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Klaus Weber

Publications and source records attributed to Klaus Weber.

At least 19 recordsLinked to original sources
PubMed2005

The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity.

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.

Animals↗
PubMed2005

Endotoxin elicits nitric oxide release in rat but prostacyclin synthesis in human and bovine vascular smooth muscle cells.

Lipopolysaccharide (LPS) exposure to cells and tissues can mimic the biochemical events leading to septic shock. Previous data demonstrated a massive upregulation of prostaglandin endoperoxide H2 synthase (PGHS-2), but not NO synthase-2 (NOS-2) in bovine smooth muscle cells (SMC) between 2 and 12 h of LPS exposure. This caused an abundant release of prostacyclin (PGI2) by constitutive PGI2-synthase as a counterregulation to a dysfunctional endothelium. We here report that human as well as bovine SMC mainly respond by the induction of PGHS-2 and the subsequent release of PGI2, whereas rat SMC exhibited a distinct induction of NOS-2 and released significantly higher amounts of *NO compared with cattle and human. The induction of either PGHS-2 or NOS-2 in the three different species investigated seems to be mutually exclusive in the time window of 2-24 h. This finding should be considered in the setup of experimental models for the investigation of septic shock.

Animals↗
PubMed2005

RNAi of FACE1 protease results in growth inhibition of human cells expressing lamin A: implications for Hutchinson-Gilford progeria syndrome.

FACE 1 is the endoprotease responsible for cleavage of prelamin A to lamin A. Transfection of HeLa cells with siRNA for human FACE 1 results in a strong phenotype. Protein and mRNA levels for FACE 1 are knocked down and cell division stops abruptly. Two populations of cells are detected. The first form aberrant mitotic spindles, arrest in mitosis and later enter apoptosis. The second show dramatic changes in nuclear morphology with extensive formation of lobulated nuclei and micronuclei. Using antibodies that specifically recognise prelamin A, but not lamin A, we show that prelamin A accumulates at the nuclear lamina in FACE1 silenced cells, whereas in control cells prelamin A is found in many small nuclear dots, but not at the nuclear lamina. In double knockdown experiments with FACE 1 and lamin A siRNAs, the results depend on which protein is knocked down first. FACE1 knockdown 24 hours prior to lamin A knockdown gives results similar to the single FACE1 knockdown. By contrast, lamin A knockdown 24 hours prior to FACE1 knockdown results in none of the changes described above. Silencing of FACE1 in HL60, a cell line that lacks lamin A, also has no effect. The combined results suggest that prelamin A is a poison in cells subjected to FACE 1 knockdown. Finally, we draw attention to similarities in phenotype between FACE1-silenced HeLa cells and fibroblasts from patients with Hutchinson-Gilford progeria syndrome containing prelamin A mutations that prevent cleavage by the FACE1 endoprotease.

Cell Line↗
PubMed2005

Terrestrial vertebrates have two keratin gene clusters; striking differences in teleost fish.

Keratins I and II form the largest subgroups of mammalian intermediate filament (IF) proteins and account as obligatory heteropolymers for the keratin filaments of epithelia. All human type I genes except for the K18 gene are clustered on chromosome 17q21, while all type II genes form a cluster on chromosome 12q13, that ends with the type I gene K18. Highly related keratin gene clusters are found in rat and mouse. Since fish seem to lack a keratin II cluster we screened the recently established draft genomes of a bird (chicken) and an amphibian (Xenopus). The results show that keratin I and II gene clusters are a feature of all terrestrial vertebrates. Because hair with its multiple hair keratins and inner root sheath keratins is a mammalian acquisition, the keratin gene clusters of chicken and Xenopus tropicalis have only about half the number of genes found in mammals. Within the type I clusters all genes have the same orientation. In type II clusters there is a rare gene of opposite orientation. Finally we show that the genes for keratins 8 and 18, which are the first expression pair in embryology, are not only adjacent in mammals, but also in Xenopus and three different fish. Thus neighboring K8 and K18 genes seem a feature shared by all vertebrates. In contrast to the two well defined keratin gene clusters of terrestrial vertebrates, three teleost fish show an excess of type I over type II genes, the lack of a keratin type II gene cluster and a striking dispersal of type I genes, that are probably the result of the teleost-specific whole genome duplication followed by a massive gene loss. This raises the question whether keratin gene clusters extend beyond the ancestral bony vertebrate to cartilage fish and lamprey. We also analyzed the complement of non-keratin IF genes of the chicken. Surprisingly, an additional nuclear lamin gene, previously overlooked by cDNA cloning, is documented on chromosome 10. The two splice variants closely resemble the lamin LIII a + b of amphibia and fish. This lamin gene is lost on the mammalian lineage.

