Publications by authors named "Ulrike Fiedler"

28 Publications

  • Page 1 of 1

First-in-Human Phase I Study of MP0250, a First-in-Class DARPin Drug Candidate Targeting VEGF and HGF, in Patients With Advanced Solid Tumors.

J Clin Oncol 2021 Jan 10;39(2):145-154. Epub 2020 Dec 10.

Cantonal Hospital St Gallen, St Gallen, Switzerland.

Purpose: A first-in-human study was performed with MP0250, a DARPin drug candidate. MP0250 specifically inhibits both vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) with the aim of disrupting the tumor microenvironment.

Patients And Methods: A multicenter, open-label, repeated-dose, phase I study was conducted to assess the safety, tolerability, and pharmacokinetics of MP0250 in 45 patients with advanced solid tumors. In the dose-escalation part, 24 patients received MP0250 as a 3-hour infusion once every 2 weeks at five different dose levels (0.5-12 mg/kg). Once the maximum tolerated dose (MTD) was established, 21 patients were treated with a 1-hour infusion (n = 13, 8 mg/kg, once every 2 weeks and n = 8, 12 mg/kg, once every 3 weeks) of MP0250 in the dose confirmation cohorts.

Results: In the dose-escalation cohort, patients treated with 12 mg/kg MP0250 once every 2 weeks experienced dose-limiting toxicities. Therefore, MTD was 8 mg/kg once every 2 weeks or 12 mg/kg once every 3 weeks. The most common adverse events (AEs) were hypertension (69%), proteinuria (51%), and diarrhea and nausea (both 36%); hypoalbuminemia was reported in 24% of patients. Most AEs were consistent with inhibition of the VEGF and HGF pathways. Exposure was dose-proportional and sustained throughout the dosing period for all patients (up to 15 months). The half-life was about 2 weeks. Signs of single-agent antitumor activity were observed: 1 unconfirmed partial response with a time to progression of 23 weeks and 24 patients with stable disease, with the longest duration of 72 weeks and a median duration of 18 weeks.

Conclusion: MP0250 is a first-in-class DARPin drug candidate with suitable tolerability and appropriate pharmacokinetic properties for further development in combination with other anticancer therapies.
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http://dx.doi.org/10.1200/JCO.20.00596DOI Listing
January 2021

Profound Functional Suppression of Tumor-Infiltrating T-Cells in Ovarian Cancer Patients Can Be Reversed Using PD-1-Blocking Antibodies or DARPin® Proteins.

J Immunol Res 2020 4;2020:7375947. Epub 2020 Aug 4.

Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Stockholm, Sweden.

PD-1/PD-L1 blockade has revolutionized the field of immunooncology. Despite the relative success, the response rate to anti-PD-1 therapy requires further improvements. Our aim was to explore the enhancement of T-cell function by using novel PD-1-blocking proteins and compare with clinically approved monoclonal antibodies (mAbs). We isolated T-cells from the ascites and tumor of 17 patients with advanced epithelial ovarian cancer (EOC) and analyzed the effects using the mAbs nivolumab and pembrolizumab and two novel engineered ankyrin repeat proteins (DARPin® proteins). PD-1 blockade with either mAb or DARPin® molecule significantly increased the release of IFN-, granzyme B, IL-2, and TNF-, demonstrating successful reinvigoration. The monovalent DARPin protein was less effective compared to its bivalent equivalent, demonstrating that bivalency brings an additional benefit to PD-1 blockade. Overall, we found a higher fold increase of lymphokine secretion in response to the PD-1 blockade by tumor-derived T-cells; however, the absolute amounts were significantly lower compared to the release from ascites-derived T-cells. Our results demonstrate that PD-1 blockade can only partially reinvigorate functionally suppressed T-cells from EOC patients. This warrants further investigation preferably in combination with other therapeutics. The study provides an early pilot proof-of-concept for the potential use of DARPin proteins as eligible alternative scaffold proteins to block PD-1.
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http://dx.doi.org/10.1155/2020/7375947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7424497PMC
August 2020

Targeting angiogenesis in multiple myeloma by the VEGF and HGF blocking DARPin protein MP0250: a preclinical study.

Oncotarget 2018 Mar 30;9(17):13366-13381. Epub 2018 Jan 30.

Department of Biomedical Sciences and Human Oncology, Unit of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy.

The investigational drug MP0250 is a multi-specific DARPin molecule that simultaneously binds and neutralizes VEGF and HGF with high specificity and affinity. Here we studied the antiangiogenic effects of the MP0250 in multiple myeloma (MM). In endothelial cells (EC) isolated from bone marrow (BM) of MM patients (MMEC) MP0250 reduces VEGFR2 and cMet phosphorylation and affects their downstream signaling cascades. MP0250 influences the secretory profile of MMEC and inhibits their in vitro angiogenic activities (spontaneous and chemotactic migration, adhesion, spreading and capillarogenesis). Compared to anti-VEGF or anti-HGF neutralizing mAbs, MP0250 strongly reduces capillary network formation and vessel-sprouting in a Matrigel angiogenesis assay. MP0250 potentiates the effect of bortezomib in the same in vitro setting. It significantly reduces the number of newly formed vessels in the choriollantoic membrane assay (CAM) and the Matrigel plug assay. In the syngeneic 5T33MM tumor model, MP0250 decreases the microvessel density (MVD) and the combination MP0250/bortezomib lowers the percentage of idiotype positive cells and the serum levels of M-protein. Overall results define MP0250 as a strong antiangiogenic agent with potential as a novel combination drug for treatment of MM patients.
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http://dx.doi.org/10.18632/oncotarget.24351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5862584PMC
March 2018

MP0250, a VEGF and HGF neutralizing DARPin molecule shows high anti-tumor efficacy in mouse xenograft and patient-derived tumor models.

