Hemagglutinating Activity Research Articles

Preliminary Characterization of Crude Lectin Fraction of the Red Alga, Acrocystis nana from Wediombo Beach of the Southern Coast of Java Island, Gunung Kidul, Yogyakarta, Indonesia

Lectins, bioactive compounds that found in algae, are bind to sugars or glycoproteins reversibly with high specificity, but are devoid of catalytic activity, and in contrast to antibodies, are not products of an immune response. Acrocystis nana is a species of red alga that is collected from the Wediombo beach, coastal region of Gunungkidul, Yogyakarta. Hemaglutination activity of a crude lectin fraction from Acrocystis nana was examined using trypsin-treated rabbit erythrocytes and its chemical properties, including its protein content, stability on pH, temperature, and divalent cations were determined as a preliminary characterization phase. Crude lectin of Acrocystis nana showed a titer of 212 on hemagglutination activity assay and the protein content was 6225.44 µg/ml. Hemagglutination activity of this crude lectin was stable after treatment at various pH from 3 to 10 and its activity was lost by heating at 50°C until 100°C. Moreover, the hemagglutination activity was slightly affected by divalent cations treatment, indicating that the presence of divalent cations may require for its activity, however, further studies are still needed for a more comprehensive understanding about its properties.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes.

We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV) virus was determined in infected silkworm Bm-N cells by fluorescence and Western blot analysis. The recombinant was inoculated into silkworm pupae and the yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3 days. Additionally, in the peptide analysis of infected pupae homogenate, it appeared approximately 300–500 μg E protein/pupa were produced. When purified the above eluates on the discontinuous sucrose density gradient centrifugation, NVLP showed a strong hemagglutination (HA) activity by using chicken red blood cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The immune antisera against NVLP strain could efficiently neutralize the plaque formation of Nakayama, Beijing-1 and Muar strains, showing tendency of much higher reaction with heterologous Muar strain than homologous Nakayama strain. Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control in many endemic areas of Asian countries as a widely effective and less expensive JE vaccine.

Purification and characterization of a novel ubiquitin-like antitumour protein with hemagglutinating and deoxyribonuclease activities from the edible mushroom Ramaria botrytis

A novel ubiquitin-like antitumour protein (RBUP) was isolated from fruiting bodies of the edible mushroom Ramaria botrytis. The protein was isolated with a purification protocol involving ion exchange chromatography on DEAE-Sepharose fast flow and gel filtration on Sephadex G-75. SDS-PAGE, Native-PAGE and ultracentrifugation analysis disclosed that RBUP was a monomeric protein with a molecular weight of 18.5 kDa. ESI–MS/MS demonstrated that it shared 69% amino acid sequence similarity with Coprinellus congregates ubiquitin (gi|136667). The protein exhibiting strong anticancer activity towards A549 cells. Analysis by employing AO/EB staining and Annexin V-FITC/PI detection indicated that the cytotoxic effect of RBUP was mediated through induction of apoptosis. Furthermore, RBUP displayed hemagglutinating and deoxyribonuclease activities. A temperature of 40 °C and pH of 7.0 were required for optimal DNase activity. Therefore, it was estimated that RBUP exerted its antitumour effect by inducing apoptosis, and its hemagglutinating and DNase activities were also thought to participate in this effect. These results demonstrated that RBUP was a multifunctional protein with potential medicinal applications.

Differential responses of Helicoverpa armigera C-type immunlectin genes to the endoparasitoid Campoletis chlorideae.

