PubMed Health⌕ Search

Biomedical subjects

A Engel

Publications and source records attributed to A Engel.

At least 37 records · Page 2Linked to original sources
PubMed2003

Membrane protein reconstitution and crystallization by controlled dilution.

Efficient reconstitution of membrane proteins for functional analyses can be achieved by dilution of a ternary mixture containing proteins, lipids and detergents. Once the dilution reaches the point where the free detergent concentration would become lower than the critical micellar concentration, detergent is recruited from the bound detergent pool, and association of proteins and lipids is initiated. Here we show that dilution is also suitable for the assembly of two-dimensional crystals. A device has been designed that allows controlled dilution of a protein-lipid-detergent mixture to induce formation of densely packed or crystalline proteoliposomes. Turbidity is used to monitor the progress of reconstitution on-line, while dilution is achieved by computer-controlled addition of buffer solution in sub-microliter steps. This system has mainly been tested with porin OmpF, a typical beta-barrel protein, and aquaporin-1, a typical alpha-helical protein. The results demonstrate that large, highly ordered two-dimensional crystals can be produced by the dilution method.

Aquaporin 1↗
PubMed2003

[Excessive hyperferritinemia as an indication of a reactive hemophagocytosis syndrome].

HISTORY AND ADMISSION FINDINGS: A 17-year-old girl with a history of a polyarthritis of unknown etiology was admitted because of acute fever and general weakness. There were palpable cervical lymph nodes and her body temperature was 39.5 degrees C. INVESTIGATIONS: GOT was raised to 282 U/1, GPT to 266 U/l lactate dehydrogenase to 1275 U/I and bilirubin to 0.6 mg/dl. The Quick value was 67%, albumin 28 mg/dl. White cell count was decreased to 1700/microl, with 43% granulocytes, 39% lymphocytes, 17% monocytes. Platelet count was 64,000/microl. Ultrasound revealed splenomegaly. Ferritin was markedly raised to 11,860 ng/ml (normal up to 150 ng/ml). An epstein-barr-virus infection was found. THERAPY AND CLINICAL COURSE: Suspecting a reactive hemophagocytosis syndrome, she was treated with prednisolone (2 mg/kg). The diagnosis was confirmed by a bone marrow aspirate. The patient's condition and laboratory values improved rapidly. CONCLUSION: Markedly increased ferritin levels in a clinically septic patient with an underlying rheumatic disease indicates a hemophagocytotic syndrome. High dosage steroid should be started before there is biopsy confirmation of the disease.

Adolescent↗
PubMed2003

Atomic force bio-analytics.

The atomic force microscope (AFM) allows biomolecules to be observed and manipulated under native conditions. It operates in buffer solution, produces molecular images with outstanding signal-to-noise ratio, and addresses single molecules. Progress in sample preparation and instrumentation has led to topographs that reveal sub-nanometer details and surface dynamics of biomolecules. Antibodies or oligonucleotides immobilized on cantilevers induce bending upon binding of the cognate biomolecule, allowing sub-picomolar concentrations to be measured. Biomolecules tethered between support and retracting AFM-tip produce force extension curves that reflect the mechanical stability of secondary structure elements. Furthermore, multifunctional tips may activate single molecules to observe them at work. In all cases, the cantilever is critical: its mechanical properties dictate the force-sensitivity and the scanning speed.

Microscopy, Atomic Force↗
PubMed2003

Removal of the surface layers of human cortical bone allografts restores in vitro osteoclast function reduced by processing and frozen storage.