Oncotarget 2017 Nov 11;8(58):98371-98383. Epub 2017 Oct 11.

Molecular Partners AG, Schlieren, Switzerland.

Background: The VEGF/VEGFR and the HGF/cMET pathways are key mediators of the interplay of tumor cells and their microenvironment. However, inhibition of VEGF has been shown to produce only limited clinical benefit and inhibition of the activation of cMET by HGF has not translated into clinical benefit in pivotal trials. MP0250, a DARPin molecule that specifically inhibits both VEGF and HGF has been developed to explore the clinical potential of dual inhibition of these pathways.

Results: MP0250 binding to VEGF and HGF inhibited downstream signalling through VEGFR2 and cMET resulting in inhibition of proliferation of VEGF- and HGF-dependent cells. Antitumor activity was demonstrated in VEGF- and HGF-dependent xenograft and syngeneic models with activity superior to that of individual VEGF- and HGF-blocking DARPin molecules. Combination therapy studies showed potentiation of the antitumor activity of chemotherapy and immunotherapy agents, including an anti-PD1 antibody.

Materials And Methods: Potency of MP0250 was assessed in cellular models and in a variety of xenograft models as monotherapy or in combination with standard-of-care drugs.

Conclusions: Dual inhibition of VEGF and HGF by MP0250 produced powerful single agent and combination antitumor activity. This, together with increasing understanding of the role of the HGF/cMET pathway in resistance to VEGF (and other agents), supports testing of MP0250 in the clinic.
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http://dx.doi.org/10.18632/oncotarget.21738DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716736PMC
November 2017

Design and characterization of MP0250, a tri-specific anti-HGF/anti-VEGF DARPin® drug candidate.

MAbs 2017 Nov/Dec;9(8):1262-1269

a Molecular Partners AG , Wagistrasse 14, Schlieren , Switzerland.

MP0250 is a multi-domain drug candidate currently being tested in clinical trials for the treatment of cancer. It comprises one anti-vascular endothelial growth factor-A (VEGF-A), one anti-hepatocyte growth factor (HGF), and two anti-human serum albumin (HSA) DARPin® domains within a single polypeptide chain. While there is first clinical validation of a single-domain DARPin® drug candidate, little is known about DARPin® drug candidates comprising multiple domains. Here, we show that MP0250 can be expressed at 15 g/L in soluble form in E. coli high cell-density fermentation, it is stable in soluble/frozen formulation for 2 years as assessed by reverse phase HPLC, it has picomolar potency in inhibiting VEGF-A and HGF in ELISA and cellular assays, and its domains are simultaneously active as shown by surface plasmon resonance. The inclusion of HSA-binding DARPin® domains leads to a favorable pharmacokinetic profile in mouse and cynomolgus monkey, with terminal half-lives of ∼ 30 hours in mouse and ∼ 5 days in cynomolgus monkey. MP0250 is thus a highly potent drug candidate that could be particularly useful in oncology. Beyond MP0250, the properties of MP0250 indicate that multi-domain DARPin® proteins can be valuable next-generation drug candidates.
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http://dx.doi.org/10.1080/19420862.2017.1305529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5680794PMC
July 2018

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

J Biol Chem 2014 Mar 28;289(12):8493-507. Epub 2014 Jan 28.

From Scil Proteins GmbH, Heinrich-Damerow-Strasse 1, 06120 Halle (Saale), Germany.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.
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http://dx.doi.org/10.1074/jbc.M113.519884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961674PMC
March 2014

Angiopoietin-2 is critical for cytokine-induced vascular leakage.

PLoS One 2013 5;8(8):e70459. Epub 2013 Aug 5.

Division of Vascular Oncology and Metastasis, German Cancer Research Center (DKFZ-ZMBH Alliance), Heidelberg, Germany.

