Search PubMed⌕ Search

Biomedical subjects

H Herrmann

Publications and source records attributed to H Herrmann.

At least 19 recordsLinked to original sources
PubMed2001

Divide-and-conquer crystallographic approach towards an atomic structure of intermediate filaments.

Intermediate filaments (IFs) represent an essential component of the cytoskeleton in higher eukaryotic cells. The elementary building block of the IF architecture is an elongated dimer with its dominant central part being a parallel double-stranded alpha-helical coiled coil. Filament formation proceeds via a specific multi-step association of the dimers into the unit-length filaments, which subsequently anneal longitudinally and finally radially compact into mature filaments. To tackle the challenge of a crystallographic structure determination, we have produced and characterised 17 overlapping soluble fragments of human IF protein vimentin. For six fragments ranging in length between 39 and 84 amino acid residues, conditions yielding macroscopic crystals could be established and X-ray diffraction data were collected to the highest resolution limit between 1.4 and 3 A. We expect that solving the crystal structures of these and further fragments will eventually allow us to patch together a molecular model for the full-length vimentin dimer. This divide-and-conquer approach will be subsequently extended to determining the crystal structures of a number of complexes formed by appropriate vimentin fragments, and will eventually allow us to establish the three- dimensional architecture of complete filaments at atomic resolution.

Amino Acid Sequence↗
PubMed2000

The 300-kDa intermediate filament-associated protein (IFAP300) is a hamster plectin ortholog.

Plectin is a high-molecular-weight cytoskeleton-associated protein that was initially identified in intermediate filament (IF)-enriched fractions of rat C6 glioma cells. At the cellular level, plectin has been found to associate with IF networks and IF-associated structures that are involved in cell-cell and cell-substrate adhesions. IFAP300 is an IF-associated protein that was initially identified in hamster cells by a monoclonal antibody directed against a high molecular weight protein present in IF-enriched cytoskeletal preparations. Plectin and IFAP300 display similar distribution patterns within cells as determined by immunofluorescence. Based upon this and the finding that their biochemical properties are similar, it has been suggested that they may actually be orthologous proteins. In this paper we demonstrate that this is the case. Cloning and sequencing of most of the hamster plectin cDNA demonstrates that plectin is found in hamster cells and that its sequence is highly conserved between species. Using immunological cross-reactivity, epitope mapping, and immunoelectron microscopy, we show that IFAP300 is actually the hamster ortholog of plectin.

Amino Acid Sequence↗
PubMed2000

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.

Animals↗
PubMed2000

The intermediate filament protein consensus motif of helix 2B: its atomic structure and contribution to assembly.

Nearly all intermediate filament proteins exhibit a highly conserved amino acid motif (YRKLLEGEE) at the C-terminal end of their central alpha-helical rod domain. We have analyzed its contribution to the various stages of assembly by using truncated forms of Xenopus vimentin and mouse desmin, VimIAT and DesIAT, which terminate exactly before this motif, by comparing them with the wild-type and tailless proteins. It is surprising that in buffers of low ionic strength and high pH where the full-length proteins form tetramers, both VimIAT and DesIAT associated into various high molecular weight complexes. After initiation of assembly, both VimIAT and DesIAT aggregated into unit-length-type filaments, which rapidly longitudinally annealed to yield filaments of around 20 nm in diameter. Mass measurements by scanning transmission electron microscopy revealed that both VimIAT and DesIAT filaments contained considerably more subunits per cross-section than standard intermediate filaments. This indicated that the YRKLLEGEE-motif is crucial for the formation of authentic tetrameric complexes and also for the control of filament width, rather than elongation, during assembly. To determine the structure of the YRKLLEGEE domain, we grew crystals of peptides containing the last 28 amino acid residues of coil 2B, chimerically fused at its amino-terminal end to the 31 amino acid-long leucine zipper domain of the yeast transcription factor GCN4 to facilitate appropriate coiled-coil formation. The atomic structure shows that starting from Tyr400 the two helices gradually separate and that the coiled coil terminates with residue Glu405 while the downstream residues fold away from the coiled-coil axis.

