes. Today known as aquaporin-1, this channel was found to be closely related to the major integral protein (MIP)1 of the eye lens. Cloning and sequencing of numerous related proteins of the MIP family revealed the widespread occurrence of such channels, suggesting an essential physiological function. Their structures hold the clues to the remarkable water channel activity, as well as to the arrangement of transmembrane segments in general. Recent medium-resolution three-dimensional electron microscopic studies determined a tetrameric complex with six tilted transmembrane helices per monomer. The helices within each monomer surround a central density formed by two interhelical loops implicated by mutagenesis in the water channel function. A combination of sequence analysis and assignment of the observed densities to predicted helices provides a basis for speculation on the nature of the water course through the protein. In particular, four highly conserved polar residues, E142-N192-N76-E17, are proposed to form a chain of key groups involved in the pathway of water flow through the channel.

Animals↗
PubMed1998

Mass Measurement in the Scanning Transmission Electron Microscope: A Powerful Tool for Studying Membrane Proteins.

The technique of mass measurement in the scanning transmission electron microscope is briefly presented. Results obtained for membrane proteins, with particular emphasis on the channel forming proteins, are discussed. The data illustrate the versatility of the technique which is applicable to particulate, filamentous, and sheet-like structures. When combined with composition analysis, the absolute mass values measured with the STEM allow protein stoichiometries to be unambiguously defined. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

2D crystallization of membrane proteins: rationales and examples.

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy. While reconstitution of membrane proteins with lipids is readily achieved, the mechanisms for crystal formation during or after reconstitution are not well understood. The nature of the detergent and lipid as well as pH and counter-ions is known to influence the crystal type and quality. Protein-protein interactions may also promote crystal stacking and aggregation of the sheet-like crystals, posing problems in data collection. Although highly promising, the number of well-studied examples is still too small to draw conclusions that would be applicable to any membrane protein of interest. Here we discuss parameters influencing the outcome of two-dimensional crystallization trials using prominent examples of channel protein crystals and highlight areas where further improvements to crystallization protocols can be made.

Bacterial Proteins↗
PubMed1998

The sodium channel has four domains surrounding a central pore.

The voltage-gated sodium channel generates the action potential. This 300-kDa protein has four homologous regions, which are also homologous to the voltage-sensitive tetrameric potassium channel. We isolated sodium channels from Electrophorus electricus electroplax by detergent solubilization and immunoaffinity chromatography and studied their structure by electron microscopy of negatively stained specimens. Different projections were aligned, classified, and averaged. In side view, the channel protein exhibits the shape of a truncated cone, 14 nm in height. One end has a diameter of 12 nm and is asymmetric, while the other is more symmetric and has a diameter of 7-10 nm. In top views, the sodium channel appears to consist of four domains of different size and to have a stain-filled pore in the center.

Animals↗
PubMed1998

Differentiation of autonomic nervous activity in different stages of coma displayed by power spectrum analysis of heart rate variability.

The analysis of heart rate (HR) variability offers a noninvasive method to investigate autonomic nervous system activity in comatose patients. We analyzed three components of the HR variability in a group of comatose patients: the low-frequency band (LF), representing mainly sympathetic influence, the mid-frequency band (MF), representing sympathetic and parasympathetic influence, and the high-frequency band (HF), representing the parasympathetic influence. A value for sympathovagal balance was defined as LF/HF and MF/HF ratio. Moreover, the skin conductance level (SCL) and the skin conductance resistance (SCR) variability were recorded. The patient group consisted of 22 patients with traumatic brain injuries. Coma depth was assessed by the Glacow Coma Scale and artifact-free HR, SCL, and SCR were measured 75 times in the patient group. The results documented a significant gain in sympathetic nervous system activity corresponding with the state of emerging from coma. This gain was most pronounced in the HF component of the HR and in the sympathovagal balance between LF/HF. The findings in SCL and SCR variability endorsed this result. It is concluded that emerging from coma is accompanied by an increasing influence of the sympathetic nervous system on HR control. This leads to a change in the sympathovagal balance, i.e., a reintegration of parasympathetic and sympathetic activity.

Adolescent↗
PubMed1998

[Labrum lesions and residual dysplasia of the hip joint. Definition and prospectives].

Labral lesions are a sign of biomechanical decompensation of the hip joint and often represent the first clinical symptom of residual hip dysplasia (RHD) in the adult. Provocation tests (impingement, apprehension) are typical but not specific. Labral lesions and concomitant findings (intra- and extraosseous ganglia, stress bone marrow oedema) can be detected by magnetic resonance arthrography (MRA) with an accuracy of 91%. Primary therapeutic goal is the normalization of the underlying pathomorphology and instability by a redirectional acetabular osteotomy. There are several concepts concerning simultaneous arthrotomy at the time of osteotomy: no arthrotomy at all, selective arthrotomy, routine arthrotomy in every case. There are more clinical studies necessary before one of these concepts can be widely accepted and recommended. Based on preliminary results, palliative arthrotomy with partial labral resection but without corrective osteotomy in osteoarthritis secondary to residual hip dysplasia gives poor results; we therefore urgently dissuade from palliative labral surgery via arthrotomy. Whether labral surgery via arthroscopy might be a useful concept in symptomatic residual hip dysplasia, is still an open question. In this review article, the "state of the art" presented at the "Vienna Labral Symposium 1997" is reflected and summarized. At the end of this article, a "common statement" of the experts is published in English and German language.

