Further screening of Aspergillus species for occurrence of lectins and their partial characterization

Fifteen species of Aspergillus were screened for occurrence of lectins. Nine of them (A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus, A. pulvinus and A. aculeatus) were found to possess lectin activity. None of the species elaborated lectin in culture supernatant. All the lectins agglutinated rat, pig and rabbit erythrocytes. A. sydowii, A. candidus, A. allahabadi, A. terricola, A. ficuum, A. sparsus, A. carneus and A. aculeatus lectins agglutinated all human type erythrocytes equally, while A. pulvinus lectin specifically agglutinated human type A and O erythrocytes. Neuraminidase and protease treatment to erythrocytes substantially augmented lectin titres manyfold. Lectins showed specificity to mucin and asialofetuin and all of them were specific to L‐arabinose except that of A. carneus. Lectins from A. sydowii, A. ficuum, A. sparsus and A. carneus displayed remarkable specificities to D‐xylose. Maximum lectin activity was expressed by 11 day old cultures of A. sydowii (titre 32), A. ficuum (titre 64) and A. sparsus (titre 1024). Lectins from A. aculeatus, A. candidus and A. terricola were expressed by 7–10 days, 6–9 days and 5–11 days old cultures, respectively. A. allahabadi cultures exhibited maximum lectin activity (titre 32) after 8–10 days of cultivation. A. carneus and A. pulvinus expressed optimal titres of 32 and 8, respectively on the 9^th day. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Purification of a potent mitogenic homodimeric Penicillium griseoroseum lectin and its characterisation

Penicillium griseoroseum lectin was 80‐fold purified by successive DEAE Sepharose anion exchange and Sephadex G‐100 gel permeation chromatography. P. griseoroseum lectin exhibited haemagglutination activity towards protease‐treated rabbit erythrocytes. It showed specificity towards various carbohydrates such as d ‐mannose, N‐acetyl‐d ‐glucosamine, mucins, and so forth. P. griseoroseum lectin was found as a glycoprotein with glycan content of 4.33%. Purified P. griseoroseum lectin is homodimeric having a molecular mass of 57 kDa with subunit molecular mass of 28.6 kDa. Haemagglutination activity of purified P. griseoroseum lectin was completely stable from 25°C to 35°C at a pH range of 6–7.5. Lectin activity was not influenced by divalent metal ions and denaturants. P. griseoroseum lectin manifested mitogenicity towards mice splenocytes and activity reached a peak at 75 μg/ml of lectin concentration. P. griseoroseum lectin in microgram concentrations stimulated proliferation of mice splenocytes. Thus, P. griseoroseum lectin exhibits potential mitogenicity, which can be exploited for further biomedical applications.     

Cold-active catalase from the psychrotolerant fungus Penicillium griseofulvum

Cold-active catalase (CAT) elicits great interest because of its vast prospective at the medical, commercial, and biotechnological levels. The study paper reports the production of cold-active CAT by the strain Penicillium griseofulvum P29 isolated from Antarctic soil. Improved enzyme production was achieved by optimization of medium and culture conditions. Maximum CAT was demonstrated under low glucose content (2%), 10% inoculum size, temperature 20°C, and dissolved oxygen concentration (DO) 40%. An effective laboratory technology based on changing the oxidative stress level through an increase of DO in the bioreactor was developed. The used strategy resulted in a 1.7- and 1.4-fold enhanced total enzyme activity and maximum enzyme productivity. The enzyme was purified and characterized. P. griseofulvum P29 CAT was most active at approximately 20°C and pH 6.0. Its thermostability was in the range between 5°C and 40°C.     

ANTI-B ACTIVITY OF A LECTIN COEXISTENT WITH BLOOD GROUP H-LIKE SUBSTANCE IN SEEDS OF EUONYMUS SIEBOLDIANA

Euonymus Sieboldiana seeds possess a lectin for human erythrocytes with anti-B specificity. Lectin fractions with strong agglutinating activity were separated by ammonium sulphate precipitation from blood group H-like substance, which is coexistent in the same seeds. The lectin has properties resembling a complete agglutinin, being non-dialysable, inactivated by heat treatment at 70°C and specific for D-galactoside.     

