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K Gast

Publications and source records attributed to K Gast.

At least 19 recordsLinked to original sources
PubMed2006

On the self-association potential of transmembrane tight junction proteins.

Tight junctions seal intercellular clefts via membrane-related strands, hence, maintaining important organ functions. We investigated the self-association of strand-forming transmembrane tight junction proteins. The regulatory tight junction protein occludin was differently tagged and cotransfected in eucaryotic cells. These occludins colocalized within the plasma membrane of the same cell, coprecipitated and exhibited fluorescence resonance energy transfer. Differently tagged strand-forming claudin-5 also colocalized in the plasma membrane of the same cell and showed fluorescence resonance energy transfer. This demonstrates self-association in intact cells both of occludin and claudin-5 in one plasma membrane. In search of dimerizing regions of occludin, dimerization of its cytosolic C-terminal coiledcoil domain was identified. In claudin-5, the second extracellular loop was detected as a dimer. Since the transmembrane junctional adhesion molecule also is known to dimerize, the assumption that homodimerization of transmembrane tight junction proteins may serve as a common structural feature in tight junction assembly is supported.

Animals↗
PubMed2005

[Does HRCT-emphysema index represent the entire lung?].

PURPOSE: : Comparison of emphysema index derived of thin section MD-CT of the entire lung volume and HRCT, simulated by calculation of every twentieth image of the whole data-set. MATERIALS AND METHODS: Pulmonary emphysema was quantified by semiautomatic, segmentation of lung borders and assessment of lung volume and emphysema volume within these borders. The emphysema index (pixel index) was calculated. Statistical analysis was done by the sign-test and Bland-Altman-analysis. RESULTS: Median lung volume, emphysema volume and emphysema index are significantly higher in simulated HRCT. Median lung volume (emphysema volume) calculated by HRCT is 5118 ml (407 ml) and 5040 ml (367 ml) calculated by the entire MD-CT data-set, representing differences of 1 and 8 % related to the median lung and emphysema volumes. Emphysema index is 0.09 (HRCT) and 0.08 (MD-CT). CONCLUSION: HRCT overrates emphysema index compared to thin section MD-CT of the entire lung volume.

Adolescent↗
PubMed2003

Effect of environmental conditions on aggregation and fibril formation of barstar.

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-beta structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state-->fibril type I-->fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.

Amyloid↗
PubMed2003

Assembly of amyloid protofibrils via critical oligomers--a novel pathway of amyloid formation.

The amyloid formation of phosphoglycerate kinase (PGK) was investigated by static and dynamic light-scattering. The time-course of the scattering intensity and the hydrodynamic radius scale with initial monomer concentration in a linear fashion over a range of about 50 in concentration. This sets limits on theories for aggregation kinetics that can be used, and points towards irreversible, cascade type models. In addition, circular dichroism (CD) was used to monitor the transition between a predominantly alpha-helical spectrum to a beta-sheet enriched one. The time-course of the CD also proves to scale linearly with initial monomer concentration. Electron microscopy shows that small oligomers as well as protofibrils are present during aggregation. The found coupling between growth of intermediates and acquisition of beta-sheet structure is interpreted in terms of a generalized diffusion-collision model, where stabilization of beta-strands takes place by intermolecular interactions.

Amyloid↗
PubMed2002

3He-MRI-based vs. conventional determination of lung volumes in patients after unilateral lung transplantation: a new approach to regional spirometry.

