Biomedical subjects

P Kussie

Publications and source records attributed to P Kussie.

13 recordsLinked to original sources

PubMed2006

Activation of FGFR1beta signaling pathway promotes survival, migration and resistance to chemotherapy in acute myeloid leukemia cells.

Fibroblast growth factors (FGFs) are important regulators of hematopoiesis and have been implicated in the tumorigenesis of solid tumors. Recent evidence suggests that FGF signaling through FGF receptors (FGFRs) may play a role in the proliferation of subsets of acute myeloid leukemias (AMLs). However, the precise mechanism and specific FGF receptors that support leukemic cell growth are not known. We show that FGF-2, through activation of FGFR1beta signaling, promotes survival, proliferation and migration of AML cells. Stimulation of FGFR1beta results in phosphoinositide 3-kinase (PI3-K)/Akt activation and inhibits chemotherapy-induced apoptosis of leukemic cells. Neutralizing FGFR1-specific antibody abrogates the physiologic and chemoprotective effects of FGF-2/FGFR1beta signaling and inhibits tumor growth in mice xenotransplanted with human AML. These data suggest that activation of FGF-2/FGFR1beta supports progression and chemoresistance in subsets of AML. Therefore, FGFR1 targeting may be of therapeutic benefit in subsets of AML.

Acute Disease↗

PubMed2003

Inhibition of human leukemia in an animal model with human antibodies directed against vascular endothelial growth factor receptor 2. Correlation between antibody affinity and biological activity.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.

Animals↗

PubMed2000

Identification of the residues in the extracellular region of KDR important for interaction with vascular endothelial growth factor and neutralizing anti-KDR antibodies.

The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.

Alanine↗

PubMed1998

Conservation of structure and mechanism between eukaryotic topoisomerase I and site-specific recombinases.

Vaccinia DNA topoisomerase breaks and rejoins DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. A C-terminal catalytic domain, Topo(81-314), suffices for transesterification chemistry. The domain contains a constellation of five amino acids, conserved in all eukaryotic type IB topoisomerases, that catalyzes attack of the tyrosine nucleophile on the scissile phosphate. The structure of the catalytic domain, consisting of ten alpha helices and a three-strand beta sheet, resembles the catalytic domains of site-specific recombinases that act via a topoisomerase IB-like mechanism. The topoisomerase catalytic pentad is conserved in the tertiary structures of the recombinases despite scant sequence similarity overall. This implies that the catalytic domains of type IB topoisomerases and recombinases derive from a common ancestral strand transferase.

Binding Sites↗

PubMed1997

Plasma kinetics of cholesteryl ester transfer protein in the rabbit. Effects of dietary cholesterol.

The plasma kinetics of recombinant human cholesteryl ester transfer protein (rCETP) were studied in six rabbits before and after cholesterol feeding (0.5% wt/wt). The rCETP, labeled with the use of the Bolton Hunter reagent, was shown to retain neutral lipid transfer activity. After intravenous infusion, labeled rCETP associated with rabbit lipoproteins to an extent similar to endogenous rabbit CETP (62% to 64% HDL associated). The plasma kinetics of CETP, modeled with the use of SAAM-II, conformed to a two-pool model, likely representing free and loosely HDL-associated CETP (fast pool) and a tightly apo (apolipoprotein) AI-associated (slow pool) CETP. The plasma residency time (chow diet) of the fast pool averaged 7.1 hours and of the slow pool, 76.3 hours. The production rate (PR) into and the fractional catabolic rate (FCR) of the fast pool were 20 and 10 times the PR and FCR, respectively, of the slow pool. In response to cholesterol feeding, CETP PR, FCR, and plasma mass increased by 416%, 60%, and 230%, respectively. There was a strong correlation (r = .95, P = .003) between the increase in rabbit plasma CETP and the modeled increase in CETP PR in response to cholesterol feeding, suggesting that labeled human rCETP is a satisfactory tracer for rabbit plasma CETP. CETP is catabolized by distinct pools, likely corresponding to an apo AI-associated (slow) pool and a free and/or loosely HDL-associated (fast) pool. Factors that alter the affinity of CETP for HDL would be predicted to result in altered CETP catabolism. The effect of dietary cholesterol on plasma CETP mass can be explained largely by the effects on CETP synthesis, consistent with the observed effects of cholesterol on tissue mRNA levels.

