Antiviral Effect of Ginsenosides rk1 against Influenza a Virus Infection by Targeting the Hemagglutinin 1-Mediated Virus Attachment.

Influenza A virus (IAV) infections have been a serious hazard to public health everywhere. With the growing concern of drug-resistant IAV strains, there is an urgent need for novel anti-IAV medications, especially those with alternative mechanisms of action. Hemagglutinin (HA), an IAV glycoprotein, plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a good target for developing anti-IAV drugs. Panax ginseng is a widely used herb in traditional medicine with extensive biological effects in various disease models, and its extract was reported to show protection in IAV-infected mice. However, the main effective anti-IAV constituents in panax ginseng remain unclear. Here, we report that ginsenoside rk1 (G-rk1) and G-rg5, out of the 23 screened ginsenosides, exhibit significant antiviral effects against 3 different IAV subtypes (H1N1, H5N1, and H3N2) in vitro. Mechanistically, G-rk1 blocked IAV binding to sialic acid in a hemagglutination inhibition (HAI) assay and an indirect ELISA assay; more importantly, we showed that G-rk1 interacted with HA1 in a dose-dependent manner in a surface plasmon resonance (SPR) analysis. Furthermore, G-rk1 treatment by intranasal inoculation effectively reduced the weight loss and mortality of mice challenged with a lethal dose of influenza virus A/Puerto Rico/8/34 (PR8). In conclusion, our findings reveal for the first time that G-rk1 possesses potent anti-IAV effects in vitro and in vivo. We have also identified and characterized with a direct binding assay a novel ginseng-derived IAV HA1 inhibitor for the first time, which could present potential approaches to prevent and treat IAV infections.

Antioxidant and Biological Activities of Proteinaceous Extract from Algerian Glycine max Plant.

OBJECTIVE: Glycine max is commonly used in Algeria for treatment of anemia deficiency and osteoporosis, it ranks first in terms of vegetal proteins. The experiment was aimed at characterizing the proteinaceous Glycine max extract and evaluating its antioxidant, biological and hematological potential. METHODOLOGY: Extraction of proteinaceous materials from Glycine max plant was undertaken using water and n-hexane as extracting media. The isolation of proteins from the crude materials was done, providing the use of ammonium sulfate. The Glycine max proteins were characterized by UV-visible and FT-IR spectroscopy and analyzed by SEM micrograph and x-ray diffraction (XRD). Rheological parameters G' and G'' were assessed. The isolated proteins were tested for their antioxidant, antimicrobial and hemagglutination activities. RESULTS: There was a gelling effect of the protein extract which can be used as an alternative in principally made vaccines with its microbiological and antifungal activities. CONCLUSION: The proteinaceous extract from Algerian Glycine max would have a potential use in biomedical application.

Isolation of a Hemagglutinin with Potent Antiproliferative Activity and a Large Antifungal Defensin from Phaseolus vulgaris cv. Hokkaido Large Pinto Beans.

Lectins (hemagglutinins) are defined as sugar-binding proteins or glycoproteins with various biological activities. A 60 kDa dimeric hemagglutinin with a blocked N-terminus was isolated in large yield (190 mg/60 g) from the common edible bean Phaseolus vulgaris cv. Hokkaido large pinto bean. Its hemagglutinating, antifungal, and antitumor activities as well as the effects of carbohydrate and metal ions on its hemagglutinating activity were examined. It inhibited the proliferation of nasopharyngeal carcinoma (CNE2), human breast cancer (MCF7), and hepatoma (HepG2) cells. The IC50 values toward HepG2, MCF7, and CNE2 cells after treatment for 48 h were 8.1, 6.07, and 7.49 µM, respectively, which were relatively low among lectins of different P. vulgaris cultivars. From the pinto beans, a 10888 Da antifungal peptide with similarity to plant defensins as revealed by mass spectroscopic analysis was also isolated with a yield of 3.2 mg of proteins from 60 g of beans. The large defensin was capable of inhibiting mycelial growth in Mycosphaerella arachidicola, Setosphaeria turcica, Bipolaris maydis, and Fusarium oxysporum but not in Valsa mali.

