Biomedical subjects

K Friedrich

Publications and source records attributed to K Friedrich.

At least 19 recordsLinked to original sources

PubMed2001

Experimental regulation of STAT gene expression reveals an involvement of STAT5 in interleukin-4-driven cell proliferation.

The precise roles of signal transducers and activators of transcription (STATs) in cytokine-triggered control of cell physiology are not sufficiently well understood. We have established cell lines in which the individual functional contributions of STAT6 and STAT5a/b to interleukin-(IL-) 3 and -4-dependent processes can be readily studied. Mutants of STAT6, STAT5a and 5b lacking the transcriptional transactivation domain were fused to the green fluorescent protein (GFP) and expressed in the murine pro-B cell line Ba/F3 in a regulatable fashion. The expression of these truncated STAT variants could be tightly controlled over a wide range by doxycycline in the medium. They specifically bound to cognate DNA elements upon cytokine stimulation and acted dominant-negatively on the transcription of respective reporter genes in response to IL-3 and -4. The system was applied to the question of STAT contributions to cytokine-dependent cell proliferation. Expression of dominant-negative STAT6 had no significant effect on cell growth in response to both IL-3 and IL-4. In contrast, truncated STAT5 interfered with cell proliferation in response to IL-3, and, interestingly, also to IL-4. The results support our earlier findings on a role of STAT5 in IL-4-induced intracellular signaling and indicate that STAT5b in particular is involved in IL-4 receptor-triggered control of cell proliferation.

Animals↗

PubMed2000

Angiotensin II directly increases transforming growth factor beta1 and osteopontin and indirectly affects collagen mRNA expression in the human heart.

OBJECTIVES: Angiotensin II (Ang II) induces fibroblast proliferation and collagen synthesis in the myocardium, but its precise mechanisms of action in human hearts are still unknown. Therefore, we investigated whether Ang II directly affects the collagen mRNA content in the human myocardium and in isolated human cardiac fibroblasts or whether the growth factors TGFbeta-1 and osteopontin are involved in this process. METHODS AND RESULTS I: In a first set of experiments, the direct effect of Ang II on collagen I, TGFbeta-1 and osteopontin mRNA content in fresh samples of human atrial myocardium was determined by the use of a short stimulation period. After 4 h, Ang II-stimulated atrial samples gave a significantly higher expression of both TGFbeta-1 (183+/-21% of control, p<0.05) and osteopontin mRNA (275+/-58%, p<0.02) than the controls. In contrast, the expression of collagen I mRNA was unchanged (95+/-8%). Stimulation with TGFbeta-1 led to an increase in collagen I and III mRNA (127+/-10%, p<0.05; 140+/-15%, p<0.02). METHODS AND RESULTS II: In a second protocol, to assess the effects of longer stimulation periods, we determined the effects of Ang II and its potential mediator TGFbeta-1 on collagen I, III and fibronectin mRNA expression and on proliferation of cultured human cardiac fibroblasts. Ang II caused a dose-dependent stimulation of proliferation but did not affect collagen I, II or fibronectin mRNA content after 24 h. In contrast, TGFbeta-1 stimulation significantly increased collagen I and III mRNA expression (124+/-5% and 128+/-5%, p<0.002). CONCLUSIONS: In the human heart, Ang II does not directly increase collagen or fibronectin mRNA, but it does increase TGFbeta-1 and osteopontin mRNA expression. Since TGFbeta-1 induces collagen I and III mRNA in atrial samples and in isolated cardiac fibroblasts, it may represent a necessary mediator of the Ang II effects in the human heart.

Aged↗

PubMed2000

DNA ploidy and chromosomal imbalances in invasive ductal breast cancer. A comparative study of DNA image cytometry and comparative genomic hybridization (CGH).

Chromosomal imbalances were analyzed in 62 breast cancers with different DNA ploidy by CGH. The results of DNA image cytometry and CGH are consistent with peridiploid and aneuploid cases. The peritetraploid tumors harbored a high number of chromosomal imbalances, as a hint for an unfavorable prognosis. The quantitative analysis of imbalances highlighted the role of different physical constituents of the chromosome, and of chromosomal losses in different DNA ploidy groups. The peritetraploid and aneuploid tumors differed from the peridiploid tumors in losses at 8p and 18q. The peritetraploid cancers exhibited more gains at 8q, the aneuploid tumors more losses at 17p than their peridiploid counterparts. The aneuploid cases differed from the peritetraploid tumors in a higher number of losses at 11q and 14q. Combinations of imbalances provide further insights into the genetic background of DNA ploidy. Hypotheses for the progression from peridiploid to nondiploid breast cancers are given.