Genetic experiments (loss-of-function and gain-of-function) have established the role of Angiopoietin/Tie ligand/receptor tyrosine kinase system as a regulator of vessel maturation and quiescence. Angiopoietin-2 (Ang-2) acts on Tie2-expressing resting endothelial cells as an antagonistic ligand to negatively interfere with the vessel stabilizing effects of constitutive Ang-1/Tie-2 signaling. Ang-2 thereby controls the vascular response to inflammation-inducing as well as angiogenesis-inducing cytokines. This study was aimed at assessing the role of Ang-2 as an autocrine (i.e. endothelial-derived) regulator of rapid vascular responses (within minutes) caused by permeability-inducing agents. Employing two independent in vivo assays to quantitatively assess vascular leakage (tracheal microsphere assay, 1-5 min and Miles assay, 20 min), the immediate vascular response to histamine, bradykinin and VEGF was analyzed in Ang-2-deficient (Ang-2(-/-)) mice. In comparison to the wild type control mice, the Ang2(-/-) mice demonstrated a significantly attenuated response. The Ang-2(-/-) phenotype was rescued by systemic administration (paracrine) of an adenovirus encoding Ang-2. Furthermore, cytokine-induced intracellular calcium influx was impaired in Ang-2(-/-) endothelioma cells, consistent with reduced phospholipase activation in vivo. Additionally, recombinant human Ang-2 (rhAng-2) alone was unable to induce vascular leakage. In summary, we report here in a definite genetic setting that Ang-2 is critical for multiple vascular permeability-inducing cytokines.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0070459PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734283PMC
March 2014

Treatment of diabetic macular edema with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study.

Am J Ophthalmol 2013 Apr 4;155(4):697-704, 704.e1-2. Epub 2012 Dec 4.

The Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287-9277, USA.

Purpose: To evaluate the safety and bioactivity of MP0112, a designed ankyrin repeat protein (DARPin) that specifically binds vascular endothelial growth factor (VEGF) in patients with diabetic macular edema (DME). DARPins are a novel class of proteins selected for specific, high-affinity binding to a target protein.

Design: Phase I/II, open-label, multicenter dose-escalation trial.

Methods: After a single intravitreal injection of MP0112, the main outcomes were safety assessments, aqueous MP0112 levels, change in best-corrected visual acuity (BCVA), and foveal thickness measured by optical coherence tomography. Six cohorts were planned, but only 3 were enrolled (0.04, 0.15, 0.4 mg), because a maximally tolerated dose of 1.0 mg was identified in a parallel age-related macular degeneration trial.

Results: Median aqueous concentration of MP0112 was 555 nM 1 week and >10 nM in 3 of 4 patients 12 weeks post injection of 0.4 mg. Median BCVA improvement at week 12 was 4, 6, and 10 letters in cohorts 1, 2, and 3. Ocular inflammation was observed in 11 patients (61%) and was severe in 1. High-resolution chromatography separated proinflammatory impurities from MP0112, resulting in a new formulation.

Conclusions: A single intraocular injection of 0.4 mg MP0112 resulted in levels above the half-maximal inhibitory concentration and neutralization of VEGF in aqueous humor for 8-12 weeks. Despite inflammation in several patients, there was prolonged edema reduction and improvement in vision in several patients. The source of the inflammation was eliminated from a new preparation that is being tested in an ongoing clinical trial.
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http://dx.doi.org/10.1016/j.ajo.2012.09.032DOI Listing
April 2013

Efficacy of sequential treatment with sunitinib-everolimus in an orthotopic mouse model of renal cell carcinoma.

Anticancer Res 2012 Jul;32(7):2399-406

Department of Medicine, Royal Marsden Hospital, Fulham Road, London SW3 6JJ, UK.

Background/aim: Sequential treatment with targeted agents is standard of care for patients with metastatic renal cell carcinoma (mRCC). However, clinical data directly comparing treatment outcomes with a mammalian target of rapamycin inhibitor or a vascular endothelial growth factor-targeted agent in the second-line setting are lacking. We evaluated sequential treatment in a syngeneic, orthotopic mouse model of mRCC.

Materials And Methods: BALB/c mice were orthotopically implanted with murine RCC (RENCA) cells expressing luciferase and randomized to vehicle, sunitinib, sunitinib followed by sorafenib, or sunitinib followed by everolimus. Tumor growth and metastases were assessed by in vivo (whole body) and ex vivo (primary tumor, lung, liver) luciferase activity and necropsies, performed on day 20 or 46 for vehicle and treatment groups, respectively.

Results: Sunitinib followed by everolimus was associated with reduced luciferase activity and primary tumor weight and volume compared with sunitinib, and sunitinib followed by sorafenib.

Conclusion: Sequential therapy with sunitinib followed by everolimus demonstrated significant antitumor and anti-metastatic effects.
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July 2012

Angiopoietin-1 mediates inhibition of hypertension-induced release of angiopoietin-2 from endothelial cells.

Cardiovasc Res 2012 Jun 14;94(3):510-8. Epub 2012 Apr 14.

Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, University of Heidelberg, Im Neuenheimer Feld 326, 69120 Heidelberg, Germany.

Aims: Adequate endothelial cell stimulation is a prerequisite for the adaptive remodelling of macro- and microvessels. A pivotal autocrine mechanism following endothelial cell activation is the release of angiopoietin-2 (Ang-2), which subsequently antagonizes the binding of Ang-1 to the Tie-2 receptor, thus sensitizing the endothelial cells to pro-angiogenic and/or pro-inflammatory stimuli. Based on the observation that hypertension in mice reduces the abundance of Ang-2 stored in arterial endothelial cells, this study was aimed at testing the hypothesis that an increase in wall stress (WS) or stretch-a hallmark of hypertension-is sufficient to release Ang-2 from endothelial cells.