Animals↗

PubMed1996

Structure-function relationships of human cholesteryl ester transfer protein: analysis using monoclonal antibodies.

Cholesteryl ester transfer protein (CETP), a 476 amino acid glycoprotein, mediates cholesteryl ester (CE), triglyceride, and phospholipid transfer among plasma lipoproteins. A monoclonal antibody (mAb), TP2, specific for an epitope within the last 26 amino acids of CETP has been shown to block all CETP-mediated lipid transfer, apparently by limiting access to lipid-binding sites in the carboxy terminal of CETP. A new panel of 16 anti-human CETP mAbs has now been used to further probe the structure-function relationships of CETP. Of the new mAbs, 9 partially inhibit CETP-mediated CE transfer (24-43%) from HDL to LDL. The corresponding epitopes were mapped within the CETP primary structure by the reactivity of the mAbs with CETP variants having deletions or amino acid substitutions. Of the 9 new, neutralizing mAbs, 6 are specific for epitopes situated between residues 410-450 and two others for epitopes between residues 184-260 and 332-366, respectively. The epitope of one neutralizing mAbs could not be mapped. Therefore, binding of mAbs to epitopes situated in four non-overlapping regions within CETP primary structure that are separated by as many as 280 residues can neutralize CETP-mediated CE transfer. Epitopes of mAbs that do not influence CE transfer activity map to the regions 184-260, 261-331, and 367-409, respectively. When pairs of mAbs were tested for their abilities to mutually compete for binding to immobilized CETP, competition was observed for mAbs specific for epitopes that are distant in CETP primary structure. The cross-competition patterns demonstrate that the carboxy terminal 60% of CETP adopts a compact structure. Together with previous mutagenesis studies, the data suggests that a carboxy terminal neutral lipid binding domain may be in close proximity to a lipoprotein binding region within native CETP.

Animals↗

PubMed1995

Identification of a plasminogen binding region in streptokinase that is necessary for the creation of a functional streptokinase-plasminogen activator complex.

Streptokinase is a plasminogen activator widely used to treat patients with myocardial infarction. However, streptokinase is not a protease, and must first bind and interact with plasminogen to form an enzymatic complex. By measuring the binding of recombinant streptokinase fragments to plasminogen, we have sought, first, to identify a plasminogen binding region in streptokinase and, second, to explore the relation between binding (via this region) and the generation of a functional streptokinase--plasminogen activator complex. Recombinant streptokinase bound in a saturable and specific manner to human Glu-plasminogen with a dissociation constant of 4.2 x 10(-10) M. Recombinant streptokinase fragments spanning amino acids 1-127 and 1-253 could not be shown to bind to Glu-plasminogen, whereas fragments spanning amino acids 1-352, 120-352, and 244-414 bound tightly to plasminogen and each fragment completely inhibited the binding of full-length streptokinase to plasminogen. Although these latter streptokinase fragments formed a complex with plasminogen, enzymatic assays indicated that none of them was capable of generating an active site. When the streptokinase region shared by these three fragments, spanning residues 244-352, was expressed, it also bound plasminogen and competitively inhibited the formation of a functional plasminogen activator complex by full-length streptokinase. Taken together, these data indicate that streptokinase binds to plasminogen with high affinity, that a primary binding region for plasminogen is located within amino acids 244-352, and that binding via this region is necessary for the generation of a functional plasminogen activator complex.

Base Sequence↗

PubMed1995

Molecular determinants of plasma cholesteryl ester transfer protein binding to high density lipoproteins.

The plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids between lipoproteins and is associated with high density lipoproteins (HDL). To understand the mechanism of interaction of CETP with HDL, we studied the binding of pure recombinant CETP to 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/apoA-I discoidal particles. Separating bound from free CETP using native gradient gel electrophoresis, complexes of CETP with 10-nm hydrodynamic diameter discoidal particles migrated with a diameter of 12-16 nm, compared with approximately 7.5 nm for CETP. At lower ratios of CETP to discs, CETP bound to discs without displacement of apoA-I. CETP alone was unable to generate discoidal complexes. Cross-linking and fluorescence resonance energy transfer experiments indicated that CETP bound to discs as monomers. Cross-linking of CETP to apoA-I in discs suggested proximity of apoA-I and CETP. By negative-stain electron microscopy, discoidal complexes containing CETP and CETP monoclonal antibody showed localization of antibody molecules to the disc edge, suggesting that CETP was bound to the disc edge. The binding of CETP to discs of different composition or size was studied. Discs (10-nm Stokes diameter) prepared with either apoA-I or apoA-II had a similar Kd (120 nM). Inclusion of 1 mol % cholesteryl oleate, 5 mol % cholesterol, or 6 mol % phosphatidylinositol increased the binding affinity of CETP 3-10 times (20-30 nM). In comparison, plasma HDL3 had a Kd of approximately 450 nM. For POPC/apoA-I discs, 10-nm discs bound CETP with much higher affinity than smaller 7.8-nm discs (Kd = 1-2 microM). 7.7-nm hydrodynamic diameter POPC/apoA-I spherical particles containing either triolein or cholesteryl oleate in their core bound CETP with higher affinity (Kd = 50-100 nM) than 7.8-nm POPC/apoA-I discs. Thus, CETP appears to bind to the perimeter of discoidal particles, possibly in a process in which flexible segments in apoA-I or apoA-II accommodate CETP at the disc edge. The binding of CETP to HDL is markedly influenced by overall particle size and shape and by lipid composition, and the increased binding affinity for cholesterol- and cholesteryl ester-containing discs suggests a higher affinity of CETP for nascent than mature HDL.

Animals↗

PubMed1995

Defective binding of neutral lipids by a carboxyl-terminal deletion mutant of cholesteryl ester transfer protein. Evidence for a carboxyl-terminal cholesteryl ester binding site essential for neutral lipid transfer activity.

The plasma cholesteryl ester transfer protein (CETP, 476 amino acids) transfers cholesteryl ester (CE) from high density lipoprotein (HDL) to triglyceride-rich lipoproteins and plays a major role in HDL catabolism. Using deletional and site-directed mutagenesis, we previously showed that the carboxyl terminus of human CETP comprises the epitope of a neutralizing monoclonal antibody and is necessary for neutral lipid transfer activity. To assess the nature of the involvement of the COOH terminus in cholesteryl ester transfer activity, we characterized a deletion mutant of CETP lacking amino acid residues 470-475 in terms of CE transfer kinetics, association with HDL, and capacity to bind CE, triglyceride (TG), and phosphatidylcholine (PC). Kinetic analysis indicated a major catalytic defect of the deletion mutant, as shown by markedly decreased maximum cholesteryl ester transfer activities (apparent Vmax) for donor (HDL) and acceptor (low density lipoprotein (LDL)) lipoproteins but there were no significant changes of concentrations of the donor and acceptor at 50% Vmax (apparent Km). The binding of CETP to HDL, as determined by native gel electrophoresis, was similar for wild-type and mutant protein. When egg PC/CE vesicles were incubated with wild type CETP and then separated by gel filtration chromatography, there was maximum binding of about 1 mol of CE/mol of CETP. Under similar conditions the mutant CETP bound 0.09-0.37 mol of CE/mol of protein. Similarly, when egg PC/TG vesicles were incubated with the CETP proteins, there was a maximum binding of 0.5 mol of triglyceride/mol of wild-type CETP, whereas there was only 0.00-0.07 mol of TG/mol of deletion mutant. The binding of phosphatidylcholine was similar for wild-type and the deletion mutant. The studies suggest that amino acids 470-475 (forming part of a COOH-terminal amphipathic helix) are involved in CE and TG binding by CETP but are not required either for the binding of PC by CETP or the association of CETP with HDL. The COOH terminus of CETP may comprise a neutral lipid binding site directly involved in the lipid transfer mechanism.

Animals↗

PubMed1994

Interfacial properties of recombinant human cholesterol ester transfer protein.

