Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.

Aspergillus niger lectin elicits MyD88 dependent proliferation and apoptosis at lower and higher doses in immortalized human corneal epithelial cells leading to pathogenesis.

An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.

Green tea polyphenols mitigate the plant lectins-induced liver inflammation and immunological reaction in C57BL/6 mice via NLRP3 and Nrf2 signaling pathways.

Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including hepatitis, inflammatory bowel disease, diabetes, and celiac disease. In this present study, we aimed to assess whether green tea polyphenols (GTPs) exerts protective effects against plant lectins-induced liver inflammation and immunological reaction in mice. The C57BL/6 mice received intragastric GTPs (200 mg/kg b.w.) once per day for 7 consecutive days prior to plant lectins stimulation (50 mg/kg b.w., intraperitoneally). GTPs supplementation alleviated the histopathological changes of liver and the disorder of serum biochemical parameters in plant lectins-challenged mice. GTPs supplementation also alleviated plant lectins-induced oxidative stress and liver inflammation, decreasing protein contents and gene expression levels of pro-inflammatory cytokines in the plasma and hepatic tissue and increasing antioxidant capacity in the liver. GTPs decreased the protein expression levels of myeloperoxidase, F4/80 and neutrophil, as determined by immunohistochemical analysis, and T lymphocytes (CD4 and CD8) contents as determined by immunofluorescence analysis, in the liver. Moreover, we found that GTPs inhibited Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome expression and increased nuclear factor erythroid 2-related factor 2 (Nrf2) pathways in the liver tissues of plant lectins-challenged mice. Taken together, these results show that GTPs alleviates hepatic inflammatory damage and immunological reaction after plant lectins challenge, and GTPs (or green tea intake) supplements can be beneficial for people exposed to plant lectins.

Moringa oleifera seed lectin inhibits Ehrlich ascites carcinoma cell growth by inducing apoptosis through the regulation of Bak and NF-κB gene expression.

A Moringa oleifera seed lectin (MOSL) was purified by using chitin column with the molecular mass of 17±1kDa. The lectin agglutinated mouse, cow and human erythrocytes and the hemagglutination activity was inhibited by methyl-α-d-mannopyranoside, methyl-ß-d-galactopyranoside, lactose and glucose. The lectin exhibited 100% hemagglutination activity at the pH range from 8.0 to 9.0 and temperature range from 30 to 60°C. Additionally, the lectin gradually lost its activity in the presence of urea but the activity abolish completely when treated with EDTA. MOSL showed mild toxicity against brine shrimp nauplii with a LC50 value of 131.0µg/ml. Antiproliferative activity was studied against Ehrlich ascites carcinoma (EAC) cells and 71.08% cell growth inhibition was observed in vitro at 200µg/ml. The lectin was injected (i.p.) into EAC mice at the doses of 2.0 and 4.0mg/kg/day for five consecutive days and 25.38% and 55% of cell growth inhibition was observed, respectively. MOSL caused the cell cycle arrest at G2/M phase as determined by FACS flow cytometry. The cell growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by cell morphological study, caspase-3 inhibitor and activation of Bak and suppression of Bcl-2 and NF-κB genes expression.

Ecotoxicity of water-soluble lectin from Moringa oleifera seeds to zebrafish (Danio rerio) embryos and larvae.

The evaluation of ecotoxicity of mosquito larvicidal agents (such as the water-soluble lectin from Moringa oleifera seeds, WSMoL) is an essential step to establish the guidelines for their use. In this sense, this work evaluated the toxicity of WSMoL to Danio rerio embryos and larvae. Embryos were exposed to waterborne WSMoL (0.0125-0.2 mg mL-1) for 96 h and lethal and sub-lethal effects were observed every 24 h. In the bioassays with larvae, the individuals were exposed to the WSMoL (0.025-0.2 mg mL-1), mortality was recorded daily, and larval swimming velocities were analyzed after 72 h and 168 h of exposure. Additionally, acetylcholinesterase (AChE) activity of larvae was determined after 168 h of exposure. WSMoL LC50 values to embryos were 0.190, 0.133 and 0.049 mg mL-1 after 48, 72 and 96 h, respectively. No toxic endpoint was observed after exposure for 24 h. In addition, hatching was delayed and larval length at 96 h was reduced compared to the control. WSMoL LC50 to larvae were 0.21 and 0.135 mg mL-1, after 24 h and 96 h, respectively. Larvae exposed to 0.1 and 0.2 mg mL-1 showed a decrease in swimming speed and a significant reduction in AChE activity. In conclusion, WSMoL at waterborne concentrations needed for its use as a larvicide to A. aegypti causes lethal and sublethal effects to zebrafish embryos and larvae. Therefore, its use in waterbodies where there are non-target organisms is not recommended.

