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Biomedical subjects

H Wiedemann

Publications and source records attributed to H Wiedemann.

At least 19 recordsLinked to original sources
PubMed2006

The effect of solvent dynamics on the low frequency collective motions of DNA in solution and unoriented films.

Infrared spectroscopy is used to probe the dynamics of in vitro samples of DNA prepared as solutions and as solid unoriented films. The lowest frequency DNA mode identified in the far-infrared spectra of the DNA samples is found to shift in frequency when the solvent influence in the hydration shell is altered. The lowest frequency mode also has characteristics that are similar to beta-relaxations identified in other glass forming polymers.

Animals↗
PubMed2005

The influence of chain dynamics on the far-infrared spectrum of liquid methanol.

Far-infrared-absorption spectroscopy is used to investigate the low-frequency (<or=100 cm-1) intermolecular interactions in liquid methanol. Using an intense source of far-infrared radiation, modes are elucidated at approximately 30 and 70 cm-1 in the absorption spectrum. These modes are believed to arise from intermolecular bending and librational motions, respectively, and are successfully reproduced in an ab initio molecular-dynamics simulation of methanol.

Computer Simulation↗
PubMed2005

The influence of chain dynamics on the far-infrared spectrum of liquid methanol-water mixtures.

Far-infrared-absorption spectroscopy has been used to study the low-frequency (<or=100 cm-1) intermolecular modes of methanol in mixtures with water. With the aid of a first-principles molecular-dynamics simulation on an equivalent system, a detailed understanding about the origin of the low-frequency IR modes has been established. The total dipole spectrum from the simulation suggests that the bands appearing in the experimental spectra at approximately 55 and 70 cm-1 in methanol and methanol-rich mixtures arise from both fluctuations and torsional motions occurring within the methanol hydrogen-bonded chains. The influence of these modes on both the solvation dynamics and the relaxation mechanisms in the liquid is discussed within the context of recent experimental and theoretical results that have emerged from studies focusing on the short-time dynamics in the methanol hydrogen bond network.

Computer Simulation↗
PubMed1997

Binding of fibulin-1 to nidogen depends on its C-terminal globular domain and a specific array of calcium-binding epidermal growth factor-like (EG) modules.

The calcium-binding basement membrane protein fibulin-1C was shown to bind nidogen in a calcium-dependent fashion. Fibulin-1C consists of small N (domain 1) and C-terminal (domain III) globular structures connected by a central rod (domain II) composed of nine epidermal growth factor (EG) modules, eight of which possess a consensus sequence for calcium binding. Several point and deletion mutants and chimeric protein constructs were used to define the nidogen binding epitope of fibulin-1C by surface plasmon resonance and solid phase assays. All recombinant products were obtained from transfected kidney cells in a folded form as shown by CD spectroscopy, electron microscopy and proteolysis. They were used to demonstrate that calcium-binding is essentially due to the EG modules possessing the consensus binding sequence. Deletion of domain III caused a 30-fold reduction in nidogen binding, whereas deletion of domain I had no effect, yet domain III alone was also inactive. Successive deletions of two to seven EG modules of domain II also caused partial of complete inactivation of binding depending on how many were deleted or their position relative to domain III. Site-directed mutagenesis within the calcium binding consensus sequences demonstrated a similar dependence. Replacement of seven of the calcium-binding modules by a similar tandem array from a related protein showed a distinct (fibulin-2) to almost complete loss of binding (fibrillin-1). This indicates a complex epitope structure involving domains II and III, which each may provide binding epitopes or stabilize each other.

Animals↗
PubMed1997

Dimer model for the microfibrillar protein fibulin-2 and identification of the connecting disulfide bridge.

Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. The entire protein and its predicted domains were obtained as recombinant products and examined by ultracentrifugation and electron microscopy. This demonstrated a disulfide-linked homodimer of 175 kDa subunits. Partial reduction to monomers identified specifically an odd Cys574 residue responsible for dimer formation in one of three anaphylatoxin-like modules that constitute the central globular domain I (13 kDa) of fibulin-2. Furthermore, a Cys574-Ser mutation abolished disulfide connection but not non-covalent dimerization of fibulin-2. The C-terminal region (85 kDa) was shown to represent a 35-nm-long rod consisting of 11 calcium-binding EGF-like modules (domain II) and a small terminal globe (domain III). The unique N-terminal domain N (55 kDa) was also rod-shaped (approximately 38 nm) and rich in galactosamine indicating extensive O-glycosylation. A dimer model is proposed indicating mainly a rod-like shape of 80 nm length based on an anti-parallel association of two subunits through their domains I. This model also implies alignment of domains II and N between different subunits. This was demonstrated by surface plasmon resonance assay which showed a distinct interaction between domains N and II with a Kd of approximately 0.7 microM.

Amino Acid Sequence↗
PubMed1997

Risks of beryllium disease related to work processes at a metal, alloy, and oxide production plant.

OBJECTIVES: To describe relative hazards in sectors of the beryllium industry, risk factors of beryllium disease and sensitisation related to work process were sought in a beryllium manufacturing plant producing pure metal, oxide, alloys, and ceramics. METHODS: All 646 active employees were interviewed; beryllium sensitisation was ascertained with the beryllium lymphocyte proliferation blood test on 627 employees; clinical evaluation and bronchoscopy were offered to people with abnormal test results; and industrial hygiene measurements related to work processes taken in 1984-93 were reviewed. RESULTS: 59 employees (9.4%) had abnormal blood tests, 47 of whom underwent bronchoscopy. 24 new cases of beryllium disease were identified, resulting in a beryllium disease prevalence of 4.6%, including five known cases (29/632). Employees who had worked in ceramics had the highest prevalence of beryllium disease (9.0%). Employees in the pebble plant (producing beryllium metal) who had been employed after 1983 also had increased risk, with a prevalence of beryllium disease of 6.4%, compared with 1.3% of other workers hired in the same period, and a prevalence of abnormal blood tests of 19.2%. Logistic regression modelling confirmed these two risk factors for beryllium disease related to work processes and the dependence on time of the risk at the pebble plant. The pebble plant was not associated with the highest gravimetric industrial hygiene measurements available since 1984. CONCLUSION: Further characterisation of exposures in beryllium metal production may be important to understanding how beryllium exposures confer high contemporary risk of beryllium disease.

Adult↗
PubMed1996

Structural and electron microscopic analysis of neurocan and recombinant neurocan fragments.

Neurocan, a nervous tissue-specific chondroitin sulfate proteoglycan of the aggrecan family which has been shown to interact with neural cell adhesion molecules and tenascin, could be visualized by rotary shadowing electron microscopy as two globular domains interconnected by an extended flexible filament of 60-90 nm. Several recombinant neurocan fragments generated in the human embryonic kidney cell line 293 represent as observed by electron microscopy the expected parts of this structure, which indicates a correct folding of these molecules. Biological activity of the recombinant N-terminal globular domain could be demonstrated by its coelution with hyaluronan in gel permeation chromatography. In addition, the modification of the recombinant fragments with certain carbohydrate structures was analyzed. High mannose oligosaccharides could be mapped to the N-terminal globular domain of the brain-derived molecule. Only recombinant fragments containing parts of the central region of the molecule were modified with chondroitin sulfate chains and with the HNK-1 epitope, and could be considerably altered in their migratory behavior on SDS-polyacrylamide gel electrophoresis by neuraminidase treatment. These findings and the electron microscopic shape indicate a mucin-like character for the central neurocan region.

Amino Acid Sequence↗
PubMed1996

Expression of fibulin-2 by fibroblasts and deposition with fibronectin into a fibrillar matrix.

