Douglas A. Lappi

Ribosome inactivating proteins from plants: present status and future prospects

Plant ribosome–inactivating proteins (RIPs) N–glycosidases which cleave the N–glycosidic bond of adenine in a specfic ribosomal RNA sequence. Most commonly RIPs are single–chain proteins (type 1 RIPs), but some (type 2 RIPs) possess a galactose–specific lectin domain that binds to cell surfaces. The latter RIPs are potent toxins, the best known of which is ricin. RIPs have antiviral and abortifacient activities, and, in a widespread application, can also be linked to antibodies or ligands to form immunotixins or conjugates specifically toxic to a given type of cell.

Immobilization of hydrogenase on glass beads.

Abstract Hydrogenase from Clostridium pasteurianum was immobilized on glass beads by four different methods. The sensitivity of the native and bound enzyme to oxygen was examined. Hydrogenase bound to succinyl glass proved to be the most stable to oxygen. All bound enzymes were active with ferredoxin as a substrate and evolved hydrogen in a chloroplast-ferredoxin-hydrogenase system driven by light.

8-(6-Aminohexyl)-amino-adenine nucleotide derivatives for affinity chromatography

Abstract 8-(6-Aminohexyl)-amino-AMP and 8-(6-aminohexyl)-amino-NAD(H) are synthesized from AMP and NAD + , respectively, in a two-step procedure via the 8-Br-adenine nucleotides. The AMP derivatives and the NAD + derivatives possess some biological activity as substrates in the adenylate kinase and as coenzymes in the alcohol and lactate dehydrogenase systems, respectively. The synthesized derivatives may be coupled to Sepharose by cyanogen bromide activation. The covalently attached ligands show strong affinities for various dehydrogenases. Examples of the selective purification of several dehydrogenases using the new affinity columns are presented.

Biological and chemical characterization of basic FGF-saporin mitotoxin

Basic fibroblast growth factor (FGF) and saporin-6, a ribosome-inactivating protein, were chemically conjugated and characterized as a cytotoxin to cells expressing the basic FGF receptor. Structural and Western blot analysis of the conjugate showed that it contained saporin and basic FGF in equimolar amounts. The conjugate inhibited protein synthesis in a cell-free system and had potent cytotoxic activity (ID50 = 25pM) for cells expressing the basic FGF receptor. It is equipotent with basic FGF in radioreceptor assays and elutes from heparin Sepharose columns with 2M NaCl. The activity of the mitotoxin can be inhibited by competition with an excess of basic FGF but not nerve growth factor. The possibility that this mitotoxin can be used as an anti-angiogenic factor in paradigms that involve basic FGF is discussed.

Mitotoxins : growth factor-targeted cytotoxic molecules

When the selective specificity and exquisite affinity of growth factors for their receptors is conferred to protein toxins, the chimeric molecules so generated become potent cytotoxins. Chimera are produced by the chemical conjugation of the two proteins or by expression of fusion proteins in bacterial expression systems. The toxic moiety, usually a ribosome-inactivating protein or a fragment of a bacterial toxin, is internalized into target cells by receptor-mediated endocytosis. Release of the receptors ligand in the endosome allows the toxic moiety to exert its action on protein synthesis. Accordingly, the potent catalytic activity of the toxin results in a sensitivity of some cells to picomolar quantities of the mitotoxin. This review discusses the numerous growth factor-toxins that have been created and describes some of their applications.

Evaluation of antihuman T lymphocyte saporin immunotoxins potentially useful in human transplantation

