Isolation and properties of N-acetyllactosamine-specific lectins from nine erythrina species

Lectins from seeds of nine species of Erythrina have been purified by affinity chromatography on columns of lactose coupled to Sepharose and their properties compared with those of the lectin from Erythrina cristagalli. All lectins are glycoproteins of M, ca 60 000 composed of two identical or nearly identical subunits. They contain between 3–10% carbohydrates comprised of N-acetylglucosamine, mannose, fucose and xylose. The amino acid composition of all Erythrina lectins is very similar. The N-terminal amino acid is valine, with the exception of the lectin from E. flabelliformis in which it is alanine. To the extent tested, identities or near identities have been found in the N-terminal sequences (up to 15 residues in some cases) of the lectins. Hapten inhibition experiments of agglutination have shown that the lectins are specific for N-acetyllactosamine, this disaccharide being 10–30 times more inhibitory than D-galactose and 10–20 times more than N-acetyl-D-galactosamine. All lectins agglutinate human erythrocytes equally well, irrespective of blood type, at minimal concentrations of 5–20 μg/ml. Six of the lectins are also very effective in agglutinating rabbit erythrocytes and are mitogenic for human peripheral blood lymphocytes, whereas three of them are considerably weaker hemagglutinins for rabbit erythrocytes, and two of these are also very weak mitogens. Our results, while demonstrating striking similarities in the molecular properties and sugar specificity of all Erythrina lectins studied, suggest the existence of differences at or close to the carbohydrate-binding site.     

A cell membrane-associated lectin of the oyster hemocyte

The presence of a lectin in association with hemocytes of the American oyster, Crassostrea virginica, has been demonstrated by utilizing a microhemagglutination assay. The plasma membrane association of this lectin is shown by its copurification with the plasma membrane fraction of disrupted hemocytes, using sucrose density gradient centrifugation, and also by the binding of ¹²⁵I-labeled glycoproteins to intact hemocytes at 4°C. Based upon agglutinating spcificity for a range of vertebrate erythrocytes, both untreated and enzyme-treated, along with hemagglutination-inhibition assays and crossed-absorption tests, it is apparent that there are also two serum (soluble) lectins, each having a distinct serological agglutination specificity, and that the hemocyte membrane-associated lectin has a specificity that is identical with one of these two serum lectins. It is proposed that the hemocyte membrane-associated lectin may be a true integral membrane protein, and therefore may function as a membrane receptor in nonself recognition by molluscan hemocytes.     

The titres of lectins in the haemolymph of Lacanobia oleracea and the effects of parasitism by the ectoparasitoid wasp Eulophus pennicornis

Serum from larvae of Lacanobia oleracea L. (Lepidoptera; Noctuidae) parasitized by Eulophus pennicornis (Hymenoptera; Eulophidae) and from normal non‐parasitized larvae is capable of agglutinating rabbit, sheep, calf, goat, chicken, horse and human erythrocytes, but not yeast. Studies with a range of inhibitory carbohydrates showed that serum lectins(s) had specificity for sugars containing galactose and for rhamnose, and for the glycosubstances fetuin and asialofetuin. Lectin activity is heat‐labile and is not dependent on calcium. Parasitism by E. pennicornis caused an increase in the agglutination titre of the serum from larvae of L. oleracea but not an increase in specific activity (titre per mg protein per ml). However, when venom from the venom gland of female wasps was injected into L. oleracea larvae, both the agglutinating activity and the specific activity of the larval serum increased. The possible causes of this increase are discussed. It is suggested that venom contains antigenic components which, when injected into the haemocoel of the L. oleracea larva, may be increasing lectin synthesis and/or release into the serum.     

The inhibition of serum opsonins by a carbohydrate and the opsonizing effect of purified agglutinin on the clearance of nonself particles from the circulation of Helix pomatia

The serum of Helix pomatia agglutinates enzyme-treated erythrocytes and also possesses opsonizing properties. The agglutinating as well as the opsonizing activity could be inhibited by N-acetylglucosamine, indicating an identicalness of these serum components. As this observation supports the hypothesis that agglutinins may function as opsonins, purified agglutinins from the albumin gland of Helix pomatia, from the sponge Axinella polypoides, and Con A were utilized to sensitize foreign cells prior to their injection into the hemocoel of H. pomatia. Helix agglutinin revealed a strong opsonic effect on the elimination of the nonself particles from the circulation of the snail. It is assumed that serum opsonins of H. pomatia may couple certain nonself materials to the surface of cells in different clearance organs and that hemocytes possess membrane-associated agglutinins which mediate their attachment to trapped foreign particles.     

