Urtica Dioica Agglutinin Research Articles

The J beta segment of the T cell receptor contributes to the V beta-specific T cell expansion caused by staphylococcal enterotoxin B and Urtica dioica superantigens.

We have used a new polymerase chain reaction-based technique to analyze at the clonal level the CDR3 diversity and the J beta usage associated with the V beta-dependent T cell receptor (TCR) recognition of two superantigens: the staphylococcal enterotoxin B and the Urtica dioica agglutinin. Our results show that subset of J beta elements is preferentially expanded in a given V beta family, independently of the nature of the superantigen. By contrast, the CDR3 loop does not contribute significantly to the T cell expansion induced by the superantigens. We conclude that the J beta segment of the TCR beta chain, but not the CDR3 region, participates in superantigen binding, presumably by influencing the quaternary structure of the TCR beta chain.

Vβ-Specific Deletion of Mature Thymocytes Induced by the Plant Superantigen Urtica Dioica Agglutinin

Urtica dioica agglutinin (UDA), a plant protein, is a superantigen activating in a MHC class II-restricted manner the Vβ8.3-bearing T-cells (Galelli and Truffa-Bachi,J. Immunol.151, 1821, 1993). Administration of UDA to adult mice provokes the clonal expansion of the responding cells which is followed by the deletion of the major fraction of the UDA-sensitive cells, whereas the remaining cells become anergic (Galelliet al., J. Immunol.154, 2600, 1995). We have analyzed the effect of UDA on thymocytes. Injection of UDA resulted in a rapid, but transient, deletion of a large fraction of the Vβ8.3-bearing mature T-cells. In contrast to other exogenous superantigens, this deletion was not preceded by the clonal expansion of the UDA-responding thymocytes. Moreover, the Vβ8.3-bearing mature T-cells escaping the deletion were not anergic to anin vitroUDA restimulation. UDA and the other superantigens also differ as the general, Vβ-unrestricted, thymic atrophy induced by classical superantigens was not observed with UDA.

Binding capacities of two immunomodulatory lectins, carrier-immobilized glycoligands and steroid hormones in lung cancer and the concentration of nitrite/nitrate in pleural effusions.

Combined analysis of the binding properties of inflammatory and tumor cells in pleural effusion, and tumor imprints for various carrier-immobilized types of ligands and lectins, and of a biochemical feature of the effusions is performed to extend the characterization of these cells and their activity. In detail, the binding of Viscum album agglutinin (VAA), Urtica dioica agglutinin (UDA), and of carrier-immobilized N-acetyl-D-glucosamine (GlcNAc), lysoganglioside GM1, estradiol, progesterone, testosterone, and hydrocortisone to native specimens consisting of 46 tumor imprints from surgically treated patients with lung cancer and 74 smears of pleural effusion (PE) cells from cancer or non-cancer patients was studied using fluorescence microscopy with Texas red-labeled streptavidin. Among the tested ligands, VAA was found to provide the most effective staining of cells (60-78.1% of positive cases). When compared with inflammatory cells from PE, cancer cells were seen to bind more frequently only two ligands, namely UDA and estradiol. Significant (P < 0.001) difference between patients with bronchial carcinoma and non-cancer patients were found, when the content of NO2-/NO3- in PE fluids was measured. Whereas the level of NO2-/NO3- in PE of non-cancer patients was 12.6 +/- 10.7 microM (n = 12), it was 37.7 +/- 19.4 microM (n = 14) in cancer patients without pleural metastases and 37.5 +/- 16.0 microM (n = 26) in patients with pleural metastases. The level of NO2-/NO3- in PE appeared to correlate with extent of staining with GM1 and GlcNAc: in non-cancer patient groups it was significantly higher (P = 0.032) for negative subjects than those binding the ligand GlcNAc, whereas in the patient group with adenocarcinoma it was significantly lower (P = 0.032) for patients without binding capacities for GlcNAc and GM1. The results obtained suggest that the combined analysis of increased levels of NO2-/NO3- in PE and of glycohistochemical properties of cancer and inflammatory cells may be useful in exploring the interrelationship of functionally important cellular characteristics.