The C-type lectins mediate nonself recognition in insects. The previous studies focused on host immunlectin response to bacterial infection; however, the molecular basis of immunlectin reactions to endoparasitoids has not been elucidated. The present study investigated the effect of parasitization by Campoletis chlorideae on hemagglutination activity (HA; defined as the ability of lectin to agglutinate erythrocytes or other cells), and transcriptional expression of C-type immunlectin genes in the larval host, Helicoverpa armigera. Parasitization induced four- to eightfold higher HA in the parasitized larvae, compared to nonparasitized larvae at days 2 and 6 postparasitization (PP), however inhibited HA at other days PP. Eight C-type lectins were differentially expressed in different host developmental stages, from feeding to wandering stage. The mRNA levels of HaCTL1, HaCTL3, HaCTL4, and HaCTL5 were upregulated and HaCTL2 and HaCTL7 were downregulated. Tissue analysis showed that HaCTLs were mainly expressed in fat body or hemocytes, while HaCTL5 was highly expressed in testes. The effects of parasitization on the lectin expression patterns differed. Lectins except HaCTL6 or HaCTL5 were significantly down- or upregulated in parasitized larvae at day 4 or 6 PP compared with that of nonparasitized larvae. We infer from our results that C-type immunlectins are involved in host-parasitoid interactions, and parasitization alter host immunlectin levels both in inhibiting and promoting host immune defenses to endoparasitoids. These immunlectin genes indicated an altered physiological status of the host insect, depending on developmental stage, tissue, and parasitization.

A Protein Isolate from Moringa oleifera Leaves Has Hypoglycemic and Antioxidant Effects in Alloxan-Induced Diabetic Mice.

Moringa oleifera has been used in traditional medicine to treat diabetes. However, few studies have been conducted to relate its antidiabetic properties to proteins. In this study, a leaf protein isolate was obtained from M. oleifera leaves, named Mo-LPI, and the hypoglycemic and antioxidant effects on alloxan-induced diabetic mice were assessed. Mo-LPI was obtained by aqueous extraction, ammonium sulphate precipitation and dialysis. The electrophoresis profile and proteolytic hydrolysis confirmed its protein nature. Mo-LPI showed hemagglutinating activity, cross-reaction with anti-insulin antibodies and precipitation after zinc addition. Single-dose intraperitoneal (i.p.) administration of Mo-LPI (500 mg/kg·bw) reduced the blood glucose level (reductions of 34.3%, 60.9% and 66.4% after 1, 3 and 5 h, respectively). The effect of Mo-LPI was also evidenced in the repeated dose test with a 56.2% reduction in the blood glucose level on the 7th day after i.p. administration. Mo-LPI did not stimulate insulin secretion in diabetic mice. Mo-LPI was also effective in reducing the oxidative stress in diabetic mice by a decrease in malondialdehyde level and increase in catalase activity. Mo-LPI (2500 mg/kg·bw) did not cause acute toxicity to mice. Mo-LPI is a promising alternative or complementary agent to treat diabetes.

Isolation, biochemical characterization and antibiofilm effect of a lectin from the marine sponge Aplysina lactuca

A new lectin was isolated from the marine sponge Aplysina lactuca (ALL) by combining ammonium sulfate precipitation and affinity chromatography on guar gum matrix. ALL showed affinity for the disaccharides α-lactose, β-lactose and lactulose (Ka=12.5, 31.9 and 145.5M−1, respectively), as well as the glycoprotein porcine stomach mucin. Its hemagglutinating activity was stable in neutral acid pH values and temperatures below 60°C. ALL is a dimeric protein formed by two covalently linked polypeptide chains. The average molecular mass, as determined by Electrospray Ionization Mass Spectrometry (ESI–MS), was 31,810±2Da. ESI–MS data also indicated the presence of three cysteines involved in one intrachain and one interchain disulfide bond. The partial amino acid sequence of ALL was determined by tandem mass spectrometry. Eight tryptic peptides presented similarity with lectin I isolated from Axinella polypoides. Its secondary structure is predominantly β-sheet, as indicated by circular dichroism (CD) spectroscopy. ALL agglutinated gram-positive and gram-negative bacterial cells, and it were able to significantly reduce the biomass of the bacterial biofilm tested at dose- dependent effect.

