Serological characterization of humoral lectin from Heterometrus granulomanus scorpion hemolymph

A lectin in the hemolymph of Indian scorpion Heterometrus granulomanus was detected by agglutination of human and animal erythrocytes. The agglutinating activity was enhanced in presence of Ca2+ ion. The lectin shows specificity for sialic acid like many other sialic acid-specific lectins such as "Limulin", as the agglutination of erythrocytes was completely abolished by treatment with Vibrio cholerae neuraminidase. However, neuraminidase-treated rat and mouse erythrocytes exhibited high titers. This result was substantiated by crossed-absorption test which suggests the adjunct specificity of the Heterometrus lectin for these cells. Hemagglutination-inhibition results of the lectin indicate that sialic acid including its derivatives and sialoglycoconjugates are the inhibitors among which glycophorin was most potent.     

Cloning and characterization of two different ficolins from the giant freshwater prawn Macrobrachium rosenbergii

Ficolins, a kind of lectin containing collagen-like and fibrinogen-related domains (FReDs, also known as FBG or FREP), are involved in the first line of host defense against pathogens. In this study, two ficolins, namely, MrFico1 and MrFico2, from the giant freshwater prawn Macrobrachium rosenbergii were identified. In contrast to other ficolins, these two ficolins have no collagen-like domain, but such ficolins contain a coiled region and a FReD domain. Phylogenetic analysis showed that MrFico1 and MrFico2, together with two ficolin-like proteins from Pacifastacus leniusculus, belonged to one group. Quantitative RT-PCR (qRT-PCR) showed that both MrFico1 and MrFico2 were expressed in hepatopancreas, stomach and intestine, with the highest expression in stomach for MrFico1, compared to the highest expression in hepatopancreas for MrFico2. qRT-PCR analysis also showed that MrFico1 was obviously upregulated upon Vibrio anguillarium challenge, while MrFico2 was upregulated after challenged by V. anguillarium or white spot syndrome virus. Bacterium-binding experiment showed that MrFico1 and MrFico2 could bind to different microbes, and sugar-binding assay revealed that these two ficolins could also bind to lipopolysaccharide and peptidoglycan, the glycoconjugates of bacteria surface. Moreover, these two ficolins could agglutinate bacteria in a calcium-dependent manner, and the results of bacteria clearance experiment showed that both ficolins could facilitate the clearance of injected bacteria in the prawn. Our results suggested that MrFico1 and MrFico2 may function as pattern-recognition receptors in the immune system of M. rosenbergii.     

The use of an ELISA-inhibition technique for the detection of humoral responses to injected antigens in the freshwater prawn Macrobrachium rosenbergii: Preliminary studies

Injection of human serum albumin (HSA) into the haemocoel of the freshwater prawn Macrobrachium rosenbergii did not appear to elicit a quantifiable humoral response in haemolymph samples taken from subject animals. Injection of human immunoglobulin G (IgG), however, appeared to elicit a response which could be shown to have activity against both HSA and IgG. Quantitation of the humoral response was achieved using an ELISA-inhibition technique. It is suggested that the choice of this methodology is appropriate to studies on inducible responses in species for which specific immunological reagents are not readily available.     

The use of an ELISA-inhibition technique for the detection of humoral responses to injected antigens in the freshwater prawn Macrobrachium rosenbergii: Preliminary studies

Injection of human serum albumin (HSA) into the haemocoel of the freshwater prawn Macrobrachium rosenbergii did not appear to elicit a quantifiable humoral response in haemolymph samples taken from subject animals. Injection of human immunoglobulin G (IgG), however, appeared to elicit a response which could be shown to have activity against both HSA and IgG. Quantitation of the humoral response was achieved using an ELISA-inhibition technique. It is suggested that the choice of this methodology is appropriate to studies on inducible responses in species for which specific immunological reagents are not readily available.     

Serological characterization of a hantavirus from Hubei, China

Hantavirus HV114, isolated from urine of a patient during epidemic of hemorrhagic fever with renal syndrome (HFRS) in China, was subjected to a detailed serological characterization using enzyme-linked immunosorbent assay (ELISA), neutralization test and indirect immunofluorescence antibody assay (IFA). It has been found that HV114 is antigenically similar to the hantavirus A9 strain isolated in China and to the Hantaan 76-118 virus (HTNV 76-118), but different from the hantaviruses isolated from Apodemus agrarius in the region endemic for HFRS.     

