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.

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.

Sialic acid-binding dwarf elder four-chain lectin displays nucleic acid N-glycosidase activity.

Sialic acid-binding dwarf elder agglutinin (SEA) present only in rhizomes of the medicinal plant Sambucus ebulus L., was found to be a tetrameric glycoprotein consisting of two covalently-associated dimers of an enzymic A chain with rRNA N-glycosidase activity (EC 3.2.2.22) linked to a B chain with agglutinin properties. The lectin inhibited protein synthesis by a cell-free system and depurinated ribosomes. Cloning of the corresponding gene and molecular modeling of the deduced amino acid sequence demonstrated that SEA has a three-dimensional structure which resembles that reported for other two tetrameric type 2 RIPs from Sambucus (SNAI and SSA). The lectin agglutinated red blood cells and displayed sugar affinity for sialic acid residues apart from d-galactose, binding to the mucin-producing gut goblet cells. Since sialic acid is present in animal cells, especially in epithelial lining gut cells, but not in plants, SEA could play a role in the defense against insect attack. The nucleotide sequence reported in this paper has been submitted to the GenBank nucleotide database under accession number AM981401.

Tumor de Abrikossoff: revisión de la literatura y presentación de tres casos / Abrikossoff’s tumor: literature review and report of three cases

El tumor de células granulares (TCG) es una lesión benigna einfrecuente, que se suele presentar como una masa asintomática de menosde dos centímetros de diámetro. Aunque su origen todavía permanece desconocido,se sospecha que es neural (célula de Schwann), debido a estudiosinmunohistoquímicos. Puede aparecer en cualquier parte del cuerpo,pero la región de la cabeza y el cuello es la más habitual (50%). En la cabezay cuello, la lengua es la localización más común del TCG, seguida porla piel, la laringe, y los sistemas respiratorio y digestivo.En el estudio histológico, es característica la presencia de hiperplasia pseudo-epiteliomatosa, y el epitelio superficial está preservado normalmente.El análisis inmunohistoquímico revela reacción positiva a la proteína S-100,enolasa neuronal y proteínas mielínicas como P0 y P2.Presentamos tres casos de TCG y un resumen breve de la literatura existente.El primer caso fue diagnosticado en una mujer de 36 años de edad,el segundo en un hombre de 45 años, y el tercero en otro hombre de 55años. Las lesiones estaban localizadas en el borde lateral, la superficie ventraly la superficie dorsal de la lengua. El tratamiento fue quirúrgico enlos tres casos y no hay evidencia de recurrencia del tumor después de unperíodo de seguimiento que varía desde los 8 meses a los 2 años(AU)

Granular cell tumor (GCT) is an uncommon bening lession,pressentig as an asympmtomatic mass, less than two centimetersin diameter. The origin is still unknown, althougt it’s suspected tobe neural (Schwann cell), because of immunohistochemical studies.It can appear in any part of the body, but the head and neck regionis the most common (50%). In the head and neck, the tongue is themost usual location for GCT, followed by the skin, larynx, respiratoryand digestive system. In the histological study is caracteristic thepresence of pseudo-epitheliomatous hyperplasia, and the overlyingephitelium is usually preserved. Immunohistochemical analisis revealspositive reaction to S-100 protein, neuronal enolase, mielinic proteinas P0 and P2. We present three cases of GCT of the tongue and abrief review of the literature. The first case was found in a 36 yearold woman, the second in a 45 year old man and the third one in a55 year old man. The lesions were placed in the lateral border, theventral surface and the dorsal surface of the tongue. The treatmentwas surgycal, and there was no evidence of recurrence after a periodfrom eight months to two years of follow up(AU)

Specific dose-dependent damage of Lieberkühn crypts promoted by large doses of type 2 ribosome-inactivating protein nigrin b intravenous injection to mice.

Nigrin b is a non-toxic type 2 ribosome-inactivating protein as active as ricin at ribosomal level but 10(5) and 5 x 10(3) times less toxic for animal cell cultures and mice, respectively, than ricin. The purpose of the present study was to analyze the effects of intravenous injection of large amounts of nigrin b to the mouse. Injection through the tail vein of 16 mg/kg body weight killed all mice studied before 2 days. Analysis of several major tissues by light microscopy did not reveal gross nigrin b-promoted changes, except in the intestines which appeared highly damaged. As a consequence of the injury, the villi and crypt structures of the small intestine disappeared, leading to profuse bleeding and death. In contrast, intravenous injection of 5 mg/kg body weight was not lethal to mice but did trigger reversible toxic effects. In both cases, lethal and sub-lethal doses, the target of nigrin b appeared to be the highly proliferating stem cells of the intestinal crypts, which had undergone apoptotic changes. In contrast to nigrin b, the injection of 3 mug/kg of ricin kills all mice in 5 days but does not trigger apoptosis in the crypts. Therefore, the effect seen with sub-lethal nigrin b concentrations seems to be specific. Nigrin b killed COLO 320 human colon adenocarcinoma cells with an IC(50) of 3.1 x 10(-8) M and the effect was parallel to the extent of DNA fragmentation of these cells. Accordingly, despite the low general toxicity exerted by nigrin b as compared with ricin, intravenous injection of large amounts of nigrin b is able to kill mouse intestinal stem cells without threatening the lives of the animals, thereby opening a door for its use for the targeting of intestinal stem cells.