Anti-leukemic activity and tolerability of anti-human CD47 monoclonal antibodies

CD47, a broadly expressed cell surface protein, inhibits cell phagocytosis via interaction with phagocyte-expressed SIRPα. A variety of hematological malignancies demonstrate elevated CD47 expression, suggesting that CD47 may mediate immune escape. We discovered three unique CD47-SIRPα blocking anti-CD47 monoclonal antibodies (mAbs) with low nano-molar affinity to human and cynomolgus monkey CD47, and no hemagglutination and platelet aggregation activity. To characterize the anti-cancer activity elicited by blocking CD47, the mAbs were cloned into effector function silent and competent Fc backbones. Effector function competent mAbs demonstrated potent activity in vitro and in vivo, while effector function silent mAbs demonstrated minimal activity, indicating that blocking CD47 only leads to a therapeutic effect in the presence of Fc effector function. A non-human primate study revealed that the effector function competent mAb IgG1 C47B222-(CHO) decreased red blood cells (RBC), hematocrit and hemoglobin by >40% at 1 mg/kg, whereas the effector function silent mAb IgG2σ C47B222-(CHO) had minimal impact on RBC indices at 1 and 10 mg/kg. Taken together, our findings suggest that targeting CD47 is an attractive therapeutic anti-cancer approach. However, the anti-cancer activity observed with anti-CD47 mAbs is Fc effector dependent as are the side effects observed on RBC indices.

Sub-Micellar Concentration of Sodium Dodecyl Sulphate Prevents Thermal Denaturation Induced Aggregation of Plant Lectin, Jacalin.

The irreversible thermal unfolding of jacalin, the lectin purified from jackfruit seeds was accompanied by aggregation, where intermolecular interactions among the subunits are favoured over intramolecular interactions. The extent of aggregation increased as a function of temperature, time and protein concentration. The anionic surfactant, sodium dodecyl sulphate (SDS) significantly suppressed the formation of aggregates as observed by turbidity measurements and Rayleigh scattering assay. Moreover, far UV-CD spectra indicate that the protein β sheet transforms into α helical structure, when denatured in the presence of 3mM SDS. Further, jacalin when heated in the presence of SDS partially retained the hemagglutination activity when jacalin-SDS mixture was diluted to 1:8 factor since 3mM SDS was found to lyse the red blood cells. Thus, SDS only altered the aggregation behaviour of jacalin by preventing intermolecular hydrogen bonding among the exposed residues but did not completely stabilize the native conformation.

Secretory Expression and Characterization of Chinese Narcissus GNA-Like Lectin in Pichia pastoris.

Narcissus tazetta lectin (NTL) is a GNA-like lectin, which has a wide potential application in medicine, agriculture, and glycobiology. In the present paper, the codon-optimized ntl gene was transformed into the yeast Pichia pastoris; SDS-PAGE gel and western blotting analysis revealed that the recombinant lectin was expressed successfully in Pichia yeast. The similarity between the recombinant NTL and the native NTL was confirmed by circular dichroism (CD) and hemagglutination assay further. In the 5-L scale fermentator, the protein yield was as high as 1.2g/L after fermentation for 96h. In addition, the effect of metal ions (K+, Mg2+, Ca2+, and Cu2+), acid, and alkaline on hemagglutinating activity of NTL was tested, which provided biochemical characterizations of the mannose-binding lectin from Chinese Narcissus.

Dimethyl-2-[(acridin-9-yl)methylidene]-malonate as fluorescent probe for histochemical analysis.