Epidemiological characterization of Neisseria gonorrhoeae by lectins

A total of 101 isolates of penicillinase-producing and non-penicillinase-producing Neisseria gonorrhoeae with known nutritional requirements, plasmid content, and serovars, were examined for lectin agglutination patterns. These isolates were from outbreaks in Georgia, California, Hawaii, and Pennsylvania. Cell suspensions made from 16- to 18-h cultures were mixed with 14 different lectins, and the resultant agglutination patterns were classified as agglutination groups. Among the 101 isolates tested, 24 different agglutination groups were demonstrated. Of the organisms tested, 55% were located in 3 of the 24 groups, and 86% of the isolates reacted with the lectins Trichosanthes kinlowii, Griffonia simplicifolia I, peanut agglutinin, soybean agglutinin, potato agglutinin, and wheat germ agglutinin. One isolate did not react with peanut or potato agglutinin, five isolates lacked reactivity with potato agglutinin, and six isolates did not react with wheat germ agglutinin. Of the wheat germ-negative isolates, four were from Pennsylvania and were identical with regard to auxotype, plasmid content, serovar, and lectin group. The other two wheat germ-negative isolates were from California and were unrelated by the same criteria to the four Pennsylvania isolates and to each other. Among the isolates tested, there were no differences in lectin groups with regard to the sex of the patient. In the Georgia collection, agglutination with one lectin group was confined to isolates of serogroup IA. This association was not observed for the other geographic areas. Some isolates showing identical auxotype, plasmid content, and serovars could be differentiated based on lectin agglutination patterns, whereas other isolates were identical by all testing criteria.     

Occurrence and characterization of lectins in actinomycetes

25 species of actinomycetes were tested for the occurrence of lectins. Using a battery of normal and desialized erythrocytes, each species was screened for 3 types of lectin activity i.e. surface bound, extracellular and intracellular. As many as 13 species showed one or more types of activity; some of them were characterized with regard to their biological action spectrum and sugar specificity.     

Kinetics of glucose transport by the perfused mid-gut of the freshwater prawn Macrobrachium rosenberg ii.

1. Mucosal influx of [3H]glucose was examined in the mid-gut of a freshwater prawn, Macrobrachium rosenbergii, using an in vitro perfusion technique. 2. [3H]glucose transfer across the apical cell membrane of the epithelium exhibited Michaelis-Menten kinetics (Jmax.in = 0-15 mumole glucose equiv/g. min, Kt = 0-17 mM). Under Na-free conditions, glucose influx was significantly reduced and a linear function of substrate concentration, indicative of either slow cellular diffusion (KD = 7-6 X 10(3) mumole glucose equiv/g. min. mM) or a facilitated process with a low carrier affinity for the sugar. 3. Phlorizin was a potent competitive inhibitor of glucose influx (K1 = 3-6 X10(-3) mM), galactose and 3-O-methylglucose (3-O-MG) were weak inhibitors, and fructose had no evident effect on glucose uptake. Azide, but not iodoacetate (IAA), significantly depressed influx. 4. Absorbed [3H]glucose was rapidly metabolized by the mid-gut. The majority of accumulated activity within the tissue was in the form of phosphorylated compounds and tritiated water (THO), while only 0-3% was recovered as a free-glucose. 5. Preliminary studies examining transmural [3-H]glucose transport, however, demonstrated a significant net mucosal to serosal free-glucose flux across the prawn mid-gut which was Na-dependent and IAA- and phlorizin-sensitive. Two alternative interpretations of the data are advanced as possible mechanisms for transepithelial glucose transport: (1) group translocation, or (2) the operation of an energized, high affinity, baso-lateral sugar transport carrier.     

D-galactose binding lectins from the tunicate Ascidia malaca: subunit characterization and hemocyte surface distribution

D-galactose specific lectins purified from Ascidia malaca serum contain a major protein component with an apparent molecular weight of about 58,000 daltons, which moves more rapidly under non-reducing conditions. Intramolecular disulfide linkages can explain this behaviour, suggesting a compact protein structure. Membrane lectins have been demonstrated on the surface of about 34% hemocytes by immunofluorescent methods using a rabbit antiserum against the isolated serum lectins. Small, medium and large hemocytes can be positive, as also shown by binding on Sepharose spherules or by rosette formation with sheep and rabbit erythrocytes. Binding is inhibited by the same sugars specific for the serum lectins. Finally, antibodies to the serum lectins specifically agglutinate the hemocytes. This evidence supports the hypothesis that a lectin with the same specificity and certain structural similarities can be found free in the serum and present on hemocyte surfaces.     

