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At least 19 recordsLinked to original sources
PubMed1999

Deficit in selective and divided attention associated with cholinergic basal forebrain immunotoxic lesion produced by 192-saporin; motoric/sensory deficit associated with Purkinje cell immunotoxic lesion produced by OX7-saporin.

The immunotoxin 192-saporin, infused intracerebroventricularly into rats, destroys cholinergic neurons in the basal forebrain nuclei. Doses required for complete cholinergic loss also kill some Purkinje cells. The immunotoxin OX7-saporin, when infused intraventricularly into rats, destroys Purkinje cells in a pattern similar to that produced by 192-saporin, without affecting cholinergic neurons. Thus, we used OX7-saporin to distinguish behavioral effects of 192-saporin due to cerebellar damage versus those due to cholinergic cell loss. Three doses of 192-saporin (1.6, 2.6, and 3.3 micrograms/rat) were chosen along with a dose of OX7-saporin (2.0 micrograms/rat) that produced Purkinje loss equivalent to the two highest doses of 192-saporin. Groups of Fischer-344 rats were trained in the multiple choice reaction time task and retested with more complex tasks after lesioning. They were also tested in the water maze, passive avoidance, acoustic startle, and open field. The OX7-saporin group exhibited changes in many tests suggesting hypermotility and sensory deficits. The 192-saporin groups differed from the OX7-saporin group when they displayed deficits in multiple choice reaction time tasks in which novel challenges were introduced, including sessions with a noise distractor, shortened and lengthened intertrial intervals, and use of nine instead of five sources of light stimulus. The 192-saporin groups showed no impairment in the other tasks. The cholinergic basal forebrain lesion may mask some of the effects of cerebellar damage up to a threshold after which effects of Purkinje cell loss predominate when 192-saporin is administered intraventricularly.

Animals↗
PubMed1995

Preclinical studies with the anti-CD19-saporin immunotoxin BU12-SAPORIN for the treatment of human-B-cell tumours.

The immunotoxin BU12-SAPORIN was constructed by covalently coupling the single-chain ribosome-inactivating protein saporin to the anti-CD19 monoclonal antibody BU12 via a disulphide linker using the heterobifunctional reagent SPDP. The immunoreactivity and specificity of BU12-SAPORIN was identical to that of unmodified native BU12 antibody. BU12-SAPORIN was selectively cytotoxic in vitro in a dose-dependent manner for the CD19+ human common acute lymphoblastic leukaemia (cALL) cell line NALM-6 but exhibited no toxicity for the CD19- T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2. The survival of severe combined immunodeficient (SCID) mice with disseminated NALM-6 leukaemia was significantly prolonged compared with sham-treated control animals by a course of therapy with BU12-SAPORIN but not with the irrelevant anti-CD7 immunotoxin HB2-SAPORIN. BU12-SAPORIN had no therapeutic effect in SCID mice with disseminated CD19- HSB-2 leukaemia. These preclinical studies have clearly demonstrated the selective cytotoxicity of BU12-SAPORIN for CD19+ target cells both in vitro and in vivo. This, taken together with the lack of expression of the CD19 molecule by any normal life-sustaining tissue and its ubiquitous and homogeneous expression by the majority of cALL and B-NHL cells, provides the rationale for undertaking a phase I trial of systemic therapy with BU12-SAPORIN.

Animals↗
PubMed1993

Targeting of saporin to CD25-positive normal and neoplastic lymphocytes by an anti-saporin/anti-CD25 bispecific monoclonal antibody: in vitro evaluation.

This study has been designed to verify the specific toxicity of saporin, a type 1 ribosome-inactivating protein (RIP), with the same activity as ricin A chain, targeted by a bispecific monoclonal antibody (bimAb) recognising both the CD25 antigen and the RIP. The CD25 antigen is expressed by lymphoid populations upon activation and by leukaemias and lymphomas with an activated membrane phenotype (Hodgkin's lymphoma, anaplastic large cell lymphoma, adult T cell leukaemia). The bimAb-saporin mixture was tested on CD25+ targets at different bimAb and saporin concentrations. Saporin, in the presence of a bimAb concentration of 10(-9) M, inhibited protein synthesis by CD25+ neoplastic lymphocytes (L540 and MT2 cell lines) with IC50S (concentrations giving 50% of inhibition) ranging from 8 x 10(-12) M to 3 x 10(-11) M. The saporin-bimAb mixture was also effective in blocking the phytohaemagglutinin-driven proliferation of normal lymphocytes, whereas it displayed the same level of toxicity exerted by saporin alone on an irrelevant CD25-negative cell line (EBV-infected B lymphoblastoid cell line). From these results it is possible to envisage a clinical use of this bimAb as a cytotoxic agent for CD25+ leukaemias and lymphomas, as well as an immunosuppressive agent for severe immune disorders such as graft-vs-host disease (GVHD) and transplanted organ rejection.

