G protein-coupled receptor kinase interactor 2 (GIT2) is a signaling scaffold protein involved in regulation of cytoskeletal dynamics and the internalization of G protein-coupled receptors (GPCRs). The short-splice form of GIT2 is expressed in peripheral T cells and thymocytes. However, the functions of GIT2 in T cells have not yet been determined. We show that treatment with Con A in a model of polyclonal T-lymphocyte activation resulted in marked inhibitions in the intrahepatic infiltration of inflammatory cells, cytokine response and acute liver failure in Git2 (-/-) mice. CD4(+) T cells from Git2 (-/-) mice showed significant impairment in proliferation, cytokine production and signal transduction upon TCR-stimulated activation. Our results suggested that GIT2 plays an important role in T-cell function in vivo and in vitro.

Hydrogen sulfide (H2S) is known to exert anti-inflammatory properties. Apoptosis and autophagy play important roles in concanavalin A (Con A)-induced acute hepatitis. The purpose of this study was to explore both the effect and mechanism of H2S on Con A-induced acute hepatitis.

Halofuginone (HF) is an active component of extracts derived from the plant alkaloid febrifugine and has shown therapeutic promise in animal models of fibrotic disease. Our main objectives were to clarify the suppressive effect of HF on concanavalin A (ConA)-induced liver fibrosis. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, while orally administration of HF significantly decreased the levels of the transaminases. In addition, the levels of hyaluronic acid (HA), procollagen III (PCIII) and TGF-β1 in the serum and collagen I, α-SMA, tissue inhibitors of metalloproteinase 2 (TIMP2) and Smad3 in the liver tissue were significantly lowered with the treatment of HF. Histological examination also demonstrated that HF significantly reduced the severity of liver fibrosis. Since ConA-induced liver fibrosis is caused by the repeated activation of T cells, immunomodulatory substances might be responsible for the suppressive effect of HF. We found that the production of nuclear factor (NF)-kB in the serum was increased in ConA-treated group, while decreased significantly with the treatment of HF. The changes of inflammatory cytokines tumor necrosis factor (TNF-α), IL-6 and IL-1β in the serum followed the same rhythm. All together, our findings indicate that orally administration HF (10ppm) would attenuate the liver fibrosis by suppressing the synthesis of collagen I and inflammation-mediated liver injury.

A novel in vitro system was employed to investigate liver tissue respiration (mitochondrial O2 consumption) in mice treated with concanavalin A (Con A). This study aimed to investigate hepatocyte bioenergetics in this well-studied hepatitis model.

Glycoproteins have immense clinical importance and comparative glycoproteomics has become a powerful tool for biomarker discovery and disease diagnosis. Seminal plasma glycoproteins participate in fertility related processes including sperm-egg recognition, modulation of capacitation and acrosome reaction inhibition. Affinity chromatography using broad specificity lectin such as Con A is widely applied for glycoproteins enrichment. More notably, Con A-interacting fraction of human seminal plasma has decapacitating activity which makes this fraction critically important. In our previous study, we isolated Con A-interacting glycoproteins from human seminal plasma and subsequently identified them by mass spectrometry. Here, we report the computational analysis of these proteins using bioinformatics tools. The analysis includes: prediction of glycosylation sites using sequence information (NetNGlyc 1.0), functional annotations to cluster these proteins into various functional groups (InterProScan and Blast2GO) and identification of protein interaction networks (STRING database). The results indicate that these proteins are involved in various biological processes including transport, morphogenesis, metabolic processes, cell differentiation and homeostasis. The clusters illustrate two major molecular functions - hydrolase activity (6) and protein (4)/carbohydrate (1)/lipid binding (1). The large interactomes of proteins point towards their versatile roles in wide range of biological processes.