Amphibians↗
PubMed2005

Critical appraisal of reports on the treatment of perioral dermatitis.

BACKGROUND: Presently, problems exist with the rationale of oral therapy and the nature and indication of topical and accompanying treatment of perioral dermatitis. OBJECTIVE: Providing the basis to overcome these problems by a quality evaluation of treatment reports and assessment of the consistency of treatment experience. METHODS: Sources were Medline (1964-2004), Embase (1966-2004), the Cochrane Central (1971-2004) and 526 references of 3 textbooks, 2 recent reviews and 30 papers on perioral dermatitis. Thirty English and German articles were selected. These studies were evaluated according to principles of evidence-based medicine and related criteria. Evaluation of 28 papers was carried out by the authors and of our own 2 papers by 2 other reviewers. Consistency of results was qualitatively assessed by the authors. RESULTS: There were only 2 therapeutic trials of medium-range quality. The other studies were of low quality. Consistency was noted concerning treatment with oral tetracycline (with 1 exception), discontinuation of topical corticosteroids and cosmetics and, to a lesser extent, regarding no therapy. There was inconsistency in respect to topical therapy. CONCLUSION: The presented data help to interpret and conduct studies on the treatment of perioral dermatitis.

Administration, Oral↗
PubMed2004

The genome of the early chordate Ciona intestinalis encodes only five cytoplasmic intermediate filament proteins including a single type I and type II keratin and a unique IF-annexin fusion protein.

We screened the recently established draft genome of the early chordate Ciona intestinalis for genes encoding cytoplasmic intermediate filament (IF) proteins. The draft of the tunicate/urochordate genome contains only the five genes (IF-A, IF-B, IF-C, IF-D and IF-F) previously established by cDNA cloning. Three of these IF proteins (IF-D, IF-C, IF-A) were shown to be orthologs of vertebrate IF subfamilies I to III while two proteins (IF-B, IF-F) seemed tunicate specific. This is now firmly established for protein IF-F since the genomic data show that it arises as a fusion protein with a C-terminal annexin domain, a feature not found before in the very large collection of metazoan IF proteins. The results also confirm the previous proposal that urochordates lack orthologs of vertebrate type IV IF proteins. We discuss the striking increase of IF complexity from 5 tunicate to 65 human genes during chordate evolution. Thus the tunicate has a single keratin pair, which is expressed in the epidermis, while the human genome has at least 25 genes each for keratins I and keratins II. Finally there are four normal Ciona annexin genes in addition to the gene encoding the IF-annexin fusion proteins (IF-F).

Amino Acid Sequence↗
PubMed2004

Altered coexpression of lectin-like receptors CD94 and CD161 on NK and T cells in HIV patients.

HIV patients either on highly active antiviral therapy (HAART) or therapy naive were analyzed for their CD56 phenotype and cytokine production in comparison to healthy controls (HC). Both NK cell populations (CD56(dim) and CD56(bright)) are found to be present in all groups with selectively decreased CD56(dim) NK cells in HAART naive patients. Patients on HAART exhibited significantly diminished numbers of CD161+CD56(dim) NK cells. CD56(dim) were equally potent in producing IFNgamma in all three groups. The number of TNFalpha+CD56(bright) NK cells from patients on HAART and TNFalpha+CD56(dim) NK cells from HAART naive patients was significantly reduced as compared to healthy controls. In summary our data revealed that functional capacities and coexpression patterns of lectin-like receptors on lymphocytes are differentially affected in HIV patients depending on the state of therapy (under HAART or HAART naive) or the cell type (NK or T cells), respectively.

Antigens, CD↗
PubMed2004

Topical cosmetics and perioral dermatitis.

