Lothar Goretzki - Academia.edu (original) (raw)

Papers by Lothar Goretzki

Sanierung historischer Stadtmauern., 2016

Springer eBooks, 1992

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activ... more Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.

Journal of Biological Chemistry, Sep 1, 2000

Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human pla... more Interactions of the developmentally regulated chondroitin sulfate proteoglycan NG2 with human plasminogen and kringle domain-containing plasminogen fragments have been analyzed by solid-phase immunoassays and by surface plasmon resonance. In immunoassays, the core protein of NG2 binds specifically and saturably to plasminogen, which consists of five kringle domains and a serine protease domain, and to angiostatin, which contains plasminogen kringle domains 1-3. Apparent dissociation constants for these interactions range from 12 to 75 nM. Additional evidence for NG2 interaction with kringle domains comes from its binding to plasminogen kringle domain 4 and to miniplasminogen (kringle domain 5 plus the protease domain) with apparent dissociation constants in the 18-71 nM range. Inhibition of plasminogen and angiostatin binding to NG2 by 6-aminohexanoic acid suggests that lysine binding sites are involved in kringle interaction with NG2. The interaction of NG2 with plasminogen and angiostatin has very interesting functional consequences. 1) Soluble NG2 significantly enhances the activation of plasminogen by urokinase type plasminogen activator. 2) The antagonistic effect of angiostatin on endothelial cell proliferation is inhibited by soluble NG2. Both of these effects of NG2 should make the proteoglycan a positive regulator of the cell migration and proliferation required for angiogenesis.

Journal of Biological Chemistry, Jun 1, 1999

NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor ... more NG2 is a transmembrane chondroitin sulfate proteoglycan that is expressed by immature progenitor cells in several developmental lineages and by some types of malignant cells. In vitro studies have suggested that NG2 participates in growth factor activation of the platelet-derived growth factor-␣ receptor. In this study the ability of recombinant NG2 core protein to interact with several different growth factors (epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF)-AA, PDGF-BB, vascular endothelial growth factor (VEGF) 165 and transforming growth factor (TGF)-␤1) was investigated using two different assay systems: enzyme-linked immunosorbent assay-type solid-phase binding and an optical biosensor (BIAcore) system. High-affinity binding of bFGF and PDGF-AA to the core protein of NG2 could be demonstrated with both types of assays. Using both the BIAcore software analysis program and nonlinear regression analysis of the solid phase binding data, K D values in the low nanomolar range were obtained for binding of each of these growth factors to NG2. The results further indicate that NG2 contains at least two binding sites for each of these two growth factors. PDGF-BB, TGF-␤1, VEGF, and EGF exhibited little or no binding to NG2 in either type of assay. These data suggest that NG2 can have an important role in organizing and presenting some types of mitogenic growth factors at the cell surface.

Molecular Diagnostics of Cancer, 1993

Two types of serine proteases, uPA (urokinase-type plasminogen activator) and tPA (tissue-type pl... more Two types of serine proteases, uPA (urokinase-type plasminogen activator) and tPA (tissue-type plasminogen activator), are known to convert plasminogen into plasmin [1]. tPA is mainly involved in intravascular thrombolysis [1,2] whereas uPA mediates pericellular proteolysis during cell migration and tissue remodeling under physiological and pathophysiological conditions [2, 3]. Although initially secreted in the form of an enzymatically inactive, single-chain proenzyme (pro-uPA), uPA exerts its proteolytic function on normal cells and tumor cells as an ectoenzyme after having bound to a specific high-affinity cell surface receptor (uPA-R) [4–6]. uPA-R on cells seems to be the reaction site for uPA-mediated plasminogen activation, also in solid tumors. Quantitative assessments of tumor tissues, invasion assays with tumor explants in experimental animals and in vitro investigations with tumor cells suggest that tumor invasion and metastasis are correlated with elevated levels of uPA and the presence of the uPA-R [2–6]. Tissues of primary cancer and/or metastases of the breast, ovary, prostate, cervix uteri, bladder, lung and gastrointestinal tract have been reported to contain high amounts of uPA compared to benign control tissues [2, 3]. uPA is produced and secreted as pro-uPA by normal cells and by tumor cells [2, 3]. Pro-uPA may be converted by small amounts of serine proteases (plasmin, kallikrein, trypsin) or cathepsin B or L into the enzymatically active, high-molecular-weight, two-chain form HMW-uPA which subsequently converts plasminogen into the serine protease plasmin [7–10].

