Biomedical subjects

M Häner

Publications and source records attributed to M Häner.

30 records · Page 2Linked to original sources

PubMed1995

Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed "twisted ribbon" structure.

The asymmetric unit membrane (AUM) forms numerous plaques covering the apical surface of mammalian urinary bladder epithelium. These plaques contain four major integral membrane proteins called uroplakins Ia, Ib, II and III, which form particles arranged in a well-ordered hexagonal lattice with p6 symmetry and a lattice constant of 16.5 nm. Bovine AUM plaques negatively stained with anionic sodium silicotungstate revealed structural detail to 3.1 nm resolution. Correlation averaging resolved each particle into 12 stain-excluding domains arranged in two concentric rings (inner ring radius (rm) = 3.7 nm, outer ring radius (rout) = 6.6 nm), each with six domains which were rotated by roughly 30 degrees relative to each other. Negative staining with cationic uranyl formate increased the resolution to 2.2 nm and unveiled distinct connections between adjacent AUM particles. These connections may provide a molecular basis for the observed insolubility of the plaques in many detergents. Examination of the luminal face of freeze-dried/unidirectionally metal-shadowed AUM plaques established a left-handed vorticity of the 16 nm protein particles, whereas the cytoplasmic face exhibited no significant surface corrugations. Three-dimensional reconstruction from sodium silicotungstate-stained specimens revealed the AUM particles to be built of six "V-shaped" subunits anchored upright in the membrane. The mass density distribution within uranyl formate-stained AUM particles was similar except that the inner tip of each V was bridged to the outer tip of an adjacent V, so that the 16 nm AUM particle appeared as a continuous, "twisted ribbon" embracing a central cavity. Finally, mass measurements of unstained/freeze-dried plaques by scanning transmission electron microscopy yielded a total mass of 1,120 kDa per membrane-bound AUM particle. By imposing constraints on the possible uroplakin stoichiometries within AUM plaques, these data provide a first glimpse of the molecular architecture of the 16 nm particles constituting the plaques.

Animals↗

PubMed1994

Mammalian uroplakins. A group of highly conserved urothelial differentiation-related membrane proteins.

The asymmetric unit membrane (AUM) forms the apical plaques of mammalian urothelium and is believed to play a role in strengthening the urothelial apical surface thus preventing the cells from rupturing during bladder distention. We have shown previously that purified bovine AUMs contain four major integral membrane proteins: the uroplakins Ia (27 kDa), Ib (28 kDa), II (15 kDa), and III (47 kDa). This contradicts some previous reports indicating that some of these proteins are absent in AUMs of several species. Using an improved procedure, we isolated AUMs from, in addition to cattle, eight mammalian species (human, monkey, sheep, pig, dog, rabbit, rat, and mouse). The AUMs of these species appear morphologically similar bearing crystalline patches of 12-nm protein particles with a center-to-center spacing of 16.5 nm. Using antibodies raised against synthetic oligopeptides or individual bovine uroplakins, we established by immunoblotting that the four uroplakins are present in AUMs of all these species. The DNA-deduced amino acid sequences of bovine and mouse uroplakin II revealed 83% identity. These results indicate that uroplakins Ia, Ib, II, and III are the major protein components of probably all mammalian urothelial plaques, and that the sequence and three-dimensional structure of uroplakin molecules are highly conserved during mammalian evolution.

Amino Acid Sequence↗

PubMed1991

Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.

Adenosine Triphosphate↗

PubMed1991

Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions.

Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.

Animals↗

PubMed1990

Malignancies in families of women with medullary, tubular and invasive ductal breast cancer.