Studies on the binding of Urtica dioica agglutinin (UDA) and other lectins in an in vitro epidermal growth factor receptor test

Urtica dioica agglutinin (UDA), a GlcN-Ac-specific lectin from the rhizomes of Urtica dioica, was found to inhibit the binding of (125)I-labelled epidermal growth factor (EGF) to its receptor (EGF-R) on A431 epidermoid cancer cells at concentrations of 500ng/ml to 100μg/ml. The effects of other lectins such as wheat germ agglutinin (WGA), which possesses the same sugar specificity, and the mannose-specific agglutinin from Canavalia ensiformis (Con A) were less pronounced. The inhibitory effect of UDA could be antagonized by the GlcN-Ac trimer (N,N',N″-triacetylchitotriose), suggesting hat UDA may be the major antiprostatic compound of Urtica dioica drug, acting by blocking the EGF-R in prostate tissues.

Different antimetabolic effects of related lectins towards nymphal stages of Nilaparvata lugens

AbstractInsect feeding trials were carried out to determine the effects of a range of mannose‐specific lectins on third instar nymphs of the rice brown planthopper (BPH), Nilaparvata lugens. Stål. Dose response curves show that Galanthus nivalis agglutinin (GNA) has the strongest toxic effect of the lectins tested, and is effective at concentrations considerably lower than those previously reported. Narcissus pseudonarcissus agglutinin (NPA) and Allium sativum agglutinin (ASA) exhibit a significant antimetabolic effect towards the insect but were less effective (on a molar basis) than GNA. LC50 values for GNA, NPA and ASA are approximately 4 μM, 11 μM and &gt;40 μM respectively. These mannose‐specific lectins are serologically identical, but differ in the number of subunits per protein molecule; ASA is a dimer, NPA is a trimer and GNA is a tetramer. The results obtained support the hypothesis, that the effectiveness of the mannose‐binding lectins as antimetabolites is determined by the number of subunits per molecule. Two N‐acetylglucosamine binding lectins, the dimeric Oryza sativa agglutinin (OSA) and the monomeric Urtica dioica agglutinin (UDA), were also tested but at a concentration of 0.1% w/v exhibited no significant antimetabolic effect towards BPH, although the related lectin wheatgerm agglutinin (WGA) has previously been demonstrated to be toxic towards the insect.

Ligand-induced perturbations in Urtica dioica agglutinin

The binding of the trisaccharide, N, N′, N″-triacetylchitotriose, to Urtica dioica agglutinin (UDA) was investigated using 1H NMR spectroscopy. UDA is a small antiviral plant lectin containing two homologous 43-amino acid domains. Carbohydrate-induced perturbations occur in one domain of UDA at trisaccharide concentrations below equimolar. Residues in the second domain are shifted at higher carbohydrate concentrations. This data confirms the presence of two binding sites of non-identical affinities per UDA monomer. Qualitative analysis of the 2D NOESY spectra indicates that UDA contains two short stretches of antiparallel β-sheet. The 1H resonance assignments for both antiparallel β-sheet sequences have been completed and there is one β-stretch per domain. A number of these β-sheet residues are perturbed in the presence of carbohydrate.

Selective expansion followed by profound deletion of mature V beta 8.3+ T cells in vivo after exposure to the superantigenic lectin Urtica dioica agglutinin.

Urtica dioica agglutinin (UDA) is a superantigen that, in vitro, binds to specific carbohydrate structures on class II and induces a sixfold enrichment of V beta 8.3+ BALB/c mice splenic T cells. Superantigens have pleiotropic effects in vivo, causing the activation, proliferation, and deletion of specific T cells, but are heterogenous in regard to their effects on T cell tolerization. We, therefore, compared the responses of peripheral T cells from adult BALB/c mice with the i.v. injection of 50 micrograms UDA or the bacterial superantigen staphylococcal enterotoxin B (SEB) that also recognizes the V beta 8.3 gene product. The data presented indicate that activation, clonal expansion, anergy, and death of V beta 8.3+ T cells occur sequentially after UDA administration. Two days after UDA injection, the proportion of V beta 8.3+ T cells in the periphery is elevated to approximately twice that of normal mice. This expansion occurs in both CD4+ and CD8+ subsets. V beta 8.3+ T cells from UDA-primed mice are anergic to UDA restimulation and fail to proliferate or to produce IL-2. Futhermore, the proliferation of V beta 8.3+ T cells is followed by their rapid disappearance concomitant with their specific elimination by apoptosis. In 1 wk, all CD4+ V beta 8.3+ peripheral T cells are deleted. The decline of V beta 8.3+ T cells in the CD4+ subset is more than in the CD8+ subset. This occurs in thymectomized and in thymus-intact animals. Two months after UDA priming, the percentage of V beta 8.3+ T cells is still lower than in control mice.