BACKGROUND: To use 3Helium (3He)-MRI in patients with unilateral lung grafts to assess the contributions of graft and native lung to total ventilated lung volume, and second to compare conventional measurements of intrapulmonary gas volume (spirometry, body plethysmography) with image-based volumetry of ventilated lung parenchyma visualized by hyperpolarized 3He-MRI. METHODS: With Ethics Committee approval, five patients with single lung transplantation (SLTX) for idiopathic pulmonary fibrosis (IPF) underwent both conventional pulmonary function testing (PFT) and 3He-MRI of the lung. Intrapulmonary gas volume (GV) during the inspiratory breathhold for 3He-MRI was calculated from measured functional residual capacity (corrected for supine position) and inspired tidal volume. Image-based global and regional lung volumetries (LV) were performed in three-dimensionally reconstructed 3He-MR images (corrected for the fraction of tissue and blood). RESULTS: Transplanted lungs were characterized by a homogeneous distribution of signal intensity, whereas the native lungs of the patients suffering from IPF displayed an inhomogeneous signal distribution pattern with numerous round or wedge-shaped ventilation defects. Total ventilated lung volume determined by 3He-MRI correlated well with PFT-based measurements, but with a systematic overestimation of the 3He-based lung volumetry of approximately 20%. Functioning lung grafts contributed 66+/-6% and their corresponding native IPF lungs 34+/-6% to total ventilated volume (P<0.05; mean+/-SD). CONCLUSION: 3Helium-MRI of the lung offers a novel approach to regional determination of ventilated lung volume, including its blood and tissue compartments. The advantage of this technique over computed tomography or ventilation scintigraphy is the lack of radiation exposure, and hence its repeatability. Follow up of SLTX patients with this new technique may allow the monitoring of functional and structural developments of grafted lungs with better sensitivity and specificity than PFT.

Female↗
PubMed2001

Study of the conformational transition of A beta(1-42) using D-amino acid replacement analogues.

A critical event in Alzheimer's disease is the transition of Abeta peptides from their soluble forms into disease-associated beta-sheet-rich conformers. Structural analysis of a complete D-amino acid replacement set of Abeta(1-42) enabled us to localize in the full-length 42-mer peptide the region responsible for the conformational switch into a beta-sheet structure. Although NMR spectroscopy of trifluoroethanol-stabilized monomeric Abeta(1-42) delineated two separated helical domains, only the destabilization of helix I, comprising residues 11-24, caused a transition to a beta-sheet structure. This conformational alpha-to-beta switch was directly accompanied by an aggregation process leading to the formation of amyloid fibrils.

Amino Acid Sequence↗
PubMed2001

Fluoroalcohol-induced structural changes of proteins: some aspects of cosolvent-protein interactions.

The conformational transitions of bovine beta-lactoglobulin A and phosphoglycerate kinase from yeast induced by hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have been studied by dynamic light scattering and circular dichroism spectroscopy in order to elucidate the potential of fluoroalcohols to bring about structural changes of proteins. Moreover, pure fluoroalcohol-water mixed solvents were investigated to prove the relation between cluster formation and the effects on proteins. The results demonstrate that cluster formation is mostly an accompanying phenomenon because important structural changes of the proteins occur well below the critical concentration of fluoroalcohol at which the formation of clusters sets in. According to our light scattering experiments, the remarkable potential of HFIP is a consequence of extensive preferential binding. Surprisingly, preferential binding seems to play a vanishing role in the case of TFE. However, the comparable Stokes radii of both proteins in the highly helical state induced by either HFIP or TFE point to a similar degree of solvation in both mixed solvents. This shows that direct binding or an indirect mechanism must be equally taken into consideration to explain the effects of alcohols on proteins. The existence of a compact helical intermediate with non-native secondary structure on the transition of beta-lactoglobulin A from the native to the highly helical state is clearly demonstrated.

Animals↗
PubMed2001

[Dynamic imaging of the nasal cavity and the paranasal sinuses with polarized 3helium MRI].