Amino Acid Motifs↗
PubMed2000

Retinoic acid differentiation of HL-60 cells promotes cytoskeletal polarization.

Retinoic acid (RA) treatment of HL-60 cells in vitro induces granulocytic differentiation, involving reorganization of the nucleus and cytoplasm, development of chemoattractant-directed migration, and eventual apoptosis. The present studies with HL-60/S4 cells document that major elements of the cytoskeleton are changed: actin increases by 50%; vimentin decreases by more than 95%. The cellular content of alpha-tubulin does not significantly change; but the centrosomal-microtubule (MT) array moves away from the lobulating nucleus. Cytoskeletal-modifying chemicals modulate this polarized reorganization: Taxol and cytochalasin D enhance centrosome movement; nocodazole reverses it. Cytoskeletal-modifying chemicals do not appear to affect nuclear lobulation or the integrity of envelope-limited chromatin sheets (ELCS). Employing bcl-2-overexpressing HL-60 cells permitted demonstration of nuclear lobulation, ELCS formation, and centrosome-MT movement concomitantly during RA-induced differentiation, implying independence between the cellular reorganization and apoptotic programs. RA appears to promote an inherent potential in HL-60 cells for cytoskeletal polarization, likely to be important for chemoattractant-directed cell migration, an established characteristic of mature granulocytes.

Actins↗
PubMed2000

In vivo observation of a nuclear channel-like system: evidence for a distinct interchromosomal domain compartment in interphase cells.

We have investigated the interchromosomal domain compartment in living cells by transfecting cDNA coding for Xenopus vimentin, engineered to contain a nuclear localization signal (NLS), coupled to the green fluorescent protein. In human vimentin-free SW13 cells, this chimeric protein was deposited in body-like "dots" both at 37 degrees C, the nonpermissive temperature for assembly of the amphibian vimentin, and 28 degrees C, the optimal temperature for Xenopus vimentin assembly, indicating that the chimeric protein was assembly incompetent. However, when transfected into a subclone stably expressing Xenopus NLS-vimentin (SW13-SC), the chimeric protein incorporated, as a fluorescent tracer, into the structures formed by NLS-vimentin and allowed us to visualize the outgrowth of the vimentin fibers after a temperature shift to 28 degrees C in living cells. In particular, we followed the time-dependent outgrowth of fibers from nuclear dots, first connecting two dots each and with time three and more, eventually generating a spatially restricted fiber system consisting of few loop-like arrays traversing the nucleus. Virtually identical results were obtained when the temperature was lowered only to 30 and 32 degrees C, respectively. An engineered human NLS-vimentin, without need for temperature shift, formed seemingly identical patterns of nuclear fibrils at 37 degrees C in three additionally transfected human cell lines: MCF-7, PLC, and HeLa. When the epithelial cytokeratin pair 8 and 18 was expressed in the nucleus via an engineered NLS in the cytokeratin 18 gene, more network-like, extended filament arrays were generated. Notably, in cotransfection experiments with Xenopus NLS-vimentin, we observed that the formation of these cytokeratin networks at 37 degrees C initiated from dots that nearly entirely colocalized with the aggregated amphibian NLS-vimentin. After a shift to 28 degrees C, extending Xenopus NLS-vimentin and cytokeratin filaments frequently followed the same path through the nucleus. These data indicate that interphase cells contain a seemingly equivalent, accessible interchromosomal space.

Actin Cytoskeleton↗
PubMed2000

Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics.

