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Biomedical subjects

A Engel

Publications and source records attributed to A Engel.

At least 55 records · Page 3Linked to original sources
PubMed2000

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle.

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.

Animals↗
PubMed2000

The human Rad52 protein exists as a heptameric ring.

The RAD52 epistasis group was identified in yeast as a group of genes required to repair DNA damaged by ionizing radiation [1]. Genetic evidence indicates that Rad52 functions in Rad51-dependent and Rad51-independent recombination pathways [2] [3] [4]. Consistent with this, purified yeast and human Rad52 proteins have been shown to promote single-strand DNA annealing [5] [6] [7] and to stimulate Rad51-mediated homologous pairing [8] [9] [10] [11]. Electron microscopic examinations of the yeast [12] and human [13] Rad52 proteins have revealed their assembly into ring-like structures in vitro. Using both conventional transmission electron microscopy and scanning transmission electron microscopy (STEM), we found that the human Rad52 protein forms heptameric rings. A three-dimensional (3D) reconstruction revealed that the heptamer has a large central channel. Like the hexameric helicases such as Escherichia coli DnaB [14] [15], bacteriophage T7 gp4b [16] [17], simian virus 40 (SV40) large T antigen [18] and papilloma virus E1 [19], the Rad52 rings show a distinctly chiral arrangement of subunits. Thus, the structures formed by the hexameric helicases may be a more general property of other proteins involved in DNA metabolism, including those, such as Rad52, that do not bind and hydrolyze ATP.

Animals↗
PubMed2000

The importance of aquaporin water channel protein structures.

The history of the water channel and recent structural and functional analyses of aquaporins are reviewed. These ubiquitous channels are important for bacteria, plants and animals, exhibit a pronounced sequence homology and share functional as well as structural similarities. Aquaporins allow water or small specific solutes to pass unhindered, but block the passage of ions to prevent dissipation of the transmembrane potential. Besides advances in structure determination, recent experiments suggest that many of these channels are regulated by pH variations, phosphorylation and binding of auxiliary proteins.

Animals↗
PubMed2000

SecYEG assembles into a tetramer to form the active protein translocation channel.

Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an approximately 5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocase-precursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes.

Bacterial Proteins↗
PubMed2000

In vitro senescence models for human T lymphocytes.

Immunosenescence is an age-associated dysregulation of immune function which may contribute to the increased susceptibility of the elderly to infectious disease. Although age-associated changes are measurable in the innate immune system, it is the adaptive arm of the immune system which is particularly susceptible to the deleterious effects of ageing, especially the T cell compartment. In this review, the characteristics of longitudinal ageing in cultured monoclonal human T cell populations will be summarized. It will be argued that parallels between this in vitro model and T cell senescence in vivo suggest the use of such models to screen for interventions ameliorating immunosenescence in vivo.

Adult↗
PubMed2000

Structural clues in the sequences of the aquaporins.

The large number of sequences available for the aquaporin family represents a valuable source of information to incorporate into three-dimensional structure determination. Phylogenetic analysis was used to define type sequences to avoid extreme over-representation of some subfamilies, and as a measure of the quality of multiple sequence alignment. Inspection of the sequence alignment suggested eight conserved segments that define the core architecture of six transmembrane helices and two functional loops, B and E, projecting into the plane of the membrane. The sum of the core segments and the minimum lengths of the interlinking loops constitute the 208 residues necessary to satisfy the aquaporin architecture. Analysis of hydrophobic and conservation periodicity and of correlated mutations across the alignment indicated the likely assignment and orientation of the helices in the bilayer. This assignment is examined with respect to the structure of the erythrocyte aquaporin 1 determined by electron crystallography. The aquaporin 1 tetramer is described as three rings of helices, each ring with a different exposure to the lipid environment. The sequence analysis clearly suggests that two helices are exposed along their whole lengths, two helices are exposed only at their N termini, and two helices are not exposed to lipid. It is further proposed that, besides loops B and E, the highly conserved motifs on helices 1 and 4, ExxxTxxF/L, could line the water channel.

Amino Acid Sequence↗
PubMed2000

The 6.9-A structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli.

The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy. The 6.9-A density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel. While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction. Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel. Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side. This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity.

Aquaporin 1↗
PubMed2000

[Effect of bFGF on regeneration of distracted mandibles after radiation].

The potential of distraction osteogenesis in mandibular reconstruction has been limited by its questionable efficacy in previously irradiated bone. The possible osteogenetic effect of recombinant human basic fibroblast growth factor (bFGF) on lengthening of irradiated mandibles was investigated in beagle dogs. We studied nine adult dogs which underwent a full course of external beam radiation therapy (60 Gy/30 fractions). Six months after completion of radiotherapy, the molars were extracted bilaterally followed by bone lengthening of the mandible using an intraoral device. On postoperative day 3 and 7 we injected 10 micrograms bFGF into the osteotomy site of each right hemimandible. The left sides were used as controls. The time course in ossification of the distracted area was evaluated at 2, 4, and 6 weeks after completion of bone lengthening. The radiographs of the newly formed bone tissue were measured by digital image analysis. Corresponding to the radiographic findings, the histological examination of the removed jaws showed an earlier and more intensive bone formation in the treated side after 2, 4, and 6 weeks compared to the control side. We conclude that bFGF promotes the ossification of distracted mandibles after radiation therapy in dogs.

Adult↗
PubMed2000

[The value of core decompression in treatment of femur head necrosis].

