Biomedical subjects

T Wirth

Publications and source records attributed to T Wirth.

At least 37 records · Page 2Linked to original sources

PubMed1995

Lymphoid- and myeloid-specific activity of the PU.1 promoter is determined by the combinatorial action of octamer and ets transcription factors.

The putative oncogene PU.1/Spi-1 is a member of the ets-family of transcription factors normally expressed in a subset of hematopoietic cell types. Here we have characterized the role of the PU.1 promoter region for the cell-type specific expression. The proximal 120 bp are sufficient to mediate a high level of activity specifically in B cells and macrophages. Three important motifs could be identified within this region. Two of them, an ets binding site (EBS) and a variant octamer motif were most important for cell-type-specific promoter activity in B cells and macrophages. An additional Sp1 motif stimulates basal activity of this promoter element. The relative contribution to overall activity of octamer motif and EBS differs in B cells and macrophages. In B cells, both octamer motif and EBS combine to mediate high level activity, whereas in macrophages the EBS predominantly confers promoter activity. Both the Oct1 and Oct2 transcription factors, presumably in combination with a B-cell-restricted coactivator, are responsible for the activity of the variant octamer motif in B cells. Interestingly, the PU.1 transcription factor can functionally interact with the EBS in its own promoter, suggesting a positive feedback regulation.

3T3 Cells↗

PubMed1995

Identification of CD36 as the first gene dependent on the B-cell differentiation factor Oct-2.

The Oct-2 transcription factor is expressed predominantly in B lymphocytes and has been shown previously to be important for the terminal phase of B-cell differentiation in mice. A number of genes specifically expressed in B cells contain Oct-2-binding sites in their regulatory regions. However, the analysis of expression levels of these genes in Oct-2-deficient B cells revealed that they were unaffected. Hence, there were no genes known that critically depend on Oct-2 for their expression. To understand the molecular basis for the Oct-2 effect on B-cell development, we searched for Oct-2 target genes by subtractive cDNA cloning. We show here that expression of the murine CD36 gene in B cells and macrophages requires a functional Oct-2 protein. Nuclear run-on experiments demonstrate that this gene is regulated transcriptionally by Oct-2. Moreover, CD36 levels correlated with the levels of Oct-2 expression in several mouse B-cell and macrophage cell lines. Finally, compared to wild-type and heterozygous mice, CD36 mRNA levels were markedly reduced in spleens and B-cell-enriched splenocyte fractions from oct-2-/- mice. The data identify CD36 as the first target gene critically dependent on Oct-2 for its expression. Because CD36 expression is also dependent on Oct-2 in vivo, it is a candidate gene through which Oct-2 could affect B-cell differentiation.

Animals↗

PubMed1995

High mobility group protein 2 functionally interacts with the POU domains of octamer transcription factors.

The octamer transcription factors Oct1 and Oct2 are involved in the transcriptional regulation of both lymphoid-specific and ubiquitously expressed genes. Their activity depends critically on their interaction with distinct cellular cofactors. Therefore, we have isolated cDNAs encoding proteins that physically interact with Oct2. Here we describe the analysis of one such clone, representing the murine homologue of high mobility group (HMG) protein 2. We have mapped the interaction domains for both proteins and have shown that HMG2 and Oct2 interact via their HMG domains and POU homeodomains, respectively. This interaction is not restricted to Oct2, as other members of the octamer transcription factor family like Oct1 and Oct6 also interact with HMG2. The interaction with HMG2 results in a marked increase in the sequence-specific DNA binding activity of the Oct proteins. Interestingly, the HMG2 protein is not present in the protein-DNA complex detected by an electrophoretic mobility shift assay. The Oct and HMG2 proteins also interact in vivo. A chimeric protein, in which the strong transactivation domain of VP16 was fused directly to the HMG domains of HMG2, stimulated the activity of an octamer-dependent reporter construct upon cotransfection. Furthermore, the expression of antisense RNA for HMG2 specifically reduces octamer-dependent transcription. These results suggest that one of the functions of HMG2 is to support the octamer transcription factors in their role as transcriptional activators.

