Toxic Moiety Research Articles

Parasporin-1, a Novel Cytotoxic Protein from Bacillus thuringiensis, Induces Ca2+ Influx and a Sustained Elevation of the Cytoplasmic Ca2+ Concentration in Toxin-sensitive Cells

Parasporin-1 is a novel non-insecticidal inclusion protein from Bacillus thuringiensis that is cytotoxic to specific mammalian cells. In this study, we investigated the effects of parasporin-1 on toxin-sensitive cell lines to elucidate the cytotoxic mechanism of parasporin-1. Parasporin-1 is not a membrane pore-forming toxin as evidenced by measurements of lactate dehydrogenase release, propidium iodide penetration, and membrane potential in parasporin-1-treated cells. Parasporin-1 decreased the level of cellular protein and DNA synthesis in parasporin-1-sensitive HeLa cells. The earliest change observed in cells treated with this toxin was a rapid elevation of the intracellular free-Ca(2+) concentration; increases in the intracellular Ca(2+) levels were observed 1-3 min following parasporin-1 treatment. Using four different cell lines, we found that the degree of cellular sensitivity to parasporin-1 was positively correlated with the size of the increase in the intracellular Ca(2+) concentration. The toxin-induced elevation of the intracellular Ca(2+) concentration was markedly decreased in low-Ca(2+) buffer and was not observed in Ca(2+)-free buffer. Accordingly, the cytotoxicity of parasporin-1 decreased in the low-Ca(2+) buffer and was restored by the addition of Ca(2+) to the extracellular medium. Suramin, which inhibits trimeric G-protein signaling, suppressed both the Ca(2+) influx and the cytotoxicity of parasporin-1. In parasporin-1-treated HeLa cells, degradation of pro-caspase-3 and poly(ADP-ribose) polymerase was observed. Furthermore, synthetic caspase inhibitors blocked the cytotoxic activity of parasporin-1. These results indicate that parasporin-1 activates apoptotic signaling in these cells as a result of the increased Ca(2+) level and that the Ca(2+) influx is the first step in the pathway that underlies parasporin-1 toxicity.

A Phase II, Double-Blind, Placebo-Controlled, Ascending-Dose Study of Eritoran (E5564), a Lipid A Antagonist, in Patients Undergoing Cardiac Surgery with Cardiopulmonary Bypass

Lipid A, the toxic moiety of endotoxin, is linked to multiple complications after cardiac surgery, including fever, vasodilation, and pulmonary and renal dysfunction. The lipid A antagonist eritoran (or E5564) prevents endotoxin-induced systemic inflammation in animals and humans. In this study we assessed the safety of eritoran administration in patients undergoing cardiac surgery and obtained preliminary efficacy data for the prophylaxis of endotoxin-mediated surgical complications. A double-blind, randomized, ascending-dose, placebo-controlled study was conducted at nine hospitals. Patients undergoing coronary artery bypass graft and/or cardiac valvular surgery with cardiopulmonary bypass were enrolled. Patients received a 4-h infusion of placebo (n = 78) vs 2 mg (n = 24), 12 mg (n = 26), or 28 mg (n = 24) of eritoran initiated approximately 1 h before cardiopulmonary bypass. No significant safety concerns were identified with continuous safety monitoring, and enrollment continued to the highest prespecified dose (28 mg). No statistically significant differences were observed in most variables related to systemic inflammation or organ dysfunction/injury. This Phase II safety study suggests that the administration of the novel lipid A antagonist, eritoran, is not associated with overt toxicity in cardiac surgical patients. Blocking lipid A with eritoran does not appear to confer any clear benefit to elective cardiac surgical patients.