Core decompression of the necrotic area for treatment of idiopathic osteonecrosis of the femoral head was developed and published by Ficat and Arlet in 1962 within the scope of their "Functional exploration of bone". The mode of action is attributed to a reduction of the intramedullary pressure in the bony compartment of the femoral head. The possibilities of repair and bone regeneration following core decompression are still discussed controversially. Core decompression is a common but not generally accepted procedure in the treatment of idiopathic osteonecrosis of the femoral head. After first publications of positive mid- and long-term effects, some subsequent studies judged it as an ineffective and high-risk method. Analysis of the literature shows that the effectiveness of core decompression depends on the stage of osteonecrosis at the time of surgical intervention. Prognosis is influenced by the extent and location of the necrotic area, the presence and amount of head depression, and continued risk factors--mainly corticoid medication. The best prognosis can be given for patients with a small, medial-centrally located necrosis without head depression. The classification according to Ficat appears to be insufficient, as the extent and localization of the necrotic area are not assessed. Magnetic resonance imaging has become a diagnostic gold standard, as radiographic diagnosis showed poor sensitivity and specificity, especially in the early stages of the disease. As an essential part, MRI was integrated into the new classification of the "Association Internationale de Recherche sur la Circulation Osseuse" (ARCO). On account of the literature and our own experience, treatment by core decompression can be recommended in cases of reversible early stages of osteonecrosis (ARCO 1), as well as in those cases of irreversible early stages (ARCO 2) that show a medial or central location of the necrosis with an extent of less than 30% of the femoral head. Once the disease reaches the irreversible early stage, complete recovery cannot be expected. In these cases only reduction of pain and retardation of the natural course of the osteonecrosis are possible to gain time until total hip replacement is unavoidable.

Decompression, Surgical↗
PubMed2000

Loss of E2F4 activity leads to abnormal development of multiple cellular lineages.

We have generated mice deficient in E2F4 activity, the major form of E2F in many cell types. Analysis of newborn pups deficient in E2F4 revealed abnormalities in hematopoietic lineage development as well as defects in the development of the gut epithelium. Specifically, we observed a deficiency of various mature hematopoietic cell types together with an increased number of immature cells in several lineages. This was associated with an increased frequency of apoptotic cells. We also found a substantial reduction in the thickness of the gut epithelium that normally gives rise to crypts as well as a reduction in the density of villi. These observations suggest a critical role for E2F4 activity in controlling the maturation of cells in a number of tissues.

Abnormalities, Multiple↗
PubMed2000

Rapid fold and structure determination of the archaeal translation elongation factor 1beta from Methanobacterium thermoautotrophicum.

The tertiary fold of the elongation factor, aEF-1beta, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1beta was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1beta structure revealed close similarity to its human analogue, eEF-1beta. In agreement with studies on EF-Ts and human EF-1beta, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1alpha. aEF-1beta was also found to bind calcium in the groove between helix alpha2 and strand beta4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.

Archaeal Proteins↗
PubMed2000

Observing single biomolecules at work with the atomic force microscope.

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.

Animals↗
PubMed2000

Prospective screening by a panfungal polymerase chain reaction assay in patients at risk for fungal infections: implications for the management of febrile neutropenia.

Invasive fungal infections are a major cause of mortality in neutropenic cancer patients. To determine whether a polymerase chain reaction (PCR)-based assay enabled the identification of patients at risk for invasive fungal infections, a prospective monitoring once per week was performed during 92 neutropenic episodes in patients receiving chemotherapy for acute leukaemia or high-dose therapy followed by allogeneic or autologous stem cell transplantation, with the investigators blinded to clinical and microbiological data. PCR positivity was documented in 34 out of 92 risk episodes. All patients developing proven invasive fungal infection were found PCR positive, and PCR was found to be the earliest indicator of invasive fungal infection preceding clinical evidence by a mean of 5.75 d (range 0-14 d). In febrile neutropenic patients without a prior history of invasive fungal infection, a sensitivity of 100% and a specificity of 73% of the PCR assay for the development of proven or probable invasive fungal infection was documented. In conclusion, panfungal PCR performed prospectively once a week enabled the identification of patients at high risk for invasive fungal infections.

Acute Disease↗
PubMed2000

The 3.7 A projection map of the glycerol facilitator GlpF: a variant of the aquaporin tetramer.

GlpF, the glycerol facilitator protein of Escherichia coli, is an archetypal member of the aquaporin superfamily. To assess its structure, recombinant histidine-tagged protein was overexpressed, solubilized in octylglucoside and purified to homogeneity. Negative stain electron microscopy of solubilized GlpF protein revealed a tetrameric structure of approximately 80 A side length. Scanning transmission electron microscopy yielded a mass of 170 kDa, corroborating the tetrameric nature of GlpF. Reconstitution of GlpF in the presence of lipids produced highly ordered two-dimensional crystals, which diffracted electrons to 3.6 A resolution. Cryoelectron microscopy provided a 3.7 A projection map exhibiting a unit cell comprised of two tetramers. In projection, GlpF is similar to AQP1, the erythrocyte water channel. However, the major density minimum within each monomer is distinctly larger in GlpF than in AQP1.

Aquaporins↗
PubMed2000

Hypoplasia of the inferior vena cava with azygous continuation presenting as recurrent leg deep vein thrombosis.

Azygous continuation of the inferior vena cava is a rare congenital malformation. It occurs because of atresia of the retrohepatic segment of the inferior vena cava. The disorder has been described mainly as an incidental finding associated with a number of congenital malformations, including cardiovascular, tracheobronchial, and other visceral malformations. Therapeutic intervention is aimed primarily at accompanying malformations. We report a case of hypoplasia of the inferior vena cava with azygous continuation in a 30-year-old man who presented with recurrent deep venous thrombosis.

Adult↗