Biological Chemistry Hoppe-Seyler, 1995

Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various... more Urokinase-type plasminogen activator (uPA) converts plasminogen to plasmin which degrades various extracellular matrix components. uPA is focused to the cell surface via binding to a specific receptor (uPAR, also termed CD87). uPAR-bound uPA mediates pericellular proteolysis in a variety of biological processes, e.g. cell migration, tissue remodeling and tumor invasion. We have developed a competitive microtiter plate-based chromogenic assay which allows the analysis of uPA/uPAR interaction. The plates are coated with recombinant uPAR expressed in Chinese hamster ovary (CHO) cells. Proteolytically active uPA (HMW-uPA) is added to the microtiter plate-attached uPAR. The amount of receptor-bound uPA is then determined indirectly via addition of plasminogen, which is activated to plasmin, followed by cleavage of a plasmin-specific chromogenic substrate. Substances interfering with binding of HMW-uPA to uPAR diminish the generation of plasmin, as indicated by a reduction of cleaved chromogenic substrate. This assay was used to analyze the inhibitory capacity of a variety of proteins and peptides, respectively, on the uPA/uPAR interaction: i) uPAR and uPAR-variants expressed in CHO cells, yeast or E. coli, ii) the aminoterminal fragment (ATF) of human uPA or yeast recombinant pro-uPA, iii) synthetic peptides derived from the sequence of the uPAR-binding region of uPA, and iv) antibodies directed against uPAR. This assay may be helpful in identifying uPA and uPAR analogues or antagonists which efficiently block uPA/uPAR interaction.

Journal of Biological Chemistry, 1999

INTRODUCTION. Cell attachment to the extracellular matrix (ECM) is mediated by a family of cell s... more INTRODUCTION. Cell attachment to the extracellular matrix (ECM) is mediated by a family of cell surface receptors, the integrins. More than 20 different integrins and several additional splice variants have been identified, a specific subset of them being expressed by each cell (1). The β1 or VLA family of integrins has the largest number of members and shares the common β1 subunit, including α2β1, a type IV collagen and type

Cancer Research, 2004

1307 Heat shock proteins (hsps) play a central role in the regulation of intracellular homeostasi... more 1307 Heat shock proteins (hsps) play a central role in the regulation of intracellular homeostasis, and differential expression of individual hsps occurs in a broad range of neoplastic processes. Hsp27 is a 27 kDa member of the heat shock protein family which has previously been studied as p29, srp27, and p24. Increasing evidence supports a role for Hsp27 in cell growth and motility. High concentrations of Hsp27 have been associated with rapid proliferation in hormonally sensitive tissues, such as breast, endometrium, and prostate. Elevated levels of Hsp27 are associated with aggressive growth and cell invasion, which often result in poor prognosis in breast, ovarian, and prostate carcinomas. An immunoassay for the quantification of the biological marker Hsp27 was developed and evaluated for its use with extracts of breast cancer tissue and total cell lysates of various cell lines. The assay is based on a murine monoclonal capture antibody and a rabbit polyclonal detector antibody. ...

Cancer Research, 2006

1988 Introduction: Interleukin-2 (IL-2; Proleukin®) is a lymphokine that is used to treat patient... more 1988 Introduction: Interleukin-2 (IL-2; Proleukin®) is a lymphokine that is used to treat patients with metastatic malignant melanoma and metastatic renal cell carcinoma. In addition to activation of signal transducer and activator of transcription (STAT) 5, IL-2 is known to signal through the Ras/MAP Kinase pathway by activating extracellular signal-regulated protein kinase (Erk) within immune cells. This signaling event mediates pleiotropic effects relating to cellular proliferation, activation, and survival. To determine the importance of Erk activation in vivo, we developed a novel dual parametric flow cytometric assay to measure phosphorylated Erk (P-Erk) within the immune effector cell subsets of normal subjects and patients receiving IL-2 therapy. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and patients with advanced renal cell carcinoma or malignant melanoma undergoing treatment with high-dose IL-2 (720,000 IU/kg i.v.) and analyzed b...