Methods And Results: In fact, stretching of isolated perfused mouse arteries or human cultured endothelial cells rapidly elicited an increased release of Ang-2. In the cultured endothelial cells, this was preceded by a transient rise in intracellular free calcium, abrogated through calcium chelation and accompanied by a decrease in Tie-2 phosphorylation. Interestingly, Ang-1 abolished the stretch-induced release of Ang-2 from both cultured and native endothelial cells through inhibiting the stretch-dependent mobilization of intracellular calcium.

Conclusion: Collectively, these results indicate that increased WS or stretch facilitates the release of Ang-2 from endothelial cell Weibel-Palade bodies, and that Ang-1 can block this by attenuating the stretch-mediated rise in intracellular calcium.
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http://dx.doi.org/10.1093/cvr/cvs124DOI Listing
June 2012

Angiopoietin-2 stimulation of endothelial cells induces alphavbeta3 integrin internalization and degradation.

J Biol Chem 2010 Jul 2;285(31):23842-9. Epub 2010 Jun 2.

Joint Research Division Vascular Biology, Medical Faculty Mannheim (CBTM), Heidelberg University, D-69120 Heidelberg, Germany.

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit beta3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, alphavbeta3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser(910) upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from alphavbeta3 integrin. The alphavbeta3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/alphavbeta3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.
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http://dx.doi.org/10.1074/jbc.M109.097543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2911308PMC
July 2010

Workflow comparison for label-free, quantitative secretome proteomics for cancer biomarker discovery: method evaluation, differential analysis, and verification in serum.

J Proteome Res 2010 Apr;9(4):1913-22

OncoProteomics Laboratory, Department of Medical Oncology, VUmc-Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands.

The cancer cell secretome has emerged as an attractive subproteome for discovery of candidate blood-based biomarkers. To choose the best performing workflow, we assessed the performance of three first-dimension separation strategies prior to nanoLC-MS/MS analysis: (1) 1D gel electrophoresis (1DGE), (2) peptide SCX chromatography, and (3) tC2 protein reversed phase chromatography. 1DGE using 4-12% gradient gels outperformed the SCX and tC2 methods with respect to number of identified proteins (1092 vs 979 and 580, respectively), reproducibility of protein identification (80% vs 70% and 72%, respectively, assessed in biological N = 3). Reproducibility of protein quantitation based on spectral counting was similar for all 3 methods (CV: 26% vs 24% and 24%, respectively). As a proof-of-concept of secretome proteomics for blood-based biomarker discovery, the gradient 1DGE workflow was subsequently applied to identify IGF1R-signaling related proteins in the secretome of mouse embryonic fibroblasts transformed with human IGF1R (MEF/Toff/IGF1R). VEGF and osteopontin were differentially detected by LC-MS/MS and verified in secretomes by ELISA. Follow-up in serum of mice bearing MEF/Toff/IGF1R-induced tumors showed an increase of osteopontin levels paralleling tumor growth, and reduction in the serum of mice in which IGF1R expression was shut off and tumor regressed.
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http://dx.doi.org/10.1021/pr901072hDOI Listing
April 2010

Therapeutic interference with EphrinB2 signalling inhibits oxygen-induced angioproliferative retinopathy.

Acta Ophthalmol 2011 Feb;89(1):82-90

University Eye Hospital, Freiburg, Germany.

Purpose: To investigate whether EphrinB2 (EfnB2) or EphB4 influence retinal angiogenesis under physiological or pathological conditions.

Methods: Using the mouse model of oxygen-induced proliferative retinopathy (OIR), the expression of EfnB2, EphB4, vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 was quantified by quantitative polymerase chain reaction (qPCR) and localized in EfnB2- and EphB4-lacZ mice. Angioproliferative retinopathy was manipulated by intravitreal injection of dimeric EfnB2 and monomeric or dimeric EphB4.

Results: Dimeric EphB4 (EphB4-Fc) and EfnB2 (EfnB2-Fc) enhanced hypoxia-induced angioproliferative retinopathy but not physiological angiogenesis. Monomeric EphB4 (sEphB4) reduced angiogenesis. The messenger RNA (mRNA) level of EfnB2 increased significantly in the hyperoxic phase (P7-P12), while EphB4, VEGF, VEGFR1 and VEGFR2 showed a significant - up to fivefold - increased expression at P14, the start of morphologically visible vasoproliferation caused by relative hypoxia.

Conclusion: The ephrin/Eph system is involved in angioproliferative retinopathy. Stimulation of EphB4 and EfnB2 signalling using EfnB2-Fc and EphB4-Fc, respectively, enhanced hypoxia-induced angiogenesis. In contrast, sEphB4 inhibited hypoxia-induced angiogenesis. Therefore, angiogenesis is enhanced by signalling through both EphB4 (forward) and EfnB2 (reverse). The distinction in the expression kinetics of EphB4 and EfnB2 indicates that they govern two different signalling pathways and are regulated in diverse ways. sEphB4 might be a useful drug for antiangiogenic therapy.
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http://dx.doi.org/10.1111/j.1755-3768.2009.01609.xDOI Listing
February 2011

Affilin molecules selected against the human papillomavirus E7 protein inhibit the proliferation of target cells.

J Mol Biol 2009 Jul 21;390(4):710-21. Epub 2009 May 21.