Acetabulum↗
PubMed1997

Mass determination, subunit organization and control of oligomerization states of keyhole limpet hemocyanin (KLH).

Analytical dark-field scanning transmission electron microscopy (STEM) of freeze-dried unstained specimens of keyhole limpet hemocyanin (KLH; from Megathura crenulata, a prosobranch gastropod) gave a molecular mass of 400 kDa for the subunit of KLH1 and of 345 kDa for the subunit of KLH2, which confirms our published values from SDS/PAGE. Within the 400-kDa KLH1 subunit we identified, by limited proteolysis, isolation of fragments and N-terminal sequencing, eight distinct 45-60 kDa functional domains (termed 1a through 1h) and determined their sequential arrangement. The KLH1 domains differ biochemically and immunologically from each other and from the previously characterized seven domains of KLH2 (termed 2a through 2g). Our partial amino acid sequences suggest that a domain, equivalent to the C-terminal domain 1h, is missing in KLH2. This deficiency is believed to be genuine and not an artifact of the subunit preparation procedure, since STEM measurements of the native didecamers yielded a mass difference of about 800 kDa between KLH1 and KLH2 (8.3 MDa versus 7.5 MDa), correlating with 20 copies of a functional 1h domain. It was also shown that the KLH1 didecamer can be rapidly split (minutes) into an almost homogeneous population of stable decamers by increasing the pH of the Tris/saline stabilizing buffer (routinely pH 7.4), which contains 5 mM CaCl2 and 5 mM MgCl2, to pH 8.5. Reformation of the didecamers occurred more slowly (days) upon dialysis against the pH 7.4 stabilizing buffer. Addition of 100 mM calcium and 100 mM magnesium ions to the pH 7.4 stabilizing buffer leads to the more rapid (overnight) formation of didecamers together with a significant number of previously unobserved KLH1 multidecamers, which could be structurally distinguished from the established multidecamers of KLH2.

Amino Acid Sequence↗
PubMed1997

The three-dimensional structure of aquaporin-1.

The entry and exit of water from cells is a fundamental process of life. Recognition of the high water permeability of red blood cells led to the proposal that specialized water pores exist in the plasma membrane. Expression in Xenopus oocytes and functional studies of an erythrocyte integral membrane protein of relative molecular mass 28,000, identified it as the mercury-sensitive water channel, aquaporin-1 (AQP1). Many related proteins, all belonging to the major intrinsic protein (MIP) family, are found throughout nature. AQP1 is a homotetramer containing four independent aqueous channels. When reconstituted into lipid bilayers, the protein forms two-dimensional lattices with a unit cell containing two tetramers in opposite orientation. Here we present the three-dimensional structure of AQP1 determined at 6A resolution by cryo-electron microscopy. Each AQP1 monomer has six tilted, bilayer-spanning alpha-helices which form a right-handed bundle surrounding a central density. These results, together with functional studies, provide a model that identifies the aqueous pore in the AQP1 molecule and indicates the organization of the tetrameric complex in the membrane.

Animals↗
PubMed1997

The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution.

The surfaces of two- and three-dimensional phi29 connector crystals were imaged in buffer solution by atomic force microscopy (AFM). Both topographies show a rectangular unit cell with dimensions of 16.5 nm x 16.5 nm. High resolution images of connectors from the two-dimensional crystal surface show two connectors per unit cell confirming the p42(1)2 symmetry. The height of the connector was estimated to be at least 7.6 nm, a value close to that found in previous studies using different techniques. The 12 subunits of the wide connector domain were clearly resolved and showed a right-handed vorticity. The channel running along the connector had a diameter of 3.7 nm in the wide domain, while it was 1.7 nm in the narrow domain end, thus suggesting a tronco-conical channel shape. Moreover, the narrow connector end appears to be rather flexible. When the force applied to the stylus was between 50 and 100 pN, the connector end was fully extended. At forces of approximately 150 pN, these ends were pushed towards the crystal surface. The complementation of the AFM data with the three-dimensional reconstruction obtained from electron microscopy not only confirmed the model proposed, but also offers new insights that may help to explain the role of the connector in DNA packing.

Bacillus Phages↗
PubMed1997

The Campylobacter jejuni porin trimers pack into different lattice types when reconstituted in the presence of lipid.