Serological characterization of black-pigmented Bacteroides endodontalis.

Serological studies on the black-pigmented Bacteroides species B. endodontalis revealed three serotypes based on capsular determinants. A common antigen (O-antigen) could be demonstrated after decapsulation. Weak cross-reactivity was found with B. asaccharolyticus, but not with B. gingivalis. Similarity between the serology of Enterobacteriaceae and black-pigmented Bacteroides spp. is discussed.     

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F. chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes.     

Serological characterization of allergens in poppy seeds

BACKGROUND AND OBJECTIVE: Poppy seeds in food can induce immediate-type allergic reactions ranging from mild local symptoms to severe anaphylactic reactions. Previous publications showed that poppy seeds cross-react with other plant-derived allergens. The IgE-binding components have not been defined so far. METHODS: We analysed sera from 11 patients with adverse reactions after ingestion of poppy seed-containing food by IgE-immunoblotting. Nine of 11 patients showed concomitant IgE binding to allergens of birch, mugwort or grass pollen in RAST-CAP, and suffered from characteristic seasonal symptoms. RESULTS: Ten of 11 patients showed IgE binding to a 45-kDa protein, 4/11 to a 34-kDa, 5/11 to a 17-kDa, 5/11 to a 14-kDa, and 3/11 to a 5-kDa component. Furthermore, individual IgE binding to proteins of 20, 25, 30 and 40 kDa proteins could be observed. Periodate treatment of blots markedly reduced the IgE binding capacity of the 40- and 45-kDa compounds, indicating the existence of IgE epitopes of the carbohydrate type. Inhibition studies indicated the presence of homologues of pollen allergens in extracts from poppy seeds, i.e. Bet v 1 and Bet v 2. CONCLUSION: The serological analysis showed IgE binding to protein and sugar components of poppy seeds. The 40- and 45-kDa allergens are glycoproteins and contain IgE binding carbohydrate moieties. Moreover, cross-reacting homologues of pollen allergens including Bet v 1 and profilin were detected in poppy seed extract.     

Development and characterization of primary cell cultures from the hematopoietic tissues of the Dublin Bay prawn, Nephrops norvegicus

Improved maintenance in vitro of the hematopoietic tissue of the Dublin Bay prawn Nephrops norvegicus (L.) resulted by using 10% (v/v) 2× Leibovitz's medium prepared in seawater (salinity = 25), and supplemented with 10% (v/v) heat inactivated fetal bovine serum plus 5% (v/v) Nephrops serum or 5% (v/v) Nephrops muscle extract, and 0.06 g/l of L-proline and 1 g/l of glucose. Pronase at 100 g/ml improved tissue dissociation and subsequent spreading of hematopoietic cell cultures. The addition of epithelial growth factor (EGF), based fibroblast growth factor (bFGF) or insulin growth factor 1 (IGF-I) did not enhance cell growth. Cell culture contained several types of maturing hemocytes, in the size range of 6–24 µm diameter. Acid phosphatase, -naphthyl butyrate esterase, -naphthyl acetate esterase, naphthyl AS-D chloroacetate esterase activity and phenoloxidase activity was demonstrated, but not so alkaline phosphatase or peroxidase. Small PAS (= Periodic acid Schiff) positive granules, unsaturated lipids and phospholipids were observed. Cultures remained functional for over two weeks. Mitosis was noticed occasionally; however, cell proliferation was not recorded by use of nuclear proliferation markers.     

Serological characterization of bovine rotaviruses isolated from dairy and beef herds in Argentina

Bovine rotaviruses isolated from beef and dairy herds in Argentina were serotyped by the immunoperoxidase focus reduction assay as previously described (G. Gerna, M. Battaglia, G. Milenesi, N. Passarani, E. Percivalle, and E. Cattaneo, Infect. Immun. 43:722-729, 1984). Three strains from beef herds were related to the UK and NCDV bovine rotavirus strains defined as serotype 6 (Y. Hoshino, R. G. Wyatt, H. B. Greenberg, J. Flores, and A. Z. Kapikian, J. Infect. Dis. 149:694-702, 1984). Two other strains from dairy herds were classified as bovine viruses related to the bovine B223 strain reported by Woode and co-workers (G. N. Woode, N. E. Kelso, T. F. Simpson, S. K. Gaul, L. E. Evans, and L. Babiuk, J. Clin. Microbiol. 18:358-364, 1983) in the United States. A serotyping antibody-capture enzyme-linked immunoassay to detect serotype 6 rotavirus using a serotype 6-specific monoclonal antibody was developed and evaluated for strain characterization. Characterization of 72 group A rotavirus-positive fecal samples from beef herds and 43 fecal samples from dairy herds showed a predominance of serotype 6 rotavirus in beef herds but both serotype 6 and non-serotype 6 rotaviruses in dairy herds. Analysis of genomic double-stranded RNA by polyacrylamide gel electrophoresis showed that when outbreaks were caused by one serotype only a single electropherotype was present in all samples.     