We have synthesized 3 immunotoxins (ITs) by covalently coupling the saporin-6 hemitoxin (SAP) to OKT11, SOT3, and SOT1a murine monoclonal antibodies that recognize human T lymphocyte CD2, CD3, and CD5 surface antigens, respectively. The resulting ITs, referred to as OKT11-SAP, SOT3-SAP, and SOT1a-SAP, are equally effective in inhibiting eukaryotic protein synthesis in a cell-free system, and all 3 ITs bind to human T lymphocytes in an almost comparable manner. However, these reagents differ markedly in their ability to kill target T lymphocytes as assessed by measuring the inhibition of DNA synthesis and growth of clonable T lymphocytes in response to mitogenic and allogeneic stimuli. Whereas the anti-CD2 IT, OKT11-SAP, shows moderate cytotoxicity against T lymphocytes, the anti-CD3 IT, SOT3-SAP, and the anti-CD5 IT, SOT1a-SAP, are highly effective in eliminating the same target cells. The concentrations inhibiting 50% (IC50) of T lymphocyte DNA synthesis are 60 nM, 4.5 nM, and 1.4 nM for OKT11-SAP, SOT3-SAP, and SOT1a-SAP, respectively. Among 3 tested lysosomotropic amines, i.e., ammonium chloride, chloroquine, and amantadine, the latter only moderately potentiates the cytotoxicity of SOT1a-SAP (IC50 0.36 nM). We show that the conditions under which T lymphocyte killing is accomplished require less than 10 min exposure of T lymphocytes to the ITs, in the absence of adjuvant molecules artificially added to the incubation medium and at physiologic culture pH. These experimental characteristics of unprecedented closeness to a physiologic in-vivo model are likely to reflect the biophysical properties of the SAP moiety of the ITs. We conclude that clinical studies are warranted to define the advantage of using SAP ITs over previously described immunoconjugates.

Basic fibroblast growth factor in cells derived from Dupuytren's contracture: Synthesis, presence, and implications for treatment of the disease☆☆☆

Dupuytrens contracture (DC) is associated with fibroblast and endothelial cell proliferation. We have identified a fibroblast and endothelial cell mitogen, basic fibroblast growth factor (FGF), in cells derived from this tissue and characterized the effects of this growth factor on DC cells. Northern blot analysis of DC cells reveals the presence of basic FGF mRNA species, and the DC cells coexpress multiple forms of basic FGF. Radioreceptor assays establish that the DC cells have high-affinity binding sites for basic FGF and proliferate in response to exogenous recombinant basic FGF. Furthermore, a conjugate between saporin (a ribosome-inactivating protein) and basic FGF, which is cytotoxic to cells possessing the basic FGF receptor, is also cytotoxic to DC cells. The possibility that basic FGF-saporin could be a potential therapeutic agent for prevention of recurrence of the disease after surgery is discussed.

Selective Elimination of Fibroblasts From Pancreatic Islet Monolayers by Basic Fibroblast Growth Factor–Saporin In Mitotoxin

Fibroblast proliferation regularly impedes the initiation and maintenance of pancreatic islet monolayers in culture. We recently characterized a specific cytotoxin to cells expressing the basic fibroblast growth factor receptor by conjugating the growth factor to saporin-6, a ribosome-inactivating protein. In contrast to untreated islets, isolated adult rat islets grown on a substrate prepared from bovine corneal endothelial cells and incubated with the mitotoxin at 10-nM concentration for 96 h were free of contaminating fibroblasts. Histological and functional studies revealed there was no damage to the islets. The results suggest that treatment of this cell type with basic fibroblast growth factor mitotoxins may be an important tool for culture of pure islets for physiological and clinical studies.

Anti-B16-F10 melanoma activity of a basic fibroblast growth factor-saporin mitotoxin.

Background. The authors attached basic fibroblast growth factor (FGF‐2), a growth factor for numerous tumors and normal cell types, to saporin (SAP), a ribosomeinactivating protein isolated from the plant Saponaria officinalis. The conjugate (FGF‐SAP) then was tested for antitumor activity using B16‐F10 melanoma cells. This rapidly growing murine melanoma cell line has been used classically as a model to screen antitumor agents.

Saporin toxins directed to basic fibroblast growth factor receptors effectively target human ovarian teratocarcinoma in an animal model

Background. The antitumor activity of the chemical conjugate and recombinant forms of the mitotoxin basic fibroblast growth factor (bFGF) saporin (SAP) and the bFGF receptor‐directed immunotoxin 11A8‐SAP against human ovarian teratocarcinoma PA‐1 was examined in athymic nude mice. Alternative administration schedules to prolong therapeutic efficacy were explored.