Purification and Characterization of a Novel Hemagglutinin with Inhibitory Activity toward Osteocarcinoma Cells from Northeast China Black Beans.

In the present study, we isolated a novel hemagglutinin from an edible legume and explored its growth-inhibitory effect on osteocarcinoma and liver cancer cells. The protein was purified by liquid chromatography techniques which entailed affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono Q, and gel filtration on Superdex 75 with an FPLC system. The hemagglutinating activity of this hemagglutinin was demonstrated to be ion dependent and stable over a wide range of temperature and pH values. Antiproliferative activity was observed in the tumor cell lines MG-63 and HepG2 but not in the normal cell line WRL 68. Osteocarcinoma cells treated with the hemagglutinin underwent obvious cell shrinkage, chromatin condensation, mitochondrial membrane depolarization, and apoptosis. The mRNA expression level of interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-1 beta (IL-1ß), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) were found to be up-regulated to different extents after treatment of this hemagglutinin.

Purification and characterization of a novel lectin from Chinese leek seeds.

A novel lectin, CLSL, was purified from Chinese leek seeds by ion exchange chromatography on SP Sephadex C-25 and gel filtration chromatography on Sephadex G50. The lectin had a molecular weight of 23.6 kDa and was composed of two identical subunits linked by disulfide bonds, a conclusion based on SDS-PAGE under reducing and nonreducing conditions. CLSL was a glycoprotein with a carbohydrate content of 3.6%. It exerted potent agglutinating activity against rat red blood cells at a concentration of 8.9 µg/mL. Hemagglutination of rat erythrocytes was inhibited by d-fructose, mannitol, and sorbose at the concentration of 20 mM. The hemagglutinating activity of CLSL was maintained at 100 °C for 60 min and under acidic pH conditions but was lost at neutral and alkaline pH conditions. The hemagglutinating activity was stimulated by Ca(2+), Fe(2+), and Cu(2+) but inactivated by Ba(2+) at a concentration of 10 mM. Ba(2+)-mediated inactivation of CLSL was caused by CLSL conformational change induced by barium ions, according to the results of circular dichroism and fluorescence spectroscopy. Deconvolution of the CLSL circular dichroism indicated that it was an α-helical lectin with α-helix and ß-fold contents of 35.8% and 8.6%, respectively. CLSL could also selectively inhibit cell proliferation.

Purification, partial characterization and immobilization of a mannose-specific lectin from seeds of Dioclea lasiophylla mart.

Lectin from the seeds of Dioclea lasiophylla (DlyL) was purified in a single step by affinity chromatography on a Sephadex® G-50 column. DlyL strongly agglutinated rabbit erythrocytes and was inhibited by monosaccharides (D-mannose and α-methyl-D-mannoside) and glycoproteins (ovalbumin and fetuin). Similar to other Diocleinae lectins, DlyL has three chains, α, ß and γ, with mass of 25,569 ± 2, 12,998 ± 1 and 12,588 ± 1 Da, respectively, and has no disulfide bonds. The hemagglutinating activity of DlyL was optimal in pH 8.0, stable at a temperature of 70 °C and decreased in EDTA solution, indicating that lectin activity is dependent on divalent metals. DlyL exhibited low toxicity on Artemia sp. nauplii, but this effect was dependent on the concentration of lectin in solution. DlyL immobilized on cyanogen bromide-activated Sepharose® 4B bound 0.917 mg of ovalbumin per cycle, showing the ability to become a tool for glycoproteomics studies.

Biological activities of aqueous and organic extracts from tropical marine sponges.

We report on screening tests of 66 extracts obtained from 35 marine sponge species from the Caribbean Sea (Curaçao) and from eight species from the Great Barrier Reef (Lizard Island). Extracts were prepared in aqueous and organic solvents and were tested for hemolytic, hemagglutinating, antibacterial and anti-acetylcholinesterase (AChE) activities, as well as their ability to inhibit or activate cell protein phosphatase 1 (PP1). The most interesting activities were obtained from extracts of Ircinia felix, Pandaros acanthifolium, Topsentia ophiraphidites, Verongula rigida and Neofibularia nolitangere. Aqueous and organic extracts of I. felix and V. rigida showed strong antibacterial activity. Topsentia aqueous and some organic extracts were strongly hemolytic, as were all organic extracts from I. felix. The strongest hemolytic activity was observed in aqueous extracts from P. acanthifolium. Organic extracts of N. nolitangere and I. felix inhibited PP1. The aqueous extract from Myrmekioderma styx possessed the strongest hemagglutinating activity, whilst AChE inhibiting activity was found only in a few sponges and was generally weak, except in the methanolic extract of T. ophiraphidites.

