Binding and intracellular routing of the plant-toxic lectins, lanceolin and stenodactylin.

BACKGROUND: The present research studied the interaction of two ribosome-inactivating proteins (RIPs) from Adenia genus with HeLa cells. Namely, lanceolin and stenodactylin were examined in comparison to volkensin, another toxic two-chain RIP from Adenia genus. METHODS: The binding, endocytosis, intracellular routing, degradation and exocytosis were investigated by measuring the distribution of radiolabelled RIP and by determining its cytotoxicity. RESULTS: Stenodactylin was the most toxic, resulting in the greater inhibition of protein synthesis and cell death. Lanceolin and stenodactylin bound to cells with comparable affinity and have a similar number of binding sites (10(5)/cell). The uptake of lanceolin and stenodactylin was 13 and 36 times greater, respectively, than that reported for volkensin. The two toxins bound to cell membrane receptors via their lectin B chain, were endocytosed through a clathrin-independent pathway, were internalised in a manner independent from endosomal acidification, and required routing through the Golgi apparatus, as reported for modeccin and volkensin. Stenodactylin showed greater uptake, exocytosis and re-uptake of non-degraded RIP than lanceolin and volkensin, whereas volkensin had the highest residual activity after being released from the cell. CONCLUSIONS: The high cytotoxicity of RIPs from the Adenia genus may depend on the following: high affinity binding to the cell and efficient endocytosis, intracellular routing that appears similar to that of other ricin-like toxic RIPs, partial resistance to proteolysis, and, regarding stenodactylin, high accumulation in cell. GENERAL SIGNIFICANCE: The data provide a model that could lead to new strategies for anti-cancer therapy and neuroscience studies.

Structure/function studies on two type 1 ribosome inactivating proteins: Bouganin and lychnin.

The three-dimensional structures of two type 1 RIPs, bouganin and lychnin, has been solved. Their adenine polynucleotide glycosylase activity was also determined together with other known RIPs: dianthin 30, PAP-R, momordin I, ricin A chain and saporin-S6. Saporin-S6 releases the highest number of adenine molecules from rat ribosomes, and poly(A), while its efficiency is similar to dianthin 30, bouganin and PAP-R on herring sperm DNA. Measures of the protein synthesis inhibitory activity confirmed that saporin-S6 is the most active. The overall structure of bouganin and lychnin is similar to the other considered RIPs and the typical RIP fold is conserved. The superimpositioning of their C(alpha) atoms highlights some differences in the N-terminal and C-terminal domains. A detailed structural analysis indicates that the efficiency of saporin-S6 on various polynucleotides can be ascribed to a negative electrostatic surface potential at the active site and several exposed positively charged residues in the region around that site. These two conditions, not present at the same time in other examined RIPs, could guarantee an efficient interaction with the substrate and an efficient catalysis.

Saporin induces multiple death pathways in lymphoma cells with different intensity and timing as compared to ricin.

Ribosome-inactivating protein (RIP)-containing immunotoxins are currently used in clinical trials as anti-tumour drugs, in particular against haematological malignancies. In cell killing-based therapies it is important to identify the death pathways induced by the cytotoxic agent. The purpose of this work was to compare the pathways of cell death induced by the RIP saporin with those carried out by ricin in the L540 human Hodgkin's lymphoma-derived cell line. Protein synthesis inhibition, activation of caspases, DNA fragmentation and loss of viability have been evaluated. The two toxins triggered a similar DNA fragmentation and cell death, at concentrations giving the same level of cell protein synthesis inhibition, although the inhibitory effect of ricin on protein synthesis was more rapid than that of saporin. Moreover, the intrinsic apoptotic pathway was equally activated by both toxins, whilst ricin activated the extrinsic caspase pathway and the effector caspase-3/7 more efficiently than saporin. The complete inhibition of caspases by Z-VAD was only partially effective in cell rescue which appeared to be time limited. Necrostatin-1, a new inhibitor of non-apoptotic death, rescued cells from death by RIPs, although the effect was also partial and temporary. Despite the high RIP doses used no necrosis was detectable by Annexin V/Propidium Iodide (PI) test. These results suggest that more than one death mechanism was elicited by both ricin and saporin, however, with different timing and strength. The perspective of modulating cell death of neoplastic lymphocytes through different pathways could add new opportunities to reduce side effects and develop combined synergic immuno-chemotherapy.