In vivo administration of plasmid DNA encoding recombinant immunotoxin DT390-IP-10 attenuates experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune demyelinating disease. The expression of chemokine receptor CXCR3 on activated T cells is crucial to direct the migration of effector cells into the inflammatory sites and initiate EAE. In this study we tested the effect of a novel recombinant immunotoxin targeting CXCR3 + cells for EAE prevention. The immunotoxin construct DT390-IP-10-SRα consisted of interferon gamma-inducible protein 10 (IP-10), a ligand of CXCR3, as the targeting moiety, and a truncated diphtheria toxin (DT390) as the toxic moiety. In vitro transfection of DT390-IP-10-SRα into NIH3T3 cells resulted in expression of DT390-IP-10 which proved highly toxic to activated T cells. To evaluate the effect of DT390-IP-10-SRα on EAE prevention in vivo, cationic liposome-embedded DT390-IP-10-SRα was injected into the muscle of hind limbs of C57BL/6 mice immunized by myelin basic protein (MBP). DT390-IP-10-SRα-treated mice showed a delayed onset of EAE and milder symptoms compared to the mice treated with empty control plasmid or PBS alone. Immunohistochemical staining detected significantly reduced infiltrating CXCR3 + cells in the inflammatory lesions of CNS from immunotoxin treated mice as compared to the controls. This study suggests that targeting CXCR3 + T cells with recombinant immunotoxin could be achieved in vivo to delay and ameliorate murine EAE.

Chloroprene: Overview of studies under consideration for the development of an IRIS assessment

Beta-chloroprene (C(4)H(5)Cl, chloroprene, 2-chloro-1,3-butadiene, CASRN 126-99-8) is a volatile, flammable liquid monomer utilized primarily in the manufacture of neoprene (polychloroprene) elastomer used in belts, hoses, gloves, wire coatings, and tubing. Absorption into the body occurs primarily via the respiratory system and may occur via the gastrointestinal tract or the skin. Once absorbed, chloroprene is widely distributed as evidenced by effects in several target organs including nose and lung, liver, and skin. Chloroprene metabolism is believed to include cytochrome P450 oxidation to a monoepoxide, hydrolysis by epoxide hydrolases, and glutathione conjugation. Similar to 1,3-butadiene, the epoxide is considered to be the toxic moiety, and species differences in metabolic capacity may influence the severity of effects as well as what tissues are affected. EPA has not previously developed an assessment of chloroprene's potential for human health effects. Existing human epidemiological studies offer little data on noncancer effects, and the associations of exposure with increased cancer (liver and lung) mortality reported are inconclusive. Recent epidemiological studies (submitted for publication) could offer information that may impact chloroprene's health assessment. Multiple-site tumors have been reported in rats and mice exposed to chloroprene by inhalation; nevertheless, there are marked differences in strain sensitivities (i.e., tumors in F344 rats versus no tumors in Wistar rats). Recently developed physiologically based toxicokinetic models may allow for the resolution of species and tissue differences and sensitivities as well as exposure-dose-response relationships relevant to humans. (This presentation does not necessarily reflect EPA policy.).

A cleavable molecular adapter reduces side effects and concomitantly enhances efficacy in tumor treatment by targeted toxins in mice

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

Relationship between CD33 Expression, P-Glycoprotein-Mediated Drug Efflux, and Clinical Outcome in Patients Treated in Phase II Trials with Gemtuzumab Ozogamicin Monotherapy.

Background: The anti-acute myeloid leukemia (AML) immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains a humanized anti-CD33 antibody (hP67.6) to facilitate uptake of the toxic calicheamicin-γ1 derivative in CD33-positive AML cells. This putative mechanism implies a critical role for the intracellular accumulation of the toxic moiety for GO-induced cytotoxicity. Indeed, drug efflux by P-glycoprotein (Pgp) mediates resistance to GO and correlates with clinical outcome after GO monotherapy. Furthermore, recent in vitro data obtained in human myeloid cell lines have unequivocally demonstrated a quantitative relationship between CD33 expression and GO-induced cytotoxicity. In light of these findings, we have now re-examined the significance of CD33 expression levels on AML blasts and relationship with Pgp activity for clinical outcome of patients treated with GO monotherapy.Methods: Pre-treatment bone marrow samples from patients enrolled in multicenter phase II protocols evaluating the safety and efficacy of GO monotherapy (generally 2 doses of 9 mg/m2 14 days apart) were used for analysis. Relative CD33 expression was quantified by flowcytometry immunophenotyping using the hP67.6 antibody, and linear fluorescence values used for calculations. Pgp function was cytofluorometrically determined by efflux of the fluorescent dye, DiOC2. Results are presented as mean values and 95% confidence intervals. Unpaired t-tests, Pearson correlations, and logistic regression models were used for statistical analysis.Results: Patients achieving a complete remission (CR) or CR with incomplete recovery of platelet counts (CRp) had statistically significantly higher mean CD33 expression levels (71.20 [57.20–85.19], n=69) compared to non-responders (54.44 [48.38–60.51], n=203; p=0.01). There was an inverse relationship between CD33 expression and Pgp efflux (r=−0.23) and this contributed to responders having a statistically significantly lower mean Pgp efflux (1.40 [1.28–1.52], n=57) compared to non-responders (1.83 [1.72–1.95], n=173; p<0.0001). The addition of Pgp statistically significantly improved a logistic regression model containing only CD33 (p<.0001), whereas the addition of CD33 did not lead to a statistically significantly improved logistic regression model containing only Pgp (p=0.14).Conclusion: These data indicate that CD33 expression levels as well as Pgp efflux are associated with clinical outcome of patients treated with GO monotherapy, and that knowledge of Pgp provides important information regarding the probability of achieving a response, even after consideration of CD33 expression level. The inverse relationship between CD33 abundance and Pgp efflux is consistent with the notion of maturation-stage dependent expression of these proteins, and offers the rationale for the use of cell-differentiation-promoting agents, for example cytokines, in combination with GO to enhance GO-induced cytotoxicity and possibly improve clinical outcome of patients undergoing GO-containing AML therapy.