Fluorescent compounds have been widely used for biomolecule labeling in cytochemistry and histochemistry analysis. Here, it is described the optical properties of dimethyl 2-[(acridin-9-yl)methylidene]-malonate (LPSF/IP-81), an acridine derivative. This compound was conjugated to Concanavalin A (Con A) lectin and applied as sugar probe in lectin histochemistry. Evaluation of luminescent properties showed that LPSF/IP-81 is photoluminescent with excitation at 360 nm and emission at 428 nm. Con A hemagglutinating activity and LPSF/IP81 photoluminescence were unaltered after conjugation. Circular dichroism of Con A-LPSF/IP81 conjugate showed the maintenance of the Con A structure. Lectin histochemistry with Con A-LPSF/IP81 conjugate demonstrated different pattern recognition studying normal, fibroadenoma, and invasive ductal carcinoma of human breast. These findings indicate that LPSF/IP-81 can be proposed as an alternative probe for histochemical analysis.

Straightforward Synthesis of N-Glycan Polymers from Free Glycans via Cyanoxyl Free Radical-Mediated Polymerization.

We report a straightforward synthesis of N-glycan polymers from free glycans via glycosylamine intermediates followed by acrylation and polymerization via cyanoxyl-mediated free radical polymerization (CMFRP) in one-pot fashion. No protection and deprotection were used in either glycomonomer or glycopolymer synthesis. A typical synthetic procedure for N-glycan polymers from free monosaccharide and disaccharide, Glc, Gal, Man, GlcNAc, and Lac, was demonstrated. In addition, enzymatic sialylation of the Lac-containing N-glycan polymers and their anti-influenza virus hemagglutination activities were investigated.

Multi-antigen avian influenza a (H7N9) virus-like particles: particulate characterizations and immunogenicity evaluation in murine and avian models

BackgroundHuman infection with avian influenza A virus (H7N9) was first reported in China in March 2013. Since then, hundreds of cases have been confirmed showing severe symptoms with a high mortality rate. The virus was transmitted from avian species to humans and has spread to many neighboring areas, raising serious concerns over its pandemic potential. Towards containing the disease, the goal of this study is to prepare a virus-like particle (VLP) that consists of hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) derived from the human isolate A/Taiwan/S02076/2013(H7N9) for potential vaccine development.ResultsFull length HA, NA, and M1 protein genes were cloned and expressed using a baculoviral expression system, and the VLPs were generated by co-infecting insect cells with three respective recombinant baculoviruses. Nanoparticle tracking analysis and transmission electron microscopy were applied to verify the VLPs’ structure and antigenicity, and the multiplicity of infection of the recombinant baculoviruses was adjusted to achieve the highest hemagglutination activity. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization showed elevated hemagglutination inhibition serum titer and antibodies against NA and M1 proteins. In addition, examination of cellular immunity showed the VLP-immunized mice and chickens exhibited an increased splenic antigen-specific cytokines production.ConclusionsThe H7N9 VLPs possess desirable immunogenicity in vivo and may serve as a candidate for vaccine development against avian influenza A (H7N9) infection.

Lectin from the Late Oyster Mushroom, Hohenbuehelia serotina (Agaricomycetes), and Its Novel Effect as an Adjuvant of the HBV DNA Vaccine.

The biological macromolecule Hohenbuehelia serotina lectin (HSL) was first isolated from dried fruiting bodies of the mushroom H. serotina and identified as a heterodimer with 2 subunits of the same molecular weight (15.6 kDa) but different isoelectric points. Lactose and d-galactose inhibited the hemagglutinating activity of HSL, whereas mental ions Mn2+ and Ca2+ could stimulate its hemagglutination. The HSL hemagglutinating activity was stable for 1 hour in NaOH and HCl solutions up to a concentration of 12.5 or 6 mmol/L. In addition, HSL was stable up to 50°C for 30 minutes; its hemagglutinating activity was halved at 60°C and totally inactivated above 90°C. HSL (10 μg) as an immune adjuvant co-inoculated with the proVAX/S2 vaccine enhanced the level of hepatitis B surface antigen in C57BL/6 mice, induced a high level of T-cell proliferation, and induced the expression of IFN- of CD4+ cells. We further illustrate that HSL, as an adjuvant upregulating the expression of major histocompatibility complex II, contributed to the maturation of dendritic cells. As the first lectin isolated from H. serotina, HSL is a potential adjuvant to chronic hepatitis B virus DNA vaccines and lays a foundation for the prevention of HBV infection.