Breast Neoplasms↗

PubMed2000

Is systematic sextant biopsy suitable for the detection of clinically significant prostate cancer?

BACKGROUND: The optimal extent of the prostate biopsy remains controversial. There is a need to avoid detection of insignificant cancer but not to miss significant and curable tumors. In alternative treatments of prostate cancer, repeated sextant biopsies are used to estimate the response. The aim of this study was to investigate the reliability of a repeated systematic sextant biopsy as the standard biopsy technique in patients with significant tumors which are being considered for curative treatment. METHODS: Systematic sextant biopsy was performed in vitro in 92 radical prostatectomy specimens. Of these patients, 81 (88.0%) had palpable lesions. RESULTS: Of the 92 investigated patients, 70 (76.1%) had potentially curable pT2-3pN0 prostate cancers. In these patients, the cancer was detected only in 72.9% of cases by a repeated in vitro biopsy. In the pT2 tumors, there was a detection rate of only 66.7%. CONCLUSIONS: This study underlines the fact that a considerable number of significant and potentially curable tumors remain undetected by the conventional sextant biopsy. A negative sextant biopsy does not rule out significant prostate cancer.

Aged↗

PubMed1999

The two subunits of the interleukin-4 receptor mediate independent and distinct patterns of ligand endocytosis.

Interleukin-4 (IL-4) triggers cellular responses by interaction with the bipartite interleukin-4 receptor (IL-4R). IL-4-responsive cells specifically endocytose IL-4. We studied the ligand internalization properties of the human IL-4R and analyzed the specific functions of its two subunits IL-4Ralpha and gammac in this process. IL-4 mutant RY, which binds to IL-4Ralpha but does not recruit gammac into the receptor complex was used as a tool to show that IL-4Ralpha can promote independent ligand uptake in human T cells. Internalization was limited, however, by rapid IL-4 dissociation, suggesting that one important function of gammac in IL-4 endocytosis is to retain the ligand sufficiently long within the ternary receptor complex. We then measured IL-4 internalization by murine Ba/F3 cells that were stably transfected with various human IL-4R constructs. Efficient IL-4 uptake required the cytoplasmic section of the receptor. The intracellular domains of IL-4Ralpha and gammac were responsible for independent endocytosis processes with distinct kinetics. IL-4Ralpha-mediated internalization resulted in long-term intracellular maintainance of IL-4, whereas gammac directed the associated radioligand to intracellular breakdown and rapid release in the form of degraded protein. Mutants of either IL-4R subunit deficient in Janus kinase activation were not impaired in internalization, indicating that IL-4 endocytosis is not functionally connected to signal transduction.

Biological Transport↗

PubMed1999

Study of ligand-receptor interactions by fluorescence correlation spectroscopy with different fluorophores: evidence that the homopentameric 5-hydroxytryptamine type 3As receptor binds only one ligand.

The 5-hydroxytryptamine receptor of type 3 was investigated by fluorescence correlation spectroscopy (FCS). Binding constants of fluorescently labeled ligands, the stoichiometry, and the mass of the receptor are readily accessible by this technique, while the duration of measurement is on the order of seconds to minutes. The receptor antagonist 1,2,3, 9-tetrahydro-3-[(5-methyl-1H-imidazol-4-yl)methyl]-9-(3-aminopropyl)- 4H-carbazol-4-one (GR-H) was labeled with the fluorophores rhodamine 6G, fluorescein, N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl], and the cyanine dye Cy5. These labels cover a large part of the visible electromagnetic spectrum. It is shown that the photophysical and chemical properties have a direct influence on the measurement quality (duration of measurement, signal-to-noise ratio) and the ligand-receptor interactions (dissociation constants), respectively. This makes it necessary to choose a suitable label or a combination of labels for receptor studies. The affinities of the fluorescently labeled ligands determined by FCS were virtually identical to the values obtained by radioligand binding experiments. Moreover, the dissociation constant of a nonfluorescent receptor ligand was determined successfully by an FCS competition assay. The experimental results showed that only one antagonist binds to the receptor, in agreement with measurements previously published [Tairi et al. (1998) Biochemistry 37, 15850-15864].

4-Chloro-7-nitrobenzofurazan↗

PubMed1999

A two-step selection approach for the identification of ligand-binding determinants in cytokine receptors.