Bases of biocontrol: Sequence predicts synthesis and mode of action of agrocin 84, the Trojan Horse antibiotic that controls crown gall

Agrobacterium radiobacter K84, used worldwide to biocontrol crown gall disease caused by Agrobacterium tumefaciens, produces an antiagrobacterial compound called agrocin 84. We report the nucleotide sequence of pAgK84, a 44.42-kb plasmid coding for production of this disubstituted adenine nucleotide antibiotic. pAgK84 encodes 36 ORFs, 17 of which (agn) code for synthesis of or immunity to agrocin 84. Two genes, agnB2 and agnA, encode aminoacyl tRNA synthetase homologues. We have shown that the toxic moiety of agrocin 84 inhibits cellular leucyl-tRNA synthetases and AgnB2, which confers immunity to the antibiotic, is a resistant form of this enzyme. AgnA, a truncated homologue of asparaginyl tRNA synthetase could catalyze the phosphoramidate bond between a precursor of the methyl pentanamide side group and the nucleotide. We propose previously undescribed chemistry, catalyzed by AgnB1, to generate the precursor necessary for this phosphoramidate linkage. AgnC7 is related to ribonucleotide reductases and could generate the 3'-deoxyarabinose moiety of the nucleoside. Bioinformatics suggest that agnC3, agnC4, and agnC6 contribute to maturation of the methyl pentanamide, whereas agnC2 may produce the glucofuranose side group bound to the adenine ring. AgnG is related to bacterial exporters. An agnG mutant accumulated agrocin 84 intracellularly but did not export the antibiotic. pAgK84 is transmissible and encodes genes for conjugative DNA processing but lacks a type IV secretion system, suggesting that pAgK84 transfers by mobilization. By sequence analysis, the deletion engineered into pAgK1026 removed the oriT and essential tra genes, confirming the enhanced environmental safety of this modified form of pAgK84.

Prevention of murine experimental autoimmune encephalomyelitis by in vivo expression of a novel recombinant immunotoxin DT390-RANTES

Experimental autoimmune encephalomyelitis (EAE) is a T-cell-mediated autoimmune disease. Chemokine receptor CCR5 has been shown to be essential for the T-cell recruitment to the inflammatory site in EAE. In this study, we assumed that an immunotoxin directed at CCR5+ cells would be able to reduce the disease activity of EAE. A recombinant immunotoxin, DT390-RANTES-SRalpha, was constructed in an eukaryotic cell expression plasmid consisting of regulated on activation normal T cells expressed and secreted (RANTES) as the targeting moiety and DT390 as the toxic moiety. DT390-RANTES was expressed in vitro and was highly toxic to activated mouse T cells with the inhibitory concentration 50 at 0.18 ng/ml. To evaluate whether DT390-RANTES was effective in preventing EAE, C57BL/6 mice were immunized with myelin basic protein, emulsified with complete Freund's adjuvant and were treated by injecting cationic liposome-embedded plasmid DNA into the muscle of hind limbs. Mice treated with DT390-RANTES-SRalpha developed a much milder EAE compared to mice treated with phosphate-buffered saline or the empty plasmid DNA. Much less CCR5+-infiltrating cells were found in the central nervous system in DT390-RANTES-SRalpha-treated mice than in the control mice. This study indicates that recombinant immunotoxin can be expressed in vivo, and targeting CCR5 can attenuate the disease activity of EAE in mice.