Type 1 ribosome-inactivating proteins - entomotoxic, oxidative and genotoxic action on Anticarsia gemmatalis (Hübner) and Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae).

Ribosome-inactivating proteins (RIPs) from plants inhibit protein synthesis by inactivating ribosomes. Some two-chain (type 2) RIPs are highly toxic and may play a role in plant defense. The lower toxicity of single-chain (type 1) RIPs reflects the lack of a protein domain able to bind to, and translocate the toxin across cell membranes. We studied the effect of single-chain RIPs, lychnin, momordin, gelonin, PAP-S and saporin S-6, in larvae of Anticarsia gemmatalis and Spodoptera frugiperda. After ingesting a total dose of 20 or 40 microg of the toxins, weight gain, survival rate, lesions in DNA and oxidative status (catalase and superoxide dismutase activities and lipidic peroxidation) of RIP-treated insects were assayed. Momordin was the less toxic in the biossays. S. frugiperda had a more pronounced weight loss on the 4th day of treatment and A. gemmatalis on the 10th day. RIP-induced mortality reached 57.13% for A. gemmatalis and 29.45% for S. frugiperda. RIP-treated insects showed a 2-3-fold increase in DNA lesions as assessed by the comet assay, but there were no correlations between stress markers and DNA damage. We conclude that single-chain RIPs are entomotoxic to lepidopteran insects causing extensive DNA lesions.

Acceptance of and discontinuation rate from erectile dysfunction oral treatment in patients following bilateral nerve-sparing radical prostatectomy.

OBJECTIVES: Assess acceptance of and discontinuation rate from erectile dysfunction (ED) treatment in patients after bilateral nerve-sparing radical retropubic prostatectomy (BNSRRP). METHODS: We analyzed acceptance and discontinuation data of 100 consecutive, age-comparable, preoperatively self-reported potent BNSRRP patients who at the discharge from the hospital received a phosphodiesterase type 5 inhibitor (PDE5-I) prescription. Patients were informed of the pharmacokinetic properties of the available compounds and the option of on-demand versus rehabilitative therapy. Thereafter, patients did not receive any specific counseling throughout the entire follow-up period and freely decided to use or not use any ED therapy. Complete preoperative data were obtained on hospital admission and included a medical and sexual history and the International Index of Erectile Function (IIEF). The IIEF was completed every 6 mo postoperatively, and patients participated in a semi-structured interview about the treatment adherence at the 18-mo follow-up. RESULTS: Forty-nine (49%) patients freely decided not to start any ED therapy (group 1). Of the remaining patients, 36 (36%) opted for an as-needed PDE5-I (group 2), whereas 15 (15%) decided to use a daily PDE5-I (group 3). At the 18-mo follow-up, the overall discontinuation rate from both treatment modalities was 72.6% (eg, 72.2% vs. 73.3% in group 2 vs. group 3; p=0.79). Treatment effect below expectations was the main reason for treatment discontinuation, followed by loss of interest in sex due to partner's causes. CONCLUSIONS: Almost 50% of BNSRRP patients freely decided not to start any ED treatment postoperatively. Roughly 73% of patients who started therapy eventually discontinued it.

Characterization of highly toxic type 2 ribosome-inactivating proteins from Adenia lanceolata and Adenia stenodactyla (Passifloraceae).

From the caudices of the Passifloraceae Adenia lanceolata and A. stenodactyla, two lectins called lanceolin and stenodactylin, respectively, were purified by affinity chromatography on CL Sepharose 6B. The lectins are glycoproteins with M(r) 61,243 (lanceolin) and 63,131 (stenodactylin), consisting of an enzymatic A chain linked to a larger B chain with lectin properties, with N-terminal amino acid sequences similar to that of volkensin, the toxic lectin from A. volkensii. The lectins agglutinate red blood cells, inhibit protein synthesis both by a cell-free system and by whole cells, and depurinate ribosomes and DNA, but not tRNA or poly(A). They are highly toxic to cells, in which they induce apoptosis, and to mice, with LD(50)s 8.16 microg/kg (lanceolin) and 2.76 microg/kg (stenodactylin) at 48 h. Thus, lanceolin and stenodactylin have all the properties of the toxic type 2 ribosome-inactivating proteins and are amongst the most potent toxins of plant origin.