Carbohydrate recognition by the rhamnose-binding lectin SUL-I with a novel three-domain structure isolated from the venom of globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus.

The globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus contains several biologically active proteins. We have cloned the cDNA of one of the toxin components, SUL-I, which is a rhamnose-binding lectin (RBL) that acts as a mitogen through binding to carbohydrate chains on target cells. Recombinant SUL-I (rSUL-I) was produced in Escherichia coli cells, and its carbohydrate-binding specificity was examined with the glycoconjugate microarray analysis, which suggested that potential target carbohydrate structures are galactose-terminated N-glycans. rSUL-I exhibited mitogenic activity for murine splenocyte cells and toxicity against Vero cells. The three-dimensional structure of the rSUL-I/l-rhamnose complex was determined by X-ray crystallographic analysis at a 1.8 Å resolution. The overall structure of rSUL-I is composed of three distinctive domains with a folding structure similar to those of CSL3, a RBL from chum salmon (Oncorhynchus keta) eggs. The bound l-rhamnose molecules are mainly recognized by rSUL-I through hydrogen bonds between its 2-, 3-, and 4-hydroxy groups and Asp, Asn, and Glu residues in the binding sites, while Tyr and Ser residues participate in the recognition mechanism. It was also inferred that SUL-I may form a dimer in solution based on the molecular size estimated via dynamic light scattering as well as possible contact regions in its crystal structure.

Lectin-Carbohydrate Interactions: Implications for the Development of New Anticancer Agents.

Lectins are a large group of proteins found in animals, plants, fungi, and bacteria that recognize specific carbohydrate targets and play an important role in cell recognition and communication, host-pathogen interactions, embryogenesis, and tissue development. Recently, lectins have emerged as important biomedical tools that have been used in the development of immunomodulatory, antipathogenic, and anticancer agents. Several lectins have been shown to have the ability to discriminate between normal cells and tumor cells as a result of their different glycosylation patterns. Furthermore, the specific binding of lectins to cancer cells has been shown to trigger mechanisms that can promote the death of these abnormal cells. Here, we review the importance of lectins-carbohydrates interactions in cancer therapy and diagnosis. We examine the use of lectins in the modification of nanoparticles (liposomes, solid lipid nanoparticles and other polymers) for anticancer drug delivery. The development of drug delivery systems (liposomes, alginate/chitosan microcapsules, alginate beads) carrying some antitumor lectins is also discussed. In these cases, the processes of cell death induced by these antitumor lectins were also showed (if available). In both cases (lectin-conjugated polymers or encapsulated lectins), these new pharmaceutical preparations showed improved intracellular delivery, bioavailability and targetability leading to enhanced therapeutic index and significantly less side effects.

Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest in Burkitt's lymphoma cells via membrane surface Gb3.

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galß1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G0/1 phase.

Cytotoxic effects of Aeromonas hydrophila culture supernatant on peripheral blood leukocytes of Nile tilapia (Oreochromis niloticus): Possible presence of a secreted cytotoxic lectin.

Number of exotoxins like haemolysin, leukocidin, aerolysin etc. were reported from Aeromonas hydrophila. In this study, we report the haemolytic and cytotoxic effect of A. hydrophila culture supernatant (CS) that is specifically inhibited by lactose and also by serum and mucus of Nile tilapia (Oreochromis niloticus). Hence, we assume the presence of a secreted lectin in the CS. CS is toxic to peripheral blood leukocytes (PBL) of O. niloticus as revealed by MTT assay and by flow cytometry. DNA laddering assay indicates that CS causes necrosis to PBL. As a result of necrosis, CS treated PBL showed increased production of reactive oxygen species as indicated by nitroblue tetrazolium and 2',7' -dichlorofluorescin diacetate assays. CS treated PBL showed reduced mRNA expression of TNF-α and IFN-γ genes. When CS was subjected to polyacrylamide gel electrophoresis, it showed a single band corresponding to the molecular weight of 45 kDa. However, upon concentrating the CS by ultrafiltration, many bands were visualized. Further studies at molecular level are required to unravel this macromolecular-leukocyte interaction which would ultimately benefit the aquaculture industry.