Animals↗
PubMed2004

Effects of hypocretin2-saporin and antidopamine-beta-hydroxylase-saporin neurotoxic lesions of the dorsolateral pons on sleep and muscle tone.

The hypocretin neurons have been implicated in regulating sleep-wake states as they are lost in patients with the sleep disorder narcolepsy. Hypocretin (HCRT) neurons are located only in the perifornical region of the posterior hypothalamus and heavily innervate pontine brainstem neurons, such as the locus coeruleus (LC), which have traditionally been implicated in promoting arousal. It is not known how the hypocretin innervation of the pons regulates sleep-wake states as pontine lesions have never been shown to increase sleep. It is likely that in previous studies specific neurons were not lesioned. Therefore, in this study, we applied saporin-based neurotoxins to the dorsolateral pons and monitored sleep in rats. Anti-dopamine-beta-hydroxylase-saporin killed the LC neurons but sleep was affected only during a two hour light-dark transition period. Application of hypocretin2-saporin killed fewer LC neurons relative to other adjacent neurons. This occurred because the LC neurons possess the hypocretin receptor 1 but the ligand hypocretin 2 binds to this receptor with less affinity relative to the hypocretin receptor 2. The hypocretin2-saporin lesioned rats compared to controls had increased sleep during the dark period and displayed increased limb movements during REM sleep. None of the lesioned rats had sleep onset REM sleep periods or cataplexy. We conclude that the hypocretin innervation to the pons functions to awaken the animal when the lights turn off (via its innervation of the LC), sustains arousal and represses sleep during the rest of the night (via a wider innervation of other pontine neurons), and modulates muscle tone.

Animals↗
PubMed1996

Hepatoxicity of ricin, saporin or a saporin immunotoxin: xanthine oxidase activity in rat liver and blood serum.

Male Wistar rats each received an i.p injection of the ribosome-inactivating proteins ricin or saporin, or a Ber-H2 (anti-CD30)-saporin immunotoxin at a dose corresponding to three times the LD50 calculated for mice. Animals were killed 24, 48 or 72 h after treatment. Histological examination showed hepatic necrosis in all treated animals, although the sinusoidal lining was affected only in ricin-poisoned rats. The activities of xanthine dehydrogenase (D-form) and oxidase (O-form) were determined spectrophotometrically in liver and serum samples. In ricin-treated animals the liver enzyme was progressively converted from the D- to the O-form, which accounted for more than 60% of total activity after 48 h of poisoning, whilst no change in the xanthine oxidase activity was found in the serum. In the liver of rats treated with free or Ber-H2-conjugated saporin, the D-form was more than 75%, as in normal animals. In the same animals the serum xanthine oxidase activity was up to three-fold control values. The determination of serum xanthine oxidase may prove helpful in the evaluation of liver damage in patients treated with immunotoxins. It may become a diagnostic tool for the differential diagnosis of liver diseases.

Animals↗
PubMed1997

Comparison of the potency and therapeutic efficacy of the anti-CD7 immunotoxin HB2-saporin constructed with one or two saporin moieties per immunotoxin molecule.

Immunotoxins that carry two toxin molecules to the target cell should in theory have a greater anti-tumour effect than those that carry just one. We have investigated the therapeutic efficacy of two anti-CD7-saporin immunotoxins constructed with one saporin (HB2-Sap 1-mer) or two saporin molecules (HB2-Sap 2-mer) per immunotoxin molecule. In vitro, the 2-mer immunotoxin was 5.6 times more effective than the 1-mer immunotoxin at inhibiting protein synthesis in the CD7+ human T-cell acute lymphoblastic leukaemia (T-ALL) cell line HSB-2 and was also more effective at inhibiting HSB-2 cell proliferation. Flow cytometry revealed that the 2-mer immunotoxin had a reduced binding capacity to HSB-2 cells compared with the 1-mer immunotoxin or native HB2 antibody. In therapy studies in SCID mice with disseminated HSB-2 human leukaemia, the 2-mer immunotoxin performed marginally better than the 1-mer immunotoxin, but log-rank analysis did not reveal any significant differences between the two therapy groups. We therefore conclude that, although the 2-mer immunotoxin performed better than the 1-mer immunotoxin against target HSB-2 cells in vitro, this improved performance was not reflected as an improved in vivo therapeutic outcome in the SCID mouse model.