Specificity of lectin activity of Bacillus thuringiensis parasporal inclusion proteins

Sheep erythrocyte-agglutinating parasporal inclusion proteins from four Bacillus thuringiensis strains (FITC-20, FITC-73, FITC-76 and IBC-1456) were examined for lectin activity against erythrocytes from four mammalian (rabbit, horse, cow and guinea pig) and an avian (chicken) species. Of the five erythrocyte species, only rabbit cells were agglutinated with the protein of the human cancer cell-killing strain IBC-1456. No haemagglutination (HA) activities were shown in other protein-erythrocyte combinations. The lectin activity of the strain IBC-1456 against rabbit cells was strongly inhibited by preincubation with D-galactose. Overall results revealed that the B. thuringiensis lectins have a preference for sheep erythrocytes.     

Effect of Lectins on Mouse Peritoneal Macrophage Phagocytic Activity

We studied the in vitro ability of lectin-treated murine peritoneal macrophages to attach and phagocytize particulate antigens. Glucose and mannose specific lectins such as Con-A and lentil lectin, as well as complex lactosamine residues specific lectins, such as Phaseolus vulgaris var. cacahuate and Phaseolus coccineus var. alubia, increased the macrophage phagocytic activity towards heterologous erythrocytes, whereas peanut agglutinin, a galactose-specific lectin, diminished the macrophage phagocytic activity. These results suggest that a galactose-N-acetyl-D galactosamine-containing structure could participate as negative modulator of the phagocytic activity.     

Screening of Aspergillus species for occurrence of lectins and their characterization

Ten species of Aspergillus were screened for occurrence of lectins. Each of the species was investigated for the occurrence of extracellular, surface-bound and intracellular lectin activities. As many as four species namely, Aspergillus niger, Aspergillus versicolor, Aspergillus rugulosus and Aspergillus nidulans, were found to possess intracellular lectin activities, while none of the species showed extracellular or surface-bound lectin activities. Each of the lectin was characterized with respect to blood group and carbohydrate specificities. All the lectins were found to agglutinate human erythrocytes, irrespective of their blood group and pig erythrocytes. However, they did not show agglutination with sheep or goat erythrocytes. Of the various carbohydrates tested, all lectins were found to be specific for inulin, mucin, asialofetuin, N-acetyl galactosamine, melibiose, D-ribose, L-fucose, D-arabinose, D-sucrose and D-mannitol. The minimum inhibitory concentration of each of the specific sugars was also determined. The lectins were partially purified using ammonium sulfate precipitation technique. Each of the lectin was found to be precipitated at 40-50% saturation of ammonium sulfate, yielding about 80% of lectin activity.     

Identification of N‐Acetylgalactosamine in Carbohydrates of Xenopus laevis Testis

Identification of glycans in amphibian testis has shown the existence of N‐acetylgalactosamine (GalNAc)‐containing carbohydrates. Labeling of the sperm acrosome with GalNAc‐binding lectins has allowed the identification of GalNAc‐containing glycans in this organelle. Futhermore, this specific labeling of the acrosome has allowed the study of acrosomal biogenesis by lectin histochemistry. However, the testis of Xenopus laevis has never been analyzed by lectin histochemistry to locate GalNAc‐containing glycoconjugates. The aim of this work was to elucidate the expression of GalNAc in glycoconjugates of Xenopus testis using five specific lectins. The results showed that most of the lectins labeled the interstitium with variable intensity. However, labeling of the different spermatogenetic germ cell types showed different labeling patterns. Some lectins produced weak or very weak staining in germ cells, for example, horse gram Dolichos biflorus agglutinin, which labeled most of the germ cell types, and lima bean Phaseolus lunatus agglutinin, which weakly labeled only spermatogonia, but did not stain other germ cells. By contrast, Maclura pomifera lectin (MPL) moderately labeled all germ cell types, except mature sperm. Labeling with other lectins was seen only at later stages, suggesting variations involved in the spermatogenetic development. Thus, snail Helix pomatia agglutinin labeled spermatids, but neither spermatogonia nor spermatocytes, while soybean Glycine max agglutinin (SBA) labeled from preleptotene spermatocytes to later stages. The periphery of the acrosome was labeled with MPL and SBA, but no specific labeling of the acrosomal content was seen with any lectin. Thus, the GalNAc‐binding lectins that have been used as acrosomal markers in some amphibians cannot be used in Xenopus testis, suggesting that acrosomal glycoconjugates in amphibians are species specific. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Reactivity of purified lectins with blood typed canine erythrocytes