Activation of fat body glycogen phosphorylase in Locusta migratoria by corpus cardiacum extract and synthetic adipokinetic hormone

Between 10 and 20 per cent of the total glycogen phosphorylase in the fat body of mature Locusta migratoria of both sexes is in the active form. Injection of an aqueous corpus cardiacum (CC) extract results in a rapid activation: within 2 min the level of active phosphorylase is significantly increased and full activation is reached within 10 to 20 min. As little as 0.002 CC gland equivalents stimulate fat body glycogen phosphorylase significantly and maximum activation is obtained with 0.05 CC gland equivalents. From experiments with known quantities of injected synthetic adipokinetic hormone (SAKH), it appears that this hormone cannot account for all the activation. This is supported by results obtained when extracts of carefully isolated storage lobes are injected; at the dose used here these have no adipokinetic activity, but activate fat body phosphorylase. Furthermore, when locusts are ‘stressed’ by rotation, although no adipokinetic hormone is released, an activation of phosphorylase occurs. Starvation causes also an increase in the active form of the enzyme. The fat body receptor sites of the locust recognise also the crustacean red pigment concentrating hormone (RPCH), whose structure closely resembles that of the locust adipokinetic hormone, leading to activation of the phosphorylase. However, RPCH is about 2.5–5 times less potent than SAKH. Crude CC extracts of a stick insect (Carausius morosus), a cockroach (Periplaneta americana) and the tobacco hornworm (Manduca sexta) activate locust fat body phosphorylase, although this last extract has no effect on lipid elevation. On the other hand, CC extracts of the death's head hawk moth (Acherontia atropos) and purified crustacean hyperglycaemic hormone from a crayfish (Orconectes limosus) have no effect.     

Opsonizing effect of serum and albumin gland extracts on the elimination of human erythrocytes from the circulation of Helix pomatia

The removal of human erythrocytes of the A₁ and B types from the circulation of the gastropod Helix pomatia follows an exponential curve. The elimination of the nonself particles is apparently dependent on serum opsonins as preincubation of A₁ and B erythrocytes in Helix serum increases the rate of their clearance. This conclusion is supported by our finding that secondary doses of nonsensitized A₁ erythrocytes injected 12–19 hr after a similar primary dose are cleared very slowly; however, the clearance rate returns to normal if erythrocytes comprising the second dose are preincubated with Helix serum. Furthermore, the elimination of second-dose A₁ erythrocytes is strongly enhanced after their pretreatment with agglutinating extracts of the albumin glands from H. pomatia and Cepaea (Helix) nemoralis. On the other hand, no opsonizing effect was obtained by pre-incubating A₁ erythrocytes in the agglutinating extract of the sponge Aaptos papillata.     

Purification and properties of d-galactose-binding lectins from some erythrina species: Comparison of properties of lectins from E. indica,E. arborescens,E. suberosa, and E. lithosperma

The lectins of the seeds of four species of the genus Erythrina, namely E. indica, E. arborescens, E. lithosperma, and E. suberosa were isolated by affinity chromatography on acid-treated ECD-Sepharose 6B. The lectins were found homogeneous in polyacrylamide gel electrophoresis and immunochemical tests. In SDS-gel electrophoresis, E. indica and E. lithosperma lectins each gave two bands with subunit molecular weights of 30,000 and 33,000 in the case of the former and 26,000 and 28,000 in the case of the latter. E. arborescens and E. suberosa gave single bands corresponding to polypetide chain molecular weight of 28,000. The lectins were found to be glycoproteins with their neutral sugar contents ranging from 4–9%. In carbohydrate specificity all the lectins were d-galactose specific. Their close similarity was also demonstrated by their homologous cross-reaction against the antiserum to E. indica lectin. In hemagglutinating activity toward human erythrocytes, E. indica and E. suberosa lectins showed higher activity toward the O group and E. arborescens toward the B group. The results show the similarity of the lectins derived from different species of the same genus in respect of immunochemical properties and carbohydrate specificity. In studies on E. indica lectin, the protein was found homogeneous by electrophoretic, immunochemical, and sedimentation experiments. Its molecular weight of 68,000 determined from sedimentation and diffusion data indicated that the molecule was a dimer of two noncovalently bound unequal subunits whose SDS-gel electrophoretic molecular weights are noted above. The lectin was devoid of cysteine and methionine and contained valine as its N-terminal amino acid. It had 9% neutral sugars and 1.5% glucosamine. Equilibrium dialysis studies with lactose showed that the values of the association constant K at different temperatures were of similar orders of magnitude to other lectins and the dimeric molecule possessed two noninteracting binding sites.     