Roles of isolectin B4-binding afferents in colorectal mechanical nociception.

Isolectin B4-binding (IB4+) dorsal root ganglion (DRG) neurons are distinct from peptidergic DRG neurons in their terminal location in the spinal cord and respective contributions to various classes and modalities of nociception. In DRG neurons innervating the mouse colon (c-DRG neurons), the reported proportion of IB4+ population is inconsistent across studies, and little is known regarding their role in colorectal mechanonociception. To address these issues, in C57BL/6J mice, we quantified IB4+ binding after labeling c-DRG neurons with Fast Blue and examined functional consequences of ablating these neurons by IB4-conjugated saporin. Sixty-one percent of Fast Blue-labeled neurons in the L6 DRG were IB4+, and 95% of these IB4+ c-DRG neurons were peptidergic. Intrathecal administration of IB4-conjugated saporin reduced the proportion of IB4+ c-DRG neurons to 37%, which was due to the loss of c-DRG neurons showing strong to medium IB4+ intensity; c-DRG neurons with weak IB4+ intensity were spared. However, this loss altered neither nociceptive behaviors to colorectal distension nor the relative proportions of stretch-sensitive colorectal afferent classes characterized by single-fiber recordings. These findings demonstrate that more than 1 half of viscerosensory L6 c-DRG neurons in C57BL/6J mouse are IB4+ and suggest, in contrast to the reported roles of IB4+/nonpeptidergic neurons in cutaneous mechanonociception, c-DRG neurons with strong-to-medium IB4+ intensity do not play a significant role in colorectal mechanonociception.

Prospecção de novas drogas contra a leishmaniose cutânea: avaliação da atividade biológica da lectina de Oreochromis niloticus / Prospecting for new drugs against cutaneousleishmaniasis: evaluation of biological activity of lectin Oreochromis niloticus

As leishmanioses constituem um grupo de doenças crônicas causadas por protozoários pertencentes ao gênero Leishmania. Tendo em vista a complexidade e ineficácia dos tratamentos atuais, o desenvolvimento de novas drogas menos tóxicas ainda é uma necessidade. Na prospecção de possíveis agentes quimioterápicos contra as leishmanioses, as lectinas apresentam-se como candidatos promissores por apresentarem um amplo espectro de atividades biológicas. No presente trabalho nós investigamos o potencial leishmanicida e imunomodulador da lectina Onil. Nossos resultados demonstraram que a Onil apresentou baixa toxidade sobre células do exsudato peritoneal (CEP) de camundongos (CC50= 317,5 ± 0,6 µg/mL), foi efetiva ao inibir o crescimento de formas promastigotas de L. braziliensis (IC50=150,58± 0,78 µg/mL), mostrou-se mais seletiva para o parasito do que para célula do hospedeiro (ISe=2,1). No entanto, não foi capaz de inibir a sobrevivência das amastigotas no interior das CEPs. A lectina Onil causou alterações ultraestruturais no flagelo, bem como mostrou um efeito sobre a divisão celular de formas promastigotas. A marcação das células tratadas com Anexina V (AV) e Iodeto de Propídio (IP) mostrou uma pequena subpopulação de células apresentava marcação para AV/IP compatíveis com o processo de morte celular por necrose/apoptose tardia. A marcação das células controles e tratadas com Onil com Rho 123 revelou que na grande maioria das células o potencial de membrana mitocondrial foi preservado. O tratamento com a lectina (75-300 µg/mL) não alterou significativamente a produção de NO e não induziu alterações na produção de citocinas em CEPs infectadas L. braziliensis. Por outro lado, uma intensa produção de citocinas associadas aos perfis Th1, Th2 e Th17 foi observada em CEPs não infectadas tratadas com 30 µg/mL da Onil. Nossos dados apontam para uma possível utilização da Onil como agente adjuvante ou como carreadora de drogas para o tratamento da leishmaniose cutânea

Leishmaniasis comprises a group of disease caused by protozoa belonging to the Leishmaniagenus. Taking in account the complexity and inefficiency of current treatments against leishmaniasis, the development of new, less toxic drugs is still needed. In a search of potential chemotherapeutic agents against leishmaniasis, lectins presented as promising candidates because for presenting a broad spectrum of biological activities. In this regard, in the present study we investigated the leishmanicidal and immunomodulatory activities of Onil in vitro. Our results demonstrated that Onil presented low toxicity to mice peritoneal exudate cells (PEC) (CC50= 317.5 ± 0.6 μg / mL), was effective to inhibit the growth of promastigotes of Leishmania braziliensis (IC50= 150.58 ± 0.78 μg / mL) and was shown to be more selective for the parasite than to the host cell, with SeI=2.1...