The assembly of intermediate filament (IF) arrays involves the recruitment of a complex set of cell-type-specific IF-associated proteins. Some of them are integral membrane proteins, others act as crosslinking proteins with vectorial binding activities, and yet others comprise motor proteins. In vivo IFs appear to be predominantly heteropolymers, although in vitro several IF proteins (e.g. vimentin, desmin, neurofilament (NF)-L and the nuclear lamins) do self-assemble into IF-like polymers. In contrast, NF-M, NF-H, nestin, synemin and paranemin, all bona fide IF proteins, are unable to self-assemble into IFs either in vitro or in vivo. The individual IF proteins of this large multigene family are chemically heterogeneous, exhibiting different assembly kinetics and yielding discrete types of filaments. The unique physical properties and interaction capabilities of these distinct IF molecular building blocks, in combination with accessory proteins, mediate the generation of a highly dynamic and interconnected, cell-type-specific cytoarchitecture.

Actin Cytoskeleton↗
PubMed2000

Current practice of temporary vena cava filter insertion: a multicenter registry.

PURPOSE: To evaluate the current practice of temporary vena cava filter placement and its complications. MATERIALS AND METHODS: A multicenter registry was conducted from May 1995 until May 1997 using a standardized questionnaire. One hundred eighty-eight patients were evaluated. Patient characteristics, filter indications, filter characteristics, and complications were registered. RESULTS: Deep vein thrombosis was proven in 95.2% of the patients. Main filter indication was thrombolysis therapy (53.1%). Average filter time was 5.4 days. An Antheor filter was inserted in 56.4%, a Guenther filter in 26.6%, and a Prolyser filter in 17.%. Transfemoral filter implantation was slightly preferred (54.8%). Four patients died of pulmonary embolism (PE) during filter protection. Major filter problems were filter thrombosis (16%) and filter dislocation (4.8%). When thrombus was found in or at the filter before explantation, additional thrombolysis was performed in 16.7%, additional filter implantation in 10%, and thrombus aspiration in 6.7%; 4.8% of filters were replaced with permanent filters. DISCUSSION: Temporary vena cava filters are placed to prevent PE in a defined patient population. Despite their presence, PEs still occur in a small percentage. Problems of filter thrombosis and dislocation have to be solved. CONCLUSION: The results of this multicenter registry support the need for innovative filter design, as well as a randomized, prospective study.

Adolescent↗
PubMed2000

Low-dose dopexamine in patients undergoing hemihepatectomy: an evaluation of effects on reduction of hepatic dysfunction and ischaemic liver injury.

BACKGROUND: Hepatic dysfunction is a common problem in patients after hemihepatectomy. Treatment with low-dose dopamine has been shown to be beneficial in hemihepatectomy patients. We hypothesized that dopexamine, a synthetic vasoactive catecholamine, due to its specific pharmocodynamic profile may be more effective in reducing hidden ischaemic episodes in the hepato-splanchnic region during and after temporary total cross-clamping of hepatic inflow in these patients. METHODS: The effects of low-dose dopexamine on hepatic venous haemoglobin oxygen saturation (ShvO2), hepatic venous lactate level, monoethylglycinxylid (MEGX) formation, hepatic synthetic function and indicators for hepatic cell damage were studied during hemihepatectomy and for 16 h postoperatively in hemihepatectomy patients and compared to those of low-dose dopamine. In a prospective, double-blind clinical study 20 patients received randomly either dopexamine (DPX) 0.5 microg kg(-1) min(-1) (n=10) or dopamine (DO) 2.5 microg kg(-1) min(-1) (n= 10). Infusions were started after induction of anaesthesia and continued 16 h postoperatively. Hepatic vein, radial and pulmonary artery were catheterized. Measurements were carried out after induction of anaesthesia, after total cross-clamping of hepatic inflow, and at 2 h and 16 h postoperatively. RESULTS: There were no differences in systemic haemodynamics, oxygenation, ShvO2, serum aminotransferases or MEGX levels between the groups. At 16 h postoperatively prothrombin and antithrombin III levels were significantly lower while hepatic venous lactate was significantly higher in the DPX group compared to the DO group. CONCLUSION: In patients undergoing hemihepatectomy, we could not reveal superior hepatoprotective effects of low-dose dopexamine compared to low-dose dopamine.

Adult↗
PubMed2000

Association of plectin with Z-discs is a prerequisite for the formation of the intermyofibrillar desmin cytoskeleton.