Breast cancer seems to represent a heterogeneous group of neoplasia originating from the parenchymal epithelium of the mammary gland. Family studies combined with genetic epidemiological analyses and histological evaluations were used to gain an insight into this nosological entity. Special emphasis was given to the type and frequency of neoplasia in close relatives. This study represents pedigrees of 36 histologically defined pure tubular and 22 pure medullary breast cancer patients as well as 171 with the invasive ductal form. The incidence of cancer in the first degree relatives of all three groups is compared to that of the local population. The first degree relatives did not have a higher risk (RR) for the neoplastic diseases. However, breast cancer occurs more frequently in the female relatives of all three groups. Other cancers have different relative risks.

Adenocarcinoma↗

PubMed1990

Effect of aluminum and other multivalent cations on neurofilaments in vitro: an electron microscopic study.

Using electron microscopy (EM) of negatively stained samples, we have systematically explored the effect of aluminum and other multivalent cations on neurofilaments (NFs) in vitro. Interactions of these cations were investigated with bovine, rabbit, and rat spinal cord native NFs, and with 10-nm filaments reconstituted from the 68-kDa subunit (NF-L) isolated from bovine spinal cord. Our results indicated that, as has been observed with other classes of intermediate filaments (IFs), all multivalent cations caused significant aggregation of native NFs, suggesting that this phenomenon is a rather general one and not limited to aluminum. In addition, all cations tried caused significant lateral aggregation of filaments reconstituted from NF-L. Aluminum lactate had an identical effect on bovine, rabbit, and rat NFs. Because aluminum causes strong aggregation of NFs in vitro, a similar phenomenon may occur in vivo leading to the observed accumulation of NFs in neuronal perikarya of rabbits after intoxication with aluminum. These in vitro observations support the concept that some human neurological diseases characterized by the accumulation of NFs may be related to abnormal levels of multivalent cations.

Aluminum↗

PubMed1989

Age-related decrease in an early step of DNA-repair of normal human lymphocytes exposed to ultraviolet-irradiation.

A monoclonal antibody-based immunoassay has been used to detect age-related changes in the rate of loss of photoproduct antigenicity from the DNA of peripheral blood lymphocytes irradiated with 10 J m-2 uv-C. Lymphocytes were obtained from 75 healthy volunteers whose ages ranged from 14 months to 82 years. The samples were divided by age decades into groups of 10 individuals, except the first decade which contained only 5 individuals. The mean loss of antigenicity +/- 1 standard deviation was determined for each group at 10, 30, and 60 min after irradiation. The data were analyzed by Mann-Whitney U test and by the Kruskal-Wallis test. After a recovery period of 10 min the loss of antigenicity was most rapid in group I (0-9 years), less rapid in group II (10-19 years), and least rapid in all other groups. The differences between groups became less at 30 min and were not significant at 60 min incubation. These data obtained from normal cells concur with our previous conclusions, that reductions in the rate of loss of antigenicity in nondiseased cells isolated from patients with melanoma and dysplastic naevus syndrome reflect genetic abnormalities in these patients.

Adolescent↗

PubMed1983

Two-dimensional crystal packing of matrix porin. A channel forming protein in Escherichia coli outer membranes.

Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981). Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm). The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72. At the higher ratio, large hexagonal lattices predominated. Higher lipid ratios did not reveal other crystal forms. The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983). The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm. The mass distribution between triplets, however, were significantly different. They are likely to correspond primarily to lipids. Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers. The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis.

Bacterial Proteins↗

PubMed1982

The wrapping phenomenon in air-dried and negatively stained preparations.

We demonstrate that the interface energies involved in the direct preparation of supramolecular structures onto supporting films leads very frequently to a smooth wrapping of the supporting film around approximately one third to one half of the structure. We conclude that in such cases the structure is more rigid than the supporting film; examples being ribosomes, small viruses and small glass fragments. Other structures are less rigid and become significantly flattened. Complete flattening is frequently observed with empty virus capsids. The sandwich technique, by which a specimen is placed between two supporting films, in general leads to increased flattening. Only in few cases (e.g. ribosomes) are biological particles rigid enough to resist flattening and become wrapped from both sides.