The extracellular matrix protein fibulin-2 was shown to be a typical product of cultured human and mouse fibroblasts by several immunological assays. It is secreted and deposited in cells and tissues as a disulfide-bonded oligomer identical in size to the previously described recombinant fibulin-2. Most of the fibroblast fibulin-2 is deposited into a dense fibrillar meshwork which requires treatment with EDTA and/or 6 M urea for solubilization. Fibulin-2 and fibronectin are synthesized at equivalent levels and both colocalize in the fibrils as shown by immunofluorescence. Metabolic labelling and pulse-chase studies demonstrated fibulin-2 oligomers in detergent extracts of cells and their rapid translocation to extracellular EDTA-sensitive assembly forms. Unlike for fibronectin and fibulin-1 only a little fibulin-2 was found in the cell culture medium. Immunogold staining of confluent human fibroblasts showed localization of fibulin-2 to a fine meshwork or bundles of amorphous microfibrils in the matrix. This also demonstrated a distinct colocalization of fibulin-2 and fibronectin at the electron microscope level, indicating that the interaction between these two protein shown in in vitro assays may also exist in situ. No distinct colocalization of both proteins could, however, be observed with cross-striated fibrils of collagen I and collagen VI microfibrils.

Animals↗
PubMed1995

Characterization of mutant forms of recombinant human properdin lacking single thrombospondin type I repeats. Identification of modules important for function.

Properdin is a serum glycoprotein that up-regulates the alternative pathway of complement by stabilizing the C3b-Bb complex. It also binds sulfated glycoconjugates, such as sulfatide, in vitro. Properdin is composed of cyclic dimers, trimers, and tetramers of a 53-kDa monomeric subunit. The monomer contains an N-terminal region of no known homology and six thrombospondin type 1 repeats (TSRs) of approximately 60 amino acids. To identify the regions of properdin important for function, we have expressed human properdin, and mutant forms each lacking a single TSR, in Chinese hamster ovary cells. In addition, limited tryptic digestion yielded "nicked" properdin by the cleavage of one peptide bond in TSR5. The structural and functional properties of these altered forms of properdin were investigated. Properdin "nicked" in TSR5 is unable to bind C3b but retains its overall structure and its ability to bind sulfatide. The removal of TSR5 prevents C3b and sulfatide binding. Properdin lacking TSR4 is unable to stabilize the C3b-Bb complex but is able to bind C3b and sulfatide, and shows the presence of monomers and dimers in an electron microscope. Properdin without TSR3 is able to stabilize the C3b-Bb complex, to bind C3b and sulfatide, and forms dimers, trimers, and tetramers. Properdin lacking TSR6 is unable to form oligomers. The N-linked carbohydrate of properdin is not required for oligomerization or stabilization of the C3b-Bb complex. The results implicate TSR5 in both C3b and sulfatide binding, and suggest that TSR4 may also be involved in stabilization of the C3b-Bb complex.

Amino Acid Sequence↗
PubMed1995

Structural properties of recombinant domain III-3 of perlecan containing a globular domain inserted into an epidermal-growth-factor-like motif.

A fragment comprising approximately domain III-3 of the basement membrane heparan sulfate proteoglycan perlecan was prepared in recombinant form from kidney cell clones. This fragment was predicted to contain a cysteine-free globular domain inserted within an epidermal-growth-factor(EGF)-like motif (L4 module) and three additional EGF-like motifs (LE module) without large inserts. This prediction was confirmed by electron microscopy, which demonstrated a globule joined to a very short rod-like segment. The globule was selectively destroyed by pepsin, which also demonstrated that its insertion into an EGF-like motif did not prevent the typical disulfide connections known for such motifs. Yet the globule was more stable against neutral proteinases. The fragment showed a distinct content (55-60%) of alpha helical and beta structure and a partially reversible melting of the conformation in 6 M guanidine. Antibodies raised against recombinant domain III-3 demonstrated a complete cross-reaction with tissue-derived perlecan but not with laminin and a distinct basement membrane staining of tissue sections. Most of the epitopes were lost after reduction and alkylation. Together the data demonstrated a proper folding of recombinant domain III-3 similar to its structure in the native protein and provided the first structural evidence for a novel globular protein motif L4 based on an EGF-like scaffold.