Animals↗
PubMed1988

Comparison of the pharmacokinetics and hepatotoxic effects of saporin and ricin A-chain immunotoxins on murine liver parenchymal cells.

Immunotoxins containing the ribosome-inactivating protein, saporin, are very effective antitumor agents but are highly toxic to mice. They induce severe necrotic lesions in the liver parenchyma of the recipients. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity of the saporin immunotoxins was found in the present study to arise from a combination of two effects. First, saporin and saporin immunotoxins were 30- and 6-fold more toxic to primary cultures of mouse liver parenchymal cells than were ricin A-chain and ricin A-chain immunotoxins, respectively. This was despite the fact that the cells bound 4- to 5-fold less saporin or saporin immunotoxins than ricin A-chain or ricin A-chain immunotoxins. The binding of ricin A-chain and its immunotoxin to the cells was mediated through the carbohydrate residues present on the A-chain whereas saporin is not glycosylated and thus must bind to other sites on the cell surface which result in transport of saporin relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half-life (t 1/2 alpha = 1.1 h; t 1/2 beta = 17.1 h) than the ricin A-chain immunotoxin (t 1/2 = 0.52 h; t 1/2 beta = 9.7 h). Analyses using a two-compartment pharmacokinetic model showed that the two immunotoxins broke down in vivo to give free antibody at a similar rate (t 1/2 = 10-12 h) but that the ricin A-chain immunotoxin was eliminated 11 times more rapidly than the saporin immunotoxin by routes other than breakdown. It was calculated that, in mice given a median lethal dose of saporin immunotoxin, the blood levels of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain our previous finding that it had antitumor activity superior to that of a ricin A-chain immunotoxin in mice.

Animals↗
PubMed1995

Behavioral and neurobiological alterations induced by the immunotoxin 192-IgG-saporin: cholinergic and non-cholinergic effects following i.c.v. injection.

192-IgG-Saporin is an anti-neuronal immunotoxin that combines the 192 monoclonal antibody to the p75 neurotrophin receptor found on terminals and cell bodies of neurons in the cholinergic basal forebrain with the ribosome-inactivating protein saporin. Bilateral intraventricular injection of the 192-saporin produced a variety of dose-related behavioral, neurochemical, and histological alterations in adult male rats. While both the 2 micrograms and 4 micrograms dose produced comparable cholinergic hypofunction only the high dose produced behavioral changes. Behavioral deficits induced by the 4 micrograms dose of 192-saporin induced alterations in rotorod performance and reactivity on the hot-plate which recovered over 8 weeks. In addition, the 4 micrograms dose produced a persistent impairment in the acquisition and performance of standard Morris water maze task as well as a cued version of the task. The neurobiological alterations induced by 192-saporin involved both cholinergic and non-cholinergic systems. Both doses of 192-saporin produced a 60-80% decrease in high affinity choline transport in the hippocampus and cortex without altering this parameter in the striatum. In addition, there was a significant dose-related decrease of norepinephrine in the hippocampus in the high dose group. 192-saporin did not alter the content of dopamine, serotonin, or their metabolites in any region examined. 192-saporin also produced a loss of Purkinje cells in the cerebellum. This cell type also expresses the p75 receptor and appears to be a target for intraventricular 192-saporin. This complex interplay of factors makes the i.c.v. model of 192-saporin very problematic for studying the functional properties of the cholinergic basal forebrain. However, recent data suggest that injection of 192-saporin directly into components of the cholinergic basal forebrain can be used to further elaborate the function of this brain system and to model disorders of cholinergic hypofunction such as Alzheimer's disease.

Animals↗
PubMed1996

Saporin, a ribosome-inactivating protein used to prepare immunotoxins, induces cell death via apoptosis.