A process for the production of a floor-cast stone having the following properties: - acid resist... more A process for the production of a floor-cast stone having the following properties: - acid resistant and alkali resistant, - resistant to conventional cleaning and polishing agents, - resistant against Auslaugerscheinungen, efflorescence and surface stains associated, - water and dirt repellent. in the preparation of the concrete stone having the aforementioned properties - the cast stone surface is first pre-treated by surface treatment such that the grains of the aggregate of the concrete stone protrude from the surface of the cementitious matrix, - and then the surface has a micro-roughness with a mean roughness value R while

Cancer Research, 2008

1544 Cell migration is crucial for embryonic development, the inflammatory immune response, wound... more 1544 Cell migration is crucial for embryonic development, the inflammatory immune response, wound repair, and tumor invasion and metastasis. Monocytes circulate in the blood stream for about one to three days and then typically migrate into tissues throughout the body. In immune responses, monocyte migration is critical for providing immunological defense and eliminating infectious agents. Circulating monocytes have the capacity to differentiate into a variety of phagocytes, including macrophages, dendritic cells, osteoclasts, microglia in the central nervous system, and Kupffer cells in the liver. To facilltate the assessment of the migratory capacity of monocytes against various chemotactic stimuli and the effect of inhibitory compounds on monocyte migration, we developed a convenient high-capacity migration/chemotaxis assay for monocytes. The Monocyte Cell Migration Assay Kit is based on the widely accepted Boyden Chamber principle and in a 96-well format. The presented assay pr...

287 We have developed a convenient and sensitive assay for the measurement of mTOR activity. A GS... more 287 We have developed a convenient and sensitive assay for the measurement of mTOR activity. A GST fusion of the classical mTOR substrate p70S6K is pre-bound to a microtiter plate coated with glutathione. mTOR-containing sample is then added to the sample well in the presence of ATP and manganese containing assay buffer. Active mTOR phosphorylates p70S6K at Thr389. The phosphorylated substrate is detected with a phosphospecific anti-p70S6K(T389) antibody, followed by detection with HRP-antibody conjugate and relative activity is determined by reading absorbance. Using the assay we are able to measure kinase activity of a partially purified mTOR standard generated from an enriched rat brain fraction. The enriched mTOR standard contains both mTOR/Raptor and mTOR/Rictor complexes. The detected mTOR activity is inhibited by the general PI-3K inhibitor wortmannin as well as rapamycin/FKBP12 (IC50 of 8 nM), a specific inhibitor of the mTOR/Raptor complex. The assay was miniaturized to a w...

3783 Galectins are a growing family of carbohydrate binding adhesion molecules (lectins) with aff... more 3783 Galectins are a growing family of carbohydrate binding adhesion molecules (lectins) with affinity for lactose and other β-galactosides. Mammals express at least 10 different galectins. Galectins are characterized by Ca 2+ independence and extensive sequence identity in the carbohydrate recognition domain. Cell adhesion involves protein-protein interaction (recognition of peptide motifs) as well as protein-carbohydrate interaction. Galectins can be found intracellularly and extracellularly. Extracellular galectins cross-link cell-surface and extracellular glycoproteins and may thereby modulate cell adhesion and induce intracellular signals. Galectin-1 is a monomeric or homodimeric prototype galectin that is expressed in a variety of cells. Galectin-1 is involved in multiple biological functions, including cell adhesion, apoptosis, and tumor metastasis. Galectin-1 can modulate cell-cell as well as cell-matrix interactions. Galectin-3 is expressed in normal and neoplastic cells an...