BACKGROUND: Perioral dermatitis (PD) is a common dermatological disease whose aetiology and pathogenesis remain speculative. Both cosmetics and topical corticosteroids have been implicated. PATIENTS AND METHODS: 75 patients with PD and 125 randomly selected control patients with negative PD records were included in a questionnaire-based study focusing on cosmetics, their use, and topical corticosteroids. The patients were assessed for atopy by clinical criteria, prick tests and specific IgE against a mixture of aeroallergens (CAP SX 1). RESULTS: There were no significant (p < 0.05) differences in facial skin cleansing between the two groups except for night-time cleansing, which was performed more often in the patient group. The use of skin care products yielded significant differences between the patient and control group in respect to skin care in the morning and the use of day creams. Otherwise, the groups did not differ in their skin care regime (number of skin care products used, type of product, frequency of care). The mean monthly expenditure on cosmetic products did not differ significantly between the two groups. 25.3% of the patients used topical corticosteroids which were initiated in all cases after the onset of the rash. Significantly (p < 0.001) higher values were found in the patient group regarding history of atopic diseases (49.3% vs. 15.2%), prick test reactivity (> or =2 reactive prick tests: 49.3% vs. 8.0%), and specific IgE against aeroallergens (CAP SX1 classes > or =2: 50.7% vs. 15.2%). CONCLUSIONS: The often heard argument that PD results mainly from external factors is no longer tenable. A variable interaction between external (e.g. cosmetics) and intrinsic factors (e.g. atopic constitution) appears to lead to PD, perhaps by subclinical repetitive irritation and final overtaxing of the reparative capacity of the epithelial barrier function.

Administration, Topical↗
PubMed2004

Comprehensive analysis of keratin gene clusters in humans and rodents.

Here, we present the comparative analysis of the two keratin (K) gene clusters in the genomes of man, mouse and rat. Overall, there is a remarkable but not perfect synteny among the clusters of the three mammalian species. The human type I keratin gene cluster consists of 27 genes and 4 pseudogenes, all in the same orientation. It is interrupted by a domain of multiple genes encoding keratin-associated proteins (KAPs). Cytokeratin, hair and inner root sheath keratin genes are grouped together in small subclusters, indicating that evolution occurred by duplication events. At the end of the rodent type I gene cluster, a novel gene related to K14 and K17 was identified, which is converted to a pseudogene in humans. The human type II cluster consists of 27 genes and 5 pseudogenes, most of which are arranged in the same orientation. Of the 26 type II murine keratin genes now known, the expression of two new genes was identified by RT-PCR. Kb20, the first gene in the cluster, was detected in lung tissue. Kb39, a new ortholog of K1, is expressed in certain stratified epithelia. It represents a candidate gene for those hyperkeratotic skin syndromes in which no K1 mutations were identified so far. Most remarkably, the human K3 gene which causes Meesmann's corneal dystrophy when mutated, lacks a counterpart in the mouse genome. While the human genome has 138 pseudogenes related to K8 and K18, the mouse and rat genomes contain only 4 and 6 such pseudogenes. Our results also provide the basis for a unified keratin nomenclature and for future functional studies.

Animals↗
PubMed2004

Most genes encoding cytoplasmic intermediate filament (IF) proteins of the nematode Caenorhabditis elegans are required in late embryogenesis.

Intestinal cells of C. elegans show an unexpectedly high complexity of cytoplasmic intermediate filament (IF) proteins. Of the 11 known IF genes six are coexpressed in the intestine, i.e. genes B2, C1, C2, D1, D2, and E1. Specific antibodies and GFP-promoter constructs show that genes B2, D1, D2, and E1 are exclusively expressed in intestinal cells. Using RNA interference (RNAi) by microinjection at 25 degrees C rather than at 20 degrees C we observe for the first time lethal phenotypes for C1 and D2. RNAi at 25 degrees C also shows that the known A1 phenotype occurs already in the late embryo after microinjection and is also observed by feeding which was not the case at 20 degrees C. Thus, RNAi at 25 degrees C may also be useful for the future analysis of other nematode genes. Finally, we show that triple RNAi at 20 degrees C is necessary for the combinations B2, D1, E1 and B2, D1, D2 to obtain a phenotype. Together with earlier results on genes A1, A2, A3, B1, and C2 RNAi phenotypes are now established for all 11IF genes except for the A4 gene. RNAi phenotypes except for A2 (early larval lethality) and C2 (adult phenotype) relate to the late embryo. We conclude that in C. elegans cytoplasmic IFs are required for tissue integrity including late embryonic stages. This is in strong contrast to the mouse, where ablation of IF genes apparently does not affect the embryo proper.

Amino Acid Sequence↗
PubMed2004

RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

3T3 Cells↗
PubMed2003

In vivo and in vitro evidence that the four essential intermediate filament (IF) proteins A1, A2, A3 and B1 of the nematode Caenorhabditis elegans form an obligate heteropolymeric IF system.