PURPOSE: Reduced or blocked ventilation of the paranasal sinuses is probably the most important factor in the development of sinusitis. Recently, the use of optically polarized noble gas isotopes has attracted increasing interest for use in a variety of promising MR applications. The aim of this study was to test the feasibility of imaging and visualization ventilation of the nasal cavity and paranasal sinus in MR by inhalation of hyperpolarized (3)helium. The goal was to evaluate ventilation defects of the paranasal sinuses. VOLUNTEERS AND METHODS: Three volunteers were enrolled in the study. (3)Helium was polarized to 40 - 50 % by direct optical pumping. 300 ml of 100 % (3)helium were administered in the left nasal vestibule through a glass tube. With a closed contralateral nasal vestibule, the Valsalva maneuver was performed twice. Using a dedicated application unit, which is also used in MR imaging of the lung, an exact amount of (3)helium gas was administered at the beginning of inspiration. Measurements were carried out on a clinical 1.5 T scanner. Coronal images of the nasal cavity and paranasal sinuses were acquired using ultrafast gradient-echo pulse sequence (TR = 2 ms, TE = 0.7 ms, FA < 2 degrees, 75 x 128, FOV = 500) with an image aquisition time of 130 ms. RESULTS: The oral cavity and nasal cavities display a very high signal intensity after inhalation of polarized (3)helium gas. The signal intensity in the left maxillary sinus was higher compared to the right one. The mean signal intensity on the left side was 526 +/- 86 and on the right side 336 +/- 102. The left and right frontal sinus and ethmoid sinus only show signal of hyperpolarized (3)helium after two Valsalva maneuvers. Because of the low signal intensity of the frontal and ethmoid cells their visualization was incomplete. The signal to noise ratio was 14.1 for the left maxillary sinus, 8.9 for the right side, 6.3 for the left ethmoid sinus, 5.8 for the right side and 6.6 for the left frontal sinus and 7.8 for the right side. CONCLUSION: (3)Helium MR allows imaging of the nasal cavity and the paranasal sinuses. Perhaps this method could be a new tool to visualize the ventilation of the maxillary sinus without ionizing radiation. Interpretations about the ventilation of the frontal and ethmoid cells remain speculative.

Administration, Inhalation↗
PubMed2000

Conversion of yeast phosphoglycerate kinase into amyloid-like structure.

Yeast phosphoglycerate kinase is a structurally well-characterized enzyme consisting of 415 amino acids without disulfide bonds. Anion-induced refolding from its acid-unfolded state gives rise to the formation of worm-like amyloid fibrils with a persistence length of 73 nm. Electron microscopy and small-angle X-ray scattering data indicate that the fibrils have an elliptical cross-section with dimensions of 10.2 nm x 5.1 nm. About half of all amino acids are organized in form of cross-beta structure which gives rise to typical infrared spectra, X-ray diffraction and yellow-green birefringence after Congo red staining. The kinetics of amyloid formation, monitored by infrared spectroscopy, dynamic light scattering and X-ray scattering, was found to be strongly dependent on protein concentration. The infrared data indicate that the formation of cross-beta structure practically comes to an end already after some hours, whereas the length-growth of the amyloid fibrils, monitored by small-angle X-ray scattering, was not yet completed after 1,300 hours.

Amyloid↗
PubMed1999

Proteins can adopt totally different folded conformations.

The three-dimensional structure of a protein is determined by interactions between its amino acids and by interactions of the amino acids with molecules of the environment. The great influence of the latter interactions is demonstrated for the enzyme phosphoglycerate kinase from yeast (PGK). In the native state, PGK is a compact, bilobal molecule; 35% and 13% of its amino acids are organised in the form of alpha-helices and beta-sheets, respectively. The molecules unfold at acidic pH and low ionic strength forming random-walk structures with a persistence length of 3 nm. More than 90% of the amino acid residues of the ensemble have phi,psi-angles corresponding to those of a straight beta-chain. Upon addition of 50% (v/v) trifluoroethanol to the acid-unfolded protein, the entire molecule is transformed into a rod-like, flexible alpha-helix. Addition of anions, such as chloride or trichloroacetate, to the acid-unfolded protein leads to the formation of amyloid-like fibres over a period of many hours when the anion concentration exceeds a critical limit. Half of the amino acid residues are then organised in beta-sheets. Both of the non-natively folded states of PGK contain more regular secondary structure than the native one. The misfolding starts in both cases from the acid-unfolded state, in which the molecules are essentially more expanded than in other denatured states, e.g. those effected by temperature or guanidine hydrochloride.