Sample multiplexing facilitates single-cell sequencing by reducing costs, revealing subtle difference between similar samples, and identifying artifacts such as cell doublets. However, universal and cost-effective strategies are rather limited. Here, we reported a concanavalin A-based sample barcoding strategy (CASB), which could be followed by both single-cell mRNA and ATAC (assay for transposase-accessible chromatin) sequencing techniques. The method involves minimal sample processing, thereby preserving intact transcriptomic or epigenomic patterns. We demonstrated its high labeling efficiency, high accuracy in assigning cells/nuclei to samples regardless of cell type and genetic background, and high sensitivity in detecting doublets by three applications: 1) CASB followed by scRNA-seq to track the transcriptomic dynamics of a cancer cell line perturbed by multiple drugs, which revealed compound-specific heterogeneous response; 2) CASB together with both snATAC-seq and scRNA-seq to illustrate the IFN-γ-mediated dynamic changes on epigenome and transcriptome profile, which identified the transcription factor underlying heterogeneous IFN-γ response; and 3) combinatorial indexing by CASB, which demonstrated its high scalability.

The effects of temperature and pH on the reversible dimer-tetramer association of concanavalin A were studied by the high-speed sedimentation equilibrium technique. Both commercial and highly purified preparations of concanavalin A were used. Equilibrium constants were analyzed as a Wyman linked function by using truncated van't Hoff temperature dependence. In the concentration range 0.1-3.0 mg/mL, from 5 to 35 degrees C and between pH 5.5 and pH 7.5 at 0.5 M ionic strength, only dimer and tetramer species were present in both preparations. For purified concanavalin A, association constants ranged from 1.5 X 10(3) to 8.0 X 10(7) M-1. Constants for our commercial preparation were approximately 10-fold lower due to the decrease competency of some subunits to self-associate. From the fit of the Wyman model to the experimental data, delta G degree, delta H degree, delta S degree, and delta Cp degree were calculated for the association and association-linked ionization reactions. From the values of the ionization thermodynamic parameters, the association is governed by the ionization of a histidine side chain on each subunit, either histidine-51 or histidine-121. The association is characterized by large entropy (66.3 cal.mol-1.deg-1 at 25 degrees C) and heat capacity (-821 cal.mol-1.deg-1) changes in accordance with the large hydrophobic association surface observed in crystallographic studies [Reeke, G. N., Jr., Becker, J. W., & Edelman, G. M. (1975) J. Biol. Chem. 250, 1525-1547]. In addition, there is a large enthalpy change (10.4 kcal.mol-1 at 25 degrees C). We propose a model for the interaction based on a more detailed thermodynamic description than was obtained in an earlier, incomplete study [Huet, M., & Clavarie, J. M. (1978) Biochemistry 17, 236-241].

Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to contribute to the inflammatory process. However, the protective effect of ethyl pyruvate on Concanavalin A (Con A)-induced autoimmune hepatitis have not been explored. Thus, the aims of this study are to investigate both the effects of ethyl pyruvate and its mechanism of protection on Con A-induced autoimmune hepatitis in mice.

Concanavalin A (ConA), a legume lectin, has been drawing increasing attention in recent years concerning its toxicity against insects and its potential application in pest management. In an attempt to evaluate the effect of ConA on potato psyllid (Bactericera cockerelli), an economically important pest of solanaceous crops, the effect of ConA on potato psyllid survival, psyllid gut nuclear morphology, and expression of psyllid caspase genes were evaluated. Our results determined that artificial diet-feeding assays using ConA had deleterious effects on potato psyllids, resulting in significant psyllid mortality following ingestion. We also found that an apoptotic response was induced by ConA in psyllid midgut cells, which was demonstrated by the DNA fragmentation and abnormal nuclear architecture in the midgut cells. Following ConA ingestion, there was also upregulation of caspase genes in the psyllid midguts. Therefore, a key mechanism behind ConA toxicity towards potato psyllid probably involves the induction of apoptosis in midgut cells. This study could provide a better understanding of the mechanisms underlying ConA toxicity in insects and be a stepping stone towards the development of new psyllid control strategies based on plant lectins.