Amino Acid Sequence↗

PubMed1995

False-positive sonographic hip examinations in newborns with congenital varus deformity of the proximal femur.

In cases of congenital varus deformity of the proximal femur, the screening of hip dysplasia can lead to misinterpretations. The false-positive ultrasound result is caused by the superiorly displaced greater trochanter in hips with coxa vara, which narrows the scan window. In this paper three typical patients are presented to demonstrate that the use of additional non-standard views cannot exclude hip dysplasia in all cases. Hence, such patients with femoral abnormalities and doubtful sonographic findings should be further evaluated by arthrography or magnetic resonance imaging to rule out additional hip dysplasia.

False Positive Reactions↗

PubMed1995

Molecular principles of Oct2-mediated gene activation in B cells.

The octamer motif is a crucial regulatory element for immunoglobulin promoter and enhancer function. We have investigated the molecular mechanisms that underlie octamer-mediated gene activation in B cells. This B cell-specific transcriptional regulation is subject to a novel type of regulatory mechanism. We could demonstrate that octamer-dependent transcription is not only regulated by specific DNA-binding transcription factors, but in addition requires the activity of B cell-restricted cofactors. Both octamer-dependent promoter and enhancer activation depend on such a combination of transcription factor and cofactors. However, the exact requirements differ for these two situations. Promoter activity can be achieved with either one of two distinct transcription factors, Oct1 and/or Oct2, together with the cofactor OCA-B1. In contrast, only Oct2 in conjunction with an additional cofactor, OCA-B2, can confer enhancer activity.

Animals↗

PubMed1994

Functional differences between the Oct2 transactivation domains determine the transactivation potential of individual Oct2 isoforms.

The lymphocyte specific transcription factor Oct2 is involved in mediating the B-cell specific transcriptional activity of the octamer motif. Mutational analyses in the context of the complete Oct2 protein had indicated that Oct2 contains two transactivation domains. These two domains appeared to be redundant for activation from a promoter proximal position, whereas stimulation from a remote enhancer position specifically required the C-terminal transactivation domain and an additional B-cell restricted activity. We have generated fusion proteins between the DNA binding domain of the yeast Gal4 transcription factor and individual Oct2 protein domains to analyze their transactivation potential separately. We show that both N- and C-terminal domains can stimulate transcription from a promoter proximal position independently. However, only the C-terminal transactivation domain activates from a distance and it can only do so in B-cells. The C-terminal transactivation domain represents a composite transactivation domain. Whereas removal of just 9 aminoacids from the extreme C-terminus lead to a complete inactivation of this domain deletions from the other side resulted in a gradual loss of activity. We also characterized the transactivation potential of different N-terminal regions of Oct2 generated by alternative splicing. We show that the N-terminus of one of the isoforms, Oct2.3, contains a negative regulatory domain (NRD), which can inactivate the neighbouring glutamine-rich transactivation in cis. The presence of this NRD affects the overall phosphorylation state of the Oct2 protein. This result suggests that the mechanism of inactivation might involve differential protein phosphorylation.

Amino Acids↗

PubMed1994

Two distinct mechanisms contribute to the constitutive activation of RelB in lymphoid cells.

The kappa B-motif is an important regulatory element both for constitutive lymphoid-specific as well as ubiquitous inducible transcriptional activity. We have shown previously that different members of the NF-kappa B/Rel family of transcription factors are responsible for these distinct functions. Whereas the p65/RelA protein is involved in inducible kappa B-dependent transcription, RelB is associated with constitutive activity in lymphoid cells. Here we have addressed the question of how RelB is constitutively activated in lymphoid cells. We demonstrate that this is achieved by two different mechanisms. Expression of relB as determined by measurement of stable RNA and protein levels is significantly enhanced in lymphoid organs compared with non-lymphoid organs. However, these observed differences in absolute amounts of RNA and protein would not suffice to explain the dramatic differences that are apparent at the level of active DNA binding complexes in extracts from the respective organs. We have therefore analyzed the interaction of RelB complexes with the I kappa B-alpha inhibitor protein. Our results show that RelB-containing complexes present in lymphoid extracts are much less susceptible to inhibition by I kappa B-alpha than RelA- or RelB-containing complexes from non-lymphoid cells. This difference might be due to post-translational modifications of the RelB protein or interaction with a lymphoid-specific cofactor for RelB.

