Ebulin-RP, a novel member of the Ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains.

BACKGROUND: Sambucus ebulus is a rich source of ribosome-inactivating proteins (RIPs) and RIP-related lectins generated from multiple genes. These proteins differ in their structure, enzymatic activity and sugar binding specificity. METHODS: We have purified and characterized ebulin-RP from S. ebulus leaves and determined the amino acid sequence by cDNA cloning. Cytotoxicity was studied in a variety of cancer cells and a comparative study of the ability of ebulin-RP to bind sugars using "in vitro" and "in silico" approaches was performed. RESULTS: Ebulin-RP is a novel heterodimeric type 2 RIP present in S. ebulus leaves together with the type 2 RIP ebulin l, which displayed rRNA N-glycosidase activity but unlike ebulin l, lacked functional sugar binding domains. As a consequence of changes in its B-chain, ebulin-RP displayed lower cytotoxicity than ebulin l towards cancer cells and induced apoptosis as the predominant pattern of cell death. CONCLUSIONS: Ebulin-RP is a novel member of the ebulin gene family with low cytotoxicity as a result of deficient sugar binding domains. Type 2 RIP genes from Sambucus have evolved to render proteins with different sugar affinities that may be related to different biological activities and could result in an advantage for the plant. GENERAL SIGNIFICANCE: The ebulin family of RIPs and lectins can serve as a good model for studying the evolutionary process which may have occurred in RIPs. The lack of cytotoxicity of ebulin-RP makes it a good candidate as a toxic moiety in the construction of immunotoxins and conjugates directed against specific targets.

The ribotoxin-like protein Ostreatin from Pleurotus ostreatus fruiting bodies: Confirmation of a novel ribonuclease family expressed in basidiomycetes.

Fungi produce several toxins active against plants, animal or humans. Among them, ribotoxins are enzymes that specifically attack ribosomes irreparably compromising protein synthesis, useful as insecticides or as anticancer agents. Here, a novel ribotoxin from the edible mushroom Pleurotus ostreatus has been purified and characterized. This ribotoxin, named Ostreatin, is a specific ribonuclease releasing α-fragment when incubated with yeast or rabbit ribosomes. Ostreatin shows IC50 of 234 pM in rabbit reticulocyte lysate, and metal dependent endonuclease activity. Following the completion of Ostreatin primary structure, we ascertained that this toxin is homologous to Ageritin, the first ribotoxin-like protein from the basidiomycete Agrocybe aegerita, with which it shares 38.8% amino acid sequence identity. Ostreatin consists of 131 amino acid residues with an experimental molecular mass of 14,263.51 Da ([M+H+]+). Homology modeling revealed that Ostreatin and Ageritin share a similar fold in which the common catalytic triad is conserved. Purified Ostreatin lacks N-terminal and C-terminal peptides, which instead are present in the Ostreatin coding sequence. Such peptides are probably involved in protein sorting and for this they could be removed. Our findings confirm the presence of ribotoxin-like proteins in basidiomycetes edible mushrooms, that we propose as novel tool for biotechnological applications.

Dangerous passengers: multidrug-resistant bacteria on hands and mobile phones.

INTRODUCTION: It is recognized that mobile phones may play a role in microorganism transmission and that hand hygiene, is considered the most important action for preventing infections and the spread of pathogens. The objective of this study was to determine presence and circulation bacteria on hands and mobile phones capable of causing infections in people and also determine if disinfection with gel-alcohol is useful to reduce the bacterial colonization. METHODS: The bacterial evaluation included 596 hands of participants and 256 mobile phones. Isolated colonies were identified by biochemical test and confirmed by gene 16S rRNA sequencing. Antimicrobial susceptibility was performed using the automated instrument Vitek®2-Compact and disk-diffusionmethod. RESULTS: In total, 92.9% of mobile phones and 98.3% of participants in study demonstrated evidence of bacterial contamination with different types of bacteria. Surprisingly, we observed that 18.6% plaques inoculated with disinfected fingers showed bacterial growth. In general, Gram negative isolates showed resistance to a higher number of antibiotics tested than Gram positive isolates. CONCLUSIONS: Our results could help to raise awareness in our society about the importance of hand hygiene, as well as frequently used devices, reducing bacterial contamination and limiting the possibility of transmission of resistant multi-drug bacteria.