Galangin is an active pharmacological ingredient from propolis and Alpinia officinarum Hance, and has been reported to have anti-inflammatory and antioxidative properties. The present study aims to reveal the effect of galangin on Concanavalin A (ConA)-induced hepatitis (CIH), a well-established animal model of immune-mediated liver injury, and to clarify the related mechanism. C57BL/6 mice were pretreated with galangin followed by ConA challenge. Results indicated that galangin inhibited ConA-induced liver damage. Mice pretreated with galangin showed more reduction of liver damage when compared with control mice pretreated with vehicle solution. In galangin-pretreated mice with induced CIH, increases in serum levels of several inflammatory cytokines, including tumor necrosis factor-α, interferon-γ, and interleukin-12 were dramatically attenuated, and chemokines and adhesion molecules like interferon inducible protein-10, macrophage inflammatory protein-1α, and inter-cellular adhesion molecule-1 messenger RNA expressions in liver were decreased. Moreover, CIH mice pretreated with galangin showed less leukocyte infiltration and T-cell activation in the liver. Further, the mechanism of the anti-inflammatory effects of galangin may be attributed to its modulation of crucial inflammatory signaling pathways, including nuclear factor kappa B and interferon-gamma/signal transducer and activator of transcription 1. Collectively, these findings suggest the preventive and therapeutic potential of galangin in immune-mediated liver injury in vivo.

Background and Aims: Acute liver failure (ALF) is a type of liver injury that is caused by multiple factors and leads to severe liver dysfunction; however, current treatments for ALF are insufficient. Magnesium isoglycyrrhizinate (MgIG), a novel glycyrrhizin extracted from the traditional Chinese medicine licorice, has a significant protective effect against concanavalin A (ConA)-induced liver injury, but its underlying therapeutic mechanism is unclear. Hence, this study aims to explore the potential therapeutic mechanism of MgIG against ConA-induced immune liver injury. Methods: ConA (20 mg/kg, i. v.) was administered for 12 h to construct an immune liver injury model, and the treatment group was given MgIG (30 mg/kg, i. p.) injection 1 h in advance. Lethality, liver injury, cytokine levels, and hepatocyte death were evaluated. The level of autophagy was evaluated by electron microscopy, RT-PCR and western blotting, and hepatocyte death was assessed in vitro by flow cytometry. Results: MgIG significantly increased the survival rate of mice and ameliorated severe liver injury mediated by ConA. The decrease in the number of autophagosomes, downregulation of LC3b expression and upregulation of p62 expression indicated that MgIG significantly inhibited ConA-induced autophagy in the liver. Reactivation of autophagy by rapamycin (RAPA) reversed the protective effect of MgIG against ConA-induced liver injury. Compared with MgIG treatment, activation of autophagy by RAPA also promoted the expression of liver inflammation markers (IL-1β, IL-6, TNF-α, CXCL-1, CXCL-2, CXCL-10, etc.) and hepatocyte death. In vitro experiments also showed that MgIG reduced ConA-induced hepatocyte death but did not decrease hepatocyte apoptosis by inhibiting autophagy. Conclusion: MgIG significantly ameliorated ConA-induced immune liver injury in mice by inhibiting autophagy. This study provides theoretical support for the ability of MgIG to protect against liver injury in clinical practice.

Kainate receptors are members of the ionotropic glutamate receptor family. They form cation-specific transmembrane channels upon binding glutamate that desensitize in the continued presence of agonists. Concanavalin A (Con-A), a lectin, stabilizes the active open-channel state of the kainate receptor and reduces the extent of desensitization. In this study, we used single-molecule fluorescence resonance energy transfer (smFRET) to investigate the conformational changes underlying kainate receptor modulation by Con-A. These studies showed that Con-A binding to GluK2 homomeric kainate receptors resulted in closer proximity of the subunits at the dimer-dimer interface at the amino-terminal domain as well as between the subunits at the dimer interface at the agonist-binding domain. Additionally, the modulation of receptor functions by monovalent ions, which bind to the dimer interface at the agonist-binding domain, was not observed in the presence of Con-A. Based on these results, we conclude that Con-A modulation of kainate receptor function is mediated by a shift in the conformation of the kainate receptor toward a tightly packed extracellular domain.

Single-cell/nucleus sequencing has been increasingly used to study specific cell populations. However, cells/nuclei often become diluted during isolation steps and are difficult to reconcentrate through centrifugation. Here, we present a protocol for sample enrichment using concanavalin A-conjugated magnetic beads. We describe steps for dissection, nuclei isolation, and fluorescence-activated cell sorting (FACS). We then detail procedures for nuclei enrichment and library preparation. This protocol enables efficient retrieval and enrichment of cells/nuclei following FACS and integrates into existing workflows of various 10× Genomics applications.