Molecular characterization and systemic induction of single-chain ribosome-inactivating proteins (RIPs) in sugar beet (Beta vulgaris) leaves.

Sugar beet (Beta vulgaris L.) leaves contain virus-inducible type 1 (single chain) ribosome-inactivating proteins that have been named beetins. The structural and functional characterization, the cellular location, and the potential role of beetins as antiviral agents are reported here. Beetins are formed of a single polypeptide chain with a varying degree of glycosylation and strongly inhibited in vitro protein synthesis in rabbit reticulocyte lysates (IC50=1.15 ng ml(-1)) and a Vicia sativa L. cell-free system (IC50=68 ng ml(-1)) through the single depurination of the large rRNA. Beetins trigger the multidepurination of tobacco mosaic virus (TMV) genomic RNA which underwent extensive degradation upon treatment with acid aniline. Beetins are extracellular proteins that were recovered from the apoplastic fluid. Induction of sugar beet RIPs with either H2O2 or artichoke mottled crinkle virus (AMCV) was observed in leaves distant from the site of application of such elicitors. The external application of purified beetin to sugar leaves prevented infection by AMCV which supports the preliminary hypothesis that beetins could be involved in plant systemic acquired resistance subjected to induction by phytopathogens.

[Refeeding syndrome. A review]. / Síndrome de realimentación. Revisión.

Refeeding syndrome is a complex clinical picture that encompass all those alterations that can occur as a consequence of the nutritional support (oral, enteral or parenteral) in malnourished patients. Refeeding syndrome is classically characterized by neurological alterations, respiratory symptoms, cardiac arrhythmias and heart failure few days after beginning of refeeding, with life-threatening outcome. Its pathogenesis includes alterations in the corporal fluids, and in some electrolytes, minerals and vitamins. In this article a review of refeeding syndrome pathogenesis and clinical manifestations is carried out, with a final series of recommendations for lowering the risk of this syndrome and for facilitate the early diagnosis and the treatment.

Síndrome de realimentación. Revisión / Refeeding syndrome. A review

El síndrome de realimentación es un cuadro clínico complejo que engloba todas aquellas alteraciones que pueden ocurrir como consecuencia del soporte nutricional (oral, enteral o parenteral) en pacientes malnutridos. Clásicamente se caracteriza por la aparición de alteraciones neurológicas, r espiratorias, arritmias e insuficiencia cardíaca que ocurren pocos días después del inicio de la realimentación y que pueden tener un desenlace fatal. En su patogenia intervienen alteraciones en los fluidos corporales, en algunos electrolitos, minerales y vitaminas. En este artículo se hace una revisión de su patogenia y características clínicas, haciendo finalmente una serie de recomendaciones encaminadas a disminuir el riesgo de aparición, facilitar el diagnóstico precoz y el tratamiento

Refeeding syndrome is a complex clinical picture that encompass all those alterations that can occuras a consequence of the nutritional support (oral, enteral or parenteral) in malnourished patients. Refeeding syndrome is classically characterized by neurological alterations, respiratory symptoms, cardiac arrhythmias and heart failure few days after beginning of refeeding, with life-threatening outcome. Its pathogenesis includes alterations in the corporal fluids, and in some electrolytes, minerals and vitamins. In this article a review of refeeding syndrome pathogenesis and clinical manifestations is carried out, with a final series of recommendations for lowering the risk of this syndrome and for facilitate the early diagnosis and the treatment

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.

Non-toxic type 2 ribosome-inactivating proteins (RIPs) from Sambucus: occurrence, cellular and molecular activities and potential uses.

Ribosome-inactivating proteins (RIPs) are a family of enzymes that trigger the catalytic inactivation of ribosomes. The most known member of the family is the highly poisonous two-chain ricin isolated from Ricinus communis L. Sambucus species contain a number of two-chain RIPs structurally and enzymatically related to ricin which have the noteworthy feature that, having an enzymatic activity on ribosomes, leading to the inhibition of protein synthesis, higher than ricin, they are lacking of the tremendous unspecific toxicity of ricin. Therefore, they have been called non-toxic type 2 RIPs. The most representative and studied members are nigrin b present in the bark of the common (black) elder Sambucus nigra L. and ebulin 1 present in the leaves of the dwarf elder Sambucus ebulus L. The molecular basis for the low unspecific activities of nigrin b and ebulin 1 as compared with ricin seems to be related with single changes of amino acids in the high affinity sugar binding sites of the B chains. These changes determine the intracellular traffic of these proteins and thus the cellular toxicity. Conjugation ofnigrin b or ebulin 1 to either transferrin or monoclonal antibodies provided highly active conjugates targeting cancer. Thus these non-toxic type 2 RIPs are promising tools for cancer therapy.