A major complication of cortical bone grafting is nonunion at the host-graft junction. Many factors are thought to be involved in successful engraftment including the quality of the graft and the host response to it. In particular, the recipient osteoclasts (OCs) play a critical role by resorbing the engrafted bone. Thus, effective engraftment may depend on the inherent biological properties of the bone graft, which subsequently correlates with early and effective OC resorption. Normally, bone grafts are stored and processed by freezing, freeze-drying, irradiation, and lipid extraction. We sought to determine whether processing and storage affected bone quality, as evaluated by OC bone resorption. Cortical bone specimens from six human donors were either fresh, frozen at -75 degrees C, or had undergone combinations of freezing at -75 degrees C, freeze-drying, lipid extraction, irradiation, and treatment with hydrogen peroxide. Bone slices of 0.5-mm thickness taken from the surface, beneath the surface, and at a depth of 7.5 mm were incubated with isolated rabbit OCs and resorption lacunae were measured. We observed highest OC activity with fresh bone followed by frozen, partially processed, and fully processed bone. When allografts were stored at -75 degrees C for 12 months, there was up to a 4.2-fold reduction in OC activity on the surface layer. Additionally, we found reduced OC activity upon the outer surface bone compared to the inner layers. Removal of more than 0.5 mm of frozen and processed bone significantly improved OC activity. These results imply that inner bone layers of stored and processed bone allografts are protected against degradation of bone matrix components, except when frozen for extended periods of time. Taken together, these data suggest that bone allografts should be stored for less than 1 year and require the removal of at least 0.5 mm from their surface prior to transplantation.

Animals↗
PubMed2002

Progress in the analysis of membrane protein structure and function.

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two-dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to-noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at sub-nanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress.

Crystallization↗
PubMed2002

Arterial occlusion after total knee arthroplasty: successful management of an uncommon complication by percutaneous thrombus aspiration.

Management of arterial occlusion after total knee arthroplasty is controversial in the literature, in part because of the relatively small number of reported cases. We report a case of perioperative popliteal artery thrombosis after total knee arthroplasty that was treated successfully with percutaneous thrombus aspiration and balloon dilation. Immediate intervention prevented ischemic muscular necrosis, resulting in full recovery of the limb.

Aged↗
PubMed2002

The SCARF osteotomy for the correction of hallux valgus deformities.

The authors report their experience with a modified SCARF osteotomy with three years follow-up. Correction of moderate to severe hallux valgus deformities was achieved using a Z step osteotomy cut to realign the first metatarsal bone. A retrospective analysis was undertaken in 89 consecutive patients (111 feet). Results were analyzed by clinical examination, a questionnaire including the AOFAS forefoot score, and plain roentgenograms. Hallux valgus and intermetatarsal angle improved at mean 19.1 degrees and 6.6 degrees, respectively. Mean forefoot score improved from 50.1 to 91 points out of 100 possible points. Satisfactory healing time was expressed by an average return back to work of 5.8 weeks and back to sport of 8.3 weeks. Persistence or recurrence of hallux valgus was seen in seven patients (6%). The complication rate was 5.4% including superficial wound infection, traumatic dislocation of the distal fragment, and hallux limitus. The presented technique provides predictable correction of moderate to severe hallux valgus deformities.

Adult↗
PubMed2001

Large-scale purification of functional recombinant human aquaporin-2.

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Amino Acid Sequence↗
PubMed2001

ATP synthase: constrained stoichiometry of the transmembrane rotor.

Recent structural data suggest that the number of identical subunits (c or III) assembled into the cation-powered rotor of F1F0 ATP synthase depends on the biological origin. Atomic force microscopy allowed individual subunits of the cylindrical transmembrane rotors from spinach chloroplast and from Ilyobacter tartaricus ATP synthase to be directly visualized in their native-like environment. Occasionally, individual rotors exhibit structural gaps of the size of one or more subunits. Complete rotors and arch-shaped fragments of incomplete rotors revealed the same diameter within one ATP synthase species. These results suggest the rotor diameter and stoichiometry to be determined by the shape of the subunits and their nearest neighbor interactions.

ATP Synthetase Complexes↗
PubMed2001

A refined structure of human aquaporin-1.

A refined structure of the human water channel aquaporin-1 is presented. The model rests on the high resolution X-ray structure of the homologous bacterial glycerol transporter GlpF, electron crystallographic data at 3.8 A resolution and a multiple sequence alignment of the aquaporin superfamily. The crystallographic R and free R values (36.7% and 37.8%) for the refined structure are significantly lower than for previous models. Improved geometry and enhanced stability in molecular dynamics simulations demonstrate a significant improvement of the aquaporin-1 structure. Comparison with previous aquaporin-1 models shows significant differences, not only in the loop regions, but also in the core of the water channel.

Aquaporin 1↗
PubMed2001

Two-dimensional crystals: a powerful approach to assess structure, function and dynamics of membrane proteins.