Effect of the chronic ethanol action on the activity of the general amino-acid permease from Saccharomyces cerevisiae var. ellipsoideus.

The presence of ethanol and cycloheximide during growth were found to inhibit the function of the general amino-acid permease of Saccharomyces cerevisiae var. ellipsoideus. Contrary to cycloheximide, the effect of ethanol upon growth in alcohol-free medium was reversible. The effect of both inhibitors could be explained in terms of reduction of the number of active carrier molecules located in the plasma membrane.

Distribution and properties of major ribosome-inactivating proteins (28 S rRNA N-glycosidases) of the plant Saponaria officinalis L. (Caryophyllaceae).

We have studied the distribution of the protein synthesis inhibitory activity in the tissues of Saponaria officinalis L. (Caryophyllaceae). Seven major saporins, ribosome-inactivating proteins, were purified to apparent homogeneity from leaves, roots and seeds using a new procedure of RIPs isolation including ion-exchange and hydrophobic chromatography. They all catalysed the depurination of rat liver ribosomes, which generate the Endo's diagnostic rRNA fragment upon treatment with acid aniline, thus indicating that A4324 from the 28S rRNA has been released (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). The molecular mass of saporins by SDS-PAGE ranged between 30.2 and 31.6 kDa and by gel-filtration between 27.5 and 30.1 kDa. Amino acid composition and amino-terminal amino acid sequence indicate that all saporins may be considered isoforms. Only two saporins present in roots were glycosylated (SO-R1 and SO-R3). All saporins are very active on cell-free translation systems derived from rabbit reticulocyte lysates, rat liver, Triticum aestivum L., Cucumis sativus L. and Vicia sativa L. However, they are poor inhibitors of an Escherichia coli translation system. They inhibit protein synthesis in HeLa, BeWo and NB 100 cells, HeLa cells being the most resistant. The enzymatic activity of at least one saporin isoform was dependent on magnesium concentration in the standard rat liver cell-free system.

Effects and molecular action of ribosome-inactivating proteins on ribosomes from Streptomyces lividans.

The effects of 29 type 1 and 2 type 2 ribosome-inactivating proteins (RIPs) from plants on polyuridylic acid-directed polyphenylalanine synthesis carried out by purified ribosomes from Streptomyces lividans were studied. Only dianthin 32, saporins R1 and R3, momordin I, trichokirin, Hura crepitans RIP 5 from latex, crotins 2 and 3, and PAPs C, R, and S, inhibited polyphenylalanine synthesis. Both the type 2 RIPs ricin and volkensin were ineffective on translation. The magnesium concentration affected the inhibition of translation to a considerable extent. Upon treatment with inhibitory RIPs, extraction of rRNA and further treatment with acid aniline, S. lividans ribosomes released an RNA fragment of about 130 nucleotides. The 5' terminal sequence of this rRNA fragment was 5'-GAGGACCGGGACGGACGAACCUCUGGUGUGCCAGUUGU-3', similar to the sequence obtained in Escherichia coli. This indicates that the most probable molecular action of these RIPs on S. lividans and E. coli ribosomes is the same: depurination of the rRNA at a site relevant to the translation mechanism and that has been highly conserved throughout evolution.

Cytotoxicity of an ebulin l-anti-human CD105 immunotoxin on mouse fibroblasts (L929) and rat myoblasts (L6E9) cells expressing human CD105.