Cyanobacterial lipopolysaccharides and human health - a review.

Cyanobacterial lipopolysaccharide/s (LPS) are frequently cited in the cyanobacteria literature as toxins responsible for a variety of heath effects in humans, from skin rashes to gastrointestinal, respiratory and allergic reactions. The attribution of toxic properties to cyanobacterial LPS dates from the 1970s, when it was thought that lipid A, the toxic moiety of LPS, was structurally and functionally conserved across all Gram-negative bacteria. However, more recent research has shown that this is not the case, and lipid A structures are now known to be very different, expressing properties ranging from LPS agonists, through weak endotoxicity to LPS antagonists. Although cyanobacterial LPS is widely cited as a putative toxin, most of the small number of formal research reports describe cyanobacterial LPS as weakly toxic compared to LPS from the Enterobacteriaceae.We systematically reviewed the literature on cyanobacterial LPS, and also examined the much lager body of literature relating to heterotrophic bacterial LPS and the atypical lipid A structures of some photosynthetic bacteria. While the literature on the biological activity of heterotrophic bacterial LPS is overwhelmingly large and therefore difficult to review for the purposes of exclusion, we were unable to find a convincing body of evidence to suggest that heterotrophic bacterial LPS, in the absence of other virulence factors, is responsible for acute gastrointestinal, dermatological or allergic reactions via natural exposure routes in humans.There is a danger that initial speculation about cyanobacterial LPS may evolve into orthodoxy without basis in research findings. No cyanobacterial lipid A structures have been described and published to date, so a recommendation is made that cyanobacteriologists should not continue to attribute such a diverse range of clinical symptoms to cyanobacterial LPS without research confirmation.

Age‐ and Gender‐Dependent In Vitro Hepatic Metabolism and acute neurotoxicity of a pyrethroid insecticide, Deltamethrin (DLM), in Rats

Metabolism is a critical determinant of DLM acute neurotoxicity as the parent compound is the toxic moiety. The aim of this study was to determine whether age- and gender-related differences in DLM metabolism by CYP450s and/or carboxylesterases (CaEs) exist and dictate the susceptibility to the chemical. Metabolism was quantified in vitro by monitoring disappearance of the parent compound from liver microsomes (via CaEs and CYP450s) obtained from 21- and 90-d-old male and female S-D rats. Intrinsic clearances (Vmax/Km) by CaEs (male−1.8±0.4 and 12.01±1.1; female−2.3±0.4 and 7.2±1.0) and CYP450s (male−16.9±1.9 and 35.5±2.9; female−10.7±2.3 and 19.7±5.4) increased with age in both sexes due to marked increases in Vmax and slight increases in Km. While there was no gender difference in young rats, intrinsic clearance of DLM by both pathways was significantly higher in adult males than that of females. CYP450s played a more important role than CaEs in DLM metabolism in rats of both sexes and ages. Preliminary results of in vivo experiments showed that young male and female rats receiving 10 mg/kg po showed severe toxic signs and 100% mortality. Male and female adult rats receiving 10 or 20 mg/kg DLM showed only mild to moderate toxicity. A gender difference was not evident at 10 mg/kg, but females showed more pronounced toxic signs and higher blood and brain DLM levels at 20 mg/kg. This study shows that limited metabolic capacity contributes to the age- and gender-dependent differences in sensitivity to acute DLM neurotoxicity. Gender dependency, however, was evident only in adults. (Supported by EPA STAR Grant R830800).