Serotonin is involved in the pathological processes of several liver diseases via the regulation of inflammatory response and oxidative stress. We aimed to investigate the role of serotonin in Concanavalin A- (Con A-) induced acute liver injury (ALI). ALI was induced in C57B/6 wild-type (WT) mice and tryptophan hydroxylase 1 (TPH1) knockout mice through tail vein injection of Con A (15 mg/kg body weight). Another group of TPH1 knockout ALI mice was supplied with 5-hydroxytryptophan (5-HTP) in advance to recover serotonin. The blood and liver tissues of mice were collected in all groups. Markedly increased serum levels of serotonin were identified after the injection of Con A. Increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and stronger hepatic tissue pathology were detected, suggesting that serotonin could mediate Con A-induced liver damage. Serotonin significantly facilitated the release of serum and intrahepatic inflammatory cytokines, including interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-17A (IL-17A), interferon-gamma (IFN-γ), and tumor necrosis-alpha (TNF-α), after the administration of Con A. In addition, serotonin significantly increased the intrahepatic levels of oxidative stress markers malonaldehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO) and decreased antioxidant stress indicator glutathione (GSH) in Con A-treated mice. Additionally, serotonin promoted hepatocyte apoptosis and autophagy based on B-cell lymphoma-2 (Bcl-2), Bcl-2-asociated X protein (Bax), and Beclin-1 levels and TUNEL staining. More importantly, serotonin activated nuclear factor kappa B (NF-κB) and upregulated the hepatic expressions of high mobility group protein B1 (HMGB1), toll-like receptor-4 (TLR4), and downstream molecules in Con A-mediated liver injury. Serotonin 2A receptor was upregulated in liver tissue after Con A injection, and serotonin 2A receptor antagonist Ketanserin protected against Con A-induced hepatitis. These results indicated that serotonin has the potential to aggravate Con A-induced ALI via the promotion of inflammatory response, oxidative stress injury, and hepatocyte apoptosis and the activation of hepatic HMGB1-TLR signaling pathway and serotonin 2A receptor.

Salidroside (Sal) is a glycoside extract from Rhodiola rosea L. with anti-inflammatory, antioxidant, anticancer and cardioprotective properties. The present study explored the protective effects and the possible mechanisms of Sal on concanavalin A (ConA)-induced liver injury in mice. Balb/C mice were divided into five groups: Normal control (injected with normal saline), ConA (25 mg/kg), Sal (10 mg/kg) +ConA, Sal (20 mg/kg) + ConA (Sal injected 2 h prior to ConA injection) and Sal (20 mg/kg) only. The serum levels of liver enzymes, pro-inflammatory cytokines, and apoptosis- and autophagy-associated marker proteins were determined at 2, 8 and 24 h after ConA injection. LY294002 was further used to verify whether the phosphoinositide 3-kinase (PI3K)/Akt pathway was activated. Primary hepatocytes were isolated to verify the effect of Sal in vitro. The results indicated that Sal was a safe agent to reduce pathological damage and serum liver enzymes in ConA-induced liver injury. Sal suppressed inflammatory reactions in serum and liver tissues, and activated the PI3K/Akt signaling pathway to inhibit apoptosis and autophagy in vivo and in vitro, which could be reversed by LY294002. In conclusion, Sal attenuated ConA-induced liver injury by modulating PI3K/Akt pathway-mediated apoptosis and autophagy in mice.

RNA interference-based gene silencing drugs are attracting attention for treating various diseases. Lipid nanoparticles (LNPs) are carriers that efficiently deliver small interfering RNA (siRNA) to the cytoplasm of target cells. Recently, we developed potent and well-tolerated biodegradable LNPs with asymmetric ionizable lipids. Here, we evaluated the effect of LNPs on immune cells in mice. After intravenous administration, LNPs were efficiently incorporated into several tissue-resident macrophages, including liver macrophages, through an apolipoprotein E (ApoE)-independent mechanism. Administration of LNP-encapsulated siRNA against Irf5, encoding the transcription factor critical for inflammatory responses, sharply reduced its expression in macrophages in vivo, and persisted for as long as 7 days. The therapeutic potential of Irf5 siRNA-loaded LNPs in inflammatory diseases was tested in a concanavalin A (Con A)-induced hepatitis model, whose pathogenic mechanisms are dependent on cytokine secretion from macrophages. We found that Con A-induced liver injury was significantly attenuated after LNP injection. Serum aspartate transaminase, alanine aminotransferase, and inflammatory cytokine levels were significantly reduced in mice injected with Irf5 siRNA-loaded LNPs compared to those injected with control siRNA-loaded LNPs. Our results suggest that administering biodegradable LNPs to deliver siRNA is a promising strategy for treating inflammatory disorders.