Tumour growth is characterised by the formation of a fine vessel network or neovasculature which nourishes tumour cells. Two kinds of novel anti-angiogenic therapies are based on the prevention of vessels growth and on the destruction of those vessels already formed. We report here on the design and construction of a novel immunotoxin formed with the non-toxic type II ribosome-inactivating protein ebulin l and the mouse anti-human CD105 monoclonal antibody 44G4. The 44G4-ebulin immunotoxin was formed by covalent linking of both proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and was purified by chromatography on Superdex 200 HiLoad. The analysis of the anti-ribosomal effects in a cell-free translation system indicated that conjugation does not affect the activity of ebulin l. The immunotoxin displays cytotoxicity with nanomolar IC50 values on human CD105+ cells like the mouse fibroblasts L929 cells transfected with the short form of human CD105 and the rat myoblasts L6E9 transfected with the long form of human CD105. In contrast, cells lacking human CD105 were 2-2.5 logs less sensitive to the immunotoxin. Free ebulin displays IC50 values in the range 10(-6) M. Since CD105 is being considered as a potential target for the anti-vascular therapy of tumours, the present immunotoxin could be a promising tool for the anticancer therapy, especially due to the very low in vivo toxicity of ebulin l as compared ricin and other toxins used for immunotoxins.

cDNA molecular cloning and seasonal accumulation of an ebulin l-related dimeric lectin of dwarf elder (Sambucus ebulus L) leaves.

SELld is a dimeric D-galactose and mucin-binding lectin (apparent Mr 68000) which coexists with the non-toxic type 2 ribosome-inactivating protein (RIP) ebulin l in dwarf elder (Sambucus ebulus L.) leaves. To ascertain a potential structural correlation with ebulin l molecular cloning of a cDNA coding for SELld was performed. SELld shared a 76% of identity with the ebulin l-B chain. Notably, it was found that SELld has Tyr present in the high affinity 2gamma sugar-binding domain of ricin which is absent in ebulin l-B chain and which seems responsible of the low cell and in vivo toxicities of ebulin l. The concentration of ebulin l in leaves decreased along the developmental stage of dwarf elder and almost disappeared in senescence while the content in SELld changed in the opposite way. Our results suggest that SELld and ebulin l play different biological roles in dwarf elder leaves.

Musarmins: three single-chain ribosome-inactivating protein isoforms from bulbs of Muscari armeniacum L. and Miller.

Three new ribosome-inactivating protein (RIP; EC 3.2.2.22) isoforms that we have named musarmins (MUs) 1, 2 and 3 have been isolated from the bulbs of Muscari armeniacum L. and Miller by ion-exchange chromatography and gel filtration. Analysis by electrophoresis revealed that they are single-chain proteins and mass spectrometry analysis afforded Mr values of 28,708, 30,003 and 27,626 for MUs 1, 2 and 3, respectively. Musarmins strongly inhibited protein synthesis carried out by mammalian ribosomes, with IC50 values in the 0.14-0.24nM range but not that carried out by plant cell-free systems or HeLa cells. MUs promote the single depurination of rabbit reticulocyte 28S rRNA. cDNA cloning of genes coding for musarmins revealed that they contain open reading frames of 298, 294 and 295 aminoacids for MU1, MU2 and MU3, respectively. Mature MU1, MU2 and MU3 contain 277, 273 and 273 aminoacids, respectively suggesting post-translational C-terminal processing. An untranslated mRNA coding for an ORF very similar to that of MU3 was detected in leaves. Each of the four MU genes contains an intron. In contrast to other RIPs, MUs are present only in bulbs and are not induced in leaves either by senescence, or by treatment of leaves with H2O2 or salicylic acid, or by growth in darkness. Therefore, these proteins could play a non-vital role in plants; for instance, as anti-pathogens and protective agents only in some stages of the plant life cycle (237).

Fusidic acid-dependent ribosomal complexes protect Escherichia coli ribosomes from the action of the type 1 ribosome-inactivating protein crotin 2.

The type 1 ribosome-inactivating protein crotin 2 depurinated Escherichia coli ribosomes which, upon treatment of the isolated rRNA with acid aniline, released a fragment of around 240 nucleotides whose 5'-end sequence was 5'-GAGGACCGGAGUGGAC-3'. The formation of fusidic acid-dependent ribosomal complexes completely prevented release of the fragment. Ribosomes from crotin 2-pretreated fusidic acid complexes were insensitive to acid aniline. They released the RNA fragment only after a second treatment with crotin 2 and acid aniline whereas unprotected ribosomes released the fragment directly after acid aniline.

Molecular action of the type 1 ribosome-inactivating protein saporin 5 on Vicia sativa ribosomes.