Agglutination tests were performed with 40 commercially available lectins and a panel of blood typed canine erythrocytes. Erythrocytes were obtained from 17 dogs (two poodles, two black labradors, five greyhounds and eight beagles). The erythrocytes expressed various combinations of dog erythrocyte antigens (DEA) 1.1, 1.2 or null, 4 and 7. Lectin reactivity with untreated, ficin treated, and neuraminidase-treated cells was determined. No correlation between lectin reactivity and the canine blood group antigens expressed on the red cell test panel was found. Results suggest that some canine erythrocytes may be differentiated from others on the basis of reactivity with Phytolacca americana and Glycine max lectins.     

Lectins as a Tool for Detecting Neural Stem/Progenitor Cells in the Adult Mouse Brain

Glycoconjugates are biopolymers that are broadly distributed in the central nervous system, including the cell surface of neural stem cells or neural precursor cells (NSCs/NPCs). Glycoconjugates can be recognized by carbohydrate‐binding proteins, lectins. Two lectins, Phaseolus vulgaris lectin agglutinin E‐form (PHA‐E4) and wheat germ agglutinin (WGA) have been reported to be useful in isolating NSCs/NPCs by fluorescence‐activated cell sorting (FACS) or immunopanning methods. In this study, we analyzed the lectin‐binding properties of NSCs/NPCs in two neurogenic regions of the adult mouse brain to determine whether PHA‐E4 and WGA exhibit specific binding patterns on sections and whether there are other lectins presenting the binding pattern similar to those of PHA‐E4 and WGA in lectin histochemistry. Among nine types of lectins, peanut agglutinin was localized to the white matter and four lectins bound to cells within the subventricular zone (SVZ) of the lateral ventricle. Lectin histochemistry combined with immunohistochemistry demonstrated that one lectin, Ricinus communis agglutinin, specifically detected type A neuronal precursors and that the remaining three lectins, Agaricus bisporus agglutinin (ABA), PHA‐E4, and WGA, recognized type B NSCs and type C transient amplifying cells in the SVZ. These three lectins also recognized type 1 quiescent neural progenitors and type 2a amplifying neural progenitors in the subgranular layer of the dentate gyrus. Lectin histochemistry of the neurosphere culture also yielded similar results. These observations suggest that, in addition to PHA‐E4 and WGA, ABA lectin may also be applicable in FACS or immunopanning for the isolation of NSCs/NPCs. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Re-evaluation of a case of progressive multifocal leukoencephalopathy previously diagnosed as simian virus 40 (SV40) etiology

A case of progressive multifocal leukoencephalopathy (PML) reported previously to be of simian virus (SV40) etiology was re-evaluated. The supernatant from a 10% homogenate of brain material was inoculated into African green monkey kidney cells and BSC-1 cells which are permissive for SV40. However no cytopathic effect (CPE) developed and no virus was isolated. The brain supernatant agglutinated human group O erythrocytes and contained 5120 units/mL. The Hirt supernatant from the brain contained three DNA bands corresponding to forms I, II and III of circular double-stranded viral DNA. Restriction endonuclease cleavage analysis revealed that this viral DNA was different from SV40 DNA, but similar to JC virus DNA. After cloning of this viral DNA into pBR322 at the BamHI site, DNA homology of this virus and of SV40 was investigated. The cloned DNA hybridized with all four HpaI/EcoRI fragments of SV40 genome at the effective temperature (Tm) of ?50°C. At Tm ?28°C, however, the cloned DNA hybridized with only HpaI/EcoRI fragment B of the SV40 genome. In contrast to this, JC virus DNA hybridized with all five EcoRI/BamHI/HindIII fragments of cloned DNA even at Tm ?28°C. Therefore, the causative agent of this PML case was not SV40 but JC virus.     

