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Biomedical subjects

H Herrmann

Publications and source records attributed to H Herrmann.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H.

A new insertion sequence (IS1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS1383 copies examined. Analysis of the integration sites of IS1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS1383 into a putative new family of insertion sequences defined as the IS1111 family.

Amino Acid Sequence↗
PubMed1999

Immunogold EM reveals a close association of plectin and the desmin cytoskeleton in human skeletal muscle.

Plectin is a multifunctional cytoskeletal linker protein with an intermediate filament-binding site and sequence elements with high homology to actin-binding domains. Mutations of the human plectin gene as well as the targeted inactivation of its murine analog cause a generalized blistering skin disorder and muscular dystrophy, thus implying its essential role in cells that are exposed to mechanical stress. In the present study we report the characterization of two new domain-specific plectin antibodies as well as ultrastructural localization of plectin in normal human skeletal muscle. Using immunogold electron microscopy, we localized plectin at three prominent sites: 1) Plectin is found at regularly spaced intervals along the cytoplasmic face of the plasma membrane. 2) It is distinctly localized at filamentous bridges between Z-lines of peripheral myofibrils and the sarcolemma and 3) at structures forming the intermyofibrillar scaffold. At the latter two locations, plectin and desmin were found to colocalize. Our ultrastructural analysis suggests that plectin may have a central role in the structural and functional organization of the intermediate filament cytoskeleton in mature human skeletal muscle.

3T3 Cells↗
PubMed1999

No evidence for an involvement of alleles of polymorphisms in the serotonin1Dbeta and 7 receptor genes in obesity, underweight or anorexia nervosa.

The serotonergic (5-hydroxytryptamine, 5-HT) system has been implicated in body weight regulation and in the etiology of anorexia nervosa (AN). Here we describe the screening of the known Phe-124-Cys polymorphism in the 5-HT1Dbeta receptor gene and of the known Pro-279-Leu polymorphism in the 5-HT7 receptor gene. For association tests allele frequencies were compared between up to 393 extremely obese children and adolescents, 142 underweight students and 84 patients with AN. None of the association tests revealed nominal P-values below 0.3. We conclude that a major role of the investigated polymorphisms in body weight regulation or AN appears unlikely.

Adolescent↗
PubMed1999

[Voluntary testing procedures of farm animal housing equipment according to the Animal Welfare Act of 1998].

Before its broad application in practice, housing equipment should be tested, in particular with regard to animal welfare. The differing positions of the German Federal Council (Bundesrat) and the German Federal Parliament (Bundestag), whether such testing should be mandatory or voluntary, have been conciliated in the amended animal welfare act by empowering the Federal Ministry of Food, Agriculture and Fisheries (BML) to fix official standards for voluntary testing procedures by regulation. On request of the BML, a report as scientific basis for a draft regulation is currently prepared by the scientific animal welfare committee of the German Agricultural Society (DLG). The scientific animal welfare committee has been appointed by the DLG in order to provide support in the effort to strengthen animal welfare aspects in the DLG-utility testing procedure of housing equipment, which is in place since 1953. The committee elaborates standards concerning testing methods, assessment criteria and the necessary size of investigations. As required, the scientific animal welfare committee may support the DLG-testing bodies in the implementation of the animal welfare part of the testing procedure. It will, moreover, be involved in the welfare assessment based on the testing results. The amendments of the already established testing procedure will help to fulfill the general requirements on an acceptable animal welfare testing procedure. While keeping in mind that there are certain limits in what can be achieved by a voluntary testing procedure, the enhanced consideration of animal welfare aspects within the DLG-utility testing procedure has the advantage to be relatively unbureaucratic and in line with EU legislation, and is, therefore, an appropriate tool for a contibrution to improved animal welfare in livestock housing.

Agriculture↗
PubMed1998

Retinoic acid induction of nuclear envelope-limited chromatin sheets in HL-60.