Structural studies on a non-toxic homologue of type II RIPs from bitter gourd: Molecular basis of non-toxicity, conformational selection and glycan structure.

The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPs, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPs of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The N-glycosylation of the lectin involves a plant-specific glycan while that in toxic type II RIPs of known structure involves a glycan which is animal as well as plant specific.

Novel hemagglutinating, hemolytic and cytotoxic activities of the intermediate subunit of Entamoeba histolytica lectin.

Galactose and N-acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin of Entamoeba histolytica, a common protozoan parasite, has roles in pathogenicity and induction of protective immunity in mouse models of amoebiasis. The lectin consists of heavy (Hgl), light (Lgl), and intermediate (Igl) subunits. Hgl has lectin activity and Lgl does not, but little is known about the activity of Igl. In this study, we assessed various regions of Igl for hemagglutinating activity using recombinant proteins expressed in Escherichia coli. We identified a weak hemagglutinating activity of the protein. Furthermore, we found novel hemolytic and cytotoxic activities of the lectin, which resided in the carboxy-terminal region of the protein. Antibodies against Igl inhibited the hemolytic activity of Entamoeba histolytica trophozoites. This is the first report showing hemagglutinating, hemolytic and cytotoxic activities of an amoebic molecule, Igl.

Schinus terebinthifolius Leaf Extract Causes Midgut Damage, Interfering with Survival and Development of Aedes aegypti Larvae.

In this study, a leaf extract from Schinus terebinthifolius was evaluated for effects on survival, development, and midgut of A. aegypti fourth instar larvae (L4), as well as for toxic effect on Artemia salina. Leaf extract was obtained using 0.15 M NaCl and evaluated for phytochemical composition and lectin activity. Early L4 larvae were incubated with the extract (0.3-1.35%, w/v) for 8 days, in presence or absence of food. Polymeric proanthocyanidins, hydrolysable tannins, heterosid and aglycone flavonoids, cinnamic acid derivatives, traces of steroids, and lectin activity were detected in the extract, which killed the larvae at an LC50 of 0.62% (unfed larvae) and 1.03% (fed larvae). Further, the larvae incubated with the extract reacted by eliminating the gut content. No larvae reached the pupal stage in treatments at concentrations between 0.5% and 1.35%, while in the control (fed larvae), 61.7% of individuals emerged as adults. The extract (1.0%) promoted intense disorganization of larval midgut epithelium, including deformation and hypertrophy of cells, disruption of microvilli, and vacuolization of cytoplasms, affecting digestive, enteroendocrine, regenerative, and proliferating cells. In addition, cells with fragmented DNA were observed. Separation of extract components by solid phase extraction revealed that cinnamic acid derivatives and flavonoids are involved in larvicidal effect of the extract, being the first most efficient in a short time after larvae treatment. The lectin present in the extract was isolated, but did not show deleterious effects on larvae. The extract and cinnamic acid derivatives were toxic to A. salina nauplii, while the flavonoids showed low toxicity. S. terebinthifolius leaf extract caused damage to the midgut of A. aegypti larvae, interfering with survival and development. The larvicidal effect of the extract can be attributed to cinnamic acid derivatives and flavonoids. The data obtained using A. salina indicates that caution should be used when employing this extract as a larvicidal agent.

[Antagonistic effect of gingerols against TNF-α release, ROS overproduction and RIP3 expression increase induced by lectin from Pinellia ternata].

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.

Molecular switch role of Akt in Polygonatum odoratum lectin-induced apoptosis and autophagy in human non-small cell lung cancer A549 cells.

Polygonatum odoratum lectin (POL), isolated from traditional Chinese medicine herb (Mill.) Druce, has drawn rising attention due to its wide biological activities. In the present study, anti-tumor effects, including apoptosis- and autophagy-inducing properties of POL, were determined by a series of cell biology methods such as MTT, cellular morphology observation, flow cytometry, immunoblotting. Herein, we found that POL could simultaneously induce apoptosis and autophagy in human non-small cell lung cancer A549 cells. POL initiated apoptosis through inhibiting Akt-NF-κB pathway, while POL triggered autophagy via suppressing Akt-mTOR pathway, suggesting the molecular switch role of Akt in regulating between POL-induced apoptosis and autophagy. Moreover, ROS was involved in POL-induced inhibition of Akt expression, and might therefore mediate both apoptosis and autophagy in A549 cells. In addition, POL displayed no significant cytotoxicity toward normal human embryonic lung fibroblast HELF cells. Due to the anti-tumor activities, POL might become a potent anti-cancer drug in future therapy, which might pave the way for exploring GNA-related lectins into effective drugs in cancer treatment.