Animals↗

PubMed1994

The POU domains of the Oct1 and Oct2 transcription factors mediate specific interaction with TBP.

We had previously shown that the ubiquitous Oct1 and the lymphoid-specific Oct2 transcription factors stimulate transcription at the level of stable preinitiation complex formation. We have therefore investigated whether the octamer binding proteins might physically interact with TBP, the TATA box binding protein component of the TFIID factor. By using several different experimental systems we show that TBP efficiently associates with Oct1 and Oct2. The interaction is direct and does not depend on the presence of DNA or additional proteins. N- and C-terminal deletions of the different proteins were used to localize the domains involved in the interaction. We show that the POU homeodomain of Oct2 and the evolutionarily conserved C-terminal core domain of TBP are both required and sufficient for the interaction. The Oct1 POU domain, which is highly homologous to the Oct2 POU domain, likewise mediates interaction with TBP. The interaction can also be observed in vivo, as TBP can be co-precipitated with Oct2 from co-transfected Cos1 cells and TBP co-immunoprecipitates with the endogenous Oct1 from HeLa cells. Co-transfection of human TBP and Oct2 expression vectors into B cells resulted in a synergistic activation of an octamer motif containing promoter.

Binding Sites↗

PubMed1994

Differential transactivation potential of Oct1 and Oct2 is determined by additional B cell-specific activities.

Cell type-specific transcriptional regulation is generally believed to be mediated by sequence-specific transcription factors that are specifically present in the corresponding cells. The interaction of the lymphoid-specific Oct2 transcription factor has been thought to be responsible for the B cell-specific activity of octamer-containing promoter and enhancer elements. Here we show that physiological concentrations of Oct2 do not suffice to generate octamer-dependent promoter activity in non-B cell lines. Furthermore, we have tested the activity of octamer-dependent promoter and enhancer elements in B cell lines that lack the endogenous Oct2 protein. Our results demonstrate that in these Oct2-deficient B cells the ubiquitous endogenous Oct1 protein is able to stimulate octamer-containing promoters to a level comparable with that of normal Oct2-positive B cells. However, reporter constructs bearing the octamer motif in a distal enhancer position are not stimulated by the Oct1 protein, but do require the presence of Oct2. The B cell-specific octamer-dependent promoter activity mediated by Oct1 correlates with the presence of a novel B cell-specific octamer-binding complex containing the Oct1 protein. From these results we conclude that B cells contain two different activities: one that interacts with both Oct1 and Oct2 and mediates promoter proximal activity of the octamer motif and a second that specifically interacts with Oct2 to confer function from a remote enhancer position.

Animals↗

PubMed1994

The implantation of cartilaginous and periosteal tissue into growth plate defects.

This experimental study reports the results of implantation of cartilaginous and periosteal tissues into growth plate defects in the tibiae of sheep. When no material was used, the defect rapidly filled with marrow-like tissue. When cartilage from the margin of the secondary centre of ossification was implanted, endochondral ossification continued and no shortening or deformity resulted. Implantation of periosteum with or without reconstructed peripheral tissues resulted in the formation of a bony bridge which led to a 32% inhibition of longitudinal growth and a 12 degrees varus deformity in the absence of peripheral connective tissues. After reconstruction with these tissues, the inhibition of longitudinal growth was 47% with a 28 degrees varus deformity. The chondroprogenitor cells in the implanted tissues cannot change phenotypic expression. Periosteum has a strong potential for bone formation after it has been implanted.

