Fruticulosin: A novel type 2 ribosome-inactivating protein from Abrus fruticulosus seeds that exhibits toxic and antileishmanial activity.

Plant ribosome-inactivating proteins (RIPs) are a family of toxins that inhibit protein synthesis. In this study, we have isolated a novel type 2 ribosome-inactivating protein (RIP) present in seeds of the Abrus fruticulosus, named of fruticulosin. Fruticulosin, shows characteristics common to other type 2 RIPs, as specificity by galactosides (d-galactose, N-acetyl-d-galactosamine, and d-lactose), mass of approximately 60 kDa and presence of the of disulfide bonds. The N-terminal amino acid sequence (26 residues) of A-chain fruticulosin, determined by Edman degradation, revealed high similarity of the A-chain with those of other type 2 RIPs. The secondary structure of fruticulosin was analysed by circular dichroism, which showed that fruticulosin contains α-helices (22.3%), ß-sheets (43.5%), and random coils and corners (34.2%). Furthermore, fruticulosin showed high toxicity in Artemia sp. (3.12 µg/mL), inhibited in vitro protein synthesis by a cell-free system and showed RNA N-glycosidase activity. Fruticulosin presented biological activities such as agglutination and antileishmanial activity on promastigote forms of Leishmania major.

Study of Subchronic Toxicity of Relatox on Sexually Immature Animals.

Intramuscular injections of Relatox in therapeutic and toxic doses to young outbred laboratory rats for 14 days caused no changes in the peripheral blood and bone marrow parameters, serum biochemical parameters, and morphology of the major viscera. In the toxic dose, the drug caused local irritation (inflammation, atrophy, and sclerosis in muscle tissue). Regeneration processes started in muscle tissue 7 days after Relatox withdrawal.

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of ß-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein.

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

Botulinum hemagglutinin-mediated selective removal of cells deviating from the undifferentiated state in hiPSC colonies.

The undifferentiated state of human induced pluripotent stem cells (hiPSCs) depends on their cell-cell and cell-substrate adhesions. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, selectively removed cells that deviated from the undifferentiated state in hiPSC colonies. After HA treatment, cell-cell adhesion was disrupted, deviated cells detached from colony centers, and dividing cells filled these spaces. Because E-cadherin-mediated adhesion was disrupted in undifferentiated cells, stress-fiber formation and focal adhesions were diminished; however, these were subsequently restored, and the cells retained expression of undifferentiated stem cell markers and their differentiation potential. In contrast, actin structures and focal adhesions were lost from deviated cells, and they subsequently died. In undifferentiated and deviated cells, the cadherin/integrin-regulator Rap1 was localized at cell-cell adhesions and in the cytoplasm, respectively. Concurrent HA and Rap1-inhibitor treatment accelerated the deviated-cell detachment and delayed the recovery of hiPSC morphology, but this effect was significantly attenuated by co-treatment with Rap1 activator. Thus, Rap1 regulated E-cadherin-integrin interplay in hiPSC colonies exhibiting deviation, while HA-mediated selective removal of these deviated cells helped maintain the undifferentiated state in the remaining hiPSCs.

Purification and partial characterization of an erythroagglutinin from the hemolymph of scorpion, Heterometrus bengalensis.

An erythroagglutinin from the hemolymph of the scorpion, Heterometrus bengalensis, has been purified by gel filtration and ion-exchange chromatography. Its homogeneity has been demonstrated by polyacrylamide gel electrophoresis. The purified agglutinin appears to be a monomeric protein having a possible molecular weight between 146,000 and 148,000. It has no divalent cation requirement for erythroagglutination. The erythroagglutination is not inhibited by saccharides, glycoproteins and mucin. Identical erythroagglutination pattern is obtained with normal as well as neuraminidase treated erythrocytes.

Haemagglutination activity of toxigenic and non-toxigenic strains of Clostridium difficile.

Cell extract of Clostridium difficile strains was fractionated by ammonium sulfate precipitation and sulfated cellulofine column chromatography to detect haemagglutination (HA) activity. HA activity without cytotoxicity was detected in fractions eluted at 0.79-0.91 M NaCl in sulfated cellulofine column chromatography of the cell extract in both toxigenic strain VPI 10463 and non-toxigenic strain KZ 1678, while toxin A was detected in fractions eluted at 0.27-0.29 M NaCl. Antisera were prepared with HA substance-containing fractions of the chromatography. Antiserum to the HA substance(s) of strain VPI 10463 neutralised the HA activity of the fractions of strains VPI 10463 and KZ 1678 at nearly the same titres. Antiserum to the HA substance(s) of strain KZ 1678 also neutralised the HA activity of both strains at nearly the same titres as above. These findings suggest that haemagglutinin(s) is commonly produced by C. difficile strains irrespective of toxin A-producing ability.

Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper).

A galactose-binding lectin, isolated from the venom of B. godmani by affinity chromatography, is an acidic protein (pI 4.9) with a subunit mol. wt of about 14,000, occurring mostly as a disulfide-linked dimer of 28,000. A small proportion of lectin appears as a monomer and as a tetramer. The lectin agglutinates erythrocytes from mice, rabbit, cow and human (all ABO types, either Rh positive or negative), but does not agglutinate horse, sheep, goat and snake (Oxybelis aeneus, Colubridae) erythrocytes. The agglutinating activity is inhibited by 1 mM EDTA. The lectin is devoid of lethal, hemorrhagic, myotoxic, proteolytic and phospholipase A2 activities. It is not mitogenic for human peripheral blood mononuclear cells. The only effect observed was a moderate induction of edema in the footpad of mice, with a minimal edema-forming dose of 22 micrograms. This effect developed rapidly, and was significantly inhibited by i.p. administration of cyproheptadine, a histamine and serotonin antagonist, before injection of the lectin. Despite the edema-forming activity observed, the low concentration of lectin in crude venom, together with its relatively low potency, suggest that this lectin is not a key component in the development of edema following envenomations by B. godmani.

Effect of hemagglutinin extracts on acetylcholinesterase activity in some brain areas of weanling rats.

Two trials involving male and female weanling albino rats (Wistar strain) were conducted to investigate the effects of cowpea and limabean hemagglutinin extracts on acetylcholinesterase (AChE) activity in different parts of the brain. The results show that AChE activity varies with brain region. Cowpea hemagglutinin significantly inhibited AChE activity in the pons of male and female rats, while limabean hemagglutinin significantly inhibited AChE activities in the pons of both male and female rats, in the hippocampus of female rats and in the medulla, cerebellum and midbrain of male rats. Inhibition of AChE activities was more pronounced in male or female rats inoculated with a mean lethal dose of either phytohemagglutinin, than in those inoculated with a sublethal dose.

[Effect of various heat treatments in the hemagglutinin content and in the protein quality of beans (Phaseolus vulgaris)]. / Efecto de diversos tratamientos térmicos en el contenido de hemaglutininas y en la calidad protéica del frijol (Phaseolus vulgaris).

The effect of pre-soaking raw seed beans upon detoxification and the biological quality of its protein were evaluated. In whole raw seed beans (Phaseolus vulgaris) var. "tórtola", the net protein utilization (NPU), true digestibility and hemagglutinin titer were determined after 60', 90' and 120' of heat treatment, with and without 14 hours of pre-soaking. It is concluded that soaking prior to cooking is not necessary to eliminate the toxicity of dry beans, but that it does contribute to the softening of seeds and reduction of cooking time. The hemagglutinin levels of six commercial bean flours were evaluated, concluding that almost all of them presented toxic levels. The effect of the cooking methods upon the toxicity of bean flours was studied. Two raw bean flours, var. "tórtola" and "burro" at 10% and 20%, were cooked employing different boiling times (5, 10, 15 and 30'). The two raw samples contained high hemagglutinin levels which were inactivated at 10% with 10' cooking. The presence of toxic levels was detected at 20% after 15' cooking and these were eliminated at 30' of cooking.

Characterization of a lectin-like protein isolated from Lachesis muta snake venom.

A lectin-like protein was isolated from L. muta venom by gel filtration on BIO Gel P-100 followed by column Chromatography on DEAE-sephades A-50. The protein eluted at 0.4 M Nacl in 0.01 Tris pH 7.3 and exhibited agglutinin activity toward 0+ human erythrocytes. The protein is a dimer with Mr 28 kDa. Amino acid analysis revealed high content of tryptophan and acid recidues and low content of cysteine and methionine residues. No neutral carbohydrates and sialic acid were detected. Circular dichroic spectrum shows 78% of B structure and 1% of alpha structure. In vitro experiments with erythrocytes from rat, rabbit and dog revealed strong agglutination while red blood cells from mice, sheep and goat were not agglutinated. In vivo experiments using anesthetized rats, a sharp and prolonged fall in the blood pressure was observed at protein dose of 1.5 mg/kg. Double dose of protein caused the death of the animal.

Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade.

Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.

The effects of purified components of Bordetella pertussis in the weight gain test for the toxicity testing of pertussis vaccines.

The effects of highly purified preparations of three Bordetella pertussis components--pertussis toxin (PT), lipopolysaccharide (LPS) and filamentous haemagglutinin (FHA)--were examined in the mouse weight gain test, a toxicity test for pertussis vaccine. When these components were administered alone, PT enhanced initial weight gains of the mice, LPS produced an initial weight loss and FHA had no detectable effect on the weights of the mice. However, testing the components in combinations revealed that the effect of PT and LPS together was not simply the sum of their individual effects. This combination generally produced lower weights than LPS alone, particularly in the later stages of the test.

Nucleotide sequence encoding the mannose-fucose-resistant hemagglutinin of Vibrio cholerae O1 and construction of a mutant.

The region of DNA encoding the mannose-fucose-resistant hemagglutinin (MFRHA) of Vibrio cholerae O1 has been localized, and the nucleotide sequence has been determined. The region contains a single open reading frame encoding 230 amino acids, corresponding to a protein of 26.9 kDa. The N terminus of this protein is atypical for a protein localized in the outer membrane. A mutant lacking MFRHA activity has been constructed by allelic exchange after inactivation via the insertion of a kanamycin resistance gene cartridge. The MFRHA-negative mutant has been assessed for virulence in the infant mouse cholera model. This mutant shows a marked defect in its ability to persist in the infant mouse gut and is incapable of competing with the wild-type organism, even when given in 25-fold excess. This defect also leads to a > 100-fold increase in the 50% lethal dose. These data suggest that the MFRHA is an important colonization factor in the infant mouse model.

The effects of purified pertussis components and lipopolysaccharide on the results of the mouse weight gain test.

The effects of highly purified components of Bordetella pertussis, that is pertussis toxin (PT) and filamentous haemagglutinin (FHA), and of lipopolysaccharide (LPS) were studied in the active mouse weight gain test (MWGT). The PT when given alone or with other components in various combinations caused weight losses and deaths 2-3 days after inoculation but FHA was not toxic in the MWGT. When FHA was given with PT, the toxic effect of PT was reduced. The LPS caused weight losses at 24 h which decreased when LPS was given with PT. The toxic effects of PT as indicated by late deaths and late weight losses or failure to gain weight continued until 14 days after inoculation. The various components had similar effects on mouse weight gain in both LACA and NIH strains of mice. The doses of PT used in the MWGT caused marked leucocytosis but FHA and LPS did not. No agglutinins appeared in the sera of mice inoculated with various purified components. The components were thus pure and did not contain agglutinogens.

Proinflammatory and proapoptotic activities associated with Bordetella pertussis filamentous hemagglutinin.

Filamentous hemagglutinin (FHA) is a dominant cell surface-associated Bordetella pertussis adhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other activities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-type B. pertussis resulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-alpha), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-alpha monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-alpha receptor 1 inhibited FHA-associated apoptosis by 47.7% (P = 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in the B. pertussis-host interaction.

Comparison of the Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase.

The soluble hemagglutinin/protease (HA/protease) produced by Vibrio cholerae and the elastase of Pseudomonas aeruginosa are both zinc/calcium-dependent proteases. In the present study the two enzymes are compared immunologically and functionally. The N-terminal amino acid sequences of the proteins had 65% identity within the first 20 amino acids. Polyclonal antisera against each purified protein recognized the enzyme of the other species in enzyme-linked immunosorbent assay, checkerboard immunoblot, and Western blot analyses and inhibited the protease activity of both enzymes in milk and elastin agars. Like the HA/protease, the elastase hemagglutinated "responder" but not "nonresponder" chicken erythrocytes, degraded ovomucin, lactoferrin, and fibronectin, and nicked the A subunit of the cholera toxin-related heat-labile enterotoxin from Escherichia coli. Whereas none of the three proteases tested (elastase, HA/protease, or pronase E) had any obvious effect in ileal loop tests in rabbits at doses up to 50 micrograms, all three produced some detectable skin reactions at a dose of 0.1 micrograms and necrosis at a higher dose (i.e., 5 micrograms). We conclude that the V. cholerae HA/protease and the P. aeruginosa elastase are structurally, functionally, and immunologically related.

Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum.

Botulism food poisoning is caused primarily by ingestion of the Clostridium botulinum neurotoxin (BoNT). The 1300 amino acid BoNT forms a progenitor toxin (PTX) that, when associated with a number of other proteins, increases its oral toxicity by protecting it from the low pH of the stomach and from intestinal proteases. One of these associated proteins, HA1, has also been suggested to be involved with internalization of the toxin into the bloodstream by binding to oligosaccharides lining the intestine. Here is reported the crystal structure of HA1 from type C Clostridium botulinum at a resolution of 1.7 Angstrom. The protein consists of two beta-trefoil domains and bears structural similarities to the lectin B-chain from the deadly plant toxin ricin. Based on structural comparison to the ricin B-chain lactose-binding sites, residues of type A HA1 were selected and mutated. The D263A and N285A mutants lost the ability to bind carbohydrates containing galactose moieties, implicating these residues in carbohydrate binding.

Molecular composition of Clostridium botulinum type A progenitor toxins.

The molecular composition of progenitor toxins produced by a Clostridium botulinum type A strain (A-NIH) was analyzed. The strain produced three types of progenitor toxins (19 S, 16 S, and 12 S) as reported previously. Purified 19 S and 16 S toxins demonstrated the same banding profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that they consist of the same protein components. The nontoxic components of the 19 S and 16 S toxins are a nontoxic non-hemagglutinin (HA) (molecular mass, 120 kDa) and HA. HA could be fractionated into five subcomponents with molecular masses of 52, 35, 20, 19, and 15 kDa in the presence of 2-mercaptoethanol. The molar ratios of neurotoxins, nontoxic non-HAs, and each HA subcomponent of the 19 S and 16 S toxins showed that only HA-35 of the 19 S toxin was approximately twice the size of that of the 16 S toxin, suggesting that the 19 S toxin is a dimer of the 16 S toxin cross-linked by the 35-kDa subcomponent. The nontoxic non-HA of the 12 S toxin, but not those of the 19 S and 16 S toxins, demonstrated two bands with molecular masses of 106 and 13 kDa on SDS-PAGE with or without 2-mercaptoethanol. It was concluded from the N-terminal amino acid sequences that 106- and 13-kDa proteins were generated by a cleavage of whole nontoxic non-HA. This may explain why the 12 S and 16 S (and 19 S) toxins exist in the same culture. We also found that the HA and its 35-kDa subcomponent exist in a free state in the culture fluid along with three types of progenitor toxins.

The interactive effects of lima bean (Phaseolus lunatus) trypsin inhibitor, hemagglutinin and cyanide on some hepatic dehydrogenases, ornithine carbamoyltransferase and intestinal disaccharidases in weanling rats.

The interactive effects of lima bean trypsin inhibitor (TI), hemagglutinin (Hgg) and cyanide (CN) when fed at the same degree of activity as found in the raw lima bean (RLB) were assessed in weanling rats using hepatic glutamate dehydrogenase (GLDH), isocitrate dehydrogenase (ICDH), ornithine carbamoyltransferase (OCT) and intestinal disaccharidases activities as the response criteria. Whereas RLB significantly (P less than 0.05) increased hepatic GLDH and decreased ICDH activities respectively, dietary CN, TI and Hgg whether acting individually or jointly had no significant influence on GLDH. Only the CN-containing diets significantly (P less than 0.05) elevated ICDH activity when compared with the control. Raw lima bean significantly (P less than 0.05) depressed OCT activity while neither the individual nor collective effects of these factors were significant. Dietary CN + TI + Hgg interaction depressed maltase activity to approximately the same extent as RLB in all the intestinal regions. These factors had neither individual nor collective effects on sucrase in the small intestine. Lactase activity in the small intestine was influenced only by the RLB diet, while CN + Hgg, and CN + TI + Hgg dietary combinations induced significant (P less than 0.05) elevations in the activities of cellobiase when compared with the control. Although synergism of action is indicated in a number of instances, it is suggested that these factors may need to combine with others within the bean, perhaps synergistically, to elicit comparable anti-nutritional influences as the RLB.