Heterostereocomplex Crystallization and Homocrystallization From the Melt in Blends of Substituted and Unsubstituted Poly(lactide)s

Poly(L ‐2‐hydroxybutyrate) [P(L ‐2HB)] and poly(D ‐lactide) (PDLA) in their blends were phase separated to form P(L ‐2HB)‐ and PDLA‐enriched domains, and heterostereocomplex (HTSC) crystallization occurred at the interface between these domains. In the blends, HTSC crystallites were formed at crystallization temperature (T_c) = 80–160 °C, and their exclusive formation without the formation of other crystalline species was observed at T_c of 150 or 160 °C. The effects of polymer blend ratio on the types of formed crystalline species were very small due to the phase separation of the blends. The presence of a small amount of P(L ‐2HB) decreased the low limit of crystallizable T_c of PDLA homocrystallites from 80 to 60 °C. The equilibrium melting temperature of HTSC crystallites was 257.6 °C.     

Differentiation of Bacillus anthracis and other Bacillus species by lectins.

Bacillus anthracis was agglutinated by several lectins, including those from Griffonia simplicifolia, Glycine max, Abrus precatorius, and Ricinus communis. Some strains of Bacillus cereus var. mycoides (B. mycoides) were strongly reactive with the lectin from Helix pomatia and weakly reactive with the G. max lectin. The differential interactions between Bacillus species and lectins afforded a means of distinguishing B. anthracis from other bacilli. B. cereus strains exhibited heterogeneity with respect to agglutination patterns by lectins but could readily be differentiated from B. anthracis and the related B. mycoides. Spores of B. anthracis and B. mycoides retained lectin receptors, although the heating of spores or vegetative cells at 100 degrees C resulted in a decrease in their ability to be specifically agglutinated. Fluorescein-conjugated lectin of G. max stained vegetative cells of B. anthracis uniformly, suggesting that the distribution of lectin receptors was continuous over the entire cellular surface. B. anthracis cells grown under conditions to promote the production of capsular poly(D-glutamyl peptide) were also readily agglutinated by the lectins, suggesting that the lectin reactive sites penetrate the polypeptide layer. Trypsin, subtilisin, lysozyme, and mutanolysin did not modify the reactivity of B. anthracis with the G. max agglutinin, although the same enzymes markedly diminished the interaction between the lectin and B. mycoides. Because the lectins which interact with B. anthracis are specific for alpha-D-galactose or 2-acetamido-2-deoxy-alpha-D-galactose residues, it is likely that the bacteria possess cell surface polymers which contain these sugars. Lectins may prove useful in the laboratory identification of B. anthracis and possibly other pathogenic Bacillus species, such as B. cereus.     

Detection and characterization of a bacteriocin,putadicin T01, produced by Pseudomonas putida isolated from hot spring water

Pseudomonas strains isolated from hot spring water were tested for bacteriocin‐like substance (BLS) production using a target panel of closely related microorganisms and other Gram‐positive and Gram‐negative bacteria. Molecular identification was carried out through specific PCR and 16S RNA sequence analysis. Isolates were identified as Brevundimonas diminuta and Pseudomonas putida, the latter exhibited antimicrobial activity. Pseudomonas putida strains produce an inhibitory substance against other Pseudomonas strains and other species including food‐borne pathogens. The BLS was sensitive to the proteolytic action of proteinase K, pronase E and trypsin but resistant to α‐amylase, RNase and lipase C, reflecting its proteinaceous nature. The BLS was stable at 100 °C and also after thermal treatment at 121 °C for 15 min. Additionally, it was stable within a wide range of pH (2–10). The substance from P. putida T01 strain was bactericidal to Escherichia coli. SDS‐PAGE analysis of the partial purified supernatant of strain T01 revealed a BLS with an approximate molecular mass of 8 kDa. Therefore, the results of this study show that P. putida strain T01 produces a BLS with a higher activity spectrum, which may find application in human medicine and in minimally processed food preservation.     