Adipokinetic and hyperglycaemic factors of different insect species: Separation with high performance liquid chromatography

Corpus cardiacum extracts from the phasmids, Carausius morosus, Cuniculina impigra, Sipyloidea sipylus, Acrophylla wuelfingi, Eurycantha goliath, Bacillus rossius and Extatosoma tiaratum, from the Orthopterans, Locusta migratoria and Gryllus bimaculatus, from the Dictyopterans, Periplaneta americana, Gromphadorrhina coquereliana and Blaberus craniifer, from the Coleopterans Tenebrio molitor and Pachnoda sp., synthetic adipokinetic hormone and synthetic crustacean red pigment-concentrating hormone (RPCH) were injected into locusts, cockroaches and ligated stick insects as bioassay systems for adipokinetic and hyperglycaemic substances, respectively. The locust and cockroach bioassay gave positive results with all corpus cardiacum material tested (however the lipid response in locusts upon injection of T. molitor corpus cardiacum extract was very poor). The stick insect bioassay was quite specific for stick insect corpus cardiacum material; only corpus cardiacum extracts from a few other species (G. bimaculatus, P. americana, G. coquereliana and Pachnoda sp.) showed weak activity. All other extracts, including synthetic adipokinetic hormone and RPCH, failed to induce a response.Separations of corpus cardiacum extracts from L. migratoria, P. americana, T. molitor, C. morosus and S. sipylus were achieved on reversed-phase high-performance liquid chromatography (RP-HPLC). Locust corpus cardiacum extract showed two absorbance peaks with adipokinetic activity, adipokinetic hormones I and II. The peaks with hyperglycaemic activity from P. americana corpus cardiacum extracts had different retention times to those of locust adipokinetic hormones I and II. Stick insect corpus cardiacum extracts revealed also 2 absorbance peaks with adipokinetic activity, the major one co-eluting with RPCH. The active compound from corpus cardiacum extracts of T. molitor appeared to elute close to locust adipokinetic hormone I.     

Lectins in the hemolymph of Japanese horseshoe crab, Tachypleus tridentatus

The hemolymph of the Japanese horsehoe crab, Tachypleus tridentatus contains lectins which agglutinate mammalian erythrocytes. Affinity chromatographic purification of the lectins using bovine submaxillary gland mucin-conjugated Sepharose resulted in the separation of the lectins into four fractions; one major and three minor lectins. Protein subunits revealed by polyacrylamide gel electrophoresis and the immunoprecipitin line of these lectins against antiserum to crude lectins were unique to each fraction. The activities of all the lectins were optimal at pH values between 6 and 8, and were destroyed by heating at 60°C. Calcium chloride augumented the activities of three lectins, but the major lectin was not influenced by the salt. Bovine erythrocytes were not agglutinated by any of the lectins and comparative agglutination titers for other erythrocytes from various sources were different among these lectins. The activities of all the lectins were inhibited by N-acetylamino sugars. They were more effectively inhibited by glycoproteins which contain sialic acid.     

Isolation and new biological properties of Arion empiricorum lectin

The lectin present in the mucus of the snail Arion empiricorum was isolated by ion exchange chromatography. Purity was demonstrated by immunelectrophoretic analysis, immunization studies, and polyacrylamide gel electrophoresis. With the latter we found a molecular weight of 43 000.Hemagglutination inhibition studies revealed that carbohydrates play a minor role in the agglutination reaction of A. empiricorum lectin. Stronger inhibition could be achieved with human serum and the serum of several animal species.These findings were clarified by the demonstration that some serum proteins were precipitated by A. empiricorum lectin. Besides its agglutinating and precipitating properties the purified A. empiricorum lectin possesses proteinase- inhibiting properties, as demonstrated by the inhibition of casein-digestion by trypsin and plasmin.     