Animals↗

PubMed1994

Early surgical treatment for congenital vertical talus.

Congenital vertical talus is characterised by a dislocated talonavicular joint in association with an equinus position of the calcaneus. We report the results in 13 operated feet in 10 children, 3 of whom (5 feet) presented with a primary neurological disorder and 2 of whom (3 feet) suffered from arthrogryposis multiplex congenita. The other children were normal. All patients were surgically treated by a one-stage procedure which included reduction of the talonavicular joint and correction of the hindfoot equinus, trying to avoid tendon lengthenings and transfers. The age at operation was between the 3rd and the 6th month of life, with one child being operated on later due to other reasons. The patients were reviewed after an average time of 3.5 +/- 2.2 years. The clinical results were good or excellent in ten feet. Two feet showed partial or complete recurrence and one foot was slightly over-corrected. Radiographic angle measurements (talo-metatarsal I angle and talocalcaneal angle on the anteroposterior radiograph; talocalcaneal, tibiotalar and tibiocalcaneal angles on the lateral radiograph) returned to normal values in the ten good or excellent feet. Early operative treatment for congenital pes vertical talus leads to very satisfactory functional and cosmetic results, usually avoiding extensive procedures including tendon lengthenings and tendon transfers.

Achilles Tendon↗

PubMed1993

Distinct NF-kappa B/Rel transcription factors are responsible for tissue-specific and inducible gene activation.

The NF-kappa B/Rel family is a growing class of transcriptional regulators whose members share the conserved Rel-homology domain, involved in specific DNA binding and dimerization. They interact with the regulatory elements of many different genes and are involved in the regulation of lymphoid-specific and inducible transcription. We tested whether these factors could alone activate a gene in transgenic mice. We report here that a minimal promoter containing three copies of a binding site for these proteins allows tissue-specific and inducible transgene activation. In lymphoid tissues constitutive transgene expression correlates with the presence of a constitutively active p50/RelB heterodimer. Other organs that only contain the p50 homodimer do not express the transgene. In contrast to this constitutive activity mediated by p50/RelB, the p50/p65 heterodimer (which is NF-kappa B) could confer inducible transgene activation in embryo fibroblasts. Thus two different members of the NF-kappa B/Rel family of transcriptional activators are involved in tissue-specific and inducible gene activation in transgenic mice.

Animals↗

PubMed1993

Analysis of transcriptional stimulation by recombinant Oct proteins in a cell-free system.

The transactivation potential of several isoforms of the lymphoid-specific transcription factor Oct2 has been analyzed using in vitro transcription. Oct2 can stimulate transcription in B-cell nuclear extracts and in HeLa nuclear extracts depleted of the ubiquitous factor Oct1 by wheat germ lectin affinity chromatography. Activity is observed from both natural and synthetic promoters containing single or multiple copies of the octamer motif ATGCAAAT. Multimerization of this motif does not result in a synergistic transcriptional stimulation, but rather leads to a linear increase in activity. To analyze the various Oct2 isoforms, they were overexpressed in HeLa cells using recombinant vaccinia virus. Although all the isoforms bind similarly to the octamer sequence, they show clear differences in their ability to transactivate transcription. This ranges from a 2-fold stimulation for Oct2.3 to the almost 20-fold effect of the most potent variant Oct2.5. In general the relative activity of the isoforms in vitro reflects that observed in vivo in cotransfection experiments. Interestingly the ubiquitous factor Oct1 is also an efficient activator of transcription in vitro, but only from promoters with multiple octamer motifs. Sarkosyl inhibition studies suggest that both Oct1 and Oct2 function in vitro by stabilizing preinitiation complexes without affecting the reinitiation rate of RNA polymerase II.

Cell-Free System↗

PubMed1993

Ultrasonography in Perthes' disease. Clinical relevance and influence on treatment.