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.

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).

Evidence for distinct cellular internalization pathways of ricin and nigrin b.

Nigrin b and ricin are type 2 (two chain) ribosome-inactivating proteins that exhibited nearly the same strong inhibitory activity on cell-free protein synthesis. Incubation of HeLa cells for 6 hr with ricin at 37 degrees C promoted protein synthesis inhibition with an IC50 of 0.2 ng/ml. Incubation of the cells for 6 hr at 18 degrees C abolished completely the inhibition. Incubation of HeLa cells with nigrin b for 6 hr at 37 degrees C was nearly 10(5) times less inhibitory than ricin. In contrast to the effects observed with ricin, incubation of HeLa cells with nigrin b at 18 degrees C slightly increased the inhibitory action on protein synthesis as compared with incubation at 37 degrees C. These results strongly support the hypothesis that the internalization of ricin and nigrin b could involve different receptors and therefore they could follow different intracellular pathways.

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.

Concordancia entre la exploración clínica y la imagen artroscópica en el diagnóstico de los desórdenes internos de la articulación temporomandibular / Concordance between clinical examination and arthroscopy image for the diagnosis of the temporomandibular joint internal derangements

Objetivos: 1. Determinar el grado de concordancia entre el diagnóstico clínico y el artroscópico en el diagnóstico de la disfunción temporomandibular (DTM). 2 Determinar la sensibilidad y especifidad de la exploración clínica en el diagnóstico de los desordenes internos de la articulación temporomandibular (ATM) Diseño. Se ha realizado un estudio experimental no randomizado, tipo ensayo clínico de efidencia a simple ciego. La muestra estaba constituida por 3O enfermos diagnosticados de DTM y sometidos a una artroscopia. Los diagnósticos clínicos y artroscópicos se emitieron según criterios internacionalmente consensuados. La concordancia entre los diagnósticos se evaluó mediante el índice Kappa de Cohen. La relación entre las variables investigadas (cualitativas) se analizó mediante la prueba de la X. Se consideraron diferencias significativas aquellas en las que el valor de p < 0,05. Resultados. La edad media de los enfermos fue de 34,7 años. El 86.1 por ciento de los pacientes eran mujeres. El diagnóstico clínico y artroscópico de desplazamiento discal con reducción, presento una concordancia importante (Kappa = 0,645), mostrando el diagnostico clínico una sensibilidad de 0,71 y una especificidad de 0,93. La concordancia observada entre el diagnóstico clínico de desplazamiento discal sin reducción y, el atroscópico fue casi completa (Kappa - 0,833) con una sensibilidad de 0,83 y una especificidad de 1. Conclusiones. En pacientes con DTM subsidiarios de tratamiento artroscópico el diagnóstico clinico es concordante con el artroscópico en la filiación de los desplazamientos discales (con o sin reduccion). La presencia de chasquidos y el tipo de trayecto de abertura oral son los dos mejores parametros clínicos para el disnóstico (AU)

Maxillary undifferentiated carcinoma with rhabdoid features.

The rhabdoid malignant tumor was individualized by Haas et al. as an independent entity within the kidney malignant neoplasms in 1981. From its histopathological characteristics, different cases of rhabdoid extra-renal tumor were documented. This aspect has been also recognized in a wide heterogeneous group of tumors (carcinoma, melanoma, mesothelioma and mesenchymal tumors). The diagnosis of extrarenal rhabdoid tumor is based on the presence of a proliferation of epithelioid cells with large nuclei, prominent nucleoli, abundant eosinophilic cytoplasm, and conspicuous cytoplasmic intermediate filaments, which compress the nuclei. In this article we present the case of a 50-year-old male patient with a malignant maxillary tumor with characteristic rhabdoid features. Its differential diagnosis will be discussed. The literature will also be reviewed.

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.

2.8-A crystal structure of a nontoxic type-II ribosome-inactivating protein, ebulin l.

Ebulin l is a type-II ribosome-inactivating protein (RIP) isolated from the leaves of Sambucus ebulus L. As with other type-II RIP, ebulin is a disulfide-linked heterodimer composed of a toxic A chain and a galactoside-specific lectin B chain. A normal level of ribosome-inactivating N-glycosidase activity, characteristic of the A chain of type-II RIP, has been demonstrated for ebulin l. However, ebulin is considered a nontoxic type-II RIP due to a reduced cytotoxicity on whole cells and animals as compared with other toxic type-II RIP like ricin. The molecular cloning, amino acid sequence, and the crystal structure of ebulin l are presented and compared with ricin. Ebulin l is shown to bind an A-chain substrate analogue, pteroic acid, in the same manner as ricin. The galactoside-binding ability of ebulin l is demonstrated crystallographically with a complex of the B chain with galactose and with lactose. The negligible cytotoxicity of ebulin l is apparently due to a reduced affinity for galactosides. An altered mode of galactoside binding in the 2gamma subdomain of the lectin B chain primarily causes the reduced affinity.