Coliphages↗

PubMedDate not supplied

Native talin is a dumbbell-shaped homodimer when it interacts with actin.

Electron microscopy of glycerol-sprayed and rotary metal-shadowed talin from human platelets reveals a dumbbell-shaped molecule with an average length of approximately 51 nm. Analytical ultracentrifugation of native talin yields a single molecular species with an apparent molecular mass of 412 (+/- 28.6) kDa and a sedimentation coefficient of S20w = 11.2. Chemical cross-linking with glutaraldehyde (GA) and corresponding SDS-PAGE analysis show that the monomer band of talin can be quantitatively converted to a dimer band at GA concentrations > or = 0.45%, indicating that there is no significant amount of monomer present in solution. These structural and biophysical data are compatible with native talin being an antiparallel homodimer. Length measurements and viscometric and fluorescent assays of actin filaments polymerized in the presence of native talin and of covalently cross-linked talin dimers all yield similar effects: namely, increased nucleation and polymerization rates and an overall reduction of actin filament length. Hence, we conclude that talin in its native biological state is a dimer when promoting nucleation of actin filaments.

Actins↗

PubMedDate not supplied

Visualization of actin filaments in keratocyte lamellipodia: negative staining compared with freeze-drying.

Depending on the method of preparation, the actin-rich lamellipodia of motile cells can show very different structural organizations. This situation has been a main contributor to differences in current ideas about the possible mechanisms of cell movement. We have here analyzed the structure of the lamellipodium in whole-mount cytoskeletons using one of the most rapid of crawling cells, the fish keratocyte, employing two procedures considered least damaging to actin filament arrays: freeze-drying and negative staining. At the front of the lamellipodium, where filaments density is the highest, freeze-dried images conveyed the impression of a cross-linked network of very short, interconnected filaments--as previously observed by others--whereas the same regions appeared as a diagonal meshwork of long, more or less straight filaments after negative staining. In general, the linearity of actin filaments was not preserved after freeze-drying, except in situations where the filaments had partially dried down onto the substrate before freezing. In the mid and posterior regions of the lamellipodium the actin filaments appeared to be up to several micrometers long by negative staining, whereas their length was impossible to discern by freeze-drying, owing to filament kinking and aggregation and to the nature of the contrasting procedure, which reveals only the upper layers of filaments. We conclude that while freeze-drying preserves the overall three-dimensional structure of the lamellipodium it also introduces fine-structural distortions in actin that obscure actin filament order. Drying in negative stain appears to stabilise the actin network.(ABSTRACT TRUNCATED AT 250 WORDS)

Actin Cytoskeleton↗

PubMedDate not supplied

Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin.

The human calcitonin (hCT) peptide hormone has a marked tendency to aggregate in aqueous solutions and to form long, thin fibrillar aggregates resulting in viscous and turbid dispersions. In this study, the in vitro aggregation products of hCT were systematically investigated using conventional transmission electron microscopy (CTEM) and in-lens field emission scanning electron microscopy. The mass per length of unstained/air-dried specimens was determined by scanning transmission electron microscopy. Irrespective of the sample preparation method and electron microscopic (EM) imaging mode employed, similar supramolecular assemblies were observed. Based on these EM data, it is proposed that hCT aggregation begins with the formation of approximately 4-nm-thick protofibrils. These protofibrils further interact via lateral association and coiling to form higher-order fibrillar assemblies, i.e., protofibril-ribbons, fibrils, fibril-ribbons, tubes, and multistranded cables. The concentration and history of the aggregated hCT solutions strongly influenced the relative frequency of the various hCT assemblies depicted. The supramolecular assemblies of hCT revealed distinct helical symmetries at their different levels of aggregation. A hypothetical mechanism assumed for aggregating solutions to form polymorphic fibrillar hCT assemblies is presented in a schematic model, and the supramolecular arrangement of hCT within the various polymorphic fibrillar aggregates is delineated.

Amino Acid Sequence↗