Agglutinated Rabbit Red Blood Cells Research Articles

Target-directed isolation and identification of a serum lectin from lamprey (Lampetra japonica) by chromatographys and MALDI-TOF/TOF

A 105-kDa polymer lectin was purified from lamprey (Lampetra japonica) serum by chromatography methods including cation ion-exchange chromatography with a SP-Sepharose™ XL column and size exclusion chromatography with a Superdex 200 column. The target fractions were collected according to the direction of hemagglutinating activity. The results revealed that the active fractions could adsorb on SP-Sepharose column and showed a 280 nm UV absorbance peak corresponding to molecular weights of 105 kDa in the following size exclusion chromatography. The target fractions with hemagglutinating activity were further checked by Native- PAGE and SDS-PAGE. Two single bands at around 105 kDa and 35 kDa were displayed by two electrophoresis methods respectively, indicating that the protein exists as a trimer in solution. After Native-PAGE and SDS-PAGE, two bands were excised from the gels respectively and further identified by MALDI-TOF/TOF as serum lectin (gi: 13094239). The lectin was able to agglutinate rabbit red blood cells (RRBCs) and sheep red blood cells (SRBCs) in vitro. The lectin isolated from lamprey serum in the current study might be helpful for deeply understanding the innate immune molecules dependent immune defence in jawless vertebrates which have been proved recently that they possess a lymphocyte-based system of anticipatory immunity with variable lymphocyte receptors as mediators.

Serum immune response of pearl oyster Pinctada fucata to xenografts and allografts

The mantle piece from the donor pearl oyster would be rejected by the immune system of recipient oyster in pearl culture practice, especially in the case that the donor and receptor are different species. Thus, investigation of the immune response of recipient oyster to grafted mantle pieces, particularly to xenografts, is of importance in creating xenograft transplantation technology for pearl culture industry. The humoral immune responses of P. fucata to allograft (mantle piece of P. fucata) and xenografts (mantle pieces of P. maxima and P. margaritifera, respectively) were studied in this paper. The oysters receiving no transplantations were served as the control group. The serum was collected from recipient P. fucata at 1 d, 2 d, 3 d, 4 d, 5 d, 7 d, 9 d, 11 d, 13 d, and 15 d, respectively after transplantation, and the serum antibacterial activity, lysozyme activity (LZM), alkaline phosphatase (AKP), acid phosphatase (ACP), total antioxidant capacity (TAC), and agglutination to rabbit red blood cells were investigated. The result indicated that serum of both the experimental groups and the control group can agglutinate rabbit red blood cells, with variation between groups and between time points, respectively. The antibacterial activity in the experimental group was significantly higher than that in the control group at 2–4 d, but lower at 5–11 d and returned back to normal at 15 d, with significant differences among experimental groups (P < 0.05). The LZM in the experimental group was significantly higher than that in the control group at 3–7 d, with significant differences in bacteriolytic activity among various groups (P < 0.05). Both the ACP and AKP activity levels in the experimental groups were higher than those in the control group at 2–9 d, with significant differences among various groups at 3–9 d (P < 0.05). The TAC level in the experimental groups was higher than that in the control group at 1–7 d, with significant differences among various groups at 4–7 d (P < 0.05). The above results indicated that all of the humoral immune factors investigated showed immune responses to both allografts and xenografts, with no specific to any of them. Thus, it is necessary to further screen immune rejection factors specific to xenografts, including cellular immune components.

Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)

In this study, two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIPs (rFIPs) were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to the well-known FIPs, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced rFIPs: rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.

Host Protein Binding and Adhesive Properties of H6 and H7 Flagella of Attaching and Effacing Escherichia coli

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.

Skin lectin and the lymphoid tissues in the leptocephalus larvae of the Japanese eel Anguilla japonica

SUMMARY: Development of skin lectin and the lymphoid tissues were studied in the leptocephalus larvae of the Japanese eel Anguilla japonica. The specimens ranging in total length from 11 to 58 mm were captured at 12–19°N, 131–137°E and at 21–23°N, 123–128°E in the Pacific Ocean. The skin of leptocephali contained extremely active lectin which agglutinated rabbit red blood cells the same as adults. Club cells, known as lectin secreting cells, were also recognized in the epidermis of leptocephali, although the shape was not an elongated club form but oval. The cells were confirmed to contain lectin in the secretory vacuole by an immunofluorescence technique. The lectin in the cells was also recognized in the 8 days post-hatch preleptocephalous larvae, which was obtained from an artificially spawned eel, suggesting the importance of the lectin in the early larval development. In contrast, lymphoid tissues concerning immune functions showed delayed development with the exception of the thymus. No blood cells were seen in the kidney and spleen, even in a large specimen. Only a few undifferentiated leukocyte-like cells could be observed; some of them showed phagocytic figures. The thymus along with many thymocytes could be seen in the smallest leptocephalus of total length (TL) 11 mm. T cells may have a function in defense mechanisms during the leptocephalus stage, although other immune cells were still underdeveloped.