A detailed description of the ultrasonographic findings in the course of Perthes' disease has recently been evolved. Synovitis was a prominent finding in the early stages. Lateral extrusion of the femoral head and signs of healing were found earlier by ultrasonography than by radiography. This has clinical implications. Immobilisation in the acute phase was shown to decrease capsular distension, and treatment using containment can be started earlier. The findings in 25 hips were evaluated in this study and their influence on treatment is discussed.

Child↗

PubMed1993

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

Transforming growth factor beta (TGF-beta) has a growth-inhibitory effect on numerous different cell types of the immune system, including T lymphocytes. We show in this study that the inhibitory action of TGF-beta on T lymphocytes is accompanied by a block of interleukin 2 (IL-2) gene expression which is mediated, at least in part, by inhibition of IL-2 promoter/enhancer activity. The functional analysis of cis-regulatory (proto-enhancer) elements of the IL-2 enhancer/promoter region showed that the most TGF-beta-responsive element maps to its so-called upstream promoter site. The proto-enhancer activity of the upstream promoter site element is also inhibited by cyclosporin A. The upstream promoter site DNA harbors two noncanonical, closely linked binding sequences for octamer and AP-1-like factors. Both sites are involved in the establishment of IL-2 enhancer activity. Since the activity of genuine octamer sites but not that of AP-1-binding sites is also impaired by TGF-beta and cyclosporin A in El4 T lymphoma cells, we conclude that both immunosuppressives interfere with the activity but not the DNA binding of octamer factors in T lymphocytes.

Animals↗

PubMed1993

[Psychometric assessment of defense mechanisms: correlation between questionnaire and expert rating. Initial study of validity].

Within the limits of an epidemiological longitudinal field survey on prevalence and course of psychogenic disorders a high-risk-population suffering from medical psychogenic impairment was investigated. The study was conducted in order to verify an etiological multi-level-model of psychogenic disorders in relation to the socialempiric variables "critical life events" and "social support" as well as the depth psychological oriented construct "personality". Besides other instruments a self rating scale based on Vallant's hierarchical model of defense, i.e. the german adaptation of the DSQ (Defense Style Questionnaire) of Bond and coworkers, was used for the accurate measurement of relevant personality parameters. Although defense processes predominantly work unconscious, manifestations of defense mechanisms could be measured indirectly by means of the rating scale. Its essential dimensions separated clinical patients from a group of healthy controls. Furthermore an immature organisation of defense was found to be related to psychogenic impairment. Concerning self- and expert-rating a significant correlation between "immature defense" and the defense mechanisms "schizoid phantasy", "projection" and "acting out" was proved.

Adult↗

PubMed1992

Functional analysis of defined mutations in the immunoglobulin heavy-chain enhancer in transgenic mice.

We have analyzed the effect of defined mutations in the mouse immunoglobulin heavy-chain enhancer after introduction into the germline of transgenic mice. We have tested a mutation of the enhancer octamer motif, a double mutation of the octamer motif and the microB-site, and a triple mutation in the microE2, microE3 and microE4-sites. All constructs are expressed in the spleen of transgenic mice. Furthermore, expression is exclusively detectable in lymphoid organs and not in several nonlymphoid tissues. Whereas mutations in the microE-sites have a more pronounced effect on transgene activity in thymocytes as compared to bone marrow and spleen cells, the octamer/microB double mutation shows significantly reduced expression levels only in B-cells. Finally, our results demonstrate that the intronic heavy-chain enhancer element does not contribute to the increase steady state levels of heavy-chain mRNA after stimulation of spleen cells with LPS.

Animals↗

PubMed1992

Synovial haemangioma of the knee joint.

Synovial haemangioma of joints are rare, but usually affect the knee joints of children and adolescents. Recurrent swelling, usually due to haemarthrosis, and intermittent pain may be present for a long time before the diagnosis is made. In this case report the common features of the condition are described, with emphasis on the occurrence of atraumatic haemarthrosis in children. The value of arthroscopy in diagnosis is discussed.

Arthroscopy↗