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T Wirth

Publications and source records attributed to T Wirth.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1992

Ultrasonography in Legg-Calvé-Perthes disease.

Ultrasonography was found to be a valuable investigation in the assessment and management of Legg-Calvé-Perthes disease (LCPD). It was used to assess 23 patients with LCPD in 25 affected hips and was compared with radiographs obtained at the same time. A chronological five-part staging of LCPD is proposed, expressing the degree of flattening and fragmentation as well as reconstitution of the femoral head as seen on ultrasound examination. Thickening of articular cartilage was documented, and associated findings of synovitis and lateral extrusion of the femoral head were evaluated. An intraarticular hip effusion was present in 74% of cases in stages I-II. Lateral extrusion increased from stage II onwards until the healing stage. The phase of reconstitution (stage IV) demonstrated both resorption of the necrotic bone and formation of new immature osteoid tissue. Lateral extrusion and the start of the healing phase can be shown earlier by ultrasonography than by radiography.

Child↗
PubMed1992

Oct2 transactivation from a remote enhancer position requires a B-cell-restricted activity.

Previous cotransfection experiments had demonstrated that ectopic expression of the lymphocyte-specific transcription factor Oct2 could efficiently activate a promoter containing an octamer motif. Oct2 expression was unable to stimulate a multimerized octamer enhancer element in HeLa cells, however. We have tested a variety of Oct2 isoforms generated by alternative splicing for the capability to activate an octamer enhancer in nonlymphoid cells and a B-cell line. Our analyses show that several Oct2 isoforms can stimulate from a remote position but that this stimulation is restricted to B cells. This result indicates the involvement of either a B-cell-specific cofactor or a specific modification of a cofactor or the Oct2 protein in Oct2-mediated enhancer activation. Mutational analyses indicate that the carboxy-terminal domain of Oct2 is critical for enhancer activation. Moreover, this domain conferred enhancing activity when fused to the Oct1 protein, which by itself was unable to stimulate from a remote position. The glutamine-rich activation domain present in the amino-terminal portion of Oct2 and the POU domain contribute only marginally to the transactivation function from a distal position.

Animals↗
PubMed1992

[Participation refusal by probands in an epidemiologic long-term study--sociodemographic, clinical and psychometric findings].

Usually little is known about probands who participated in an epidemiological longitudinal field survey but refused participation in follow-up investigations. For reasons of data protection and on account of the fact that investigative instruments used in longterm field surveys or panel studies are more focused on well defined issues (opinions, attitudes, assessment of behaviors) and less on personality variables, differentiated statements on probands who explicitly refused cooperation can hardly be made. In our epidemiological longitudinal field study on prevalence and course of psychogenic disorders we have a different situation. Within the limits of our study we had the unique opportunity to gain far-reaching information on probands who refused to participate in preceding investigations in regard to sociodemographic, psychometric and clinical variables. The clientele of refusers we present in our paper mainly comprises elderly, married, rather obsessive-compulsive structured, lower-class females. According to our data interactive aspects are equally responsible for reduced cooperativeness. The significance of our findings for the planning and carrying out of epidemiological longitudinal field surveys is discussed.

Adult↗
PubMed1991

Anti-IgM antibodies down modulate mu-enhancer activity and OTF2 levels in LPS-stimulated mouse splenic B-cells.

Stimulation of small, resting, splenic B cells with bacterial lipopolysaccharide (LPS) induces proliferation, differentiation to plasma cell formation, and the expression of immunoglobulin heavy chain (IgH). When this is combined with agents which crosslink surface Ig, differentiation and the induction of surface immunoglobulin are suppressed even though proliferation proceeds. We find that anti-mu antibodies suppresses Ig gene expression of transfected mu constructs, even if either the membrane or secretory segments have been deleted. We examined the effects of anti-mu treatment on the IgH enhancer (IgHE) attached to a heterologous test gene (CAT). Indeed the IgH enhancer alone was subject to anti-mu suppression, while the SV40 enhancer was insensitive. To determine what was responsible for suppression of enhancer function by anti-mu we examined nuclear extracts from stimulated splenic B cells for the presence of sequence-specific DNA binding activities to various sites within the enhancer. We found two specific differences--an induction in mu E5 binding activity, and a reduction in octamer transcription factor 2 (OTF2) binding activity, after anti-mu treatment. Analysis of these cells by in situ immunofluorescence with anti-OTF2 antibodies suggests that the nuclear localization of OTF2 in anti-mu treated cells may change, as well as its absolute level.

Animals↗