Anions↗
PubMed1999

Secondary structure and oligomerization behavior of equilibrium unfolding intermediates of the lambda cro repressor.

The thermal unfolding of the wild-type Cro repressor, its disulfide-bridged mutant Cro-V55C (with the Val-55 --> Cys single amino acid substitution), and a CNBr-fragment (13-66)2 of Cro-V55C was studied by Fourier transform infrared spectroscopy and dynamic light scattering. The combined approach reveals that thermal denaturation of Cro-WT and Cro-V55C proceeds in two steps through equilibrium unfolding intermediates. The first thermal transition of the Cro-V55C dimer involves the melting of the alpha-helices and the short beta-strand localized in the N-terminal part of the molecule. This event is accompanied by the formation of tetramers, and also impacts on the hydrogen-bonding interactions of the C-terminal beta-strands. The beta-sheet formed by the C-terminal parts of each polypeptide chain is the major structural feature of the intermediate state of Cro-V55C and unfolds during a second thermal transition, which is accompanied by the dissociation of the tetramers. Cutting of 12 amino acids in the N-terminal region is sufficient to prevent the formation of alpha-helical structure in the CNBr-fragment of Cro-V55C, and to induce tetramerization already at room temperature. The tetramers may persist over a broad temperature range, and start to dissociate only upon thermal unfolding of the beta-sheet structure formed by the C-terminal regions. The wild-type protein is a dimer at room temperature and at protein concentrations of 1.8-5.8 mg/mL. At lower concentrations, the dimers are stable until the onset of thermal unfolding, which is accompanied by the dissociation of the dimers into monomers. At higher protein concentrations, the unfolding is more complex and involves the formation of tetramers at intermediate temperatures. At these intermediate temperatures, the Cro-WT has lost all of its alpha-helical structure and also most of its native beta-sheet structure. Upon further temperature increase, a tendency for an intermolecular association of the beta-strands is observed, which may result in irreversible beta-aggregation at high protein concentrations.

Amino Acid Substitution↗
PubMed1999

Trifluoroethanol-induced conformational transitions of proteins: insights gained from the differences between alpha-lactalbumin and ribonuclease A.

The trifluoroethanol (TFE)-induced structural changes of two proteins widely used in folding experiments, bovine alpha-lactalbumin, and bovine pancreatic ribonuclease A, have been investigated. The experiments were performed using circular dichroism spectroscopy in the far- and near-UV region to monitor changes in the secondary and tertiary structures, respectively, and dynamic light scattering to measure the hydrodynamic dimensions and the intermolecular interactions of the proteins in different conformational states. Both proteins behave rather differently under the influence of TFE: alpha-lactalbumin exhibits a molten globule state at low TFE concentrations before it reaches the so-called TFE state, whereas ribonuclease A is directly transformed into the TFE state at TFE concentrations above 40% (v/v). The properties of the TFE-induced states are compared with those of equilibrium and kinetic intermediate states known from previous work to rationalize the use of TFE in yielding information about the folding of proteins. Additionally, we report on the properties of TFE/water and TFE/buffer mixtures derived from dynamic light scattering investigations under conditions used in our experiments.

Animals↗
PubMed1998

Folding of firefly (Photinus pyralis) luciferase: aggregation and reactivation of unfolding intermediates.

The guanidine-induced unfolding of firefly (Photinus pyralis) luciferase involves two inactive equilibrium intermediates and is freely reversible at low protein concentration and low temperature. However, reactivation is exceedingly slow so that the equilibrium is attained only after several days of incubation and reactivation yields decrease strongly with increasing protein concentration, suggesting that aggregation is a competing side reaction [Herbst et al. (1997) J. Biol. Chem. 272, 7099-7105]. We investigated the role of the equilibrium intermediates in the aggregation process using size-exclusion chromatography and dynamic light scattering to monitor their association state. Although the more unfolded intermediate aggregated much more rapidly, both intermediates associated irreversibly without a conformational change visible by fluorescence or circular dichroism, forming small oligomers which remained soluble in the presence of the denaturant. The association kinetics are compatible with a nucleated polymerization mechanism. Unfolding kinetics at 1 M denaturant indicated the presence of a further inactive intermediate capable to reactivate rapidly with kinetics similar to those observed for luciferase reactivation in the presence of cell extracts. The data suggest a kinetic trap in luciferase refolding that is accessible from both equilibrium intermediate conformations and is avoided in the presence of molecular chaperones.