Immunocytochemical differentiation between adipokinetic hormone (AKH)-like peptides in neurons and glandular cells in the corpus cardiacum of Locusta migratoria and Periplaneta americana with C-terminal and N-terminal specific antisera to AKH

Summary An immunocytochemical method was used to differentiate between immunoreactive substances in glandular cells in the corpora cardiaca (CC) and in certain cerebral neurons in 2 insect species, Locusta migratoria migratorioides and Periplaneta americana. The staining properties of antisera raised to different parts of the decapeptide adipokinetic hormone (AKH) were compared and their specificity was determined by preabsorption with AKH and related peptides. Antibodies raised to the N-terminal part of AKH (serum 433) and the central and C-terminal part (serum 241) were found to have different staining properties.In the CC of the locust both antisera show a strong immunoreactivity with glandular cells, we therefore suggest that at least one of the compounds revealed is AKH. Some of the glandular cells in the locust and large numbers of glandular cells in the CC of the cockroach are revealed by the N-terminal specific antiserum. On the other hand, neurons in the central nervous system are revealed only by the C-terminal specific antiserum. The possible identity of the various substances revealed by these two antisera is discussed.     

In vivo and in vitro inhibition of lipid-linked saccharide and glycoprotein synthesis in plants by amphomycin

The effect of the polypeptide antibiotic, amphomycin, on the in vitro and in vivo synthesis of polyprenyl-linked sugars and glycoproteins in plants was examined. This antibiotic blocked the transfer of mannose from GDP-[¹⁴C]mannose into mannosyl-phos-phoryl-dolichol by a particulate enzyme preparation from mung beans and also inhibited the transfer of GlcNAc from UDP-[³H]GlcNAc to GlcNAc-pyrophosphoryl-polyisoprenol. The in vitro incorporation of these sugars into trichloroacetic acid-insoluble material was also markedly inhibited by this antibiotic. Since most of the radioactivity incorporated into this insoluble material is rendered water-soluble by treatment with pronase, it seems likely that these sugars are incorporated into glycoproteins whose synthesis is sensitive to amphomycin. Amphomycin also inhibited the transfer of glucose from UDP-[¹⁴C]glucose to steryl glucosides, although this system was less sensitive to antibiotic than was synthesis of the polyprenyl-linked sugars. The antibiotic did not block the in vitro transfer of glucose from UDP-[¹⁴C]glucose to β-glucans. In carrot slice cultures, amphomycin also inhibited the incorporation of [¹⁴C]mannose into glycolipid and glycoprotein, but it did not prevent the incorporation of [¹⁴C]lysine into protein.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

A new survey of Brazilian marine algae for agglutinins

Aqueous protein extracts from 30 Brazilian marine algae were examined for haemagglutinating activity using native and enzyme-treated rabbit, chicken, sheep and human erythrocytes. Most extracts agglutinated at least one of the blood cells used. Sheep and rabbit erythrocytes were more suitable for detection of the agglutinating activity. The minimum protein concentration necessary to produce positive agglutination was usually lower with enzyme-treated erythrocytes than native ones. The five algal protein extracts showing the greatest haemagglutination titre were tested for sugar-binding specificity. Only the activity present in the green alga Cauler pacupressoides was inhibited by simple sugars and not by the glycoproteins tested. The activity of the other four extracts was inhibited by at least one of the glycoproteins utilised. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of intestinal erythrocyte agglutination on the feeding performance of Triatoma brasiliensis (Hemiptera: Reduviidae)

Triatoma brasiliensis is an important vector of Trypanosoma cruzi in Brazil. The feeding efficiency on its hosts depends on several parameters including the maintenance of the ingested blood at low viscosity, which could be modulated by the anterior midgut (crop) anticoagulant and haemagglutinant activities. In the present study, we characterized T. brasiliensis crop haemagglutination activity and evaluated its importance in the feeding process. Soluble crop contents (SCC) of T. brasiliensis were able to agglutinate rat, mouse and rabbit eryhtrocytes, but had no activity on cattle and Thrichomys apereoides, a rodent species commonly associated with T. brasiliensis in the wild. The haemagglutination was characterized by the immediate formation of several clusters of erythrocytes connected by flexible elastic-like fibers. The feeding efficiency of T. brasiliensis on rat (agglutinated by SCC) was almost double that from T. apereoides (not agglutinated by SCC). The influence of haemagglutination on feeding was confirmed by artificially feeding bugs on a diet composed of cattle or rat erythrocytes. The bugs fed on cattle erythrocytes had lower ingestion rates in comparison to those fed on rats. The results indicate that, in addition to other parameters, haemagglutination brought about by SCC has an important role in the feeding efficiency of T. brasiliensis.     

Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: Interspecific and intraspecific differences in infectivity for insects

The effectiveness of various dosages of different species/strains of nematodes was compared for Galleria mellonella and various pest insects that live in or pupate in soil. Neoaplectana feltiae (= carpocapsae), the only nematode species tested by most other workers, was never the most infective for any of the insect species tested and was least infective for two. All species/strains of nematode were able to kill insects of each species. The degree of infectivity of each of the nematode species/strains for different hosts varied considerably, and no one species/strain of nematode was the most infective for all insect species. This indicates the importance of testing a number of nematode species against any particular insect before commencing field evaluations for biological control.     

Serum opsonins and the passive transfer of protection in Babesia rodhaini infections of rats

Serum opsonins and the passive transfer of protection in Babesia rodhaini infections of rats. International Journal for Parasitology4: 197–201. An investigation into the protective activity of serum from rats immune to B. rodhaini and the role played by opsonins in that activity was undertaken. One, three and six infections with B. rodhaini resulted in corresponding increases in the titre of specific protective antibody demonstrable by the administration of immune serum to rats. Drug control of infection resulted in a lower level of protective activity than that which developed when rats controlled infection unaided. Protective activity following recovery from a single drug controlled infection was undiminished 20 weeks after infection.Serum opsonins were detected in an in vitro culture system of normal rat peritoneal macrophages and these antibodies were specific for parasitized erythrocytes. It is suggested that opsonins were largely responsible for the protective effect demonstrated by assay in rats but that their importance, relative to other antibodies with a possible protective function, in the development of acquired immunity remains to be determined.     

Performance and thermal sensitivity of the southernmost lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus

Locomotor activity performance of reptiles is largely temperature dependent and, in harsh environments with short activity periods during the day and throughout the year, plays a vital role in the fitness of the species. This particular study focuses on the performance and the thermal sensitivity for running, at different body temperatures, of the two southernmost species of lizards in the world, Liolaemus sarmientoi and Liolaemus magellanicus, studied at two locations in the south of Santa Cruz province, Argentina (51°S, 70°W and 50°S, 72°W; 133 m asl). The speed of sprint and long runs was measured in the field to determine the physiological performance of lizards at different air temperatures. In both species speed increases with the temperature, and they reach the highest performance at high temperatures. The difference between activity and thermal optima suggests that L. magellanicus has colonized its actual environment recently, and that it has not had enough time for its physiological mechanisms to evolve and achieve a maximum performance at the cold temperatures they have to tolerate at present. In contrast, L. sarmientoi achieved a high performance over a wider range of temperatures that included temperatures lower than the preferred temperatures in the lab, which they can generally find in their environment.     

Haemagglutinin activity in Acrididae (grasshopper) haemolymph

The haemolymph of Acrididae causes haemagglutination of human and animal erythrocytes. Thirteen of seventeen species tested had detectable activity and gave agglutination titres in the range 2–64, Melanoplus bivittatus, and M. sanguinipes showed greatest activity. Haemagglutinin activity is continuously present in male and female insects from 4th instar and throughout adulthood. Females contain slightly more activity than do males. M. sanguinipes haemolymph agglutinates rabbit, calf, human (all ABO types) guinea pig, mouse, chicken, cat, pig and sheep erythrocytes. Rabbit red cells are agglutinated most strongly and sheep and chicken cells least. M. sanguinipes haemolymph also agglutinates the protozoan Nosema locustae, a natural grasshopper pathogen. Preabsorption of haemolymph with different erythrocyte types selectively removes haemagglutinin activity suggesting the presence of multiple or heteroagglutinins. M. sanguinipes haemagglutinin is inhibited by glycoproteins, simple carbohydrates and carbohydrate derivatives. The inhibitory pattern is complex and among the sugars tested only mannose and derivatives of mannose are exclusively non-inhibitory. Haemolymph haemagglutinin activity is destroyed by heat and EDTA. It is totally precipitated by dialysis against water and may be partially recovered in phosphate or Tris buffer. Activity is stable in frozen haemolymph.