Institute of Biochemistry/Biotechnology, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.

Intracellular binding proteins can be applied as research tools for target validation and study of protein function in cells and potentially as therapeutics. The success of intracellular binding reagents depends on their affinity and specificity for target molecules, although their stability and functionality in the intracellular environment actually determine their usefulness for such application. Alternative binding proteins derived from scaffolds devoid of disulfide bonds are well suited for intracellular use, as their folding and stability are usually not impaired under reducing conditions. Here, we describe the generation of intracellular binding reagents called Affilin, based on the human gammaB-crystallin scaffold. The target was human papillomavirus E7 protein implicated in the development of cervical cancer. E7 binders were selected from the combinatorial gammaB-crystallin library by conventional phage display technique. Affilin variants specifically bound the E7 protein with affinities in the nanomolar range. Intracellular expression of Affilin molecules in E7-positive cells led to inhibition of cellular proliferation. The effect was specific, as the growth of E7-negative cells or cells expressing the wild-type gammaB-crystallin scaffold remained unaffected. These results demonstrate that the gammaB-crystallin scaffold allows the de novo generation of alternative binding proteins, which are suitable for intracellular applications as they retain their functionality in the reducing environment of mammalian cells.
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http://dx.doi.org/10.1016/j.jmb.2009.05.027DOI Listing
July 2009

Host-derived angiopoietin-2 affects early stages of tumor development and vessel maturation but is dispensable for later stages of tumor growth.

Cancer Res 2009 Feb 10;69(4):1324-33. Epub 2009 Feb 10.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Freiburg, Germany.

The angiopoietin/Tie2 system has been identified as the second vascular-specific receptor tyrosine kinase system controlling vessel assembly, maturation, and quiescence. Angiopoietin-2 (Ang-2) is prominently up-regulated in the host-derived vasculature of most tumors, making it an attractive candidate for antiangiogenic intervention. Yet, the net outcome of Ang-2 functions on tumor angiogenesis is believed to be contextual depending on the local cytokine milieu. Correspondingly, Ang-2 manipulatory therapies have been shown to exert protumorigenic as well as antitumorigenic effects. To clarify the role of Ang-2 for angiogenesis and tumor growth in a definite genetic experimental setting, the present study was aimed at comparatively studying the growth of different tumors in wild-type and Ang-2-deficient mice. Lewis lung carcinomas, MT-ret melanomas, and B16F10 melanomas all grew slower in Ang-2-deficient mice. Yet, tumor growth in wild-type and Ang-2-deficient mice dissociated during early stages of tumor development, whereas tumor growth rates during later stages of primary tumor progression were similar. Analysis of the intratumoral vascular architecture revealed no major differences in microvessel density and perfusion characteristics. However, diameters of intratumoral microvessels were smaller in tumors grown in Ang-2-deficient mice, and the vasculature had an altered pattern of pericyte recruitment and maturation. Ang-2-deficient tumor vessels had higher pericyte coverage indices. Recruited pericytes were desmin and NG2 positive and predominately alpha-smooth muscle actin negative, indicative of a more mature pericyte phenotype. Collectively, the experiments define the role of Ang-2 during tumor angiogenesis and establish a better rationale for combination therapies involving Ang-2 manipulatory therapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-3030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514474PMC
February 2009

Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.

J Mol Biol 2007 Sep 22;372(1):172-85. Epub 2007 Jun 22.

Scil Proteins GmbH, Heinrich Damerow Str. 1, 06120 Halle (Saale), Germany.

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.
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http://dx.doi.org/10.1016/j.jmb.2007.06.045DOI Listing
September 2007

High efficient adenoviral-mediated VEGF and Ang-1 gene delivery into osteogenically differentiated human mesenchymal stem cells.

Microvasc Res 2008 Jan 10;75(1):83-90. Epub 2007 May 10.

Department of Plastic and Hand Surgery, University of Freiburg Medical Center, Freiburg, Germany.

Survival of ex vivo constructed tissues after transplantation is limited by insufficient oxygen and nutrient supply. Therefore, strategies aiming at improvement of neovascularization of engineered tissues are a key issue in tissue engineering applications. This in vitro study aimed at exploring the usability of osteogenically differentiated human mesenchymal stem cells (MSCs) as carriers of the angiogenic growth factor genes vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) for therapeutic angiogenesis in bone tissue engineering. The ex vivo adenoviral vector mediated transduction into osteogenically differentiated MSCs revealed a highly efficient and long lasting expression of the transgenes. Biological activity of VEGF and Ang-1 secreted from transduced cells was confirmed by analyzing the sprouting, proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) in response to conditioned medium obtained from transduced cells. The transduced osteogenically differentiated MSCs described in this report may be suitable for inducing neovascularization in bone tissue engineering applications.
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http://dx.doi.org/10.1016/j.mvr.2007.04.010DOI Listing
January 2008

Angiopoietins: a link between angiogenesis and inflammation.

Trends Immunol 2006 Dec 12;27(12):552-8. Epub 2006 Oct 12.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Freiburg 79106, Germany.