Cytotoxicity of Acetaldehyde‐Derived Advanced Glycation End‐Products (AA‐AGE) in Alcoholic‐Induced Neuronal Degeneration

The Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients, in aging and in neurodegenerative processes. We hypothesize that acetaldehyde (AA), one of the main metabolites of alcohol, may be involved in alcohol-induced neurotoxicity in vivo by formation of AA-derived AGEs (AA-AGE) with brain proteins. AA-AGE-bovine serum albumin (BSA) and AA-AGE-rabbit serum albumin (RSA) were prepared as described previously. Antibody specific for AA-AGE was isolated from rabbit antiserum by affinity chromatography. Primary cortical neuronal cell cultures were prepared as described previously. Incubation of cortical neurons with AA-AGE produced a dose-dependent increase in neuronal cell-death, and the neurotoxicity of AA-AGE was neutralized by the addition of an anti-AA-AGE specific antibody, but not by anti-N-ethyllysine (NEL) antibody. The AA-AGE epitope was detected in human brain of alcoholism. We propose that the structural epitope AA-AGE is an important toxic moiety for neuronal cells in alcoholism.

MITOCHONDRIAL OXIDANT STRESS IN EXPERIMENTAL NON‐ALCOHOLIC STEATOHEPATITIS (NASH)

Background and Hypothesis: The hypothetical two hits leading to NASH are insulin resistance and oxidative stress related to steatosis. Experimental NASH can be produced by feeding mice a methionine and choline deficient (MCD) diet, and exposing mouse AML-12 hepatocytes in vitro to MCD medium stimulates PI3-K dependent steatosis and release of ALT, both without global oxidative stress. Early mitochondrial swelling suggests that focal oxidative stress may be involved. We hypothesize that mitochondrial reactive oxygen species (mROS) generation leads to impaired beta-oxidation, accumulation of toxic acyl-CoA moieties and subsequent mitochondrial dysfunction. Further toxic acyl-CoA moieties may be a therapeutic target for supplementation with carnitine. Methods: AML-12 hepatocytes were exposed to MCD and control (MC+) media ± 0.5 mM L-carnitine (CARN), and mROS generation was evaluated by co-staining with fluorescent dyes, dihydroethidium (DHE, a ROS specific vital dye) and DAPI for double-stranded DNA. ROS generation was confirmed by a cell permeable superoxide scavenger, Manganese benzoyl porphyrin chloride (MnTBAP). Female A/J mice were fed MCD or MC+ diet ± 0.5% CARN in drinking water. mROS was measured by DHE staining in frozen liver sections obtained on days 3, 7 and 14. Results: DHE staining showed marked ↑ mROS activity within 30 min of exposure of AML-12 hepatocytes to MCD medium, which dissipated by 3 hr MCD exposure to levels in MC+ medium treated cells. Incubating with MnTBAP before DHE staining reduced fluorescence, confirming mROS generation. Supplementing with CARN substantially reduced mROS generation in MCD treated cells as evidenced by ↓ DHE fluorescence. ↑ hepatic mROS in MCD diet fed mice at all time points was reduced by CARN supplementation as evidenced by ↓ DHE fluorescence. Conclusions: 1. Focal mitochondrial oxidative stress due to ↑ mROS generation has a potentially important mechanistic role in experimental NASH. 2. CARN may provide a targeted strategy to protect from mROS stress and thus retard progression of NASH.

Protein toxins: intracellular trafficking for targeted therapy

The immunotoxin approach is based on the use of tumor-targeting ligands or antibodies that are linked to the catalytic (toxic) moieties of bacterial or plant protein toxins. In this review, we first discuss the current state of clinical development of immunotoxin approaches describing the results obtained with the two toxins most frequently used: diphtheria and Pseudomonas toxin-derived proteins. In the second part of the review, a novel concept will be presented in which the roles are inverted: nontoxic receptor-binding toxin moieties are used for the targeting of therapeutic and diagnostic compounds to cancer or immune cells. The cell biological basis of these novel types of toxin-based therapeutics will be discussed, and we will summarize ongoing preclinical and clinical testing.