Genomic Recombination Enhances Pathogenic Factors in the Periodontopathogenic Bacterium <i>Eikenella corrodens</i>

We reported previously that plasmid-mediated genomic recombination at the pilin gene locus increased hemagglutination activity, growth rate, biofilm formation, hemolytic activity, and adherence to epithelial cells in Eikenella corrodens 23834. To determine whether these enhancements were common in this bacterium, we introduced the recombinase gene ORF4 into seven clinically isolated strains. Genomic recombination at the type IV pilin gene locus was observed in strains 1080, L9B6, L8Ao3, and RV2 (group A), but not in strains 261-2, 612-L, and 257-4 (group B). Similarly, group A strains displayed changed colony morphology following loss of type IV pili, which was not observed in group B. Group A strains showed also enhanced hemagglutination activity, growth rate, hemolytic, activity and biofilm formation. These results suggest that ORF4-induced genomic recombination at the pilin gene locus is a general phenomenon in a part of E. corrodens, which likely stimulates patho-genicity and virulence.

Interaction of Glucose/Sucrose Binding Lectin Isolated from Nigeria Wild Bean with <i>E. coli</i> and <i>S. aureus</i>

Lectin purified from wild underutilized local bean—Otili, Feregede, Pakalai was comparatively characterized and further evaluated for interaction with gastrointestinal bacteria—Esherichia coli and Staphylococcus aureus. The purified lectin in all the bean samples showed to be glucose and sucrose binding. The hemagglutinating activity, was non selective to type of blood group (A, B, AB and O). Anti-bacteria interaction with Escherichia coli showed clear zone of inhibition of about 1.5 ± 0.5 mm with lectin from Feregede and Otili while there was slight agglutination with lectin from Pakala. Staphylococcus aureus sensitivity to the lectin extracted from Otili with clear zone of inhibition of 2.0 ± 0.5 mm was also found in the control chloramphenicol. However there was pronounced agglutination with lectin from Feregede and Pakala with Staphylococcus auereus. This may be a clear indication that lectin from local underutilized wild bean understudy will agglutinate and interact with a gram positive bacteria more than gram negative bacteria.

Antimicrobial Activities of Lectins Extracted from Some Cultivars of Phaseolus vulgaris Seeds

Lectins of five cultivars of Phaseolus vulgaris seeds were isolated using ammonium sulfate precipitation followed by dialysis and their molecular characterization were determined using SDS-PAGE. The suggested regions of all isolated lectins ranged between 31 to 34 kDa. The isolated lectins demonstrated remarkable hemagglutination activity to all human blood groups (A, B, AB and O). The antimicrobial activity was studied using agar-well diffusion method and minimum inhibitory concentration (MIC) was determined. The results showed that the lectins of all tested Phaseolus vulgaris seeds had a potent antibacterial activity against all bacterial strains studied (Staphylococcus aureus ATCC 6538, and Streptococcus mutants ATCC 25175, Pseudomonas aeruginosa ATCC 10145 and Klebsiella pneumonia) with the exception of Escherichia coli 0157: HZ ATCC 51659 that was not affected. All tested lectins showed antifungal activity against Candida albicans. As far as we are aware, our work is the first approach that showed photographs of scanning electron microscope (SEM) before and after treatment of 90% saturated fraction of the lectins extracted from the Egyptian Shalatine cv. seeds against Staphylococcus aureus and Pseudomonas aeruginosa, where a potential agglutination of bacterial cells was shown.