C-reactive protein in eel: purification and agglutinating activity

C-reactive protein was highly purified from Japanese eel ( Anguilla japonica) serum by precipitation with phosphatidylcholine and Ca 2+. On SDS-PAGE, eel C-reactive protein (eCRP) migrated as a single band with a molecular weight of 24 000 under reducing and 23 500 under non-reducing conditions. The molecular weight of native eCRP was estimated to be 120 000 by Sephacryl S-300 gel filtration. The eCRP was detected within the albumin region on immunoelectrophoresis. The eCRP showed an agglutining activity against Streptococcus pneumoniae in the presence of Ca 2+, and the activity was inhibited by 1 mM EDTA or 1 mM phosphorylcholine (PC). The eCRP also agglutinated rabbit red blood cells (RRBC), but not human and five other kinds of red blood cell. The hemagglutinating activity was inhibited by glucosamine or mannose. The eCRP formed a precipitin line with histone, protamine, poly( l-lysine and poly( l-arginine) in agarose gel. The serum levels of eCRP were distributed in 6.8 ng/ml-5.3 mg/ml, n=187, the mean value being 834 ng/ml.

Cloning of the carbohydrate-binding portion of the toxin A gene ofClostridium difficile

A portion of the toxin A gene ofClostridium difficile was cloned into pBR322 withEscherichia coli Chi 1776 as the host. Five identical clones, each containing a 4.7-kbPstI restriction endonuclease fragment and producing toxin A antigens, were detected with affinity-purified, monospecific antibodies against toxin A. Digestion of the cloned DNA withPstI revealed as internal restriction site that resulted in two fragments (2.1 and 2.6 kb in size). Probe DNA from either of these fragments hybridized with DNA in the 4.7 kb region ofPstI-digested, high-molecular-weight DNA from the sourceC. difficile strain, indicating that the internalPstI site is protected. The probe DNA also hybridized with restriction-digested DNA from five additional toxigenic strains, but it did not hybridize with DNA from four nontoxigenic strains. In addition, a DNA fragment from a toxigenic strain ofClostridium sordellii, whose toxin cross-reacts with antibody toC. difficile toxin A, hybridized with the clonedC. difficile DNA. Unlike native toxin A, the cell lysate from the recombinant clone was not cytotoxic to Chinese hamster ovary cells or enterotoxic in hamsters. It did agglutinate rabbit red blood cells, a characteristic of toxin A. The cell lysate also exhibited a line of partial identity when compared with purified toxin A in Ouchterlony assays, and it reacted with monoclonal antibody to toxin A in an enzyme-linked immunosorbent assay. The cloned DNA appears to code for a nontoxic binding portion of toxin A, which is responsible for binding to galactose-α1-3-galactose-β1-4-N-acetylglucosamine.

Demonstration of the mucous hemagglutinin in the club cells of eel skin

Eel epidermis was subjected to immunocytochemical study in order to clarify the origin of mucus hemagglutinin. Skin mucus extract was applied on a Sephacryl S-400 column. The active fractions of the hemagglutinin, which agglutinated rabbit red blood cells, were collected. These fractions were contaminated with a hemolysin. Therefore, after inactivation of the hemolysin by heating at 45°C for 30min, they were reacted with rabbit red blood cells. The agglutinated cells were hemolysed to make a hemagglutinin-cell membrane complex. Rabbits were immunized with the complex, and antisera against the hemagglutinin were obtained. Skin sections of eel were stained immunocytochemically by a peroxidase-antiperoxidase (PAP) method using the rabbit anti-hemagglutinin serum. Positive reaction for the hemagglutinin was seen in club cells, especially in secretory vacuoles. This fact indicates the mucous hemagglutinin is derived from the club cells in eel.

The lectin-like activity of Helix aspersa mucus

1. 1. Helix mucus agglutinated rabbit red blood cells but not ox or human types A, B or O. Sheep cells were weakly agglutinated. The agglutination was inhibited by d -Ga1NAc and to a lesser extent d -GlcNAc and d -Gal. 2. 2. The lectin-like activity was lost above 80°C. Treatment at 60°C raised the activity several-fold over levels exhibited between -20 and 50°C. 3. 3. The activity was reduced by treatment of mucus with trypsin or pronase. The activity is ascribed to a protein component of the mucus and expressed as a titre based on protein content. The titre of mucus from individual snails varied widely. 4. 4. Mucus activity was stable for periods of up to 6 months. The protein content also remained stable providing microorganism growth was inhibited. Contrary to a previous report, no proteolytic activity associated with mucus was detected. Rehydration of dry mucus restored activity. 5. 5. The lectin-like activity was not an expression of an α-galactosidase-lectin.