A New Titanium Complex Having Two Phenoxy‐Imine Chelate Ligands for Ethylene Polymerization

A new titanium complex having two phenoxy‐imine chelate ligands, bis[N‐(3‐tert‐butylsalicylidene)anilinato]titanium(IV)dichloride 1 , was synthesized and its structure determined by X‐ray analysis. Density functional theory (DFT) calculations and X‐ray analysis suggest that complex 1 , when activated, possesses two available cis‐located sites needed for ethylene polymerization. Complex 1 /methylaluminoxane (MAO) in toluene or heptane solvent displayed very high ethylene polymerization activities (2 280–4 150 (kg PE)·(mol cat)^–1·h^–1) with high molecular weight values (M̄_v = 288 000–881 000) at 25–75°C under atmospheric pressure. The activity values displayed by complex 1 /MAO are some of the highest values exhibited by any titanium complex with no cyclopentadienyl (Cp) ligand(s). Alternatively, complex 1 /ⁱBu₃Al/Ph₃CB(C₆F₅)₄ in toluene solvent displayed high ethylene polymerization activities (190–670 (kg PE)· (mol cat)^–1·h^–1) with exceptionally high molecular weight values (M̄_v = 3 920 000–5 860 000) at 25–75°C under atmospheric pressure. The molecular weight values displayed by complex 1 /ⁱBu₃Al/Ph₃CB(C₆F₅)₄ are some of the largest values displayed by homogeneous olefin polymerization catalysts including the group 4 metallocenes. The high potential of complex 1 for ethylene polymerization has therefore been demonstrated.     

Mixed (ionic and electronic) conductive solid polymer matrix. Synthesis and characterization of homopolymers of 3‐(2,5,8,11,14,17,20,23‐octaoxyhexadodecyl)thiophene and copolymers with 3‐methylthiophene

The macromonomer, 3‐(2,5,8,11,14,17,20,23‐octaoxyhexadodecyl)thiophene, abbreviated as (3‐OHDT), was synthesized. The macromonomer has a glass transition temperature of –77°C, which corresponds to the soft oligooxyethylene side chain attached to the thiophene. The homopolymer, poly[3‐(2,5,8,11,14,17,20,23‐octaoxyhexadodecyl)thiophene], abbreviated to P[3‐(OHDT)], has been synthesized via oxidative coupling of the macromonomer using FeCl₃. Two distinct glass transition temperatures are observed for the homopolymer. The T_g at –47°C corresponds to the soft oligooxyethylene phase and the T_g value at 72°C corresponds to the hard thiophene backbone. The homopolymer P[3‐(OHDT)] can be doped with LiClO₄ and I₂ to generate ionic and electronic conductive phases, respectively. For the P[3‐(OHDT)]/LiClO₄ system, with an ethylene oxide to lithium mole ratio of 8 : 1, an ionic conductivity value of 1.7×10^–6 S cm^–1 is observed at 25°C. The room temperature electronic conductivity of P[3‐(OHDT)]/doped with I₂ is 3.5x10^–4 S cm^–1. Several random copolymers of 3‐methylthiophene and 3‐(2,5,8,11,14,17,20,23‐octaoxyhexadodecyl)thiophene were also synthesized. Depending on the copolymer composition, glass transition temperatures ranged from 67°C to 76°C, the higher glass transition temperature corresponding to higher 3‐methylthiophene content. The copolymers may be appropriately doped to generate conductive matrices, with a maximum ionic conductivity (ethylene oxide/Li⁺ mole ratio of 8 : 1) of 1.0×10^–7 S cm^–1 and electronic conductivity around 6.7×10^–1 S cm^–1 at 25°C.     

Mitogenic response and cytokine production induced by cramoll 1,4 lectin in splenocytes of inoculated mice

Cramoll 1,4 is a lectin with specific glucose/mannose binding, which is extracted from seeds of Cratylia mollis Mart. Many assays have shown the cytokine expression activity and anti‐inflammatory profile of this lectin. The aim of this study was to evaluate the immunostimulatory response, in vitro, of splenocytes in mice previously inoculated, in vivo, with C. mollis (Cramoll 1,4) and Canavalia ensiformis (Con A) lectins. Results demonstrated higher proliferation indexes induced by Cramoll 1,4 than Con A lectin in relation to all experimental groups. Cramoll 1,4 and Con A also induced high levels of IL‐2, IL‐6, IFN‐γ and nitric oxide production. Moreover, Cramoll 1,4 did not induce apoptosis and stimulated a significant number of cells in the S phase of the cell cycle. Results showed that Cramoll 1,4 lectin induces proliferative response and suggested that this lectin can be used as a mitogenic agent in immunostimulatory assays.     