We have developed a novel cell-based method for the isolation and selection of mutant cytokine receptors with defects in ligand binding and applied it to the human interleukin-4 receptor. The experimental procedure is based upon the functional heterologous expression of receptor mutants in eukaryotic cells followed by a two-step selection procedure. Positive selection for cells that express receptor variants is achieved by means of an agonistic antibody that mediates cell survival through receptor dimerization. An IL-4-coupled toxin is subsequently used to select against cells expressing wild-type receptors. Cells expressing mutant receptors that are unable to bind the cytotoxic ligand survive and can be amplified. The procedure allows the isolation of rare receptor variants from cell pools containing predominantly wild-type cells. This method, which should be equally applicable to similar receptor systems, was used to demonstrate the importance of a critical charged amino acid residue in the human IL-4 receptor alpha-subunit for IL-4-induced receptor activation.

Animals↗

PubMed1999

Detection of genomic DNA fragmentation during apoptosis (DNA ladder) and the simultaneous isolation of RNA from low cell numbers.

In the present paper we describe a rapid and sensitive method for the simultaneous isolation of total RNA and genomic plus low-molecular-weight DNA from apoptotic cells. Using this method, we were able to detect a DNA ladder from as low as 30,000 apoptotic cells in only 45 min including gel electrophoresis. In addition, RNA can be readily obtained from the same specimen to assess gene expression during apoptosis. This method therefore appears to be advantageous when sensitivity and low amounts of sample material are a limiting factor.

Adsorption↗

PubMed1999

Annular erythema caused by impending pacemaker extrusion.

A 72-year-old patient with an implanted cardiac pacemaker presented with a circumscribed erythematous area on his chest. It was only several months later, after he had developed positional, localized pain in this area, that the diagnosis of an impending pacemaker extrusion became evident. This case illustrates the diagnostic difficulties in patients with pacemaker-associated skin lesions. Regular follow-up examinations and close co-operation of dermatologists, cardiologists and cardiothoracic surgeons are of major importance in view of the potentially life-threatening complications.

Aged↗

PubMed1999

Activation of STAT5 by IL-4 relies on Janus kinase function but not on receptor tyrosine phosphorylation, and can contribute to both cell proliferation and gene regulation.

We have investigated mechanisms and consequences of STAT5 activation through the human IL-4 receptor (IL-4R). By functionally expressing receptor mutants in the murine pro-B cell line Ba/F3, we could show that phosphorylated tyrosine residues within the IL-4R alpha chain are dispensable for IL-4-induced STAT5 activity. However, disruption of a membrane-proximal proline-rich sequence motif ('box1') in either subunit of the bipartite IL-4R abolished not only ligand-induced tyrosine phosphorylation of Janus kinases JAK1 and JAK3, but also IL-4-triggered activation of STAT5 and concomitant cell proliferation. A dominant-negative version of STAT5b, but not of STAT5a, interfered with IL-4-induced DNA synthesis in Ba/F3 cells, suggesting an involvement of STAT5b in the control of cell proliferation through IL-4R. Reporter gene experiments finally showed that transcription from promoters of STAT5 target genes can be specifically induced by challenging cells with IL-4, and that both STAT5a and STAT5b can contribute to IL-4-triggered transcriptional control.

B-Lymphocytes↗

PubMed1998

The interleukin-4 receptor activates STAT5 by a mechanism that relies upon common gamma-chain.

Interleukin (IL)-4 signaling proceeds via cytoplasmic activation of the Janus kinases JAK1 and JAK3 and the signal transducer and activator of transcription STAT6. We show that the IL-4 receptor, like other cytokine receptor systems utilizing the common receptor gamma-chain (gammac), is also connected to a signaling pathway that involves STAT5. Both STAT5a and STAT5b become tyrosine-phosphorylated and acquire specific DNA-binding properties in response to IL-4 receptor stimulation in the murine pro-B cell line Ba/F3. In preactivated human T cells, STAT5 became activated in an IL-4-dependent fashion as assayed by IL-4-induced STAT5 translocation from the cytoplasm to the cell nucleus and by binding to cognate DNA. Moreover, stimulation of preactivated human T cells by IL-4 led to specific transcriptional up-regulation of STAT5 target genes. IL-4 receptor-mediated STAT5 activation is dependent on the presence of gammac and JAK3 within the receptor complex. In COS-7 cells, the JAK/STAT pathway leading from the IL-4 receptor to STAT5-dependent regulation of a reporter gene relied largely on coexpression of JAK3. In Ba/F3 cells, studies on signal transduction evoked by directed specific receptor homo- or heterodimerization revealed that STAT5 activation can be triggered exclusively by IL-4R heterodimers containing gammac.