The in vitro polymerization and tissue-specific expression patterns of the four essential intermediate filament (IF) proteins (A1, A2, A3, B1) and the non-essential IF protein A4 were analyzed. Recombinant B1, used as a probe in blot overlay assays of the 11 Caenorhabditis elegans IF proteins, reacted strongly with proteins A1 to A4, indicating a heterotypic interaction. Obligate heteropolymeric filament assembly in vitro was confirmed by electron microscopy. Protein B1 formed long IF when mixed with an equimolar amount of A1, A2 or A3. Developmentally regulated coexpression of B1 and one or more members of the A family was found with GFP-promoter reporters. This coexpression pattern argues for a heteropolymer system in vivo. One or both splice variants of the B1 gene are always coexpressed in a tissue-specific manner with at least one member of the A family in hypodermis, pharynx, pharyngeal-intestinal valve, excretory cells, uterus, vulva and rectum. Interestingly, while the intestine normally lacks a B1/A pair, the dauer larva shows intestinal B1 and A4. These results are in line with similar postembryonic phenotypes of the hypodermis induced by RNA interference (RNAi) of genes B1, A2 and A3. Similarly, defects of the pharynx and its A1-GFP containing tonofilaments observed in the postembryonic B1 RNAi phenotype are consistent with the coexpression of B1 and A1 in the marginal cells. Thus RNAi analyses provide independent evidence for the existence of the B1/A obligate heteropolymer system in vivo. Proteins A1 and B1 have a similar and rather slow turnover rate in photobleaching experiments of the pharynx tonofilaments.

Amino Acid Sequence↗
PubMed2003

Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid.

Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.

Aspartic Acid↗
PubMed2003

Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells.

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.

Adaptor Protein Complex alpha Subunits↗
PubMed2003

Effects of expressing lamin A mutant protein causing Emery-Dreifuss muscular dystrophy and familial partial lipodystrophy in HeLa cells.

Patients with the autosomal dominant form of Emery-Dreifuss muscular dystrophy (EDMD) or familial partial lipodystrophy (FPLD) have specific mutations in the lamin A gene. Three such point mutations, G465D (FPLD), R482L, (FPLD), or R527P (EDMD), were introduced by site-specific mutagenesis in the C-terminal tail domain of a FLAG-tagged full-length lamin A construct. HeLa cells were transfected with mutant and wild-type constructs. Lamin A accumulated in nuclear aggregates and the number of cells with aggregates increased with time after transfection. At 72 h post transfection 60-80% of cells transfected with the mutant lamin A constructs had aggregates, while only 35% of the cells transfected with wild-type lamin A revealed aggregates. Mutant transfected cells expressed 10-24x, and wild-type transfected cells 20x, the normal levels of lamin A. Lamins C, B1 and B2, Nup153, LAP2, and emerin were recruited into aggregates, resulting in a decrease of these proteins at the nuclear rim. Aggregates were also characterized by electron microscopy and found to be preferentially associated with the inner nuclear membrane. Aggregates from mutant constructs were larger than those formed by the wild-type constructs, both in immunofluorescence and electron microscopy. The combined results suggest that aggregate formation is in part due to overexpression, but that there are also mutant-specific effects.

Animals↗
PubMed2003

Genes coding for intermediate filament proteins: common features and unexpected differences in the genomes of humans and the teleost fish Fugu rubripes.

We screened the genomic sequences of the teleost fish Fugu rubripes for genes that encode cytoplasmic intermediate filament (IF) proteins. Here, we compare the number of genes per subfamily (I to IV) as well as the gene mapping in the human and fish genomes. There are several unexpected differences. F. rubripes has a sizeable excess of keratin type I genes over keratin type II genes. Four of the six keratin type II genes map close to four keratin type I genes. Thus, a single keratin II gene cluster (as in mammals) seems excluded. Although a continuous genome sequence is not yet available for F. rubripes, it is difficult to see how all 19 keratin type I genes can be collected as in the human genome into a single cluster without the presence of type II genes and various unrelated genes. F. rubripes has more type III and type IV genes than humans. Some of the type IV genes acquired additional novel intron positions. One gene even harbors (in addition to the two type IV introns) three novel introns and three introns usually present only in mammalian and F. rubripes type I-III genes. This mixture of type IV and type I-III intron positions poses a problem for the traditional view that the first type IV gene arose in evolution by a mRNA-mediated translocation event. In the 42 F. rubripes genes analysed here, there are several differences in intron patterns compared with mammalian genes. Most correspond to additional introns in the fish genes. A search for genes encoding nuclear lamins reveals the four established fish lamins (A, B1, B2 and LIII) as well as an unexpected second lamin A.

Amino Acid Sequence↗
PubMed2003

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity.

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.

Amino Acid Sequence↗