Characterization of a neutral pectin lyase produced by Oidiodendron echinulatum MTCC 1356 in solid state fermentation

A neutral pectin lyase produced by a new fungal strain Oidiodendron echinulatum MTCC 1356 under solid state fermentation using wheat bran as agro waste has been studied. The enzyme was purified by ammonium sulphate precipitation (30–60%), DEAE anion exchange and Sephadex G‐100 column chromatographies. The SDS‐PAGE and native PAGE revealed two bands of sizes 42 and 47 kDa. The enzyme was purified 37 fold with specific activity of 4.5 U/mg and 2.25% yield. The K_m and V_max values determined using citrus pectin were 1.2 mg/ml and 0.36 IU/min respectively. The pH and temperature optima were pH 7.0 and 50 °C, respectively. The pH stability was around 5.0 for 24 h at 20 °C. The purified enzyme retained maximum activity for 30 min upto 50 °C. The activation energy for thermal denaturation of the purified enzyme was found to be 60.0 kJ/Mol. The effects of various metal ions and protein inhibitors on enzyme activity have revealed total inhibition of the enzyme activity in the presence of Ag⁺and Cu⁺and KMnO₄ at 1 mM. The neutral pectin lyase showed retting of Crotalaria juncea fibre in the presence of EDTA. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Agglutination of murine and guinea pig peritoneal cells by α-L-fucose-binding lectin: Evonymus europaea

Among lectins from Lotus tetragonolobus, Ulex europaeus I and Evonymus europaea, agglutinating cells with blood group H determinants containing L -fucose α1→2-linked to subterminal D -galactose, only the last lectin agglutinates thioglycolate- and paraffin oil-stimulated murine and guinea pig peritoneal exudate cells (PEC). The agglutination is inhibited by specific inhibitors of Evonymus lectin only: lacto-N-fucopentaose I and lactose. These results suggest the presence of a determinant on the surface of PEC, containing L -fucose α1-linked at the nonreducing end which is different from blood group H determinants. Nonstimulated murine peritoneal cells (PC) are not agglutinated by the lectin but become agglutinable after neuraminidase treatment. Unstimulated guinea pig PC from different animals are agglutinated to a different extent by the same lectin concentration and show increased agglutinability after neuraminidase digestion. These results show that receptor for Evonymus lectin also exists on the nonstimulated PC but access to it is hindered by sialic acid. Trypsin- and pronase-digested PEC show increased agglutinability with Evonymus lectin. These results suggest that the lectin receptor is a glycolipid. Since α-linked L -fucose has been suggested as a part of the macrophage receptor for migration inhibitory factor in the guinea pig (Remold, J. Exp. Med. 1973. 138: 1065), the effect of Evonymus europaea lectin on the migration of PEC was studied. It was found that lectin inhibits the migration of PEC in the capillary tube assay up to 80%.     

COMPOSITION AND IMMUNOCHEMICAL PROPERTIES OF GLYCOPROTEINS WITH ANTI-B AGGLUTININ ACTIVITY ISOLATED FROM EUONYMUS SIEBOLDIANA SEEDS

Five major glycoproteins with anti-B agglutinin activity were isolated from seeds of Euonymus Sieboldiana by a procedure based on precipitation with ammonium sulphate, Sepharose 4B gel filtration, CM- and DEAE-Sepharose chromatography and Sephacryl S-200 gel filtration. The purified glycoproteins each gave a single symmetrical peak on Sephacryl S-200 gel filtration with elution volumes corresponding to molecular weights of approximately 15,000 to 130,000, each forming a single precipitin line on gel diffusion plates with anti-E. Sieboldiana antibody. These anti-B glycoproteins were rich in acidic amino acids without cysteine and methionine and contained about 8-36% carbohydrate, of which galactose, arabinose and glucose were the predominant sugars, with small amounts of glucosamine, rhamnose, fucose, xylose, mannose and ribose. The most anti-B active lectin agglutinated human B red blood cells at a concentration of 2 μg/ml and was strongly inhibited by melibiose. The three other lectins with anti-B agglutinin activity, however, were not inhibited by any galactose-containing glycosides.