A comparative review of apomorphine formulations for erectile dysfunction : recommendations for use in the elderly.

Erectile dysfunction (ED) is a common medical condition that affects the sexual life of millions of men worldwide. First-line oral therapy for ED includes the use of phosphodiesterase type 5 inhibitors (sildenafil, tadalafil and vardenafil) and sublingual apomorphine. Apomorphine is a dopamine D(1) and D(2) receptor agonist that has been approved for marketing in Europe. Different apomorphine formulations have been tested, such as sublingual, subcutaneous and intranasal. However, the sublingual formulation has shown the best results in terms of efficacy, safety and tolerability, especially the 2mg and 3mg doses. Although clinical studies of the efficacy and tolerability of apomorphine sublingual (SL) have included older patients, who are more likely to have ED, no study has specifically assessed the efficacy and tolerability of different doses of apomorphine SL in aging men. Therefore, a MEDLINE search was conducted from January 1987 to November 2005 to identify studies of the efficacy, safety (in particular cardiovascular safety) and tolerability of different apomorphine formulations and doses as treatments for ED in the subcohort of aging men. On the basis of the most recent peer-reviewed publications, the first part of this article critically evaluates data regarding the epidemiology of ED in the aging population. The second part of the article focuses on the mechanism of action and pharmacokinetics of apomorphine both in the general and the elderly population. Finally, a critical analysis of the efficacy and safety of different apomorphine formulations and doses for the treatment of ED is reported. Apomorphine represents a first-line oral treatment for ED. Available formulations include only sublingual administration. Few studies have assessed the efficacy and safety of apomorphine in the elderly population. However, in clinical practice, older patients with multiple vascular risk factors and systematic vascular damage show poor overall response to apomorphine SL for the treatment of ED.

Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata.

The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml(-1)) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml(-1), all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.

Clinical update on phosphodiesterase type-5 inhibitors for erectile dysfunction.

Erectile dysfunction (ED) affects the sexual lives of millions of men. The first-line oral pharmacotherapy for most ED patients is phosphodiesterase type-5 (PDE-5) inhibitors, of which three are available. Sildenafil is the most widely prescribed oral agent for ED and has a very satisfactory efficacy-safety profile in all patient categories. Tadalafil and vardenafil were introduced in the European Union and in the United States in 2003 and 2004, respectively. The three PDE-5 inhibitors share many pharmacological and clinical characteristics, and each has unique features. This review, which is based on the contemporary literature on PDE-5 inhibitors, describes the chemical, pharmacological, and clinical features of sildenafil, vardenafil, and tadalafil. The first section reviews the pathophysiology of penile erection and PDE-5 inhibitor pharmacology. The second section summarizes data regarding efficacy and safety of the three drugs in treating ED in the general population as well as in selected patient categories.

Ribosome-inactivating proteins and other lectins from Adenia (Passifloraceae).

The caudices of 10 Adenia species contain galactose-binding lectins that were purified by affinity chromatography. All lectins but three agglutinate human erythrocytes. Six lectins consist of two unequal chains, which can be separated by reduction, and inhibit protein synthesis both by a rabbit reticulocyte lysate and by HeLa and Raji cells. The lectins from A. goetzii, A. lanceolata and A. stenodactyla had the highest cytotoxicity, inhibiting cell protein synthesis with IC50s (concentration inhibiting by 50%) below 0.1 ng/ml, and deadenylate DNA, thus being type 2 ribosome-inactivating proteins.

Vardenafil for the treatment of erectile dysfunction: a critical review of the literature based on personal clinical experience.

OBJECTIVE: To critically review the literature on vardenafil in the treatment of erectile dysfunction while integrating the clinical findings with the personal experience of the authors. METHODS: Analysis of published full-length papers that were identified through Medline search from January 2000 through May 2004. Abstracts published in peer-reviewed journals from the same period were also considered. RESULTS: Efficacy, tolerability and safety, as reported in the peer-reviewed literature compares well with the authors' personal experience. Authors' personal observations include discussions on potency, selectivity, selection of initial dose, counselling for patients characteristically considered difficult-to-treat (diabetes, prostatectomy, depression), including the determination of the maximal efficacious dose and the possible role of daily dosing, optimisation of the use of vardenafil according to its pharmacokinetic and pharmacodynamic profiles (onset and reliability), and management of ED patients with or at risk for cardiovascular disease. CONCLUSIONS: Extensive experience with vardenafil as reported in peer reviewed literature confirms the important role of vardenafil in the management of patients with ED. The development of each physician's own experience with vardenafil is key to optimise overall satisfaction of this therapy by the patient and his partner.