Isolation and characterization of a French bean hemagglutinin with antitumor, antifungal, and anti-HIV-1 reverse transcriptase activities and an exceptionally high yield.

A dimeric 64-kDa hemagglutinin was isolated with a high yield from dried Phaseolus vulgaris cultivar "French bean number 35" seeds using a chromatographic protocol that involved Blue-Sepharose, Q-Sepharose, and Superdex 75. The yield was exceptionally high (1.1g hemagglutinin per 100g seed), which is around 10-85 times higher than other Phaseolus cultivars. Its N-terminal sequence resembled those of other Phaseolus hemagglutinins. The hemagglutinating activity of the hemagglutinin was stable in the pH range 6-8, and in the temperature range 0 degrees C-50 degrees C. It inhibited HIV-1 reverse transcriptase with an IC50 of 2microM. It suppressed mycelial growth in Valsa mali with an IC50 of 10microM. It inhibited proliferation of hepatoma HepG2 cells and breast cancer MCF-7 cells with an IC50 of 100 and 2microM, respectively. It had no antiproliferative effect on normal embryonic liver WRL68 cells.

Effect of gamma-immunoglobulin on the asaccharolytic growth of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVES: A minimal medium is indispensable for examining the growth properties of the asaccharolytic bacterium, Porphyromonas gingivalis. The purpose of the present study was to improve the widely used KGB medium to support good growth of P. gingivalis. MATERIAL AND METHODS: Growth of P. gingivalis (W50, W83, and ATCC33277) in a minimal medium was monitored by measuring the optical density of the culture during incubation. RESULTS: W50, W83, and ATCC33277 grew poorly with bovine serum albumin as the sole carbon and nitrogen source, and alpha-ketoglutarate had little or no effect on this poor growth. In contrast, FeCl3 improved the growth of W83 and ATCC33277; however, the use of a high concentration of FeCl3 elicited black pigmentation of the cells. Bovine gamma-immunoglobulin greatly recovered the growth defect. None of alpha-ketoglutarate, citrate, or trace metal ions, when used to supplement KGB medium, was required for growth. We determined the optimal conditions for growth, and developed a new simple minimal medium for P. gingivalis (GA medium). Growth of ATCC33277 in GA medium was dependent on gingipains; Arg-gingipains and Lys-gingipain contributed comparably to proliferation of the bacterium. CONCLUSION: These data indicate that GA medium is currently the most reliable minimal medium for examining the growth properties of P. gingivalis.

A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus.

A dimeric lectin, composed of subunits with a molecular weight of 40 and 41 kDa, respectively, and demonstrating similarity in N-terminal sequence to each other and to Aleuria aurantia lectin, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus ostreatus. The lectin was unadsorbed on DEAE-cellulose in 10 mmol/L phosphate buffer (pH 7.5), subsequently adsorbed on CM-cellulose in 10 mmol/L NH(4)OAc (pH 5.4), and came off in the first peak from a Superose 12 column during fast protein liquid chromatography. The lectin was acid-labile, alkali-labile, and heat-labile. Its hemagglutinating activity was sensitive to inhibition by CaCl(2), MgCl(2), MnCL(2) and FeCl(3) and potentiation by AlCl(3). Melibiose, lactose, d-galactose, alpha-methyl-d-galactopyranoside, N-acetylneuraminic acid, raffinose, and inulin were capable of inhibiting its hemagglutinating activity, with melibiose being the most potent. The lectin exerted potent antitumor activity in mice bearing sarcoma S-180 and hepatoma H-22. Survival in these mice was prolonged and body weight increase reduced after lectin treatment.

Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation.

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.