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1-Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel-Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang-Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.
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http://dx.doi.org/10.1016/j.it.2006.10.004DOI Listing
December 2006

Emerging roles of the Angiopoietin-Tie and the ephrin-Eph systems as regulators of cell trafficking.

J Leukoc Biol 2006 Oct 24;80(4):719-26. Epub 2006 Jul 24.

Department of Vascular Oncology and Metastasis, University of Heidelberg, Germany.

Vascular receptor tyrosine kinases (RTK) have been identified as critical regulatory signaling molecules of developmental and adult vascular morphogenic processes [vascular endothelial growth factor (VEGF) receptors=sprouting; EphB receptors=assembly; Tie2 receptor=maturation and quiescence]. It is intriguing that the same molecules that control the growth of blood and lymphatic vessels play critical roles in the adult to regulate maintenance functions related to vascular homeostasis. VEGF is among the most potent inducers of vascular permeability. The second vascular RTK system, the interaction of paracrine-acting Angiopoietin-1 with its cognate receptor Tie2, acts as an endothelial maintenance and survival-mediating molecular system, which stabilizes the vessel wall and controls endothelial cell quiescence. The third vascular RTK system, the interaction of Eph receptors with their Eph family receptor-interacting protein (ephrin) ligands, transduces positional guidance cues on outgrowing vascular sprouts, which are critical for proper arteriovenous assembly and establishment of blood flow. As such, Eph-ephrin interactions act as an important regulator of cell-cell interactions, exerting propulsive and repulsive functions on neighboring cells and mediating adhesive functions. This review summarizes recent findings related to the roles of the Angiopoietin-Tie and the Eph-ephrin systems as regulators of cell trafficking in the vascular system. The recognition of vascular homeostatic functions of vascular RTKs marks an important change of paradigm in the field of angiogenesis research as it relates angiogenesis-inducing molecules to vascular maintenance functions in the adult. This may also broaden the scope of vascular RTK-targeted therapies.
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http://dx.doi.org/10.1189/jlb.1105652DOI Listing
October 2006

The sialomucin CD34 is a marker of lymphatic endothelial cells in human tumors.

Am J Pathol 2006 Mar;168(3):1045-53

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany.

The mechanisms of lymphangiogenesis have been increasingly understood in recent years. Yet, the contribution of lymphangiogenesis versus lymphatic cooption in human tumors and the functionality of tumor lymphatics are still controversial. Furthermore, despite the identification of lymphatic endothelial cell (LEC) markers such as Prox1, podoplanin, LYVE-1, and VEGFR-3, no activation marker for tumor-associated LECs has been identified. Applying double-staining techniques with established LEC markers, we have screened endothelial cell differentiation antigens for their expression in LECs. These experiments identified the sialomucin CD34 as being exclusively expressed by LECs in human tumors but not in corresponding normal tissues. CD34 is expressed by LYVE-1(+)/podoplanin(+)/Prox1(+) tumor-associated LECs in colon, breast, lung, and skin tumors. More than 60% of analyzed tumors contained detectable intratumoral lymphatics. Of these, more than 80% showed complete co-localization of CD34 with LEC markers. In contrast, LECs in all analyzed normal organs did not express CD34. Corresponding analyses of experimental tumors revealed that mouse tumor-associated LECs do not express CD34. Taken together, these experiments identify CD34 as the first differentially expressed LEC antigen that is selectively expressed by tumor-associated LECs. The data warrant further exploration of CD34 in tumor-associated LECs as a prognostic tumor marker.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1606520PMC
http://dx.doi.org/10.2353/ajpath.2006.050554DOI Listing
March 2006

Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation.

Nat Med 2006 Feb 5;12(2):235-9. Epub 2006 Feb 5.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Freiburg 79106, Germany.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the receptor tyrosine kinase Tie-2 (refs. 1,2). Paracrine Ang-1-mediated activation of Tie-2 acts as a regulator of vessel maturation and vascular quiescence. In turn, the antagonistic ligand Ang-2 acts by an autocrine mechanism and is stored in endothelial Weibel-Palade bodies from where it can be rapidly released upon stimulation. The rapid release of Ang-2 implies functions of the angiopoietin-Tie system beyond its established role during vascular morphogenesis as a regulator of rapid vascular responses. Here we show that mice deficient in Ang-2 (encoded by the gene Angpt2) cannot elicit an inflammatory response in thioglycollate-induced or Staphylococcus aureus-induced peritonitis, or in the dorsal skinfold chamber model. Recombinant Ang-2 restores the inflammation defect in Angpt2(-/-) mice. Intravital microscopy showed normal TNF-alpha-induced leukocyte rolling in the vasculature of Angpt2(-/-)mice, but rolling cells did not firmly adhere to activated endothelium. Cellular experiments showed that Ang-2 promotes adhesion by sensitizing endothelial cells toward TNF-alpha and modulating TNF-alpha-induced expression of endothelial cell adhesion molecules. Together, these findings identify Ang-2 as an autocrine regulator of endothelial cell inflammatory responses. Ang-2 thereby acts as a switch of vascular responsiveness exerting a permissive role for the activities of proinflammatory cytokines.
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http://dx.doi.org/10.1038/nm1351DOI Listing
February 2006

A single local injection of recombinant VEGF receptor 2 but not of Tie2 inhibits retinal neovascularization in the mouse.