Exposure of the human leukemic cell line (HL-60) to 1 microM retinoic acid (RA) induces in vitro granulopoiesis, including the development of lobulated nuclei. Ultrastructural studies, presented here, demonstrate the formation of extensive quantities of nuclear envelope-limited chromatin sheets (ELCS), in addition to nuclear lobulation, following treatment with RA. ELCS contain DNA, as shown by the Feulgen-like electron microscope stain osmium ammine-B. Lamin B was demonstrated in ELCS by immunoelectron microscopy with colloidal gold-labeled antibody. Formation of ELCS occurred in Bcl2-overexpressing HL-60 cell sublines with suppressed apoptotic cell death, indicating separable mechanisms for ELCS formation and apoptosis. Immunofluorescent and immunoblotting procedures demonstrated modulations in the amounts and distribution of nuclear envelope-associated components. Total amounts of lamins A/C and cytoplasmic vimentin were reduced by RA treatment. The amounts of lamin B, lamin B receptor (LBR), and lamina-associated polypeptide 2 (LAP2) did not exhibit significant quantitative changes, but acquired heterogeneous staining patterns on the nuclear envelope. RA induced the appearance of low-molecular-weight LBR-related proteins. This study demonstrated the parallel induction of lobulated nuclei and of ELCS and the modulation of nuclear envelope components following exposure of HL-60 to retinoic acid.

Apoptosis↗
PubMed1998

Single-tube nested competitive PCR with homologous competitor for quantitation of DNA target sequences: theoretical description of heteroduplex formation, evaluation of sensitivity, precision and linear range of the method.

Competitive PCR is a frequently used technique for quantitation of DNA and mRNA. However, the application of the most favourable homologous mutated competitors is impeded by the formation of heteroduplex molecules which complicates the data evaluation and may lead to quantitation errors. Moreover, in most cases a single quantitation of an unknown sample requires multiple competitive reactions for identification of the equivalence point. In the present study, a highly efficient and reliable method as well as the underlying theoretical model is described. The mathematical solutions of this model provide the basis for single-tube quantitation using a homologous mutated competitor. For quantitation of Human Papilloma Virus 16-DNA, it is shown that single tube quantitations using simple PAGE separation and video evaluation for signal analysis permit linear detection within more than two orders of magnitude. In addition, repeated single-tube competitive PCRs exhibited good precision (average standard deviation 5%), even if carried out as nested high cycle PCR for quantitation of low abundant sequences (intraassay sensitivity <2 x 10(2) copies). This evaluation method can be applied to any DNA separation and detection method which is capable of resolving the heteroduplex fraction from both homoduplex molecules.

Animals↗
PubMed1998

[Cardiotropic DNA viruses and bacteria in the pathogenesis of dilated cardiomyopathy with or without inflammation].

In the report of the 1995 WHO/ISFC task force on the definition and classification of cardiomyopathies a new entity within the dilated cardiomyopathies was introduced as "inflammatory cardiomyopathy". It is defined as myocarditis associated with cardiac dysfunction. Idiopathic, autoimmune and infectious forms of inflammatory cardiomyopathy are now recognized through this definition. Dilated cardiomyopathy with inflammation (DCMi, chronic myocarditis) was also defined by a recent ISFC task force as > 14 lymphocytes/macrophages/mm3. Enteroviruses, adenoviruses and cytomegaloviruses are considered as main etiopathogenetic factors in the pathogenesis of inflammatory heart disease and have been demonstrated as important trigger for inflammatory cardiac disease. They may also cause dilated cardiomyopathy by viral persistence or secondary immunopathogenesis due to antigenic or molecular mimicry. For the detection of viral persistence the investigation of endomyocardial biopsies in patients with cardiomyopathy by the use of polymerase chain reaction and southern blot analysis is an important step for the standardization of diagnostic criteria on virally induced inflammatory cardiomyopathy. Present studies indicate an incidence of cytomegalovirus-DNA in patients with inflammatory cardiomyopathy in 10%, adenoviral-DNA in 17% and borreliosis only in rare cases (< 1%). In dilated cardiomyopathy without inflammation the respective incidences were for cytomegalovirus 12%, 15% for adenovirus and only 0.5% of cases for borreliosis. In addition the results of immunohistochemical analysis and molecular biological investigations of endomyocardial biopsies may have implications for future therapeutic studies. Depending on the etiology of the disease, immunosuppression may have benefit for patients with virus-negative cardiomyopathy with inflammation in contrast to patients with cytomegalo-, adenovirus-DNA or enteroviral persistence, in whom immunomodulation with hyperimmunoglobulins or immunoglobulins may be a feasible therapeutic option. Patients with a positive PCR for Borrelia burgdorferi should be treated with 3rd generation cephalosporines and/or sublactam.

Biopsy↗
PubMed1998

The transposable elements resident on the plasmids of Pseudomonas putida strain H, Tn5501 and Tn5502, are cryptic transposons of the Tn3 family.