Plectin is a high-molecular mass protein (approximately 500 kd) that binds actin, intermediate filaments, and microtubules. Mutations of the plectin gene cause a generalized blistering skin disorder and muscular dystrophy. In adult muscle, plectin is colocalized with desmin at structures forming the intermyofibrillar scaffold and beneath the plasma membrane. To study the involvement of plectin in myofibrillogenesis, we analyzed the spatial and temporal expression patterns of plectin in cultured differentiating human skeletal muscle cells and its relationship to desmin intermediate filaments during this process. Northern and Western blot analyses demonstrated that at least two different plectin isoforms are expressed at all developmental stages from proliferating myoblasts to mature myotubes. Using immunocytochemistry, we show that the localization of plectin dramatically changes from a network-like distribution into a cross-striated distribution during maturation of myocytes. Double immunofluorescence experiments revealed that desmin and plectin are colocalized in premyofibrillar stages and in mature myotubes. Interestingly, plectin was often found to localize to the periphery of Z-discs during the actual alignment of neighboring myofibrils, and an obvious cross-striated plectin staining pattern was observed before desmin was localized in the Z-disc region. We conclude that the association of plectin with Z-discs is an early event in the lateral alignment of myofibrils that precedes the formation of the intermyofibrillar desmin cytoskeleton.

Cells, Cultured↗
PubMed2000

Physical characterization of plakophilin 1 reconstituted with and without zinc.

Plakophilin 1 (PKP1) belongs to the arm-repeat protein family which is characterized by the presence of a conserved 42-amino-acid motif. Despite individual members of the family containing a similar type of structural domain, they exhibit diverse cellular functions. PKP1 is ubiquitously expressed in human tissues and, depending on the type of cell, found prominently in the karyoplasm and/or in desmosomes. In surface plasmon resonance detection experiments, we noticed that PKP1 specifically bound zinc but not calcium or magnesium. Therefore we have used circular dichroism spectroscopy, limited proteolysis, analytical ultracentrifugation, electron microscopy and dynamic light scattering to establish the physical properties of recombinant PKP1 depending on the presence or absence of zinc. The alpha helix content of PKP1 was considerably higher when reconstituted with zinc than without. By atomic absorption spectroscopy 7.3 atoms zinc were shown to be tightly associated with one molecule of wild-type PKP1. The zinc-reconstituted protein formed globular particles of 21.9 +/- 8.4 nm diameter, as measured by electron microscopy after glycerol spraying/rotary metal shadowing. In parallel, the average sedimentation coefficient (s20, w) for zinc-containing PKP1 was 41S and its diffusion coefficient, as obtained by dynamic light scattering, 1.48 x 10-7 cm2.s-1. The molecular mass of 2.44 x 106 obtained from s and D yields an average stoichiometry of 30 for the PKP1 oligomer. In contrast, PKP1, reconstituted without zinc, contained no significant amount of zinc, sedimented with 4.6S, and was present in monomeric form as determined by sedimentation equilibrium centrifugation.

Circular Dichroism↗
PubMed2000

Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis.

Caspase 8 plays an essential role in the execution of death receptor-mediated apoptosis. To determine the localization of endogenous caspase 8, we used a panel of subunit-specific anti-caspase 8 monoclonal antibodies in confocal immunofluorescence microscopy. In the human breast carcinoma cell line MCF7, caspase 8 predominantly colocalized with and bound to mitochondria. After induction of apoptosis through CD95 or tumor necrosis factor receptor I, active caspase 8 translocated to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase 8 prodomain remained bound to mitochondria. Plectin was quantitatively cleaved by caspase 8 at Asp 2395 in the center of the molecule in all cells tested. Cleavage of plectin clearly preceded that of other caspase substrates such as poly(ADP-ribose) polymerase, gelsolin, cytokeratins, or lamin B. In primary fibroblasts from plectin-deficient mice, apoptosis-induced reorganization of the actin cytoskeleton, as seen in wild-type cells, was severely impaired, suggesting that during apoptosis, plectin is required for the reorganization of the microfilament system.