Curr Eye Res 2005 Apr;30(4):249-57

Department of Ophthalmology, University of Freiburg, Freiburg Germany.

Purpose: The purpose of this study was to develop pharmacological therapeutic alternatives for ischemia-induced proliferative retinopathy.

Methods: C57BL/6J mice were placed in 76% oxygen on postnatal day 7 (P7) for 5 days. On P12 recombinant, chimeric vascular endothelial growth factor (sVEGF-R2) or sTie2 was injected intravitreally in one eye. The fellow eye received a control injection. On P17, retinal wholemounts were prepared after perfusion with fluorescein-dextran to quantify the retinopathy.

Results: A single intravitreal injection of sVEGF-R2 reduced pathologic vascular changes significantly (p = 0.02). No significant effect was observed after intravitreal application of sTie2 (p = 0.07), although Ang-2 was upregulated in control animals without treatment as neovascularization developed and Ang-1 was constantly transcribed (ratio PCR).

Conclusions: sVEGF-R2 interferes with VEGF signaling via VEGF-R2 receptor. Thus, local application of soluble receptors for angiogenic factors is a possible therapy for proliferative retinopathy. Receptors with a wide range of ligands might prove more useful for local application than those binding few or antagonistic ligands.
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http://dx.doi.org/10.1080/02713680590923249DOI Listing
April 2005

The Tie-2 ligand angiopoietin-2 destabilizes quiescent endothelium through an internal autocrine loop mechanism.

J Cell Sci 2005 Feb 1;118(Pt 4):771-80. Epub 2005 Feb 1.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, 79106 Freiburg, Germany.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands of the endothelial receptor tyrosine kinase Tie-2, which controls vascular assembly and endothelial quiescence. The largely complementary phenotypes of Ang-1-deficient mice and Ang-2-overexpressing mice have led to an antagonistic model in which Ang-1 acts as Tie-2-activating agonist and Ang-2 acts as a Tie-2-inhibiting antagonist. To date, no mechanistic equivalent of the antagonistic Ang-1/Ang-2 model has been established and the mechanisms of Ang-2 function in particular remain mysterious. We have studied the effector functions of Ang-1 and Ang-2 on quiescent endothelial cells using a three-dimensional co-culture model of endothelial cells and smooth-muscle cells. Endothelial-cell monolayer integrity in this model is dependent on Tie-2 signaling, as evidenced by detaching endothelial cells following exposure to the small molecular weight Tie-2 inhibitor A-422885.66, which cannot be overcome by exogenous Ang-1. Accordingly, exogenous Ang-2 rapidly destabilizes the endothelial layer, which can be observed within 30-60 minutes and leads to prominent endothelial-cell detachment within 4 hours. Exogenous Ang-2-mediated endothelial-cell detachment can be rescued by Ang-1, soluble Tie-2 and vascular endothelial growth factor. Similar findings were obtained in an umbilical-vein explant model. Ang-2 is mainly produced by endothelial cells and therefore acts primarily in an autocrine manner. Thus, stimulated release of endogenous Ang-2 or overexpression of Ang-2 in endothelial cells perturbs co-culture spheroid integrity, which can be rescued by exogenous Ang-1 and vascular endothelial growth factor. However, autocrine Ang-2-mediated endothelial-cell detachment cannot be blocked by soluble Tie-2. Taken together, the data demonstrate for the first time the antagonistic Ang-1/Ang-2 concept in a defined cellular model and identify Ang-2 as a rapidly acting autocrine regulator of the endothelium that acts through an internal autocrine loop mechanism.
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http://dx.doi.org/10.1242/jcs.01653DOI Listing
February 2005

Expression of angiopoietin-2 in endothelial cells is controlled by positive and negative regulatory promoter elements.

Arterioscler Thromb Vasc Biol 2004 Oct 29;24(10):1803-9. Epub 2004 Jul 29.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, D-79106 Freiburg, Germany.

Objective: Angiopoietin-2 (Ang-2) is a non-signal transducing ligand of the endothelial receptor tyrosine kinase Tie-2. Ang-2 is produced by endothelial cells and acts as an autocrine regulator mediating vascular destabilization by inhibiting Angiopoietin-1-mediated Tie-2 activation. To examine the transcriptional regulation of Ang-2, we studied the Ang-2 promoter in endothelial cells and nonendothelial cells.

Methods And Results: The human Ang-2 promoter contains a 585-bp region around the transcriptional start site (-109 to +476) that is sufficient to control endothelial cell-specific and cytokine-dependent Ang-2 expression. Strong repressor elements of Ang-2-promoter activity are located in the 5'-region of the promoter and in the first intron. The Ets family transcription factors Ets-1 and Elf-1 act as strong enhancers of endothelial cell Ang-2-promoter activity. Ets-binding sites -4 and -7 act as positive regulators, whereas Ets-binding site -3 acts as negative regulator. Demethylation experiments revealed that the Ang-2 gene (in contrast to the Tie-2 gene) is not controlled by imprinting.