Amino Acid Sequence↗
PubMed1995

Structural comparison of cuticle and interstitial collagens from annelids living in shallow sea-water and at deep-sea hydrothermal vents.

Two types of annelid collagens of different sizes were purified, one from acetic acid extracts of the cuticle (length 2.5 microns) and the other, after pepsin digestion, from interstitial spaces of the body wall (0.3 micron). They were obtained from Alvinella pompejana, Alvinella caudata and Paralvinella grasslei collected at 2600 m depth around anoxic hydrothermal vents and from Arenicola marina and Nereis diversicolor living in shallow sea-water habitats. The length of the corresponding collagens from different species and their amino acid compositions including the hydroxylation of proline were remarkably similar. The melting point of the triple helix, however, differed between the Alvinella species (approximately 45 degrees C), Paralvinella (approximately 35 degrees C) and the shallow sea-water annelids (approximately 28 degrees C), indicating adaption to habitats with different temperatures. The cuticle collagens of the annelids possess a globular domain, which is apparently involved in oligomer formation, and show similar fragment pattern. Almost identical cross-striation patterns of segment-long-spacing segments of the interstitial collagens indicated sequence similarity, which was confirmed by partial Edman degradation of alpha-chains. These data showed almost complete identity between the two Alvinella species and a lower sequence identity with Paralvinella (approximately 95%), Arenicola (67 to 72%) and the vent vestimentiferan Riftia pachyptila (64 to 71%). The data suggest a close evolutionary relationship between these worms, despite a clear separation of habitat preference and thermal stability of the collagens.

Amino Acid Sequence↗
PubMed1995

Comparative study of the structural and functional properties of a bovine plasma C-type lectin, collectin-43, with other collectins.

Collectin-43 (CL-43) is a recently described bovine plasma protein containing both collagenous regions and C-type-lectin domains [Holmskov, Teisner, Willis, Reid and Jensenius (1993) J. Biol. Chem. 268, 10120-10125; Lim, Willis, Reid, Lu, Laursen, Jensenius and Holmskov (1994) J. Biol. Chem. 269, 11820-11824]. CL-43 was purified by affinity chromatography on mannan-Sepharose. On SDS/PAGE under reducing conditions the purified lectin showed a double band at about 43 kDa, with the upper band representing the intact molecule and the lower band a truncated form that lacked the N-terminal nine amino acid residues. Under non-reducing conditions, only one band was seen at 120 kDa. Analytical gel chromatography and sucrose-density-gradient centrifugation of the purified molecule, showed a Stokes radius of 9.1 +/- 0.3 nm (91 +/- 3 A) and a sedimentation coefficient (s20,w) of 3.6 +/- 0.1 S. These values correspond to a molecular mass of 119-138 kDa under non-denaturing condition in solution. The frictional coefficient (f/f0) was 2.7, indicating extreme elongation due to the collagenous segment. Only monomer subunits, with 37.4 +/- 1.7-nm-long rods, were seen by electron microscopy. These findings indicate that CL-43, in contrast with the other circulating collectins, is found only as a single subunit composed of three polypeptide chains. Two-dimensional gel electrophoresis showed that CL-43 has two isoforms, with pI values of 4.9 and 5.3, corresponding to the native form and the truncated form of the molecule respectively. CL-43, like conglutinin, lung surfactant protein A and mannan-binding protein (MBP), was shown to bind to the collectin receptor. Bovine MBP caused the activation of the complement system as revealed by the deposition of complement component C4 upon incubation of diluted serum in wells containing MBP bound to solid-phase mannan. CL-43, lung surfactant protein D (SP-D) and conglutinin showed no complement-activating properties under the same conditions. Conglutinin binds fluid- and solid-phase iC3b, while CL-43 and MBP do not show such reactivity.

Animals↗