Animals↗

PubMed1998

Constitutive association of JAK1 and STAT5 in pro-B cells is dissolved by interleukin-4-induced tyrosine phosphorylation of both proteins.

The bipartite human interleukin-4 (IL-4) receptor was functionally expressed in murine pro-B cells and activated by human IL-4 to evoke intracellular signaling. Mutual association of signal transducing proteins within the receptor complex was then studied in dependence of ligand stimulation. Besides ligand-induced receptor heterodimerization and contacts of the two IL-4 receptor subunits alpha and gamma with Janus kinases JAK1 and JAK3 a prominent constitutive binding between JAK1 and signal transducer and activator of transcription STAT5 was detected. Since both these proteins become phosphorylated in response to IL-4 receptor stimulation, the influence of tyrosine phosphorylation on their mutual contact was analyzed. Association of JAK1 and STAT5 was found to occur exclusively between unphosphorylated proteins.

B-Lymphocytes↗

PubMed1998

Inhibition of the cAMP-dependent protein kinase by synthetic A-helix peptides.

The catalytic subunit of the cAMP-dependent protein kinase from Dictyostelium discoideum, PkaC, displays the same properties as its mammalian counterpart, except for being about twice as large in size. Sequence comparisons indicated the presence of a conserved alpha-helix (A-helix) within the N-terminal region of PkaC which could potentially establish close contacts with the catalytic core [Véron, M., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10618-10622]. We show in this report that a synthetic peptide with the A-helix sequence inhibits PKA activity, whereas unrelated peptides display no inhibitory activity. The inhibition seems competitive with respect to the kemptide substrate rather than due to binding to a secondary site. We further show by amino acid replacements that the last lysine of the A-helix sequence is involved in this specific inhibition. A model is proposed for the possible role of the A-helix.

Amino Acid Sequence↗

PubMed1998

Signalling by the oncogenic receptor tyrosine kinase Xmrk leads to activation of STAT5 in Xiphophorus melanoma.

Overexpression of the mutationally activated Xmrk receptor initiates the formation of hereditary malignant melanoma in the fish Xiphophorus. In addition to transcriptional overexpression a cell-type specific signal transduction is essential for Xmrk mediated tumor formation. To elucidate the consequence of Xmrk signalling and to identify target proteins that characterize the tumor phenotype, we analysed proteins that are strongly tyrosine phosphorylated in the fish melanoma cell line PSM. One of the most prominent phosphotyrosine proteins was found to be the signal transducer and activator of transcription STAT5. In a heterologous cell system (murine pro B-cells), activation of the Xmrk kinase in a chimeric receptor induced tyrosine phosphorylation, nuclear translocation and DNA binding of STAT5. Following receptor stimulation, expression of the STAT5 specific target genes cis, osm and pim-1 was induced. In Xiphophorus PSM cells STAT5 was found to be preferentially localized in the nucleus, but treatment with tyrphostin AG555, a specific Xmrk kinase-inhibitor, blocked nuclear localization. In these cells as well as in Xiphophorus melanoma expression of pim-1 and constitutive DNA-binding activity of STAT5 was detectable. This constitutive activity was higher in malignant than in benign melanomas, indicating that STAT5 activation is correlated with the malignancy of these tumors.

Animals↗

PubMed1998

Activation of STAT6 is not dependent on phosphotyrosine-mediated docking to the interleukin-4 receptor and can be blocked by dominant-negative mutants of both receptor subunits.

Stimulation of susceptible cells by interleukin-4 leads to activation of signal transducer and activator of transcription (STAT6) through tyrosine phosphorylation and dimerisation, thus directing it to the cell nucleus and rendering it a sequence-specific transcription factor. We functionally reconstituted human interleukin-4 receptor complexes with intracellular truncations of either the alpha or gamma subunits and demonstrate the requirement for elements from both receptor chains for STAT6 activation induced by interleukin-4. By assaying the signalling properties of human interleukin-4-receptor alpha-chain-deletion constructs in both Ba/F3 cells and COS-7 cells, we show that all its cytoplasmic tyrosine residues can be removed without affecting the capability of the receptor complex to trigger STAT6 function with regard to tyrosine phosphorylation, DNA binding, and specific gene transcription. The activation of both STAT6 and janus kinase 1 (JAK1) by the interleukin-4 receptor was completely abolished by disruption of the membrane-proximal 'box1' motif in the interleukin-4 receptor alpha chain. Our results indicate a redundant role of the previously defined phosphotyrosine-containing STAT6 docking site and suggest a mechanism of immediate activation of STAT6 by receptor-associated janus kinase(s). In addition, we demonstrate that dominant negative versions of both interleukin-4 receptor subunits are able to block interleukin-4 induced signalling via STAT6.