The plant toxin saporin is a ribosome-inactivating protein which inhibits protein synthesis and growth of both normal and tumour cells. Its cytotoxic activity can be increased by coupling with antibodies recognizing cell surface antigens. In this work we performed experiments to test the hypothesis that saporin induces cell death via apoptosis. Exposure to saporin induced apoptosis in different cellular models, such as human peripheral blood B lymphocytes and neutrophils, in the Daudi B-cell line, and in the haemopoietic cell lines HL-60 and TF-1. This was indicated by: (a) the appearance of typical morphological features such as chromatin condensation, nuclear fragmentation and blebbing of plasma membranes: (b) DNA degradation into oligonucleosomal fragments: (c) the appearance of apoptotic cells on DNA flow cytometry as a cell population with reduced DNA content (A0 region). The fraction of cells showing features of apoptosis ranged from 19 +/- 5% for TF-1 cells to 35 +/- 8% for neutrophils. In experiments with normal peripheral blood B lymphocytes or with Daudi cells, we compared the activity of native saporin with that of an immunotoxin hybrid molecule obtained by binding the toxin to two bispecific antibodies recognizing both saporin and the B lymphocyte-specific antigen CD22 (Sap/BsAb complexes). Saporin bound to the antibodies was 2-3 logs more effective than native saporin in inducing apoptosis, with maximal inhibitions being observed at concentrations of 10(-6) M for native saporin and 10(-9)-10(-8) M for the hybrid molecules. These findings indicate that treatment with saporin results in apoptosis of target cells and suggest that this may be relevant to the therapeutic use of saporin-containing immunotoxins. In fact, if used in vivo as an immunotoxin, its cytotoxic activity could be devoid of more extensive and non-specific tissue damaging effects as would be the case if saporin induced necrosis of target cells.

Antibodies, Monoclonal↗
PubMed2006

Substance P-saporin down-regulates substance P receptor immunoreactive sensory dorsal root ganglion neurons innervating the lumbar intervertebral discs in rats.

STUDY DESIGN: To examine changes in substance P receptors on dorsal root ganglion cells innervating the rat lumbar intervertebral discs using immunohistochemistry and a retrograde neurotracing method. OBJECTIVE: We evaluated the effects of intradiscal administration of substance P-saporin, a toxin selective for cells expressing substance P receptors. SUMMARY OF BACKGROUND DATA: The rat L5/6 intervertebral disc is multi-segmentally innervated from the L1-L6 dorsal root ganglions. Substance P and the neurokinin-1 receptor contribute to inflammatory pain transmission. Substance P immunoreactive-sensory nerve fibers in human intervertebral discs and immunoreactive-dorsal root ganglion neurons innervating rat intervertebral discs have been reported to be important in the transmission of discogenic low back pain. In the current study, we evaluated the effects of intradiscal administration of substance P-saporin, a toxin selective for cells expressing substance P receptor. METHODS: Sixteen rats were used (control group, n = 8; substance P-saporin group, n = 8). To detect dorsal root ganglion neurons innervating the L5/6 intervertebral disc, neurotracer (fluoro-gold crystals) was placed into the intervertebral disc. Seven days after fluoro-gold application, the L5/6 intervertebral disc was exposed and injected with 175 ng of sterile substance P-saporin (substance P-saporin group, n = 8). Fourteen days after the first operation, each dorsal root ganglion was harvested, sectioned, and processed for neurokinin-1 immunohistochemistry using rabbit antibody to neurokinin-1. The numbers of fluoro-gold labeled neurons, and fluoro-gold labeled and neurokinin-1 immunoreactive neurons were counted in both groups. RESULTS: Neurons innervating the L5/6 intervertebral discs, retrogradely labeled with fluoro-gold, were distributed throughout dorsal root ganglions from L1 to L6 in both groups. Of fluoro-gold labeled neurons, the proportion of neurokinin-1 immunoreactive neurons was 35% in the control group. However, the proportion of neurokinin-1 immunoreactive neurons was 8% after administration of substance P-saporin into the intervertebral discs (substance P-saporin group). Substance P-saporin significantly decreased the ratio of neurokinin-1 immunoreactive neurons. CONCLUSION: Substance P-saporin decreased the ratio of neurokinin-1 immunoreactive neurons innervating the disc related to discogenic low back pain. Substance P-saporin may be a useful tool to investigate the mechanism of discogenic low back pain.