The type 1 ribosome-inactivating protein (RIP) saporin 5 isolated from seeds of Saponaria officinalis L. strongly inhibited translation carried out by Vicia sativa L. purified ribosomes. The toxin multidepurinated V. sativa rRNA, which upon treatment with acid aniline releases several RNA fragments including an RNA fragment of approximately 370 nucleotides the 5'-end sequence of which was 5'-GAGGAACG-3'.

Molecular mechanism of inhibition of mammalian protein synthesis by some four-chain agglutinins. Proposal of an extended classification of plant ribosome-inactivating proteins (rRNA N-glycosidases).

The four chain agglutinins from Abrus precatorius, Viscum album and Ricinus communis promote depurination of the 28 S rRNA from rabbit reticulocyte ribosomes characteristic of the common ribosome-inactivating proteins (RIPs). These agglutinins inhibited mammalian protein synthesis at nanomolar concentrations but they do not affect plant protein synthesis under the same conditions. Therefore, they should also be considered as true RIPs but of a new class, the four-chain RIPs. An extended classification of RIPs is presented based on the former one from Stirpe et al. [Bio/technology 10 (1992) 405-412].

Isolation and partial characterization of a novel and uncommon two-chain 64-kDa ribosome-inactivating protein from the bark of elder (Sambucus nigra L.).

A novel, strongly basic, two-chain ribosome-inactivating protein (RIP) with an apparent Mr of 64000 by SDS-PAGE and 63469 by mass spectrometry analysis, that we have named basic nigrin b, has been found in the bark of elder (Sambucus nigra L.). The new protein does not agglutinate red blood cells, even at high concentrations and displays an unusually and extremely high activity towards animal ribosomes (IC50 of 18 pg/ml for translation by rabbit reticulocyte lysates). However, it is inactive against plant and HeLa cells protein synthesis. Our functional and structural data are consistent with a heterodimeric structure for basic nigrin b of the type A-B*, B* being a truncated lectin lacking functional binding domains equivalent to the B (lectin) chain of the type 2 RIP SNA I and nigrin b present also in elder bark.

Ebulitins: a new family of type 1 ribosome-inactivating proteins (rRNA N-glycosidases) from leaves of Sambucus ebulus L. that coexist with the type 2 ribosome-inactivating protein ebulin 1.

A new family of single chain (type 1) ribosome-inactivating proteins (RIPs), that we have named ebulitins, have been found in mature leaves of Sambucus ebulus L., a caprifoliaceae plant also known to contain a non-toxic two chain (type 2) RIP named ebulin I in its leaves. Ebulitins are basic proteins of M(r) 32,000, 29,000 and 29,000 for ebulitins alpha, beta and gamma, respectively. The simultaneous presence of different basic type 1 and acidic type 2 RIPs in the same plant and in the same tissue is described here for the first time and opens a new door in research into RIPs.

Constitutive and inducible type 1 ribosome-inactivating proteins (RIPs) in elderberry (Sambucus nigra L.).

Two novel highly basic type 1 (single chain) ribosome-inactivating proteins (RIPs) with N-glycosidase activity have been found in elderberries (the fruits of Sambucus nigra L.). Mass spectrometry of these RIPs, which we named nigritins f1 and f2, gave Mr values of 24095 and 23 565, respectively. Both proteins strongly inhibited protein synthesis in rabbit reticulocyte lysates but were inactive against plant ribosomes. Both nigritins have a similar topological activity on pBlueScript SK+ DNA as that displayed by dianthin 30. Nigritin f1 is a constitutive RIP since it is present in both green and mature intact elderberries at nearly the same proportion with respect to total fruit protein. By contrast, nigritin f2 is inducible and only appeared in mature intact elderberries. Elderberries also contain two isoforms of a basic nigrin equivalent to the recently found basic nigrin b in elder bark (De Benito et al., FEBS Letters 413 (1997) 85-91). Our results indicate that probably not all plant RIPs exert the same biological function and that this may be determined by the physiological state of the tissue.