Animals↗
PubMed1998

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.

Animals↗
PubMed1998

Pulmonary nodules: detection with low-dose vs conventional-dose spiral CT.

The purpose of the study was the evaluation of low-dose spiral CT in the detection and assessment of contours of pulmonary nodules. In a prospective investigation 71 consecutive chest CT examinations were acquired both at 30 and 200 mA. Films were interpreted independently by two radiologists. According to the size, nodules were divided into four categories: </= 3, 4-5, 6-10, and > 10 mm; nodule shape was registered. With both protocols, 240 nodules were detected. The correlation coefficient for both methods was 0.89. Discrepancies were found most frequently in nodules near to pulmonary vessels. Nodule size estimation did not differ more than one size category. Eight spiculated nodules were identified by both techniques. Low-dose spiral CT of the chest has a high sensitivity in the detection of pulmonary nodules. If clinical circumstances require dose minimization, low-dose spiral CT may be advocated as an alternative screening method to conventional dose spiral CT.

Adolescent↗
PubMed1998

Initial hydrophobic collapse is not necessary for folding RNase A.

BACKGROUND: One of the main distinctions between different theories describing protein folding is the predicted sequence of secondary structure formation and compaction during the folding process. Whether secondary structure formation precedes compaction of the protein molecules or secondary structure formation is driven by a hydrophobic collapse cannot be decided unequivocally on the basis of existing experimental data. RESULTS: In this study, we investigate the refolding of chemically denatured, disulfide-intact ribonuclease A (RNase A) by monitoring compaction and secondary structure formation using stopped-flow dynamic light scattering and stopped-flow CD, respectively. Our data reveal the formation of a considerable amount of secondary structure early in the refolding of the slow folding species of RNase A without a significant compaction of the molecules. A simultaneous formation of secondary structure and compaction is observed in the subsequent rate-limiting step of folding. CONCLUSIONS: During folding of RNase A an initial global hydrophobicity is not observed, which contradicts the view that this is a general requirement for protein folding. This folding behavior could be typical of similar, moderately hydrophobic proteins.

Circular Dichroism↗
PubMed1998

Denatured states of yeast phosphoglycerate kinase.

Structures of proteins in unfolded states have important implications for the protein folding problem and for the translocation of polypeptide chains. Acid-denatured, cold-denatured, and 6 M guanidine hydrochloride (GuHCl) denatured yeast phosphoglycerate kinase (PGK) are ensembles of flexible unfolded molecules with rapidly interconverting structures of the individual polypeptide chains. They differ, however, in their physical properties, such as in coil size and in stiffness over a short distance along the chain. These properties of polypeptide chains can be described well by persistence statistics. A solution containing 0.7 M GuHCl at 4.5 degrees C is nearly a Theta-solvent for PGK. By contrast, 6 M GuHCl is a good solvent for PGK. Acid-denatured PGK at low ionic strength has the most expanded and stiffest chains. The conformation of heat-denatured PGK should be more compact than that of random walk chains at the Theta-point, as can be inferred from measurements on other proteins. Investigations of heat-denatured PGK by scattering methods are unfeasible due to aggregation of the protein. The persistence length as a measure of chain stiffness varies between a = 1.74 nm for cold-denatured PGK and a = 3.0 nm for acid-denatured PGK. The distribution functions of the gyration radii were calculated from the X-ray scattering data for all unfolded states and compared with the radius of gyration of the natively folded molecule.

Models, Molecular↗