Animals↗
PubMed1988

Bispecific F(ab' gamma)2 antibody for the delivery of saporin in the treatment of lymphoma.

This work describes the successful use of bispecific F(ab' gamma)2 antibody (Ab) in combination with a ribosome-inactivating protein (RIP), saporin, for the treatment of neoplastic disease in vivo. A total of three thioether-linked F(ab' gamma)2 heterodimers were prepared, each having dual specificities for saporin and the guinea pig lymphoblastic leukemia, L2C. In all three cases specificity for the L2C cells was provided by a high affinity mouse anti-idiotype (anti-Id) mAb, whereas the antisaporin activity came from either one of two mouse mAb or an affinity-purified rabbit polyclonal Ab. In vitro studies, measuring protein synthesis, showed that all three derivatives were extremely efficient at delivering saporin to L2C cells, to the extent that addition of the rabbit Fab' gamma-containing bispecific Ab to cell culture at 1 microgram/ml increased the toxicity of saporin (50% inhibiting concentration) by close to 90,000-fold. Similarly, in leukemic guinea pigs, a small dose of saporin (10 micrograms) which by itself showed no therapeutic effect, was able to completely eradicate Id-positive tumor when given in combination with an excess of bispecific Ab. Although tumors did eventually emerge in most of these animals, immunofluorescence analysis showed that in almost all instances the escaping cells were Id- variants of the L2C. Experiments to define the optimal treatment regimen in this model showed that, although the administration of saporin and bispecific Ab at separate sites could be therapeutically effective, mixing the Ab and saporin to form immune complexes before injection did generally enhance their performance. A molar surplus of bispecific Ab in these mixtures both extended the metabolic survival of the saporin and enhanced the therapeutic performance, with molar ratios above 3:1 generally being required for optimum treatment when using saporin at 10 micrograms. Derivatives containing polyclonal antisaporin were more efficient than those containing mAb, yielding optimum therapeutic results with a molar ratio of 1.5:1 when combined with 10 micrograms saporin. These findings have shown that bispecific F(ab' gamma)2 Ab, as well as being straightforward to prepare, can also function as an extremely efficient vector for delivering cytotoxic agents such as ribosome-inactivating protein to unwanted cells in vivo.

Animals↗
PubMed2001

Effects of hypocretin-saporin injections into the medial septum on sleep and hippocampal theta.

Neurons containing the peptide hypocretin, also known as orexin, were recently implicated in the human sleep disorder narcolepsy. Hypocretin neurons are located only in the lateral hypothalamus from where they innervate virtually the entire brain and spinal cord. This peptide is believed to be involved in regulating feeding and wakefulness. However, to fully understand what other behaviors are regulated by this peptide it is necessary to investigate each hypocretin target site. In the present study, we focus on one hypocretin target site, the medial septum, where there is a dense collection of hypocretin-2 receptor-containing cells, and degenerating axons are present here in canines with narcolepsy [J. Neurosci. 19 (1999) 248]. We utilize a saporin toxin conjugated to the hypocretin receptor binding ligand, hypocretin-2, and find that when this toxin is injected into the medial septum, it lesions the parvalbumin and cholinergic neurons. We contrast the effects of the hypocretin-saporin with another saporin conjugated toxin, 192 IgG-saporin, that lesions only the cholinergic neurons in the basal forebrain. 192 IgG-saporin reduced theta activity, a finding consistent with previous reports [J. Neurophysiol. 79 (1998) 1633; Neurodegeneration 4 (1995) 61; Neuroscience 62 (1994) 1033]. However, hypocretin-saporin completely eliminated hippocampal theta activity by day 12, indicating that parvalbumin-containing cells in the medial septum generate theta. The daily amount of sleep and wakefulness were not different between hypocretin-saporin, 192 IgG-saporin, or saline-treated rats. The homeostatic response to 12 h prolonged wakefulness was also not affected in hypocretin-saporin lesioned rats. These findings suggest that hypocretin neurons could facilitate theta generation during episodes of purposeful behavior by activating GABAergic neurons in the MS/VDB. In this way, hypocretin, which is implicated in feeding, energy metabolism and wakefulness, serves to influence cognitive processes critical for the animal's survival.