Genes for (methyl)phenol degradation in Pseudomonas putida strain H (phl genes) are located on the plasmid pPGH1. Adjacent to the phl catabolic operon we identified a cryptic transposon, Tn5501, of the Tn3 family (class II transposons). The genes encoding the resolvase and the transposase are transcribed in the same direction, as is common for the Tn501 subfamily. The enzymes encoded by Tn5501, however, show only the overall homology characteristic for resolvases/integrases and transposases of Tn3-type transposons. Therefore it is likely that Tn5501 is not a member of one of the previously defined subfamilies. Inactivation of the conditional lethal sacB gene was used to detect transposition of Tn5501. While screening for transposition events we found another transposon integrated into sacB in one of the sucrose-resistant survivors. This element, Tn5502, is a composite transposon consisting of Tn5501 and an additional DNA fragment. It is flanked by inverted repeats identical to those of Tn5501 and the additional fragment is separated from the Tn5501 portion by an internal repeat (identical to the left terminal repeat). Transposition of phenol degradation genes could not be detected. Analysis of sequence data revealed that the phl genes are not located on a Tn5501-like transposon.

Base Sequence↗
PubMed1998

Zebrafish vimentin: molecular characterization, assembly properties and developmental expression.

To provide a basis for the investigation of the intermediate filament (IF) protein vimentin in one of the most promising experimental vertebrate systems, the zebrafish (Danio rerio), we have isolated a cDNA clone of high sequence identity to and with the characteristic features of human vimentin. Using this clone we produced recombinant zebrafish vimentin and studied its assembly behaviour. Unlike other vimentins, zebrafish vimentin formed unusually thick filaments when assembled at temperatures below 21 degrees C. At 37 degrees C few filaments were observed, which often also terminated in aggregated masses, indicating that its assembly was severely disturbed at this temperature. Between 21 and 34 degrees C apparently normal IFs were generated. By viscometry, the temperature optimum of assembly was determined to be around 28 degrees C. At this temperature, zebrafish vimentin partially rescued, in mixing experiments, the temperature-dependent assembly defect of trout vimentin. Therefore it is apparently able to "instruct" the misorganized trout vimentin such that it can enter normal IFs. This feature, that assembly is best at the normal body temperature of various species, puts more weight on the assumption that vimentin is vital for some aspects of generating functional adult tissues. Remarkably, like in most other vertebrates, zebrafish vimentin appears to be an abundant factor in the lens and the retina as well as transiently, during development, in various parts of the central and peripheral nervous system. Therefore, promising cell biological investigations may now be performed with cells involved in the generation of the vertebrate eye and brain, and, in particular, the retina. Moreover, the power of genetics of the zebrafish system may be employed to investigate functional properties of vimentin in vivo.

Amino Acid Sequence↗
PubMed1998

Intermediate filament assembly: fibrillogenesis is driven by decisive dimer-dimer interactions.

Intermediate filaments are built from one to several members of a multigene family encoding fibrous proteins that share a highly conserved hierarchic assembly plan for the formation of multistranded filaments from distinctly structured extended coiled coils. Despite the rather low primary sequence identity, intermediate filaments form apparently similar filaments with regard to their spatial dimensions and physical properties. Over the past few years, substantial progress has been made in the elucidation of the complex expression patterns and clinically relevant phenotypes of intermediate filaments. The key question of how these filaments assemble and what the molecular architecture of their distinct assembly intermediates comprises, however, has still not been answered to the extent that has been achieved for microfilaments and microtubules.

Animals↗
PubMed1998

Identification of an interchromosomal compartment by polymerization of nuclear-targeted vimentin.

A number of structural and functional subnuclear compartments have been described, including regions exclusive of chromosomes previously hypothesized to form a reactive nuclear space. We have now explored this accessible nuclear space and interchromosomal nucleoplasmic domains experimentally using Xenopus vimentin engineered to contain a nuclear localization signal (NLS-vimentin). In stably transfected human cells incubated at 37 degrees C, the NLS-vimentin formed a restricted number of intranuclear speckles. At 28 degrees C, the optimal temperature for assembly of the amphibian protein, NLS-vimentin progressively extended with time out from the speckles into strictly orientated intranuclear filamentous arrays. This enabled us to observe the development of a system of interconnecting channel-like areas. Quantitative analysis based on 3-D imaging microscopy revealed that these arrays were localized almost exclusively outside of chromosome territories. During mitosis the filaments disassembled and dispersed throughout the cytoplasm, while in anaphase-telophase the vimentin was recruited back into the nucleus and reassembled into filaments at the chromosome surfaces, in distributions virtually identical to those observed in the previous interphase. The filaments also colocalized with specific nuclear RNAs, coiled bodies and PML bodies, all situated outside of chromosome territories, thereby interlinking these structures. This strongly implies that these nuclear entities coexist in the same interconnected nuclear compartment. The assembling NLS-vimentin is restricted to and can be used to delineate, at least in part, the formerly proposed reticular interchromosomal domain compartment (ICD). The properties of NLS-vimentin make it an excellent tool for performing structural and functional studies on this compartment.