The gene for stinging nettle lectin (Urtica dioica agglutinin) encodes both a lectin and a chitinase.

Chitin-binding proteins are present in a wide range of plant species, including both monocots and dicots, even though these plants contain no chitin. To investigate the relationship between in vitro antifungal and insecticidal activities of chitin-binding proteins and their unknown endogenous functions, the stinging nettle lectin (Urtica dioica agglutinin, UDA) cDNA was cloned using a synthetic gene as the probe. The nettle lectin cDNA clone contained an open reading frame encoding 374 amino acids. Analysis of the deduced amino acid sequence revealed a 21-amino acid putative signal sequence and the 86 amino acids encoding the two chitin-binding domains of nettle lectin. These domains were fused to a 19-amino acid "spacer" domain and a 244-amino acid carboxyl extension with partial identity to a chitinase catalytic domain. The authenticity of the cDNA clone was confirmed by deduced amino acid sequence identity with sequence data obtained from tryptic digests, RNA gel blot, and polymerase chain reaction analyses. RNA gel blot analysis also showed the nettle lectin message was present primarily in rhizomes and inflorescence (with immature seeds) but not in leaves or stems. Chitinase enzymatic activity was found when the chitinase-like domain alone or the chitinase-like domain with the chitin-binding domains were expressed in Escherichia coli. This is the first example of a chitin-binding protein with both a duplication of the 43-amino acid chitin-binding domain and a fusion of the chitin-binding domains to a structurally unrelated domain, the chitinase domain.

Effect of the Urtica dioica agglutinin on germination and cell wall formation of Phycomyces blakesleeanus Burgeff

The lectin from stinging nettle rhizomes, Urtica dioica agglutinin (UDA), did not affect the evolution of wet and dry weight, protein, nucleic acid, ATP, cAMP and glycerol content during early germination of Phycomyces blakesleeanus spores. However, earlier investigations established a strongly reduced mycelial growth of several phytopathogenic fungi by this small plant lectin. Total uptake and incorporation of radioactive precursors showed no differences between UDA or control hyphae, but UDA significantly altered the distribution patterns of [14C]-glucose incorporated into the walls of Phycomyces blakesleeanus (more label was recovered in the chitin fraction). Moreover, a small but significant stimulation of chitin synthase and a similar inhibition of chitin deacetylase was found in cell wall preparations. These observations could lead to a better understanding of plant-pathogen interrelationships and to a further elucidation of cell wall structure in fungi.

The primary structure of stinging nettle ( Urtica dioica) agglutinin A two-domain member of the hevein family

The primary structure of stinging nettle ( Urtica dioica) agglutinin has been determined by sequence analysis of peptides obtained from three overlapping proteolytic digests. The sequence of 89 residues consists of two hevein-like domains with the same spacing or half-cystine residues and several other conserved residues as observed earlier in other proteins with hevein-like domains, The hinge region between the two domains is four residues longer than those between the four domains in cereal lectins like wheat germ agglutinin.

Mouse T-lymphocyte activation by Urtica dioica agglutinin : I. — Delineation of two lymphocyte subsets