Rapid assays for lectin toxicity and binding changes that reflect altered glycosylation in mammalian cells.

Glycosylation engineering is used to generate glycoproteins, glycolipids, or proteoglycans with a more defined complement of glycans on their glycoconjugates. For example, a mammalian cell glycosylation mutant lacking a specific glycosyltransferase generates glycoproteins, and/or glycolipids, and/or proteoglycans with truncated glycans missing the sugar transferred by that glycosyltransferase, as well as those sugars that would be added subsequently. In some cases, an alternative glycosyltransferase may then use the truncated glycans as acceptors, thereby generating a new or different glycan subset in the mutant cell. Another type of glycosylation mutant arises from gain-of-function mutations that, for example, activate a silent glycosyltransferase gene. In this case, glycoconjugates will have glycans with additional sugar(s) that are more elaborate than the glycans of wild type cells. Mutations in other genes that affect glycosylation, such as nucleotide sugar synthases or transporters, will alter the glycan complement in more general ways that usually affect several types of glycoconjugates. There are now many strategies for generating a precise mutation in a glycosylation gene in a mammalian cell. Large-volume cultures of mammalian cells may also generate spontaneous mutants in glycosylation pathways. This article will focus on how to rapidly characterize mammalian cells with an altered glycosylation activity. The key reagents for the protocols described are plant lectins that bind mammalian glycans with varying avidities, depending on the specific structure of those glycans. Cells with altered glycosylation generally become resistant or hypersensitive to lectin toxicity, and have reduced or increased lectin or antibody binding. Here we describe rapid assays to compare the cytotoxicity of lectins in a lectin resistance test, and the binding of lectins or antibodies by flow cytometry in a glycan-binding assay. Based on these tests, glycosylation changes expressed by a cell can be revealed, and glycosylation mutants classified into phenotypic groups that may reflect a loss-of-function or gain-of-function mutation in a specific gene involved in glycan synthesis.

Insights into embryo defenses of the invasive apple snail Pomacea canaliculata: egg mass ingestion affects rat intestine morphology and growth.

BACKGROUND: The spread of the invasive snail Pomacea canaliculata is expanding the rat lungworm disease beyond its native range. Their toxic eggs have virtually no predators and unusual defenses including a neurotoxic lectin and a proteinase inhibitor, presumably advertised by a warning coloration. We explored the effect of egg perivitellin fluid (PVF) ingestion on the rat small intestine morphology and physiology. METHODOLOGY/PRINCIPAL FINDINGS: Through a combination of biochemical, histochemical, histopathological, scanning electron microscopy, cell culture and feeding experiments, we analyzed intestinal morphology, growth rate, hemaglutinating activity, cytotoxicity and cell proliferation after oral administration of PVF to rats. PVF adversely affects small intestine metabolism and morphology and consequently the standard growth rate, presumably by lectin-like proteins, as suggested by PVF hemaglutinating activity and its cytotoxic effect on Caco-2 cell culture. Short-term effects of ingested PVF were studied in growing rats. PVF-supplemented diet induced the appearance of shorter and wider villi as well as fused villi. This was associated with changes in glycoconjugate expression, increased cell proliferation at crypt base, and hypertrophic mucosal growth. This resulted in a decreased absorptive surface after 3 days of treatment and a diminished rat growth rate that reverted to normal after the fourth day of treatment. Longer exposure to PVF induced a time-dependent lengthening of the small intestine while switching to a control diet restored intestine length and morphology after 4 days. CONCLUSIONS/SIGNIFICANCE: Ingestion of PVF rapidly limits the ability of potential predators to absorb nutrients by inducing large, reversible changes in intestinal morphology and growth rate. The occurrence of toxins that affect intestinal morphology and absorption is a strategy against predation not recognized among animals before. Remarkably, this defense is rather similar to the toxic effect of plant antipredator strategies. This defense mechanism may explain the near absence of predators of apple snail eggs.

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.