Purified major outer membrane protein of Campylobacter jejuni exhibited different classes of molecules by SDS/PAGE and immunoblotting. A high-molecular-mass product (120-140 kDa) was observed under mild conditions of solubilization, a folded monomeric form of 35 kDa was seen when treated at high SDS concentrations and finally, a single band around 45 kDa occurred when the sample was heated to 96 degrees C [Bolla, J. M., Loret, E., Zalewski. M. & Pages, J. M. (1995) J. Bacteriol. 177, 4266-4271]. The high-molecular-mass product was reconstituted into two-dimensional crystals in the presence of phospholipids and Mg2+. The C. jejuni porin required different conditions for successful reconstitution into two-dimensional crystals than the Escherichia coli porin OmpF. Electron microscopy and digital image processing of negatively stained specimens revealed a rectangular lattice with a unit cells size of a = 8.9 nm, b = 14.9 nm, an oblique lattice with a = 8.9 nm, b = 30.1 nm, gamma = 98 degrees, and a trigonal lattice with a = b = 9.6 nm. Projection maps were calculated to a resolution of 2 nm, and exhibited a trimeric protein with three stain-filled indentations.

Bacterial Outer Membrane Proteins↗
PubMed1997

[Effectiveness of selective hepatic artery embolization in a child after blunt hepatic trauma].

A 9-year-old boy was admitted after a bicycle fall. Abdominal CT-scan revealed severe liver injury (stage IV according to the liver injury scale of the American Association for Surgery Trauma), including ruptured intraparenchymal hematoma with active bleeding. The patient was hemodynamically stable and was treated conservatively for the first 2 days. On the 3rd day selective hepatic artery angiography was performed because of abdominal distension and the need for 7 pints of packed red blood cells. Active right hepatic artery bleeding was identified and treated successfully by embolization. We think that early angiography and selective embolization should always be considered for acute or continuous bleeding after liver injury.

Child↗
PubMed1997

Polymorphic fibrillar assembly of human amylin.

Human amylin forms fibrillar amyloid between pancreatic islet cells in patients with non-insulin-dependent (type 2) diabetes mellitus. Fibrillar assemblies also form in vitro in aqueous solutions of synthetic human amylin. We now report on the structural polymorphism of these fibrils. The thinnest fibril, referred to as the protofibril, has an apparent width of 5 nm but is only rarely observed by itself. These protofibrils spontaneously assemble into higher order fibrillar structures with distinct morphologies. Prominent among these is an 8-nm fibril with a distinct 25-nm axial crossover repeat which is formed by left-handed coiling of two 5-nm protofibrils. Coiling of more than two 5-nm protofibrils results in cable-like structures of variable width depending on the number of protofibrils involved. Lateral (side-by-side) assembly of 5-nm protofibrils is also observed and produces ribbons which may contain two, three, four, or more protofibrils and occasionally large single-layered sheets. The mass-per-length (MPL) of the 5-nm protofibril is 10 kDa/nm. This has been established in two ways: first, the 8-nm fibril, which is formed by coiling two 5-nm protofibrils around each other, has an MPL of 20 kDa/nm. Second, higher order fibrils differ by increments of 10 kDa/nm. Hence, about 2.6 human amylin molecules (3904 Da) are packed in 1 nm of protofibril length. Similarities exist between amylin fibrils and those formed from other amyloid proteins, suggesting that the in vitro assembly of synthetic protein may serve as a useful model system in advancing our understanding of amyloid formation in disease.

Amyloid↗
PubMed1997

Adsorption of biological molecules to a solid support for scanning probe microscopy.

Scanning probe microscopes are now established tools to study the surface structure of biological macromolecules under physiological conditions. Sample preparation methods for this microscopy all have the objective to attach the specimen firmly to a support. Here we analyse the commonly used method of adsorbing biological specimens to freshly cleaved mica. This is facilitated by adjusting the electrolyte concentration and the pH of the buffer solution. Native macromolecular systems absorbed to mica in this way can be reproducibly imaged at submolecular resolution.

Adsorption↗
PubMed1997

Structural changes in native membrane proteins monitored at subnanometer resolution with the atomic force microscope: a review.

Three membrane proteins, OmpF porin from Escherichia coli, bacteriorhodopsin from Halobacterium salinarium, and the hexagonally packed intermediate (HPI) layer from Deinoccocus radiodurans, were investigated with the atomic force microscope in buffer solution. A resolution of up to 0.8 nm allowed structural differences of individual proteins to be detected. OmpF porin exhibits different static conformations on the outer surface, which possibly represent the two conductive states of the ion channels. Reversible structural changes in the cytoplasmic surface of purple membrane have been induced by changing the force applied to the scanning stylus: doughnut-shaped bacteriorhodopsin trimers transformed into a structure with three pronounced protrusions when the force was reduced from 300 to 100 pN. Furthermore, individual pores of the inner surface of the HPI layer were observed to switch from an "open" to a "closed" state. Together, the structural changes in proteins monitored under physiological conditions suggest that direct observation of function-related conformational changes of biomolecules with the atomic force microscope is feasible.

Bacterial Outer Membrane Proteins↗