Conclusions: The data determine unique positive and negative regulatory mechanisms of endothelial cell Ang-2 expression and provide further evidence for the critical role of Ang-2 as a key autocrine regulator of vascular stability and responsiveness.
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http://dx.doi.org/10.1161/01.ATV.0000140819.81839.0eDOI Listing
October 2004

The Tie-2 ligand angiopoietin-2 is stored in and rapidly released upon stimulation from endothelial cell Weibel-Palade bodies.

Blood 2004 Jun 19;103(11):4150-6. Epub 2004 Feb 19.

Department of Vascular Biology and Angiogenesis Research Tumor Biology Center, Breisacher Str 117, 79106 Freiburg, Germany.

The angiopoietins Ang-1 and Ang-2 have been identified as ligands with opposing functions of the receptor tyrosine kinase Tie-2 regulating endothelial cell survival and vascular maturation. Ang-1 acts in a paracrine agonistic manner, whereas Ang-2 appears to act primarily as an autocrine antagonistic regulator. To shed further light on the complexity of autocrine/paracrine agonistic/antagonistic functions of the angiopoietin/Tie-2 system, we have studied Ang-2 synthesis and secretion in different populations of wild-type and retrovirally Ang-2-transduced endothelial cells. Endogenous and overexpressed endothelial cell Ang-2 is expressed in a characteristic granular pattern indicative of a cytoplasmic storage granule. Light and electron microscopic double staining revealed Ang-2 colocalization with von Willebrand factor, identifying Ang-2 as a Weibel-Palade body molecule. Costaining with P-selectin showed that storage of Ang-2 and P-selectin in Weibel-Palade bodies is mutually exclusive. Stored Ang-2 has a long half-life of more than 18 hours and can be secreted within minutes of stimulation (eg, by phorbol 12-myristate 13-acetate [PMA], thrombin, and histamine). Collectively, the identification of Ang-2 as a stored, rapidly available molecule in endothelial cells strongly suggests functions of the angiopoietin/Tie-2 system beyond the established roles during angiogenesis likely to be involved in rapid vascular homeostatic reactions such as inflammation and coagulation.
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http://dx.doi.org/10.1182/blood-2003-10-3685DOI Listing
June 2004

Angiopoietin-1 and angiopoietin-2 share the same binding domains in the Tie-2 receptor involving the first Ig-like loop and the epidermal growth factor-like repeats.

J Biol Chem 2003 Jan 9;278(3):1721-7. Epub 2002 Nov 9.

Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, 79106 Freiburg, Germany.

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) have been identified as ligands with different effector functions of the vascular assembly and maturation-mediating receptor tyrosine kinase Tie-2. To understand the molecular interactions of the angiopoietins with their receptor, we have studied the binding of Ang-1 and Ang-2 to the Tie-2 receptor. Enzyme-linked immunosorbent assay-based competition assays and co-immunoprecipitation experiments analyzing the binding of Ang-1 and Ang-2 to truncation mutants of the extracellular domain of Tie-2 showed that the first Ig-like loop of Tie-2 in combination with the epidermal growth factor (EGF)-like repeats (amino acids 1-360) is required for angiopoietin binding. The first Ig-like domain or the EGF-like repeats alone are not capable of binding Ang-1 and Ang-2. Concomitantly, we made the surprising finding that Tie-2 exon-2 knockout mice do express a mutated Tie-2 protein that lacks 104 amino acids of the first Ig-like domain. This mutant Tie-2 receptor is functionally inactive as shown by the lack of ligand binding and receptor phosphorylation. Collectively, the data show that the first 104 amino acids of the Tie-2 receptor are essential but not sufficient for angiopoietin binding. Conversely, the first 360 amino acids (Ig-like domain plus EGF-like repeats) of the Tie-2 receptor are necessary and sufficient to bind both Ang-1 and Ang-2, which suggests that differential receptor binding is not likely to be responsible for the different functions of Ang-1 and Ang-2.
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http://dx.doi.org/10.1074/jbc.M208550200DOI Listing
January 2003

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata.

Planta 2002 Jul 14;215(3):413-23. Epub 2002 May 14.

Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben, Germany.

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.
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http://dx.doi.org/10.1007/s00425-002-0771-zDOI Listing
July 2002

RNA polymerase II complexes in the very early phase of transcription are not susceptible to TFIIS-induced exonucleolytic cleavage.

Nucleic Acids Res 2002 Jun;30(11):2290-8

Laboratory for Physiological Chemistry, UMCU, Stratenum, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands.

TFIIS is a transcription elongation factor for RNA polymerase II (pol II), which can suppress ribonucleotide misincorporation. We reconstituted transcription complexes in a highly purified pol II system on adenovirus Major-Late promoter constructs. We noted that these complexes have a high propensity for read-through upon GTP omission. Read-through occurred during the early stages at all registers analyzed. Addition of TFIIS reversed read-through of productive elongation complexes, which indicated that it was due to misincorporation. However, before register 13 transcription complexes were insensitive to TFIIS. These findings are discussed with respect to the structural models for pol II and we propose that TFIIS action is linked to the RNA:DNA hybrid.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC117193PMC
http://dx.doi.org/10.1093/nar/30.11.2290DOI Listing
June 2002