Mutational studies of the amino acid residues in the combining site of Erythrina corallodendron lectin.

High-resolution X-ray crystallography of the complex of the Gal/GalNAc-specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccharides and oligosaccharides by the lectin was examined by site-directed mutagenesis. Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contrary to an earlier assumption. Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N-acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide. Comparison with other legume lectins specific for mannose/glucose, galactose, N-acetylgalactosamine, L-fucose or N-acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.

[Soluble beta-galactoside-binding lectins]. / Lectinas solubles beta-galactosídicas.

Animal lectins are classified on the basis of structural and functional studies in two types: the C-type, characterized by their dependence on calcium ions and the S-type which are not calcium-dependent, but thiol-dependent. In this late one, a group has been extensively studied as the S-Lac type. They are extracted with saline buffers added with lactose in presence of thiol agents, and constitute a family of structurally related protein which contain a series of conserved amino acids. They specifically bind to complementary glicoconjugates, and their biosynthesis and localization are developmentally regulated. Their role could be related to several biological activities in different organs.

Properties of hemagglutinins newly separated from toxic phytoplankton.

Unique substances of the hemagglutinating activity were separated from unialgal cultures of the toxic phytoflagellates, Chattonella marina and Gymnodinium sp. Molecular masses of the substances were estimated to be 14,000 in Chattonella and 15,000 in Gymnodinium. These two substances contained phosphorus, hexose (galactose) and glycerol in the following molar ratios; 1.00:1.18:1.24 (Chattonella) and 1.00:1.07:1.10 (Gymnodinium). Hemagglutinating titres using rabbit red blood cells were inferred to be 25,000 in both preparations. In the presence of Chattonella hemagglutinin (0.10-0.50 microM), the growth of C. marina in Provasoli's ES media was little affected during 24 h of incubation, whereas a marked suppression took place in growth in Gymnodinium sp. (50% inhibition, 0.12 microM) or the diatom Nitzschia closterium (0.17 microM). On the other hand, Gymnodinium hemagglutinin inhibited the growth of the three species of phytoplankton. A 50% inhibition occurred at 0.12 microM in C. marina, at 0.22 microM in N. closterium or at 0.50 microM in Gymnodinium sp.

Efficient induction of superoxide release from human neutrophils by the galactoside-specific lectin from Viscum album.

The immunomodulatory galactoside-specific lectin from Viscum album (VAA) induces superoxide anion (O2.-) release from human neutrophils. Among twelve tested lectins, VAA, has the highest activity, clearly surpassing the effect of the human beta-galactoside-specific lectin (galaptin). Its reactivity is blocked in the presence of lactose and is strictly dependent on the carbohydrate-binding B-subunit. The toxic A-subunit of the lectin does not elicit a response. The VAA-induced respiratory burst is less sensitive to addition of adenosine and theophylline than the concanavalin A-mediated reaction. Other modulators like amiloride (up to 100 microM), trifluoperazine and N-ethylmaleimide reveal less pronounced differences, and indomethacin, colchicine (up to 100 microM) as well as cytochalasin B act as stimulators of lectin-induced O2.- release from neutrophils. The O2.- production in the presence of small concentrations of VAA (0.1-20 micrograms/ml) is increased by addition of N-formylmethionyl-leucyl-phenylalanine (FMLP), phorbol 12-myristate 13-acetate (PMA) or digitonin, respectively, whereas concanavalin A (Con A) or wheat germ agglutinin (WGA) fail to affect the VAA-dependent extent of the respiratory burst. These results substantiate that the VAA-induced O2.- release from human neutrophils can be enhanced by other classes of inductors.

Stimulation of vascular cell proliferation by beta-galactoside specific lectins.