Serological characterization of Proteus penneri species novum.

Serological characterization of the first collection of the 20 Proteus penneri strains is presented. All anti-0 sera were examined in microagglutination, semi-quantitative precipitation and passive hemagglutination tests. Some P. penneri lipopolysaccharides showed strong cross-reactivity in passive hemagglutination additionally confirmed by inhibition in this test. Serological similarity between species within genus Proteus is discussed.     

Serological characterization of the O-specific polysaccharide of Providencia alcalifaciens O23

INTRODUCTION: The genus Providencia belongs to the Enterobacteriaceae family and currently consists of five species: P. alcalifaciens, P. heimbachae, P. rettgerii, P. rustigianii and P. stuartii. The serological classification scheme of P. alcalifaciens, P. rustigianii and P. stuartii includes 63 O-serogroups and 30 H-serogroups. The O-antigenic specificity is defined by the structure of the O-antigen (O-specific polysaccharide--OPS), a part of the lipopolysaccharide (LPS, endotoxin), one of the major components of the outer membrane of gram-negative bacteria and an important virulence factor of these bacteria. Among the bacteria of the Enterobacteriaceae family, the genus Providencia is one of the least studied in respect to its LPS structure and antigenic specificity. Studies of the chemical structures and the serological specificity of the O-antigens aim at the elucidation of the molecular basis of the serological classification of Providencia sp. MATERIALS AND METHODS: LPS and alkali-treated LPS of P. alcalifaciens O23 and serologically related P. rustigianii O14, P. mirabilis O13 and P. myxofaciens as well as O-antiserum against P. alcalifaciens O23 were used. Serological characterization of P. alcalifaciens O23 O-specific polysaccharide was done by use enzyme immunosorbent assay (EIA), passive hemolysis test (PHT) as well as by inhibition and sodium deoxycholate polyacrylamide gel electrophoresis (DOC-PAGE) of LPS and Western blot. RESULTS AND CONCLUSIONS: The OPS of P. alcalifaciens, O23, contains an N-(D-glucuronoyl)-N-[(R)-1-carboxyethyl]-L-lysine residue (GlcAAlaLys). The LPS of P. alcalifaciens, O23, and other LPSs containing AlaLys from Providencia and Proteus strains were tested with rabbit anti-P. alcalifiaciens O23 serum. The serological data showed that a GlcAAlaLys-associated epitope plays a role as an antigenic determinant in the P. alcalifaciens O23 OPS and revealed the particular importance of glucuronic acid and the carboxyethyl group for the binding of O23-specific antibodies.     

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.     

Screening of Penicillium species for occurrence of lectins and their characterization

Out of 15 Penicillium species screened for lectin activities, P. griseofulvum and P. thomii were found to possess mycelial lectin activity. None of the species displayed extracellular or cell surface‐bound lectin activity. Both species agglutinated rabbit erythrocytes. P. griseofulvum lectin showed specificity to human type O erythrocytes. While P. thomii lectin specifically agglutinated human type A erythrocytes. Highest lectin activities from P. thomii and P. griseofulvum were expressed after 8 and 7 days of growth, respectively. Lectins from both the species displayed a high binding affinity to chondroitin‐6‐sulphate, mucin, asialofetuin, D‐sucrose, and D‐trehalose. Ammonium sulphate at 50% saturation yielded 80% of the total lectin activity. Dialysis and ultrafiltration of the precipitates resulted in 1.79 and 3.46 fold purification of P. griseofulvum and P. thomii lectins, respectively. Both lectins showed pH optima between 7.0–8.0 and were stable near the neutral pH after 2 h. P. thomii lectin exhibited optimal activity at 35–40 °C, and P. griseofulvum lectin at 30–40 °C. P. thomii lectin showed a complete loss of activity above 40 °C, P. griseofulvum lectin was stable at or below 35 °C. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)