Adrenal Cortex Neoplasms↗
PubMed1997

Studies on spontaneous promoter-up mutations in the transcriptional activator-encoding gene phIR and their effects on the degradation of phenol in Escherichia coli and Pseudomonas putida.

The activator-encoding gene phlR was identified upstream of the plasmid-encoded operon for phenol degradation in Pseudomonas putida strain H by cassette mutagenesis and DNA sequence analysis. The deduced amino acid sequence of PHLR shows high homology to DmpR of P. putida sp. CF600 and to the chromosomally encoded PhhR of P. putida P35X reported previously. Trans-activation of phenol degradation was observed when phlR was overexpressed in a phlR insertion mutant. Transconjugants of Escherichia coli carrying pPGH11, which contains the complete set of phl genes, are unable to grow on phenol as carbon source. However, two types of mutants were selected for further characterization that were able to metabolize phenol as sole source of carbon and energy. In both types of mutants enhanced expression of phlR is responsible for the Phl+ phenotype. In type I (pPGH13) a deletion of 1 bp made the -35 region and the spacing between the -35 and -10 regions of the phlR promoter more similar to the consensus structure. In type II (pPGH14) a duplication of the phlR 5' region was identified that includes part of the -35 motif and reduces the spacing between the -35 and -10 regions. In addition, due to the duplication of part of phlR, the distance from the phlR promoter to the catabolic phl operon is increased. Different transcriptional start sites have been identified by primer extension analysis in clones harboring pPGH14 or the wild type phlR. Quantitative primer extension analysis revealed that the greatest amount of phlR transcript is expressed from the partial, phlR duplication. Growth on phenol and phenol hydroxylase activity reflect the high level of phlR transcript in E. coli transconjugants. Overexpression of PhlR was also observed when pPGH14 was transferred into P. putida, and results in earlier induction of the phenol degradation operon relative to the wild-type strain.

Bacterial Proteins↗
PubMed1996

Structure and assembly properties of the intermediate filament protein vimentin: the role of its head, rod and tail domains.

We have investigated the functional role of the non-helical end domains of vimentin on its assembly properties using truncated Xenopus and human recombinant proteins. Removal of the amino-terminal "head" domain yielded a molecule that did not assemble into 10 nm filaments but remained in a soluble oligomeric particle form with a sedimentation coefficient considerably smaller than that of wild-type vimentin (Vim(wt)). In contrast, removal of the carboxy-terminal "tail" domain had no obvious effect on the sedimentation characteristics. In particular, sedimentation equilibrium analysis under low ionic strength conditions yielded oligomeric particle species of Mr 135,000 to 360,000, indistinguishable from those obtained with Vim(wt). When induced to form filaments from this state by rapid dilution into filament forming buffer, Vim(wt) and Vim(deltaT) protein generated similar viscosity profiles. However, as determined by scanning transmission electron microscopy, under these conditions Vim(deltaT) formed filaments of heterogeneous diameter, corresponding to various distinct mass-per-length (MPL) values: whereas Vim(wt) yielded MPL values peaking between 40 and 45 kDa/nm, Vim(deltaT) filaments produced histograms which could be fitted by three Gaussian curves peaking between 37 and 131 kDa/nm. In contrast, when dialyzed against, instead of being rapidly diluted into, filament forming buffer, Vim(deltaT) gave histograms with one major peak at about 54 kDa/nm. The MPL heterogeneity observed for Vim(deltaT) was already evident at the earliest stages of assembly. For example, ten seconds after initiation, "unit-length" filament segments (58 to 63 nm) were formed with both wt and deltaT proteins, but the diameters were considerably larger for Vim(deltaT) compared to Vim(wt) (20(+/- 3) nm versus 16(+/- 3)nm), indicating a distinct role of the carboxy-terminal tail domain in the width control during unit-length filament formation. Despite this difference both Vim(deltaT) and Vim(wt) filaments appeared to grow stepwise in a modular fashion from such unit-length filament segments. This suggests that assembly occurred by a principally similar mechanism involving the end-on-fusion or annealing of unit-length filaments.

Amino Acid Sequence↗