Comparison of the effect of sildenafil and apomorphine SL on nocturnal erections in healthy volunteers: a placebo-controlled study.

OBJECTIVE: To compare the effects of sildenafil and sublingual (sl) apomorphine on nocturnal erections. METHODS: In a prospective, single-blinded, placebo-controlled at-home study we compared the effect of apomorphine sl and sildenafil on sleep-related erectile activity in 30 healthy potent volunteers (mean age, 26.4 years; range, 23-35 years; Erectile Function domain of the International Index of Erectile Function > or =26), not reporting any subjective sleep abnormality during 4 consecutive nights with rigidity monitoring (NPTR) with the RigiScantrade mark device. After the first night of adaptation, they were randomized to receive placebo or apomorphine sl 2mg or sildenafil 50mg taken at bedtime during the following 3 consecutive nights. RESULTS: Sildenafil increased the mean+/-SE number of erections, overall length of erectile events, and the time of erections with rigidity >60% at both tip and base, as compared to placebo. The same parameters were reduced after administration of apomorphine sl. Similar findings were observed with regards to Rigidity Activity Units and Tumescence Activity Units. CONCLUSIONS: Our results confirm that sildenafil taken at bedtime might increase nocturnal erectile activity also in young potent men as compared to placebo. Apomorphine sl taken at bedtime seems to have the opposite effect.

Shiga toxin 1 and ricin inhibit the repair of H2O2-induced DNA single strand breaks in cultured mammalian cells.

A growing body of evidence suggests that ribosome-inactivating proteins (RIPs) remove adenine moieties not only from rRNA, but also from DNA--an effect leading to DNA damage in cultured cells. We herein report that two distinct RIPs of bacterial (shiga toxin 1, Stx1) and plant (ricin) origin, inhibit the repair of the DNA lesions generated by hydrogen peroxide in cultured human cells. This effect is unrelated either to inhibition of protein synthesis or to depletion of cellular antioxidant defenses and is likely to derive from direct interactions with cellular DNA repair machinery. Therefore, the genotoxicity of these toxins on mammalian cells seems to be a complex phenomenon resulting from the balance between direct (DNA damaging activity), indirect (DNA repair inhibition) effects and the eventual presence of other DNA damaging species. In particular, with regard to Stx1, it could be hypothesized that Stx-producing bacteria increase the risk of transformation of surrounding, inflamed tissues in the course of human infections.

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

Isolation and characterization of an RIP (ribosome-inactivating protein)-like protein from tobacco with dual enzymatic activity.

Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a protein termed tobacco RIP (TRIP) was isolated from tobacco (Nicotiana tabacum) leaves and purified using ion exchange and gel filtration chromatography in combination with yeast ribosome depurination assays. TRIP has a molecular mass of 26 kD as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed strong N-glycosidase activity as manifested by the depurination of yeast rRNA. Purified TRIP showed immunoreactivity with antibodies of RIPs from Mirabilis expansa. TRIP released fewer amounts of adenine residues from ribosomal (Artemia sp. and rat ribosomes) and non-ribosomal substrates (herring sperm DNA, rRNA, and tRNA) compared with other RIPs. TRIP inhibited translation in wheat (Triticum aestivum) germ more efficiently than in rabbit reticulocytes, showing an IC50 at 30 ng in the former system. Antimicrobial assays using highly purified TRIP (50 microg mL(-1)) conducted against various fungi and bacterial pathogens showed the strongest inhibitory activity against Trichoderma reesei and Pseudomonas solancearum. A 15-amino acid internal polypeptide sequence of TRIP was identical with the internal sequences of the iron-superoxide dismutase (Fe-SOD) from wild tobacco (Nicotiana plumbaginifolia), Arabidopsis, and potato (Solanum tuberosum). Purified TRIP showed SOD activity, and Escherichia coli Fe-SOD was observed to have RIP activity too. Thus, TRIP may be considered a dual activity enzyme showing RIP-like activity and Fe-SOD characteristics.