Targeting cancer cells with transferrin conjugates containing the non-toxic type 2 ribosome-inactivating proteins nigrin b or ebulin l.

Nigrin b and ebulin l are type 2 ribosome-inactivating proteins (RIPs) with 10(4) times less cellular and in vivo toxicity than ricin that are currently being considered for the construction of anti-cancer conjugates. Here we provide evidence that both RIPs can be used for the construction of conjugates directed to a target such as the transferrin receptor (TfR), which is over-expressed in cancer cells. Nigrin b- and ebulin l-transferrin conjugates were constructed with no substantial reduction in the translational inhibitory molecular activity of either RIPs. Conjugation with transferrin decreased the IC(50) of the proteins from 3 x 10(-7)M (nigrin b) and 1.5 x 10(-8)M (ebulin l) to 3.5 x 10(-10)M in HeLa cells. Thus, both conjugates could be considered as useful tools for targeting TfR-over-expressing cancer cells.

Sensitivity of cancer cell lines to the novel non-toxic type 2 ribosome-inactivating protein nigrin b.

The cytotoxicity of the type 2 ribosome-inactivating proteins (RIPs) ricin and nigrin b was determined in a variety of cancer cells. Nigrin b, considered to be a novel non-toxic type 2 RIP as compared with ricin, was approximately 10(4)-10(5) times less toxic than ricin in all cancer cells studied, with the exception of melanoma cells. Cancer cells displayed considerable heterogeneity in their sensitivity to ricin, melanoma cells being the least sensitive. Rabbit polyclonal anti-nigrin b antibodies did not cross-react with ricin as analyzed by enzyme-linked immunosorbent assays. The low non-specific toxicity of nigrin b as compared with that of ricin and the lack of immunological cross-reaction between anti-nigrin b antibodies and ricin supports the use of nigrin b in the construction of cytotoxic conjugates as an alternative to ricin when anti-ricin antibodies are produced during cancer therapy.

Adaptation of in vitro rat brain protein synthesis to long-term ingestion of n-butanol.

Long-term treatment of rats with n-butanol leads to a change in in vitro brain protein synthesis which increases the resistance of this process to either ethanol or isopropanol. The change seems to be related to ribosomal events since the synthesis of aminoacyl-tRNA was not affected in the same conditions.

A Cucumis sativus cell-free translation system: preparation, optimization and sensitivity to some antibiotics and ribosome inactivating proteins.

A cell-free translation system was prepared from 3- to 5-day-old embryonic axes of gherkin (Cucumis sativus L.). The system was optimized for Mg2+ , K+ , NH+4 , high speed supernatants, tRNA mixture from wheat germ, time and temperature. The system translates efficiently both endogenous mRNA (using a 30000 g supernatant) and polyuridylic acid (using either a 30000 g supernatant or a 100000 g supernatant supplemented with purified ribosomes). Translation by gherkin ribosomes was inhibited by several well-known eukaryotic inhibitors, antibiotics and ribosome-inactivating proteins. A translational inhibitory activity found in Cucumis sativus L. dry seeds acted on polypeptide synthesis carried out by cell-free systems from several mammals and plants, including gherkin embryonic axes. Our results indicate that the inhibitor is located in the seed bark and cotyledons, and is either blocked or absent in the embryonic axes, thus allowing the isolation of active gherkin ribosomes. The presence of the putative inhibitor appeared to be unevenly distributed in developing plants.

Isolation and characterization of a new d-galactose-binding lectin from Sambucus racemosa L.

A new acidic lectin from red elder (Sambucus racemosa L.) bark has been isolated by affinity chromatography and gel filtration. Noteworthy, and in contrast to other Sambucus species, red elder bark lacks acidic non-toxic type 2 ribosome-inactivating proteins but has basic ribosome-inactivating protein activities. The new lectin (SRLbm) shows specificity for N-Ac-Galactosamine/D-Galactose and has an apparent Mr of 30,000. The N-terminal amino acid sequence displays a close homology with other lectins and B chains of non-toxic type 2 ribosome-inactivating proteins nigrins and ebulins present in other Sambucus species. SRLbm triggers red blood cell agglutination in the range 4-12 micro g/ml.