CD33-directed therapy with gemtuzumab ozogamicin in acute myeloid leukemia: progress in understanding cytotoxicity and potential mechanisms of drug resistance

CD33 is expressed on the malignant blast cells in most cases of acute myeloid leukemia (AML) but not on normal hematopoietic pluripotent stem cells. Antibody-based therapies for AML have, therefore, focused on CD33 as a suitable tumor-associated target antigen. The most promising results have been obtained with gemtuzumab ozogamicin (GO, Mylotarg), a humanized IgG(4) anti-CD33 monoclonal antibody joined to a calicheamicin-gamma(1) derivative. Engagement of CD33 by GO results in immunoconjugate internalization and hydrolytic release of the toxic calicheamicin moiety, which, in turn, causes DNA damage and cell death. Since 2000, when GO was approved for clinical use, treatment trials and pilot studies have revealed potential expanded applications along with additional limitations. At the same time, correlative biological and in vitro functional studies have further characterized CD33 expression patterns in AML, the significance of CD33-antibody interactions, pathways involved in GO-induced cytotoxicity and potential drug resistance mechanisms. This review summarizes the recent data addressing mechanisms of GO action and discusses their relevance with regard to clinical applications and the limitations of using experimental model systems to mimic in vivo conditions. As the first drug conjugate approved for clinical use, GO serves as an important paradigm for other immunoconjugates against internalizing tumor antigens.

Structural Determinants of Substrate Specificity in Family 1 β-Glucosidases: NOVEL INSIGHTS FROM THE CRYSTAL STRUCTURE OF SORGHUM DHURRINASE-1, A PLANT β-GLUCOSIDASE WITH STRICT SPECIFICITY, IN COMPLEX WITH ITS NATURAL SUBSTRATE

Plant beta-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, beta-glucosides to release toxic aglycone moieties. The Sorghum bicolor beta-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-D-glucoside), whereas its close homolog, the maize beta-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-beta-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme.substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn(259), Phe(261), and Ser(462), located in the aglycone-binding site of S. bicolor Dhr1, are crucial for aglycone recognition and binding. The tight binding of the aglycone moiety of dhurrin promotes the stabilization of the reaction intermediate in which the glycone moiety is in a deformed (1)S(3) conformation within the glycone-binding site, ready for nucleophilic attack to occur. Compared with the broad specificity maize beta-glucosidase, this different binding mode explains the narrow specificity of sorghum dhurrinase-1.

A dominant linear B-cell epitope of ricin A-chain is the target of a neutralizing antibody response in Hodgkin's lymphoma patients treated with an anti-CD25 immunotoxin.

Hodgkin's lymphoma patients treated with an anti-CD25 Ricin toxin A-chain (RTA)-based Immunotoxin (RFT5.dgA) develop an immune response against the toxic moiety of the immunoconjugate. The anti-RTA antibody response of 15 patients showing different clinical features and receiving different total amounts of RFT5.dgA was therefore studied in detail, considering antibody titre, IgG and IgM content, average binding efficacy and ability to inhibit in vitro the cytotoxicity of a RTA-based Immunotoxin. No correlations were found between these parameters and the clinical features of the patients or the total amount of Immunotoxin administered. However, using a peptide scan approach we have identified a continuous epitope recognized by all patients studied, located within the stretch L161-I175 of the RTA primary sequence, close to a previously identified T-cell epitope. The ability of anti-L161-I175 antibodies to recognize folded RTA and to affect the biological activity of RTA by inhibiting RTA-IT cytotoxicity in vitro revealed that they may exert an important role in IT neutralization in vivo. Discovery of RTA immunodominant epitopes which are the target of anti-RTA immune response may lead to the development of immunomodulating strategies and to more successful treatment schedules.

Involvement of Advanced Glycation End-products (AGEs) in Alzheimers Disease

The advanced stage of the glycation process (one of the post-translational modifications of proteins) leads to the formation of advanced glycation end-products (AGEs) and plays an important role in the pathogenesis of angiopathy in diabetic patients. It has recently become clear that AGEs also influence physiological aging and neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Recently we have provided direct immunochemical evidence for the existence of six distinct AGE structures within the AGE-modified proteins and peptides that circulate in the serum of diabetic patients on hemodialysis (DM-HD). We showed a direct toxic effect of the synthetic AGE-2 (glyceraldehyde-derived AGEs) on cortical neuronal cells and provided evidence for a toxic effect of AGE-2 present in DM-HD serum. These results indicate that of the various types of AGE structures that can form in vivo, the AGE-2 structure is likely to play an important role in the pathophysiological processes associated with AGE formation. In AD brains, AGE-2 epitope was mainly present in the cytosol of neurons in the hippocampus and para-hippocampal gyrus. Protein cross-linking by AGE structures results in the formation of protease-resistant aggregates. Such protein aggregates may interfere with both axonal transport and intracellular protein traffic in neuron. In this review, we provide an outline of AGEs formation in vivo and propose that the novel structural epitope AGE-2 is an important toxic moiety for neuronal cells in AD.