Actins↗
PubMed2000

Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis.

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

Cell Extracts↗
PubMed2000

Grids and high kilo-volt-peak-setting in bedside chest radiographic examinations.

Bedside chest radiographic examinations in intensive care units with grids are impaired by artefacts caused by angulation of the grid (grid cut-off). Two different grids--a grid with a high strip density of 70 lines per cm and the "InSight portable imaging system"--were examined in an intensive care unit with respect to their susceptibility to angulation, image quality and handling of the grid. Five radiologists compared 50 radiographs of each grid considering ten image quality criteria. Using the "InSight portable imaging system" major artefacts were undetectable even at an angulation of 10%; no adjustment of the grid was required, which reduced the amount of time needed to take the radiograph by 26%. The increase in dosage demanded by the employment of the grids at low kilovolt peak setting could be partially compensated by the use of high kilovolt peak setting. The image quality of the "InSight portable imaging system" together with a high kilovolt peak setting is satisfactory, and due to its simplified handling, the portable imaging system has proved to be suitable for bedside chest radiography in intensive care.

Artifacts↗
PubMed1999

Intermediate filament assembly: temperature sensitivity and polymorphism.

Intermediate filament (IF) proteins are encoded by a large multigene family and form polymers with a uniform diameter of approximately 10 nm. However, although the cytoplasmic representatives all confirm to a unit-type structural principle leading to the formation of extended coiled coils, it is becoming increasingly clear that subunit arrangements and physical properties vary among the different filaments. Thus, the intricate tissue-specific expression pattern of individual IF proteins (especially, their co-expression with other members of the IF protein family or with IF-associated proteins to form obligatory heteropolymers) points to distinct functions acquired during evolution relevant to cellular homeostasis in various tissues.

Amino Acid Sequence↗
PubMed1999

Characterization of distinct early assembly units of different intermediate filament proteins.

We have determined the mass-per-length (MPL) composition of distinct early assembly products of recombinant intermediate filament (IF) proteins from the four cytoplasmic sequence homology classes, and compared these values with those of the corresponding mature filaments. After two seconds under standard assembly conditions (i.e. 25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 37 degrees C), vimentin, desmin and the neurofilament triplet protein NF-L aggregated into similar types of "unit-length filaments" (ULFs), whereas cytokeratins (CKs) 8/18 already yielded long IFs at this time point, so the ionic strength had to be reduced. The number of molecules per filament cross-section, as deduced from the MPL values, was lowest for CK8/18, i.e. 16 and 25 at two seconds compared to 16 and 21 at one hour. NF-L exhibited corresponding values of 26 and 30. Vimentin ULFs yielded a pronounced heterogeneity, with major peak values of 32 and 45 at two seconds and 30, 37 and 44 after one hour. Desmin formed filaments of distinctly higher mass with 47 molecules per cross-section, at two seconds and after one hour of assembly. This indicates that individual types of IF proteins generate filaments with distinctly different numbers of molecules per cross-section. Also, the observed significant reduction of apparent filament diameter of ULFs compared to the corresponding mature IFs is the result of a "conservative" radial compaction-type reorganization within the filament, as concluded from the fact that both the immature and mature filaments contain very similar numbers of subunits per cross-section. Moreover, the MPL composition of filaments is strikingly dependent on the assembly conditions employed. For example, vimentin fibers formed in 0.7 mM phosphate (pH 7.5), 2.5 mM MgCl2, yield a significantly increased number of molecules per cross-section (56 and 84) compared to assembly under standard conditions. Temperature also strongly influences assembly: above a certain threshold temperature "pathological" ULFs form that are arrested in this state, indicating that the system is forced into strong but unproductive interactions between subunits. Similar "dead-end" structures were obtained with vimentins mutated to introduce principal alterations in subdomains presumed to be of general structural importance, indicating that these sequence changes led to new modes of intermolecular interactions.

Amino Acid Sequence↗