Animals↗

PubMed1997

Receptor-associated constitutive protein tyrosine phosphatase activity controls the kinase function of JAK1.

Exposure of cells to protein tyrosine phosphatase (PTP) inhibitors causes an increase in the phosphotyrosine content of many cellular proteins. However, the level at which the primary signaling event is affected is still unclear. We show that Jaks are activated by tyrosine phosphorylation in cells that are briefly exposed to the PTP inhibitor pervanadate (PV), resulting in tyrosine phosphorylation and functional activation of Stat6 (in addition to other Stats). Mutant cell lines that lack Jak1 activity fail to support PV-mediated [or interleukin 4 (IL-4)-dependent] activation of Stat6 but can be rescued by complementation with functional Jak1. The docking sites for both Jak1 and Stat6 reside in the cytoplasmic domain of the IL-4 receptor alpha-chain (IL-4Ralpha). The glioblastoma-derived cell lines T98G, GRE, and M007, which do not express the IL-4Ralpha chain, fail to support Stat6 activation in response to either IL-4 or PV. Complementation of T98G cells with the IL-4Ralpha restores both PV-mediated and IL-4-dependent Stat6 activation. Murine L929 cells, which do not express the gamma common chain of the IL-4 receptor, support PV-mediated but not IL-4-dependent Stat6 activation. Thus, Stat6 activation by PV is an IL-4Ralpha-mediated, Jak1-dependent event that is independent of receptor dimerization. We propose that receptor-associated constitutive PTP activity functions to down-regulate persistent, receptor-linked kinase activity. Inhibition or deletion of PTP activity results in constitutive activation of cytokine signaling pathways.

Amino Acid Sequence↗

PubMed1997

X-SCID B cell responses to interleukin-4 and interleukin-13 are mediated by a receptor complex that includes the interleukin-4 receptor alpha chain (p140) but not the gamma c chain.

This study investigates the effect of interleukin (IL)-4 mutant proteins and a monoclonal antibody to the IL-4 receptor alpha chain on IL-4 and IL-13 response by B cells from X-linked severe combined immunodeficiency (X-SCID) patients in which the common gamma chain (gamma c chain) gene mutations have been fully characterized and no gamma c chain expression was detected. In this gamma c chain gene knockout model, it was confirmed that the gamma c chain is essential for B cell responses to IL-2 but not for IL-4 or IL-13. Dose-response curves for X-SCID and normal B cell responses to IL-4 were indistinguishable, showing that the loss of the gamma c chain did not diminish the sensitivity of B cells to IL-4. The mutant protein IL-4(Y124D) and an antibody to the IL-4R alpha chain both inhibited responses of X-SCID B cells to IL-4 and IL-13, showing that X-SCID B cell responses to these cytokines are mediated by a receptor complex that includes the IL-4R alpha chain but not the gamma c chain. Another mutant protein, IL-4(R88D), which has greatly reduced affinity for IL-4R alpha, was found to inhibit responses by normal B cells to IL-4 but not to IL-13. IL-4(R88D), did not, however, inhibit X-SCID B cell responses to IL-4. This result is consistent with IL-4(R88D) inhibition of responses mediated by receptor complexes that include the gamma c chain. We propose that X-SCID B cells responses to IL-4 are mediated by an IL-13 receptor complex comprised of the IL-4R alpha chain associated with the recently cloned IL-13R binding protein. This model has major implications for understanding normal B cell responses to IL-4.

Animals↗

PubMed1997

Electron beam dose calculations with the VMC algorithm and the verification data of the NCI working group.

The accuracy of the Monte Carlo algorithm for fast electron dose calculation, VMC, is demonstrated by comparing calculations with measurements performed by a working group of the National Cancer Institute (NCI) of the USA. For both energies investigated, 9 and 20 MeV, the measurements in water are taken to determine the energy spectra of the Varian Clinac 1800 accelerator. For the majority of the experiments a good agreement is found between measurements and VMC calculations. However, in some cases deviations have been observed, which could be explained by the incompletely known geometry on the one hand and by inconsistent data on the other hand. As a reference, dose distributions calculated by the MDAH pencil-beam algorithm are also presented. It is shown that, especially near low- or high-density inhomogeneities, large dose overestimations and underestimations are calculated by using a pencil-beam approach, whereas VMC is able to reproduce the correct doses for these cases also.

Algorithms↗