Cytochrome c oxidase inhibition in the rice weevil Sitophilus oryzae (L.) by formate, the toxic metabolite of volatile alkyl formates

Volatile alkyl formates are potential replacements for the ozone-depleting fumigant, methyl bromide, as postharvest insecticides and here we have investigated their mode of insecticidal action. Firstly, a range of alkyl esters, ethanol and formic acid were tested in mortality bioassays with adults of the rice weevil, Sitophilus oryzae (L.) and the grain borer, Rhyzopertha dominica (F.) to determine whether the intact ester or one of its components was the toxic moiety. Volatile alkyl formates and formic acid caused similar levels of mortality (LC 50 131–165 μmol l −1) to S. oryzae and were more potent than non-formate containing alkyl esters and ethanol (LC 50>275 μmol l −1). The order of potency was the same in R. dominica. Ethyl formate was rapidly metabolised in vitro to formic acid when incubated with insect homogenates, presumably through the action of esterases. S. oryzae and R. dominica fumigated with a lethal dose of ethyl formate had eight and 17-fold higher concentrations of formic acid, respectively, in their bodies than untreated controls. When tested against isolated mitochondria from S. oryzae, alkyl esters, alcohols, acetate and propionate salts were not inhibitory towards cytochrome c oxidase (EC 1.9.3.1), but sodium cyanide and sodium formate were inhibitory with IC 50 values of 0.0015 mM and 59 mM, respectively. Volatile formate esters were more toxic than other alkyl esters, and this was found to be due, at least in part, to their hydrolysis to formic acid and its inhibition of cytochrome c oxidase.

Plant toxic proteins and their current significance for warfare and medicine

Abrin, ricin, viscumin, modeccin, and volkensin are very potent toxins derived from plants. They are glycoproteins composed of two polypeptide chains linked by a disulphide bridge. The A-chain is the enzymatic toxic moiety and B-chain is responsible for bonding to the target cell and internalization of toxin. The toxic part of the toxin molecule removes an adenine from a specific adenosine residue in ribosomal RNA and block proteosynthesis. That is the reason of extreme toxicity of these compounds and their capacity to be used as biological warfare agents or terrorist weapon. Therefore all these compounds are in the schedules of controlled biological agents and toxins. Contrariwise, plant ribosome-inactivating proteins are studied intensive as possible chemotherapeutic agents.

Spherical aggregates of beta-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/glycogen synthase kinase-3beta.

beta-Amyloid (Abeta) acquires toxicity by self-aggregation. To identify and characterize the toxic form(s) of Abeta aggregates, we examined in vitro aggregation conditions by using large quantities of homogenous, chemically synthesized Abeta1-40 peptide. We found that slow rotation of Abeta1-40 solution reproducibly gave self-aggregated Abeta1-40 containing a stable and highly toxic moiety. Examination of the aggregates purified by glycerol-gradient centrifugation by atomic force microscopy and transmission electron microscopy revealed that the toxic moiety is a perfect sphere, which we call amylospheroid (ASPD). Other Abeta1-40 aggregates, including fibrils, were nontoxic. Correlation studies between toxicity and sphere size indicate that 10- to 15-nm ASPD was highly toxic, whereas ASPD <10 nm was nontoxic. A positive correlation between the toxicity and ASPD >10 nm also appeared to exist when Abeta1-42 formed ASPD by slow rotation. However, Abeta1-42-ASPD formed more rapidly, killed neurons at lower concentrations, and showed approximately 100-fold-higher toxicity than Abeta1-40-ASPD. The toxic ASPD was associated with SDS-resistant oligomeric bands in immunoblotting, which were absent in nontoxic ASPD. Because the formation of ASPD was not disturbed by pentapeptides that break beta-sheet interactions, Abeta may form ASPD through a pathway that is at least partly distinct from that of fibril formation. Inhibition experiments with lithium suggest the involvement of tau protein kinase I/glycogen synthase kinase-3beta in the early stages of ASPD-induced neurodegeneration. Here we describe the identification and characterization of ASPD and discuss its possible role in the neurodegeneration in Alzheimer's disease.