S-Propargyl-cysteine (SPRC), an endogenous H2S modulator, exerts anti-inflammatory effects on cardiovascular and neurodegenerative disease, but it remains unknown whether SPRC can prevent autoimmune hepatitis.

The main biologically active components of plants belonging to the genus Allium, responsible for their biological activities, including anti-inflammatory, antioxidant and immunomodulatory, are organosulfur compounds. The aim of this study was to synthetize the mixture of dipropyl polysulfides (DPPS) and to test their biological activity in acute hepatitis. C57BL/6 mice were administered orally with DPPS 6 h before intravenous injection of Concanavalin A (ConA). Liver inflammation, necrosis and hepatocytes apoptosis were determined by histological analyses. Cytokines in liver tissue were determined by ELISA, expression of adhesive molecules and enzymes by RT PCR, while liver mononuclear cells were analyzed by flow cytometry. DPPS pretreatment significantly attenuated liver inflammation and injury, as evidenced by biochemical and histopathological observations. In DPPS-pretreated mice, messenger RNA levels of adhesion molecules and NADPH oxidase complex were significantly reduced, while the expression of SOD enzymes was enhanced. DPPS pretreatment decreased protein level of inflammatory cytokines and increased percentage of T regulatory cells in the livers of ConA mice. DPPS showed hepatoprotective effects in ConA-induced hepatitis, characterized by attenuation of inflammation and affection of Th17/Treg balance in favor of T regulatory cells and implicating potential therapeutic usage of DPPS mixture in inflammatory liver diseases.

Herein, we report that the concanavalin A binding of Tip60 (a target of the human immunodeficiency virus type 1-encoded transactivator Tat interacting protein 60 KD; a histone acetyltransferase; HAT) is enhanced as the result of endoplasmic reticulum (ER) stress. The cell expression of Tip60 combined with site-directed mutagenesis analysis was used to identify the glutamine 324 residue as the lecithin binding (Concanavalin A; Con A) site. The Tip60 N324A mutant strain, which seems to be the Con A binding-deficient, was attenuated the protein-protein interactions with FE65 and its protein stability, but its ability of G0-G1 cell cycle arrest was not interrupted. Interestingly, both HAT activity and the nuclear localization of Tip60 N324A mutant were enhanced than those of Tip60 WT. Thus, our results indicate that the Con A binding deficient of Tip60 seems to be one of the most pivotal posttranslational modifications (such as N-glycosylation) for its functional regulation signal, which is generated in response to ER stress.

Tissue culture monolayers of seven human intracranial tumours comprising 2 astrocytomas, 3 meningiomas, 1 secondary squamous cell carcinoma and 1 secondary adenocarcinoma were examined by a double immunofluorescent staining technique to demonstrate Concanavalin A (Con A) surface receptors and cytoplasmic actin in the same cell. Tumour cells, treated with fluoresceinisothiocyanate-labelled Con A (FITC-Con A) showed staining in cell margins or in a random distribution over the cell surface. Incubating the cells with FITC-Con A at 37 degrees for increasing periods of time resulted first in staining of clusters and later of perinuclear globules. Cells, pretreated with 4% paraformaldehyde at 4 degrees for 10 min or with cytochalasin B at 37 degrees for 30 min showed staining restricted to cell margins. In the cytochalasin B-treated cells, the peripheral staining was in the form of coarse clusters. Double fluorochrome studies showed that the anti-actin antibody (AAA) staining occurred in sites closely related to those stained by FITC-Con A both in untreated as well as in cytochalasin B-treated cells. The findings suggest that Con A receptors, as an example of a stable cell membrane determinant in human tumour cells, are associated with actin and that their mobility on the cell surface is dependent on an intact cytoplasmic actin system.