We investigated the interfacial behavior of recombinant human cholesterol ester transfer protein (rCETP) using monolayer and surface balance techniques. rCETP bound to egg phosphatidylcholine monolayers spread at the air/water interface with a maximum surface pressure of 23 millinewtons (mN)/m at subphase concentrations between 3 and 5 x 10(-5) g/dl; the estimated dissociation constant was 7.5 x 10(-6) g/dl or 1 nM. The binding of rCETP to the lipid interface decreased linearly with increasing initial surface pressure; rCETP was excluded at pressures greater than 31 mN/m. rCETP catalyzed the desorption of [14C]cholesterol oleate from mixed lipid monolayers in a concentration dependent fashion. Similar studies with apolipoproteins A-I and A-IV established that cholesterol ester desorption was not caused by changes in surface pressure or cholesterol ester solubility. The desorption rate was proportional to subphase rCETP concentration, but at all concentrations surface radioactivity remained constant until surface pressure reached a plateau. The calculated binding stochiometry was one molecule of cholesterol ester desorbed for every 1000 molecules of rCETP in the subphase. We conclude that rCETP is surface active, binds to phospholipid monolayers with an affinity equivalent to that of the plasma apolipoproteins, and effects the desorption of cholesterol ester molecules from phospholipid monolayers by a carrier mechanism. Moreover, the relatively low equilibrium surface pressure of rCETP suggests that when bound to lipid the entire rCETP molecule may not penetrate the interface.

Adsorption↗

PubMed1993

A functional analysis of the antigenicity of streptokinase using monoclonal antibody mapping and recombinant streptokinase fragments.

Streptokinase (SK), a bacterial product of pathogenic Streptococcus species, is now widely used as an effective therapy for the treatment of heart attacks. Because naturally occurring antibody to SK is ubiquitous, serious allergic reactions to SK therapy are common. To begin to identify regions of the molecule that are important for the antigenicity of SK we performed studies using a panel of 51 hybridomas producing anti-SK antibodies, recombinant SK fragments, and assays of SK activity. Antibodies generated from mice hyperimmunized with wild-type SK were shown to fall into six distinct complementation groups by competitive binding studies. Recombinant SK fragments were used to determine the peptide regions recognized by these complementation groups. Correlation of the effects of the mAb on SK function, with knowledge of their SK fragment-binding pattern, suggested regions of the SK molecule that are important for the construction and the catalytic function of the SK-plasminogen activator complex.

Antibodies, Monoclonal↗

PubMed1993

A missense mutation in the cholesteryl ester transfer protein gene with possible dominant effects on plasma high density lipoproteins.

Plasma HDL are a negative risk factor for atherosclerosis. Cholesteryl ester transfer protein (CETP; 476 amino acids) transfers cholesteryl ester from HDL to other lipoproteins. Subjects with homozygous CETP deficiency caused by a gene splicing defect have markedly elevated HDL; however, heterozygotes have only mild increases in HDL. We describe two probands with a CETP missense mutation (442 D:G). Although heterozygous, they have threefold increases in HDL concentration and markedly decreased plasma CETP mass and activity, suggesting that the mutation has dominant effects on CETP and HDL in vivo. Cellular expression of mutant cDNA results in secretion of only 30% of wild type CETP activity. Moreover, coexpression of wild type and mutant cDNAs leads to inhibition of wild type secretion and activity. The dominant effects of the CETP missense mutation during cellular expression probably explains why the probands have markedly increased HDL in the heterozygous state, and suggests that the active molecular species of CETP may be multimeric.

Aged↗

PubMed1991

Inhibition of acetylcholinesterase by caffeine, anabasine, methyl pyrrolidine and their derivatives.

The inhibition of acetylcholinesterase (AChE) by caffeine, anabasine, methylpyrrolidine and several derivatives was examined. Most of the compounds had moderate inhibitory activity with I50 values in the range of 87-480 microM. The inhibition of AChE by these compounds has not been previously reported. A structural feature common to these compounds is the N-methyl determinant of the pyrrolidine ring which may be important in binding to the AChE.

Acetylcholinesterase↗