Ribosome-inactivating proteins depurinate poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase and have transforming activity for 3T3 fibroblasts.

It has been known that ribosome-inactivating proteins (RIPs) from plants damage ribosomes by removing adenine from a precise position of rRNA. Subsequently it was observed that all tested RIPs depurinate DNA, and some of them also non-ribosomal RNAs and poly(A), hence the denomination of adenine polynucleotide glycosylases was proposed. We report now that ricin, saporin-L2, saporin-S6, gelonin and momordin depurinate also poly(ADP-ribosyl)ated poly(ADP-ribose) polymerase (auto modified enzyme), an enzyme involved in DNA repair. We observed also that all RIPs but gelonin induce transformation of fibroblasts, possibly as a consequence of damage to DNA and of the altered DNA repair system.

Conjugates of nucleoside analogs with lactosaminated human albumin to selectively increase the drug levels in liver blood: requirements for a regional chemotherapy.

Nucleoside analogs (NAs) conjugated with galactosyl terminating peptides selectively enter hepatocytes via the asialoglycoprotein receptor and, after intracellular release from the carrier, partly exit from these cells into the bloodstream, resulting in higher concentrations in liver blood than in systemic circulation. Therefore, conjugates of anticancer NAs can be exploited to accomplish a loco-regional noninvasive treatment of liver micrometastases. In the present experiments we studied whether the enhancement of drug levels in liver blood achieved when NAs are given in the coupled form depends on the rate of drug elimination from the bloodstream. Three NAs, adenine arabinoside (ara-A), 5-fluoro-2'-deoxyuridine (FUdR), and 2',2'-difluorodeoxycytidine, were coupled with lactosaminated human albumin, a galactosyl terminating carrier. In rats that received an intravenous bolus injection of these conjugates, we compared the drug concentrations in liver blood to those in the systemic circulation. We found that enhanced levels of NAs in liver blood were only achieved by administering the conjugates of the drugs (ara-A and FUdR), which are rapidly cleared from the bloodstream. Increased drug levels also were obtained when ara-A and FUdR conjugates were slowly infused (a way of administration often used for anticancer drugs). The experiments also showed that galactosyl terminating conjugates of NAs might have the potential to produce a therapeutic effect only when the coupled drugs are active at low blood concentrations, since the amounts of drugs introduced into hepatocytes and released by these cells in the bloodstream cannot be increased when the receptor for the hepatic uptake of galactosyl terminating peptides is saturated.

Damage to nuclear DNA induced by Shiga toxin 1 and ricin in human endothelial cells.

Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.

Ribosome-inactivating and adenine polynucleotide glycosylase activities in Mirabilis jalapa L. tissues.

Several tissues of Mirabilis jalapa L. (Nyctaginaceae) were assayed for inhibition of translation by a rabbit reticulocyte lysate (as a signal of ribosome-inactivating activity) and for adenine DNA glycosylase activity, activities that are both due to the presence of a class of enzymes called ribosome-inactivating proteins (RIPs), currently classified as rRNA N-glycosylases (EC ). These activities were highest in seed; intermediate in flower bud, immature seed, sepal + gynoecium, leaf, and root; and very low in all other tissues. By cation-exchange chromatography, four protein peaks with inhibitory activity on cell-free translation were identified in extracts from seeds, and two proteins were isolated from peaks 1 and 4, all of which have the properties of single-chain type 1 RIP. One is Mirabilis antiviral protein (MAP), so far purified only from roots. The second is a new protein that we propose to call MAP-4. The distribution of MAP and MAP-4 in several tissues was determined with a novel experimental approach based on liquid chromatography/mass spectrometry. The direct enzymatic activity of MAP on several substrates is described here for the first time. MAP depurinated not only rRNA in intact ribosomes, thus inhibiting protein synthesis, but also other polynucleotides such as poly(A), DNA, and tobacco mosaic virus RNA. Autologous DNA was depurinated more extensively than other polynucleotides. Therefore, the enzymatic activity of this protein may be better described as adenine polynucleotide glycosylase activity rather than rRNA N-glycosylase activity. Finally, MAP does not cross-react immunologically with other commonly utilized RIPs.