Protein fractionation and its Invitro hemagglutinating activity of Star anise extracts

Star anise ( Illicium verum ) is an aromatic evergreen tree bearing purple-red flowers and anise-scented star-shaped fruit. It carry chemical compounds known to have antioxidant, disease preventing and health promoting properties. It's application is in many traditional medicines such as antispasmodic, digestive stimulant and toxic agent. The objective of the study was to fractionate proteins based on solubility and assess its haemgglutinating activity. Protein fractionation was carried out using different solvent such as aqueous, 1.0 M Nacl, 70% ethanol and 0.2% NaOH to extract albumin, globulin, prolamin and glutelins respectively.. The amount of proteins was quantitatively measured. The result of protein fractionation indicated increase in glutelin and decrease in albumin content. The obtained protein fractions were used for determining hemagglutination activity. Agglutination was observed in prolamin fractions and not for the other fractions.

Isolation and Characterization of Lectin from the Artist's Conk Medicinal Mushroom, Ganoderma applanatum (Agaricomycetes), and Evaluation of Its Antiproliferative Activity in HT-29 Colon Cancer Cells.

The growth and lectin production of Ganoderma applanatum, a white rot fungus, was optimized in broth cultures. The fungus was found to have a higher growth rate and higher lectin activity when grown in a medium adjusted to pH 6.5 at 26°C under stationary conditions. Expression of lectin activity started in 5-day-old mycelial culture; maximum activity was expressed after the 15th day of incubation. Among the various carbon and nitrogen sources tested, the carbon source sucrose and the nitrogen source yeast extract support maximum growth and lectin production. Lectin from G. applanatum was purified by ammonium sulfate precipitation and ion exchange chromatography. The purified fraction revealed a single band with a molecular weight of 35.0 kDa. Moreover, carbohydrates such as mannitol, glucose, sucrose, maltose, mannose, galactose, sorbose, and fructose were found to inhibit the hemagglutinating activity of the lectin. The purified lectins from G. applanatum contain cytotoxic and proapoptotic activities against HT-29 colon adenocarcinoma cells.

Properties of induced antimicrobial activity in Musca domestica larvae.

Insects produce antimicrobial molecules that contribute to their innate immune responses to eliminate invading microorganisms. To explore the potential utility of these antimicrobial molecules, we focused on larvae of the house fly Musca domestica, which is an efficient processor of organic waste and a good resource of protein and oil for animal feeding. The induction of hemagglutinating activity, which is usually accompanied by activation of innate immune responses in fly larvae, was observed in the hemolymph following needle injury. Hemolymph collected from injured larvae demonstrated potent antimicrobial activities against both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa. Furthermore, the antimicrobial activity was significantly retained in hemolymph after heat-treatments, suggesting that pasteurization of animal feed prepared from fly larvae would be a useful sterilization method. These observations indicate that injured Musca domestica larvae are a source of antimicrobial agents, and highlight the utility of preparing animal feed from these larvae.

Steric exclusion chromatography for purification of cell culture-derived influenza A virus using regenerated cellulose membranes and polyethylene glycol

Steric exclusion chromatography has been used for the purification of proteins and bacteriophages using monoliths. The operation is carried out by mixing a crude sample containing the target species with a predetermined concentration and molecular weight of polyethylene glycol (PEG) and loading it onto a non-reactive hydrophilic surface. Product capture occurs by the mutual steric exclusion of PEG between the product and the matrix. Selectivity is significantly influenced by target product size. Product elution is achieved by decreasing the PEG concentration. In this study, a 75cm2 cellulose membrane adsorber was used for the purification of a clarified and inactivated influenza A virus broth produced in a 5L bioreactor using suspension Madin Darby canine kidney cells. Product recovery was above 95% based on hemagglutination activity and single radial immunodiffusion assays. Maximum depletion of double stranded host cell DNA and total protein was 99.7% and 92.4%, respectively. Purified virus particles showed no aggregation with a monodisperse peak around 84nm. 250mL of the clarified inactivated virus broth was purified within 40min. The surface area productivity based on the recovery of the viral hemagglutinin antigen was 28–50mgm−2h−1 depending on the feed and loading conditions.