Acetylcholine↗
PubMed1991

Characteristics and performance of a bispecific F (ab'gamma)2 antibody for delivering saporin to a CD7+ human acute T-cell leukaemia cell line.

We have investigated the efficacy of a F(ab'gamma)2 bispecific antibody (BsAb) with dual specificity for the CD7 molecule in one Fab arm and for the ribosome inactivating protein (rip) saporin in the other arm, for delivering saporin to the acute T-cell leukaemia cell line HSB-2. Saporin titration experiments revealed that BsAb increased the toxicity of saporin 435-fold for HSB-2 cells, reducing the IC50 for saporin alone from 0.1 mumol to 0.23 nmol when BsAb was included. The rate of protein synthesis inactivation brought about by BsAb-mediated toxin delivery to HSB-2 cells was very similar to that described for conventional immunotoxins (IT's) with a t10 (time taken for a one log inhibition of protein synthesis compared with controls) of 46 h obtained at a saporin concentration of 1 nmol and 226 h at 0.1 nmol. BsAb titration studies demonstrated a clear dose response effect of BsAb concentration on target cell protein synthesis inhibition and cell proliferation. The absolute specificity of toxin delivery was unequivocally demonstrated by a failure of BsAb to deliver an effective dose of saporin to the CD7- cell line HL60 and by the blocking of BsAb-mediated delivery of saporin to HSB-2 cells with an excess of F(ab)2 fragments of the anti-CD7 antibody, HB2. These studies have clearly demonstrated the effectiveness of this BsAb for delivering saporin to a T-ALL cell line utilising CD7 as the target molecule on the cell surface. BsAb's would therefore appear to offer a realistic alternative to IT's for toxin delivery to tumour cells and may even offer certain advantages over conventional IT's for clinical use.

Antibodies, Monoclonal↗
PubMed2006

The differential catalytic activity of ribosome-inactivating proteins saporin 5 and 6 is due to a single substitution at position 162.

Saporin, a type I ribosome-inactivating protein produced by the soapwort plant Saponaria officinalis belongs to a multigene family that encodes its several isoforms. The saporin seed isoform 6 has significantly higher N-glycosidase and cytotoxic activities compared with the seed isoform 5, although the two have identical active sites. In the present study, we have investigated the contribution of non-conservative amino acid changes outside the active sites of these isoforms towards their differential catalytic activity. The saporin 6 residues Lys134, Leu147, Phe149, Asn162, Thr188 and Asp196 were replaced by the corresponding saporin 5 residues, Gln134, Ser147, Ser149, Asp162, Ile188 and Asn196, to generate six variants of saporin 6, K134Q, L147S, F149S, N162D, T188I and D196N. By functional characterization, we show that the change in amino acid Asn162 in saporin 6 to aspartic acid residue of saporin 5 contributes mainly to the lower catalytic activity of saporin 5 compared with saporin 6. The non-involvement of other non-conservative amino acids in the differential catalytic activity of these isoforms was confirmed with the help of the double mutations N162D/K134Q, N162D/L147S, N162D/F149S, N162D/T188I and N162D/D196N.

Amino Acid Substitution↗
PubMed1997

Characterization of a saporin isoform with lower ribosome-inhibiting activity.

We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in vitro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to alpha2-macroglobulin receptor (alpha2MR; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the alpha2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem. 232, 165-171]. Here we report that SAP-C bound to alpha2MR equally well as native saporin. However, the same isoform had about ten times lower cytotoxicity than the other saporin isoforms towards different cell lines. This indicates that the lower cell-killing ability of the SAP-C isoform is presumably due to its altered interaction with the protein synthesis machinery of target cells. Since saporin binding to the alpha2MR is competed by heparin, we also tested in cell-killing experiments Chinese hamster ovary cell lines defective for expression of either heparan sulphates or proteoglycans. No differences were observed in cytotoxicity using native saporin or the recombinant isoforms. Therefore saporin binding to the cell surface should not be mediated by interaction with proteoglycans, as is the case for other alpha2MR ligands.

Amino Acid Sequence↗
PubMed1995

Cytotoxic effects of FGF2-saporin on bovine epithelial lens cells in vitro.