Fibrinolysis and Proteolysis, 1997

Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in nume... more Urokinase-type plasminogen activator (uPA) is a serine protease which has been implicated in numerous physiological and pathological processes, e.g. tissue remodelling, embryogenesis, fibrinolysis, and tumour spread, uPA protease binds to a specific high-affinity receptor (uPAR; CD87) on normal and tumour cells. This binding is mediated by the growth factor domain of uPA and thus independent of its proteolytic activity. An ELISA-type, solid-phase microtitre plate assay is presented, designed for the quantitation of such uPA molecules capable of binding to the receptor uPAR. This solid-phase uPA-ligand binding assay makes use of the specific, high-affinity interaction of uPA with uPAR. This assay format is different from the common uPA-ELISA which measures uPA antigen but not uPAR-reactivity. Recombinant soluble uPAR (CHO-uPAR), attached to the well of a microtitre plate, serves as the capture molecule for uPA. uPA-containing samples are added to allow binding of uPA to immobilized uPAR. Receptor-bound uPA is then detected by reaction of uPA with biotinylated monoclonal antibody no. 377 (American Diagnostica, Greenwich, CT, USA) directed to the kringle domain of uPA followed by avidin-peroxidase. The solid-phase uPA-ligand binding assay detects various forms of uPA: pro-uPA, HMW-uPA, ATF, GFD but does not react with the low molecular weight form of uPA (LMW-uPA) lacking the uPAR-reactive domain, uPA molecules in which the uPAR-binding domain has been impaired by proteolysis and uPA/uPAR complexes are also excluded from detection. The high sensitivity of the solid-phase uPAligand binding assay (lower limit 2 pM = 0.1 ng uPA/ml) allowed to measure reactive uPA (and fractions thereof) in the supernatants of cultured ovarian cancer cells, in extracts of ovarian and breast cancer tissues, in placenta tissue extracts and in malignant ascites. The solid-phase uPA-ligand binding assay was also used to screen the receptor binding reactivity of recombinant human uPA-polypeptides synthesized by yeast cells and that of synthetic uPA-peptides. The uPAR-blocking capability of uPA-peptides uPA14-32, uPA14-32/H29A, and uPA14-32/N32A, determined by the solidphase uPA-ligand binding assay, was confirmed by flow cytofluorometric analysis employing fluorescent pro-uPA (FITCpro-uPA) and the uPAR-rich promyeloid cell line U937.

FEBS Letters, 1992

Increased levels of both the cysteine protease, cathepsin L, and the serinc protease, uPA (urokin... more Increased levels of both the cysteine protease, cathepsin L, and the serinc protease, uPA (urokinase-typc plasminogen activator), are present in solid tumors and are correlated with malignancy, uPA is released by tumor cells as an inactive single.chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protcase, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro.uPA, As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys~K-lle~9 peptidc bond, a common activation site of serine proteases such as plasmin and kallikrcin. Similar to cathepsin B (Kobayashi et al,, J. Biol, Chem, (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most efl'~ctive at acidic pH (molar ratio ofcathepsin L to pro-uPA of 1:2,000), Nevertheless, even at pH 7,0, pro-uPA was activated by cathepsin L, althougla a 10-fold higher concentration of cathep~in L was required, As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autoeatalytic intrinsic activation of pro-uPA; (it) activation by serine protcases (plhsmin, kallikrein, Factor Xlla); and (iii) activation by cysteine proteases (cathepsin B and L),

Biochemical Society Transactions, 1992

208 Flow cytofluorometry in tumour cell receptor analysis. Survey of the literature and recent de... more 208 Flow cytofluorometry in tumour cell receptor analysis. Survey of the literature and recent developments concerning the urokinase-type plasminogen activator ( u W Nicolaus Chucholowski. Manfred Schmitt, Lothar Goretzki. Elisabeth Schuren, Nobuhiko Moniwa, Ulrich Weidle,t Michael Kramer,$ Bettina Wagner,* Fritz Janicke and Henner Graeff Frauenklinik der Technischen Universitat Munchen, Ktinikum rechts der Isar, Munchen, FRG, *Leica, Heidelberg, FRG, tBoehringer-Mannheim, Penzberg, FRG and lnstitut fur Imrnunologie, Universitat Heidelberg, FRG

Hemostasis and Circulation, 1992