Urtica dioica agglutinin (UDA) is a mouse T-lymphocyte-specific mitogen endowed with proliferative characteristics different from ConA, the prototypic T-lymphocyte mitogen. In particular, UDA induces 2-3-fold-reduced thymidine incorporation as compared to ConA. In an attempt to define the basis of this reduced proliferation, we analysed whether UDA binds to a unique subset of T lymphocytes or whether it activates only a T-cell subset. Cytofluorimetric analysis showed that this lectin binds uniformly to all T lymphocytes and does not, on this criterion, distinguish a particular T-cell subset. We next analysed whether UDA provokes the activation of all T lymphocytes. This was carried out by measuring the increase in cell size and the induction of the p55 chain of the IL2 receptor. The analysis showed that, throughout the kinetics of cell activation, only one subset of T lymphocytes increased in size and expressed the p55 chain of the IL2 receptor, suggesting that UDA activates only a subpopulation of T cells. This conclusion was strengthened by the analysis of 5-bromo-2-deoxyuridine (BrdU) incorporation into the DNA of UDA-activated cells. Two populations were easily identifiable: a BrdU-negative subset consisting of all the small p55-negative lymphocytes, and a BrdU-labelled subset including all the large p55-positive cells. BrdU was incorporated in both CD4+ and CD8+ cells, indicating that UDA did not distinguish helper from cytotoxic T lymphocytes. In addition to the p55 chain of the IL2R, all cycling cells expressed the Pgp-1 activation marker. The T lymphocytes, which bound UDA but did not proliferate, remained fully susceptible to subsequent stimulation by ConA. In conclusion, the capacity to proliferate upon UDA binding differentiates a UDA-sensitive from a UDA-refractory subset among splenic mouse T lymphocytes.

Mouse T-lymphocyte activation by Urtica dioica agglutinin : II. — Original pattern of cell activation and cytokine production induced by UDA

Urtica dioica agglutinin (UDA) is a T-lymphocyte-specific polyclonal activator that differs from ConA, the classical mouse T-cell mitogen, by inducing a late and limited proliferation of a distinct T-cell subset recruited among both CD4+ and CD8+ lymphocytes. We investigated the possibility that the particular kinetics may originate from UDA-specific activation processes in which the known early mandatory signals were completed only after an extended delay. We report that the time of contact required between lectin and the cell membrane to acquire the capacity to proceed into cell cycle was much longer (36-40 h) for UDA than for ConA (8-10 h). Addition of phorbol ester, which artificially induces PKC translocation, or ionomycin, which provokes Ca2+ mobilization, did not accelerate the proliferative kinetics, suggesting that these early mandatory signals are not the limiting factors in the delayed proliferation. The induction of c-myc was retarded in the UDA group, and there was a good correlation between the kinetics of c-myc induction and the kinetics of cell proliferation. The comparison of the level of transcription of the genes encoding different cytokines revealed additional differences between the two mitogens: the whole wave of cytokine gene expression was delayed with UDA. In particular, IL2, IL3 and IFN gamma gene expression was retarded compared to the ConA-induced single wave. An even later transcriptional wave took place at around 72 h for IL4 and IL5. Finally, this particular kinetics corresponded to an unusually high level of IL3 and IFN gamma and a low level of IL4 and IL5 gene transcripts.(ABSTRACT TRUNCATED AT 250 WORDS)

A technique for detecting antifungal activity of proteins separated by polyacrylamide gel electrophoresis

A technique was developed for the detection of antifungal activity of proteins after discontinuous polyacrylamide gel electrophoresis under native conditions. The antifungal activity is detected as growth inhibition zones in a homogeneous fungal lawn, grown in an agar layer spread on top of the polyacrylamide gel. The position of proteins with antifungal activity can be determined on a diffusion blot prepared from the same gel. The technique is illustrated for three antifungal plant proteins, i.e. alpha-purothionin, Urtica dioica agglutinin, and tobacco chitinase.

Urtica dioica agglutinin, a new mitogen for murine T lymphocytes: Unaltered interleukin-1 production but late interleukin 2-mediated proliferation

Urtica dioica agglutinin, a small-molecular-weight lectin purified from stinging nettle rhizomes, induces murine cell proliferation. U. dioica agglutinin is a specific T-cell mitogen for both thymocytes and spleen T lymphocytes; its mitogenic properties are strictly dependent on the presence of accessory cells. The kinetics of proliferation are markedly different from those of the classical T-cell mitogen concanavalin A, with a 2 to 3-day delay for both splenic and thymic populations and a rate of DNA synthesis twofold lower than that observed with concanavalin A. The late T-lymphocyte proliferation induced by U. dioica agglutinin correlates well with (i) the observed late interleukin-2 production and interleukin-2 receptor expression, and (ii) the long-lasting cyclosporin A-sensitive early activation period. In contrast, the production of interleukin-1 is not different, both in terms of concentration and kinetics, from that observed with concanavalin A.