An investigation was conducted to assess the effects of various beta-galactoside specific lectins on the growth of vascular cells in vitro. The plant lectins from peanut (Arachis hypogaea), mushroom (Agaricus bisporus), and coral tree (Erythrina corallodendron) were used in these studies with the ultimate purpose of comparing those findings with data derived with the lectin isolated from rat lung. Peanut lectin was added to confluent and subconfluent cultures of smooth muscle cells (SMC), pulmonary arterial (PEC), and aortic endothelial cells (BAEC) at concentrations of 2, 3.5, and 7.0 micrograms/ml. There was a dose-dependent increase in cell proliferation for both confluent and subconfluent SMC, with maximal stimulation noted between 3.5 and 7 micrograms/ml of peanut lectin. A dose-dependent stimulation of PEC proliferation was also found with maximal stimulation between 3.5 and 7.0 micrograms/ml. Peanut lectin did not stimulate BAEC to multiply. The stimulation of PEC and SMC by peanut lectin could be prevented by the addition of 50 mM lactose. Peanut and mushroom lectin stimulated the proliferation of sparse cultures of SMC in a dose-dependent fashion in both standard (10% fetal bovine serum, or FBS) or low (0.5% FBS) serum to about the same degree. Coral tree lectin did not have a significant stimulation of proliferation under either serum conditions. The incorporation of [3H]thymidine into the DNA of PEC was increased 30 and 150% by peanut lectin and lung galaptin, respectively, under standard serum conditions. However, under low serum conditions, both lectins increased incorporation by about the same extent (93 and 78% for peanut lectin and galaptin, respectively). Both lectins produced a 30% increase in DNA synthesis by SMC under standard serum conditions, and about a 200% increase under low serum conditions. These studies indicate that beta-galactoside specific lectins such as lung galaptin have mitogenic activity toward vascular cells.

Increased secretion of tumor necrosis factors alpha, interleukin 1, and interleukin 6 by human mononuclear cells exposed to beta-galactoside-specific lectin from clinically applied mistletoe extract.

A beta-galactoside-specific lectin from proprietary mistletoe extract, recently reported to exhibit immunomodulatory potency in vivo (T. Hajto, K. Hostanska, and H J. Gabius, Cancer Res. 49: 4803-4808, 1989), induced increased secretion of tumor necrosis factor alpha, interleukin 1, and interleukin 6 in cultures of human peripheral blood mononuclear cells. The enhancement of secretion, determined independently by bioassays and enzyme-linked immunosorbent assay-based quantitation, was caused by selective protein-carbohydrate interaction, as revealed by the strict dependence on the presence of the carbohydrate-binding subunit of the specific lectin-binding sugar as well as anti-lectin antibodies. Increased cytokine levels in serum of patients after injection of optimal lectin doses corroborated the in vitro results. Thus, these data provide an explanation for the increases in cellular parameters of the host defense system in vivo, which presents a further step toward their potentially beneficial clinical exploitation in standardized regimens.

Effect of Trichosanthes kirilowii lectin on lipolysis and lipogenesis in isolated rat and hamster adipocytes.

The galactose-binding lectin from the Chinese herb Trichosanthes kirilowii (Tianhuafen) stimulated the incorporation of D-[3-3H]glucose into lipids in isolated rat epididymal adipocytes. The lectin, however, did not inhibit lipolysis induced by either epinephrine or corticotropin in these adipocytes. Similarly, it did not suppress corticotropin-induced lipolysis in isolated hamster adipocytes. At a dose that did not stimulate lipolysis on its own, the lectin slightly potentiated the lipolytic effects of corticotropin and epinephrine. These findings are discussed in relation to previous observations on other galactose-binding lectins.

Ulex seed extracts: lymphocyte growth inhibition and the anti-H hemagglutinins.

Extracts produced from the seeds of Ulex europaeus are commonly used for their ability to react with the H basic substance present on erythrocytes and secreted in body fluids. Such extracts were found to also contain a potent inhibitor of human and murine lymphocyte growth. Inhibition of growth does not result from cytotoxicity and is easily reversible. Ulex seed extract (USE) solutions were modified in various ways to produce reagents in which the anti-H hemagglutinins were either retained or removed. The fractionated solutions were then analyzed for hemagglutination and lymphocyte growth-inhibiting activity. Such studies clearly indicated that these two biological functions resulted from the action of different materials. The lymphocyte growth inhibitor is not a glycoprotein lectin. It does not mediate its effect through the H basic substance and is a heat-stable, small molecule. The data suggest that plant seed extracts employed for their lectin content may contain an additional class of biologically active agents potentially useful in man.