Electron crystallography and atomic force microscopy allow the study of two-dimensional membrane protein crystals. While electron crystallography provides atomic scale three-dimensional density maps, atomic force microscopy gives insight into the surface structure and dynamics at sub-nanometer resolution. Importantly, the membrane protein studied is in its native environment and its function can be assessed directly. The approach allows both the atomic structure of the membrane protein and the dynamics of its surface to be analyzed. In this way, the function-related conformational changes can be assessed, thus providing a detailed insight on the molecular mechanisms of essential biological processes.

Cell Membrane↗
PubMed2001

Multilayer neural networks with extensively many hidden units.

The information processing abilities of a multilayer neural network with a number of hidden units scaling as the input dimension are studied using statistical mechanics methods. The mapping from the input layer to the hidden units is performed by general symmetric Boolean functions, whereas the hidden layer is connected to the output by either discrete or continuous couplings. Introducing an overlap in the space of Boolean functions as order parameter, the storage capacity is found to scale with the logarithm of the number of implementable Boolean functions. The generalization behavior is smooth for continuous couplings and shows a discontinuous transition to perfect generalization for discrete ones.

Models, Statistical↗
PubMed2001

Pattern and wave number selection in magnetic fluids.

The formation of patterns of peaks on the free surface of a magnetic fluid subject to a magnetic field normal to the undisturbed interface is investigated theoretically. The relative stability of ridge, square, and hexagon planforms is studied using a perturbative energy minimization procedure. Extending previous studies the finite depth of the fluid layer is taken into account. Moreover, adding the wave number modulus k to the set of variational parameters also the wave number selection problem is addressed. The results are compared with previous investigations and recent experimental findings.

Journal Article↗
PubMed2001

High-resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2.

Light-harvesting complexes 2 (LH2) are the accessory antenna proteins in the bacterial photosynthetic apparatus and are built up of alphabeta-heterodimers containing three bacteriochlorophylls and one carotenoid each. We have used atomic force microscopy (AFM) to investigate reconstituted LH2 from Rubrivivax gelatinosus, which has a C-terminal hydrophobic extension of 21 amino acids on the alpha-subunit. High-resolution topographs revealed a nonameric organization of the regularly packed cylindrical complexes incorporated into the membrane in both orientations. Native LH2 showed one surface which protruded by approximately 6 A and one that protruded by approximately 14 A from the membrane. Topographs of samples reconstituted with thermolysin-digested LH2 revealed a height reduction of the strongly protruding surface to approximately 9 A, and a change of its surface appearance. These results suggested that the alpha-subunit of R.gelatinosus comprises a single transmembrane helix and an extrinsic C-terminus, and allowed the periplasmic surface to be assigned. Occasionally, large rings ( approximately 120 A diameter) surrounded by LH2 rings were observed. Their diameter and appearance suggest the large rings to be LH1 complexes.

Amino Acid Sequence↗
PubMed2001

The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides.

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA(+) proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg(2+) and GTP, GDP or GTP-gamma-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB(L) and McrB(S) form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.

Bacterial Proteins↗
PubMed2001

Molecular mass, stoichiometry, and assembly of 20 S particles.

N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.

Amino Acid Sequence↗
PubMed2001

The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities.

Voltage-sensitive membrane channels, the sodium channel, the potassium channel and the calcium channel operate together to amplify, transmit and generate electric pulses in higher forms of life. Sodium and calcium channels are involved in cell excitation, neuronal transmission, muscle contraction and many functions that relate directly to human diseases. Sodium channels--glycosylated proteins with a relative molecular mass of about 300,000 (ref. 5)--are responsible for signal transduction and amplification, and are chief targets of anaesthetic drugs and neurotoxins. Here we present the three-dimensional structure of the voltage-sensitive sodium channel from the eel Electrophorus electricus. The 19 A structure was determined by helium-cooled cryo-electron microscopy and single-particle image analysis of the solubilized sodium channel. The channel has a bell-shaped outer surface of 135 A in height and 100 A in side length at the square-shaped bottom, and a spherical top with a diameter of 65 A. Several inner cavities are connected to four small holes and eight orifices close to the extracellular and cytoplasmic membrane surfaces. Homologous voltage-sensitive calcium and tetrameric potassium channels, which regulate secretory processes and the membrane potential, may possess a related structure.

Animals↗