PURPOSE: To test the ability of two preparations of FGF2-saporin, either FGF2 chemically conjugated to saporin (FGF2-SAP) or genetically engineered FGF2-saporin (rFGF2-SAP) to inhibit the growth of bovine epithelial lens (BEL) cells in vitro when in solution and when immobilized on heparin surface-modified (HSM) polymethylmethacrylate (PMMA) intraocular lenses (IOLs). METHOD: Bovine epithelial lens cells were incubated with various concentrations FGF2-saporin for as long as 4 days. The number of surviving cells was determined by counting the number of nuclei. Because FGF2 binds to heparin, FGF2-saporin was incubated with HSM PMMA IOLs; excess toxin was washed off, and the BEL cells were grown on the FGF2-saporin-treated IOLs (HSM and non-HSM) for 4 days. Cell density was determined by image analysis. RESULTS: Both FGF2-SAP and rFGF2-SAP were highly cytotoxic (nM range), with rFGF2-SAP 10 times less active than FGF2-SAP. FGF2-saporin bound to the surface of HSM IOLs and eluted by 2M NaCl retained its activity. Toxin bound to HSM IOLs killed more than 90% of the BEL cells placed on the IOL surface within 4 days. The ability of FGF2-saporin to prevent the growth of cells on the IOL surface was strictly dependent on the presence of heparin on the IOL. CONCLUSIONS: FGF2-saporin is bound to HSM PMMA IOLs and prevents the growth of epithelial cells on the surface of the lens.

Animals↗
PubMed2003

Cytotoxicity of ribosome-inactivating protein saporin is not mediated through alpha2-macroglobulin receptor.

Saporin is a single chain ribosome-inactivating protein produced by the plant Saponaria officinalis. Several isoforms of saporin have been isolated from various parts of the plant. In the present study recombinant saporin isoforms 5 and 6 were produced in Escherichia coli. Saporin-6 was found to be more active than saporin-5 in its N-glycosidase, cytotoxic, and genomic DNA fragmentation activities. Earlier, saporin has been shown to bind low-density lipoprotein receptor-related protein (LRP), however, in this study the sensitivities of LRP-negative and LRP-positive cell lines were found to be similar towards saporin-6 toxicity suggesting the internalization of saporin not to be solely dependent on the expression of LRP on eukaryotic cells.

Animals↗
PubMed2003

Effects of lateral hypothalamic lesion with the neurotoxin hypocretin-2-saporin on sleep in Long-Evans rats.

Narcolepsy, a disabling neurological disorder characterized by excessive daytime sleepiness, sleep attacks, sleep fragmentation, cataplexy, sleep-onset rapid eye movement sleep periods and hypnagogic hallucinations was recently linked to a loss of neurons containing the neuropeptide hypocretin. There is considerable variability in the severity of symptoms between narcoleptic patients, which could be related to the extent of neuronal loss in the lateral hypothalamus. To investigate this possibility, we administered two concentrations (90 ng or 490 ng in a volume of 0.5 microl) of the neurotoxin hypocretin-2-saporin, unconjugated saporin or saline directly to the lateral hypothalamus and monitored sleep, the entrained and free-running rhythm of core body temperature and activity. Neurons stained for hypocretin or for the neuronal specific marker were counted in the perifornical area, dorsomedial and ventromedial nucleus of the hypothalamus. More neuronal nuclei (NeuN) cells were destroyed by the higher concentration of hypocretin-2-saporin (-55%) compared with the lower concentration (-34%) in the perifornical area, although both concentrations lesioned the hypocretin neurons almost equally well (high concentration=91%; low concentration=88%). The high concentration of hypocretin-2-saporin also lesioned neurons in the dorsomedial nucleus of the hypothalamus and ventromedial nucleus of the hypothalamus. Narcoleptic-like sleep behavior was produced by both concentrations of the hypocretin-2-saporin. The high concentration produced a larger increase in non-rapid eye movement sleep amounts during the normally active night cycle than low concentration. Neither concentration of hypocretin-2-saporin disrupted the phase or period of the core temperature or activity rhythms. The low concentration of unconjugated saporin did not significantly lesion hypocretin or neurons and did not alter sleep. The high concentration of unconjugated saporin produced some loss of neuronal nuclei-immunoreactive (NeuN-ir) neurons and hypocretin immunoreactive neurons, but only a transient increase in non-rapid eye movement sleep. These results led us to conclude that the extent of hypocretin neuronal loss together with an accompanying loss of cells in the lateral hypothalamus may explain the differences in severity of symptoms seen in human narcolepsy.

Animals↗